WO2015089465A1 - Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders - Google Patents

Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders Download PDF

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Publication number
WO2015089465A1
WO2015089465A1 PCT/US2014/070135 US2014070135W WO2015089465A1 WO 2015089465 A1 WO2015089465 A1 WO 2015089465A1 US 2014070135 W US2014070135 W US 2014070135W WO 2015089465 A1 WO2015089465 A1 WO 2015089465A1
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WIPO (PCT)
Prior art keywords
sequence
crispr
target
cas
enzyme
Prior art date
Application number
PCT/US2014/070135
Other languages
French (fr)
Inventor
Sangeeta Bhatia
Charles Rice
Feng Zhang
David Benjamin Turitz COX
Vyas RAMANAN
Robert Schwartz
Amir SHLOMAI
Original Assignee
The Broad Institute Inc.
Massachusetts Institute Of Technology
The Rockefeller University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority to MX2016007324A priority Critical patent/MX2016007324A/en
Priority to BR112016013207A priority patent/BR112016013207A2/en
Priority to EP14830911.5A priority patent/EP3080261B1/en
Priority to CA2932479A priority patent/CA2932479A1/en
Priority to RU2016128077A priority patent/RU2016128077A/en
Priority to EP22182408.9A priority patent/EP4183876A1/en
Priority to EP19168543.7A priority patent/EP3540051B1/en
Priority to KR1020167018647A priority patent/KR20160089530A/en
Application filed by The Broad Institute Inc., Massachusetts Institute Of Technology, The Rockefeller University filed Critical The Broad Institute Inc.
Priority to CN201480072878.4A priority patent/CN105899658B/en
Priority to AU2014361784A priority patent/AU2014361784A1/en
Priority to JP2016539225A priority patent/JP2017527256A/en
Publication of WO2015089465A1 publication Critical patent/WO2015089465A1/en
Priority to IL246117A priority patent/IL246117B/en
Priority to US15/179,938 priority patent/US20160317677A1/en
Priority to US17/002,262 priority patent/US20200389425A1/en

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Definitions

  • the present invention generally relates to the delivery, engineering, optimization and therapeutic applications of systems, methods, and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.
  • sequence targeting such as genome perturbation or gene-editing
  • CRISPR Clustered Regularly Interspaced Short Palindromic Repeats
  • the CRISPR-Cas system does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target.
  • Adding the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significantly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases.
  • cccDNA a dsDNA structure that arises during the propagation of HBV in the cell nucleus and can remain permanently present in infected subjects.
  • An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within the target polynucleotide.
  • the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.
  • the invention provides methods for using one or more elements of a CRISPR-Cas system.
  • the CRISPR complex of the invention provides an effective means for modifying a target polynucleotide.
  • the CRISPR complex of the invention has a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types in various tissues and organs.
  • modifying e.g., deleting, inserting, translocating, inactivating, activating
  • a target polynucleotide in a multiplicity of cell types in various tissues and organs.
  • the CRISPR complex of the invention has a broad spectrum of applications in, e.g., gene or genome editing, gene therapy, drug discovery, drug screening, disease diagnosis, and prognosis.
  • aspects of the invention relate to Cas9 enzymes having improved targeting specificity in a CRISPR-Cas9 system having guide RNAs having optimal activity, smaller in length than wild-type Cas9 enzymes and nucleic acid molecules coding therefor, and chimeric Cas9 enzymes, as well as methods of improving the target specificity of a Cas9 enzyme or of designing a CRISPR-Cas9 system comprising designing or preparing guide RNAs having optimal activity and/or selecting or preparing a Cas9 enzyme having a smaller size or length than wild-type Cas9 whereby packaging a nucleic acid coding therefor into a delivery vector is more advanced as there is less coding therefor in the delivery vector than for wild-type Cas9, and/or generating chimeric Cas9 enzymes.
  • a Cas9 enzyme may comprise one or more mutations and may be used as a generic DNA binding protein with or without fusion to a functional domain.
  • the mutations may be artificially introduced mutations or gain- or loss-of- function mutations.
  • the mutations may include but are not limited to mutations in one of the catalytic domains (D10 and H840) in the RuvC and HNH catalytic domains, respectively. Further mutations have been characterized.
  • the transcriptional activation domain may be VP64.
  • the transcriptional repressor domain may be KRAB or SID4X.
  • mutated Cas9 enzyme being fused to domains which include but are not limited to a transcriptional activator, repressor, a recombinase, a transposase, a histone remodeler, a demethylase, a DNA methyltransferase, a cryptochrome, a light inducible/controllable domain or a chemically inducible/controllable domain.
  • the invention provides for methods to generate mutant tracrRNA and direct repeat sequences or mutant chimeric guide sequences that allow for enhancing performance of these RNAs in cells. Aspects of the invention also provide for selection of said sequences. [0014] Aspects of the invention also provide for methods of simplifying the cloning and delivery of components of the CRISPR complex.
  • a suitable promoter such as the U6 promoter, is amplified with a DNA oligo and added onto the guide RNA. The resulting PCR product can then be transfected into cells to drive expression of the guide RNA.
  • aspects of the invention also relate to the guide RNA being transcribed in vitro or ordered from a synthesis company and directly transfected.
  • the invention provides for methods to improve activity by using a more active polymerase.
  • the expression of guide RNAs under the control of the T7 promoter is driven by the expression of the T7 polymerase in the cell.
  • the cell is a eukaryotic cell.
  • the eukaryotic cell is a human cell.
  • the human cell is a patient specific cell, e.g., a cell removed from a patient that may be modified and/or expanded into a cell population or a modified cell population, for instance, for re-administration to the patient..
  • the invention provides for methods of reducing the toxicity of Cas enzymes.
  • the Cas enzyme is any Cas9 as described herein, for instance any naturally-occurring bacterial Cas9 as well as any chimaeras, mutants, homologs or orthologs.
  • the Cas enzyme is a nickase.
  • the Cas9 is delivered into the cell in the form of mRNA. This allows for the transient expression of the enzyme thereby reducing toxicity.
  • the Cas9 is delivered into the cell in the nucleotide construct that encodes and expresses the Cas9 enzyme.
  • the invention also provides for methods of expressing Cas9 under the control of an inducible promoter, and the constructs used therein.
  • the invention provides for methods of improving the in vivo applications of the CRISPR-Cas system.
  • the Cas enzyme is wildtype Cas9 or any of the modified versions described herein, including any naturally- occurring bacterial Cas9 as well as any chimaeras, mutants, homologs or orthologs. .
  • the CRISPR enzyme comprises one or more mutations in one of the catalytic domains.
  • the Cas enzyme is a nickase.
  • An advantageous aspect of the invention provides for the selection of Cas9 homologs that are easily packaged into viral vectors for delivery.
  • Cas9 orthologs typically share the general organization of 3-4 RuvC domains and a HNH domain.
  • the 5' most RuvC domain cleaves the non-complementary strand, and the HNH domain cleaves the complementary strand. All notations are in reference to the guide sequence.
  • the catalytic residue in the 5' RuvC domain is identified through homology comparison of the Cas9 of interest with other Cas9 orthologs (from S. pyogenes type II CRISPR locus, S. thermophilus CRISPR locus 1, S. thermophilus CRISPR locus 3, and Franciscilla novicida type II CRISPR locus), and the conserved Asp residue (D10) is mutated to alanine to convert Cas9 into a complementary-strand nicking enzyme.
  • the conserved His and Asn residues in the HNH domains are mutated to Alanine to convert Cas9 into a non- complementary-strand nicking enzyme.
  • both sets of mutations may be made, to convert Cas9 into a non-cutting enzyme.
  • the CRISPR enzyme is a type I or III CRISPR enzyme, preferably a type II CRISPR enzyme.
  • This type II CRISPR enzyme may be any Cas enzyme.
  • a preferred Cas enzyme may be identified as Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system.
  • the Cas9 enzyme is from, or is derived from, spCas9 or saCas9.
  • Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as described herein
  • Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent.
  • residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.
  • this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCas9, St1Cas9 and so forth. Further examples are provided herein.
  • the skilled person will be able to determine appropriate corresponding residues in Cas9 enzymes other than SpCas9 by comparison of the relevant amino acid sequences.
  • a specific amino acid replacement is referred to using the SpCas9 numbering, then, unless the context makes it apparent this is not intended to refer to other Cas9 enzymes, the disclosure is intended to encompass corresponding modifications in other Cas9 enzymes.
  • the invention provides for methods of enhancing the function of Cas9 by generating chimeric Cas9 proteins.
  • Chimeric Cas9 proteins chimeric Cas9s may be new Cas9 containing fragments from more than one naturally occurring Cas9. These methods may comprise fusing N-terminal fragments of one Cas9 homolog with C-terminal fragments of another Cas9 homolog. These methods also allow for the selection of new properties displayed by the chimeric Cas9 proteins.
  • the modification may occur ex vivo or in vitro, for instance in a cell culture and in some instances not in vivo. In other embodiments, it may occur in vivo.
  • the invention provides a method of modifying an organism or a non- human organism by manipulation of a target sequence in a genomic locus of interest (e.g. an integrated viral sequence) comprising:
  • a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence wherein the polynucleotide sequence comprises:
  • a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences
  • the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence
  • the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA
  • the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence
  • the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA.
  • any or all of the polynucleotide sequence encoding a CRISPR enzyme, guide sequence, tracr mate sequence or tracr sequence may be RNA.
  • the polynucleotides encoding the sequence encoding a CRISPR enzyme, the guide sequence, tracr mate sequence or tracr sequence may be RNA and may be delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
  • RNA sequence includes the feature.
  • the DNA sequence is or can be transcribed into the RNA including the feature at issue.
  • the feature is a protein, such as the CRISPR enzyme
  • the DNA or RNA sequence referred to is, or can be, translated (and in the case of DNA transcribed first).
  • the invention provides a method of modifying an organism, e.g., mammal including human or a non-human mammal or organism by manipulation of a target sequence in a genomic locus of interest comprising delivering a non- naturally occurring or engineered composition comprising a viral or plasmid vector system comprising one or more viral or plasmid vectors operably encoding a composition for expression thereof, wherein the composition comprises: (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I.
  • a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II.
  • chiRNA CRISPR-Cas system chimeric RNA
  • a first regulatory element operably linked to (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and III.
  • a third regulatory element operably linked to a tracr sequence wherein components I, II and III are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence.
  • components I, II and III are located on the same vector. In other embodiments, components I and II are located on the same vector, while component III is located on another vector.
  • components I and III are located on the same vector, while component II is located on another vector. In other embodiments, components II and III are located on the same vector, while component I is located on another vector. In other embodiments, each of components I, II and III is located on different vectors.
  • the invention also provides a viral or plasmid vector system as described herein.
  • the vector is a viral vector, such as a lenti- or baculo- or preferably adeno- viral/adeno-associated viral vectors, but other means of delivery are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided.
  • a viral vector such as a lenti- or baculo- or preferably adeno- viral/adeno-associated viral vectors, but other means of delivery are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided.
  • one or more of the viral or plasmid vectors may be delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
  • Applicants By manipulation of a target sequence, Applicants also mean the epigenetic manipulation of a target sequence. This may be of the chromatin state of a target sequence, such as by modification of the methylation state of the target sequence (i.e. addition or removal of methylation or methylation patterns or CpG islands), histone modification, increasing or reducing accessibility to the target sequence, or by promoting 3D folding. In relation to treatment of viral infections, however, excision of integrated viral genome sequences is the manipulation of primary interest.
  • the invention provides a method of treating or inhibiting a condition caused by the presence of an integrated viral sequence in a genomic locus of interest in a subject (e.g., mammal or human) or a non-human subject (e.g., mammal) in need thereof comprising modifying the subject or a non-human subject by manipulation of a target sequence in the integrated viral sequence and wherein the condition is susceptible to treatment or inhibition by manipulation of the target sequence comprising providing treatment comprising: delivering a non-naturally occurring or engineered composition comprising an AAV or lentivirus vector system comprising one or more AAV or lentivirus vectors operably encoding a composition for expression thereof, wherein the target sequence is manipulated by the composition when expressed, wherein the composition comprises: (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I.
  • a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II.
  • chiRNA CRISPR-Cas system chimeric RNA
  • a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (or optionally at least one or more nuclear localization sequences as some embodiments can involve no NLS, i.e., there can be zero NLSs but advantageously there is greater than zero NLSs, such as one or more or advantageously two or more NLSs, and thus the invention comprehends embodiments wherein there is 0, 1, 2, 3, or more NLSs) wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate
  • a first regulatory element operably linked to (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and III.
  • a third regulatory element operably linked to a tracr sequence wherein components I, II and III are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence.
  • components I, II and III are located on the same vector. In other embodiments, components I and II are located on the same vector, while component III is located on another vector.
  • components I and III are located on the same vector, while component II is located on another vector. In other embodiments, components II and III are located on the same vector, while component I is located on another vector. In other embodiments, each of components I, II and III is located on different vectors.
  • the invention also provides a viral (e.g. AAV or lentivirus) vector system as described herein.
  • Delivery therefore can be via a vector, such as a viral vector, e.g., a recombinant viral vector delivery system; and, this system can be an AAV or lentivirus or derived from an AAV or a lentivirus (e.g., a recombinant AAV or lentivirus that expresses that which is foreign, heterologous or that which is not homologous or native to the virus may make some consider the virus“derived from” is parent virus).
  • the viral vector is a lentivirus-derived vector.
  • the viral vector is an Agrobacterium Ti or Ri plasmid for use in plants.
  • the organism or subject is a eukaryote (including mammal including human) or a non-human eukaryote or a non-human animal or a non-human mammal.
  • the organism or subject is a non-human animal, and may be an arthropod, for example, an insect, or may be a nematode.
  • the organism or subject is a plant.
  • the organism or subject is a mammal or a non-human mammal.
  • a non-human mammal may be for example a rodent (preferably a mouse or a rat), an ungulate, or a primate.
  • the organism or subject is a plant or algae, including microalgae, or is a fungus.
  • the viral vector is an AAV or a lentivirus, and can be part of a vector system as described herein.
  • the CRISPR enzyme is a Cas9.
  • the expression of the guide sequence is under the control of the T7 promoter and is driven by the expression of T7 polymerase.
  • the expression of the guide sequence is under the control of a U6 promoter.
  • the CRISPR enzyme comprises one or more mutations in one of the catalytic domains.
  • the CRISPR enzyme is a Cas9 nickase.
  • the invention in some embodiments comprehends a method of delivering a CRISPR enzyme comprising delivering to a cell a nucleic acid molecule, e.g., DNA, RNA, mRNA encoding the CRISPR enzyme.
  • the CRISPR enzyme is a Cas9. This allows for the transient expression of the enzyme thereby reducing toxicity.
  • the Cas9 is delivered into the cell in the nucleotide construct that encodes and expresses the Cas9 enzyme.
  • the invention also provides methods of preparing the vector systems of the invention, in particular the viral vector systems as described herein.
  • the invention in some embodiments comprehends a method of preparing the AAV of the invention comprising transfecting plasmid(s) containing or consisting essentially of nucleic acid molecule(s) coding for the AAV into AAV-infected cells, and supplying AAV rep and/or cap obligatory for replication and packaging of the AAV.
  • the AAV rep and/or cap obligatory for replication and packaging of the AAV are supplied by transfecting the cells with helper plasmid(s) or helper virus(es).
  • the helper virus is a poxvirus, adenovirus, herpesvirus or baculovirus.
  • the poxvirus is a vaccinia virus.
  • the cells are mammalian cells. And in some embodiments the cells are insect cells and the helper virus is baculovirus. In other embodiments, the virus is a lentivirus.
  • the invention further comprehends a composition of the invention or a CRISPR enzyme thereof (including or alternatively mRNA encoding the CRISPR enzyme) for use in medicine or in therapy.
  • the invention comprehends a composition according to the invention or a CRISPR enzyme thereof (including or alternatively a nucleic acid molecule, e.g., mRNA encoding the CRISPR enzyme) for use in a method according to the invention.
  • the invention provides for the use of a composition of the invention or a CRISPR enzyme thereof (including or alternatively mRNA encoding the CRISPR enzyme) in ex vivo gene or genome editing.
  • the invention comprehends use of a composition of the invention or a CRISPR enzyme thereof (including or alternatively mRNA encoding the CRISPR enzyme) in the manufacture of a medicament for ex vivo gene or genome editing or for use in a method according of the invention.
  • the invention thus also envisions a CRISPR-Cas Complex or a component thereof of any of any description herein for use in delivery to and/or a method of treating tissue, or tissue containing cells having a viral infection, such as Hepatitis B Virus; or in preparing or formulating a medicament or pharmaceutical composition for such treatment.
  • a CRISPR-Cas Complex or a component thereof of any of any description herein for use in delivery to and/or a method of treating tissue, or tissue containing cells having a viral infection, such as Hepatitis B Virus; or in preparing or formulating a medicament or pharmaceutical composition for such treatment.
  • the invention comprehends in some embodiments a composition of the invention or a CRISPR enzyme thereof (including or alternatively mRNA encoding the CRISPR enzyme), wherein the target sequence is flanked at its 3’ end by a 5’ motif termed a proto-spacer adjacent motif or PAM, especially where the Cas9 is (or is derived from) S. pyogenes or S. aureus Cas9.
  • a suitable PAM is 5'-NRG or 5'-NNGRR (where N is any Nucleotide) for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively, as mentioned below.
  • a suitable PAM is 5'-NRG.
  • SpCas9 or SaCas9 are those from or derived from S. pyogenes or S. aureus Cas9.
  • Apects of the invention comprehend improving the specificity of a CRISPR enzyme, e.g. Cas9, mediated gene targeting and reducing the likelihood of off-target modification by the CRISPR enzyme, e.g. Cas9.
  • the invention in some embodiments comprehends a method of modifying an organism or a non-human organism by minimizing off-target modifications by manipulation of a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a cell comprising delivering a non-naturally occurring or engineered composition comprising:
  • a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence wherein the first polynucleotide sequence comprises: (a) a first guide sequence capable of hybridizing to the first target sequence,
  • a second CRISPR-Cas system chiRNA polynucleotide sequence wherein the second polynucleotide sequence comprises:
  • a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences and comprising one or more mutations wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation, wherein when transcribed, the first and the second tracr mate sequence hybridize to the first and second tracr sequence respectively and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively, wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized or hybridizable to the first target sequence, and (2) the first tracr mate sequence that is hybridized or hybridizable to the first tracr sequence, wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized or hybridizable to the second target sequence, and (2) the second tracr mate sequence that is hybridized or
  • the first nick and the second nick in the DNA is offset relative to each other by at least one base pair of the duplex. In one aspect, the first nick and the second nick are offset relative to each other so that the resulting DNA break has a 3’ overhang. In one aspct, the first nick and the second nick are offset relative to each other so that the resulting DNA break has a 5’ overhang. In one aspect, the first nick and the second nick are positioned relative to each other such that the overhang is at least 1 nucleotide (nt), at least 10 nt, at least 15 nt, at least 26 nt, at least 30 nt, at least 50 nt or more that at least 50 nt. Additional aspects of the invention comprising the resulting offset double nicked DNA strand can be appreciated by one skilled in the art, and exemplary uses of the double nick system are provided herein.
  • any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence is/are RNA.
  • the polynucleotides encoding the sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence is/are RNA and are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
  • the first and second tracr mate sequence share 100% identity and/or the first and second tracr sequence share 100% identity.
  • the polynucleotides may be comprised within a vector system comprising one or more vectors.
  • the CRISPR enzyme is a Cas9 enzyme, e.g. SpCas9.
  • the CRISPR enzyme comprises one or more mutations in a catalytic domain, wherein the one or more mutations are selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A.
  • the CRISPR enzyme has the D10A mutation.
  • the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme
  • the second CRISPR enzyme has one or more mutations such that the enzyme is a non-complementary strand nicking enzyme.
  • the first enzyme may be a non-complementary strand nicking enzyme
  • the second enzyme may be a complementary strand nicking enzyme.
  • mutations of the CRISPR enzyme when the enzyme is not SpCas9, mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools).
  • any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged.
  • the invention provides as to any or each or all embodiments herein-discussed wherein the CRISPR enzyme comprises at least one or more, or at least two or more mutations, wherein the at least one or more mutation or the at least two or more mutations is as to D10, E762, H840, N854, N863, or D986 according to SpCas9 protein, e.g., D10A, E762A, H840A, N854A, N863A and/or D986A as to SpCas9, or N580 according to SaCas9, e.g., N580A as to SaCas9, or any corresponding mutation(s) in a Cas9 of an ortholog to Sp or Sa, or the CRISPR enzyme comprises at least one mutation wherein at least H840 or N863A as to Sp Cas9 or N580A as to Sa Cas9 is mutated; e.g., wherein the CRISPR enzyme comprises H840A, or D10A, where
  • the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of the other strand near the second target sequence results in a 5’ overhang.
  • the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs.
  • the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs.
  • the invention in some embodiments comprehends a method of modifying an organism or a non-human organism by minimizing off-target modifications by manipulation of a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a cell comprising delivering a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
  • components I, II, III and IV are located on the same or different vectors of the system, when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the first and the second guide sequence direct sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively, wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized or hybridizable to the first target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized or hybridizable to the second target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs cleavage of one strand of the DNA
  • the invention also provides a vector system as described herein.
  • the system may comprise one, two, three or four different vectors.
  • Components I, II, III and IV may thus be located on one, two, three or four different vectors, and all combinations for possible locations of the components are herein envisaged, for example: components I, II, III and IV can be located on the same vector; components I, II, III and IV can each be located on different vectors; components I, II, II I and IV may be located on a total of two or three different vectors, with all combinations of locations envisaged, etc.
  • any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence is/are RNA.
  • the first and second tracr mate sequence share 100% identity and/or the first and second tracr sequence share 100% identity.
  • the CRISPR enzyme is a Cas9 enzyme, e.g. SpCas9.
  • the CRISPR enzyme comprises one or more mutations in a catalytic domain, wherein the one or more mutations are selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A.
  • the CRISPR enzyme has the D10A mutation.
  • the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme
  • the second CRISPR enzyme has one or more mutations such that the enzyme is a non-complementary strand nicking enzyme.
  • the first enzyme may be a non-complementary strand nicking enzyme
  • the second enzyme may be a complementary strand nicking enzyme.
  • one or more of the viral vectors are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
  • the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a 5’ overhang.
  • the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs.
  • the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs.
  • the invention in some embodiments comprehends a method of modifying a genomic locus of interest by minimizing off-target modifications by introducing into a cell containing and expressing a double stranded DNA molecule encoding a gene product of interest an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein having one or more mutations and two guide RNAs that target a first strand and a second strand of the DNA molecule respectively, whereby the guide RNAs target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the Cas protein and the two guide RNAs do not naturally occur together.
  • the Cas protein nicking each of the first strand and the second strand of the DNA molecule encoding the gene product results in a 5’ overhang.
  • the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs.
  • the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs.
  • Embodiments of the invention also comprehend the guide RNAs comprising a guide sequence fused to a tracr mate sequence and a tracr sequence.
  • the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell. As explained in more detail below, codon usage can even be optimized for expression in particular cell types e.g. for liver cells.
  • the Cas protein is a type II CRISPR-Cas protein, e.g. a Cas9 protein.
  • the Cas protein is a Cas9 protein, e.g. SpCas9.
  • the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A.
  • the Cas protein has the D10A mutation.
  • aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased.
  • the gene product is a protein.
  • the invention also comprehends an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein having one or more mutations and two guide RNAs that target a first strand and a second strand respectively of a double stranded DNA molecule encoding a gene product in a cell, whereby the guide RNAs target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the Cas protein and the two guide RNAs do not naturally occur together.
  • the guide RNAs may comprise a guide sequence fused to a tracr mate sequence and a tracr sequence.
  • the Cas protein is a type II CRISPR-Cas protein.
  • the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell.
  • the Cas protein is a type II CRISPR-Cas protein, e.g. a Cas9 protein.
  • the Cas protein is a Cas9 protein, e.g. SpCas9.
  • the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A.
  • the Cas protein has the D10A mutation.
  • aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased.
  • the gene product is a protein.
  • the invention also comprehends an engineered, non-naturally occurring vector system comprising one or more vectors comprising:
  • the guide RNAs may comprise a guide sequence fused to a tracr mate sequence and a tracr sequence.
  • the Cas protein is a type II CRISPR-Cas protein.
  • the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell.
  • the Cas protein is a type II CRISPR-Cas protein, e.g. a Cas9 protein.
  • the Cas protein is a Cas9 protein, e.g. SpCas9.
  • the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A.
  • the Cas protein has the D10A mutation.
  • aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased.
  • the gene product is a protein.
  • the vectors of the system are viral vectors.
  • the vectors of the system are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
  • the invention provides a method of modifying a target polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • said cleavage comprises cleaving one or two strands at the location of the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence.
  • the method further comprises delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors drive expression of one or more of: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence.
  • said vectors are delivered to the eukaryotic cell in a subject.
  • said modifying takes place in said eukaryotic cell in a cell culture.
  • the method further comprises isolating said eukaryotic cell from a subject prior to said modifying.
  • the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.
  • the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors drive expression of one or more of: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence.
  • the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • this invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises decreasing expression of a target polynucleotide by using a CRISPR complex that binds to the polynucleotide.
  • one or more vectors comprising a tracr sequence, a guide sequence linked to the tracr mate sequence, a sequence encoding a CRISPR enzyme is delivered to a cell.
  • the one or more vectors comprises a regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence; and a regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting a guide sequence upstream of the tracr mate sequence.
  • the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a cell.
  • the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence.
  • a target polynucleotide can be inactivated to effect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild-type sequence does. For example, a protein or microRNA coding sequence may be inactivated such that the protein or microRNA is not produced.
  • the CRISPR enzyme comprises one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A or D986A and/or the one or more mutations is in a RuvC1 or HNH domain of the CRISPR enzyme or is a mutation as otherwise as discussed herein.
  • the CRISPR enzyme has one or more mutations in a catalytic domain, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the enzyme further comprises a functional domain.
  • the functional domain is a transcriptional activation domain, preferably VP64. In some embodiments, the functional domain is a transcription repression domain, preferably KRAB. In some embodiments, the transcription repression domain is SID, or concatemers of SID (eg SID4X). In some embodiments, the functional domain is an epigenetic modifying domain, such that an epigenetic modifying enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be the P65 activation domain. Thus, in some embodiments a mutated Cas9 enzyme may be fused to a protein domain or functional domain.
  • the CRISPR enzyme is a type I or III CRISPR enzyme, but is preferably a type II CRISPR enzyme.
  • This type II CRISPR enzyme may be any Cas enzyme.
  • a Cas enzyme may be identified as Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system.
  • the Cas9 enzyme is from, or is derived from, spCas9 or saCas9.
  • Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent.
  • residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.
  • this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCa9, St1Cas9 and so forth.
  • delivery is in the form of a vector which may be a viral vector, such as a lenti- or baculo- or preferably adeno-viral/adeno-associated viral vectors, but other means of delivery are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided.
  • a vector may mean not only a viral or yeast system (for instance, where the nucleic acids of interest may be operably linked to and under the control of (in terms of expression, such as to ultimately provide a processed RNA) a promoter), but also direct delivery of nucleic acids into a host cell.
  • the vector may be a viral vector and this is advantageously an AAV
  • viral vectors as herein discussed can be employed, such as lentivirus.
  • baculoviruses may be used for expression in insect cells. These insect cells may, in turn be useful for producing large quantities of further vectors, such as AAV or lentivirus vectors adapted for delivery of the present invention.
  • a method of delivering the present CRISPR enzyme comprising delivering to a cell mRNA encoding the CRISPR enzyme.
  • the CRISPR enzyme is truncated, and/or comprised of less than one thousand amino acids or less than four thousand amino acids, and/or is a nuclease or nickase, and/or is codon-optimized, and/or comprises one or more mutations, and/or comprises a chimeric CRISPR enzyme, and/or the other options as herein discussed.
  • AAV and lentiviral vectors are preferred.
  • the target sequence is flanked or followed, at its 3’ end, by a PAM suitable for the CRISPR enzyme, typically a Cas and in particular a Cas9.
  • a suitable PAM is 5'-NRG or 5'-NNGRR for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively.
  • the invention also relates to a method of modifying a cell of a eukaryotic organism by manipulating at least one target viral nucleic acid within the cell, the method comprising introducing into the cell an exogenous composition capable of forming a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex, the composition comprising:
  • a tracr sequence wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing to the tracr mate sequence; and (B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme, wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
  • the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the invention also relates to an exogenous composition which, when introduced into a cell of a eukaryotic organism, is capable of forming at least one Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex, wherein the complex is capable of modifying the cell by manipulating of at least one target viral nucleic acid within the cell, the composition comprising:
  • CRISPR Clustered Regularly Interspersed Short Palindromic Repeats
  • Cas CRISPR-Cas
  • system polynucleotide sequences comprising:
  • a guide sequence which when transcribed is capable of hybridizing to a sequence of the at least one target viral nucleic acid to be manipulated;
  • a trans-activating CRISPR RNA (tracr) mate sequence which is linked to the guide sequence;
  • a tracr sequence wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and (B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme, wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
  • the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the invention also relates to a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex which, when introduced into a cell of a eukaryotic organism, is capable of modifying the cell by manipulating a target viral nucleic acid within the cell, the complex comprising:
  • RNA polynucleotide sequences comprising:
  • the tracr mate sequence is hybridized to the tracr sequence or portion thereof;
  • the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and
  • the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the invention also relates to a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA) which, when introduced into a cell of a eukaryotic organism, is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex, wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell;
  • the chiRNA comprising:
  • the tracr mate sequence hybridizes to the tracr sequence or portion thereof; b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex;
  • the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the invention also relates to a DNA polynucleotide molecule comprising sequences encoding a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR- associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA), wherein upon introduction of said chiRNA into a cell of a eukaryotic organism said chiRNA is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell; the chiRNA comprising:
  • the tracr mate sequence hybridizes to the tracr sequence or portion thereof; b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex;
  • the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the guide sequence as described herein which is capable of hybridizing to a sequence of a target nucleic acid to be manipulated, and the tracr mate sequence, as described herein, may preferably be linked in a tandem arrangement, wherein the tracr mate sequence comprises a region of sense sequence.
  • the tracr sequence, as described herein may comprise a region of antisense sequence which is capable of hybridizing with the region of sense sequence of the tracr mate sequence.
  • the region of antisense sequence is hybridized to the region of sense sequence thereby forming a dual RNA molecule; and wherein when said dual RNA molecule binds within the cell to the CRISPR/Cas enzyme so forming a CRISPR-Cas complex, the guide sequence hybridizes to a sequence of the target nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the target nucleic acid, whereupon manipulation of said sequence of said target nucleic acid is effected by the CRISPR/Cas enzyme of the complex.
  • certain embodiments may optionally comprise a chimeric single guide RNA molecule (sgRNA).
  • sgRNA molecules may preferably comprise, in a tandem arrangement:
  • a guide sequence as described herein, which is capable of hybridizing to a sequence of the target nucleic acid to be manipulated
  • a tracr mate sequence as described herein, comprising a region of sense sequence
  • a tracr sequence as described herein, comprising a region of antisense sequence which is positioned adjacent the linker sequence and which is capable of hybridizing with the region of sense sequence thereby forming a stem-loop.
  • the linker may be a polynucleotide linker, optionally comprising GAAA.
  • Other linkers such as those described herein, are envisaged.
  • the region of antisense sequence is hybridized to the region of sense sequence thereby forming the stem-loop; and wherein when said sgRNA molecule binds within the cell to the CRISPR/Cas enzyme so forming a CRISPR- Cas complex, the guide sequence hybridizes to a sequence of the target nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the target nucleic acid, whereupon manipulation of said sequence of said target nucleic acid is effected by the CRISPR/Cas enzyme of the complex.
  • the guide sequence, the trans-activating CRISPR RNA (tracr) mate sequence and the tracr sequence may be non-coding sequences.
  • the tracr mate sequence may not be capable of hybridizing to a sequence of the at least one target viral nucleic acid to be manipulated, in contrast to the guide sequence.
  • target viral nucleic acids may be performed by the CRISPR/Cas complexes described herein.
  • Preferred manipulations of target viral nucleic acids include cleavage of viral DNA, as described in more detail herein.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein manipulation of at least one target viral nucleic acid within the cell is performed.
  • multiplexing methods are described and exemplified wherein multiple target viral nucleic acid sequences are manipulated by CRISPR/Cas complexes targeting different sequences of the target viral nucleic acid.
  • an“exogenous composition” is an engineered or non-naturally occurring composition.
  • the target sequence may be flanked or followed, at its 3’ end, by a protospacer adjacent motif (PAM) suitable for recognition by the CRISPR enzyme of the complex, typically a Cas enzyme and more typically a Cas9 enzyme.
  • PAM protospacer adjacent motif
  • a suitable PAM is 5'-NRG or 5'-NNGRR for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively.
  • Other PAMs, such as are described herein, may be recognized in association with the target sequence depending upon the specific CRISPR enzyme used.
  • the CRISPR/Cas enzyme of the exogenous composition may be provided as a polynucleotide sequence which comprises either (a) RNA or (b) DNA wherein the polynucleotide sequence is operably linked to a regulatory element capable of directing expression of RNA encoding the CRISPR/Cas enzyme.
  • any of the CRISPR/Cas system polynucleotide sequences of the exogenous composition may comprise either (a) RNA or (b) DNA wherein the polynucleotide sequences are operably linked to one or more regulatory elements capable of directing expression of CRISPR/Cas system RNA polynucleotide sequences.
  • Each of the CRISPR/Cas system polynucleotide sequences of the exogenous composition may consist of RNA and wherein the CRISPR/Cas system polynucleotide sequences may comprise a chimeric RNA polynucleotide molecule comprising the guide sequence, the tracr mate sequence and the tracr sequence.
  • Each of the CRISPR/Cas system polynucleotide sequences of the exogenous composition may be provided as DNA polynucleotide sequences further comprising at least one regulatory element operably linked to polynucleotide sequences encoding CRISPR/Cas system RNA polynucleotide sequences and capable of directing expression thereof, and wherein the CRISPR/Cas system RNA polynucleotide sequences may comprise a chimeric RNA polynucleotide (chiRNA) molecule comprising the guide sequence, the tracr mate sequence and the tracr sequence.
  • chiRNA chimeric RNA polynucleotide
  • compositions, complexes, chiRNA or DNA polynucleotide molecules each of the guide sequence, the tracr mate sequences and the tracr sequence may be arranged in a 5’ to 3’ orientation; or each of the guide sequence, the tracr mate sequences and the tracr sequence may be arranged in a 3’ to 5’ orientation.
  • the CRISPR/Cas system polynucleotide sequences or polynucleotide sequences encoding the CRISPR/Cas system polynucleotide sequences and/or (b) polynucleotide sequences encoding the CRISPR/Cas enzyme may be comprised in one or more recombinant viral vectors.
  • the polynucleotide sequences of (a) may be located on the same or different recombinant viral vector as polynucleotide sequences of (b).
  • chiRNAs or the DNA polynucleotide molecules described herein may be comprised in a recombinant viral vector.
  • the viral vector may be a retroviral vector, optionally a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adenoassociated viral (AAV) vector such as AAV8 vector, or a poxvirus such as a vaccinia virus.
  • retroviral vector optionally a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adenoassociated viral (AAV) vector such as AAV8 vector, or a poxvirus such as a vaccinia virus.
  • the CRISPR/Cas system polynucleotide sequences or polynucleotide sequences encoding the CRISPR/Cas system polynucleotide sequences and/or (b) polynucleotide sequences encoding the CRISPR/Cas enzyme may be delivered to the cell of the organism via liposomes, nanoparticles, exosomes, microvesicles or a gene-gun.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein hybridization between the tracr sequence and the tracr mate sequence may produce a transcript having secondary structure, preferably a hairpin.
  • the tracr sequence may comprise one or more regions capable of forming secondary structure, preferably a hairpin.
  • the tracr sequence may comprise one or more hairpins, two or more hairpins, three or more hairpins, four or more hairpins, five or more hairpins, or at most five hairpins.
  • the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme.
  • preferred CRISPR/Cas enzymes are Type II CRISPR/Cas enzymes, preferably Type II Cas9 CRISPR/Cas enzymes or biologically active fragments or derivatives thereof.
  • the guide sequence may be 10 to 30 nucleotides in length.
  • the CRISPR/Cas enzyme may be a Cas9 enzyme of Streptococcus pyogenes or a Cas9 enzyme of Streptococcus aureus, or a biologically active fragment or derivative thereof. Described herein are specific NLS sequences which may be applied to the CRISPR/Cas enzymes.
  • the CRISPR/Cas enzyme may further comprise one or more nuclear localization sequences (NLSs) capable of driving the accumulation of the CRISPR/Cas enzyme to a detectible amount in the nucleus of the cell of the organism.
  • the CRISPR/Cas enzyme may comprise two or more NLSs, three or more NLSs, four or more NLSs, five or more NLSs, six or more NLSs, seven or more NLSs, eight or more NLSs, nine or more NLSs, or ten or more NLSs.
  • the CRISPR/Cas enzyme may comprise at least one NLS at or near the amino-terminus of the CRISPR/Cas enzyme and/or at least one NLS at or near the carboxy-terminus the CRISPR/Cas enzyme.
  • the guide sequence when present as RNA within the cell the guide sequence may be capable of hybridizing to a sequence of a target viral nucleic acid which is an episomal nucleic acid molecule which is not integrated into the genome of the organism and wherein said manipulation is a manipulation of the episomal viral nucleic acid molecule, preferably wherein the episomal nucleic acid molecule is a double-stranded DNA polynucleotide molecule.
  • the double-stranded DNA polynucleotide may be an episomal viral nucleic acid which is a covalently closed circular DNA (cccDNA).
  • the double-stranded DNA polynucleotide may preferably be an episomal viral nucleic acid which is a cccDNA.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be capable of reducing the amount of episomal viral nucleic acid molecule in a cell of the organism compared to the amount of episomal viral nucleic acid molecule in a cell of the organism in the absence of providing the complex.
  • the CRISPR/Cas complex may be capable of manipulating the episomal nucleic acid molecule to promote degradation of the episomal nucleic acid molecule.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein when present as RNA within in the cell the guide sequence may be capable of hybridizing to a sequence of the target viral nucleic acid which is integrated into the genome of the organism and wherein said manipulation is a manipulation of the integrated target nucleic acid.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein when formed within the cell the CRISPR/Cas complex may be capable of manipulating the integrated nucleic acid to promote excision of all or part of the target viral nucleic acid from the genome of the organism.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be used in the manipulation of at least one target viral nucleic acid within the cell of a eukaryotic organism as described herein. Such a use may be in vitro and ex vivo.
  • At least one target viral nucleic acid may be comprised in a double-stranded DNA molecule polynucleotide cccDNA and/or viral DNA integrated into the genome of the organism, in which case the manipulation of the at least one target viral nucleic acid by the CRISPR-Cas complex comprises cleavage of viral cccDNA and/or integrated viral DNA.
  • the cleavage may comprise one or more double-strand break(s) introduced into the viral cccDNA and/or integrated viral DNA, optionally at least two double-strand break(s).
  • the cleavage may comprise one or more single-strand break(s) introduced into the viral cccDNA and/or integrated viral DNA, optionally at least two single-strand break(s).
  • one or more double-strand break(s) and/or one or more single-strand break(s) may lead to the formation of one or more insertion and deletion mutations (INDELs) in the target viral cccDNA sequences and/or target integrated viral DNA sequences.
  • INDELs insertion and deletion mutations
  • cleavage of the viral cccDNA sequences or viral DNA sequences integrated in the genome of the organism may lead to excision of viral polynucleotide sequences from the cccDNA thereby reducing viral infection or excision of viral DNA sequences from the genome of the organism thereby reducing viral infection.
  • the composition may comprise components of at least two types of CRISPR/Cas complex, wherein each type of complex comprises a guide sequence capable of hybridizing to different sequences of the target nucleic acid, wherein said cleavage is cleavage of first and second strands of the viral DNA via at least two double-strand breaks introduced into the viral cccDNA and/or into or adjacent viral DNA integrated into the genome of the organism;
  • a first double-strand break is introduced at a first position of the DNA by manipulating a first target sequence and a second double-strand break is introduced at a second position of the DNA by manipulating a second target sequence;
  • first and second double-strand breaks viral sequences between first and second double-strand breaks are excised from cccDNA and/or from the genomic DNA of the organism.
  • the composition may comprise components of at least four types of CRISPR/Cas complex, wherein each type of complex comprises a guide sequence capable of hybridizing to different sequences of the target nucleic acid, wherein said cleavage is via at least two pairs of single-strand breaks introduced into the viral cccDNA and/or introduced into or adjacent viral DNA integrated into the genome of the organism;
  • a first single-strand break is introduced into a first strand of DNA by manipulating a first target sequence to create a first nick and a second single-strand break is introduced into the opposite strand of DNA by manipulating a second target sequence to create a second nick;
  • a third single-strand break is introduced into said first strand of DNA by manipulating a third target sequence to create a third nick and a fourth single-strand break is introduced into said opposite strand of DNA by manipulating a fourth target sequence to create a fourth nick; wherein upon introduction of first and second pairs of single-strand breaks viral sequences between first and second pairs of single-strand breaks are excised from cccDNA and/or from the genomic DNA of the organism.
  • the first and second nicks may be offset relative to each other by at least one base pair of the duplex creating a first overhang, and wherein third and fourth nicks are offset relative to each other by at least one base pair of the duplex creating a second overhang.
  • the ends of the cleaved first strand of DNA may be ligated together and the ends of the cleaved second strand of DNA may be ligated together thus reforming unbroken first and second strands.
  • the single-strand break(s) may be introduced into DNA by a nickase enzyme which is a modified Cas9 enzyme comprising a substitution leading to catalytic inactivation of the HNH nuclease domain or the RuvC nuclease domain of Cas9; optionally wherein the substitution is at position D10 of SpCas9, preferably a D10A substitution or substitution of a residue corresponding to position D10 in a SpCas9-related enzyme, or wherein the substitution is at position H840 of SpCas9, preferably a H840A substitution or substitution of a residue corresponding to position H840 in a SpCas9-related enzyme.
  • a nickase enzyme which is a modified Cas9 enzyme comprising a substitution leading to catalytic inactivation of the HNH nuclease domain or the RuvC nuclease domain of Cas9; optionally wherein the substitution is at position D10 of SpCas9, preferably a D10A substitution or
  • target viral nucleic acid may be cccDNA and/or viral DNA integrated into the genome of the organism and wherein said manipulation comprises insertion of one or more nucleotides into or adjacent viral cccDNA sequences or into or adjacent integrated viral DNA sequences, deletion of one or more nucleotides of viral cccDNA or of integrated viral DNA, translocation of viral cccDNA sequences or of integrated viral DNA sequences, repression of transcription of viral cccDNA sequences or of integrated viral DNA sequences, and/or inactivation of viral cccDNA sequences or of integrated viral DNA sequences.
  • Repression of transcription of viral cccDNA sequences and/or integrated viral DNA sequences may be effected by the action of a CRISPR-Cas system comprising a CRISPR enzyme fused to one or more transcriptional repressor domains, optionally wherein the one or more transcriptional repressor domains comprises KRAB, SID and/or SID4X, preferably wherein the CRISPR enzyme is a Cas9 enzyme.
  • the manipulation may comprise activation of genes carried by viral cccDNA, e.g.
  • a CRISPR-Cas system comprising a deactivated CRISPR enzyme fused to one or more transcriptional activation domains such as VP64, preferably the viral cccDNA is HBV and activation results in increased activity of APOBEC3A and/or APOBEC3B, and or other viral interferon-stimulated genes (ISGs), thereby leading to a reduction in HBV cccDNA.
  • a CRISPR-Cas system comprising a deactivated CRISPR enzyme fused to one or more transcriptional activation domains such as VP64, preferably the viral cccDNA is HBV and activation results in increased activity of APOBEC3A and/or APOBEC3B, and or other viral interferon-stimulated genes (ISGs), thereby leading to a reduction in HBV cccDNA.
  • Manipulation of nucleotide sequences of viral cccDNA or integrated viral DNA may lead to disruption of one or more viral open reading
  • manipulation of said viral cccDNA may lead to a reduction in the level of one or more of viral rcDNA, viral cccDNA and viral ssDNA compared to the level in the absence of the CRISPR/Cas complex.
  • the effect of said manipulation may comprise inhibiting the production of new virions.
  • the effect of said modifying may comprise removing viral sequences from said organism thereby reducing viral infection.
  • the described composition may further comprises components of one or more additional CRISPR/Cas complexes, or components required for the assembly of one or more additional CRISPR/Cas complexes, wherein each type of complex comprises a different guide sequence capable hybridizing to a different sequence of the target nucleic acid within the cell.
  • any of the methods and compositions described herein may be additionally characterized by one or more additional CRISPR/Cas complexes each of which one or more additional CRISPR/Cas complexes may be characterized as described herein.
  • the target viral nucleic acid may be a hepatitis B virus (HBV) nucleic acid.
  • HBV hepatitis B virus
  • the cell of the organism is a cell capable of being infected by HBV.
  • the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP).
  • NTCP sodium taurocholate cotransporting polypeptide
  • the cell may be a hepatocyte, preferably a primary hepatocyte, more preferably a human hepatocyte or a human primary hepatocyte, a HepG2.2.15 or a HepG2-hNTCP cell.
  • the guide sequence may be capable of hybridizing with target viral nucleic acids of HBV ORF S, ORF C, ORF P, or ORF X, preferably ORF C, optionally wherein the sequence of the guide sequence comprises 5'-gggcgcacctctctttacg-3', 5'-cctctgccgatccatactg-3' or 5’-taaagaatttggagctactg-3’.
  • the target viral nucleic acid may be a human papillomavirus (HPV) nucleic acid, an Epstein Barr virus (EBV) nucleic acid, a herpes simplex virus (HSV) nucleic acid, or a varicella zoster virus (VZV) nucleic acid.
  • HPV human papillomavirus
  • EBV Epstein Barr virus
  • HSV herpes simplex virus
  • VZV varicella zoster virus
  • compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein said manipulation may be performed in vitro or ex vivo.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use as a medicament.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use in the treatment of a viral infection.
  • a treatment may be of a viral infection wherein target viral sequences are comprised in an episomal nucleic acid molecule which is not integrated into the genome of the organism, such as a covalently closed circular DNA (cccDNA).
  • the viral infection may caused by hepatitis B virus (HBV), human papillomavirus (HPV), Epstein Barr virus (EBV), herpes simplex virus (HSV) or varicella zoster virus (VZV).
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use as a medicament or for use in the treatment of a viral infection wherein the organism is a mammal such as a human.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use in the manufacture of a medicament.
  • compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use in the manufacture of a medicament for the treatment of a viral infection.
  • a treatment may be of a viral infection wherein target viral sequences are comprised in an episomal nucleic acid molecule which is not integrated into the genome of the organism, such as a covalently closed circular DNA (cccDNA).
  • the viral infection may be caused by hepatitis B virus (HBV) or the viral infection may be caused by human papillomavirus (HPV), Epstein Barr virus (EBV), herpes simplex virus (HSV), or varicella zoster virus (VZV).
  • HBV hepatitis B virus
  • HPV human papillomavirus
  • EBV Epstein Barr virus
  • HSV herpes simplex virus
  • VZV varicella zoster virus
  • the organism may be a mammal such as a human.
  • the invention also relates to a method of modifying a cell, of a eukaryotic organism by manipulating at least one target viral nucleic acid within the cell, the method comprising introducing into the cell an exogenous composition capable of forming a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex, the composition comprising: (A) CRISPR-Cas system polynucleotide sequences comprising:
  • a tracr sequence wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing to the tracr mate sequence; and (B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme, wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
  • the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA).
  • cccDNA HBV double-stranded covalently closed circular DNA
  • the invention also relates to an exogenous composition which, when introduced into a cell of a eukaryotic organism, is capable of forming at least one Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex, wherein the complex is capable of modifying the cell by manipulating at least one target viral nucleic acid within the cell, the composition comprising:
  • CRISPR Clustered Regularly Interspersed Short Palindromic Repeats
  • Cas CRISPR-Cas
  • system polynucleotide sequences comprising:
  • a tracr sequence wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and (B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme, wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
  • the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA).
  • cccDNA HBV double-stranded covalently closed circular DNA
  • the invention also relates to a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex which, when introduced into a cell of a eukaryotic organism, is capable of modifying the cell by manipulating a target viral nucleic acid within the cell, the complex comprising:
  • RNA polynucleotide sequences comprising:
  • the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA).
  • cccDNA HBV double-stranded covalently closed circular DNA
  • the invention also relates to a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA) which, when introduced into a cell of a eukaryotic organism, is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex, wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell;
  • the chiRNA comprising:
  • the tracr mate sequence hybridizes to the tracr sequence or portion thereof; b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex;
  • the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA).
  • a chiRNA may be additionally characterized, as appropriate, in accordance with any of the further and particular features as described herein.
  • the invention also relates to a DNA polynucleotide molecule comprising sequences encoding a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR- associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA) wherein upon introduction of said chiRNA into a cell of a eukaryotic organism said chiRNA is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex, wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell; the chiRNA comprising:
  • the tracr mate sequence hybridizes to the tracr sequence or portion thereof; b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex;
  • the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
  • the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA).
  • cccDNA HBV double-stranded covalently closed circular DNA
  • FIG. 1 shows a schematic model of the CRISPR system.
  • the Cas9 nuclease from Streptococcus pyogenes (yellow) is targeted to genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue) and a scaffold (red).
  • the guide sequence base-pairs with the DNA target (blue), directly upstream of a requisite 5’-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) ⁇ 3 bp upstream of the PAM (red triangle).
  • PAM magenta
  • Figure 2A-F shows an exemplary CRISPR system, a possible mechanism of action, an example adaptation for expression in eukaryotic cells, and results of tests assessing nuclear localization and CRISPR activity.
  • Figure 3A-D shows results of an evaluation of SpCas9 specificity for an example target.
  • Figure 4A-G show an exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells.
  • Figure 5 provides a table of protospacer sequences and summarizes modification efficiency results for protospacer targets designed based on exemplary S. pyogenes and S. thermophilus CRISPR systems with corresponding PAMs against loci in human and mouse genomes.
  • Figure 6A-C shows a comparison of different tracrRNA transcripts for Cas9- mediated gene targeting.
  • Figure 7 shows a schematic of a surveyor nuclease assay for detection of double strand break-induced micro-insertions and–deletions.
  • Figure 8A-B shows exemplary bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cells.
  • Figure 9A-C shows histograms of distances between adjacent S. pyogenes SF370 locus 1 PAM (NGG) ( Figure 9A) and S. thermophilus LMD9 locus 2 PAM (NNAGAAW) ( Figure 9B) in the human genome; and distances for each PAM by chromosome (Chr) ( Figure 9C).
  • Figure 10A-D shows an exemplary CRISPR system, an example adaptation for expression in eukaryotic cells, and results of tests assessing CRISPR activity.
  • Figure 11A-C shows exemplary manipulations of a CRISPR system for targeting of genomic loci in mammalian cells.
  • Figure 12A-B shows the results of a Northern blot analysis of crRNA processing in mammalian cells.
  • Figure 13A-B shows an exemplary selection of protospacers in the human PVALB and mouse Th loci.
  • Figure 14 shows example protospacer and corresponding PAM sequence targets of the S. thermophilus CRISPR system in the human EMX1 locus.
  • Figure 15 provides a table of sequences for primers and probes used for Surveyor, RFLP, genomic sequencing, and Northern blot assays.
  • Figure 16A-C shows exemplary manipulation of a CRISPR system with chimeric RNAs and results of SURVEYOR assays for system activity in eukaryotic cells.
  • Figure 17A-B shows a graphical representation of the results of SURVEYOR assays for CRISPR system activity in eukaryotic cells.
  • Figure 18 shows an exemplary visualization of some S. pyogenes Cas9 target sites in the human genome using the UCSC genome browser.
  • Figure 19A-D shows a circular depiction of the phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s ( ⁇ 1400 amino acids) and two of small Cas9s ( ⁇ 1100 amino acids).
  • Figure 20A-F shows the linear depiction of the phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s ( ⁇ 1400 amino acids) and two of small Cas9s ( ⁇ 1100 amino acids).
  • Figure 21A-D shows genome editing via homologous recombination.
  • Figure 22A-B shows single vector designs for SpCas9.
  • Figure 23 shows a graph representing the length distribution of Cas9 orthologs.
  • Figure 24A-M shows sequences where the mutation points are located within the SpCas9 gene.
  • Figure 25A shows the Conditional Cas9, Rosa26 targeting vector map.
  • Figure 25B shows the Constitutive Cas9, Rosa26 targeting vector map.
  • Figure 26 shows a schematic of the important elements in the Constitutive and Conditional Cas9 constructs.
  • Figure 27 shows delivery and in vivo mouse brain Cas9 expression data.
  • Figure 28 shows RNA delivery of Cas9 and chimeric RNA into cells
  • A Delivery of a GFP reporter as either DNA or mRNA into Neuro-2A cells.
  • B Delivery of Cas9 and chimeric RNA against the Icam2 gene as RNA results in cutting for one of two spacers tested.
  • C Delivery of Cas9 and chimeric RNA against the F7 gene as RNA results in cutting for one of two spacers tested.
  • Figure 29 shows how DNA double-strand break (DSB) repair promotes gene editing.
  • NHEJ error-prone non-homologous end joining
  • Indel random insertion/deletion
  • a repair template in the form of a plasmid or single-stranded oligodeoxynucleotides (ssODN) can be supplied to leverage the homology-directed repair (HDR) pathway, which allows high fidelity and precise editing.
  • FIG. 30A-C shows anticipated results for HDR in HEK and HUES9 cells.
  • Either a targeting plasmid or an ssODN (sense or antisense) with homology arms can be used to edit the sequence at a target genomic locus cleaved by Cas9 (red triangle).
  • a targeting plasmid or an ssODN (sense or antisense) with homology arms can be used to edit the sequence at a target genomic locus cleaved by Cas9 (red triangle).
  • Applicants introduced a HindIII site (red bar) into the target locus, which was PCR- amplified with primers that anneal outside of the region of homology. Digestion of the PCR product with HindIII reveals the occurrence of HDR events.
  • ssODNs oriented in either the sense or the antisense (s or a) direction relative to the locus of interest, can be used in combination with Cas9 to achieve efficient HDR-mediated editing at the target locus.
  • Example of the effect of ssODNs on HDR in the EMX1 locus is shown using both wild-type Cas9 and Cas9 nickase (D10A). Each ssODN contains homology arms of 90 bp flanking a 12-bp insertion of two restriction sites.
  • Figure 31A-C shows the repair strategy for Cystic Fibrosis delta F508 mutation.
  • Figure 32A-B (a) shows a schematic of the GAA repeat expansion in FXN intron 1 and (b) shows a schematic of the strategy adopted to excise the GAA expansion region using the CRISPR/Cas system.
  • Figure 33 shows a screen for efficient SpCas9 mediated targeting of Tet1 -3 and Dnmt1, 3a and 3b gene loci. Surveyor assay on DNA from transfected N2A cells demonstrates efficient DNA cleavage by using different gRNAs.
  • Figure 34 shows a strategy of multiplex genome targeting using a 2-vector system in an AAV1/2 delivery system. Tet1-3 and Dnmt1, 3a and 3b gRNA under the control of the U6 promoter. GFP-KASH under the control of the human synapsin promoter. Restriction sides shows simple gRNA replacement strategy by subcloning. HA-tagged SpCas9 flanked by two nuclear localization signals (NLS) is shown. Both vectors are delivered into the brain by AAV1/2 virus in a 1:1 ratio.
  • NLS nuclear localization signals
  • Figure 35 shows verification of multiplex DNMT targeting vector #1 functionality using Surveyor assay.
  • N2A cells were co-transfected with the DNMT targeting vector #1 (+) and the SpCas9 encoding vector for testing SpCas9 mediated cleavage of DNMTs genes family loci.
  • gRNA only (-) is negative control. Cells were harvested for DNA purification and downstream processing 48 h after transfection.
  • Figure 36 shows guide RNA design for HBV targeting CRISPR constructs. Cleavage sites were optimized for low homology to human genomic DNA and conservation (guides 13-24)
  • Figure 37 shows a scheme for quantifying cccDNA in response to Cas9 treatment, 1st round of experiments.
  • Figure 38 shows qPCR results from 1st round of HepG2.2.15 experiments.
  • Figure 39 shows a Surveyor assay for nuclease activity. Indels form as the result of imperfect NHEJ events due to repeated DSB formation from nuclease activity. For Cas9 targeted genomic loci indel formation at rates 10-30% are often observed and can approach 50%.
  • Figure 40 shows representative surveyor for 1st set of HepG2.2.15 experiments.
  • Figure 41 shows HepG2.2.15 HBV quantification scheme, an experimental design motivated by noise of initial data sets.
  • Figure 42 shows HepG2.2.15 results using sorting based normalization.
  • Figure 43 shows low levels of indels observed with guides targeting conserved HBV sequences in 2nd round of HepG2.2.15 experiments.
  • Figure 44 shows HepG2 co-transfection experiments.
  • Figure 45 shows HDD data for Cohort 1.
  • Figure 46 shows HDD data for Cohort 2.
  • Figure 47 shows HDD data for Cohort 2.
  • Figure 48 shows HDD data for Cohort 2.
  • Figure 49 shows Cohort 2-liver analysis 9d post HDD.
  • Figure 50 shows Cohort 2-liver analysis 9d post HDD.
  • Figure 51 shows low/no indels formed during HDD experiments. Predicted band sizes for guide 21 formation: 235 + 272 + 507 bp (undigested PCR product).
  • Figure 52 shows HDD Cohort 3 Results: HBsAg.
  • Figure 53 shows HDD Cohort 3 Results: Viremia.
  • Figure 54 shows HDD Cohort 3 Results: HBV in Liver
  • Figure 55 shows HDD Cohort 3 Results: Luciferase normalized to GAPDH.
  • Figure 56 shows despite low/no indel formation, effects on HBV are dependent on Cas9 nuclease activity.
  • Figure 57A-B shows (a) schematic of HBV life cycle and putative anti-HBV effect of CRISPR constructs; Cas9-mediated DSB formation should linearize the small, episomal cccDNA repeatedly, potentially leading to indel formation (generating less-fit viral mutants) or even degradation. (b) (left) HBV genome organization and location of target sequences for several tested guide RNA constructs, (right) Table of all possible CRISPR target sites in each HBV ORF, including number of possible target sites in conserved genomic regions.
  • Figure 58A-D shows (a) guide RNAs targeting conserved regions target large majority of patient-derived virus genomes. All whole-genome sequences from HBV isolates were queried from GenBank to determine the conservation of 23 nt target sequence (20 nt spacer + 3 nt PAM) for 3 guides (6, 17, and 21).
  • x-axis denotes number of allowed mismatches
  • y- axis denotes the percentage of sequenced isolates that fall within this number of mismatches to native sgRNA target site
  • Figure 59A-C shows (a) Experimental schematic for hydrodynamic injection experiments of (b-c): 1.3x WT HBV and sgRNA/Cas9-2A-mCherry are delivered to the livers of immunodeficient NRG mice via hydrodynamic injection, and (b) HBsAg and (c) secreted HBV titer are quantified in mouse blood at 2 and 4 days post injection.
  • 21M guide RNA with 5 bp mismatch from g21. Data shown are from one representative experiment, and consistent across multiple experiments.
  • UT ‘untargeted’ guide RNA (no target sequence in HBV genome).
  • FIG. 60A-B shows (a) the HBV life cycle within HepG2.2.15 cells.
  • HepG2.2.15 cells contain genomically integrated linear 1.3x WT HBV sequences, from which viral proteins and cccDNA are constitutively produced via transcription followed by translation (proteins) or reverse transcription and nuclear re-import (cccDNA).
  • the persistent HBV production in this system enables assay of the long-term anti-HBV effects of CRISPR/Cas systems targeting viral DNA; and (b) schematic of lentiviral vector and experimental strategy for sustained CRISPR expression within HepG2.2.15 cells.
  • Figure 61A-B shows that HBV-targeting CRISPR reduces HBV DNA and cccDNA dependent upon HBV-specific guide RNA and Cas9 activity.
  • Figure 62A-C shows HBV products are reduced upon long-term CRISPR/Cas expression.
  • Stable lines of HepG2.2.15 cells expressing 3 different on-target guides with nuclease-active or nuclease-dead Cas9, along with 3 non-HBV targeting guides, were seeded at consistent cell densities (20,000 cells/cm2) and allowed to secrete virions and viral proteins into the supernatant. 72h later, supernatant was collected and (a) viral titer and (b) HBeAg (a secreted protein produced from the C ORF, used clinically as a marker of active viral replication) were quantified (c).
  • HBeAg a secreted protein produced from the C ORF, used clinically as a marker of active viral replication
  • FIG. 63A-B shows that CRISPR constructs targeting HBV cause large and progressive reduction in (a) cccDNA and (b) total HBV DNA levels that is dependent on successful targeting of viral DNA.
  • Figure 64A-B shows HBV DNA and cccDNA reductions upon long-term CRISPR/Cas expression are produced with multiple guides.
  • Figure 65 shows Southern blot of HBV DNA.
  • Figure 66 shows Southern blot of HBV DNA.
  • Figure 67 shows Surveyor assay to detect indel formation in total HBV DNA (top) and episomal HBV DNA, enriched by treatment with plasmid-safe DNase (bottom); lentiviral transduction enables high levels of cutting of HBV.
  • Expected PCR product sizes for g6, g17 and g21 are respectively 599, 946 and 507 bp.
  • Approximate sizes of surveyor digestion products for g6, g17 and g21 are respectively: 429 + 170, 570 + 376, 275 + 232.
  • Figure 68 shows Immunofluorescent imaging of HBV Core protein demonstrates large reduction in Core staining upon targeting by g17 specifically against the Core ORF.
  • FIG. 69 shows schematics for de novo infection experiments.
  • Hep-NTCP cells were transduced with Cas9/gRNA constructs containing either g17 or g17M (mutant of g17, resulting in 5bp DNA bulge upon complexation to HBV DNA target), and either WT or dead Cas9, and then selected with puromycin to generate stable lines.
  • (Left) These cells were seeded in coculture with HepG2.2.15 cells, which produce infectious HBV virions that then infect the transduced Hep-NTCP cells. After transient coculture, HepG2.2.15 cells were killed by puromycin selection, and Hep-NTCP cells were cultured for several days and then harvested to assay viral parameters.
  • Light These cells were infected with HBV virions derived from HBV+ patient plasma, then cultured and harvested to assay viral parameters.
  • Figure 70A-D shows HBsAg secretion (a), cccDNA copies (b), levels of HBV 3.5kb RNA relative to 5 bp mismatch control (c), and titer of HBV DNA in culture medium (d).
  • the data show that Cas9/g17 reduce HBV infection in de novo infection context.
  • 17M 5 bp mismatch control.
  • 17D dead Cas9 with g17. Data shown are from one representative experiment, and consistent across experiments.
  • FIG 71A-C shows CRISPR/Cas-mediated disruption of HBV in patient-derived virus model system.
  • Hep-NTCP cells were infected with HBV from infected patient serum upon transduction of guide 17 and active or nuclease-dead Cas9. 9 days after infection, the cells were harvested and viral products were quantified. Nuclease-active Cas9 caused decreases in HBV 3.5kb RNA (a), cccDNA (b), and total DNA levels (c).
  • Figure 72 shows Surveyor assay performed on DNA untreated (left) or treated (right) with plasmid-Safe DNase to remove non-episomal viral forms. Arrowheads indicate shorter amplicons resulting from indel formation. The low levels of indel formation in 17M likely result from inefficient cutting enabled by the 5 bp bulge between target DNA and gRNA, recently appreciated in Lin, Y et al., (2014). (b-c) *p ⁇ 0.05 for selected comparison; **p ⁇ 0.01 for selected comparison, as assessed by one-way ANOVA with Tukey post-hoc test.
  • Figure 73 depicts a plot where the columns labeled‘D’ signify where Applicants used a nuclease-deficient Cas9 as an internal control.
  • the 29 dpt corresponds to 29 days post transduction, where a single lentiviral vector encoding U6-sgRNA and EFS-hSpCas9-2A-Puro was transduced into HepG2.2.15 cells followed by selection with puromycin.
  • Jiang et al. used the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli.
  • CRISPR clustered, regularly interspaced, short palindromic repeats
  • the approach relied on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems.
  • the study reported reprogramming dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates.
  • Konermann et al. addressed the need in the art for versatile and robust technologies that enable optical and chemical modulation of DNA-binding domains based CRISPR Cas9 enzyme and also Transcriptional Activator Like Effectors
  • Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis.
  • Ran et al. described an approach that combined a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage.
  • Hsu et al. characterized SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects.
  • the authors further showed that SpCas9- mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification.
  • the authors reported providing a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
  • Nishimasu et al. reported the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A° resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and non-complementary strands of the target DNA, respectively.
  • the nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • Hsu 2014 is a review article that discusses generally CRISPR-Cas9 history from yogurt to genome editing, including genetic screening of cells, that is in the information, data and findings of the applications in the lineage of this specification filed prior to June 5, 2014.
  • the general teachings of Hsu 2014 do not involve the specific models, animals of the instant specification.
  • CRISPR-Cas or CRISPR system is as used in the foregoing documents, such as WO 2014/093622 (PCT/US2013/074667) and refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g.
  • RNA(s) as that term is herein used (e.g., RNA(s) to guide Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus.
  • RNA(s) to guide Cas9 e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)
  • sgRNA single guide RNA
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • direct repeats may be identified in silico by searching for repetitive motifs that fulfill any or all of the following criteria: 1. found in a 2Kb window of genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Transform e.g. the Burrows Wheeler Aligner
  • ClustalW Clustal X
  • BLAT Novoalign
  • ELAND Illumina, San Diego, CA
  • SOAP available at soap.genomics.org.cn
  • Maq available at maq.sourceforge.net.
  • a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably the guide sequence is 10 - 30 nucleotides long. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • a guide sequence may be selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell.
  • Exemplary target sequences include those that are unique in the target genome.
  • a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • a unique target sequence in a genome may include an S.
  • a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXAGAAW where NNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome.
  • a unique target sequence in a genome may include an S.
  • a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNNNNNNNNNNXGGXG where NNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • a unique target sequence in a genome may include an S.
  • pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNNNXGGXG where NNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • N is A, G, T, or C; and X can be anything
  • a guide sequence is selected to reduce the degree secondary structure within the guide sequence. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the guide sequence participate in self-complementary base pairing when optimally folded.
  • Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence.
  • degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences.
  • Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence.
  • the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.
  • the transcript or transcribed polynucleotide sequence has at least two or more hairpins.
  • the transcript has two, three, four or five hairpins.
  • the transcript has at most five hairpins.
  • the portion of the sequence 5’ of the final“N” and upstream of the loop corresponds to the tracr mate sequence
  • the portion of the sequence 3’ of the loop corresponds to the tracr sequence
  • “N” represents a base of a guide sequence
  • the first block of lower case letters represent the tracr mate sequence
  • the second block of lower case letters represent the tracr sequence
  • the final poly-T sequence represents the transcription terminator: (1) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNgttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtttcgtatttttttt
  • sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1.
  • sequences (4) to (6) are used in combination with Cas9 from S. pyogenes.
  • the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.
  • candidate tracrRNA may be subsequently predicted by sequences that fulfill any or all of the following criteria: 1. sequence homology to direct repeats (motif search in Geneious with up to 18-bp mismatches); 2. presence of a predicted Rho- independent transcriptional terminator in direction of transcription; and 3. stable hairpin secondary structure between tracrRNA and direct repeat.
  • 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3.
  • all 3 criteria may be used.
  • chimeric synthetic guide RNAs may incorporate at least 12 bp of duplex structure between the direct repeat and tracrRNA.
  • CRISPR enzyme mRNA and guide RNA For minimization of toxicity and off-target effect, it will be important to control the concentration of CRISPR enzyme mRNA and guide RNA delivered. Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci.
  • deep sequencing can be used to assess the level of modification at the following two off-target loci, 1: 5’-GAGTCCTAGCAGGAGAAGAA-3’ and 2: 5’-GAGTCTAAGCAGAAGAAGAA- 3’.
  • concentration that gives the highest level of on-target modification while minimizing the level of off-target modification should be chosen for in vivo delivery.
  • CRISPR enzyme nickase mRNA for example S.
  • pyogenes Cas9 with the D10A mutation can be delivered with a pair of guide RNAs targeting a site of interest.
  • the two guide RNAs need to be spaced as follows.
  • Guide sequences and strategies to mimize toxicity and off-target effects can be as in WO 2014/093622 (PCT/US2013/074667).
  • the CRISPR system is derived advantageously from a type II CRISPR system.
  • one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes DNA cleavage.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.
  • the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9.
  • the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence.
  • the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.
  • two or more catalytic domains of Cas9 may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity.
  • a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity.
  • a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the non- mutated form of the enzyme; an example can be when the DNA cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form.
  • the enzyme is not SpCas9
  • mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools).
  • any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged.
  • the same (or conservative substitutions of these mutations) at corresponding positions in other Cas9s are also preferred.
  • Particularly preferred are D10 and H840 in SpCas9.
  • residues corresponding to SpCas9 D10 and H840 are also preferred.
  • Orthologs of SpCas9 can be used in the practice of the invention.
  • a Cas enzyme may be identified Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system.
  • the Cas9 enzyme is from, or is derived from, spCas9 (S. pyogenes Cas9) or saCas9 (S. aureus Cas9).
  • StCas9 refers to wild type Cas9 from S. thermophilus, the protein sequence of which is given in the SwissProt database under accession number G3ECR1.
  • S pyogenes Cas9 or spCas9 is included in SwissProt under accession number Q99ZW2.
  • Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent.
  • residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.
  • this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCa9, St1Cas9 and so forth.
  • Enzymatic action by Cas9 derived from Streptococcus pyogenes or any closely related Cas9 generates double stranded breaks at target site sequences which hybridize to 20 nucleotides of the guide sequence and that have a protospacer-adjacent motif (PAM) sequence (examples include NGG/NRG or a PAM that can be determined as described herein) following the 20 nucleotides of the target sequence.
  • PAM protospacer-adjacent motif
  • the CRISPR system small RNA- guided defence in bacteria and archaea, Mole Cell 2010, January 15; 37(1): 7.
  • the type II CRISPR locus from Streptococcus pyogenes SF370 which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30bp each).
  • DSB DNA double-strand break
  • RNAs two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus.
  • tracrRNA hybridizes to the direct repeats of pre-crRNA, which is then processed into mature crRNAs containing individual spacer sequences.
  • the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA.
  • Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer.
  • Cas9 may be constitutively present or inducibly present or conditionally present or administered or delivered. Cas9 optimization may be used to enhance function or to develop new functions, one can generate chimeric Cas9 proteins. And Cas9 may be used as a generic DNA binding protein.
  • a CRISPR complex comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins
  • formation of a CRISPR complex results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • the tracr sequence which may comprise or consist of all or a portion of a wild- type tracr sequence (e.g.
  • a wild-type tracr sequence may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence.
  • a codon optimized sequence is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans), or for another eukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 human codon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known.
  • an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate.
  • processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes may be excluded.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g.
  • Codon bias differences in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • genes can be tailored for optimal gene expression in a given organism based on codon optimization.
  • Codon usage tables are readily available, for example, at the“Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y., et al.“Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).
  • Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50,
  • a vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
  • the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus).
  • the CRISPR enzyme comprises at most 6 NLSs.
  • an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV; the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from importin-alpha; the sequences VSRKRPRP and PPKKARED of the myoma T protein; the sequence POPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-abl IV; the sequences DRLRR and PKQKKRK of the influenza
  • the one or more NLSs are of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.
  • strength of nuclear localization activity may derive from the number of NLSs in the CRISPR enzyme, the particular NLS(s) used, or a combination of these factors.
  • Detection of accumulation in the nucleus may be performed by any suitable technique.
  • a detectable marker may be fused to the CRISPR enzyme, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI).
  • Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity), as compared to a control no exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the one or more NLSs.
  • an assay for the effect of CRISPR complex formation e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity
  • aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased.
  • the gene product is a protein. Only sgRNA pairs creating 5’ overhangs with less than 8bp overlap between the guide sequences (offset greater than -8 bp) were able to mediate detectable indel formation.
  • each guide used in these assays is able to efficiently induce indels when paired with wildtype Cas9, indicating that the relative positions of the guide pairs are the most important parameters in predicting double nicking activity.
  • Cas9n and Cas9H840A nick opposite strands of DNA
  • substitution of Cas9n with Cas9H840A with a given sgRNA pair should have resulted in the inversion of the overhang type; but no indel formation is observed as with Cas9H840A indicating that Cas9H840A is a CRISPR enzyme substantially lacking all DNA cleavage activity (which is when the DNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the non- mutated form of the enzyme; whereby an example can be when the DNA cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form, e
  • a recombination template is also provided.
  • a recombination template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide.
  • a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex.
  • a template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length.
  • the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence.
  • a template polynucleotide When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.
  • one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors.
  • RNA(s) of the CRISPR System can be delivered to a transgenic Cas9 animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses Cas9; or an animal or mammal that is otherwise expressing Cas9 or has cells containing Cas9, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo Cas9.
  • a transgenic Cas9 animal or mammal e.g., an animal or mammal that constitutively or inducibly or conditionally expresses Cas9; or an animal or mammal that is otherwise expressing Cas9 or has cells containing Cas9, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo Cas9.
  • two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector.
  • CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element.
  • the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
  • a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron).
  • the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.
  • Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a CRISPR system are as used in the foregoing documents, such as WO 2014/093622 (PCT/US2013/074667).
  • a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a“cloning site”).
  • one or more insertion sites e.g.
  • a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence- specific binding of a CRISPR complex to a target sequence in a eukaryotic cell.
  • a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site.
  • the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these.
  • a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.
  • a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.
  • a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.
  • CRISPR enzyme or CRISPR enzyme mRNA or CRISPR guide RNA or RNA(s) can be delivered separately; and advantageously at least one of these is delivered via a nanoparticle complex.
  • CRISPR enzyme mRNA can be delivered prior to the guide RNA to give time for CRISPR enzyme to be expressed.
  • CRISPR enzyme mRNA might be administered 1 -12 hours (preferably around 2-6 hours) prior to the administration of guide RNA.
  • CRISPR enzyme mRNA and guide RNA can be administered together.
  • a second booster dose of guide RNA can be administered 1 -12 hours (preferably around 2-6 hours) after the initial administration of CRISPR enzyme mRNA + guide RNA. Additional administrations of CRISPR enzyme mRNA and/or guide RNA might be useful to achieve the most efficient levels of genome modification.
  • the invention provides methods for using one or more elements of a CRISPR system.
  • the CRISPR complex of the invention provides an effective means for modifying a target polynucleotide.
  • the CRISPR complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types.
  • the CRISPR complex of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis.
  • An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide.
  • the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.
  • this invention provides a method of cleaving a target polynucleotide.
  • the method comprises modifying a target polynucleotide using a CRISPR complex that binds to the target polynucleotide and effect cleavage of said target polynucleotide.
  • the CRISPR complex of the invention when introduced into a cell, creates a break (e.g., a single or a double strand break) in the genome sequence.
  • the method can be used to cleave a disease gene in a cell.
  • the break created by the CRISPR complex can be repaired by a repair processes such as the error prone non-homologous end joining (NHEJ) pathway or the high fidelity homology-directed repair (HDR).
  • NHEJ error prone non-homologous end joining
  • HDR high fidelity homology-directed repair
  • an exogenous polynucleotide template can be introduced into the genome sequence.
  • the HDR process is used modify genome sequence.
  • an exogenous polynucleotide template comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell.
  • the upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome.
  • a donor polynucleotide can be DNA, e.g., a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • the exogenous polynucleotide template comprises a sequence to be integrated (e.g., a mutated gene).
  • the sequence for integration may be a sequence endogenous or exogenous to the cell.
  • sequences to be integrated include polynucleotides encoding a protein or a non-coding RNA (e.g., a microRNA).
  • the sequence for integration may be operably linked to an appropriate control sequence or sequences.
  • the sequence to be integrated may provide a regulatory function.
  • the upstream and downstream sequences in the exogenous polynucleotide template are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide.
  • the upstream sequence is a nucleic acid sequence that shares sequence similarity with the genome sequence upstream of the targeted site for integration.
  • the downstream sequence is a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the targeted site of integration.
  • the upstream and downstream sequences in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted genome sequence.
  • the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted genome sequence.
  • the upstream and downstream sequences in the exogenous polynucleotide template have about 99% or 100% sequence identity with the targeted genome sequence.
  • An upstream or downstream sequence may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp.
  • the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp.
  • the exogenous polynucleotide template may further comprise a marker. Such a marker may make it easy to screen for targeted integrations.
  • exogenous polynucleotide template of the invention can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996).
  • a double stranded break is introduced into the genome sequence by the CRISPR complex, the break is repaired via homologous recombination an exogenous polynucleotide template such that the template is integrated into the genome.
  • the presence of a double-stranded break facilitates integration of the template.
  • this invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises increasing or decreasing expression of a target polynucleotide by using a CRISPR complex that binds to the polynucleotide.
  • a target polynucleotide can be inactivated to effect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild-type sequence does.
  • a protein or microRNA coding sequence may be inactivated such that the protein or microRNA or pre-microRNA transcript is not produced.
  • a control sequence can be inactivated such that it no longer functions as a control sequence.
  • control sequence refers to any nucleic acid sequence that effects the transcription, translation, or accessibility of a nucleic acid sequence. Examples of a control sequence include, a promoter, a transcription terminator, and an enhancer are control sequences.
  • the target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway- associated gene or polynucleotide.
  • target polynucleotides include a disease associated gene or polynucleotide.
  • A“disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease.
  • a disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease.
  • the transcribed or translated products may be known or unknown, and may be at a normal or abnormal level.
  • the target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell.
  • the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell.
  • the target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA).
  • the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the CRISPR complex.
  • PAM protospacer adjacent motif
  • the precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence) Examples of PAM sequences are given in the examples section below, and the skilled person will be able to identify further PAM sequences for use with a given CRISPR enzyme.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • Similar considerations and conditions apply as above for methods of modifying a target polynucleotide. In fact, these sampling, culturing and re-introduction options apply across the aspects of the present invention.
  • the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a cell or population of cells from a human or non- human animal, and modifying the cell or cells. Culturing may occur at any stage ex vivo.
  • the cell or cells may even be re-introduced into the non-human animal or plant. For re-introduced cells it is particularly preferred that the cells are stem cells.
  • the CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence, wherein said guide sequence may be linked to a tracr mate sequence which in turn may hybridize to a tracr sequence.
  • the invention relates to the engineering and optimization of systems, methods and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to the CRISPR-Cas system and components thereof.
  • the Cas enzyme is Cas9.
  • Cas9 optimization may be used to enhance function or to develop new functions, one can generate chimeric Cas9 proteins, as demonstrated in the Examples.
  • Chimeric Cas9 proteins can be made by combining fragments from different Cas9 homologs.
  • two example chimeric Cas9 proteins from the Cas9s described herein. For example, Applicants fused the N- term of St1Cas9 (fragment from this protein is in bold) with C-term of SpCas9.
  • chimeric Cas9s include any or all of: reduced toxicity; improved expression in eukaryotic cells; enhanced specificity; reduced molecular weight of protein, for example, making the protein smaller by combining the smallest domains from different Cas9 homologs; and/or altering the PAM sequence requirement.
  • the Cas9 may be used as a generic DNA binding protein as demonstrated in the Examples. Applicants used Cas9 as a generic DNA binding protein by mutating the two catalytic domains (D10 and H840) responsible for cleaving both strands of the DNA target. In order to upregulate gene transcription at a target locus Applicants fused a transcriptional activation domain (VP64) to Cas9. Other transcriptional activation domains are known. As shown in the Examples transcriptional activation is possible as well as gene repression using a Cas9 repressor (DNA-binding domain) that binds to the target gene sequence, thus repressing its activity.
  • Cas9 repressor DNA-binding domain
  • Cas9 and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, US Patents Nos. 8,454,972 (formulations, doses for adenovirus), 8,404,658 (formulations, doses for AAV) and 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus.
  • AAV the route of administration, formulation and dose can be as in US Patent No. 8,454,972 and as in clinical trials involving AAV.
  • the route of administration, formulation and dose can be as in US Patent No. 8,404,658 and as in clinical trials involving adenovirus.
  • the route of administration, formulation and dose can be as in US Patent No 5,846,946 and as in clinical studies involving plasmids.
  • Doses may be based on or extrapolated to an average 70 kg individual (e.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.
  • the viral vectors can be injected into the tissue of interest.
  • the expression of Cas9 can be driven by a cell-type specific promoter.
  • liver-specific expression might use the Albumin promoter
  • neuron-specific expression for viruses which may be latent in the brain
  • Synapsin I promoter for viruses which may be latent in the brain
  • Transgenic animals are also provided.
  • Preferred examples include animals comprising Cas9, in terms of polynucleotides encoding Cas9 or the protein itself. Mice, rats and rabbits are preferred.
  • To generate transgenic mice with the constructs as exemplified herein one may inject pure, linear DNA into the pronucleus of a zygote from a pseudo pregnant female, e.g. a CB56 female. Founders may then be identified, genotyped, and backcrossed to CB57 mice. The constructs may then be cloned and optionally verified, for instance by Sanger sequencing. Knock outs are envisaged where for instance one or more genes are knocked out in a model.
  • knockins are also envisaged (alone or in combination).
  • An example knockin Cas9 mouse was generated and this is exemplified, but Cas9 knockins are preferred.
  • To generate a Cas9 knock in mice one may target the same constitutive and conditional constructs to the Rosa26 locus, as described herein (Figs. 25A-B and 26).
  • Methods of US Patent Publication Nos. 20120017290 and 20110265198 assigned to Sangamo BioSciences, Inc. directed to targeting the Rosa locus may be modified to utilize the CRISPR Cas system of the present invention.
  • the methods of US Patent Publication No. 20130236946 assigned to Cellectis directed to targeting the Rosa locus may also be modified to utilize the CRISPR Cas system of the present invention.
  • Vector delivery e.g., plasmid, viral delivery:
  • the CRISPR enzyme for instance a Cas9, and/or any of the present RNAs, for instance a guide RNA, can be delivered using any suitable vector, e.g., plasmid or viral vectors, such as adeno associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof.
  • Cas9 and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vectors.
  • the vector e.g., plasmid or viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses.
  • the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choice, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.
  • Such a dosage may further contain, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), a pharmaceutically-acceptable excipient, and/or other compounds known in the art.
  • a carrier water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.
  • a pharmaceutically-acceptable carrier e.g., phosphate-buffered saline
  • a pharmaceutically-acceptable excipient e.g., phosphate-buffered saline
  • the dosage may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc.
  • auxiliary substances such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein.
  • Suitable exemplary ingredients include microcrystalline cellulose, carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof.
  • the delivery is via an adenovirus, which may be at a single booster dose containing at least 1 x 10 5 particles (also referred to as particle units, pu) of adenoviral vector.
  • the dose preferably is at least about 1 x 10 6 particles (for example, about 1 x 10 6 -1 x 10 12 particles), more preferably at least about 1 x 10 7 particles, more preferably at least about 1 x 10 8 particles (e.g., about 1 x 10 8 -1 x 10 11 particles or about 1 x 10 8 -1 x 10 12 particles), and most preferably at least about 1 x 10 0 particles (e.g., about 1 x 10 9 -1 x 10 10 particles or about 1 x 10 9 -1 x 10 12 particles), or even at least about 1 x 10 10 particles (e.g., about 1 x 10 10 -1 x 10 12 particles) of the adenoviral vector.
  • the dose comprises no more than about 1 x 10 14 particles, preferably no more than about 1 x 10 13 particles, even more preferably no more than about 1 x 10 12 particles, even more preferably no more than about 1 x 10 11 particles, and most preferably no more than about 1 x 10 10 particles (e.g., no more than about 1 x 10 9 articles).
  • the dose may contain a single dose of adenoviral vector with, for example, about 1 x 10 6 particle units (pu), about 2 x 10 6 pu, about 4 x 10 6 pu, about 1 x 10 7 pu, about 2 x 10 7 pu, about 4 x 10 7 pu, about 1 x 10 8 pu, about 2 x 10 8 pu, about 4 x 10 8 pu, about 1 x 10 9 pu, about 2 x 10 9 pu, about 4 x 10 9 pu, about 1 x 10 10 pu, about 2 x 10 10 pu, about 4 x 10 10 pu, about 1 x 10 11 pu, about 2 x 10 11 pu, about 4 x 10 11 pu, about 1 x 10 12 pu, about 2 x 10 12 pu, or about 4 x 10 12 pu of adenoviral vector.
  • adenoviral vector with, for example, about 1 x 10 6 particle units (pu), about 2 x 10 6 pu, about 4 x 10 6 pu, about 1 x 10 7 pu, about 2 x 10 7 pu
  • the adenoviral vectors in U.S. Patent No. 8,454,972 B2 to Nabel, et. al., granted on June 4, 2013; incorporated by reference herein, and the dosages at col 29, lines 36-58 thereof.
  • the adenovirus is delivered via multiple doses.
  • the delivery is via an AAV.
  • a therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1 x 10 10 to about 1 x 10 10 functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects.
  • the AAV dose is generally in the range of concentrations of from about 1 x 10 5 to 1 x 10 50 genomes AAV, from about 1 x 10 8 to 1 x 10 20 genomes AAV, from about 1 x 10 10 to about 1 x 10 16 genomes, or about 1 x 10 11 to about 1 x 10 16 genomes AAV.
  • a human dosage may be about 1 x 10 13 genomes AAV. Such concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, U.S. Patent No. 8,404,658 B2 to Hajjar, et al., granted on March 26, 2013, at col. 27, lines 45-60.
  • the delivery is via a plasmid.
  • the dosage should be a sufficient amount of plasmid to elicit a response.
  • suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg, or from about 1 g to about 10 g per 70 kg individual.
  • Plasmids of the invention will generally comprise (i) a promoter; (ii) a sequence encoding a CRISPR enzyme, operably linked to said promoter; (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii).
  • the plasmid can also encode the RNA components of a CRISPR complex, but one or more of these may instead be encoded on a different vector.
  • the doses herein are based on an average 70 kg individual.
  • the frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), or scientist skilled in the art. It is also noted that mice used in experiments are typically about 20g and from mice experiments one can scale up to a 70 kg individual.
  • RNA molecules of the invention are delivered in liposome or lipofectin formulations and the like and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference. Delivery systems aimed specifically at the enhanced and improved delivery of siRNA into mammalian cells have been developed, (see, for example, Shen et al FEBS Let. 2003, 539:111 -114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision.
  • siRNA has recently been successfully used for inhibition of gene expression in primates (see for example. Tolentino et al., Retina 24(4):660 which may also be applied to the present invention.
  • RNA delivery is a useful method of in vivo delivery. It is possible to deliver Cas9 and gRNA (and, for instance, HR repair template) into cells using liposomes or nanoparticles.
  • delivery of the CRISPR enzyme, such as a Cas9 and/or delivery of the RNAs of the invention may be in RNA form and via microvesicles, liposomes or nanoparticles.
  • Cas9 mRNA and gRNA can be packaged into liposomal particles for delivery in vivo.
  • Liposomal transfection reagents such as lipofectamine from Life Technologies and other reagents on the market can effectively deliver RNA molecules into the liver.
  • Means of delivery of RNA also preferred include delivery of RNA via nanoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells, Advanced Functional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A., Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-based nanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID: 20059641).
  • exosomes have been shown to be particularly useful in delivery siRNA, a system with some parallels to the CRISPR system.
  • El-Andaloussi S, et al. (“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc. 2012 Dec;7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012 Nov 15.) describe how exosomes are promising tools for drug delivery across different biological barriers and can be harnessed for delivery of siRNA in vitro and in vivo.
  • Their approach is to generate targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand.
  • RNA is loaded into the exosomes.
  • Delivery or administration according to the invention can be performed with exosomes, in particular but not limited to the brain.
  • Vitamin E ⁇ -tocopherol
  • CRISPR Cas may be conjugated with CRISPR Cas and delivered to the brain along with high density lipoprotein (HDL), for example in a similar manner as was done by Uno et al. (HUMAN GENE THERAPY 22:711–719 (June 2011)) for delivering short-interfering RNA (siRNA) to the brain.
  • HDL high density lipoprotein
  • Mice were infused via Osmotic minipumps (model 1007D; Alzet, Cupertino, CA) filled with phosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL and connected with Brain Infusion Kit 3 (Alzet).
  • PBS phosphate-buffered saline
  • a brain- infusion cannula was placed about 0.5mm posterior to the bregma at midline for infusion into the dorsal third ventricle.
  • Uno et al. found that as little as 3 nmol of Toc-siRNA with HDL could induce a target reduction in comparable degree by the same ICV infusion method.
  • a similar dosage of CRISPR Cas conjugated to ⁇ -tocopherol and co-administered with HDL targeted to the brain may be contemplated for humans in the present invention, for example, about 3 nmol to about 3 mol of CRISPR Cas targeted to the brain may be contemplated.
  • Zou et al. (HUMAN GENE THERAPY 22:465-475 (April 2011)) describes a method of lentiviral-mediated delivery of short-hairpin RNAs targeting PKC ⁇ for in vivo gene silencing in the spinal cord of rats. Zou et al.
  • a similar dosage of CRISPR Cas expressed in a lentiviral vector targeted to the brain may be contemplated for humans in the present invention, for example, about 10-50 ml of CRISPR Cas targeted to the brain in a lentivirus having a titer of 1 x 10 9 transducing units (TU)/ml may be contemplated.
  • material can be delivered intrastriatally e.g. by injection. Injection can be performed stereotactically via a craniotomy.
  • Enhancing NHEJ or HR efficiency is also helpful for delivery. It is preferred that NHEJ efficiency is enhanced by co-expressing end-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011 August; 188(4): 787–797). It is preferred that HR efficiency is increased by transiently inhibiting NHEJ machineries such as Ku70 and Ku86. HR efficiency can also be increased by co-expressing prokaryotic or eukaryotic homologous recombination enzymes such as RecBCD, RecA. Packaging and Promoters generally
  • Ways to package Cas9 coding nucleic acid molecules, e.g., DNA, into vectors, e.g., viral vectors, to mediate genome modification in vivo include:
  • Vector 1 containing one expression cassette for driving the expression of Cas9 Promoter-Cas9 coding nucleic acid molecule-terminator
  • Vector 2 containing one more expression cassettes for driving the expression of one or more guideRNAs
  • Promoter-gRNA(N)-terminator up to size limit of vector
  • an additional vector is used to deliver a homology-direct repair template.
  • the promoter used to drive Cas9 coding nucleic acid molecule expression can include:
  • AAV ITR can serve as a promoter: this is advantageous for eliminating the need for an additional promoter element (which can take up space in the vector). The additional space freed up can be used to drive the expression of additional elements (gRNA, etc.). Also, ITR activity is relatively weaker, so can be used to reduce potential toxicity due to over expression of Cas9.
  • promoters CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.
  • promoters For brain or other CNS expression, can use promoters: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.
  • For liver expression can use Albumin promoter
  • For lung expression can use SP-B.
  • ICAM ICAM
  • hematopoietic cells can use IFNbeta or CD45.
  • the promoter used to drive guide RNA can include:
  • Pol III promoters such as U6 or H1
  • AAV Adeno associated virus
  • Cas9 and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, US Patents Nos. 8,454,972 (formulations, doses for adenovirus), 8,404,658 (formulations, doses for AAV) and 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus.
  • AAV the route of administration, formulation and dose can be as in US Patent No. 8,454,972 and as in clinical trials involving AAV.
  • the route of administration, formulation and dose can be as in US Patent No. 8,404,658 and as in clinical trials involving adenovirus.
  • the route of administration, formulation and dose can be as in US Patent No 5,846,946 and as in clinical studies involving plasmids.
  • Doses may be based on or extrapolated to an average 70 kg individual (e.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.
  • the viral vectors can be injected into the tissue of interest.
  • the expression of Cas9 can be driven by a cell-type specific promoter.
  • liver-specific expression might use the Albumin promoter and neuron- specific expression (e.g. for targeting CNS disorders) might use the Synapsin I promoter.
  • AAV is advantageous over other viral vectors for a couple of reasons:
  • AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cas9 as well as a promoter and transcription terminator have to be all fit into the same viral vector. Constructs larger than 4.5 or 4.75 Kb will lead to significantly reduced virus production. SpCas9 is quite large, the gene itself is over 4.1 Kb, which makes it difficult for packing into AAV. Therefore embodiments of the invention include utilizing homologs of Cas9 that are shorter. For example:
  • the AAV can be AAV1, AAV2, AAV5 or any combination thereof.
  • AAV8 is useful for delivery to the liver. The herein promoters and vectors are preferred individually.
  • a tabulation of certain AAV serotypes as to these cells is as follows:
  • Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells.
  • the most commonly known lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types.
  • HIV human immunodeficiency virus
  • lentiviral transfer plasmid pCasES10
  • pMD2.G VSV-g pseudotype
  • psPAX2 gag/pol/rev/tat
  • Transfection was done in 4mL OptiMEM with a cationic lipid delivery agent (50uL Lipofectamine 2000 and 100ul Plus reagent). After 6 hours, the media was changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods use serum during cell culture, but serum-free methods are preferred.
  • Lentivirus may be purified as follows. Viral supernatants were harvested after 48 hours. Supernatants were first cleared of debris and filtered through a 0.45um low protein binding (PVDF) filter. They were then spun in a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets were resuspended in 50ul of DMEM overnight at 4C. They were then aliquotted and immediately frozen at -80°C.
  • PVDF 0.45um low protein binding
  • minimal non-primate lentiviral vectors based on the equine infectious anemia virus are also contemplated, especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8: 275– 285).
  • RetinoStat® an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the web form of age related macular degeneration is also contemplated (see e g Binley et al HUMAN GENE THERAPY 23:980–991 (September 2012)) and this vector may be modified for the CRISPR-Cas system of the present invention.
  • self-inactivating lentiviral vectors with an siRNA targeting a common exon shared by HIV tat/rev, a nucleolar-localizing TAR decoy, and an anti–CCR5- specific hammerhead ribozyme may be used/and or adapted to the CRISPR-Cas system of the present invention.
  • a minimum of 2.5 ⁇ 10 6 CD34+ cells per kilogram patient weight may be collected and prestimulated for 16 to 20 hours in X-VIVO 15 medium (Lonza) containing 2 mol/L-glutamine, stem cell factor (100 ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml) (CellGenix) at a density of 2 ⁇ 10 6 cells/ml.
  • Prestimulated cells may be transduced with lentiviral at a multiplicity of infection of 5 for 16 to 24 hours in 75-cm 2 tissue culture flasks coated with fibronectin (25 mg/cm 2 ) (RetroNectin,Takara Bio Inc.).
  • Lentiviral vectors have been disclosed as in the treatment for Parkinson’s Disease, see, e.g., US Patent Publication No. 20120295960 and US Patent Nos. 7303910 and 7351585. Lentiviral vectors have also been disclosed for the treatment of ocular diseases, see e.g., US Patent Publication Nos. 20060281180, 20090007284, US20110117189; US20090017543; US20070054961, US20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., US Patent Publication Nos. US20110293571; US20110293571, US20040013648, US20070025970, US20090111106 and US Patent No. US7259015.
  • RNA delivery The CRISPR enzyme, for instance a Cas9, and/or any of the present RNAs, for instance a guide RNA, can also be delivered in the form of RNA.
  • Cas9 mRNA can be generated using in vitro transcription.
  • Cas9 mRNA can be synthesized using a PCR cassette containing the following elements: T7_promoter-kozak sequence (GCCACC)-Cas9-3’ UTR from beta globin-polyA tail (a string of 120 or more adenines).
  • the cassette can be used for transcription by T7 polymerase.
  • Guide RNAs can also be transcribed using in vitro transcription from a cassette containing T7_promoter-GG-guide RNA sequence.
  • the CRISPR enzyme-coding sequence and/or the guide RNA can be modified to include one or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.
  • RNA delivery methods are especially promising for liver delivery currently.
  • Much clinical work on RNA delivery has focused on RNAi or antisense, but these systems can be adapted for delivery of RNA for implementing the present invention. References below to RNAi etc. should be read accordingly.
  • CRISPR enzyme mRNA and guide RNA may be delivered simultaneously using nanoparticles or lipid envelopes.
  • Su X, Fricke J, Kavanagh DG, Irvine DJ (“In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles” Mol Pharm. 2011 Jun 6;8(3):774-87. doi: 10.1021/mp100390w. Epub 2011 Apr 1) describes biodegradable core-shell structured nanoparticles with a poly( ⁇ -amino ester) (PBAE) core enveloped by a phospholipid bilayer shell. These were developed for in vivo mRNA delivery.
  • the pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Such are, therefore, preferred for delivering RNA of the present invention.
  • nanoparticles based on self assembling bioadhesive polymers are contemplated, which may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, all to the brain.
  • Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated.
  • the molecular envelope technology involves an engineered polymer envelope which is protected and delivered to the site of the disease (see, e.g., Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. Mol Pharm, 2012. 9(1):14-28; Lalatsa, A., et al.
  • nanoparticles that can deliver RNA to a cancer cell to stop tumor growth developed by Dan Anderson’s lab at MIT may be used/and or adapted to the CRISPR Cas system of the present invention.
  • the Anderson lab developed fully automated, combinatorial systems for the synthesis, purification, characterization, and formulation of new biomaterials and nanoformulations. See, e.g., Alabi et al., Proc Natl Acad Sci U S A. 2013 Aug 6;110(32):12881-6; Zhang et al., Adv Mater. 2013 Sep 6;25(33):4641-5; Jiang et al., Nano Lett.
  • US patent application 20110293703 relates to lipidoid compounds are also particularly useful in the administration of polynucleotides, which may be applied to deliver the CRISPR Cas system of the present invention.
  • the aminoalcohol lipidoid compounds are combined with an agent to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles.
  • the agent to be delivered by the particles, liposomes, or micelles may be in the form of a gas, liquid, or solid, and the agent may be a polynucleotide, protein, peptide, or small molecule.
  • the minoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition.
  • US Patent Publication No. 20110293703 also provides methods of preparing the aminoalcohol lipidoid compounds.
  • One or more equivalents of an amine are allowed to react with one or more equivalents of an epoxide-terminated compound under suitable conditions to form an aminoalcohol lipidoid compound of the present invention.
  • all the amino groups of the amine are fully reacted with the epoxide-terminated compound to form tertiary amines.
  • all the amino groups of the amine are not fully reacted with the epoxide-terminated compound to form tertiary amines thereby resulting in primary or secondary amines in the aminoalcohol lipidoid compound.
  • These primary or secondary amines are left as is or may be reacted with another electrophile such as a different epoxide-terminated compound.
  • amines may be fully functionalized with two epoxide-derived compound tails while other molecules will not be completely functionalized with epoxide-derived compound tails.
  • a diamine or polyamine may include one, two, three, or four epoxide-derived compound tails off the various amino moieties of the molecule resulting in primary, secondary, and tertiary amines. In certain embodiments, all the amino groups are not fully functionalized.
  • two of the same types of epoxide-terminated compounds are used.
  • two or more different epoxide- terminated compounds are used.
  • the synthesis of the aminoalcohol lipidoid compounds is performed with or without solvent, and the synthesis may be performed at higher temperatures ranging from 30.-100 C., preferably at approximately 50.-90 C.
  • the prepared aminoalcohol lipidoid compounds may be optionally purified.
  • the mixture of aminoalcohol lipidoid compounds may be purified to yield an aminoalcohol lipidoid compound with a particular number of epoxide-derived compound tails.
  • the mixture may be purified to yield a particular stereo- or regioisomer.
  • the aminoalcohol lipidoid compounds may also be alkylated using an alkyl halide (e.g., methyl iodide) or other alkylating agent, and/or they may be acylated.
  • US Patent Publication No. 20110293703 also provides libraries of aminoalcohol lipidoid compounds prepared by the inventive methods. These aminoalcohol lipidoid compounds may be prepared and/or screened using high-throughput techniques involving liquid handlers, robots, microtiter plates, computers, etc. In certain embodiments, the aminoalcohol lipidoid compounds are screened for their ability to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into the cell.
  • agents e.g., proteins, peptides, small molecules
  • US Patent Publication No. 20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) has been prepared using combinatorial polymerization.
  • PBAAs poly(beta-amino alcohols)
  • the inventive PBAAs may be used in biotechnology and biomedical applications as coatings (such as coatings of films or multilayer films for medical devices or implants), additives, materials, excipients, non-biofouling agents, micropatterning agents, and cellular encapsulation agents.
  • coatings such as coatings of films or multilayer films for medical devices or implants
  • additives such as coatings of films or multilayer films for medical devices or implants
  • materials such as coatings of films or multilayer films for medical devices or implants
  • additives such as coatings of films or multilayer films for medical devices or implants
  • materials such as coatings of films or multilayer films for medical devices or implants
  • excipients such as coatings of films or multilayer films for medical devices or implants
  • these coatings reduce the recruitment of inflammatory cells, and reduce fibrosis, following the subcutaneous implantation of carboxylated polystyrene microparticles.
  • These polymers may be used to form polyelectrolyte complex capsules for cell encapsulation.
  • the invention may also have many other biological applications such as antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering.
  • US Patent Publication No. 20130302401 may be applied to the CRISPR Cas system of the present invention.
  • lipid nanoparticles are contemplated.
  • an antitransthyretin small interfering RNA encapsulated in lipid nanoparticles may be applied to the CRISPR Cas system of the present invention.
  • Doses of about 0.01 to about 1 mg per kg of body weight administered intravenously are contemplated.
  • Medications to reduce the risk of infusion-related reactions are contemplated, such as dexamethasone, acetampinophen, diphenhydramine or cetirizine, and ranitidine are contemplated.
  • LNPs have been shown to be highly effective in delivering siRNAs to the liver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470) and are therefore contemplated for delivering RNA encoding CRISPR Cas to the liver.
  • a dosage of about four doses of 6 mg/kg of the LNP every two weeks may be contemplated.
  • ionizable cationic lipids with pKa values below 7 were developed (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011).
  • Negatively charged polymers such asiRNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge.
  • pH 4 e.g., pH 4
  • the LNPs exhibit a low surface charge compatible with longer circulation times.
  • ionizable cationic lipids Four species of ionizable cationic lipids have been focused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA).
  • DLinDAP 1,2-dilineoyl-3-dimethylammonium-propane
  • DLinDMA 1,2-dilinoleyloxy-3-N,N- dimethylaminopropane
  • DLinKDMA 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-a
  • LNP siRNA systems containing these lipids exhibit remarkably different gene silencing properties in hepatocytes in vivo, with potencies varying according to the series DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII gene silencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011).
  • a dosage of 1 g/ml levels may be contemplated, especially for a formulation containing DLinKC2-DMA.
  • Preparation of LNPs and CRISPR Cas encapsulation may be used/and or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011).
  • the cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3- o-[2 ⁇ -(methoxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[((
  • Cholesterol may be purchased from Sigma (St Louis, MO).
  • the specific CRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios).
  • 0.2% SP-DiOC18 Invitrogen, Burlington, Canada
  • Encapsulation may be performed by dissolving lipid mixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanol to a final lipid concentration of 10 mmol/l.
  • This ethanol solution of lipid may be added drop-wise to 50 mmol/l citrate, pH 4.0 to form multilamellar vesicles to produce a final concentration of 30% ethanol vol/vol.
  • Large unilamellar vesicles may be formed following extrusion of multilamellar vesicles through two stacked 80 nm Nuclepore polycarbonate filters using the Extruder (Northern Lipids, Vancouver, Canada).
  • Encapsulation may be achieved by adding RNA dissolved at 2 mg/ml in 50 mmol/l citrate, pH 4.0 containing 30% ethanol vol/vol drop-wise to extruded preformed large unilamellar vesicles and incubation at 31 °C for 30 minutes with constant mixing to a final RNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol and neutralization of formulation buffer were performed by dialysis against phosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulose dialysis membranes.
  • PBS phosphate-buffered saline
  • Nanoparticle size distribution may be determined by dynamic light scattering using a NICOMP 370 particle sizer, the vesicle/intensity modes, and Gaussian fitting (Nicomp Particle Sizing, Santa Barbara, CA). The particle size for all three LNP systems may be ⁇ 70 nm in diameter.
  • siRNA encapsulation efficiency may be determined by removal of free siRNA using VivaPureD MiniH columns (Sartorius Stedim Biotech) from samples collected before and after dialysis. The encapsulated RNA may be extracted from the eluted nanoparticles and quantified at 260 nm.
  • siRNA to lipid ratio was determined by measurement of cholesterol content in vesicles using the Cholesterol E enzymatic assay from Wako Chemicals USA (Richmond, VA). Preparation of large LNPs may be used/and or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011.
  • a lipid premix solution (20.4 mg/ml total lipid concentration) may be prepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at 50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premix at a molar ratio of 0.75:1 (sodium acetate:DLinKC2- DMA).
  • the lipids may be subsequently hydrated by combining the mixture with 1.85 volumes of citrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting in spontaneous liposome formation in aqueous buffer containing 35% ethanol.
  • the liposome solution may be incubated at 37 °C to allow for time-dependent increase in particle size. Aliquots may be removed at various times during incubation to investigate changes in liposome size by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK).
  • the liposomes should their size, effectively quenching further growth.
  • RNA may then be added to the empty liposomes at an siRNA to total lipid ratio of approximately 1:10 (wt:wt), followed by incubation for 30 minutes at 37 °C to form loaded LNPs. The mixture may be subsequently dialyzed overnight in PBS and filtered with a 0.45- m syringe filter.
  • Spherical Nucleic Acid (SNATM) constructs and other nanoparticles (particularly gold nanoparticles) are also contemplate as a means to delivery CRISPR/Cas system to intended targets.
  • Significant data show that AuraSense Therapeutics' Spherical Nucleic Acid (SNATM) constructs, based upon nucleic acid-functionalized gold nanoparticles, are superior to alternative platforms based on multiple key success factors, such as: High in vivo stability: Due to their dense loading, a majority of cargo (DNA or siRNA) remains bound to the constructs inside cells, conferring nucleic acid stability and resistance to enzymatic degradation.
  • Deliverability For all cell types studied (e.g., neurons, tumor cell lines, etc.) the constructs demonstrate a transfection efficiency of 99% with no need for carriers or transfection agents.
  • Therapeutic targeting The unique target binding affinity and specificity of the constructs allowaki specificity for matched target sequences (i.e., limited off-target effects). Superior efficacy: The constructs significantly outperform leading conventional transfection reagents (Lipofectamine 2000 and Cytofectin). Low toxicity: The constructs can enter a variety of cultured cells, primary cells, and tissues with no apparent toxicity. No significant immune response: The constructs elicit minimal changes in global gene expression as measured by whole-genome microarray studies and cytokine-specific protein assays.
  • Any number of single or combinatorial agents can be used to tailor the surface of the constructs.
  • This platform for nucleic acid-based therapeutics may be applicable to numerous disease states, including inflammation and infectious disease, cancer, skin disorders and cardiovascular disease.
  • Citable literature includes: Cutler et al.,. J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962- 6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al.,. Nano Lett.
  • Self-assembling nanoparticles with siRNA may be constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG), for example, as a means to target tumor neovasculature expressing integrins and used to deliver siRNA inhibiting vascular endothelial growth factor receptor-2 (VEGF R2) expression and thereby tumor angiogenesis (see, e.g., Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19).
  • PEI polyethyleneimine
  • RGD Arg-Gly-Asp
  • VEGF R2 vascular endothelial growth factor receptor-2
  • Nanoplexes may be prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid to give a net molar excess of ionizable nitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.
  • the electrostatic interactions between cationic polymers and nucleic acid resulted in the formation of polyplexes with average particle size distribution of about 100 nm, hence referred to here as nanoplexes.
  • a dosage of about 100 to 200 mg of CRISPR Cas is envisioned for delivery in the self-assembling nanoparticles of Schiffelers et al.
  • the nanoplexes of Bartlett et al. may also be applied to the present invention.
  • the nanoplexes of Bartlett et al. are prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid to give a net molar excess of ionizable nitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6.
  • the electrostatic interactions between cationic polymers and nucleic acid resulted in the formation of polyplexes with average particle size distribution of about 100 nm, hence referred to here as nanoplexes.
  • DOTA- NHSester 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester)
  • DOTA- NHSester 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester)
  • the amine modified RNA sense strand with a 100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) was added to a microcentrifuge tube. The contents were reacted by stirring for 4 h at room temperature.
  • the DOTA-RNAsense conjugate was ethanol-precipitated, resuspended in water, and annealed to the unmodified antisense strand to yield DOTA-siRNA.
  • Tf-targeted and nontargeted siRNA nanoparticles may be formed by using cyclodextrin-containing polycations. Typically, nanoparticles were formed in water at a charge ratio of 3 (+/-) and an siRNA concentration of 0.5 g/liter. One percent of the adamantane-PEG molecules on the surface of the targeted nanoparticles were modified with Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5% (wt/vol) glucose carrier solution for injection.
  • the nanoparticles consist of a synthetic delivery system containing: (1) a linear, cyclodextrin- based polymer (CDP), (2) a human transferrin protein (TF) targeting ligand displayed on the exterior of the nanoparticle to engage TF receptors (TFR) on the surface of the cancer cells, (3) a hydrophilic polymer (polyethylene glycol (PEG) used to promote nanoparticle stability in biological fluids), and (4) siRNA designed to reduce the expression of the RRM2 (sequence used in the clinic was previously denoted siR2B+5).
  • CDP linear, cyclodextrin- based polymer
  • TF human transferrin protein
  • TFR TF receptors
  • siRNA designed to reduce the expression of the RRM2 (sequence used in the clinic was previously denoted siR2B+5).
  • the TFR has long been known to be upregulated in malignant cells, and RRM2 is an established anti-cancer target.
  • nanoparticles (clinical version denoted as CALAA-01) have been shown to be well tolerated in multi-dosing studies in non-human primates.
  • Davis et al. s clinical trial is the initial human trial to systemically deliver siRNA with a targeted delivery system and to treat patients with solid cancer.
  • Davis et al. investigated biopsies from three patients from three different dosing cohorts; patients A, B and C, all of whom had metastatic melanoma and received CALAA-01 doses of 18, 24 and 30 mg m -2 siRNA, respectively.
  • CRISPR Cas system of the present invention Similar doses may also be contemplated for the CRISPR Cas system of the present invention.
  • the delivery of the invention may be achieved with nanoparticles containing a linear, cyclodextrin-based polymer (CDP), a human transferrin protein (TF) targeting ligand displayed on the exterior of the nanoparticle to engage TF receptors (TFR) on the surface of the cancer cells and/or a hydrophilic polymer (for example, polyethylene glycol (PEG) used to promote nanoparticle stability in biological fluids).
  • CDP linear, cyclodextrin-based polymer
  • TF human transferrin protein
  • TFR TF receptors
  • hydrophilic polymer for example, polyethylene glycol (PEG) used to promote nanoparticle stability in biological fluids.
  • Exosomes for example, polyethylene glycol (PEG) used to promote nanoparticle stability in biological fluids.
  • Exosomes are endogenous nano-vesicles that transport RNAs and proteins which can deliver short interfering (si)RNA to the brain and other target organs.
  • siRNAs and proteins which can deliver short interfering (si)RNA to the brain and other target organs.
  • Alvarez-Erviti et al. 2011, Nat Biotechnol 29: 341
  • Lamp2b an exosomal membrane protein, fused to the neuron-specific RVG peptide3.
  • Purified exosomes were loaded with exogenous siRNA by electroporation.
  • RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease. To obtain a pool of immunologically inert exosomes, Alvarez-Erviti et al.
  • MHC major histocompatibility complex
  • T-cell activators such as MHC-II and CD86
  • GM-CSF granulocyte/macrophage-colony stimulating factor
  • Exosomes were purified from the culture supernatant the following day using well-established ultracentrifugation protocols. The exosomes produced were physically homogenous, with a size distribution peaking at 80 nm in diameter as determined by nanoparticle tracking analysis (NTA) and electron microscopy.
  • NTA nanoparticle tracking analysis
  • Alvarez-Erviti et al. obtained 6-12 g of exosomes (measured based on protein concentration) per 10 6 cells.
  • Alvarez-Erviti et al. investigated the possibility of loading modified exosomes with exogenous cargoes using electroporation protocols adapted for nanoscale applications.
  • electroporation for membrane particles at the nanometer scale is not well-characterized, nonspecific Cy5-labeled siRNA was used for the empirical optimization of the electroporation protocol.
  • the amount of encapsulated siRNA was assayed after ultracentrifugation and lysis of exosomes. Electroporation at 400 V and 125 F resulted in the greatest retention of siRNA and was used for all subsequent experiments.
  • Alvarez-Erviti et al. carried out 5'-rapid amplification of cDNA ends (RACE) on BACE1 cleavage product, which provided evidence of RNAi-mediated knockdown by the siRNA.
  • RACE 5'-rapid amplification of cDNA ends
  • siRNA-RVG exosomes induced immune responses in vivo by assessing IL-6, IP-10, TNF ⁇ and IFN- ⁇ serum concentrations.
  • siRNA-RVG exosome treatment nonsignificant changes in all cytokines were registered similar to siRNA-transfection reagent treatment in contrast to siRNA-RVG-9R, which potently stimulated IL-6 secretion, confirming the immunologically inert profile of the exosome treatment.
  • delivery with RVG-exosome appears to be more efficient than RVG-9R delivery as comparable mRNA knockdown and greater protein knockdown was achieved with fivefold less siRNA without the corresponding level of immune stimulation.
  • the exosome delivery system of Alvarez-Erviti et al. may be applied to deliver the CRISPR-Cas system of the present invention to therapeutic targets, especially neurodegenerative diseases.
  • a dosage of about 100 to 1000 mg of CRISPR Cas encapsulated in about 100 to 1000 mg of RVG exosomes may be contemplated for the present invention.
  • El-Andaloussi et al. discloses how exosomes derived from cultured cells can be harnessed for delivery of siRNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. Next, El- Andaloussi et al. explain how to purify and characterize exosomes from transfected cell supernatant. Next, El-Andaloussi et al. detail crucial steps for loading siRNA into exosomes. Finally, El-Andaloussi et al.
  • exosome-mediated siRNA delivery is evaluated by functional assays and imaging.
  • the entire protocol takes 3 weeks. Delivery or administration according to the invention may be performed using exosomes produced from self-derived dendritic cells.
  • Exosomes are nano-sized vesicles (30– 90nm in size) produced by many cell types, including dendritic cells (DC), B cells, T cells, mast cells, epithelial cells and tumor cells. These vesicles are formed by inward budding of late endosomes and are then released to the extracellular environment upon fusion with the plasma membrane. Because exosomes naturally carry RNA between cells, this property might be useful in gene therapy.
  • DC dendritic cells
  • B cells B cells
  • T cells T cells
  • mast cells epithelial cells
  • tumor cells tumor cells.
  • Exosomes from plasma are prepared by centrifugation of buffy coat at 900g for 20 min to isolate the plasma followed by harvesting cell supernatants, centrifuging at 300g for 10 min to eliminate cells and at 16 500g for 30 min followed by filtration through a 0.22 mm filter. Exosomes are pelleted by ultracentrifugation at 120 000g for70 min. Chemical transfection of siRNA into exosomes is carried out according to the manufacturer’s instructions in RNAi Human/Mouse Starter Kit (Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a final concentration of 2 mmol/ml.
  • exosomes are re- isolated using aldehyde/sulfate latex beads.
  • the chemical transfection of CRISPR Cas into exosomes may be conducted similarly to siRNA.
  • the exosomes may be co-cultured with monocytes and lymphocytes isolated from the peripheral blood of healthy donors. Therefore, it may be contemplated that exosomes containing CRISPR Cas may be introduced to monocytes and lymphocytes of and autologously reintroduced into a human. Accordingly, delivery or administration according to the invention may beperformed using plasma exosomes.
  • Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes have gained considerable attention as drug delivery carriers because they are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
  • BBB blood brain barrier
  • Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). Several other additives may be added to liposomes in order to modify their structure and properties.
  • liposomes are prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol, and dicetyl phosphate, and their mean vesicle sizes were adjusted to about 50 and 100 ⁇ nm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review).
  • a liposome formulation may be mainly comprised of natural phospholipids and lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside. Since this formulation is made up of phospholipids only, liposomal formulations have encountered many challenges, one of the ones being the instability in plasma. Several attempts to overcome these challenges have been made, specifically in the manipulation of the lipid membrane. One of these attempts focused on the manipulation of cholesterol.
  • DSPC 1,2-distearoryl-sn-glycero-3-phosphatidyl choline
  • the CRISPR Cas system may be administered in liposomes, such as a stable nucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005).
  • SNALP stable nucleic-acid-lipid particle
  • a specific CRISPR Cas targeted in a SNALP daily intravenous injections of about 1, 3 or 5 mg/kg/day of a specific CRISPR Cas targeted in a SNALP are contemplated.
  • the daily treatment may be over about three days and then weekly for about five weeks.
  • a specific CRISPR Cas encapsulated SNALP) administered by intravenous injection to at doses of about 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006).
  • the SNALP formulation may contain the lipids 3-N- [(wmethoxypoly(ethylene glycol) 2000) carbamoyl] -1 ,2-dimyristyloxy-propylamine (PEG-C- DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40:10:48 molar per cent ratio (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006).
  • SNALPs stable nucleic-acid-lipid particles
  • the SNALP liposomes may be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA.
  • DSPC distearoylphosphatidylcholine
  • Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA.
  • the resulted SNALP liposomes are about 80–100 nm in size.
  • a SNALP may comprise synthetic cholesterol (Sigma- Aldrich, St Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2- dimyrestyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g., Geisbert et al., Lancet 2010; 375: 1896-905).
  • a dosage of about 2 mg/kg total CRISPR Cas per dose administered as, for example, a bolus intravenous infusion may be contemplated.
  • a SNALP may comprise synthetic cholesterol (Sigma- Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG- cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)).
  • Formulations used for in vivo studies may comprise a final lipid/RNA mass ratio of about 9:1.
  • the stability profile of RNAi nanomedicines has been reviewed by Barros and Gollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug Delivery Reviews 64 (2012) 1730– 1737).
  • the stable nucleic acid lipid particle is comprised of four different lipids— an ionizable lipid (DLinDMA) that is cationic at low pH, a neutral helper lipid, cholesterol, and a diffusible polyethylene glycol (PEG)-lipid.
  • the particle is approximately 80 nm in diameter and is charge-neutral at physiologic pH.
  • the ionizable lipid serves to condense lipid with the anionic siRNA during particle formation.
  • the ionizable lipid When positively charged under increasingly acidic endosomal conditions, the ionizable lipid also mediates the fusion of SNALP with the endosomal membrane enabling release of siRNA into the cytoplasm.
  • the PEG-lipid stabilizes the particle and reduces aggregation during formulation, and subsequently provides a neutral hydrophilic exterior that improves pharmacokinetic properties.
  • ALN-TTR01 which employs the SNALP technology described above and targets hepatocyte production of both mutant and wild-type TTR to treat TTR amyloidosis (ATTR).
  • TTR amyloidosis TTR amyloidosis
  • FAP familial amyloidotic polyneuropathy
  • FAC familial amyloidotic cardiomyopathy
  • SSA senile systemic amyloidosis
  • ALN-TTR01 was administered as a 15-minute IV infusion to 31 patients (23 with study drug and 8 with placebo) within a dose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatmentwaswell tolerated with no significant increases in liver function tests. Infusion-related reactions were noted in 3 of 23 patients at 0.4 mg/kg; all responded to slowing of the infusion rate and all continued on study. Minimal and transient elevations of serum cytokines IL-6, IP-10 and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (as anticipated from preclinical and NHP studies). Lowering of serum TTR, the expected pharmacodynamics effect of ALN-TTR01, was observed at 1 mg/kg.
  • a SNALP may be made by solubilizing a cationic lipid, DSPC, cholesterol and PEG-lipid were solubilized in ethanol at a molar ratio of 40:10:40:10, respectively (see, Semple et al., Nature Niotechnology, Volume 28 Number 2 February 2010, pp. 172-177).
  • the lipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) with mixing to a final ethanol and lipid concentration of 30% (vol/vol) and 6.1 mg/ml, respectively, and allowed to equilibrate at 22 °C for 2 min before extrusion.
  • the hydrated lipids were extruded through two stacked 80 nm pore-sized filters (Nuclepore) at 22 °C using a Lipex Extruder (Northern Lipids) until a vesicle diameter of 70–90 nm, as determined by dynamic light scattering analysis, was obtained. This generally required 1–3 passes.
  • the siRNA (solubilized in a 50 mM citrate, pH 4 aqueous solution containing 30% ethanol) was added to the pre- equilibrated (35 °C) vesicles at a rate of ⁇ 5 ml/min with mixing.
  • siRNA/lipid ratio 0.06 (wt/wt) was reached, the mixture was incubated for a further 30 min at 35 °C to allow vesicle reorganization and encapsulation of the siRNA.
  • the ethanol was then removed and the external buffer replaced with PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5) by either dialysis or tangential flow diafiltration.
  • siRNA were encapsulated in SNALP using a controlled step-wise dilution method process.
  • the lipid constituents of KC2-SNALP were DLin-KC2-DMA (cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti Polar Lipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molar ratio of 57.1:7.1:34.3:1.4.
  • SNALP were dialyzed against PBS and filter sterilized through a 0.2 m filter before use.
  • Mean particle sizes were 75–85 nm and 90–95% of the siRNA was encapsulated within the lipid particles.
  • the final siRNA/lipid ratio in formulations used for in vivo testing was ⁇ 0.15 (wt/wt).
  • LNP-siRNA systems containing Factor VII siRNA were diluted to the appropriate concentrations in sterile PBS immediately before use and the formulations were administered intravenously through the lateral tail vein in a total volume of 10 ml/kg. This method may be extrapolated to the CRISPR Cas system of the present invention.
  • DLin-KC2-DMA amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane
  • DLin-KC2-DMA amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane
  • a preformed vesicle with the following lipid composition may be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy) propyl-1- (methoxy poly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10, respectively, and a FVII siRNA/total lipid ratio of approximately 0.05 (w/w).
  • the particles may be extruded up to three times through 80 nm membranes prior to adding the CRISPR Cas RNA.
  • Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) which may be further optimized to enhance in vivo activity.
  • Michael S D Kormann et al. (“Expression of therapeutic proteins after delivery of chemically modified mRNA in mice: Nature Biotechnology, Volume:29, Pages: 154–157 (2011)) describes the use of lipid envelopes to deliver RNA. Use of lipid envelopes is also preferred in the present invention.
  • lipids may be formulated with the CRISPR Cas system of the present invention to form lipid nanoparticles (LNPs).
  • Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG- DMG may be formulated with CRISPR Cas instead of siRNA (see, e.g., Novobrantseva, Molecular Therapy–Nucleic Acids (2012) 1, e4; doi:10.1038/mtna.2011.3) using a spontaneous vesicle formation procedure.
  • the component molar ratio may be about 50/10/38.5/1.5 (DLin- KC2-DMA or C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG).
  • the final lipid:siRNA weight ratio may be ⁇ 12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipid nanoparticles (LNPs), respectively.
  • the formulations may have mean particle diameters of ⁇ 80 nm with >90% entrapment efficiency. A 3 mg/kg dose may be contemplated.
  • Tekmira has a portfolio of approximately 95 patent families, in the U.S. and abroad, that are directed to various aspects of LNPs and LNP formulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos .1766035; 1519714; 1781593 and 1664316), all of which may be used and/or adapted to the present invention.
  • the CRISPR Cas system may be delivered encapsulated in PLGA Microspheres such as that further described in US published applications 20130252281 and 20130245107 and 20130244279 (assigned to Moderna Therapeutics) which relate to aspects of formulation of compositions comprising modified nucleic acid molecules which may encode a protein, a protein precursor, or a partially or fully processed form of the protein or a protein precursor.
  • the formulation may have a molar ratio 50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEG lipid).
  • the PEG lipid may be selected from, but is not limited to PEG-c- DOMG, PEG-DMG.
  • the fusogenic lipid may be DSPC. See also, Schrum et al., Delivery and Formulation of Engineered Nucleic Acids, US published application 20120251618.
  • Nanomerics’ technology addresses bioavailability challenges for a broad range of therapeutics, including low molecular weight hydrophobic drugs, peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA).
  • Specific administration routes for which the technology has demonstrated clear advantages include the oral route, transport across the blood- brain-barrier, delivery to solid tumours, as well as to the eye. See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb 26;7(2):1016-26; Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al., 2012, J Control Release. 2012 Jul 20;161(2):523-36. US Patent Publication No.
  • cationic dendrimers for delivering bioactive molecules, such as polynucleotide molecules, peptides and polypeptides and/or pharmaceutical agents, to a mammalian body.
  • the dendrimers are suitable for targeting the delivery of the bioactive molecules to, for example, the liver, spleen, lung, kidney or heart.
  • Dendrimers are synthetic 3- dimensional macromolecules that are prepared in a step-wise fashion from simple branched monomer units, the nature and functionality of which can be easily controlled and varied.
  • Dendrimers are synthesised from the repeated addition of building blocks to a multifunctional core (divergent approach to synthesis), or towards a multifunctional core (convergent approach to synthesis) and each addition of a 3-dimensional shell of building blocks leads to the formation of a higher generation of the dendrimers.
  • Polypropylenimine dendrimers start from a diaminobutane core to which is added twice the number of amino groups by a double Michael addition of acrylonitrile to the primary amines followed by the hydrogenation of the nitriles. This results in a doubling of the amino groups.
  • Polypropylenimine dendrimers contain 100% protonable nitrogens and up to 64 terminal amino groups (generation 5, DAB 64).
  • Protonable groups are usually amine groups which are able to accept protons at neutral pH.
  • the use of dendrimers as gene delivery agents has largely focused on the use of the polyamidoamine. and phosphorous containing compounds with a mixture of amine/amide or N--P(O 2 )S as the conjugating units respectively with no work being reported on the use of the lower generation polypropylenimine dendrimers for gene delivery.
  • Polypropylenimine dendrimers have also been studied as pH sensitive controlled release systems for drug delivery and for their encapsulation of guest molecules when chemically modified by peripheral amino acid groups. The cytotoxicity and interaction of polypropylenimine dendrimers with DNA as well as the transfection efficacy of DAB 64 has also been studied.
  • cationic dendrimers such as polypropylenimine dendrimers
  • display suitable properties such as specific targeting and low toxicity, for use in the targeted delivery of bioactive molecules, such as genetic material.
  • derivatives of the cationic dendrimer also display suitable properties for the targeted delivery of bioactive molecules.
  • Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. David Liu’s lab reported the creation and characterization of supercharged proteins in 2007 (Lawrence et al., 2007, Journal of the American Chemical Society 129, 10110– 10112).
  • +36 GFP is an effective plasmid delivery reagent in a range of cells.
  • plasmid DNA is a larger cargo than siRNA, proportionately more +36 GFP protein is required to effectively complex plasmids.
  • Applicants have developed a variant of +36 GFP bearing a C-terminal HA2 peptide tag, a known endosome-disrupting peptide derived from the influenza virus hemagglutinin protein. The following protocol has been effective in a variety of cells, but as above it is advised that plasmid DNA and supercharged protein doses be optimized for specific cell lines and delivery applications.
  • implantable devices are also contemplated for delivery of the CRISPR Cas system.
  • US Patent Publication 20110195123 discloses an implantable medical device which elutes a drug locally and in prolonged period is provided, including several types of such a device, the treatment modes of implementation and methods of implantation.
  • the device comprising of polymeric substrate, such as a matrix for example, that is used as the device body, and drugs, and in some cases additional scaffolding materials, such as metals or additional polymers, and materials to enhance visibility and imaging.
  • RNA interference RNA interference
  • si RNA si RNA
  • sh RNA sh RNA
  • antisense RNA/DNA ribozyme and nucleoside analogs. Therefore, this system may be used/and or adapted to the CRISPR Cas system of the present invention.
  • the modes of implantation in some embodiments are existing implantation procedures that are developed and used today for other treatments, including brachytherapy and needle biopsy.
  • a drug delivery implantable or insertable system including systems applicable to a cavity such as the abdominal cavity and/or any other type of administration in which the drug delivery system is not anchored or attached, comprising a biostable and/or degradable and/or bioabsorbable polymeric substrate, which may for example optionally be a matrix.
  • insertion also includes implantation.
  • the drug delivery system is preferably implemented as a "Loder” as described in US Patent Publication 20110195123.
  • the polymer or plurality of polymers are biocompatible, incorporating an agent and/or plurality of agents, enabling the release of agent at a controlled rate, wherein the total volume of the polymeric substrate, such as a matrix for example, in some embodiments is optionally and preferably no greater than a maximum volume that permits a therapeutic level of the agent to be reached. As a non-limiting example, such a volume is preferably within the range of 0.1 m 3 to 1000 mm 3 , as required by the volume for the agent load.
  • the Loder may optionally be larger, for example when incorporated with a device whose size is determined by functionality, for example and without limitation, a knee joint, an intra-uterine or cervical ring and the like.
  • the drug delivery system (for delivering the composition) is designed in some embodiments to preferably employ degradable polymers, wherein the main release mechanism is bulk erosion; or in some embodiments, non degradable, or slowly degraded polymers are used, wherein the main release mechanism is diffusion rather than bulk erosion, so that the outer part functions as membrane, and its internal part functions as a drug reservoir, which practically is not affected by the surroundings for an extended period (for example from about a week to about a few months). Combinations of different polymers with different release mechanisms may also optionally be used.
  • the concentration gradient at the surface is preferably maintained effectively constant during a significant period of the total drug releasing period, and therefore the diffusion rate is effectively constant (termed "zero mode" diffusion).
  • the term “constant” it is meant a diffusion rate that is preferably maintained above the lower threshold of therapeutic effectiveness, but which may still optionally feature an initial burst and/or fluctuate, for example increasing and decreasing to a certain degree.
  • the diffusion rate is preferably so maintained for a prolonged period, and it can be considered constant to a certain level to optimize the therapeutically effective period, for example the effective silencing period.
  • the drug delivery system optionally and preferably is designed to shield the nucleotide based therapeutic agent from degradation, whether chemical in nature or due to attack from enzymes and other factors in the body of the subject.
  • the drug delivery system as described in US Patent Publication 20110195123 is optionally associated with sensing and/or activation appliances that are operated at and/or after implantation of the device, by non and/or minimally invasive methods of activation and/or acceleration/deceleration, for example optionally including but not limited to thermal heating and cooling, laser beams, and ultrasonic, including focused ultrasound and/or RF (radiofrequency) methods or devices.
  • sensing and/or activation appliances that are operated at and/or after implantation of the device, by non and/or minimally invasive methods of activation and/or acceleration/deceleration, for example optionally including but not limited to thermal heating and cooling, laser beams, and ultrasonic, including focused ultrasound and/or RF (radiofrequency) methods or devices.
  • RF radiofrequency
  • the site for local delivery may optionally include target sites characterized by high abnormal proliferation of cells, and suppressed apoptosis, including tumors, active and or chronic inflammation and infection including autoimmune diseases states, degenerating tissue including muscle and nervous tissue, chronic pain, degenerative sites, and location of bone fractures and other wound locations for enhancement of regeneration of tissue, and injured cardiac, smooth and striated muscle.
  • the site for implantation of the composition, or target site preferably features a radius, area and/or volume that is sufficiently small for targeted local delivery.
  • the target site optionally has a diameter in a range of from about 0.1 mm to about 5 cm.
  • the location of the target site is preferably selected for maximum therapeutic efficacy.
  • the composition of the drug delivery system (optionally with a device for implantation as described above) is optionally and preferably implanted within or in the proximity of a tumor environment, or the blood supply associated thereof.
  • the composition (optionally with the device) is optionally implanted within or in the proximity to pancreas, prostate, breast, liver, via the nipple, within the vascular system and so forth.
  • the target location is optionally selected from the group consisting of (as non-limiting examples only, as optionally any site within the body may be suitable for implanting a Loder): 1. brain at degenerative sites like in Parkinson or Alzheimer disease at the basal ganglia, white and gray matter; 2. spine as in the case of amyotrophic lateral sclerosis (ALS); 3.
  • Intra vaginal 10. Inner ear--auditory system, labyrinth of the inner ear, vestibular system; 11. Intra tracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder; 14. biliary system; 15. parenchymal tissue including and not limited to the kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18. dental gums; 19. Intra-articular (into joints); 20.
  • insertion of the system is associated with injection of material to the ECM at the target site and the vicinity of that site to affect local pH and/or temperature and/or other biological factors affecting the diffusion of the drug and/or drug kinetics in the ECM, of the target site and the vicinity of such a site.
  • the release of said agent could be associated with sensing and/or activation appliances that are operated prior and/or at and/or after insertion, by non and/or minimally invasive and/or else methods of activation and/or acceleration/deceleration, including laser beam, radiation, thermal heating and cooling, and ultrasonic, including focused ultrasound and/or RF (radiofrequency) methods or devices, and chemical activators.
  • the drug preferably comprises a gene silencing biological RNAi drug, for example for localized cancer cases in breast, pancreas, brain, kidney, bladder, lung, and prostate as described below.
  • RNAi RNA binds to drugs other than siRNA
  • the Loder substrate such as a matrix for example
  • this system may be used and/or adapted to deliver the CRISPR Cas system of the present invention.
  • the method of insertion may optionally already be used for other types of tissue implantation and/or for insertions and/or for sampling tissues, optionally without modifications, or alternatively optionally only with non-major modifications in such methods.
  • Such methods optionally include but are not limited to brachytherapy methods, biopsy, endoscopy with and/or without ultrasound, such as ERCP, stereotactic methods into the brain tissue, Laparoscopy, including implantation with a laparoscope into joints, abdominal organs, the bladder wall and body cavities.
  • CRISPR enzyme mRNA and guide RNA might also be delivered separately.
  • CRISPR enzyme mRNA can be delivered prior to the guide RNA to give time for CRISPR enzyme to be expressed.
  • CRISPR enzyme mRNA might be administered 1-12 hours (preferably around 2-6 hours) prior to the administration of guide RNA.
  • CRISPR enzyme mRNA and guide RNA can be administered together.
  • a second booster dose of guide RNA can be administered 1-12 hours (preferably around 2-6 hours) after the initial administration of CRISPR enzyme mRNA + guide RNA. Additional administrations of CRISPR enzyme mRNA and/or guide RNA might be useful to achieve the most efficient levels of genome modification.
  • CRISPR enzyme mRNA and guide RNA delivered For minimization of toxicity and off-target effect, it will be important to control the concentration of CRISPR enzyme mRNA and guide RNA delivered.
  • Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing different concentrations in a cellular or animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci. For example, for the guide sequence targeting 5’- GAGTCCGAGCAGAAGAAGAA-3’ in the EMX1 gene of the human genome, deep sequencing can be used to assess the level of modification at the following two off-target loci, 1: 5’-GAGTCCTAGCAGGAGAAGAA-3’ and 2: 5’-GAGTCTAAGCAGAAGAAGAA-3’.
  • CRISPR enzyme nickase mRNA for example S. pyogenes Cas9 with the D10A mutation
  • CRISPR enzyme nickase mRNA can be delivered with a pair of guide RNAs targeting a site of interest.
  • the two guide RNAs need to be spaced as follows. Guide sequences in red (single underline) and blue (double underline) respectively (these examples are based on the PAM requirement for Streptococcus pyogenes Cas9).
  • the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs.
  • the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs or 1-34 base pairs.
  • the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a blunt cut or a 3’ overhang.
  • the 3’ overhang is at most 150, 100 or 25 base pairs or at least 15, 10 or 1 base pairs. In preferred embodiments the 3’ overhang is 1- 100 basepairs.
  • aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased.
  • the gene product is a protein. sgRNA pairs creating 5’ overhangs with less than 8bp overlap between the guide sequences (offset greater than -8 bp) were able to mediate detectable indel formation.
  • each guide used in these assays is able to efficiently induce indels when paired with wildtype Cas9, indicating that the relative positions of the guide pairs are the most important parameters in predicting double nicking activity.
  • Cas9n and Cas9H840A nick opposite strands of DNA
  • substitution of Cas9n with Cas9H840A with a given sgRNA pair should result in the inversion of the overhang type.
  • a pair of sgRNAs that will generate a 5’ overhang with Cas9n should in principle generate the corresponding 3’ overhang instead. Therefore, sgRNA pairs that lead to the generation of a 3’ overhang with Cas9n might be used with Cas9H840A to generate a 5’ overhang.
  • PCSK9 liver, proprotein convertase subtilisin kexin 9
  • Bailey et al. J Mol Med (Berl). 1999 Jan;77(1):244-9) discloses insulin delivery by ex-vivo somatic cell gene therapy involves the removal of non-B-cell somatic cells (e.g. fibroblasts) from a diabetic patient, and genetically altering them in vitro to produce and secrete insulin.
  • the cells can be grown in culture and returned to the donor as a source of insulin replacement. Cells modified in this way could be evaluated before implantation, and reserve stocks could be cryopreserved.
  • the procedure should obviate the need for immunosuppression and overcome the problem of tissue supply, while avoiding a recurrence of cell destruction.
  • Ex-vivo somatic cell gene therapy requires an accessible and robust cell type that is amenable to multiple transfections and subject to controlled proliferation.
  • Special problems associated with the use of non-B-cell somatic cells include the processing of proinsulin to insulin, and the conferment of sensitivity to glucose-stimulated proinsulin biosynthesis and regulated insulin release.
  • Preliminary studies using fibroblasts, pituitary cells, kidney (COS) cells and ovarian (CHO) cells suggest that these challenges could be met, and that ex-vivo somatic cell gene therapy offers a feasible approach to insulin replacement therapy.
  • the system of Bailey et al. may be used/and or adapted to the CRISPR Cas system of the present invention for delivery to the liver.
  • Cationic liposomes (Lipotrust) containing O,O’-ditetradecanoyl-N-(a- trimethylammonioacetyl) diethanolamine chloride (DC-6-14) as a cationic lipid, cholesterol and dioleoylphosphatidylethanolamine at a molar ratio of 4:3:3 (which has shown high transfection efficiency under serumcontaining conditions for in vitro and in vivo gene delivery) were purchased from Hokkaido System Science. The liposomes were manufactured using a freeze- dried empty liposomes method and prepared at a concentration of 1 mM (DC-16-4) by addition of double-distilled water (DDW) to the lyophilized lipid mixture under vortexing before use.
  • DC-6-14 O,O’-ditetradecanoyl-N-(a- trimethylammonioacetyl) diethanolamine chloride
  • VA-coupled liposomes 200 nmol of vitamin A (retinol, Sigma) dissolved in DMSO was mixed with the liposome suspensions (100 nmol as DC-16-4) by vortexing in a 1.5 ml tube at 25 1C.
  • VA-lip-siRNAgp46 a solution of siRNAgp46 (580 pmol/ml in DDW) was added to the retinol-coupled liposome solution with stirring at 25 C.
  • the ratio of siRNA to DC-16-4 was 1:11.5 (mol/mol) and the siRNA to liposome ratio (wt/wt) was 1:1.
  • any free vitamin A or siRNA that was not taken up by liposomes were separated from liposomal preparations using a micropartition system (VIVASPIN 2 concentrator 30,000 MWCO PES, VIVASCIENCE).
  • the liposomal suspension was added to the filters and centrifuged at 1,500g for 5 min 3 times at 25 1C. Fractions were collected and the material trapped in the filter was reconstituted with PBS to achieve the desired dose for in vitro or in vivo use.
  • Three injections of 0.75 mg/kg siRNA were given every other day to rats.
  • the system of Sato et al. may be used/and or adapted to the CRISPR Cas system of the present invention for delivery to the liver by delivering about 0.5 to 1 mg/kg of CRISPR Cas RNA in the liposomes as described by Sato et al. to humans.
  • SATA-modified siRNAs are synthesized by reaction of 5’ aminemodified siRNA with 1 weight equivalents (wt eq) of Nsuccinimidyl-S-acetylthioacetate (SATA) reagent (Pierce) and 0.36 wt eq of NaHCO 3 in water at 4°C for 16 h.
  • SATA Nsuccinimidyl-S-acetylthioacetate
  • NaHCO 3 NaHCO 3
  • PBAVE (30 mg/ml in 5mMTAPS, pH 9) is modified by addition of 1.5 wt % SMPT (Pierce). After a 1-h incubation, 0.8 mg of SMPT-PBAVE was added to 400 l of isotonic glucose solution containing 5 mM TAPS (pH 9). To this solution was added 50 g of SATA-modified siRNA. For the dose– response experiments where [PBAVE] was constant, different amounts of siRNA are added. The mixture is then incubated for 16 h. To the solution is then added 5.6 mg of Hepes free base followed by a mixture of 3.7 mg ofCDM-NAGand 1.9mg of CDM-PEG.
  • CDM-PEG and CDM-NAG are synthesized from the acid chloride generated by using oxalyl chloride.
  • To the acid chloride is added 1.1 molar equivalents polyethylene glycol monomethyl ether (molecular weight average of 450) to generate CDM-PEG or (aminoethoxy)ethoxy-2-(acetylamino)-2-deoxy- ⁇ -D- glucopyranoside to generate CDM-NAG.
  • the final product is purified by using reverse-phase HPLC with a 0.1% TFA water/acetonitrile gradient. About 25 to 50 g of siRNA was delivered to mice.
  • the system of Rozema et al. may be applied to the CRISPR Cas system of the present invention for delivery to the liver, for example by envisioning a dosage of about 50 to about 200 mg of CRISPR Cas for delivery to a human.
  • Delivery options for the brain include encapsulation of CRISPR enzyme and guide RNA in the form of either DNA or RNA into liposomes and conjugating to molecular Trojan horses for trans-blood brain barrier (BBB) delivery.
  • BBB trans-blood brain barrier
  • Molecular Trojan horses have been shown to be effective for delivery of B-gal expression vectors into the brain of non-human primates.
  • the same approach can be used to delivery vectors containing CRISPR enzyme and guide RNA.
  • Xia CF and Boado RJ, Pardridge WM Antibody-mediated targeting of siRNA via the human insulin receptor using avidin-biotin technology. Mol Pharm. 2009 May- Jun;6(3):747-51.
  • siRNA short interfering RNA
  • mAb monoclonal antibody
  • avidin-biotin a receptor-specific monoclonal antibody
  • the authors also report that because the bond between the targeting mAb and the siRNA is stable with avidin-biotin technology, and RNAi effects at distant sites such as brain are observed in vivo following an intravenous administration of the targeted siRNA.
  • Zhang et al. (Mol Ther. 2003 Jan;7(1):11-8.) describe how expression plasmids encoding reporters such as luciferase were encapsulated in the interior of an "artificial virus" comprised of an 85 nm pegylated immunoliposome, which was targeted to the rhesus monkey brain in vivo with a monoclonal antibody (MAb) to the human insulin receptor (HIR).
  • MAb monoclonal antibody
  • HIR human insulin receptor
  • the HIRMAb enables the liposome carrying the exogenous gene to undergo transcytosis across the blood-brain barrier and endocytosis across the neuronal plasma membrane following intravenous injection.
  • the level of luciferase gene expression in the brain was 50-fold higher in the rhesus monkey as compared to the rat.
  • Widespread neuronal expression of the beta-galactosidase gene in primate brain was demonstrated by both histochemistry and confocal microscopy. The authors indicate that this approach makes feasible reversible adult transgenics in 24 hours. Accordingly, the use of immunoliposome is preferred. These may be used in conjunction with antibodies to target specific tissues or cell surface proteins.
  • RNA Ribonucleic acid
  • nanoparticles Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells, Advanced Functional Materials, 19: 3112-3118, 2010
  • exosomes Schroeder, A., Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-based nanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID: 20059641).
  • exosomes have been shown to be particularly useful in delivery siRNA, a system with some parallels to the CRISPR system.
  • El-Andaloussi S, et al. (“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc. 2012 Dec;7(12):2112- 26. doi: 10.1038/nprot.2012.131. Epub 2012 Nov 15.) describe how exosomes are promising tools for drug delivery across different biological barriers and can be harnessed for delivery of siRNA in vitro and in vivo.
  • Their approach is to generate targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand.
  • exosomes are then purify and characterized from transfected cell supernatant, then siRNA is loaded into the exosomes.
  • Delivery or administration according to the invention can be performed with exosomes, in particular but not limited to the brain.
  • Vitamin E ⁇ -tocopherol
  • CRISPR Cas may be conjugated with CRISPR Cas and delivered to the brain along with high density lipoprotein (HDL), for example in a similar manner as was done by Uno et al. (HUMAN GENE THERAPY 22:711–719 (June 2011)) for delivering short-interfering RNA (siRNA) to the brain.
  • HDL high density lipoprotein
  • Mice were infused via Osmotic minipumps (model 1007D; Alzet, Cupertino, CA) filled with phosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL and connected with Brain Infusion Kit 3 (Alzet).
  • PBS phosphate-buffered saline
  • a brain-infusion cannula was placed about 0.5mm posterior to the bregma at midline for infusion into the dorsal third ventricle.
  • Uno et al. found that as little as 3 nmol of Toc-siRNA with HDL could induce a target reduction in comparable degree by the same ICV infusion method.
  • a similar dosage of CRISPR Cas conjugated to ⁇ -tocopherol and co- administered with HDL targeted to the brain may be contemplated for humans in the present invention, for example, about 3 nmol to about 3 mol of CRISPR Cas targeted to the brain may becontemplated.
  • Zou et al. (HUMAN GENE THERAPY 22:465-475 (April 2011)) describes a method of lentiviral-mediated delivery of short-hairpin RNAs targeting PKC ⁇ for in vivo gene silencing in the spinal cord of rats.
  • Zou et al. administered about 10 l of a recombinant lentivirus having a titer of 1 x 10 9 transducing units (TU)/ml by an intrathecal catheter.
  • a similar dosage of CRISPR Cas expressed in a lentiviral vector targeted to the brain may be contemplated for humans in the present invention, for example, about 10-50 ml of CRISPR Cas targeted to the brain in a lentivirus having a titer of 1 x 10 9 transducing units (TU)/ml may be contemplated.
  • TU transducing units
  • material can be delivered intrastriatally e.g. by injection. Injection can be performed stereotactically via a craniotomy.
  • Targeted deletion of viral genes or other viral elements is preferred. Examples are exemplified in Example 18. Preferred are, therefore, latent viral genes. As exemplified here, Applicants prefer gene delivery of a CRISPR-Cas system to the liver, brain, ocular, epithelial, hematopoetic, or another tissue of a subject or a patient in need thereof, suffering from latent viral infections, using either viral or nonviral (e.g. nanoparticle) delivery system.
  • a CRISPR-Cas system to the liver, brain, ocular, epithelial, hematopoetic, or another tissue of a subject or a patient in need thereof, suffering from latent viral infections, using either viral or nonviral (e.g. nanoparticle) delivery system.
  • Therapeutic applications of the CRISPR-Cas system include treatment of viral infections, such as HBV infection.
  • the immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes. Reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity (Tangri S, et al. (“Rationally engineered therapeutic proteins with reduced immunogenicity” J Immunol. 2005 Mar 15;174(6):3187-96.)
  • the immunogenicity of the CRISPR enzyme in particular may be reduced following the approach first set out in Tangri et al with respect to erythropoietin and subsequently developed.
  • directed evolution or rational design may be used to reduce the immunogenicity of the CRISPR enzyme (for instance a Cas9) in the host species (human or other species).
  • CRISPR enzyme for instance a Cas9
  • Applicants used 3 guideRNAs of interest and able to visualize efficient DNA cleavage in vivo occurring only in a small subset of cells. Essentially, what Applicants have shown here is targeted in vivo cleavage. In particular, this provides proof of concept that specific targeting in higher organisms such as mammals can also be achieved. It also highlights multiplex aspect in that multiple guide sequences (i.e. separate targets) can be used simultaneously (in the sense of co-delivery). In other words, Applicants used a multiple approach, with several different sequences targeted at the same time, but independently.
  • a suitable example of a protocol for producing AAV, a preferred vector of the invention is provided in the Examples.
  • the present invention also contemplates delivering the CRISPR-Cas system to the blood.
  • the plasma exosomes of Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) were previously described and may be utilized to deliver the CRISPR Cas system to the blood.
  • the present invention also contemplates delivering the CRISPR-Cas system to the heart.
  • a myocardium tropic adena-associated virus AAVM
  • AAVM41 which showed preferential gene transfer in the heart (see, e.g., Lin-Yanga et al., PNAS, March 10, 2009, vol. 106, no. 10).
  • Administration may be systemic or local.
  • a dosage of about 1-10 x 10 14 vector genomes are contemplated for systemic administration. See also, e.g., Eulalio et al. (2012) Nature 492: 376 and Somasuntharam et al. (2013) Biomaterials 34: 7790.
  • the present invention also contemplates delivering the CRISPR-Cas system to the kidney.
  • Delivery strategies to induce cellular uptake of the therapeutic nucleic acid include physical force or vector systems such as viral-, lipid- or complex- based delivery, or nanocarriers. From the initial applications with less possible clinical relevance, when nucleic acids were addressed to renal cells with hydrodynamic high pressure injection systemically, a wide range of gene therapeutic viral and non-viral carriers have been applied already to target posttranscriptional events in different animal kidney disease models in vivo (Csaba Révész and Péter Hamar (2011). Delivery Methods to Target RNAs in the Kidney, Gene Therapy Applications, Prof.
  • the present invention also contemplates delivering the CRISPR-Cas system to oneor both lungs.
  • AAV-2-based vectors were originally proposed for CFTR delivery to CF airways, other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9 exhibit improved gene transfer efficiency in a variety of models of the lung epithelium (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009).
  • AAV-1 was demonstrated to be ⁇ 100-fold more efficient than AAV-2 and AAV-5 at transducing human airway epithelial cells in vitro,5 although AAV-1 transduced murine tracheal airway epithelia in vivo with an efficiency equal to that of AAV-5.
  • Other studies have shown that AAV-5 is 50-fold more efficient than AAV-2 at gene delivery to human airway epithelium (HAE) in vitro and significantly more efficient in the mouse lung airway epithelium in vivo.
  • AAV-6 has also been shown to be more efficient than AAV-2 in human airway epithelial cells in vitro and murine airways in vivo.8
  • AAV-9 The more recent isolate, AAV-9, was shown to display greater gene transfer efficiency than AAV-5 in murine nasal and alveolar epithelia in vivo with gene expression detected for over 9 months suggesting AAV may enable long-term gene expression in vivo, a desirable property for a CFTR gene delivery vector.
  • AAV-9 could be readministered to the murine lung with no loss of CFTR expression and minimal immune consequences.
  • CF and non- CF HAE cultures may be inoculated on the apical surface with 100 l of AAV vectors for hours (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009).
  • the MOI may vary from 1 ⁇ 10 3 to 4 ⁇ 10 5 vector genomes/cell, depending on virus concentration and purposes of the experiments.
  • the above cited vectors are contemplated for the delivery and/or administration of the invention.
  • Zamora et al. (Am J Respir Crit Care Med Vol 183. pp 531–538, 2011) reported an example of the application of an RNA interference therapeutic to the treatment of human infectious disease and also a randomized trial of an antiviral drug in respiratory syncytial virus (RSV)-infected lung transplant recipients.
  • RSV respiratory syncytial virus
  • Zamora et al. performed a randomized, double-blind, placebocontrolled trial in LTX recipients with RSV respiratory tract infection. Patients were permitted to receive standard of care for RSV. Aerosolized ALN-RSV01 (0.6 mg/kg) or placebo was administered daily for 3 days. This study demonstrates that an RNAi therapeutic targeting RSV can be safely administered to LTX recipients with RSV infection.
  • Three daily doses of ALN-RSV01 did not result in any exacerbation of respiratory tract symptoms or impairment of lung function and did not exhibit any systemic proinflammatory effects, such as induction of cytokines or CRP.
  • Pharmacokinetics showed only low, transient systemic exposure after inhalation, consistent with preclinical animal data showing that ALN-RSV01, administered intravenously or by inhalation, is rapidly cleared from the circulation through exonucleasemediated digestion and renal excretion.
  • the method of Zamora et al. may be applied to the CRISPR Cas system of the present invention and an aerosolized CRISPR Cas, for example with a dosage of 0.6 mg/kg, may be contemplated for the present invention.
  • the present invention also contemplates delivering the CRISPR-Cas system to muscle(s).
  • the present invention also contemplates delivering the CRISPR-Cas system to the skin.
  • Hickerson et al. Molecular Therapy—Nucleic Acids (2013) 2, e129
  • a motorized microneedle array skin delivery device for delivering self-delivery (sd)-siRNA to human and murine skin.
  • the primary challenge to translating siRNA-based skin therapeutics to the clinic is the development of effective delivery systems.
  • Substantial effort has been invested in a variety of skin delivery technologies with limited success.
  • the naked pain associated with the hypodermic needle injection precluded enrollment of additional patients in the trial, highlighting the need for improved, more“patient-friendly” (i.e., little or no pain) delivery approaches.
  • Microneedles represent an efficient way to deliver large charged cargos including siRNAs across the primary barrier, the stratum corneum, and are generally regarded as less painful than conventional hypodermic needles.
  • Motorized“stamp type” microneedle devices including the motorized microneedle array (MMNA) device used by Hickerson et al., have been shown to be safe in hairless mice studies and cause little or no pain as evidenced by (i) widespread use in the cosmetic industry and (ii) limited testing in which nearly all volunteers found use of the device to be much less painful than a flushot, suggesting siRNA delivery using this device will result in much less pain than was experienced in the previous clinical trial using hypodermic needle injections.
  • MMNA motorized microneedle array
  • the MMNA device (marketed as Triple-M or Tri-M by Bomtech Electronic Co, Seoul, South Korea) was adapted for delivery of siRNA to mouse and human skin.
  • sd-siRNA solution up to 300 l of 0.1 mg/ml RNA
  • the disposable Tri-M needle cartridge (Bomtech), which was set to a depth of 0.1 mm.
  • deidentified skin obtained immediately following surgical procedures
  • All intradermal injections were performed using an insulin syringe with a 28-gauge 0.5-inch needle.
  • the MMNA device and method of Hickerson et al. could be used and/or adapted to deliver the CRISPR Cas of the present invention, for example, at a dosage of up to 300 l of 0.1 mg/ml CRISPR Cas to the skin.
  • the present invention may also be applied to treat latent or chronic viral infections.
  • Viral latency is the abiity of a pathogenic virus to remain latent or dormant within a cell during the lysogenic part of its life cycle.
  • Latent infection is distinct from chronic infection in which a virus continues to replicate and proliferate. Instead, proliferation of the virus ceases but the viral genome is not eradicated, and so it can reactivate and again result in production of viral progeny (lytic part of the life cycle) without requiring reinfection of the host.
  • the invention provides the use of a CRISPR-Cas system to inactivate a virus within a eukaryotic cell, and in particular a latent form of the virus.
  • the CRISPR-Cas system can be used to excise an integrated provirus from a cell’s genome and/or to inactivate a latent virus which is present in episomal form (e.g. to cleave a cccDNA form).
  • the invention provides a CRISPR-Cas system comprising one or more vectors for delivery to a eukaryotic cell, wherein the vector(s) encode(s): (i) a CRISPR enzyme; (ii) a guide RNA capable of hybridizing to a target sequence in a viral genome within the cell; and (iii); a tracr mate sequence; and (iv) a tracr sequence, wherein, when expressed within the cell, the guide RNA directs sequence-specific binding of a CRISPR complex to the target sequence, and the CRISPR complex comprises (a) the tracr mate sequence hybridised to the tracr sequence and (b) a CRISPR enzyme bound to the guide RNA, such that the guide RNA can hybridise to its target sequence in the viral genome.
  • the vector(s) encode(s): (i) a CRISPR enzyme; (ii) a guide RNA capable of hybridizing to a target sequence in a viral genome within the cell; and (iii
  • the guide sequences can be part of a chiRNA sequence which provides the guide, tracr mate and tracr sequences within a single RNA, such that the system can encode (i) a CRISPR enzyme; (ii) a chiRNA comprising a sequence capable of hybridizing to a target sequence in a viral genome, a tracr mate sequence, and a tracr sequence.
  • the enzyme can include one or more NLS, etc.
  • Latent viruses can exist in episomal form or integrated proviral form, and the invention can be used to treat both types.
  • the invention is particularly useful with DNA viruses, and especially viruses with a double-stranded DNA genome.
  • Examples of pathogenic DNA viruses which have an episomal latent form herpes simplex virus (HSV) 1 and 2, human papillomavirus (HPV) of any type, Epstein Barr virus (EBV), and varicella zoster virus (VZV).
  • HSV herpes simplex virus
  • HPV human papillomavirus
  • EBV Epstein Barr virus
  • VZV varicella zoster virus
  • the invention can be used to treat infections with any of these viruses.
  • Some plant viruses also exhibit a latent form and the invention can be used to eliminate these as well.
  • CRISPR systems of the invention can be targeted to cell types which maintain a latent virus e.g.
  • Genomic sequences for viruses are widely available, and guide sequences for targeting the viral genome can thus be designed without difficulty. Where a virus has various sequence variants (e.g. different subtypes of HBV) it is useful to design guide sequences to target regions of the genome which are conserved, thereby providing broad activity. It is preferred to use guide RNAs against more than one site in the viral genome.
  • a key advantage of CRISPR technology when compared to ZFNs or TALENs is the relative ease by which multiple sequences can be targeted. Targeting multiple sites in a viral genome offers two main advantages.
  • a CRISPR system can target multiple genes or ORFs within a viral genome.
  • CRISPR systems can be used to target chronic viral infections by viruses whose life cycles include a dsDNA form which can be bound by CRISPR complexes.
  • the CRISPR system can be used in conjunction with an antiviral compound, such as tenofovir (HBV), entecavir (HBV), aciclovir (HSV, VZV), etc.
  • the present invention may also be applied to treat hepatitis B virus (HBV).
  • HBV hepatitis B virus
  • the invention provides the use of a CRISPR-Cas system to inactivate HBV within a mammalian cell, and in particular a latent form of HBV.
  • the CRISPR-Cas system can be used to excise integrated HBV provirus from a cell’s genome (a rare occurrence) and/or to inactivate latent HBV which is present in covalently closed circular DNA (cccDNA) form.
  • HBV genomic sequences are widely available, and guide sequences for targeting the HBV genome can thus be designed without difficulty.
  • HBV exists in several serological subtypes (e.g.
  • adw, ayw, ady, adr which differ by >8% in primary sequence, and it is useful to design guide sequences to target regions of the genome which are conserved between multiple subtypes.
  • Applicants have designed 24 guide RNAs to target the HBV genome. These include targets which are highly conserved within the HBV genome; and the location of 9 of these guide sequences is mapped against the HBV genome (See Figures 36, 57). It is preferred to use guide RNAs against more than one site in the HBV genome.
  • CRISPR systems for treating HBV are ideally delivered to liver cells, and in particular to hepatocytes.
  • ORF P which encodes the polymerase
  • ORF X which encodes HBX protein
  • the EnhI enhancer regulatory element and/or the EnhII enhancer regulatory element.
  • CRISPR systems for treating HBV are ideally delivered to liver cells, and in particular to hepatocytes.
  • an AAV8 vector may be useful.
  • expression of the components of the CRISPR system are ideally under the transcriptional control of a liver-specific or hepatocyte-specific promoter.
  • the CRISPR system can be used in conjunction with an anti-HBV compound such as tenofovir or entecavir.
  • an anti-HBV compound such as tenofovir or entecavir.
  • co-treatment with epigenetic modifiers for example Class I and Class III HDAC inhibitors trichostatin A (TSA), valproate, and nicotinamide (NAM), and Type I interferons
  • TSA trichostatin A
  • NAM nicotinamide
  • Type I interferons Type I interferons
  • the CRISPR Cas system For treating HBV in practice, the CRISPR Cas system must avoid the shortcomings of RNAi, such as the risk of oversaturating endogenous small RNA pathways, by for example, optimizing dose and sequence (see, e.g., Grimm et al., Nature vol. 441, 26 May 2006). For example, low doses, such as about 1-10 x 10 14 particles per human are contemplated.
  • the CRISPR Cas system directed against HBV may be administered in liposomes, such as a stable nucleic-acid- lipid particle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005).
  • SNALP stable nucleic-acid- lipid particle
  • dsAAV2/8 Daily intravenous injections of about 1, 3 or 5 mg/kg/day of CRISPR Cas targeted to HBV RNA in a SNALP are contemplated.
  • the daily treatment may be over about three days and then weekly for about five weeks.
  • the system of Chen et al. (Gene Therapy (2007) 14, 11–19) may be used/and or adapted for the CRISPR Cas system of the present invention.
  • Chen et al. use a double-stranded adenoassociated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA.
  • dsAAV2/8 double-stranded adenoassociated virus 8-pseudotyped vector
  • a CRISPR Cas system directed to HBV may be cloned into an AAV vector, such as a dsAAV2/8 vector and administered to a human, for example, at a dosage of about 1 x 10 15 vector genomes to about 1 x 10 16 vector genomes per human.
  • Wooddell et al. (Molecular Therapy vol. 21 no. 5, 973–985 May 2013) may be used/and or adapted to the CRISPR Cas system of the present invention.
  • Woodell et al. show that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines.
  • chol-siRNA liver-tropic cholesterol-conjugated siRNA
  • F7 coagulation factor VII
  • Intraveinous coinjections for example, of about 6 mg/kg of NAG-MLP and 6 mg/kg of HBV specific CRISPR Cas may be envisioned for the present invention.
  • about 3 mg/kg of NAG-MLP and 3 mg/kg of HBV specific CRISPR Cas may be delivered on day one, followed by administration of about about 2-3 mg/kg of NAG- MLP and 2-3 mg/kg of HBV specific CRISPR Cas two weeks later.
  • the present invention may also be applied to treat hepatitis C virus (HCV).
  • HCV hepatitis C virus
  • the methods of Roelvinki et al. may be applied to the CRISPR Cas system.
  • an AAV vector such as AAV8 may be a contemplated vector and for example a dosage of about 1.25 ⁇ 10 11 to 1.25 ⁇ 10 13 vector genomes per kilogram body weight (vg/kg) may be contemplated.
  • the invention uses nucleic acids to bind target DNA sequences. This is advantageous as nucleic acids are much easier and cheaper to produce than proteins, and the specificity can be varied according to the length of the stretch where homology is sought. Complex 3-D positioning of multiple fingers, for example is not required.
  • polynucleotide refers to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown.
  • polynucleotides coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers.
  • loci locus defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched poly
  • a polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer.
  • the sequence of nucleotides may be interrupted by non-nucleotide components.
  • a polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
  • wild type is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
  • nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
  • “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non- traditional types.
  • a percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence.
  • “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
  • stringent conditions for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences.
  • Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence.
  • Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology- Hybridization With Nucleic Acid Probes Part I, Second Chapter“Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y.
  • complementary or partially complementary sequences are also envisaged. These are preferably capable of hybridising to the reference sequence under highly stringent conditions.
  • relatively low-stringency hybridization conditions are selected: about 20 to 25° C lower than the thermal melting point (T m ).
  • T m is the temperature at which 50% of specific target sequence hybridizes to a perfectly complementary probe in solution at a defined ionic strength and pH.
  • highly stringent washing conditions are selected to be about 5 to 15° C lower than the T m .
  • moderately-stringent washing conditions are selected to be about 15 to 30° C lower than the T m .
  • Highly permissive (very low stringency) washing conditions may be as low as 50° C below the T m , allowing a high level of mis-matching between hybridized or hybridizable sequences.
  • Other physical and chemical parameters in the hybridization and wash stages can also be altered to affect the outcome of a detectable hybridization signal from a specific level of homology between target and probe sequences.
  • Preferred highly stringent conditions comprise incubation in 50% formamide, 5 ⁇ SSC, and 1% SDS at 42° C, or incubation in 5 ⁇ SSC and 1% SDS at 65° C, with wash in 0.2 ⁇ SSC and 0.1% SDS at 65° C.
  • Hybridization refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues.
  • the hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • the complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self- hybridizing strand, or any combination of these.
  • a hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme.
  • a sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.
  • the term“genomic locus” or“locus” is the specific location of a gene or DNA sequence on a chromosome.
  • A“gene” refers to stretches of DNA or RNA that encode a polypeptide or an RNA chain that has functional role to play in an organism and hence is the molecular unit of heredity in living organisms.
  • genes include regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences.
  • a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
  • “expression of a genomic locus” or“gene expression” is the process by which information from a gene is used in the synthesis of a functional gene product.
  • the products of gene expression are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is functional RNA.
  • the process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea) and viruses to generate functional products to survive.
  • "expression" of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context.
  • expression also refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins.
  • Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
  • polypeptide “peptide” and“protein” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non amino acids.
  • the terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • amino acid includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
  • domain or“protein domain” refers to a part of a protein sequence that may exist and function independently of the rest of the protein chain.
  • sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.
  • the capping region of the dTALEs described herein have sequences that are at least 95% identical or share identity to the capping region amino acid sequences provided herein.
  • Sequence homologies may be generated by any of a number of computer programs known in the art, for example BLAST or FASTA, etc.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387).
  • Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid– Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
  • Percentage (%) sequence homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an“ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
  • BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program.
  • a new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999177(1): 187-8 and the website of the National Center for Biotechnology information at the website of the National Institutes for Health).
  • the final % homology may be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pair-wise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table, if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
  • percentage homologies may be calculated using the multiple alignment feature in DNASIS TM (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244).
  • % homology preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
  • sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups.
  • Amino acids may be grouped together based on the properties of their side chains alone. However, it is more useful to include mutation data as well.
  • the sets of amino acids thus derived are likely to be conserved for structural reasons. These sets may be described in the form of a Venn diagram (Livingstone C.D.
  • Embodiments of the invention include sequences (both polynucleotide or polypeptide) which may comprise homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue or nucleotide, with an alternative residue or nucleotide) that may occur i.e., like-for-like substitution in the case of amino acids such as basic for basic, acidic for acidic, polar for polar, etc.
  • Non-homologous substitution may also occur i.e., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
  • Z ornithine
  • B diaminobutyric acid ornithine
  • O norleucine ornithine
  • pyriylalanine pyriylalanine
  • thienylalanine thienylalanine
  • naphthylalanine phenylglycine
  • Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or E-alanine residues.
  • alkyl groups such as methyl, ethyl or propyl groups
  • amino acid spacers such as glycine or E-alanine residues.
  • a further form of variation which involves the presence of one or more amino acid residues in peptoid form, may be well understood by those skilled in the art.
  • the peptoid form is used to refer to variant amino acid residues wherein the D-carbon substituent group is on the residue’s nitrogen atom rather than the D-carbon.
  • the invention provides for vectors that are used in the engineering and optimization of CRISPR-Cas systems.
  • a“vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment.
  • a vector is capable of replication when associated with the proper control elements.
  • the term“vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art.
  • a“plasmid” refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques.
  • viral vector wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g.
  • Viral vectors also include polynucleotides carried by a virus for transfection into a host cell.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • Other vectors e.g., non-episomal mammalian vectors are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as“expression vectors.”
  • expression vectors Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed.
  • “operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • aspects of the invention relate to bicistronic vectors for chimeric RNA and Cas9.
  • Bicistronic expression vectors for chimeric RNA and Cas9 are preferred.
  • Cas9 is preferably driven by the CBh promoter.
  • the chimeric RNA may preferably be driven by a U6 promoter. Ideally the two are combined.
  • the chimeric guide RNA typically consists of a 20bp guide sequence (Ns) and this may be joined to the tracr sequence (running from the first“U” of the lower strand to the end of the transcript). The tracr sequence may be truncated at various positions as indicated.
  • the guide and tracr sequences are separated by the tracr-mate sequence, which may be GUUUUAGAGCUA. This may be followed by the loop sequence GAAA as shown. Both of these are preferred examples.
  • Applicants have demonstrated Cas9-mediated indels at the human EMX1 and PVALB loci by SURVEYOR assays.
  • ChiRNAs are indicated by their“+n” designation, and crRNA refers to a hybrid RNA where guide and tracr sequences are expressed as separate transcripts.
  • chimeric RNA may also be called single guide, or synthetic guide RNA (sgRNA).
  • the loop is preferably GAAA, but it is not limited to this sequence or indeed to being only 4bp in length.
  • preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences.
  • the sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG.
  • the term“regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences).
  • Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences).
  • tissue-specific regulatory sequences may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes).
  • a vector comprises one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof.
  • pol III promoters include, but are not limited to, U6 and H1 promoters.
  • pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521 -530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the ⁇ -actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1 ⁇ promoter.
  • RSV Rous sarcoma virus
  • CMV cytomegalovirus
  • PGK phosphoglycerol kinase
  • enhancer elements such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit ⁇ -globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981).
  • WPRE WPRE
  • CMV enhancers the R-U5’ segment in LTR of HTLV-I
  • SV40 enhancer SV40 enhancer
  • the intron sequence between exons 2 and 3 of rabbit ⁇ -globin Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981.
  • a vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.).
  • CRISPR clustered regularly interspersed short palindromic repeats
  • Vectors can be designed for expression of CRISPR transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells.
  • CRISPR transcripts e.g. nucleic acid transcripts, proteins, or enzymes
  • CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase (although any suitable DNA-dependent RNA polymerase can be used, such as SP6, T3 or T7).
  • T7 RNA polymerase any suitable DNA-dependent RNA polymerase can be used, such as SP6, T3 or T7.
  • Amplification by T7 RNA polymerase requires the presence of suitable promoters in the RNA-coding DNA. Sequence requirements for polymerase binding sites are well known in the art.
  • Various T7 RNA polymerase promoter sequences are known, including natural sequences and artificial ones. Different T7 RNA polymerases can have different promoter sequence preferences, and mutant T7 RNA polymerases have been produced to match specific promoters (e.g.
  • T7 RNA polymerases and promoter sequences can easily match any particular T7 RNA polymerase to its preferred promoter sequence.
  • the consensus 23 base-pair T7 DNA promoter is classically divided into two domains, an upstream binding domain (-17 to -5, numbered relative to the start of transcription), and a downstream initiation domain (-4 to +6).
  • One strand of this 23mer is 5'-TAATACGACTCACTATAGGGAGA-3'.
  • the minimum sequence required for efficient transcription is the first 19mer of this 23mer.
  • Vectors may be introduced and propagated in a prokaryote or prokaryotic cell.
  • a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system).
  • a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism.
  • Fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein.
  • Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein.
  • Such enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988.
  • GST glutathione S-transferase
  • Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
  • a vector is a yeast expression vector.
  • yeast Saccharomyces cerivisae examples include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
  • a vector drives protein expression in insect cells using baculovirus expression vectors.
  • Baculovirus vectors available for expression of proteins in cultured insect cells include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
  • a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195).
  • the expression vector’s control functions are typically provided by one or more regulatory elements.
  • commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art.
  • the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific regulatory elements are known in the art.
  • suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J.
  • a regulatory element is operably linked to one or more elements of a CRISPR system so as to drive expression of the one or more elements of the CRISPR system.
  • CRISPRs Clustered Regularly Interspaced Short Palindromic Repeats
  • SPIDRs Sacer Interspersed Direct Repeats
  • the CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; and Nakata et al., J.
  • the CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]).
  • SRSRs short regularly spaced repeats
  • the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., [2000], supra).
  • the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J.
  • CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575 [2002]; and Mojica et al., [2005]) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomon
  • CRISPR system refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a“direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus.
  • a tracr trans-activating CRISPR
  • tracr-mate sequence encompassing a“direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system
  • guide sequence also referred to as a“spacer” in the context of an endogenous CRISPR system
  • one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system.
  • one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes.
  • a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system).
  • target sequence refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex.
  • a target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides.
  • a target sequence is located in the nucleus or cytoplasm of a cell.
  • direct repeats may be identified in silico by searching for repetitive motifs that fulfill any or all of the following criteria: 1. found in a 2Kb window of genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.
  • candidate tracrRNA may be subsequently predicted by sequences that fulfill any or all of the following criteria: 1. sequence homology to direct repeats (motif search in Geneious with up to 18-bp mismatches); 2.
  • chimeric synthetic guide RNAs designs may incorporate at least 12 bp of duplex structure between the direct repeat and tracrRNA.
  • the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes DNA cleavage.
  • Enzymatic action by Cas9 derived from Streptococcus pyogenes or any closely related Cas9 generates double stranded breaks at target site sequences which hybridize to 20 nucleotides of the guide sequence and that have a protospacer-adjacent motif (PAM) sequence (examples include NGG/NRG or a PAM that can be determined as described herein) following the 20 nucleotides of the target sequence.
  • PAM protospacer-adjacent motif
  • CRISPR activity through Cas9 for site- specific DNA recognition and cleavage is defined by the guide sequence, the tracr sequence that hybridizes in part to the guide sequence and the PAM sequence. More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defence in bacteria and archaea, Mole Cell 2010, January 15; 37(1): 7.
  • the type II CRISPR locus from Streptococcus pyogenes SF370 which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30bp each).
  • DSB targeted DNA double-strand break
  • tracrRNA hybridizes to the direct repeats of pre-crRNA, which is then processed into mature crRNAs containing individual spacer sequences.
  • the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA.
  • Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer (Fig. 2A).
  • Fig. 2B demonstrates the nuclear localization of the codon optimized Cas9.
  • the RNA polymerase III-based U6 promoter was selected to drive the expression of tracrRNA (Fig. 2C).
  • a U6 promoter-based construct was developed to express a pre-crRNA array consisting of a single spacer flanked by two direct repeats (DRs, also encompassed by the term“tracr-mate sequences”; Fig. 2C).
  • the initial spacer was designed to target a 33-base-pair (bp) target site (30-bp protospacer plus a 3-bp CRISPR motif (PAM) sequence satisfying the NGG recognition motif of Cas9) in the human EMX1 locus (Fig. 2C), a key gene in the development of the cerebral cortex.
  • bp 33-base-pair
  • PAM 3-bp CRISPR motif
  • a CRISPR complex comprising a guide sequence hybridized or hybridizable to a target sequence and complexed with one or more Cas proteins
  • formation of a CRISPR complex results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence.
  • the tracr sequence which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g.
  • one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites.
  • a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors.
  • two or more of the elements expressed from the same or different regulatory elements may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector.
  • CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element.
  • the coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction.
  • a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron).
  • the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.
  • a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a“cloning site”).
  • one or more insertion sites e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites are located upstream and/or downstream of one or more sequence elements of one or more vectors.
  • a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell.
  • a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site.
  • the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these.
  • a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell.
  • a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.
  • a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein.
  • Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof, or modified versions thereof, or modified versions thereof,
  • the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9.
  • the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence.
  • an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand).
  • mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A.
  • two or more catalytic domains of Cas9 may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity.
  • a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity.
  • a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respect to its non-mutated form.
  • the enzyme is not SpCas9
  • mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools .
  • any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged.
  • the same (or conservative substitutions of these mutations) at corresponding positions in other Cas9s are also preferred.
  • Particularly preferred are D10 and H840 in SpCas9 .
  • residues corresponding to SpCas9 D10 and H840 are also preferred.
  • a Cas enzyme may be identified Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the Cas9 enzyme is from, or is derived from, spCas9 or saCas9. By derived, Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as described herein.
  • Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent.
  • residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes.
  • this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCa9, St1Cas9 and so forth.
  • an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells.
  • the eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non- human mammal or primate.
  • processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes may be excluded.
  • codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ (visited Jul. 9, 2002), and these tables can be adapted in a number of ways. See Nakamura, Y., et al.“Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000).
  • codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.
  • one or more codons e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.
  • Codon usage can even be optimized for expression in particular cell types e.g. for brain cells.
  • Plotkin et al. (2004) PNAS USA 101:12588-91 reports on tissue-specific codon usage and notes, for instance, that brain-specific genes show a characteristically different codon usage than liver-specific genes.
  • a protein-coding sequence can be codon-optimised for expression in a target cell type of interest e.g. for expression in the liver.
  • NLSs Nuclear localization sequences
  • a vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs.
  • the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus).
  • the CRISPR enzyme comprises at most 6 NLSs.
  • an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus.
  • Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV; the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from importin-alpha; the sequences VSRKRPRP and PPKKARED of the myoma T protein; the sequence POPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-abl IV; the sequences DRLRR and PKQKKRK of the influenza
  • the one or more NLSs are of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell.
  • strength of nuclear localization activity may derive from the number of NLSs in the CRISPR enzyme, the particular NLS(s) used, or a combination of these factors.
  • Detection of accumulation in the nucleus may be performed by any suitable technique.
  • a detectable marker may be fused to the CRISPR enzyme, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI).
  • Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity), as compared to a control no exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the one or more NLSs.
  • an assay for the effect of CRISPR complex formation e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity
  • a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net).
  • Burrows-Wheeler Transform e.g. the Burrows Wheeler Aligner
  • ClustalW Clustal X
  • BLAT Novoalign
  • ELAND Illumina, San Diego, CA
  • SOAP available at soap.genomics.org.cn
  • Maq available at maq.sourceforge.net.
  • a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • a guide sequence may be selected to target any target sequence.
  • the target sequence is a sequence within a genome of a cell.
  • Exemplary target sequences include those that are unique in the target genome.
  • a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNXGG (N is A G T or C; and X can be anything) has a single occurrence in the genome.
  • a unique target sequence in a genome may include an S.
  • a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXAGAAW where NNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome.
  • a unique target sequence in a genome may include an S.
  • a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNNNNNNNNNNNXGGXG where NNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • a unique target sequence in a genome may include an S.
  • pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNNNXGGXG where NNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome.
  • N is A, G, T, or C; and X can be anything
  • a guide sequence is selected to reduce the degree secondary structure within the guide sequence. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the guide sequence participate in self-complementary base pairing when optimally folded.
  • Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized or hybridizable to the tracr sequence.
  • degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences.
  • Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence.
  • the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher.
  • the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length.
  • the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin.
  • the transcript or transcribed polynucleotide sequence has at least two or more hairpins.
  • the transcript has two, three, four or five hairpins.
  • the transcript has at most five hairpins.
  • the portion of the sequence 5’ of the final“N” and upstream of the loop corresponds to the tracr mate sequence
  • the portion of the sequence 3’ of the loop corresponds to the tracr sequence
  • “N” represents a base of a guide sequence
  • the first block of lower case letters represent the tracr mate sequence
  • the second block of lower case letters represent the tracr sequence
  • the final poly-T sequence represents the transcription terminator: (1) NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNgttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgtttcgtatttttttt
  • sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1.
  • sequences (4) to (6) are used in combination with Cas9 from S. pyogenes.
  • the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.
  • a recombination template is also provided.
  • a recombination template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide.
  • a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex.
  • a template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length.
  • the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence.
  • a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, or more nucleotides).
  • the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.
  • the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme).
  • a CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains.
  • protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity.
  • epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags.
  • reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP).
  • GST glutathione-S-transferase
  • HRP horseradish peroxidase
  • CAT chloramphenicol acetyltransferase
  • beta-galactosidase beta-galactosidase
  • beta-glucuronidase beta-galactosidase
  • luciferase green fluorescent protein
  • GFP green fluorescent protein
  • HcRed HcRed
  • DsRed cyan fluorescent protein
  • a CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.
  • MBP maltose binding protein
  • DBD Lex A DNA binding domain
  • HSV herpes simplex virus
  • a CRISPR enzyme may form a component of an inducible system.
  • the inducible nature of the system would allow for spatiotemporal control of gene editing or gene expression using a form of energy.
  • the form of energy may include but is not limited to electromagnetic radiation, sound energy, chemical energy and thermal energy.
  • inducible system examples include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc), or light inducible systems (Phytochrome, LOV domains, or cryptochrome).
  • the CRISPR enzyme may be a part of a Light Inducible Transcriptional Effector (LITE) to direct changes in transcriptional activity in a sequence-specific manner.
  • the components of a light may include a CRISPR enzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsis thaliana), and a transcriptional activation/repression domain.
  • inducible DNA binding proteins and methods for their use are provided in US 61/736465 and US 61/721,283, which is hereby incorporated by reference in its entirety (see also Konerman et al. (2013) Nature doi:10.1038/nature12466).
  • the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell.
  • the invention further provides cells produced by such methods, and animals comprising or produced from such cells.
  • a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to a cell.
  • Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism.
  • Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome.
  • Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell.
  • Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA.
  • Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., TransfectamTM and LipofectinTM).
  • Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
  • lipid:nucleic acid complexes including targeted liposomes such as immunolipid complexes
  • Boese et al. Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
  • RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus.
  • Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo).
  • Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
  • Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression.
  • Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); WO94/26877).
  • MiLV murine leukemia virus
  • GaLV gibbon ape leukemia virus
  • SIV Simian Immuno deficiency virus
  • HAV human immuno deficiency virus
  • Cocal vesiculovirus envelope pseudotyped retroviral vector particles are contemplated (see, e.g., US Patent Publication No. 20120164118 assigned to the Fred Hutchinson Cancer Research Center).
  • Cocal virus is in the Vesiculovirus genus, and is a causative agent of vesicular stomatitis in mammals.
  • Cocal virus was originally isolated from mites in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)), and infections have been identified in Trinidad, Brazil, and Argentina from insects, cattle, and horses.
  • the Cocal vesiculovirus envelope pseudotyped retroviral vector particles may include for example, lentiviral, alpharetroviral, betaretroviral, gammaretroviral, deltaretroviral, and epsilonretroviral vector particles that may comprise retroviral Gag, Pol, and/or one or more accessory protein(s) and a Cocal vesiculovirus envelope protein.
  • the Gag, Pol, and accessory proteins are lentiviral and/or gammaretroviral.
  • adenoviral based systems may be used.
  • Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
  • Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No.
  • Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 or PER.C6 cells, which package adenovirus, and 2 cells or PA317 cells, which package retrovirus.
  • Viral vectors used in gene therapy are usually generated by producer a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome.
  • Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences.
  • the cell line may also infected with adenovirus as a helper.
  • the helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid.
  • the helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Accordingly, AAV is considered an ideal candidate for use as a transducing vector.
  • Such AAV transducing vectors can comprise sufficient cis-acting functions to replicate in the presence of adenovirus or herpesvirus or poxvirus (e.g., vaccinia virus) helper functions provided in trans.
  • Recombinant AAV rAAV
  • rAAV Recombinant AAV
  • the AAV cap and/or rep genes are deleted from the viral genome and replaced with a DNA segment of choice.
  • Current AAV vectors may accommodate up to 4300 bases of inserted DNA.
  • plasmid(s) containing or consisting essentially of the desired viral construct are transfected into AAV-infected cells or into a packaging cell.
  • a second or additional helper plasmid is cotransfected into these cells to provide the AAV rep and/or cap genes which are obligatory for replication and packaging of the recombinant viral construct.
  • the rep and/or cap proteins of AAV act in trans to stimulate replication and packaging of the rAAV construct. Two to Three days after transfection, rAAV is harvested.
  • rAAV is harvested from the cells along with adenovirus. The contaminating adenovirus is then inactivated by heat treatment. In the instant invention, rAAV is advantageously harvested not from the cells themselves, but from cell supernatant.
  • rAAV can be prepared by a method that comprises or consists essentially of: infecting susceptible cells with a rAAV containing exogenous DNA including DNA for expression, and helper virus (e.g., adenovirus, herpesvirus, poxvirus such as vaccinia virus) wherein the rAAV lacks functioning cap and/or rep (and the helper virus (e.g., adenovirus, herpesvirus, poxvirus such as vaccinia virus) provides the cap and/or rev function that the rAAV lacks); or infecting susceptible cells with a rAAV containing exogenous DNA including DNA for expression, wherein the recombinant lacks functioning cap and/or rep, and transfecting said cells with a plasmid supplying cap and/or rep function that the rAAV lacks; or infecting susceptible cells with a rAAV containing ex
  • the rAAV can be from an AAV as herein described, and advantageously can be an rAAV1, rAAV2, AAV5 or rAAV having hybrid or capsid which may comprise AAV1, AAV2, AAV5 or any combination thereof.
  • the invention provides rAAV that contains or consists essentially of an exogenous nucleic acid molecule encoding a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, e.g., a plurality of cassettes comprising or consisting a first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a CRISPR-associated (Cas) protein (putative nuclease or helicase proteins), e.g., Cas9 and a terminator, and a two, or more, advantageously up to the packaging size limit of the vector, e.g., in total (including the first cassette) five, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator ...
  • CRISPR Clustered Regularly
  • Promoter- gRNA(N)-terminator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector), or two or more individual rAAVs, each containing one or more than one cassette of a CRISPR system, e.g., a first rAAV containing the first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding Cas, e.g., Cas9 and a terminator, and a second rAAV containing a plurality, four, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator ...
  • gRNA nucleic acid molecule encoding guide RNA
  • Promoter-gRNA(N)-terminator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector).
  • N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector.
  • the promoter is in some embodiments advantageously human Synapsin I promoter (hSyn).
  • a host cell is transiently or non-transiently transfected with one or more vectors described herein.
  • a cell is transfected as it naturally occurs in a subject.
  • a cell that is transfected is taken from a subject.
  • the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art.
  • cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB
  • a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences.
  • a cell transiently transfected with the components of a CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence.
  • cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.
  • a fluid delivery device with an array of needles may be contemplated for delivery of CRISPR Cas to solid tissue.
  • a device of US Patent Publication No. 20110230839 for delivery of a fluid to a solid tissue may comprise a plurality of needles arranged in an array; a plurality of reservoirs, each in fluid communication with a respective one of the plurality of needles; and a plurality of actuators operatively coupled to respective ones of the plurality of reservoirs and configured to control a fluid pressure within the reservoir.
  • each of the plurality of actuators may comprise one of a plurality of plungers, a first end of each of the plurality of plungers being received in a respective one of the plurality of reservoirs, and in certain further embodiments the plungers of the plurality of plungers are operatively coupled together at respective second ends so as to be simultaneously depressable. Certain still further embodiments may comprise a plunger driver configured to depress all of the plurality of plungers at a selectively variable rate. In other embodiments each of the plurality of actuators may comprise one of a plurality of fluid transmission lines having first and second ends, a first end of each of the plurality of fluid transmission lines being coupled to a respective one of the plurality of reservoirs.
  • the device may comprise a fluid pressure source, and each of the plurality of actuators comprises a fluid coupling between the fluid pressure source and a respective one of the plurality of reservoirs.
  • the fluid pressure source may comprise at least one of a compressor, a vacuum accumulator, a peristaltic pump, a master cylinder, a microfluidic pump, and a valve.
  • each of the plurality of needles may comprise a plurality of ports distributed along its length.
  • the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro.
  • the method comprises sampling a cell or population of cells from a human or non- human animal, or a plant, and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant. For re- introduced cells it is particularly preferred that the cells are stem cells.
  • the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
  • the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. Similar considerations and conditions apply as above for methods of modifying a target polynucleotide.
  • the CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence, wherein said guide sequence may be linked to a tracr mate sequence which in turn may hybridize to a tracr sequence. Similar considerations and conditions apply as above for methods of modifying a target polynucleotide.
  • kits containing any one or more of the elements disclosed in the above methods and compositions. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. In some embodiments, the kit includes instructions in one or more languages, for example in more than one language. In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers.
  • Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form).
  • a buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof.
  • the buffer is alkaline.
  • the buffer has a pH from about 7 to about 10.
  • the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element.
  • the kit comprises a homologous recombination template polynucleotide. In some embodiments, the kit comprises one or more of the vectors and/or one or more of the polynucleotides described herein. The kit may advantageously allows to provide all elements of the systems of the invention.
  • the invention provides methods for using one or more elements of a CRISPR system.
  • the CRISPR complex of the invention provides an effective means for modifying a target polynucleotide.
  • the CRISPR complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating) a target polynucleotide in a multiplicity of cell types.
  • An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within the target polynucleotide.
  • the guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.
  • this invention provides a method of cleaving a target polynucleotide.
  • the method comprises modifying a target polynucleotide using a CRISPR complex that binds to the target polynucleotide and effect cleavage of said target polynucleotide.
  • the CRISPR complex of the invention when introduced into a cell, creates a break (e.g., a single or a double strand break) in the genome sequence.
  • the method can be used to cleave an integrated viral gene in a cell.
  • the break created by the CRISPR complex can be repaired by a repair processes such as the error prone non-homologous end joining (NHEJ) pathway or the high fidelity homology-directed repair (HDR) (Fig. 29).
  • NHEJ error prone non-homologous end joining
  • HDR high fidelity homology-directed repair
  • an exogenous polynucleotide template can be introduced into the genome sequence.
  • the HDR process is used modify genome sequence.
  • an exogenous polynucleotide template comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell.
  • the upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome.
  • a donor polynucleotide can be DNA, e.g., a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer.
  • the exogenous polynucleotide template comprises a sequence to be integrated (e.g., a mutated gene).
  • the sequence for integration may be a sequence endogenous or exogenous to the cell.
  • sequences to be integrated include polynucleotides encoding a protein or a non-coding RNA (e.g., a microRNA).
  • the sequence for integration may be operably linked to an appropriate control sequence or sequences.
  • the sequence to be integrated may provide a regulatory function.
  • the upstream and downstream sequences in the exogenous polynucleotide template are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide.
  • the upstream sequence is a nucleic acid sequence that shares sequence similarity with the genome sequence upstream of the targeted site for integration.
  • the downstream sequence is a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the targeted site of integration.
  • the upstream and downstream sequences in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted genome sequence.
  • the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted genome sequence.
  • the upstream and downstream sequences in the exogenous polynucleotide template have about 99% or 100% sequence identity with the targeted genome sequence.
  • An upstream or downstream sequence may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp.
  • the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp.
  • the exogenous polynucleotide template may further comprise a marker. Such a marker may make it easy to screen for targeted integrations.
  • exogenous polynucleotide template of the invention can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996).
  • a double stranded break is introduced into the genome sequence by the CRISPR complex, the break is repaired via homologous recombination an exogenous polynucleotide template such that the template is integrated into the genome.
  • the presence of a double-stranded break facilitates integration of the template.
  • this invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell.
  • the method comprises decreasing expression of a target polynucleotide by using a CRISPR complex that binds to the polynucleotide.
  • a target polynucleotide can be inactivated to effect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein or RNA is not produced, or the sequence does not function as the wild-type sequence does.
  • control sequence refers to any nucleic acid sequence that effects the transcription, translation, or accessibility of a nucleic acid sequence. Examples of a control sequence include, a promoter, a transcription terminator, and an enhancer are control sequences.
  • the inactivated target sequence may include a deletion mutation (i.e., deletion of one or more nucleotides), an insertion mutation (i.e., insertion of one or more nucleotides), or a nonsense mutation (i.e., substitution of a single nucleotide for another nucleotide such that a stop codon is introduced).
  • a deletion mutation i.e., deletion of one or more nucleotides
  • an insertion mutation i.e., insertion of one or more nucleotides
  • a nonsense mutation i.e., substitution of a single nucleotide for another nucleotide such that a stop codon is introduced.
  • wild type StCas9 refers to wild type Cas9 from S thermophilus, the protein sequence of which is given in the SwissProt database under accession number G3ECR1. Similarly, S pyogenes Cas9 is included in SwissProt under accession number Q99ZW2.
  • a first step is to redesign and produce sgRNA sequences compatible with the SaCas9 PAM. Then, the sgRNAs are cloned into a viral vector (likely lentiviral for initial in vitro cell line studies) and these new guides are screened for their ability to cleave cccDNA in de novo infections. These experiments use the HepG2-hNTCP knockin cell lines and HBV virions purified from HepG2.2.15 cells.
  • the screen is performed either with guides in individual wells side-by-side, or using a pooled format in which lentivirus is produced with a pool of all possible sgRNA sequences against HBV, and then deep sequencing identifes regions of the HBV genome most susceptible to cleavage by SaCas9.
  • Rationale for choosing appropriate sgRNAs for anti-HBV CRISPR/Cas9 systems The process is a multistep one in which several parameters should be optimized: efficacy of the sgRNA sequence, targeting to an accessible part of cccDNA, conservation of the target sequence across viral genotypes, and minimization of target sequence homology to the human genome. These criteria should be general criteria across all episomal viruses, although the specifics may differ (for example, HBV cccDNA seems to be most accessible to cleavage in the ORF for Core, and it is possible that latent HSV may be most accessible in the region encoding latency- associated transcript LAT).
  • the general workflow is as follows:
  • the columns labeled‘D’ signify where Applicants used a nuclease-deficient Cas9 as an internal control.
  • the 29 dpt corresponds to 29 days post transduction, where a single lentiviral vector encoding U6-sgRNA and EFS-hSpCas9-2A-Puro was transduced into HepG2.2.15 cells followed by selection with puromycin.
  • the CRISPR-based activation system can specifically upregulate targeted genes, it is possible to target APOBEC3A, APOBEC3B, and/or other antiviral interferon-stimulated genes (ISGs) using this system in order to target HBV cccDNA for degradation. While this is an indirect approach, the advantage here may be that the use of a nuclease-competent Cas9 is not required, potentially reducing the chance of deleterious off- target effects.
  • ISGs antiviral interferon-stimulated genes
  • An example type II CRISPR system is the type II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30bp each).
  • DSB targeted DNA double-strand break
  • tracrRNA hybridizes to the direct repeats of pre- crRNA, which is then processed into mature crRNAs containing individual spacer sequences.
  • the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA.
  • Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer (Fig. 2A).
  • This example describes an example process for adapting this RNA-programmable nuclease system to direct CRISPR complex activity in the nuclei of eukaryotic cells.
  • HEK cell line HEK 293FT Human embryonic kidney (HEK) cell line HEK 293FT (Life Technologies) was maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2mM GlutaMAX (Life Technologies), 100U/mL penicillin, and 100 g/mL streptomycin at 37oC with 5% CO 2 incubation.
  • DMEM Dulbecco’s modified Eagle’s Medium
  • HEK 293FT or N2A cells were seeded into 24-well plates (Corning) one day prior to transfection at a density of 200,000 cells per well. Cells were transfected using Lipofectamine 2000 (Life Technologies) following the manufacturer’s recommended protocol. For each well of a 24-well plate a total of 800ng of plasmids were used.
  • HEK 293FT or N2A cells were transfected with plasmid DNA as described above. After transfection, the cells were incubated at 37oC for 72 hours before genomic DNA extraction. Genomic DNA was extracted using the QuickExtract DNA extraction kit (Epicentre) following the manufacturer’s protocol. Briefly, cells were resuspended in QuickExtract solution and incubated at 65oC for 15 minutes and 98oC for 10 minutes. Extracted genomic DNA was immediately processed or stored at–20oC.
  • the genomic region surrounding a CRISPR target site for each gene was PCR amplified, and products were purified using QiaQuick Spin Column (Qiagen) following manufacturer’s protocol.
  • a total of 400ng of the purified PCR products were mixed with 2 l 10X Taq polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 20 l, and subjected to a re-annealing process to enable heteroduplex formation: 95oC for 10min, 95oC to 85oC ramping at– 2oC/s, 85oC to 25oC at– 0.25oC/s, and 25oC hold for 1 minute.
  • HEK 293FT and N2A cells were transfected with plasmid DNA, and incubated at 37°C for 72 hours before genomic DNA extraction as described above.
  • the target genomic region was PCR amplified using primers outside the homology arms of the homologous recombination (HR) template.
  • PCR products were separated on a 1% agarose gel and extracted with MinElute GelExtraction Kit (Qiagen). Purified products were digested with HindIII (Fermentas) and analyzed on a 6% Novex TBE poly-acrylamide gel (Life Technologies).
  • RNA secondary structure prediction was performed using the online webserver RNAfold developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
  • HEK 293FT cells were maintained and transfected as stated above. Cells were harvested by trypsinization followed by washing in phosphate buffered saline (PBS). Total cell RNA was extracted with TRI reagent (Sigma) following manufacturer’s protocol. Extracted total RNA was quantified using Naonodrop (Thermo Scientific) and normalized to same concentration.
  • RNAs were mixed with equal volumes of 2X loading buffer (Ambion), heated to 95qC for 5 min, chilled on ice for 1 min, and then loaded onto 8% denaturing polyacrylamide gels (SequaGel, National Diagnostics) after pre-running the gel for at least 30 minutes. The samples were electrophoresed for 1.5 hours at 40W limit. Afterwards, the RNA was transferred to Hybond N+ membrane (GE Healthcare) at 300 mA in a semi-dry transfer apparatus (Bio-rad) at room temperature for 1.5 hours. The RNA was crosslinked to the membrane using autocrosslink button on Stratagene UV Crosslinker the Stratalinker (Stratagene).
  • the membrane was pre-hybridized in ULTRAhyb-Oligo Hybridization Buffer (Ambion) for 30 min with rotation at 42qC, and probes were then added and hybridized overnight. Probes were ordered from IDT and labeled with [gamma- 32 P] ATP (Perkin Elmer) with T4 polynucleotide kinase (New England Biolabs). The membrane was washed once with pre-warmed (42qC) 2xSSC, 0.5% SDS for 1 min followed by two 30 minute washes at 42qC. The membrane was exposed to a phosphor screen for one hour or overnight at room temperature and then scanned with a phosphorimager (Typhoon).
  • CRISPR locus elements including tracrRNA, Cas9, and leader were PCR amplified from Streptococcus pyogenes SF370 genomic DNA with flanking homology arms for Gibson Assembly. Two BsaI type IIS sites were introduced in between two direct repeats to facilitate easy insertion of spacers (Fig. 8). PCR products were cloned into EcoRV-digested pACYC184 downstream of the tet promoter using Gibson Assembly Master Mix (NEB). Other endogenous CRISPR system elements were omitted, with the exception of the last 50bp of Csn2.
  • Oligos Integrated DNA Technology
  • pDC000 BsaI-digested vector pDC000
  • T7 ligase Enzymatics
  • 6C shows results of a Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying long or short tracrRNA, as well as SpCas9 and DR-EMX1(1)-DR.
  • Left and right panels are from 293FT cells transfected without or with SpRNase III, respectively.
  • U6 indicate loading control blotted with a probe targeting human U6 snRNA Transfection of the short tracrRNA expression construct led to abundant levels of the processed form of tracrRNA ( ⁇ 75bp). Very low amounts of long tracrRNA are detected on the Northern blot.
  • RNA polymerase III-based U6 promoter was selected to drive the expression of tracrRNA (Fig. 2C).
  • a U6 promoter- based construct was developed to express a pre-crRNA array consisting of a single spacer flanked by two direct repeats (DRs, also encompassed by the term“tracr-mate sequences”; Fig. 2C).
  • the initial spacer was designed to target a 33-base-pair (bp) target site (30-bp protospacer plus a 3-bp CRISPR motif (PAM) sequence satisfying the NGG recognition motif of Cas9) in the human EMX1 locus (Fig. 2C), a key gene in the development of the cerebral cortex.
  • bp 33-base-pair
  • PAM 3-bp CRISPR motif
  • HEK 293FT cells were transfected with combinations of CRISPR components. Since DSBs in mammalian nuclei are partially repaired by the non-homologous end joining (NHEJ) pathway, which leads to the formation of indels, the Surveyor assay was used to detect potential cleavage activity at the target EMX1 locus (Fig. 7) (see e.g. Guschin et al., 2010, Methods Mol Biol 649: 247).
  • NHEJ non-homologous end joining
  • Fig. 12 provides an additional Northern blot analysis of crRNA processing in mammalian cells.
  • Fig. 12A illustrates a schematic showing the expression vector for a single spacer flanked by two direct repeats (DR-EMX1(1)-DR). The 30bp spacer targeting the human EMX1 locus protospacer 1 (see Fig. 6) and the direct repeat sequences are shown in the sequence beneath Fig. 12A. The line indicates the region whose reverse-complement sequence was used to generate Northern blot probes for EMX1(1) crRNA detection.
  • Fig. 12B shows a Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying DR-EMX1(1)-DR.
  • DR-EMX1(1)-DR was processed into mature crRNAs only in the presence of SpCas9 and short tracrRNA and was not dependent on the presence of SpRNase III.
  • the mature crRNA detected from transfected 293FT total RNA is ⁇ 33bp and is shorter than the 39-42bp mature crRNA from S. pyogenes.
  • Fig. 2 illustrates the bacterial CRISPR system described in this example.
  • Fig. 2A illustrates a schematic showing the CRISPR locus 1 from Streptococcus pyogenes SF370 and a proposed mechanism of CRISPR-mediated DNA cleavage by this system.
  • Mature crRNA processed from the direct repeat-spacer array directs Cas9 to genomic targets consisting of complimentary protospacers and a protospacer-adjacent motif (PAM).
  • PAM protospacer-adjacent motif
  • Cas9 mediates a double-strand break in the target DNA.
  • Fig. 2B illustrates engineering of S.
  • Fig. 2C illustrates mammalian expression of SpCas9 and SpRNase III driven by the constitutive EF1a promoter and tracrRNA and pre- crRNA array (DR-Spacer-DR) driven by the RNA Pol3 promoter U6 to promote precise transcription initiation and termination.
  • DR-Spacer-DR pre- crRNA array
  • a protospacer from the human EMX1 locus with a satisfactory PAM sequence is used as the spacer in the pre-crRNA array.
  • Fig. 2D illustrates surveyor nuclease assay for SpCas9-mediated minor insertions and deletions.
  • SpCas9 was expressed with and without SpRNase III, tracrRNA, and a pre-crRNA array carrying the EMX1- target spacer.
  • Fig. 2E illustrates a schematic representation of base pairing between target locus and EMX1-targeting crRNA, as well as an example chromatogram showing a micro deletion adjacent to the SpCas9 cleavage site.
  • a chimeric crRNA-tracrRNA hybrid design was adapted, where a mature crRNA (comprising a guide sequence) may be fused to a partial tracrRNA via a stem-loop to mimic the natural crRNA:tracrRNA duplex.
  • a bicistronic expression vector was created to drive co-expression of a chimeric RNA and SpCas9 in transfected cells.
  • the bicistronic vectors were used to express a pre-crRNA (DR-guide sequence-DR) with SpCas9, to induce processing into crRNA with a separately expressed tracrRNA (compare Fig. 11B top and bottom).
  • FIG. 8 provides schematic illustrations of bicistronic expression vectors for pre-crRNA array ( Figure 8A) or chimeric crRNA (represented by the short line downstream of the guide sequence insertion site and upstream of the EF1 ⁇ promoter in Fig. 8B) with hSpCas9, showing location of various elements and the point of guide sequence insertion.
  • the expanded sequence around the location of the guide sequence insertion site in Fig. 8B also shows a partial DR sequence (GTTTAGAGCTA) and a partial tracrRNA sequence (TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT).
  • Guide sequences can be inserted between BbsI sites using annealed oligonucleotides.
  • RNA design for the oligonucleotides are shown below the schematic illustrations in Fig. 8, with appropriate ligation adapters indicated.
  • WPRE represents the Woodchuck hepatitis virus post-transcriptional regulatory element.
  • the efficiency of chimeric RNA-mediated cleavage was tested by targeting the same EMX1 locus described above. Using both Surveyor assay and Sanger sequencing of amplicons, Applicants confirmed that the chimeric RNA design facilitates cleavage of human EMX1 locus with approximately a 4.7% modification rate (Fig. 3).
  • Fig. 13 illustrates the selection of some additional targeted protospacers in human PVALB (Fig. 13A) and mouse Th (Fig. 13B) loci. Schematics of the gene loci and the location of three protospacers within the last exon of each are provided.
  • the underlined sequences include 30bp of protospacer sequence and 3bp at the 3’ end corresponding to the PAM sequences.
  • FIG. 11A provides a schematic of the human EMX1 locus showing the location of five protospacers, indicated by the underlined sequences.
  • Fig. 11B provides a schematic of the pre-crRNA/trcrRNA complex showing hybridization between the direct repeat region of the pre-crRNA and tracrRNA (top), and a schematic of a chimeric RNA design comprising a 20bp guide sequence, and tracr mate and tracr sequences consisting of partial direct repeat and tracrRNA sequences hybridized in a hairpin structure (bottom). Results of a Surveyor assay comparing the efficacy of Cas9-mediated cleavage at five protospacers in the human EMX1 locus is illustrated in Fig. 11C. Each protospacer is targeted using either processed pre-crRNA/tracrRNA complex (crRNA) or chimeric RNA (chiRNA).
  • crRNA processed pre-crRNA/tracrRNA complex
  • chiRNA chimeric RNA
  • RNA Since the secondary structure of RNA can be crucial for intermolecular interactions, a structure prediction algorithm based on minimum free energy and Boltzmann-weighted structure ensemble was used to compare the putative secondary structure of all guide sequences used in the genome targeting experiment (see e.g. Gruber et al., 2008, Nucleic Acids Research, 36: W70). Analysis revealed that in most cases, the effective guide sequences in the chimeric crRNA context were substantially free of secondary structure motifs, whereas the ineffective guide sequences were more likely to form internal secondary structures that could prevent base pairing with the target protospacer DNA. It is thus possible that variability in the spacer secondary structure might impact the efficiency of CRISPR-mediated interference when using a chimeric crRNA.
  • FIG. 22 illustrates single expression vectors incorporating a U6 promoter linked to an insertion site for a guide oligo, and a Cbh promoter linked to SpCas9 coding sequence.
  • the vector shown in Fig. 22b includes a tracrRNA coding sequence linked to an H1 promoter.
  • Fig. 3A illustrates results of a Surveyor nuclease assay comparing the cleavage efficiency of Cas9 when paired with different mutant chimeric RNAs.
  • Single-base mismatch up to 12-bp 5’ of the PAM substantially abrogated genomic cleavage by SpCas9, whereas spacers with mutations at farther upstream positions retained activity against the original protospacer target (Fig. 3B).
  • SpCas9 has single-base specificity within the last 12- bp of the spacer. Furthermore, CRISPR is able to mediate genomic cleavage as efficiently as a pair of TALE nucleases (TALEN) targeting the same EMX1 protospacer.
  • TALEN TALE nucleases
  • Fig. 3C provides a schematic showing the design of TALENs targeting EMX1
  • Fig. 4C provides a schematic illustration of the HR strategy, with relative locations of recombination points and primer annealing sequences (arrows). SpCas9 and SpCas9n indeed catalyzed integration of the HR template into the EMX1 locus.
  • RNA to program sequence-specific DNA cleavage defines a new class of genome engineering tools for a variety of research and industrial applications.
  • CRISPR system can be further improved to increase the efficiency and versatility of CRISPR targeting.
  • Optimal Cas9 activity may depend on the availability of free Mg 2+ at levels higher than that present in the mammalian nucleus (see e.g. Jinek et al., 2012, Science, 337:816), and the preference for an NGG motif immediately downstream of the protospacer restricts the ability to target on average every 12-bp in the human genome (Fig. 9, evaluating both plus and minus strands of human chromosomal sequences).
  • FIG. 10 illustrates adaptation of the Type II CRISPR system from CRISPR 1 of Streptococcus thermophilus LMD-9 for heterologous expression in mammalian cells to achieve CRISPR-mediated genome editing.
  • Fig. 10A provides a Schematic illustration of CRISPR 1 from S. thermophilus LMD-9.
  • Figure 10B illustrates the design of an expression system for the S. thermophilus CRISPR system.
  • Human codon-optimized hStCas9 is expressed using a constitutive EF1 ⁇ promoter. Mature versions of tracrRNA and crRNA are expressed using the U6 promoter to promote precise transcription initiation. Sequences from the mature crRNA and tracrRNA are illustrated. A single base indicated by the lower case“a” in the crRNA sequence is used to remove the polyU sequence, which serves as a RNA polIII transcriptional terminator.
  • Fig. 10C provides a schematic showing guide sequences targeting the human EMX1 locus.
  • Fig. 10D shows the results of hStCas9-mediated cleavage in the target locus using the Surveyor assay. RNA guide spacers 1 and 2 induced 14% and 6.4%, respectively.
  • Fig. 14 provides a schematic of additional protospacer and corresponding PAM sequence targets of the S. thermophilus CRISPR system in the human EMX1 locus. Two protospacer sequences are highlighted and their corresponding PAM sequences satisfying NNAGAAW motif are indicated by underlining 3’ with respect to the corresponding highlighted sequence. Both protospacers target the anti-sense strand.
  • Example 3 Sample target sequence selection algorithm
  • a software program is designed to identify candidate CRISPR target sequences on both strands of an input DNA sequence based on desired guide sequence length and a CRISPR motif sequence (PAM) for a specified CRISPR enzyme.
  • PAM CRISPR motif sequence
  • target sites for Cas9 from S. pyogenes, with PAM sequences NGG may be identified by searching for 5’-N x -NGG-3’ both on the input sequence and on the reverse-complement of the input.
  • target sites for Cas9 of S. thermophilus CRISPR1, with PAM sequence NNAGAAW may be identified by searching for 5’-N x -NNAGAAW-3’ both on the input sequence and on the reverse-complement of the input.
  • target sites for Cas9 of S. thermophilus CRISPR3, with PAM sequence NGGNG may be identified by searching for 5’-N x -NGGNG-3’ both on the input sequence and on the reverse-complement of the input.
  • the value“x” in N x may be fixed by the program or specified by the user, such as 20.

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Abstract

The invention provides for delivery, engineering and optimization of systems, methods, and compositions for manipulation of sequences and/or activities of target sequences. Provided are delivery systems and tissues or organ which are targeted as sites for delivery. Also provided are vectors and vector systems some of which encode one or more components of a CRISPR complex, as well as methods for the design and use of such vectors. Also provided are methods of directing CRISPR complex formation in eukaryotic cells to ensure enhanced specificity for target recognition and avoidance of toxicity and to edit or modify a target site in a genomic locus of interest to alter or improve the status of a disease or a condition.

Description

DELIVERY, USE AND THERAPEUTIC APPLICATIONS OF
THE CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR
HBV AND VIRAL DISEASES AND DISORDERS RELATED APPLICATIONS AND INCORPORATION BY REFERENCE
[0001] This application claims priority from US provisional application 61/915,301, filed December 12, 2013 and US provisional application 62/010,329, filed June 10, 2014.
[0002] The foregoing application(s), and all documents cited therein or during its or their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, and all documents cited or referenced herein (“herein cited documents”), and all documents cited or referenced in herein cited documents, together with any manufacturer’s instructions, descriptions, product specifications, and product sheets for any products mentioned herein or in any document incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. More specifically, all referenced documents are incorporated by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
FIELD OF THE INVENTION
[0003] The present invention generally relates to the delivery, engineering, optimization and therapeutic applications of systems, methods, and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.
STATEMENT AS TO FEDERALLY SPONSORED RESEARCH
[0004] This invention was made with government support under the NIH Pioneer Award (1DP1MH100706) awarded by the National Institutes of Health. The government has certain rights in the invention. The project described was supported by award Number T32GM007753 from the National Institute of General Medical Sciences.
BACKGROUND OF THE INVENTION
[0005] Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. Precise genome targeting technologies are needed to enable systematic reverse engineering of causal genetic variations by allowing selective perturbation of individual genetic elements, as well as to advance synthetic biology, biotechnological, and medical applications. Although genome-editing techniques such as designer zinc fingers, transcription activator-like effectors (TALEs), or homing meganucleases are available for producing targeted genome perturbations, there remains a need for new genome engineering technologies that are affordable, easy to set up, scalable, and amenable to targeting multiple positions within the eukaryotic genome.
SUMMARY OF THE INVENTION
[0006] The CRISPR-Cas system does not require the generation of customized proteins to target specific sequences but rather a single Cas enzyme can be programmed by a short RNA molecule to recognize a specific DNA target. Adding the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significantly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse range of biological functions and diseases. To utilize the CRISPR-Cas system effectively for genome editing without deleterious effects, it is critical to understand aspects of engineering, optimization and cell-type/tissue/organ specific delivery of these genome engineering tools, which are aspects of the claimed invention.
[0007] There exists a pressing need for alternative and robust systems and techniques for nucleic sequence targeting with a wide array of applications. Aspects of this invention address this need and provide related advantages, particularly in relation to treatment of viral infections which persist due to integration of a virus into a host’s genome and/or by maintenance of an episomal form (e.g. hepatitis B virus, HBV, which maintains extraordinary persistence in the nucleus of human hepatocytes by means of a long-lived episomal double-stranded DNA form called covalent closed circular DNA, or cccDNA). Applicants have shown that it is possible to directly cleave and reduce the abundance of this episomal form of the virus (cccDNA: a dsDNA structure that arises during the propagation of HBV in the cell nucleus and can remain permanently present in infected subjects).
[0008] An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence.
[0009] In one aspect, the invention provides methods for using one or more elements of a CRISPR-Cas system. The CRISPR complex of the invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the invention has a wide variety of utilities including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types in various tissues and organs. As such the CRISPR complex of the invention has a broad spectrum of applications in, e.g., gene or genome editing, gene therapy, drug discovery, drug screening, disease diagnosis, and prognosis.
[0010] Aspects of the invention relate to Cas9 enzymes having improved targeting specificity in a CRISPR-Cas9 system having guide RNAs having optimal activity, smaller in length than wild-type Cas9 enzymes and nucleic acid molecules coding therefor, and chimeric Cas9 enzymes, as well as methods of improving the target specificity of a Cas9 enzyme or of designing a CRISPR-Cas9 system comprising designing or preparing guide RNAs having optimal activity and/or selecting or preparing a Cas9 enzyme having a smaller size or length than wild-type Cas9 whereby packaging a nucleic acid coding therefor into a delivery vector is more advanced as there is less coding therefor in the delivery vector than for wild-type Cas9, and/or generating chimeric Cas9 enzymes.
[0011] Also provided are uses of the present sequences, vectors, enzymes or systems, in medicine. Also provided are uses of the same in gene or genome editing.
[0012] In an additional aspect of the invention, a Cas9 enzyme may comprise one or more mutations and may be used as a generic DNA binding protein with or without fusion to a functional domain. The mutations may be artificially introduced mutations or gain- or loss-of- function mutations. The mutations may include but are not limited to mutations in one of the catalytic domains (D10 and H840) in the RuvC and HNH catalytic domains, respectively. Further mutations have been characterized. In one aspect of the invention, the transcriptional activation domain may be VP64. In other aspects of the invention, the transcriptional repressor domain may be KRAB or SID4X. Other aspects of the invention relate to the mutated Cas9 enzyme being fused to domains which include but are not limited to a transcriptional activator, repressor, a recombinase, a transposase, a histone remodeler, a demethylase, a DNA methyltransferase, a cryptochrome, a light inducible/controllable domain or a chemically inducible/controllable domain.
[0013] In a further embodiment, the invention provides for methods to generate mutant tracrRNA and direct repeat sequences or mutant chimeric guide sequences that allow for enhancing performance of these RNAs in cells. Aspects of the invention also provide for selection of said sequences. [0014] Aspects of the invention also provide for methods of simplifying the cloning and delivery of components of the CRISPR complex. In the preferred embodiment of the invention, a suitable promoter, such as the U6 promoter, is amplified with a DNA oligo and added onto the guide RNA. The resulting PCR product can then be transfected into cells to drive expression of the guide RNA. Aspects of the invention also relate to the guide RNA being transcribed in vitro or ordered from a synthesis company and directly transfected.
[0015] In one aspect, the invention provides for methods to improve activity by using a more active polymerase. In a preferred embodiment, the expression of guide RNAs under the control of the T7 promoter is driven by the expression of the T7 polymerase in the cell. In an advantageous embodiment, the cell is a eukaryotic cell. In a preferred embodiment the eukaryotic cell is a human cell. In a more preferred embodiment the human cell is a patient specific cell, e.g., a cell removed from a patient that may be modified and/or expanded into a cell population or a modified cell population, for instance, for re-administration to the patient..
[0016] In one aspect, the invention provides for methods of reducing the toxicity of Cas enzymes. In certain aspects, the Cas enzyme is any Cas9 as described herein, for instance any naturally-occurring bacterial Cas9 as well as any chimaeras, mutants, homologs or orthologs. In one aspect, the Cas enzyme is a nickase. In a preferred embodiment, the Cas9 is delivered into the cell in the form of mRNA. This allows for the transient expression of the enzyme thereby reducing toxicity. In another embodiment, the Cas9 is delivered into the cell in the nucleotide construct that encodes and expresses the Cas9 enzyme. In another preferred embodiment, the invention also provides for methods of expressing Cas9 under the control of an inducible promoter, and the constructs used therein.
[0017] In another aspect, the invention provides for methods of improving the in vivo applications of the CRISPR-Cas system. In the preferred embodiment, the Cas enzyme is wildtype Cas9 or any of the modified versions described herein, including any naturally- occurring bacterial Cas9 as well as any chimaeras, mutants, homologs or orthologs. . In some methods of the invention the CRISPR enzyme comprises one or more mutations in one of the catalytic domains. In one aspect, the Cas enzyme is a nickase. An advantageous aspect of the invention provides for the selection of Cas9 homologs that are easily packaged into viral vectors for delivery. Cas9 orthologs typically share the general organization of 3-4 RuvC domains and a HNH domain. The 5' most RuvC domain cleaves the non-complementary strand, and the HNH domain cleaves the complementary strand. All notations are in reference to the guide sequence. [0018] The catalytic residue in the 5' RuvC domain is identified through homology comparison of the Cas9 of interest with other Cas9 orthologs (from S. pyogenes type II CRISPR locus, S. thermophilus CRISPR locus 1, S. thermophilus CRISPR locus 3, and Franciscilla novicida type II CRISPR locus), and the conserved Asp residue (D10) is mutated to alanine to convert Cas9 into a complementary-strand nicking enzyme. Similarly, the conserved His and Asn residues in the HNH domains are mutated to Alanine to convert Cas9 into a non- complementary-strand nicking enzyme. In some embodiments, both sets of mutations may be made, to convert Cas9 into a non-cutting enzyme.
[0019] In some embodiments, the CRISPR enzyme is a type I or III CRISPR enzyme, preferably a type II CRISPR enzyme. This type II CRISPR enzyme may be any Cas enzyme. A preferred Cas enzyme may be identified as Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the Cas9 enzyme is from, or is derived from, spCas9 or saCas9. By derived, Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as described herein
[0020] It will be appreciated that the terms Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent. As mentioned above, many of the residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes. However, it will be appreciated that this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCas9, St1Cas9 and so forth. Further examples are provided herein. The skilled person will be able to determine appropriate corresponding residues in Cas9 enzymes other than SpCas9 by comparison of the relevant amino acid sequences. Thus, where a specific amino acid replacement is referred to using the SpCas9 numbering, then, unless the context makes it apparent this is not intended to refer to other Cas9 enzymes, the disclosure is intended to encompass corresponding modifications in other Cas9 enzymes.
[0021] An example of a codon optimized sequence, in this instance optimized for humans (i.e. being optimized for expression in humans) is provided herein, see the SaCas9 human codon optimized sequence. Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species is known.
[0022] In further embodiments, the invention provides for methods of enhancing the function of Cas9 by generating chimeric Cas9 proteins. Chimeric Cas9 proteins chimeric Cas9s may be new Cas9 containing fragments from more than one naturally occurring Cas9. These methods may comprise fusing N-terminal fragments of one Cas9 homolog with C-terminal fragments of another Cas9 homolog. These methods also allow for the selection of new properties displayed by the chimeric Cas9 proteins.
[0023] It will be appreciated that in the present methods, where the organism is an animal or a plant, the modification may occur ex vivo or in vitro, for instance in a cell culture and in some instances not in vivo. In other embodiments, it may occur in vivo.
[0024] In one aspect, the invention provides a method of modifying an organism or a non- human organism by manipulation of a target sequence in a genomic locus of interest (e.g. an integrated viral sequence) comprising:
delivering a non-naturally occurring or engineered composition comprising:
A) - I. a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises:
(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell,
(b) a tracr mate sequence, and
(c) a tracr sequence, and
II. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences,
wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation,
wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,
or
(B) I. polynucleotides comprising:
(a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and
(b) at least one or more tracr mate sequences,
II. a polynucleotide sequence encoding a CRISPR enzyme, and III. a polynucleotide sequence comprising a tracr sequence,
wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA.
[0025] Any or all of the polynucleotide sequence encoding a CRISPR enzyme, guide sequence, tracr mate sequence or tracr sequence, may be RNA. The polynucleotides encoding the sequence encoding a CRISPR enzyme, the guide sequence, tracr mate sequence or tracr sequence may be RNA and may be delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
[0026] It will be appreciated that where reference is made to a polynucleotide, which is RNA and is said to‘comprise’ a feature such a tracr mate sequence, the RNA sequence includes the feature. Where the polynucleotide is DNA and is said to comprise a feature such a tracr mate sequence, the DNA sequence is or can be transcribed into the RNA including the feature at issue. Where the feature is a protein, such as the CRISPR enzyme, the DNA or RNA sequence referred to is, or can be, translated (and in the case of DNA transcribed first).
[0027] Accordingly, in certain embodiments the invention provides a method of modifying an organism, e.g., mammal including human or a non-human mammal or organism by manipulation of a target sequence in a genomic locus of interest comprising delivering a non- naturally occurring or engineered composition comprising a viral or plasmid vector system comprising one or more viral or plasmid vectors operably encoding a composition for expression thereof, wherein the composition comprises: (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (or optionally at least one or more nuclear localization sequences as some embodiments can involve no NLS), wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, or (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence. In some embodiments, components I, II and III are located on the same vector. In other embodiments, components I and II are located on the same vector, while component III is located on another vector. In other embodiments, components I and III are located on the same vector, while component II is located on another vector. In other embodiments, components II and III are located on the same vector, while component I is located on another vector. In other embodiments, each of components I, II and III is located on different vectors. The invention also provides a viral or plasmid vector system as described herein.
[0028] Preferably, the vector is a viral vector, such as a lenti- or baculo- or preferably adeno- viral/adeno-associated viral vectors, but other means of delivery are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided. In some embodiments, one or more of the viral or plasmid vectors may be delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
[0029] By manipulation of a target sequence, Applicants also mean the epigenetic manipulation of a target sequence. This may be of the chromatin state of a target sequence, such as by modification of the methylation state of the target sequence (i.e. addition or removal of methylation or methylation patterns or CpG islands), histone modification, increasing or reducing accessibility to the target sequence, or by promoting 3D folding. In relation to treatment of viral infections, however, excision of integrated viral genome sequences is the manipulation of primary interest.
[0030] It will be appreciated that where reference is made to a method of modifying an organism or mammal including human or a non-human mammal or organism by manipulation of a target sequence in a genomic locus of interest, this may apply to the organism (or mammal) as a whole or just a single cell or population of cells from that organism. In the case of humans, for instance, Applicants envisage, inter alia, a single cell or a population of cells and these may preferably be modified ex vivo and then re-introduced. In this case, a biopsy or other tissue or biological fluid sample may be necessary. Stem cells are also particularly preferred in this regard. But, of course, in vivo embodiments are also envisaged.
[0031] In certain embodiments the invention provides a method of treating or inhibiting a condition caused by the presence of an integrated viral sequence in a genomic locus of interest in a subject (e.g., mammal or human) or a non-human subject (e.g., mammal) in need thereof comprising modifying the subject or a non-human subject by manipulation of a target sequence in the integrated viral sequence and wherein the condition is susceptible to treatment or inhibition by manipulation of the target sequence comprising providing treatment comprising: delivering a non-naturally occurring or engineered composition comprising an AAV or lentivirus vector system comprising one or more AAV or lentivirus vectors operably encoding a composition for expression thereof, wherein the target sequence is manipulated by the composition when expressed, wherein the composition comprises: (A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, (b) a tracr mate sequence, and (c) a tracr sequence, and II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences (or optionally at least one or more nuclear localization sequences as some embodiments can involve no NLS, i.e., there can be zero NLSs but advantageously there is greater than zero NLSs, such as one or more or advantageously two or more NLSs, and thus the invention comprehends embodiments wherein there is 0, 1, 2, 3, or more NLSs) wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation, wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, or (B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising I. a first regulatory element operably linked to (a) a guide sequence capable of hybridizing to a target sequence in a eukaryotic cell, and (b) at least one or more tracr mate sequences, II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence. In some embodiments, components I, II and III are located on the same vector. In other embodiments, components I and II are located on the same vector, while component III is located on another vector. In other embodiments, components I and III are located on the same vector, while component II is located on another vector. In other embodiments, components II and III are located on the same vector, while component I is located on another vector. In other embodiments, each of components I, II and III is located on different vectors. The invention also provides a viral (e.g. AAV or lentivirus) vector system as described herein. Delivery therefore can be via a vector, such as a viral vector, e.g., a recombinant viral vector delivery system; and, this system can be an AAV or lentivirus or derived from an AAV or a lentivirus (e.g., a recombinant AAV or lentivirus that expresses that which is foreign, heterologous or that which is not homologous or native to the virus may make some consider the virus“derived from” is parent virus). In some methods of the invention the viral vector is a lentivirus-derived vector. In some methods of the invention the viral vector is an Agrobacterium Ti or Ri plasmid for use in plants.
[0032] The organism or subject is a eukaryote (including mammal including human) or a non-human eukaryote or a non-human animal or a non-human mammal. In some embodiments, the organism or subject is a non-human animal, and may be an arthropod, for example, an insect, or may be a nematode. In some methods of the invention the organism or subject is a plant. In some methods of the invention the organism or subject is a mammal or a non-human mammal. A non-human mammal may be for example a rodent (preferably a mouse or a rat), an ungulate, or a primate. In some methods of the invention the organism or subject is a plant or algae, including microalgae, or is a fungus. In some methods of the invention the viral vector is an AAV or a lentivirus, and can be part of a vector system as described herein. In some methods of the invention the CRISPR enzyme is a Cas9. In some methods of the invention the expression of the guide sequence is under the control of the T7 promoter and is driven by the expression of T7 polymerase. In some methods of the invention the expression of the guide sequence is under the control of a U6 promoter. In some methods of the invention the CRISPR enzyme comprises one or more mutations in one of the catalytic domains. In some methods of the invention the CRISPR enzyme is a Cas9 nickase.
[0033] The invention in some embodiments comprehends a method of delivering a CRISPR enzyme comprising delivering to a cell a nucleic acid molecule, e.g., DNA, RNA, mRNA encoding the CRISPR enzyme. In some of these methods the CRISPR enzyme is a Cas9. This allows for the transient expression of the enzyme thereby reducing toxicity. In another embodiment, the Cas9 is delivered into the cell in the nucleotide construct that encodes and expresses the Cas9 enzyme.
[0034] The invention also provides methods of preparing the vector systems of the invention, in particular the viral vector systems as described herein. The invention in some embodiments comprehends a method of preparing the AAV of the invention comprising transfecting plasmid(s) containing or consisting essentially of nucleic acid molecule(s) coding for the AAV into AAV-infected cells, and supplying AAV rep and/or cap obligatory for replication and packaging of the AAV. In some embodiments the AAV rep and/or cap obligatory for replication and packaging of the AAV are supplied by transfecting the cells with helper plasmid(s) or helper virus(es). In some embodiments the helper virus is a poxvirus, adenovirus, herpesvirus or baculovirus. In some embodiments the poxvirus is a vaccinia virus. In some embodiments the cells are mammalian cells. And in some embodiments the cells are insect cells and the helper virus is baculovirus. In other embodiments, the virus is a lentivirus.
[0035] In plants, viral pathogens are often host-specific, but this is not always the case. For instance, citrus tristeza virus infects only a few species in the Citrus genus, whereas cucumber mosaic virus infects over 1000 species in 85 plant families..Plants have existing and induced defenses to resist most pathogens, but the invention offers new ways to clear viral infections from plants. [0036] The invention further comprehends a composition of the invention or a CRISPR enzyme thereof (including or alternatively mRNA encoding the CRISPR enzyme) for use in medicine or in therapy. In some embodiments the invention comprehends a composition according to the invention or a CRISPR enzyme thereof (including or alternatively a nucleic acid molecule, e.g., mRNA encoding the CRISPR enzyme) for use in a method according to the invention. In some embodiments the invention provides for the use of a composition of the invention or a CRISPR enzyme thereof (including or alternatively mRNA encoding the CRISPR enzyme) in ex vivo gene or genome editing. In certain embodiments the invention comprehends use of a composition of the invention or a CRISPR enzyme thereof (including or alternatively mRNA encoding the CRISPR enzyme) in the manufacture of a medicament for ex vivo gene or genome editing or for use in a method according of the invention. The invention thus also envisions a CRISPR-Cas Complex or a component thereof of any of any description herein for use in delivery to and/or a method of treating tissue, or tissue containing cells having a viral infection, such as Hepatitis B Virus; or in preparing or formulating a medicament or pharmaceutical composition for such treatment.
[0037] The invention comprehends in some embodiments a composition of the invention or a CRISPR enzyme thereof (including or alternatively mRNA encoding the CRISPR enzyme), wherein the target sequence is flanked at its 3’ end by a 5’ motif termed a proto-spacer adjacent motif or PAM, especially where the Cas9 is (or is derived from) S. pyogenes or S. aureus Cas9. For example, a suitable PAM is 5'-NRG or 5'-NNGRR (where N is any Nucleotide) for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively, as mentioned below. For S. pyrogenes Cas9 or derived enzymes, a suitable PAM is 5'-NRG.
[0038] It will be appreciated that SpCas9 or SaCas9 are those from or derived from S. pyogenes or S. aureus Cas9.
[0039] Apects of the invention comprehend improving the specificity of a CRISPR enzyme, e.g. Cas9, mediated gene targeting and reducing the likelihood of off-target modification by the CRISPR enzyme, e.g. Cas9. The invention in some embodiments comprehends a method of modifying an organism or a non-human organism by minimizing off-target modifications by manipulation of a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a cell comprising delivering a non-naturally occurring or engineered composition comprising:
[0040] I. a first CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the first polynucleotide sequence comprises: (a) a first guide sequence capable of hybridizing to the first target sequence,
(b) a first tracr mate sequence, and
(c) a first tracr sequence,
[0041] II. a second CRISPR-Cas system chiRNA polynucleotide sequence, wherein the second polynucleotide sequence comprises:
(a) a second guide sequence capable of hybridizing to the second target sequence, (b) a second tracr mate sequence, and
(c) a second tracr sequence, and
[0042] III. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences and comprising one or more mutations, wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation, wherein when transcribed, the first and the second tracr mate sequence hybridize to the first and second tracr sequence respectively and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively, wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized or hybridizable to the first target sequence, and (2) the first tracr mate sequence that is hybridized or hybridizable to the first tracr sequence, wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized or hybridizable to the second target sequence, and (2) the second tracr mate sequence that is hybridized or hybridizable to the second tracr sequence, wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of the other strand near the second target sequence inducing a double strand break, thereby modifying the organism or the non-human organism by minimizing off- target modifications. In one aspect, the first nick and the second nick in the DNA is offset relative to each other by at least one base pair of the duplex. In one aspect, the first nick and the second nick are offset relative to each other so that the resulting DNA break has a 3’ overhang. In one aspct, the first nick and the second nick are offset relative to each other so that the resulting DNA break has a 5’ overhang. In one aspect, the first nick and the second nick are positioned relative to each other such that the overhang is at least 1 nucleotide (nt), at least 10 nt, at least 15 nt, at least 26 nt, at least 30 nt, at least 50 nt or more that at least 50 nt. Additional aspects of the invention comprising the resulting offset double nicked DNA strand can be appreciated by one skilled in the art, and exemplary uses of the double nick system are provided herein.
[0043] In some methods of the invention any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA. In further embodiments of the invention the polynucleotides encoding the sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA and are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun. In certain embodiments of the invention, the first and second tracr mate sequence share 100% identity and/or the first and second tracr sequence share 100% identity. In some embodiments, the polynucleotides may be comprised within a vector system comprising one or more vectors. In preferred embodiments of the invention the CRISPR enzyme is a Cas9 enzyme, e.g. SpCas9. In an aspect of the invention the CRISPR enzyme comprises one or more mutations in a catalytic domain, wherein the one or more mutations are selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A. In a highly preferred embodiment the CRISPR enzyme has the D10A mutation. In preferred embodiments, the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme, and the second CRISPR enzyme has one or more mutations such that the enzyme is a non-complementary strand nicking enzyme. Alternatively the first enzyme may be a non-complementary strand nicking enzyme, and the second enzyme may be a complementary strand nicking enzyme.
[0044] With respect to mutations of the CRISPR enzyme, when the enzyme is not SpCas9, mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools). In particular, any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged. In an aspect the invention provides as to any or each or all embodiments herein-discussed wherein the CRISPR enzyme comprises at least one or more, or at least two or more mutations, wherein the at least one or more mutation or the at least two or more mutations is as to D10, E762, H840, N854, N863, or D986 according to SpCas9 protein, e.g., D10A, E762A, H840A, N854A, N863A and/or D986A as to SpCas9, or N580 according to SaCas9, e.g., N580A as to SaCas9, or any corresponding mutation(s) in a Cas9 of an ortholog to Sp or Sa, or the CRISPR enzyme comprises at least one mutation wherein at least H840 or N863A as to Sp Cas9 or N580A as to Sa Cas9 is mutated; e.g., wherein the CRISPR enzyme comprises H840A, or D10A and H840A, or D10A and N863A, according to SpCas9 protein, or any corresponding mutation(s) in a Cas9 of an ortholog to Sp protein or Sa protein.
[0045] In preferred methods of the invention the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of the other strand near the second target sequence results in a 5’ overhang. In embodiments of the invention the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs.
[0046] The invention in some embodiments comprehends a method of modifying an organism or a non-human organism by minimizing off-target modifications by manipulation of a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a cell comprising delivering a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
[0047] I. a first regulatory element operably linked to
(a) a first guide sequence capable of hybridizing to the first target sequence, and (b) at least one or more tracr mate sequences,
[0048] II. a second regulatory element operably linked to
(a) a second guide sequence capable of hybridizing to the second target sequence, and (b) at least one or more tracr mate sequences,
[0049] III. a third regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and
[0050] IV. a fourth regulatory element operably linked to a tracr sequence,
[0051] wherein components I, II, III and IV are located on the same or different vectors of the system, when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the first and the second guide sequence direct sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively, wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized or hybridizable to the first target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized or hybridizable to the second target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of the other strand near the second target sequence inducing a double strand break, thereby modifying the organism or the non-human organism by minimizing off-target modifications.
[0052] The invention also provides a vector system as described herein. The system may comprise one, two, three or four different vectors. Components I, II, III and IV may thus be located on one, two, three or four different vectors, and all combinations for possible locations of the components are herein envisaged, for example: components I, II, III and IV can be located on the same vector; components I, II, III and IV can each be located on different vectors; components I, II, II I and IV may be located on a total of two or three different vectors, with all combinations of locations envisaged, etc.
[0053] In some methods of the invention any or all of the polynucleotide sequence encoding the CRISPR enzyme, the first and the second guide sequence, the first and the second tracr mate sequence or the first and the second tracr sequence, is/are RNA. In further embodiments of the invention the first and second tracr mate sequence share 100% identity and/or the first and second tracr sequence share 100% identity. In preferred embodiments of the invention the CRISPR enzyme is a Cas9 enzyme, e.g. SpCas9. In an aspect of the invention the CRISPR enzyme comprises one or more mutations in a catalytic domain, wherein the one or more mutations are selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A. In a highly preferred embodiment the CRISPR enzyme has the D10A mutation. In preferred embodiments, the first CRISPR enzyme has one or more mutations such that the enzyme is a complementary strand nicking enzyme, and the second CRISPR enzyme has one or more mutations such that the enzyme is a non-complementary strand nicking enzyme. Alternatively the first enzyme may be a non-complementary strand nicking enzyme, and the second enzyme may be a complementary strand nicking enzyme. In a further embodiment of the invention, one or more of the viral vectors are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
[0054] In preferred methods of the invention the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a 5’ overhang. In embodiments of the invention the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs.
[0055] The invention in some embodiments comprehends a method of modifying a genomic locus of interest by minimizing off-target modifications by introducing into a cell containing and expressing a double stranded DNA molecule encoding a gene product of interest an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein having one or more mutations and two guide RNAs that target a first strand and a second strand of the DNA molecule respectively, whereby the guide RNAs target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the Cas protein and the two guide RNAs do not naturally occur together.
[0056] In preferred methods of the invention the Cas protein nicking each of the first strand and the second strand of the DNA molecule encoding the gene product results in a 5’ overhang. In embodiments of the invention the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs.
[0057] Embodiments of the invention also comprehend the guide RNAs comprising a guide sequence fused to a tracr mate sequence and a tracr sequence. In an aspect of the invention the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell. As explained in more detail below, codon usage can even be optimized for expression in particular cell types e.g. for liver cells. In further embodiments of the invention the Cas protein is a type II CRISPR-Cas protein, e.g. a Cas9 protein. In a highly preferred embodiment the Cas protein is a Cas9 protein, e.g. SpCas9. In aspects of the invention the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A. In a highly preferred embodiment the Cas protein has the D10A mutation.
[0058] Aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased. In an embodiment of the invention, the gene product is a protein. [0059] The invention also comprehends an engineered, non-naturally occurring CRISPR-Cas system comprising a Cas protein having one or more mutations and two guide RNAs that target a first strand and a second strand respectively of a double stranded DNA molecule encoding a gene product in a cell, whereby the guide RNAs target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the Cas protein and the two guide RNAs do not naturally occur together.
[0060] In aspects of the invention the guide RNAs may comprise a guide sequence fused to a tracr mate sequence and a tracr sequence. In an embodiment of the invention the Cas protein is a type II CRISPR-Cas protein. In an aspect of the invention the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell. In further embodiments of the invention the Cas protein is a type II CRISPR-Cas protein, e.g. a Cas9 protein. In a highly preferred embodiment the Cas protein is a Cas9 protein, e.g. SpCas9. In aspects of the invention the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A. In a highly preferred embodiment the Cas protein has the D10A mutation.
[0061] Aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased. In an embodiment of the invention, the gene product is a protein.
[0062] The invention also comprehends an engineered, non-naturally occurring vector system comprising one or more vectors comprising:
a) a first regulatory element operably linked to each of two CRISPR-Cas system guide RNAs that target a first strand and a second strand respectively of a double stranded DNA molecule encoding a gene product, b) a second regulatory element operably linked to a Cas protein,
wherein components (a) and (b) are located on same or different vectors of the system, whereby the guide RNAs target the DNA molecule encoding the gene product and the Cas protein nicks each of the first strand and the second strand of the DNA molecule encoding the gene product, whereby expression of the gene product is altered; and, wherein the Cas protein and the two guide RNAs do not naturally occur together. [0063] In aspects of the invention the guide RNAs may comprise a guide sequence fused to a tracr mate sequence and a tracr sequence. In an embodiment of the invention the Cas protein is a type II CRISPR-Cas protein. In an aspect of the invention the Cas protein is codon optimized for expression in a eukaryotic cell, preferably a mammalian cell or a human cell. In further embodiments of the invention the Cas protein is a type II CRISPR-Cas protein, e.g. a Cas9 protein. In a highly preferred embodiment the Cas protein is a Cas9 protein, e.g. SpCas9. In aspects of the invention the Cas protein has one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A and D986A. In a highly preferred embodiment the Cas protein has the D10A mutation.
[0064] Aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased. In an embodiment of the invention, the gene product is a protein. In preferred embodiments of the invention the vectors of the system are viral vectors. In a further embodiment, the vectors of the system are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
[0065] In one aspect, the invention provides a method of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. In some embodiments, said cleavage comprises cleaving one or two strands at the location of the target sequence by said CRISPR enzyme. In some embodiments, said cleavage results in decreased transcription of a target gene. In some embodiments, the method further comprises repairing said cleaved target polynucleotide by homologous recombination with an exogenous template polynucleotide, wherein said repair results in a mutation comprising an insertion, deletion, or substitution of one or more nucleotides of said target polynucleotide. In some embodiments, said mutation results in one or more amino acid changes in a protein expressed from a gene comprising the target sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cell, wherein the one or more vectors drive expression of one or more of: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence. In some embodiments, said vectors are delivered to the eukaryotic cell in a subject. In some embodiments, said modifying takes place in said eukaryotic cell in a cell culture. In some embodiments, the method further comprises isolating said eukaryotic cell from a subject prior to said modifying. In some embodiments, the method further comprises returning said eukaryotic cell and/or cells derived therefrom to said subject.
[0066] In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotic cells, wherein the one or more vectors drive expression of one or more of: the CRISPR enzyme, the guide sequence linked to the tracr mate sequence, and the tracr sequence.
[0067] In one aspect, the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
[0068] In other embodiments, this invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. The method comprises decreasing expression of a target polynucleotide by using a CRISPR complex that binds to the polynucleotide.
[0069] Where desired, to effect the modification of the expression in a cell, one or more vectors comprising a tracr sequence, a guide sequence linked to the tracr mate sequence, a sequence encoding a CRISPR enzyme is delivered to a cell. In some methods, the one or more vectors comprises a regulatory element operably linked to an enzyme-coding sequence encoding said CRISPR enzyme comprising a nuclear localization sequence; and a regulatory element operably linked to a tracr mate sequence and one or more insertion sites for inserting a guide sequence upstream of the tracr mate sequence. When expressed, the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a cell. Typically, the CRISPR complex comprises a CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence.
[0070] In some methods, a target polynucleotide can be inactivated to effect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild-type sequence does. For example, a protein or microRNA coding sequence may be inactivated such that the protein or microRNA is not produced.
[0071] In certain embodiments, the CRISPR enzyme comprises one or more mutations selected from the group consisting of D10A, E762A, H840A, N854A, N863A or D986A and/or the one or more mutations is in a RuvC1 or HNH domain of the CRISPR enzyme or is a mutation as otherwise as discussed herein. In some embodiments, the CRISPR enzyme has one or more mutations in a catalytic domain, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target sequence, and wherein the enzyme further comprises a functional domain. In some embodiments, the functional domain is a transcriptional activation domain, preferably VP64. In some embodiments, the functional domain is a transcription repression domain, preferably KRAB. In some embodiments, the transcription repression domain is SID, or concatemers of SID (eg SID4X). In some embodiments, the functional domain is an epigenetic modifying domain, such that an epigenetic modifying enzyme is provided. In some embodiments, the functional domain is an activation domain, which may be the P65 activation domain. Thus, in some embodiments a mutated Cas9 enzyme may be fused to a protein domain or functional domain.
[0072] In some embodiments, the CRISPR enzyme is a type I or III CRISPR enzyme, but is preferably a type II CRISPR enzyme. This type II CRISPR enzyme may be any Cas enzyme. A Cas enzyme may be identified as Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the Cas9 enzyme is from, or is derived from, spCas9 or saCas9. By derived, Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as described herein. [0073] It will be appreciated that the terms Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent. As mentioned above, many of the residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes. However, it will be appreciated that this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCa9, St1Cas9 and so forth.
[0074] An example of a codon optimized sequence, in this instance optimized for humans (i.e. being optimized for expression in humans) is provided herein, see the SaCas9 human codon optimized sequence. Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species is known.
[0075] Preferably, delivery is in the form of a vector which may be a viral vector, such as a lenti- or baculo- or preferably adeno-viral/adeno-associated viral vectors, but other means of delivery are known (such as yeast systems, microvesicles, gene guns/means of attaching vectors to gold nanoparticles) and are provided. A vector may mean not only a viral or yeast system (for instance, where the nucleic acids of interest may be operably linked to and under the control of (in terms of expression, such as to ultimately provide a processed RNA) a promoter), but also direct delivery of nucleic acids into a host cell. While in herein methods the vector may be a viral vector and this is advantageously an AAV, other viral vectors as herein discussed can be employed, such as lentivirus. For example, baculoviruses may be used for expression in insect cells. These insect cells may, in turn be useful for producing large quantities of further vectors, such as AAV or lentivirus vectors adapted for delivery of the present invention. Also envisaged is a method of delivering the present CRISPR enzyme comprising delivering to a cell mRNA encoding the CRISPR enzyme. It will be appreciated that in certain embodiments the CRISPR enzyme is truncated, and/or comprised of less than one thousand amino acids or less than four thousand amino acids, and/or is a nuclease or nickase, and/or is codon-optimized, and/or comprises one or more mutations, and/or comprises a chimeric CRISPR enzyme, and/or the other options as herein discussed. AAV and lentiviral vectors are preferred.
[0076] In certain embodiments, the target sequence is flanked or followed, at its 3’ end, by a PAM suitable for the CRISPR enzyme, typically a Cas and in particular a Cas9.
[0077] For example, a suitable PAM is 5'-NRG or 5'-NNGRR for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively.
[0078] The invention also relates to a method of modifying a cell of a eukaryotic organism by manipulating at least one target viral nucleic acid within the cell, the method comprising introducing into the cell an exogenous composition capable of forming a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex, the composition comprising:
(A) CRISPR-Cas system polynucleotide sequences comprising:
(i) a guide sequence, which when transcribed is capable of hybridizing to a sequence of the at least one target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing to the tracr mate sequence; and (B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme, wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
(i) the tracr mate sequence is hybridized to the tracr sequence or portion thereof;
(ii) the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0079] The invention also relates to an exogenous composition which, when introduced into a cell of a eukaryotic organism, is capable of forming at least one Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex, wherein the complex is capable of modifying the cell by manipulating of at least one target viral nucleic acid within the cell, the composition comprising:
(A) Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR associated (Cas) (CRISPR-Cas) system polynucleotide sequences comprising:
(i) a guide sequence, which when transcribed is capable of hybridizing to a sequence of the at least one target viral nucleic acid to be manipulated; (ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and (B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme, wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
(i) the tracr mate sequence is hybridized to the tracr sequence or portion thereof;
(ii) the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0080] The invention also relates to a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex which, when introduced into a cell of a eukaryotic organism, is capable of modifying the cell by manipulating a target viral nucleic acid within the cell, the complex comprising:
(A) CRISPR-Cas system RNA polynucleotide sequences comprising:
(i) a guide sequence, which is capable of hybridizing to a sequence of the target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
(B) a CRISPR/Cas enzyme,
wherein when the CRISPR/Cas system RNA polynucleotide sequences and the CRISPR/Cas enzyme are present within the cell:
(i) the tracr mate sequence is hybridized to the tracr sequence or portion thereof; (ii) the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0081] The invention also relates to a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA) which, when introduced into a cell of a eukaryotic organism, is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex, wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell; the chiRNA comprising:
(i) a guide sequence, which is capable of hybridizing to a sequence of the target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
wherein when the chiRNA and the CRISPR/Cas enzyme are present within the cell:
a) the tracr mate sequence hybridizes to the tracr sequence or portion thereof; b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex; and
c) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0082] The invention also relates to a DNA polynucleotide molecule comprising sequences encoding a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR- associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA), wherein upon introduction of said chiRNA into a cell of a eukaryotic organism said chiRNA is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell; the chiRNA comprising:
(i) a guide sequence, which is capable of hybridizing to a sequence of the target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
wherein when the chiRNA and the CRISPR/Cas enzyme are present within the cell:
a) the tracr mate sequence hybridizes to the tracr sequence or portion thereof; b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex; and
c) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0083] In embodiments described herein the guide sequence as described herein, which is capable of hybridizing to a sequence of a target nucleic acid to be manipulated, and the tracr mate sequence, as described herein, may preferably be linked in a tandem arrangement, wherein the tracr mate sequence comprises a region of sense sequence. The tracr sequence, as described herein, may comprise a region of antisense sequence which is capable of hybridizing with the region of sense sequence of the tracr mate sequence.
[0084] Preferably, when the tracr mate sequence (linked to the guide sequence) and the tracr sequence are present within the cell, the region of antisense sequence is hybridized to the region of sense sequence thereby forming a dual RNA molecule; and wherein when said dual RNA molecule binds within the cell to the CRISPR/Cas enzyme so forming a CRISPR-Cas complex, the guide sequence hybridizes to a sequence of the target nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the target nucleic acid, whereupon manipulation of said sequence of said target nucleic acid is effected by the CRISPR/Cas enzyme of the complex. [0085] As described herein certain embodiments may optionally comprise a chimeric single guide RNA molecule (sgRNA). Such sgRNA molecules may preferably comprise, in a tandem arrangement:
I. a guide sequence, as described herein, which is capable of hybridizing to a sequence of the target nucleic acid to be manipulated;
II. a tracr mate sequence, as described herein, comprising a region of sense sequence;
III. a linker sequence; and
IV. a tracr sequence, as described herein, comprising a region of antisense sequence which is positioned adjacent the linker sequence and which is capable of hybridizing with the region of sense sequence thereby forming a stem-loop.
[0086] In such cases the linker may be a polynucleotide linker, optionally comprising GAAA. Other linkers, such as those described herein, are envisaged. Preferably, in any such cases when the sgRNA molecule is present within the cell, the region of antisense sequence is hybridized to the region of sense sequence thereby forming the stem-loop; and wherein when said sgRNA molecule binds within the cell to the CRISPR/Cas enzyme so forming a CRISPR- Cas complex, the guide sequence hybridizes to a sequence of the target nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the target nucleic acid, whereupon manipulation of said sequence of said target nucleic acid is effected by the CRISPR/Cas enzyme of the complex.
[0087] In the embodiments described herein the guide sequence, the trans-activating CRISPR RNA (tracr) mate sequence and the tracr sequence may be non-coding sequences. When transcribed, the tracr mate sequence may not be capable of hybridizing to a sequence of the at least one target viral nucleic acid to be manipulated, in contrast to the guide sequence.
[0088] As described herein, various manipulations of target viral nucleic acids may be performed by the CRISPR/Cas complexes described herein. Preferred manipulations of target viral nucleic acids include cleavage of viral DNA, as described in more detail herein.
[0089] In certain methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein, manipulation of at least one target viral nucleic acid within the cell is performed. However, as will be apparent, multiplexing methods are described and exemplified wherein multiple target viral nucleic acid sequences are manipulated by CRISPR/Cas complexes targeting different sequences of the target viral nucleic acid. [0090] In the methods, compositions, complexes, chiRNAs or DNA polynucleotides described herein an“exogenous composition” is an engineered or non-naturally occurring composition.
[0091] As noted herein, the target sequence may be flanked or followed, at its 3’ end, by a protospacer adjacent motif (PAM) suitable for recognition by the CRISPR enzyme of the complex, typically a Cas enzyme and more typically a Cas9 enzyme. For example, a suitable PAM is 5'-NRG or 5'-NNGRR for SpCas9 or SaCas9 enzymes (or derived enzymes), respectively. Other PAMs, such as are described herein, may be recognized in association with the target sequence depending upon the specific CRISPR enzyme used.
[0092] The CRISPR/Cas enzyme of the exogenous composition may be provided as a polynucleotide sequence which comprises either (a) RNA or (b) DNA wherein the polynucleotide sequence is operably linked to a regulatory element capable of directing expression of RNA encoding the CRISPR/Cas enzyme.
[0093] Any of the CRISPR/Cas system polynucleotide sequences of the exogenous composition may comprise either (a) RNA or (b) DNA wherein the polynucleotide sequences are operably linked to one or more regulatory elements capable of directing expression of CRISPR/Cas system RNA polynucleotide sequences.
[0094] Each of the CRISPR/Cas system polynucleotide sequences of the exogenous composition may consist of RNA and wherein the CRISPR/Cas system polynucleotide sequences may comprise a chimeric RNA polynucleotide molecule comprising the guide sequence, the tracr mate sequence and the tracr sequence.
[0095] Each of the CRISPR/Cas system polynucleotide sequences of the exogenous composition may be provided as DNA polynucleotide sequences further comprising at least one regulatory element operably linked to polynucleotide sequences encoding CRISPR/Cas system RNA polynucleotide sequences and capable of directing expression thereof, and wherein the CRISPR/Cas system RNA polynucleotide sequences may comprise a chimeric RNA polynucleotide (chiRNA) molecule comprising the guide sequence, the tracr mate sequence and the tracr sequence.
[0096] In the above-described methods, compositions, complexes, chiRNA or DNA polynucleotide molecules each of the guide sequence, the tracr mate sequences and the tracr sequence may be arranged in a 5’ to 3’ orientation; or each of the guide sequence, the tracr mate sequences and the tracr sequence may be arranged in a 3’ to 5’ orientation. [0097] In the methods or compositions described herein (a) the CRISPR/Cas system polynucleotide sequences or polynucleotide sequences encoding the CRISPR/Cas system polynucleotide sequences and/or (b) polynucleotide sequences encoding the CRISPR/Cas enzyme may be comprised in one or more recombinant viral vectors. The polynucleotide sequences of (a) may be located on the same or different recombinant viral vector as polynucleotide sequences of (b).
[0098] The chiRNAs or the DNA polynucleotide molecules described herein may be comprised in a recombinant viral vector.
[0099] In the methods, compositions, complexes, chiRNAs or DNA polynucleotides described herein which utilize viral vectors, the viral vector may be a retroviral vector, optionally a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adenoassociated viral (AAV) vector such as AAV8 vector, or a poxvirus such as a vaccinia virus.
[0100] In the methods described herein (a) the CRISPR/Cas system polynucleotide sequences or polynucleotide sequences encoding the CRISPR/Cas system polynucleotide sequences and/or (b) polynucleotide sequences encoding the CRISPR/Cas enzyme may be delivered to the cell of the organism via liposomes, nanoparticles, exosomes, microvesicles or a gene-gun.
[0101] In the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein the tracr sequence may be 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length.
[0102] In the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein hybridization between the tracr sequence and the tracr mate sequence may produce a transcript having secondary structure, preferably a hairpin. The tracr sequence may comprise one or more regions capable of forming secondary structure, preferably a hairpin. The tracr sequence may comprise one or more hairpins, two or more hairpins, three or more hairpins, four or more hairpins, five or more hairpins, or at most five hairpins.
[0103] In some embodiments it may be preferred in a CRISPR complex that the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme.
[0104] As described herein, preferred CRISPR/Cas enzymes are Type II CRISPR/Cas enzymes, preferably Type II Cas9 CRISPR/Cas enzymes or biologically active fragments or derivatives thereof. [0105] In the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein, the guide sequence may be 10 to 30 nucleotides in length.
[0106] In any of the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein the CRISPR/Cas enzyme may be a Cas9 enzyme of Streptococcus pyogenes or a Cas9 enzyme of Streptococcus aureus, or a biologically active fragment or derivative thereof. Described herein are specific NLS sequences which may be applied to the CRISPR/Cas enzymes.
[0107] In any of the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein the CRISPR/Cas enzyme may further comprise one or more nuclear localization sequences (NLSs) capable of driving the accumulation of the CRISPR/Cas enzyme to a detectible amount in the nucleus of the cell of the organism. The CRISPR/Cas enzyme may comprise two or more NLSs, three or more NLSs, four or more NLSs, five or more NLSs, six or more NLSs, seven or more NLSs, eight or more NLSs, nine or more NLSs, or ten or more NLSs. The CRISPR/Cas enzyme may comprise at least one NLS at or near the amino-terminus of the CRISPR/Cas enzyme and/or at least one NLS at or near the carboxy-terminus the CRISPR/Cas enzyme.
[0108] In any of the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein when present as RNA within the cell the guide sequence may be capable of hybridizing to a sequence of a target viral nucleic acid which is an episomal nucleic acid molecule which is not integrated into the genome of the organism and wherein said manipulation is a manipulation of the episomal viral nucleic acid molecule, preferably wherein the episomal nucleic acid molecule is a double-stranded DNA polynucleotide molecule. The double-stranded DNA polynucleotide may be an episomal viral nucleic acid which is a covalently closed circular DNA (cccDNA). Where the target viral nucleic acid is a hepatitis B virus (HBV) nucleic acid, the double-stranded DNA polynucleotide may preferably be an episomal viral nucleic acid which is a cccDNA.
[0109] In any of the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein the CRISPR/Cas complex may be capable of reducing the amount of episomal viral nucleic acid molecule in a cell of the organism compared to the amount of episomal viral nucleic acid molecule in a cell of the organism in the absence of providing the complex. [0110] In any of the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein the CRISPR/Cas complex may be capable of manipulating the episomal nucleic acid molecule to promote degradation of the episomal nucleic acid molecule.
[0111] In any of the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein, when present as RNA within in the cell the guide sequence may be capable of hybridizing to a sequence of the target viral nucleic acid which is integrated into the genome of the organism and wherein said manipulation is a manipulation of the integrated target nucleic acid.
[0112] In any of the methods, compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein, when formed within the cell the CRISPR/Cas complex may be capable of manipulating the integrated nucleic acid to promote excision of all or part of the target viral nucleic acid from the genome of the organism.
[0113] Any of the compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be used in the manipulation of at least one target viral nucleic acid within the cell of a eukaryotic organism as described herein. Such a use may be in vitro and ex vivo.
[0114] In any of the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein at least one target viral nucleic acid may be comprised in a double-stranded DNA molecule polynucleotide cccDNA and/or viral DNA integrated into the genome of the organism, in which case the manipulation of the at least one target viral nucleic acid by the CRISPR-Cas complex comprises cleavage of viral cccDNA and/or integrated viral DNA. The cleavage may comprise one or more double-strand break(s) introduced into the viral cccDNA and/or integrated viral DNA, optionally at least two double-strand break(s). The cleavage may comprise one or more single-strand break(s) introduced into the viral cccDNA and/or integrated viral DNA, optionally at least two single-strand break(s).
[0115] In any of the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein, one or more double-strand break(s) and/or one or more single-strand break(s) may lead to the formation of one or more insertion and deletion mutations (INDELs) in the target viral cccDNA sequences and/or target integrated viral DNA sequences. The presence of INDELs may be assessed by SURVEYOR assay as described herein.
[0116] In any of the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein, cleavage of the viral cccDNA sequences or viral DNA sequences integrated in the genome of the organism may lead to excision of viral polynucleotide sequences from the cccDNA thereby reducing viral infection or excision of viral DNA sequences from the genome of the organism thereby reducing viral infection.
[0117] In methods or compositions or other embodiments of the invention which promote the formation of double-strand breaks the composition may comprise components of at least two types of CRISPR/Cas complex, wherein each type of complex comprises a guide sequence capable of hybridizing to different sequences of the target nucleic acid, wherein said cleavage is cleavage of first and second strands of the viral DNA via at least two double-strand breaks introduced into the viral cccDNA and/or into or adjacent viral DNA integrated into the genome of the organism;
wherein a first double-strand break is introduced at a first position of the DNA by manipulating a first target sequence and a second double-strand break is introduced at a second position of the DNA by manipulating a second target sequence;
wherein upon introduction of first and second double-strand breaks viral sequences between first and second double-strand breaks are excised from cccDNA and/or from the genomic DNA of the organism.
[0118] In methods or compositions or other embodiments of the invention which promote the formation of single-strand breaks, the composition may comprise components of at least four types of CRISPR/Cas complex, wherein each type of complex comprises a guide sequence capable of hybridizing to different sequences of the target nucleic acid, wherein said cleavage is via at least two pairs of single-strand breaks introduced into the viral cccDNA and/or introduced into or adjacent viral DNA integrated into the genome of the organism;
wherein to introduce a first pair of single-strand breaks a first single-strand break is introduced into a first strand of DNA by manipulating a first target sequence to create a first nick and a second single-strand break is introduced into the opposite strand of DNA by manipulating a second target sequence to create a second nick;
wherein to introduce a second pair of single-strand breaks a third single-strand break is introduced into said first strand of DNA by manipulating a third target sequence to create a third nick and a fourth single-strand break is introduced into said opposite strand of DNA by manipulating a fourth target sequence to create a fourth nick; wherein upon introduction of first and second pairs of single-strand breaks viral sequences between first and second pairs of single-strand breaks are excised from cccDNA and/or from the genomic DNA of the organism.
[0119] The first and second nicks may be offset relative to each other by at least one base pair of the duplex creating a first overhang, and wherein third and fourth nicks are offset relative to each other by at least one base pair of the duplex creating a second overhang. Following excision of viral sequences the ends of the cleaved first strand of DNA may be ligated together and the ends of the cleaved second strand of DNA may be ligated together thus reforming unbroken first and second strands.
[0120] In methods or compositions or other embodiments of the invention which promote the formation of single-strand breaks the single-strand break(s) may be introduced into DNA by a nickase enzyme which is a modified Cas9 enzyme comprising a substitution leading to catalytic inactivation of the HNH nuclease domain or the RuvC nuclease domain of Cas9; optionally wherein the substitution is at position D10 of SpCas9, preferably a D10A substitution or substitution of a residue corresponding to position D10 in a SpCas9-related enzyme, or wherein the substitution is at position H840 of SpCas9, preferably a H840A substitution or substitution of a residue corresponding to position H840 in a SpCas9-related enzyme.
[0121] In any of the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein, target viral nucleic acid may be cccDNA and/or viral DNA integrated into the genome of the organism and wherein said manipulation comprises insertion of one or more nucleotides into or adjacent viral cccDNA sequences or into or adjacent integrated viral DNA sequences, deletion of one or more nucleotides of viral cccDNA or of integrated viral DNA, translocation of viral cccDNA sequences or of integrated viral DNA sequences, repression of transcription of viral cccDNA sequences or of integrated viral DNA sequences, and/or inactivation of viral cccDNA sequences or of integrated viral DNA sequences. Repression of transcription of viral cccDNA sequences and/or integrated viral DNA sequences may be effected by the action of a CRISPR-Cas system comprising a CRISPR enzyme fused to one or more transcriptional repressor domains, optionally wherein the one or more transcriptional repressor domains comprises KRAB, SID and/or SID4X, preferably wherein the CRISPR enzyme is a Cas9 enzyme. The manipulation may comprise activation of genes carried by viral cccDNA, e.g. by the action of a CRISPR-Cas system comprising a deactivated CRISPR enzyme fused to one or more transcriptional activation domains such as VP64, preferably the viral cccDNA is HBV and activation results in increased activity of APOBEC3A and/or APOBEC3B, and or other viral interferon-stimulated genes (ISGs), thereby leading to a reduction in HBV cccDNA. Manipulation of nucleotide sequences of viral cccDNA or integrated viral DNA may lead to disruption of one or more viral open reading frames, disruption of viral mRNA expression and/or inhibition of the production of functional virions.
[0122] In any of the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein, manipulation of said viral cccDNA may lead to a reduction in the level of one or more of viral rcDNA, viral cccDNA and viral ssDNA compared to the level in the absence of the CRISPR/Cas complex.
[0123] In any of the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein, the effect of said manipulation may comprise inhibiting the production of new virions.
[0124] In any of the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein, the effect of said modifying may comprise removing viral sequences from said organism thereby reducing viral infection.
[0125] In any of the methods and compositions described herein, the described composition may further comprises components of one or more additional CRISPR/Cas complexes, or components required for the assembly of one or more additional CRISPR/Cas complexes, wherein each type of complex comprises a different guide sequence capable hybridizing to a different sequence of the target nucleic acid within the cell. Thus, any of the methods and compositions described herein may be additionally characterized by one or more additional CRISPR/Cas complexes each of which one or more additional CRISPR/Cas complexes may be characterized as described herein.
[0126] In any of the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein, the target viral nucleic acid may be a hepatitis B virus (HBV) nucleic acid. Where the target viral nucleic acid is a hepatitis B virus (HBV) nucleic acid, the cell of the organism is a cell capable of being infected by HBV. The cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP). The cell may be a hepatocyte, preferably a primary hepatocyte, more preferably a human hepatocyte or a human primary hepatocyte, a HepG2.2.15 or a HepG2-hNTCP cell.
[0127] When the target viral nucleic acid is a hepatitis B virus (HBV) nucleic acid the guide sequence may be capable of hybridizing with target viral nucleic acids of HBV ORF S, ORF C, ORF P, or ORF X, preferably ORF C, optionally wherein the sequence of the guide sequence comprises 5'-gggcgcacctctctttacg-3', 5'-cctctgccgatccatactg-3' or 5’-taaagaatttggagctactg-3’. [0128] In the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein, the target viral nucleic acid may be a human papillomavirus (HPV) nucleic acid, an Epstein Barr virus (EBV) nucleic acid, a herpes simplex virus (HSV) nucleic acid, or a varicella zoster virus (VZV) nucleic acid.
[0129] In any of the methods, compositions, complexes, chiRNAs, DNA polynucleotide molecules or uses described herein, said manipulation may be performed in vitro or ex vivo.
[0130] Any of the compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use as a medicament.
[0131] Any of the compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use in the treatment of a viral infection. Such a treatment may be of a viral infection wherein target viral sequences are comprised in an episomal nucleic acid molecule which is not integrated into the genome of the organism, such as a covalently closed circular DNA (cccDNA). The viral infection may caused by hepatitis B virus (HBV), human papillomavirus (HPV), Epstein Barr virus (EBV), herpes simplex virus (HSV) or varicella zoster virus (VZV).
[0132] Any of the compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use as a medicament or for use in the treatment of a viral infection wherein the organism is a mammal such as a human.
[0133] Any of the compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use in the manufacture of a medicament.
[0134] Any of the compositions, complexes, chiRNAs or DNA polynucleotide molecules described herein may be described for use in the manufacture of a medicament for the treatment of a viral infection. Such a treatment may be of a viral infection wherein target viral sequences are comprised in an episomal nucleic acid molecule which is not integrated into the genome of the organism, such as a covalently closed circular DNA (cccDNA). The viral infection may be caused by hepatitis B virus (HBV) or the viral infection may be caused by human papillomavirus (HPV), Epstein Barr virus (EBV), herpes simplex virus (HSV), or varicella zoster virus (VZV). In any such uses the organism may be a mammal such as a human.
[0135] The invention also relates to a method of modifying a cell, of a eukaryotic organism by manipulating at least one target viral nucleic acid within the cell, the method comprising introducing into the cell an exogenous composition capable of forming a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex, the composition comprising: (A) CRISPR-Cas system polynucleotide sequences comprising:
(i) a guide sequence, which when transcribed is capable of hybridizing to a sequence of the at least one target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing to the tracr mate sequence; and (B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme, wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
(i) the tracr mate sequence is hybridized to the tracr sequence or portion thereof;
(ii) the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0136] In relation to such a method the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA). Such a method may be additionally characterized, as appropriate, in accordance with any of the further and particular features as described herein.
[0137] The invention also relates to an exogenous composition which, when introduced into a cell of a eukaryotic organism, is capable of forming at least one Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex, wherein the complex is capable of modifying the cell by manipulating at least one target viral nucleic acid within the cell, the composition comprising:
(A) Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR associated (Cas) (CRISPR-Cas) system polynucleotide sequences comprising:
(i) a guide sequence, which when transcribed is capable of hybridizing to a sequence of the at least one target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and (B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme, wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
(i) the tracr mate sequence is hybridized to the tracr sequence or portion thereof;
(ii) the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0138] In relation to such a composition the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA). Such a composition may be additionally characterized, as appropriate, in accordance with any of the further and particular features as described herein. [0139] The invention also relates to a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex which, when introduced into a cell of a eukaryotic organism, is capable of modifying the cell by manipulating a target viral nucleic acid within the cell, the complex comprising:
(A) CRISPR-Cas system RNA polynucleotide sequences comprising:
(i) a guide sequence, which is capable of hybridizing to a sequence of the target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
(B) a CRISPR/Cas enzyme,
wherein when the CRISPR/Cas system RNA polynucleotide sequences and the CRISPR/Cas enzyme are present within the cell:
(i) the tracr mate sequence is hybridized to the tracr sequence or portion thereof;
(ii) the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and (iii) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0140] In relation to such a complex the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA). Such a complex may be additionally characterized, as appropriate, in accordance with any of the further and particular features as described herein. [0141] The invention also relates to a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA) which, when introduced into a cell of a eukaryotic organism, is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex, wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell; the chiRNA comprising:
(i) a guide sequence, which is capable of hybridizing to a sequence of the target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
wherein when the chiRNA and the CRISPR/Cas enzyme are present within the cell:
a) the tracr mate sequence hybridizes to the tracr sequence or portion thereof; b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex; and
c) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0142] In relation to such a chiRNA, the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA). Such a chiRNA may be additionally characterized, as appropriate, in accordance with any of the further and particular features as described herein.
[0143] The invention also relates to a DNA polynucleotide molecule comprising sequences encoding a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR- associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA) wherein upon introduction of said chiRNA into a cell of a eukaryotic organism said chiRNA is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex, wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell; the chiRNA comprising:
(i) a guide sequence, which is capable of hybridizing to a sequence of the target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
wherein when the chiRNA and the CRISPR/Cas enzyme are present within the cell:
a) the tracr mate sequence hybridizes to the tracr sequence or portion thereof; b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex; and
c) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
[0144] In relation to such a DNA, the cell may be a cell which expresses the sodium taurocholate cotransporting polypeptide (NTCP), preferably the cell may be a hepatocyte; the tracr sequence has one or more hairpins and is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length; the guide sequence is between 10 to 30 nucleotides in length, the CRISPR/Cas enzyme is a Type II Cas9 enzyme; the target viral nucleic acid is comprised in an HBV episomal nucleic acid which is not integrated into the genome of the organism and is an HBV double-stranded covalently closed circular DNA (cccDNA). Such a DNA may be additionally characterized, as appropriate, in accordance with any of the further and particular features as described herein.
[0145] Certain methods, products and uses described herein may not be applied in situations which result in the destruction of a human embryo and in situations which result in the modification of the germ line genetic identity of humans. Methods, products and uses described herein may be used for non-therapeutic purposes. Furthermore, any of the methods described herein may be applied in vitro and ex vivo [0146] It will be appreciated that the invention described herein involves various components which may display variations in their specific characteristics. It will be appreciated that any combination of features described above and herein, as appropriate, are contemplated as a means for implementing the invention.
[0147] Furthermore, it is an object of the invention to not encompass within the invention any previously known product, process of making the product, or method of using the product such that Applicants reserve the right and hereby disclose a disclaimer of any previously known product, process, or method. It is further noted that the invention does not intend to encompass within the scope of the invention any product, process, or making of the product or method of using the product, which does not meet the written description and enablement requirements of the USPTO (35 U.S.C. §112, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any such cubject matter.
[0148] It is noted that in this disclosure and particularly in the claims and/or paragraphs, terms such as“comprises”,“comprised”,“comprising” and the like can have the meaning attributed to it in U.S. Patent law; e.g., they can mean“includes”,“included”,“including”, and the like; and that terms such as“consisting essentially of” and“consists essentially of” have the meaning ascribed to them in U.S. Patent law, e.g., they allow for elements not explicitly recited, but exclude elements that are found in the prior art or that affect a basic or novel characteristic of the invention. It may be advantageous in the practice of the invention to be in compliance with Art. 53(c) EPC and Rule 28(b) and (c) EPC. Nothing herein is intended as a promise.
[0149] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.
BRIEF DESCRIPTION OF THE DRAWINGS
[0150] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:
[0151] Figure 1 shows a schematic model of the CRISPR system. The Cas9 nuclease from Streptococcus pyogenes (yellow) is targeted to genomic DNA by a synthetic guide RNA (sgRNA) consisting of a 20-nt guide sequence (blue) and a scaffold (red). The guide sequence base-pairs with the DNA target (blue), directly upstream of a requisite 5’-NGG protospacer adjacent motif (PAM; magenta), and Cas9 mediates a double-stranded break (DSB) ~3 bp upstream of the PAM (red triangle).
[0152] Figure 2A-F shows an exemplary CRISPR system, a possible mechanism of action, an example adaptation for expression in eukaryotic cells, and results of tests assessing nuclear localization and CRISPR activity.
[0153] Figure 3A-D shows results of an evaluation of SpCas9 specificity for an example target.
[0154] Figure 4A-G show an exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells.
[0155] Figure 5 provides a table of protospacer sequences and summarizes modification efficiency results for protospacer targets designed based on exemplary S. pyogenes and S. thermophilus CRISPR systems with corresponding PAMs against loci in human and mouse genomes. Cells were transfected with Cas9 and either pre-crRNA/tracrRNA or chimeric RNA, and analyzed 72 hours after transfection. Percent indels are calculated based on Surveyor assay results from indicated cell lines (N=3 for all protospacer targets, errors are S.E.M., N.D. indicates not detectable using the Surveyor assay, and N.T. indicates not tested in this study).
[0156] Figure 6A-C shows a comparison of different tracrRNA transcripts for Cas9- mediated gene targeting.
[0157] Figure 7 shows a schematic of a surveyor nuclease assay for detection of double strand break-induced micro-insertions and–deletions.
[0158] Figure 8A-B shows exemplary bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cells.
[0159] Figure 9A-C shows histograms of distances between adjacent S. pyogenes SF370 locus 1 PAM (NGG) (Figure 9A) and S. thermophilus LMD9 locus 2 PAM (NNAGAAW) (Figure 9B) in the human genome; and distances for each PAM by chromosome (Chr) (Figure 9C).
[0160] Figure 10A-D shows an exemplary CRISPR system, an example adaptation for expression in eukaryotic cells, and results of tests assessing CRISPR activity.
[0161] Figure 11A-C shows exemplary manipulations of a CRISPR system for targeting of genomic loci in mammalian cells.
[0162] Figure 12A-B shows the results of a Northern blot analysis of crRNA processing in mammalian cells. [0163] Figure 13A-B shows an exemplary selection of protospacers in the human PVALB and mouse Th loci.
[0164] Figure 14 shows example protospacer and corresponding PAM sequence targets of the S. thermophilus CRISPR system in the human EMX1 locus.
[0165] Figure 15 provides a table of sequences for primers and probes used for Surveyor, RFLP, genomic sequencing, and Northern blot assays.
[0166] Figure 16A-C shows exemplary manipulation of a CRISPR system with chimeric RNAs and results of SURVEYOR assays for system activity in eukaryotic cells.
[0167] Figure 17A-B shows a graphical representation of the results of SURVEYOR assays for CRISPR system activity in eukaryotic cells.
[0168] Figure 18 shows an exemplary visualization of some S. pyogenes Cas9 target sites in the human genome using the UCSC genome browser.
[0169] Figure 19A-D shows a circular depiction of the phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (~1400 amino acids) and two of small Cas9s (~1100 amino acids).
[0170] Figure 20A-F shows the linear depiction of the phylogenetic analysis revealing five families of Cas9s, including three groups of large Cas9s (~1400 amino acids) and two of small Cas9s (~1100 amino acids).
[0171] Figure 21A-D shows genome editing via homologous recombination. (a) Schematic of SpCas9 nickase, with D10A mutation in the RuvC I catalytic domain. (b) Schematic representing homologous recombination (HR) at the human EMX1 locus using either sense or antisense single stranded oligonucleotides as repair templates. Red arrow above indicates sgRNA cleavage site; PCR primers for genotyping (Tables J and K) are indicated as arrows in right panel. (c) Sequence of region modified by HR. d, SURVEYOR assay for wildtype (wt) and nickase (D10A) SpCas9-mediated indels at the EMX1 target 1 locus (n=3). Arrows indicate positions of expected fragment sizes.
[0172] Figure 22A-B shows single vector designs for SpCas9.
[0173] Figure 23 shows a graph representing the length distribution of Cas9 orthologs.
[0174] Figure 24A-M shows sequences where the mutation points are located within the SpCas9 gene.
[0175] Figure 25A shows the Conditional Cas9, Rosa26 targeting vector map.
[0176] Figure 25B shows the Constitutive Cas9, Rosa26 targeting vector map. [0177] Figure 26 shows a schematic of the important elements in the Constitutive and Conditional Cas9 constructs.
[0178] Figure 27 shows delivery and in vivo mouse brain Cas9 expression data.
[0179] Figure 28 shows RNA delivery of Cas9 and chimeric RNA into cells (A) Delivery of a GFP reporter as either DNA or mRNA into Neuro-2A cells. (B) Delivery of Cas9 and chimeric RNA against the Icam2 gene as RNA results in cutting for one of two spacers tested. (C) Delivery of Cas9 and chimeric RNA against the F7 gene as RNA results in cutting for one of two spacers tested.
[0180] Figure 29 shows how DNA double-strand break (DSB) repair promotes gene editing. In the error-prone non-homologous end joining (NHEJ) pathway, the ends of a DSB are processed by endogenous DNA repair machineries and rejoined together, which can result in random insertion/deletion (indel) mutations at the site of junction. Indel mutations occurring within the coding region of a gene can result in frame-shift and a premature stop codon, leading to gene knockout. Alternatively, a repair template in the form of a plasmid or single-stranded oligodeoxynucleotides (ssODN) can be supplied to leverage the homology-directed repair (HDR) pathway, which allows high fidelity and precise editing.
[0181] Figure 30A-C shows anticipated results for HDR in HEK and HUES9 cells. (a) Either a targeting plasmid or an ssODN (sense or antisense) with homology arms can be used to edit the sequence at a target genomic locus cleaved by Cas9 (red triangle). To assay the efficiency of HDR, Applicants introduced a HindIII site (red bar) into the target locus, which was PCR- amplified with primers that anneal outside of the region of homology. Digestion of the PCR product with HindIII reveals the occurrence of HDR events. (b) ssODNs, oriented in either the sense or the antisense (s or a) direction relative to the locus of interest, can be used in combination with Cas9 to achieve efficient HDR-mediated editing at the target locus. A minimal homology region of 40 bp, and preferably 90 bp, is recommended on either side of the modification (red bar). (c) Example of the effect of ssODNs on HDR in the EMX1 locus is shown using both wild-type Cas9 and Cas9 nickase (D10A). Each ssODN contains homology arms of 90 bp flanking a 12-bp insertion of two restriction sites.
[0182] Figure 31A-C shows the repair strategy for Cystic Fibrosis delta F508 mutation.
[0183] Figure 32A-B (a) shows a schematic of the GAA repeat expansion in FXN intron 1 and (b) shows a schematic of the strategy adopted to excise the GAA expansion region using the CRISPR/Cas system. [0184] Figure 33 shows a screen for efficient SpCas9 mediated targeting of Tet1 -3 and Dnmt1, 3a and 3b gene loci. Surveyor assay on DNA from transfected N2A cells demonstrates efficient DNA cleavage by using different gRNAs.
[0185] Figure 34 shows a strategy of multiplex genome targeting using a 2-vector system in an AAV1/2 delivery system. Tet1-3 and Dnmt1, 3a and 3b gRNA under the control of the U6 promoter. GFP-KASH under the control of the human synapsin promoter. Restriction sides shows simple gRNA replacement strategy by subcloning. HA-tagged SpCas9 flanked by two nuclear localization signals (NLS) is shown. Both vectors are delivered into the brain by AAV1/2 virus in a 1:1 ratio.
[0186] Figure 35 shows verification of multiplex DNMT targeting vector #1 functionality using Surveyor assay. N2A cells were co-transfected with the DNMT targeting vector #1 (+) and the SpCas9 encoding vector for testing SpCas9 mediated cleavage of DNMTs genes family loci. gRNA only (-) is negative control. Cells were harvested for DNA purification and downstream processing 48 h after transfection.
[0187] Figure 36 shows guide RNA design for HBV targeting CRISPR constructs. Cleavage sites were optimized for low homology to human genomic DNA and conservation (guides 13-24)
[0188] Figure 37 shows a scheme for quantifying cccDNA in response to Cas9 treatment, 1st round of experiments.
[0189] Figure 38 shows qPCR results from 1st round of HepG2.2.15 experiments.
[0190] Figure 39 shows a Surveyor assay for nuclease activity. Indels form as the result of imperfect NHEJ events due to repeated DSB formation from nuclease activity. For Cas9 targeted genomic loci indel formation at rates 10-30% are often observed and can approach 50%.
[0191] Figure 40 shows representative surveyor for 1st set of HepG2.2.15 experiments.
[0192] Figure 41 shows HepG2.2.15 HBV quantification scheme, an experimental design motivated by noise of initial data sets.
[0193] Figure 42 shows HepG2.2.15 results using sorting based normalization.
[0194] Figure 43 shows low levels of indels observed with guides targeting conserved HBV sequences in 2nd round of HepG2.2.15 experiments.
[0195] Figure 44 shows HepG2 co-transfection experiments.
[0196] Figure 45 shows HDD data for Cohort 1.
[0197] Figure 46 shows HDD data for Cohort 2.
[0198] Figure 47 shows HDD data for Cohort 2.
[0199] Figure 48 shows HDD data for Cohort 2. [0200] Figure 49 shows Cohort 2-liver analysis 9d post HDD.
[0201] Figure 50 shows Cohort 2-liver analysis 9d post HDD.
[0202] Figure 51 shows low/no indels formed during HDD experiments. Predicted band sizes for guide 21 formation: 235 + 272 + 507 bp (undigested PCR product).
[0203] Figure 52 shows HDD Cohort 3 Results: HBsAg.
[0204] Figure 53 shows HDD Cohort 3 Results: Viremia.
[0205] Figure 54 shows HDD Cohort 3 Results: HBV in Liver
[0206] Figure 55 shows HDD Cohort 3 Results: Luciferase normalized to GAPDH.
[0207] Figure 56 shows despite low/no indel formation, effects on HBV are dependent on Cas9 nuclease activity.
[0208] Figure 57A-B shows (a) schematic of HBV life cycle and putative anti-HBV effect of CRISPR constructs; Cas9-mediated DSB formation should linearize the small, episomal cccDNA repeatedly, potentially leading to indel formation (generating less-fit viral mutants) or even degradation. (b) (left) HBV genome organization and location of target sequences for several tested guide RNA constructs, (right) Table of all possible CRISPR target sites in each HBV ORF, including number of possible target sites in conserved genomic regions.
[0209] Figure 58A-D shows (a) guide RNAs targeting conserved regions target large majority of patient-derived virus genomes. All whole-genome sequences from HBV isolates were queried from GenBank to determine the conservation of 23 nt target sequence (20 nt spacer + 3 nt PAM) for 3 guides (6, 17, and 21). x-axis denotes number of allowed mismatches, and y- axis denotes the percentage of sequenced isolates that fall within this number of mismatches to native sgRNA target site; (b) schematic for HepG2 transfection experiments and (c-d): HepG2 cells co-transfected with 1.3x WT HBV and sgRNA/Cas9-2A-mCherry construct showing effect on HBsAg (c) and HBV 3.5kb RNA (d).
[0210] Figure 59A-C shows (a) Experimental schematic for hydrodynamic injection experiments of (b-c): 1.3x WT HBV and sgRNA/Cas9-2A-mCherry are delivered to the livers of immunodeficient NRG mice via hydrodynamic injection, and (b) HBsAg and (c) secreted HBV titer are quantified in mouse blood at 2 and 4 days post injection. 21M: guide RNA with 5 bp mismatch from g21. Data shown are from one representative experiment, and consistent across multiple experiments. UT:‘untargeted’ guide RNA (no target sequence in HBV genome). *p < 0.05 for selected comparison; **p < 0.01 for selected comparison; ***p < 0.001 for selected comparison as assessed by two-tailed t-test. [0211] Figure 60A-B shows (a) the HBV life cycle within HepG2.2.15 cells. HepG2.2.15 cells contain genomically integrated linear 1.3x WT HBV sequences, from which viral proteins and cccDNA are constitutively produced via transcription followed by translation (proteins) or reverse transcription and nuclear re-import (cccDNA). The persistent HBV production in this system enables assay of the long-term anti-HBV effects of CRISPR/Cas systems targeting viral DNA; and (b) schematic of lentiviral vector and experimental strategy for sustained CRISPR expression within HepG2.2.15 cells.
[0212] Figure 61A-B shows that HBV-targeting CRISPR reduces HBV DNA and cccDNA dependent upon HBV-specific guide RNA and Cas9 activity.
[0213] Figure 62A-C shows HBV products are reduced upon long-term CRISPR/Cas expression. Stable lines of HepG2.2.15 cells expressing 3 different on-target guides with nuclease-active or nuclease-dead Cas9, along with 3 non-HBV targeting guides, were seeded at consistent cell densities (20,000 cells/cm2) and allowed to secrete virions and viral proteins into the supernatant. 72h later, supernatant was collected and (a) viral titer and (b) HBeAg (a secreted protein produced from the C ORF, used clinically as a marker of active viral replication) were quantified (c). Total HBV RNA and 3.5kb RNA (consisting of pregenomic RNA and the longest translated HBV RNA species, which are difficult to distinguish) were quantified at 36 days post transduction, with HBV RNA suppression continuing out to this late time point. (a-b) *p < 0.05 vs. UT; **p < 0.01 vs. UT; ***p < 0.001 vs. UT as assessed by one-way ANOVA followed by Dunnett’s post-hoc test.
[0214] Figure 63A-B shows that CRISPR constructs targeting HBV cause large and progressive reduction in (a) cccDNA and (b) total HBV DNA levels that is dependent on successful targeting of viral DNA.
[0215] Figure 64A-B shows HBV DNA and cccDNA reductions upon long-term CRISPR/Cas expression are produced with multiple guides. (a) cccDNA reductions at 21 and 36 days post transduction across 3 guides (6, 17, and 21); large reductions are seen in each. (b) Total HBV DNA reductions at 21 and 36 days post transduction are also large across these 3 guide RNAs.
[0216] Figure 65 shows Southern blot of HBV DNA.
[0217] Figure 66 shows Southern blot of HBV DNA.
[0218] Figure 67 shows Surveyor assay to detect indel formation in total HBV DNA (top) and episomal HBV DNA, enriched by treatment with plasmid-safe DNase (bottom); lentiviral transduction enables high levels of cutting of HBV. Arrowheads depict surveyor digestion products resulting from indel formation. Expected PCR product sizes for g6, g17 and g21 are respectively 599, 946 and 507 bp. Approximate sizes of surveyor digestion products for g6, g17 and g21 are respectively: 429 + 170, 570 + 376, 275 + 232.
[0219] Figure 68 shows Immunofluorescent imaging of HBV Core protein demonstrates large reduction in Core staining upon targeting by g17 specifically against the Core ORF.
[0220] Figure 69 shows schematics for de novo infection experiments. Hep-NTCP cells were transduced with Cas9/gRNA constructs containing either g17 or g17M (mutant of g17, resulting in 5bp DNA bulge upon complexation to HBV DNA target), and either WT or dead Cas9, and then selected with puromycin to generate stable lines. (Left) These cells were seeded in coculture with HepG2.2.15 cells, which produce infectious HBV virions that then infect the transduced Hep-NTCP cells. After transient coculture, HepG2.2.15 cells were killed by puromycin selection, and Hep-NTCP cells were cultured for several days and then harvested to assay viral parameters. (Right) These cells were infected with HBV virions derived from HBV+ patient plasma, then cultured and harvested to assay viral parameters.
[0221] Figure 70A-D shows HBsAg secretion (a), cccDNA copies (b), levels of HBV 3.5kb RNA relative to 5 bp mismatch control (c), and titer of HBV DNA in culture medium (d). The data show that Cas9/g17 reduce HBV infection in de novo infection context. 17M: 5 bp mismatch control. 17D: dead Cas9 with g17. Data shown are from one representative experiment, and consistent across experiments.
[0222] Figure 71A-C shows CRISPR/Cas-mediated disruption of HBV in patient-derived virus model system. Hep-NTCP cells were infected with HBV from infected patient serum upon transduction of guide 17 and active or nuclease-dead Cas9. 9 days after infection, the cells were harvested and viral products were quantified. Nuclease-active Cas9 caused decreases in HBV 3.5kb RNA (a), cccDNA (b), and total DNA levels (c).
[0223] Figure 72 shows Surveyor assay performed on DNA untreated (left) or treated (right) with plasmid-Safe DNase to remove non-episomal viral forms. Arrowheads indicate shorter amplicons resulting from indel formation. The low levels of indel formation in 17M likely result from inefficient cutting enabled by the 5 bp bulge between target DNA and gRNA, recently appreciated in Lin, Y et al., (2014). (b-c) *p < 0.05 for selected comparison; **p < 0.01 for selected comparison, as assessed by one-way ANOVA with Tukey post-hoc test.
[0224] Figure 73 depicts a plot where the columns labeled‘D’ signify where Applicants used a nuclease-deficient Cas9 as an internal control. The 29 dpt corresponds to 29 days post transduction, where a single lentiviral vector encoding U6-sgRNA and EFS-hSpCas9-2A-Puro was transduced into HepG2.2.15 cells followed by selection with puromycin.
[0225] The figures herein are for illustrative purposes only and are not necessarily drawn to scale.
DETAILED DESCRIPTION OF THE INVENTION
[0226] With respect to general information on CRISPR-Cas Systems, components thereof, and delivery of such components, including methods, materials, delivery vehicles, vectors, particles, AAV, and making and using thereof, including as to amounts and formulations, all useful in the practice of the instant invention, reference is made to: US Patents Nos. 8,697,359, 8,771,945, 8,795,965, 8,865,406, 8,871,445, 8,889,356, 8,889,418 and 8,895,308; US Patent Publications US 2014-0310830 (US APP. Ser. No. 14/105,031), US 2014-0287938 A1 (U.S. App. Ser. No. 14/213,991), US 2014-0273234 A1 (U.S. App. Ser. No. 14/293,674), US2014- 0273232 A1 (U.S. App. Ser. No. 14/290,575), US 2014-0273231 (U.S. App. Ser. No. 14/259,420), US 2014-0256046 A1 (U.S. App. Ser. No. 14/226,274), US 2014-0248702 A1 (U.S. App. Ser. No. 14/258,458), US 2014-0242700 A1 (U.S. App. Ser. No. 14/222,930), US 2014-0242699 A1 (U.S. App. Ser. No. 14/183,512), US 2014-0242664 A1 (U.S. App. Ser. No. 14/104,990), US 2014-0234972 A1 (U.S. App. Ser. No. 14/183,471), US 2014-0227787 A1 (U.S. App. Ser. No. 14/256,912), US 2014-0189896 A1 (U.S. App. Ser. No. 14/105,035), US 2014-0186958 (U.S. App. Ser. No. 14/105,017), US 2014-0186919 A1 (U.S. App. Ser. No. 14/104,977), US 2014-0186843 A1 (U.S. App. Ser. No. 14/104,900), US 2014-0179770 A1 (U.S. App. Ser. No. 14/104,837) and US 2014-0179006 A1 (U.S. App. Ser. No. 14/183,486), US 2014-0170753 (US App Ser No 14/183,429); European Patent Applications EP 2 771 468 (EP13818570.7), EP 2 764 103 (EP13824232.6), and EP 2 784 162 (EP14170383.5); and PCT Patent Publications WO 2014/093661 (PCT/US2013/074743), WO 2014/093694 (PCT/US2013/074790), WO 2014/093595 (PCT/US2013/074611), WO 2014/093718 (PCT/US2013/074825), WO 2014/093709 (PCT/US2013/074812), WO 2014/093622 (PCT/US2013/074667), WO 2014/093635 (PCT/US2013/074691), WO 2014/093655 (PCT/US2013/074736), WO 2014/093712 (PCT/US2013/074819), WO2014/093701 (PCT/US2013/074800), and WO2014/018423 (PCT/US2013/051418). Reference is also made to US provisional patent applications 61/758,468; 61/802,174; 61/806,375; 61/814,263; 61/819,803 and 61/828,130, filed on January 30, 2013; March 15, 2013; March 28, 2013; April 20, 2013; May 6, 2013 and May 28, 2013 respectively. Reference is also made to US provisional patent application 61/836,123, filed on June 17, 2013. Reference is additionally made to US provisional patent applications 61/835,931, 61/835,936, 61/836,127, 61/836, 101, 61/836,080 and 61/835,973, each filed June 17, 2013. Further reference is made to US provisional patent applications 61/862,468 and 61/862,355 filed on August 5, 2013; 61/871,301 filed on August 28, 2013; 61/960,777 filed on September 25, 2013 and 61/961,980 filed on October 28, 2013. Reference is yet further made to: PCT Patent applications Nos: PCT/US2014/041803, PCT/US2014/041800, PCT/US2014/041809, PCT/US2014/041804 and PCT/US2014/041806, each filed June 10, 2014 6/10/14; PCT/US2014/041808 filed June 11, 2014; and PCT/US2014/62558 filed October 28, 2014, and US Provisional Patent Applications Serial Nos.: 61/915,150, 61/915,301, 61/915,267 and 61/915,260, each filed December 12, 2013; 61/757,972 and 61/768,959, filed on January 29, 2013 and February 25, 2013; 61/835,936, 61/836,127, 61/836,101, 61/836,080, 61/835,973, and 61/835,931, filed June 17, 2013; 62/010,888 and 62/010,879, both filed June 11, 2014; 62/010,329 and 62/010,441, each filed June 10, 2014; 61/939,228 and 61/939,242, each filed February 12, 2014; 61/980,012, filed April 15,2014; 62/038,358, filed August 17, 2014; 62/054,490, 62/055,484, 62/055,460 and 62/055,487, each filed September 25, 2014; and 62/069,243, filed October 27, 2014. Reference is also made to US provisional patent applications Nos. 62/055,484, 62/055,460, and 62/055,487, filed September 25, 2014; US provisional patent application 61/980,012, filed April 15, 2014; and US provisional patent application 61/939,242 filed February 12, 2014. Reference is made to PCT application designating, inter alia, the United States, application No. PCT/US14/41806, filed June 10, 2014. Reference is made to US provisional patent application 61/930,214 filed on January 22, 2014. Reference is made to US provisional patent applications 61/915,251; 61/915,260 and 61/915,267, each filed on December 12, 2013. Reference is made to US provisional patent application USSN 61/980,012 filed April 15, 2014. Reference is made to PCT application designating, inter alia, the United States, application No. PCT/US14/41806, filed June 10, 2014. Reference is made to US provisional patent application 61/930,214 filed on January 22, 2014. Reference is made to US provisional patent applications 61/915,251; 61/915,260 and 61/915,267, each filed on December 12, 2013. Each of these patents, patent publications, and applications, and all documents cited therein or during their prosecution (“appln cited documents”) and all documents cited or referenced in the appln cited documents, together with any instructions, descriptions, product specifications, and product sheets for any products mentioned therein or in any document therein and incorporated by reference herein, are hereby incorporated herein by reference, and may be employed in the practice of the invention. All documents (e.g., these patents, patent publications and applications and the appln cited documents) are incorporated herein by reference to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference.
[0227] Also with respect to general information on CRISPR-Cas Systems, mention is made of the following (also hereby incorporated herein by reference):
Æ Multiplex genome engineering using CRISPR/Cas systems. Cong, L., Ran, F.A., Cox, D., Lin, S., Barretto, R., Habib, N., Hsu, P.D., Wu, X., Jiang, W., Marraffini, L.A., & Zhang, F. Science Feb 15;339(6121):819-23 (2013);
Æ RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Jiang W., Bikard D., Cox D., Zhang F, Marraffini LA. Nat Biotechnol Mar;31(3):233-9 (2013);
Æ One-Step Generation of Mice Carrying Mutations in Multiple Genes by CRISPR/Cas-Mediated Genome Engineering. Wang H., Yang H., Shivalila CS., Dawlaty MM., Cheng AW., Zhang F., Jaenisch R. Cell May 9;153(4):910-8 (2013);
Æ Optical control of mammalian endogenous transcription and epigenetic states. Konermann S, Brigham MD, Trevino AE, Hsu PD, Heidenreich M, Cong L, Platt RJ, Scott DA, Church GM, Zhang F. Nature. 2013 Aug 22;500(7463):472-6. doi: 10.1038/Nature12466. Epub 2013 Aug 23;
Æ Double Nicking by RNA-Guided CRISPR Cas9 for Enhanced Genome Editing Specificity. Ran, FA., Hsu, PD., Lin, CY., Gootenberg, JS., Konermann, S., Trevino, AE., Scott, DA., Inoue, A., Matoba, S., Zhang, Y., & Zhang, F. Cell Aug 28. pii: S0092-8674(13)01015-5. (2013);
Æ DNA targeting specificity of RNA-guided Cas9 nucleases. Hsu, P., Scott, D., Weinstein, J., Ran, FA., Konermann, S., Agarwala, V., Li, Y., Fine, E., Wu, X., Shalem, O., Cradick, TJ., Marraffini, LA., Bao, G., & Zhang, F. Nat Biotechnol doi:10.1038/nbt.2647 (2013);
Æ Genome engineering using the CRISPR-Cas9 system. Ran, FA., Hsu, PD., Wright, J., Agarwala, V., Scott, DA., Zhang, F. Nature Protocols Nov;8(11):2281-308. (2013);
Æ Genome-Scale CRISPR-Cas9 Knockout Screening in Human Cells. Shalem, O., Sanjana, NE., Hartenian, E., Shi, X., Scott, DA., Mikkelson, T., Heckl, D., Ebert, BL., Root, DE., Doench, JG., Zhang, F. Science Dec 12. (2013). [Epub ahead of print];
Æ Crystal structure of cas9 in complex with guide RNA and target DNA.
Nishimasu, H., Ran, FA., Hsu, PD., Konermann, S., Shehata, SI., Dohmae, N., Ishitani, R., Zhang, F., Nureki, O. Cell Feb 27. (2014). 156(5):935-49;
Æ Genome-wide binding of the CRISPR endonuclease Cas9 in mammalian cells.
Wu X., Scott DA., Kriz AJ., Chiu AC., Hsu PD., Dadon DB., Cheng AW., Trevino AE., Konermann S., Chen S., Jaenisch R., Zhang F., Sharp PA. Nat Biotechnol. (2014) Apr 20. doi: 10.1038/nbt.2889,
Æ CRISPR-Cas9 Knockin Mice for Genome Editing and Cancer Modeling, Platt et al., Cell 159(2): 440-455 (2014) DOI: 10.1016/j.cell.2014.09.014,
Æ Development and Applications of CRISPR-Cas9 for Genome Engineering, Hsu et al, Cell 157, 1262-1278 (June 5, 2014) (Hsu 2014), Æ Genetic screens in human cells using the CRISPR/Cas9 system, Wang et al., Science. 2014 January 3; 343(6166): 80–84. doi:10.1126/science.1246981, Æ Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation, Doench et al., Nature Biotechnology published online 3 September 2014; doi:10.1038/nbt.3026, and
Æ In vivo interrogation of gene function in the mammalian brain using CRISPR- Cas9, Swiech et al, Nature Biotechnology ; published online 19 October 2014; doi:10.1038/nbt.3055.
each of which is incorporated herein by reference, and discussed briefly below:
[0228] Cong et al. engineered type II CRISPR/Cas systems for use in eukaryotic cells based on both Streptococcus thermophilus Cas9 and also Streptoccocus pyogenes Cas9 and demonstrated that Cas9 nucleases can be directed by short RNAs to induce precise cleavage of DNA in human and mouse cells. Their study further showed that Cas9 as converted into a nicking enzyme can be used to facilitate homology-directed repair in eukaryotic cells with minimal mutagenic activity. Additionally, their study demonstrated that multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several at endogenous genomic loci sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology. This ability to use RNA to program sequence specific DNA cleavage in cells defined a new class of genome engineering tools These studies further showed that other CRISPR loci are likely to be transplantable into mammalian cells and can also mediate mammalian genome cleavage. Importantly, it can be envisaged that several aspects of the CRISPR/Cas system can be further improved to increase its efficiency and versatility.
[0229] Jiang et al. used the clustered, regularly interspaced, short palindromic repeats (CRISPR)–associated Cas9 endonuclease complexed with dual-RNAs to introduce precise mutations in the genomes of Streptococcus pneumoniae and Escherichia coli. The approach relied on dual-RNA:Cas9-directed cleavage at the targeted genomic site to kill unmutated cells and circumvents the need for selectable markers or counter-selection systems. The study reported reprogramming dual-RNA:Cas9 specificity by changing the sequence of short CRISPR RNA (crRNA) to make single- and multinucleotide changes carried on editing templates. The study showed that simultaneous use of two crRNAs enabled multiplex mutagenesis. Furthermore, when the approach was used in combination with recombineering, in S. pneumoniae, nearly 100% of cells that were recovered using the described approach contained the desired mutation, and in E. coli, 65% that were recovered contained the mutation.
[0230] Konermann et al. addressed the need in the art for versatile and robust technologies that enable optical and chemical modulation of DNA-binding domains based CRISPR Cas9 enzyme and also Transcriptional Activator Like Effectors
[0231] Cas9 nuclease from the microbial CRISPR-Cas system is targeted to specific genomic loci by a 20 nt guide sequence, which can tolerate certain mismatches to the DNA target and thereby promote undesired off-target mutagenesis. To address this, Ran et al. described an approach that combined a Cas9 nickase mutant with paired guide RNAs to introduce targeted double-strand breaks. Because individual nicks in the genome are repaired with high fidelity, simultaneous nicking via appropriately offset guide RNAs is required for double-stranded breaks and extends the number of specifically recognized bases for target cleavage. The authors demonstrated that using paired nicking can reduce off-target activity by 50- to 1,500-fold in cell lines and to facilitate gene knockout in mouse zygotes without sacrificing on-target cleavage efficiency. This versatile strategy enables a wide variety of genome editing applications that require high specificity.
[0232] Hsu et al. characterized SpCas9 targeting specificity in human cells to inform the selection of target sites and avoid off-target effects. The study evaluated >700 guide RNA variants and SpCas9-induced indel mutation levels at >100 predicted genomic off-target loci in 293T and 293FT cells. The authors reported that SpCas9 tolerates mismatches between guide RNA and target DNA at different positions in a sequence-dependent manner, sensitive to the number, position and distribution of mismatches. The authors further showed that SpCas9- mediated cleavage is unaffected by DNA methylation and that the dosage of SpCas9 and sgRNA can be titrated to minimize off-target modification. Additionally, to facilitate mammalian genome engineering applications, the authors reported providing a web-based software tool to guide the selection and validation of target sequences as well as off-target analyses.
[0233] Ran et al. described a set of tools for Cas9-mediated genome editing via non- homologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off- target cleavage, the authors further described a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. The protocol provided by the authors experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off- target activity. The studies showed that beginning with target design, gene modifications can be achieved within as little as 1–2 weeks, and modified clonal cell lines can be derived within 2–3 weeks.
[0234] Shalem et al. described a new way to interrogate gene function on a genome-wide scale. Their studies showed that delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeted 18,080 genes with 64,751 unique guide sequences enabled both negative and positive selection screening in human cells. First, the authors showed use of the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, the authors screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic that inhibits mutant protein kinase BRAF. Their studies showed that the highest-ranking candidates included previously validated genes NF1 and MED12 as well as novel hits NF2, CUL3, TADA2B, and TADA1. The authors observed a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, and thus demonstrated the promise of genome-scale screening with Cas9.
[0235] Nishimasu et al. reported the crystal structure of Streptococcus pyogenes Cas9 in complex with sgRNA and its target DNA at 2.5 A° resolution. The structure revealed a bilobed architecture composed of target recognition and nuclease lobes, accommodating the sgRNA:DNA heteroduplex in a positively charged groove at their interface. Whereas the recognition lobe is essential for binding sgRNA and DNA, the nuclease lobe contains the HNH and RuvC nuclease domains, which are properly positioned for cleavage of the complementary and non-complementary strands of the target DNA, respectively. The nuclease lobe also contains a carboxyl-terminal domain responsible for the interaction with the protospacer adjacent motif (PAM). This high-resolution structure and accompanying functional analyses have revealed the molecular mechanism of RNA-guided DNA targeting by Cas9, thus paving the way for the rational design of new, versatile genome-editing technologies.
[0236] Wu et al. mapped genome-wide binding sites of a catalytically inactive Cas9 (dCas9) from Streptococcus pyogenes loaded with single guide RNAs (sgRNAs) in mouse embryonic stem cells (mESCs). The authors showed that each of the four sgRNAs tested targets dCas9 to between tens and thousands of genomic sites, frequently characterized by a 5-nucleotide seed region in the sgRNA and an NGG protospacer adjacent motif (PAM). Chromatin inaccessibility decreases dCas9 binding to other sites with matching seed sequences; thus 70% of off-target sites are associated with genes. The authors showed that targeted sequencing of 295 dCas9 binding sites in mESCs transfected with catalytically active Cas9 identified only one site mutated above background levels. The authors proposed a two-state model for Cas9 binding and cleavage, in which a seed match triggers binding but extensive pairing with target DNA is required for cleavage.
[0237] Hsu 2014 is a review article that discusses generally CRISPR-Cas9 history from yogurt to genome editing, including genetic screening of cells, that is in the information, data and findings of the applications in the lineage of this specification filed prior to June 5, 2014. The general teachings of Hsu 2014 do not involve the specific models, animals of the instant specification.
[0238] Mention is also made of Tsai et al,“Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing,” Nature Biotechnology 32(6): 569-77 (2014) which is not believed to be prior art to the instant invention or application, but which may be considered in the practice of the instant invention.
[0239] In general, the CRISPR-Cas or CRISPR system is as used in the foregoing documents, such as WO 2014/093622 (PCT/US2013/074667) and refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR- associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a“direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or“RNA(s)” as that term is herein used (e.g., RNA(s) to guide Cas9, e.g. CRISPR RNA and transactivating (tracr) RNA or a single guide RNA (sgRNA) (chimeric RNA)) or other sequences and transcripts from a CRISPR locus. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex,“target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell. In some embodiments, direct repeats may be identified in silico by searching for repetitive motifs that fulfill any or all of the following criteria: 1. found in a 2Kb window of genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.
[0240] In embodiments of the invention the terms guide sequence and guide RNA are used interchangeably as in foregoing cited documents such as WO 2014/093622 (PCT/US2013/074667). In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. Preferably the guide sequence is 10 - 30 nucleotides long. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG where NNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. For the S. thermophilus CRISPR1 Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. A unique target sequence in a genome may include an S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. For the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG where NNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. In each of these sequences“M” may be A, G, T, or C, and need not be considered in identifying a sequence as unique. In some embodiments, a guide sequence is selected to reduce the degree secondary structure within the guide sequence. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the guide sequence participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
[0241] In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In an embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has at most five hairpins. In a hairpin structure the portion of the sequence 5’ of the final“N” and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3’ of the loop corresponds to the tracr sequence Further non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5’ to 3’), where“N” represents a base of a guide sequence, the first block of lower case letters represent the tracr mate sequence, and the second block of lower case letters represent the tracr sequence, and the final poly-T sequence represents the transcription terminator: (1) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (2) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (3) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgtTTTTTT; (4) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaactt gaaaaagtggcaccgagtcggtgcTTTTTT; (5) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaac ttgaaaaagtgTTTTTTT; and (6) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTT TTTTTT. In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.
[0242] In some embodiments, candidate tracrRNA may be subsequently predicted by sequences that fulfill any or all of the following criteria: 1. sequence homology to direct repeats (motif search in Geneious with up to 18-bp mismatches); 2. presence of a predicted Rho- independent transcriptional terminator in direction of transcription; and 3. stable hairpin secondary structure between tracrRNA and direct repeat. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used.
[0243] In some embodiments, chimeric synthetic guide RNAs (sgRNAs) designs may incorporate at least 12 bp of duplex structure between the direct repeat and tracrRNA.
[0244] For minimization of toxicity and off-target effect, it will be important to control the concentration of CRISPR enzyme mRNA and guide RNA delivered. Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing different concentrations in a cellular or non-human eukaryote animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci. For example, for the guide sequence targeting 5’-GAGTCCGAGCAGAAGAAGAA-3’ in the EMX1 gene of the human genome, deep sequencing can be used to assess the level of modification at the following two off-target loci, 1: 5’-GAGTCCTAGCAGGAGAAGAA-3’ and 2: 5’-GAGTCTAAGCAGAAGAAGAA- 3’. The concentration that gives the highest level of on-target modification while minimizing the level of off-target modification should be chosen for in vivo delivery. Alternatively, to minimize the level of toxicity and off-target effect, CRISPR enzyme nickase mRNA (for example S. pyogenes Cas9 with the D10A mutation) can be delivered with a pair of guide RNAs targeting a site of interest. The two guide RNAs need to be spaced as follows. Guide sequences and strategies to mimize toxicity and off-target effects can be as in WO 2014/093622 (PCT/US2013/074667).
[0245] The CRISPR system is derived advantageously from a type II CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In preferred embodiments of the invention, the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes DNA cleavage. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof.
[0246] In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III or the HNH domain) may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity. In some embodiments, a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the non- mutated form of the enzyme; an example can be when the DNA cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form. Where the enzyme is not SpCas9, mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools). In particular, any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged. The same (or conservative substitutions of these mutations) at corresponding positions in other Cas9s are also preferred. Particularly preferred are D10 and H840 in SpCas9. However, in other Cas9s, residues corresponding to SpCas9 D10 and H840 are also preferred. Orthologs of SpCas9 can be used in the practice of the invention. A Cas enzyme may be identified Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the Cas9 enzyme is from, or is derived from, spCas9 (S. pyogenes Cas9) or saCas9 (S. aureus Cas9). StCas9” refers to wild type Cas9 from S. thermophilus, the protein sequence of which is given in the SwissProt database under accession number G3ECR1. Similarly, S pyogenes Cas9 or spCas9 is included in SwissProt under accession number Q99ZW2. By derived, Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as described herein. It will be appreciated that the terms Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent. As mentioned above, many of the residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes. However, it will be appreciated that this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCa9, St1Cas9 and so forth. Enzymatic action by Cas9 derived from Streptococcus pyogenes or any closely related Cas9 generates double stranded breaks at target site sequences which hybridize to 20 nucleotides of the guide sequence and that have a protospacer-adjacent motif (PAM) sequence (examples include NGG/NRG or a PAM that can be determined as described herein) following the 20 nucleotides of the target sequence. CRISPR activity through Cas9 for site-specific DNA recognition and cleavage is defined by the guide sequence, the tracr sequence that hybridizes in part to the guide sequence and the PAM sequence. More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA- guided defence in bacteria and archaea, Mole Cell 2010, January 15; 37(1): 7. The type II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30bp each). In this system, targeted DNA double-strand break (DSB) is generated in four sequential steps. First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats of pre-crRNA, which is then processed into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer. A pre-crRNA array consisting of a single spacer flanked by two direct repeats (DRs) is also encompassed by the term“tracr-mate sequences”). In certain embodiments, Cas9 may be constitutively present or inducibly present or conditionally present or administered or delivered. Cas9 optimization may be used to enhance function or to develop new functions, one can generate chimeric Cas9 proteins. And Cas9 may be used as a generic DNA binding protein.
[0247] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild- type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence.
[0248] An example of a codon optimized sequence, is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans), or for another eukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 human codon optimized sequence in WO 2014/093622 (PCT/US2013/074667). Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known. In some embodiments, an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, or non-human eukaryote or animal or mammal as herein discussed, e.g., mouse, rat, rabbit, dog, livestock, or non-human mammal or primate. In some embodiments, processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes, may be excluded. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the“Codon Usage Database” available at www.kazusa.orjp/codon/ and these tables can be adapted in a number of ways. See Nakamura, Y., et al.“Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.
[0249] In some embodiments, a vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. zero or at least one or more NLS at the amino-terminus and zero or at one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In a preferred embodiment of the invention, the CRISPR enzyme comprises at most 6 NLSs. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV; the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from importin-alpha; the sequences VSRKRPRP and PPKKARED of the myoma T protein; the sequence POPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-abl IV; the sequences DRLRR and PKQKKRK of the influenza virus NS1; the sequence RKLKKKIKKL of the Hepatitis virus delta antigen; the sequence REKKKFLKRR of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK of the steroid hormone receptors (human) glucocorticoid. In general, the one or more NLSs are of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the CRISPR enzyme, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the CRISPR enzyme, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity), as compared to a control no exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the one or more NLSs.
[0250] Aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased. In an embodiment of the invention, the gene product is a protein. Only sgRNA pairs creating 5’ overhangs with less than 8bp overlap between the guide sequences (offset greater than -8 bp) were able to mediate detectable indel formation. Importantly, each guide used in these assays is able to efficiently induce indels when paired with wildtype Cas9, indicating that the relative positions of the guide pairs are the most important parameters in predicting double nicking activity. Since Cas9n and Cas9H840A nick opposite strands of DNA, substitution of Cas9n with Cas9H840A with a given sgRNA pair should have resulted in the inversion of the overhang type; but no indel formation is observed as with Cas9H840A indicating that Cas9H840A is a CRISPR enzyme substantially lacking all DNA cleavage activity (which is when the DNA cleavage activity of the mutated enzyme is about no more than 25%, 10%, 5%, 1%, 0.1%, 0.01%, or less of the DNA cleavage activity of the non- mutated form of the enzyme; whereby an example can be when the DNA cleavage activity of the mutated form is nil or negligible as compared with the non-mutated form, e.g., when no indel formation is observed as with Cas9H840A in the eukaryotic system in contrast to the biochemical or prokaryotic systems). Nonetheless, a pair of sgRNAs that will generate a 5’ overhang with Cas9n should in principle generate the corresponding 3’ overhang instead, and double nicking. Therefore, sgRNA pairs that lead to the generation of a 3’ overhang with Cas9n can be used with another mutated Cas9 to generate a 5’ overhang, and double nicking. Accordingly, in some embodiments, a recombination template is also provided. A recombination template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex. A template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.
[0251] In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Or, RNA(s) of the CRISPR System can be delivered to a transgenic Cas9 animal or mammal, e.g., an animal or mammal that constitutively or inducibly or conditionally expresses Cas9; or an animal or mammal that is otherwise expressing Cas9 or has cells containing Cas9, such as by way of prior administration thereto of a vector or vectors that code for and express in vivo Cas9. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter. Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a CRISPR system are as used in the foregoing documents, such as WO 2014/093622 (PCT/US2013/074667). In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a“cloning site”). In some embodiments, one or more insertion sites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and/or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence- specific binding of a CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell. In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. CRISPR enzyme or CRISPR enzyme mRNA or CRISPR guide RNA or RNA(s) can be delivered separately; and advantageously at least one of these is delivered via a nanoparticle complex. CRISPR enzyme mRNA can be delivered prior to the guide RNA to give time for CRISPR enzyme to be expressed. CRISPR enzyme mRNA might be administered 1 -12 hours (preferably around 2-6 hours) prior to the administration of guide RNA. Alternatively, CRISPR enzyme mRNA and guide RNA can be administered together. Advantageously, a second booster dose of guide RNA can be administered 1 -12 hours (preferably around 2-6 hours) after the initial administration of CRISPR enzyme mRNA + guide RNA. Additional administrations of CRISPR enzyme mRNA and/or guide RNA might be useful to achieve the most efficient levels of genome modification.
[0252] In one aspect, the invention provides methods for using one or more elements of a CRISPR system. The CRISPR complex of the invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating, activating) a target polynucleotide in a multiplicity of cell types. As such the CRISPR complex of the invention has a broad spectrum of applications in, e.g., gene therapy, drug screening, disease diagnosis, and prognosis. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence. In one embodiment, this invention provides a method of cleaving a target polynucleotide. The method comprises modifying a target polynucleotide using a CRISPR complex that binds to the target polynucleotide and effect cleavage of said target polynucleotide. Typically, the CRISPR complex of the invention, when introduced into a cell, creates a break (e.g., a single or a double strand break) in the genome sequence. For example, the method can be used to cleave a disease gene in a cell. The break created by the CRISPR complex can be repaired by a repair processes such as the error prone non-homologous end joining (NHEJ) pathway or the high fidelity homology-directed repair (HDR). During these repair process, an exogenous polynucleotide template can be introduced into the genome sequence. In some methods, the HDR process is used modify genome sequence. For example, an exogenous polynucleotide template comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell. The upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome. Where desired, a donor polynucleotide can be DNA, e.g., a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. The exogenous polynucleotide template comprises a sequence to be integrated (e.g., a mutated gene). The sequence for integration may be a sequence endogenous or exogenous to the cell. Examples of a sequence to be integrated include polynucleotides encoding a protein or a non-coding RNA (e.g., a microRNA). Thus, the sequence for integration may be operably linked to an appropriate control sequence or sequences. Alternatively, the sequence to be integrated may provide a regulatory function. The upstream and downstream sequences in the exogenous polynucleotide template are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide. The upstream sequence is a nucleic acid sequence that shares sequence similarity with the genome sequence upstream of the targeted site for integration. Similarly, the downstream sequence is a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the targeted site of integration. The upstream and downstream sequences in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted genome sequence. Preferably, the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted genome sequence. In some methods, the upstream and downstream sequences in the exogenous polynucleotide template have about 99% or 100% sequence identity with the targeted genome sequence. An upstream or downstream sequence may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp. In some methods, the exogenous polynucleotide template may further comprise a marker. Such a marker may make it easy to screen for targeted integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous polynucleotide template of the invention can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996). In a method for modifying a target polynucleotide by integrating an exogenous polynucleotide template, a double stranded break is introduced into the genome sequence by the CRISPR complex, the break is repaired via homologous recombination an exogenous polynucleotide template such that the template is integrated into the genome. The presence of a double-stranded break facilitates integration of the template. In other embodiments, this invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. The method comprises increasing or decreasing expression of a target polynucleotide by using a CRISPR complex that binds to the polynucleotide. In some methods, a target polynucleotide can be inactivated to effect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein is not produced, or the sequence does not function as the wild-type sequence does. For example, a protein or microRNA coding sequence may be inactivated such that the protein or microRNA or pre-microRNA transcript is not produced. In some methods, a control sequence can be inactivated such that it no longer functions as a control sequence. As used herein,“control sequence” refers to any nucleic acid sequence that effects the transcription, translation, or accessibility of a nucleic acid sequence. Examples of a control sequence include, a promoter, a transcription terminator, and an enhancer are control sequences. The target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell. The target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). Examples of target polynucleotides include a sequence associated with a signaling biochemical pathway, e.g., a signaling biochemical pathway- associated gene or polynucleotide. Examples of target polynucleotides include a disease associated gene or polynucleotide. A“disease-associated” gene or polynucleotide refers to any gene or polynucleotide which is yielding transcription or translation products at an abnormal level or in an abnormal form in cells derived from a disease-affected tissues compared with tissues or cells of a non disease control. It may be a gene that becomes expressed at an abnormally high level; it may be a gene that becomes expressed at an abnormally low level, where the altered expression correlates with the occurrence and/or progression of the disease. A disease-associated gene also refers to a gene possessing mutation(s) or genetic variation that is directly responsible or is in linkage disequilibrium with a gene(s) that is responsible for the etiology of a disease. The transcribed or translated products may be known or unknown, and may be at a normal or abnormal level. The target polynucleotide of a CRISPR complex can be any polynucleotide endogenous or exogenous to the eukaryotic cell. For example, the target polynucleotide can be a polynucleotide residing in the nucleus of the eukaryotic cell. The target polynucleotide can be a sequence coding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide or a junk DNA). Without wishing to be bound by theory, it is believed that the target sequence should be associated with a PAM (protospacer adjacent motif); that is, a short sequence recognized by the CRISPR complex. The precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence) Examples of PAM sequences are given in the examples section below, and the skilled person will be able to identify further PAM sequences for use with a given CRISPR enzyme. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. Similar considerations and conditions apply as above for methods of modifying a target polynucleotide. In fact, these sampling, culturing and re-introduction options apply across the aspects of the present invention. In one aspect, the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non- human animal, and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant. For re-introduced cells it is particularly preferred that the cells are stem cells.
[0253] Indeed, in any aspect of the invention, the CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized to a target sequence, wherein said guide sequence may be linked to a tracr mate sequence which in turn may hybridize to a tracr sequence.
[0254] The invention relates to the engineering and optimization of systems, methods and compositions used for the control of gene expression involving sequence targeting, such as genome perturbation or gene-editing, that relate to the CRISPR-Cas system and components thereof. In advantageous embodiments, the Cas enzyme is Cas9. An advantage of the present methods is that the CRISPR system minimizes or avoids off-target binding and its resulting side effects. This is achieved using systems arranged to have a high degree of sequence specificity for the target DNA.
Cas9
[0255] Cas9 optimization may be used to enhance function or to develop new functions, one can generate chimeric Cas9 proteins, as demonstrated in the Examples. Chimeric Cas9 proteins can be made by combining fragments from different Cas9 homologs. For example, two example chimeric Cas9 proteins from the Cas9s described herein. For example, Applicants fused the N- term of St1Cas9 (fragment from this protein is in bold) with C-term of SpCas9. The benefit of making chimeric Cas9s include any or all of: reduced toxicity; improved expression in eukaryotic cells; enhanced specificity; reduced molecular weight of protein, for example, making the protein smaller by combining the smallest domains from different Cas9 homologs; and/or altering the PAM sequence requirement.
[0256] The Cas9 may be used as a generic DNA binding protein as demonstrated in the Examples. Applicants used Cas9 as a generic DNA binding protein by mutating the two catalytic domains (D10 and H840) responsible for cleaving both strands of the DNA target. In order to upregulate gene transcription at a target locus Applicants fused a transcriptional activation domain (VP64) to Cas9. Other transcriptional activation domains are known. As shown in the Examples transcriptional activation is possible as well as gene repression using a Cas9 repressor (DNA-binding domain) that binds to the target gene sequence, thus repressing its activity.
[0257] Cas9 and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, US Patents Nos. 8,454,972 (formulations, doses for adenovirus), 8,404,658 (formulations, doses for AAV) and 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For examples, for AAV, the route of administration, formulation and dose can be as in US Patent No. 8,454,972 and as in clinical trials involving AAV. For Adenovirus, the route of administration, formulation and dose can be as in US Patent No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as in US Patent No 5,846,946 and as in clinical studies involving plasmids. Doses may be based on or extrapolated to an average 70 kg individual (e.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed.
[0258] The viral vectors can be injected into the tissue of interest. For cell-type specific genome modification, the expression of Cas9 can be driven by a cell-type specific promoter. For example, liver-specific expression might use the Albumin promoter whereas neuron-specific expression (for viruses which may be latent in the brain) might use the Synapsin I promoter.
Transgenic animals and plants
[0259] Transgenic animals are also provided. Preferred examples include animals comprising Cas9, in terms of polynucleotides encoding Cas9 or the protein itself. Mice, rats and rabbits are preferred. To generate transgenic mice with the constructs, as exemplified herein one may inject pure, linear DNA into the pronucleus of a zygote from a pseudo pregnant female, e.g. a CB56 female. Founders may then be identified, genotyped, and backcrossed to CB57 mice. The constructs may then be cloned and optionally verified, for instance by Sanger sequencing. Knock outs are envisaged where for instance one or more genes are knocked out in a model. However, are knockins are also envisaged (alone or in combination). An example knockin Cas9 mouse was generated and this is exemplified, but Cas9 knockins are preferred. To generate a Cas9 knock in mice one may target the same constitutive and conditional constructs to the Rosa26 locus, as described herein (Figs. 25A-B and 26). Methods of US Patent Publication Nos. 20120017290 and 20110265198 assigned to Sangamo BioSciences, Inc. directed to targeting the Rosa locus may be modified to utilize the CRISPR Cas system of the present invention. In another embodiment, the methods of US Patent Publication No. 20130236946 assigned to Cellectis directed to targeting the Rosa locus may also be modified to utilize the CRISPR Cas system of the present invention.
[0260] Utility of the conditional Cas9 mouse: Applicants have shown in 293 cells that the Cas9 conditional expression construct can be activated by co-expression with Cre. Applicants also show that the correctly targeted R1 mESCs can have active Cas9 when Cre is expressed. Because Cas9 is followed by the P2A peptide cleavage sequence and then EGFP Applicants identify successful expression by observing EGFP. Applicants have shown Cas9 activation in mESCs. This same concept is what makes the conditional Cas9 mouse so useful. Applicants may cross their conditional Cas9 mouse with a mouse that ubiquitously expresses Cre (ACTB-Cre line) and may arrive at a mouse that expresses Cas9 in every cell. It should only take the delivery of chimeric RNA to induce genome editing in embryonic or adult mice. Interestingly, if the conditional Cas9 mouse is crossed with a mouse expressing Cre under a tissue specific promoter, there should only be Cas9 in the tissues that also express Cre. This approach may be used to edit the genome in only precise tissues by delivering chimeric RNA to the same tissue.
Delivery generally
[0261] Vector delivery, e.g., plasmid, viral delivery: The CRISPR enzyme, for instance a Cas9, and/or any of the present RNAs, for instance a guide RNA, can be delivered using any suitable vector, e.g., plasmid or viral vectors, such as adeno associated virus (AAV), lentivirus, adenovirus or other viral vector types, or combinations thereof. Cas9 and one or more guide RNAs can be packaged into one or more vectors, e.g., plasmid or viral vectors. In some embodiments, the vector, e.g., plasmid or viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such delivery may be either via a single dose, or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein may vary greatly depending upon a variety of factors, such as the vector choice, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation/modification sought, the administration route, the administration mode, the type of transformation/modification sought, etc.
[0262] Such a dosage may further contain, for example, a carrier (water, saline, ethanol, glycerol, lactose, sucrose, calcium phosphate, gelatin, dextran, agar, pectin, peanut oil, sesame oil, etc.), a diluent, a pharmaceutically-acceptable carrier (e.g., phosphate-buffered saline), a pharmaceutically-acceptable excipient, and/or other compounds known in the art. The dosage may further contain one or more pharmaceutically acceptable salts such as, for example, a mineral acid salt such as a hydrochloride, a hydrobromide, a phosphate, a sulfate, etc.; and the salts of organic acids such as acetates, propionates, malonates, benzoates, etc. Additionally, auxiliary substances, such as wetting or emulsifying agents, pH buffering substances, gels or gelling materials, flavorings, colorants, microspheres, polymers, suspension agents, etc. may also be present herein. In addition, one or more other conventional pharmaceutical ingredients, such as preservatives, humectants, suspending agents, surfactants, antioxidants, anticaking agents, fillers, chelating agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reconstitutable form. Suitable exemplary ingredients include microcrystalline cellulose, carboxymethylcellulose sodium, polysorbate 80, phenylethyl alcohol, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, the parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, gelatin, albumin and a combination thereof. A thorough discussion of pharmaceutically acceptable excipients is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which is incorporated by reference herein.
[0263] In an embodiment herein the delivery is via an adenovirus, which may be at a single booster dose containing at least 1 x 105 particles (also referred to as particle units, pu) of adenoviral vector. In an embodiment herein, the dose preferably is at least about 1 x 106 particles (for example, about 1 x 106-1 x 1012 particles), more preferably at least about 1 x 107 particles, more preferably at least about 1 x 108 particles (e.g., about 1 x 108-1 x 1011 particles or about 1 x 108-1 x 1012 particles), and most preferably at least about 1 x 100 particles (e.g., about 1 x 109-1 x 1010 particles or about 1 x 109-1 x 1012 particles), or even at least about 1 x 1010 particles (e.g., about 1 x 1010-1 x 1012 particles) of the adenoviral vector. Alternatively, the dose comprises no more than about 1 x 1014 particles, preferably no more than about 1 x 1013 particles, even more preferably no more than about 1 x 1012 particles, even more preferably no more than about 1 x 1011 particles, and most preferably no more than about 1 x 1010 particles (e.g., no more than about 1 x 109 articles). Thus, the dose may contain a single dose of adenoviral vector with, for example, about 1 x 106 particle units (pu), about 2 x 106 pu, about 4 x 106 pu, about 1 x 107 pu, about 2 x 107 pu, about 4 x 107 pu, about 1 x 108 pu, about 2 x 108 pu, about 4 x 108 pu, about 1 x 109 pu, about 2 x 109 pu, about 4 x 109 pu, about 1 x 1010 pu, about 2 x 1010 pu, about 4 x 1010 pu, about 1 x 1011 pu, about 2 x 1011 pu, about 4 x 1011 pu, about 1 x 1012 pu, about 2 x 1012 pu, or about 4 x 1012 pu of adenoviral vector. See, for example, the adenoviral vectors in U.S. Patent No. 8,454,972 B2 to Nabel, et. al., granted on June 4, 2013; incorporated by reference herein, and the dosages at col 29, lines 36-58 thereof. In an embodiment herein, the adenovirus is delivered via multiple doses.
[0264] In an embodiment herein, the delivery is via an AAV. A therapeutically effective dosage for in vivo delivery of the AAV to a human is believed to be in the range of from about 20 to about 50 ml of saline solution containing from about 1 x 1010 to about 1 x 1010 functional AAV/ml solution. The dosage may be adjusted to balance the therapeutic benefit against any side effects. In an embodiment herein, the AAV dose is generally in the range of concentrations of from about 1 x 105 to 1 x 1050 genomes AAV, from about 1 x 108 to 1 x 1020 genomes AAV, from about 1 x 1010 to about 1 x 1016 genomes, or about 1 x 1011 to about 1 x 1016 genomes AAV. A human dosage may be about 1 x 1013 genomes AAV. Such concentrations may be delivered in from about 0.001 ml to about 100 ml, about 0.05 to about 50 ml, or about 10 to about 25 ml of a carrier solution. Other effective dosages can be readily established by one of ordinary skill in the art through routine trials establishing dose response curves. See, for example, U.S. Patent No. 8,404,658 B2 to Hajjar, et al., granted on March 26, 2013, at col. 27, lines 45-60.
[0265] In an embodiment herein the delivery is via a plasmid. In such plasmid compositions, the dosage should be a sufficient amount of plasmid to elicit a response. For instance, suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg, or from about 1 g to about 10 g per 70 kg individual. Plasmids of the invention will generally comprise (i) a promoter; (ii) a sequence encoding a CRISPR enzyme, operably linked to said promoter; (iii) a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii). The plasmid can also encode the RNA components of a CRISPR complex, but one or more of these may instead be encoded on a different vector. [0266] The doses herein are based on an average 70 kg individual. The frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), or scientist skilled in the art. It is also noted that mice used in experiments are typically about 20g and from mice experiments one can scale up to a 70 kg individual.
[0267] In some embodiments the RNA molecules of the invention are delivered in liposome or lipofectin formulations and the like and can be prepared by methods well known to those skilled in the art. Such methods are described, for example, in U.S. Pat. Nos. 5,593,972, 5,589,466, and 5,580,859, which are herein incorporated by reference. Delivery systems aimed specifically at the enhanced and improved delivery of siRNA into mammalian cells have been developed, (see, for example, Shen et al FEBS Let. 2003, 539:111 -114; Xia et al., Nat. Biotech. 2002, 20:1006-1010; Reich et al., Mol. Vision. 2003, 9: 210-216; Sorensen et al., J. Mol. Biol. 2003, 327: 761-766; Lewis et al., Nat. Gen. 2002, 32: 107-108 and Simeoni et al., NAR 2003, 31, 11: 2717-2724) and may be applied to the present invention. siRNA has recently been successfully used for inhibition of gene expression in primates (see for example. Tolentino et al., Retina 24(4):660 which may also be applied to the present invention.
[0268] Indeed, RNA delivery is a useful method of in vivo delivery. It is possible to deliver Cas9 and gRNA (and, for instance, HR repair template) into cells using liposomes or nanoparticles. Thus delivery of the CRISPR enzyme, such as a Cas9 and/or delivery of the RNAs of the invention may be in RNA form and via microvesicles, liposomes or nanoparticles. For example, Cas9 mRNA and gRNA can be packaged into liposomal particles for delivery in vivo. Liposomal transfection reagents such as lipofectamine from Life Technologies and other reagents on the market can effectively deliver RNA molecules into the liver.
[0269] Means of delivery of RNA also preferred include delivery of RNA via nanoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells, Advanced Functional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A., Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-based nanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID: 20059641). Indeed, exosomes have been shown to be particularly useful in delivery siRNA, a system with some parallels to the CRISPR system. For instance, El-Andaloussi S, et al. (“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc. 2012 Dec;7(12):2112-26. doi: 10.1038/nprot.2012.131. Epub 2012 Nov 15.) describe how exosomes are promising tools for drug delivery across different biological barriers and can be harnessed for delivery of siRNA in vitro and in vivo. Their approach is to generate targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. The exosomes are then purify and characterized from transfected cell supernatant, then RNA is loaded into the exosomes. Delivery or administration according to the invention can be performed with exosomes, in particular but not limited to the brain. Vitamin E (Į -tocopherol) may be conjugated with CRISPR Cas and delivered to the brain along with high density lipoprotein (HDL), for example in a similar manner as was done by Uno et al. (HUMAN GENE THERAPY 22:711–719 (June 2011)) for delivering short-interfering RNA (siRNA) to the brain. Mice were infused via Osmotic minipumps (model 1007D; Alzet, Cupertino, CA) filled with phosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL and connected with Brain Infusion Kit 3 (Alzet). A brain- infusion cannula was placed about 0.5mm posterior to the bregma at midline for infusion into the dorsal third ventricle. Uno et al. found that as little as 3 nmol of Toc-siRNA with HDL could induce a target reduction in comparable degree by the same ICV infusion method. A similar dosage of CRISPR Cas conjugated to Į -tocopherol and co-administered with HDL targeted to the brain may be contemplated for humans in the present invention, for example, about 3 nmol to about 3 mol of CRISPR Cas targeted to the brain may be contemplated. Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April 2011)) describes a method of lentiviral-mediated delivery of short-hairpin RNAs targeting PKCȖ for in vivo gene silencing in the spinal cord of rats. Zou et al. administered about 10 l of a recombinant lentivirus having a titer of 1 x 109 transducing units (TU)/ml by an intrathecal catheter. A similar dosage of CRISPR Cas expressed in a lentiviral vector targeted to the brain may be contemplated for humans in the present invention, for example, about 10-50 ml of CRISPR Cas targeted to the brain in a lentivirus having a titer of 1 x 109 transducing units (TU)/ml may be contemplated.
[0270] In terms of local delivery to the brain, this can be achieved in various ways. For instance, material can be delivered intrastriatally e.g. by injection. Injection can be performed stereotactically via a craniotomy.
[0271] Enhancing NHEJ or HR efficiency is also helpful for delivery. It is preferred that NHEJ efficiency is enhanced by co-expressing end-processing enzymes such as Trex2 (Dumitrache et al. Genetics. 2011 August; 188(4): 787–797). It is preferred that HR efficiency is increased by transiently inhibiting NHEJ machineries such as Ku70 and Ku86. HR efficiency can also be increased by co-expressing prokaryotic or eukaryotic homologous recombination enzymes such as RecBCD, RecA. Packaging and Promoters generally
[0272] Ways to package Cas9 coding nucleic acid molecules, e.g., DNA, into vectors, e.g., viral vectors, to mediate genome modification in vivo include:
To achieve NHEJ-mediated gene knockout:
Single virus vector:
Vector containing two or more expression cassettes:
Promoter-Cas9 coding nucleic acid molecule -terminator
Promoter-gRNA1-terminator
Promoter-gRNA2-terminator
Promoter-gRNA(N)-terminator (up to size limit of vector) Double virus vector:
Vector 1 containing one expression cassette for driving the expression of Cas9 Promoter-Cas9 coding nucleic acid molecule-terminator
Vector 2 containing one more expression cassettes for driving the expression of one or more guideRNAs
Promoter-gRNA1-terminator
Promoter-gRNA(N)-terminator (up to size limit of vector)
To mediate homology-directed repair.
[0273] In addition to the single and double virus vector approaches described above, an additional vector is used to deliver a homology-direct repair template.
[0274] The promoter used to drive Cas9 coding nucleic acid molecule expression can include:
AAV ITR can serve as a promoter: this is advantageous for eliminating the need for an additional promoter element (which can take up space in the vector). The additional space freed up can be used to drive the expression of additional elements (gRNA, etc.). Also, ITR activity is relatively weaker, so can be used to reduce potential toxicity due to over expression of Cas9.
For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.
For brain or other CNS expression, can use promoters: SynapsinI for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for GABAergic neurons, etc.
For liver expression can use Albumin promoter For lung expression, can use SP-B.
For endothelial cells, can use ICAM.
For hematopoietic cells can use IFNbeta or CD45.
For Osteoblasts can use OG-2.
[0275] The promoter used to drive guide RNA can include:
Pol III promoters such as U6 or H1
Use of Pol II promoter and intronic cassettes to express gRNA
Adeno associated virus (AAV)
[0276] Cas9 and one or more guide RNA can be delivered using adeno associated virus (AAV), lentivirus, adenovirus or other plasmid or viral vector types, in particular, using formulations and doses from, for example, US Patents Nos. 8,454,972 (formulations, doses for adenovirus), 8,404,658 (formulations, doses for AAV) and 5,846,946 (formulations, doses for DNA plasmids) and from clinical trials and publications regarding the clinical trials involving lentivirus, AAV and adenovirus. For examples, for AAV, the route of administration, formulation and dose can be as in US Patent No. 8,454,972 and as in clinical trials involving AAV. For Adenovirus, the route of administration, formulation and dose can be as in US Patent No. 8,404,658 and as in clinical trials involving adenovirus. For plasmid delivery, the route of administration, formulation and dose can be as in US Patent No 5,846,946 and as in clinical studies involving plasmids. Doses may be based on or extrapolated to an average 70 kg individual (e.g. a male adult human), and can be adjusted for patients, subjects, mammals of different weight and species. Frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian), depending on usual factors including the age, sex, general health, other conditions of the patient or subject and the particular condition or symptoms being addressed. The viral vectors can be injected into the tissue of interest. For cell- type specific genome modification, the expression of Cas9 can be driven by a cell-type specific promoter. For example, liver-specific expression might use the Albumin promoter and neuron- specific expression (e.g. for targeting CNS disorders) might use the Synapsin I promoter.
[0277] In terms of in vivo delivery, AAV is advantageous over other viral vectors for a couple of reasons:
Low toxicity (this may be due to the purification method not requiring ultra centrifugation of cell particles that can activate the immune response) Low probability of causing insertional mutagenesis because it doesn’t integrate into the host genome.
[0278] AAV has a packaging limit of 4.5 or 4.75 Kb. This means that Cas9 as well as a promoter and transcription terminator have to be all fit into the same viral vector. Constructs larger than 4.5 or 4.75 Kb will lead to significantly reduced virus production. SpCas9 is quite large, the gene itself is over 4.1 Kb, which makes it difficult for packing into AAV. Therefore embodiments of the invention include utilizing homologs of Cas9 that are shorter. For example:
Species Cas9 Size Corynebacter diphtheriae 3252 Eubacterium ventriosum 3321 Streptococcus pasteurianus 3390 Lactobacillus farciminis 3378 Sphaerochaeta globus 3537 Azospirillum B510 3504 Gluconacetobacter diazotrophicus 3150 Neisseria cinerea 3246 Roseburia intestinalis 3420 Parvibaculum lavamentivorans 3111 Staphylococcus aureus 3159 Nitratifractor salsuginis DSM 16511 3396 Campylobacter lari CF89-12 3009 Streptococcus thermophilus LMD-9 3396 [0279] These species are therefore, in general, preferred Cas9 species.
[0280] As to AAV, the AAV can be AAV1, AAV2, AAV5 or any combination thereof. One can select the AAV of the AAV with regard to the cells to be targeted; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAV8 is useful for delivery to the liver. The herein promoters and vectors are preferred individually. A tabulation of certain AAV serotypes as to these cells (see Grimm, D. et al, J. Virol. 82: 5887- 5911 (2008)) is as follows:
Figure imgf000082_0001
Figure imgf000083_0001
Lentivirus
[0281] Lentiviruses are complex retroviruses that have the ability to infect and express their genes in both mitotic and post-mitotic cells. The most commonly known lentivirus is the human immunodeficiency virus (HIV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types.
[0282] Lentiviruses may be prepared as follows. After cloning pCasES10 (which contains a lentiviral transfer plasmid backbone), HEK293FT at low passage (p=5) were seeded in a T-75 flask to 50% confluence the day before transfection in DMEM with 10% fetal bovine serum and without antibiotics. After 20 hours, media was changed to OptiMEM (serum-free) media and transfection was done 4 hours later. Cells were transfected with 10 g of lentiviral transfer plasmid (pCasES10) and the following packaging plasmids: 5 g of pMD2.G (VSV-g pseudotype), and 7.5ug of psPAX2 (gag/pol/rev/tat). Transfection was done in 4mL OptiMEM with a cationic lipid delivery agent (50uL Lipofectamine 2000 and 100ul Plus reagent). After 6 hours, the media was changed to antibiotic-free DMEM with 10% fetal bovine serum. These methods use serum during cell culture, but serum-free methods are preferred.
[0283] Lentivirus may be purified as follows. Viral supernatants were harvested after 48 hours. Supernatants were first cleared of debris and filtered through a 0.45um low protein binding (PVDF) filter. They were then spun in a ultracentrifuge for 2 hours at 24,000 rpm. Viral pellets were resuspended in 50ul of DMEM overnight at 4C. They were then aliquotted and immediately frozen at -80°C.
[0284] In another embodiment, minimal non-primate lentiviral vectors based on the equine infectious anemia virus (EIAV) are also contemplated, especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8: 275– 285). In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostatin and angiostatin that is delivered via a subretinal injection for the treatment of the web form of age related macular degeneration is also contemplated (see e g Binley et al HUMAN GENE THERAPY 23:980–991 (September 2012)) and this vector may be modified for the CRISPR-Cas system of the present invention.
[0285] In another embodiment, self-inactivating lentiviral vectors with an siRNA targeting a common exon shared by HIV tat/rev, a nucleolar-localizing TAR decoy, and an anti–CCR5- specific hammerhead ribozyme (see, e.g., DiGiusto et al. (2010) Sci Transl Med 2:36ra43) may be used/and or adapted to the CRISPR-Cas system of the present invention. A minimum of 2.5 × 106 CD34+ cells per kilogram patient weight may be collected and prestimulated for 16 to 20 hours in X-VIVO 15 medium (Lonza) containing 2 mol/L-glutamine, stem cell factor (100 ng/ml), Flt-3 ligand (Flt-3L) (100 ng/ml), and thrombopoietin (10 ng/ml) (CellGenix) at a density of 2 × 106 cells/ml. Prestimulated cells may be transduced with lentiviral at a multiplicity of infection of 5 for 16 to 24 hours in 75-cm2 tissue culture flasks coated with fibronectin (25 mg/cm2) (RetroNectin,Takara Bio Inc.).
[0286] Lentiviral vectors have been disclosed as in the treatment for Parkinson’s Disease, see, e.g., US Patent Publication No. 20120295960 and US Patent Nos. 7303910 and 7351585. Lentiviral vectors have also been disclosed for the treatment of ocular diseases, see e.g., US Patent Publication Nos. 20060281180, 20090007284, US20110117189; US20090017543; US20070054961, US20100317109. Lentiviral vectors have also been disclosed for delivery to the brain, see, e.g., US Patent Publication Nos. US20110293571; US20110293571, US20040013648, US20070025970, US20090111106 and US Patent No. US7259015.
RNA delivery
[0287] RNA delivery: The CRISPR enzyme, for instance a Cas9, and/or any of the present RNAs, for instance a guide RNA, can also be delivered in the form of RNA. Cas9 mRNA can be generated using in vitro transcription. For example, Cas9 mRNA can be synthesized using a PCR cassette containing the following elements: T7_promoter-kozak sequence (GCCACC)-Cas9-3’ UTR from beta globin-polyA tail (a string of 120 or more adenines). The cassette can be used for transcription by T7 polymerase. Guide RNAs can also be transcribed using in vitro transcription from a cassette containing T7_promoter-GG-guide RNA sequence.
[0288] To enhance expression and reduce possible toxicity, the CRISPR enzyme-coding sequence and/or the guide RNA can be modified to include one or more modified nucleoside e.g. using pseudo-U or 5-Methyl-C.
[0289] mRNA delivery methods are especially promising for liver delivery currently. [0290] Much clinical work on RNA delivery has focused on RNAi or antisense, but these systems can be adapted for delivery of RNA for implementing the present invention. References below to RNAi etc. should be read accordingly.
Nanoparticles
[0291] CRISPR enzyme mRNA and guide RNA may be delivered simultaneously using nanoparticles or lipid envelopes. For example, Su X, Fricke J, Kavanagh DG, Irvine DJ (“In vitro and in vivo mRNA delivery using lipid-enveloped pH-responsive polymer nanoparticles” Mol Pharm. 2011 Jun 6;8(3):774-87. doi: 10.1021/mp100390w. Epub 2011 Apr 1) describes biodegradable core-shell structured nanoparticles with a poly(ȕ-amino ester) (PBAE) core enveloped by a phospholipid bilayer shell. These were developed for in vivo mRNA delivery. The pH-responsive PBAE component was chosen to promote endosome disruption, while the lipid surface layer was selected to minimize toxicity of the polycation core. Such are, therefore, preferred for delivering RNA of the present invention.
[0292] In one embodiment, nanoparticles based on self assembling bioadhesive polymers are contemplated, which may be applied to oral delivery of peptides, intravenous delivery of peptides and nasal delivery of peptides, all to the brain. Other embodiments, such as oral absorption and ocular delivery of hydrophobic drugs are also contemplated. The molecular envelope technology involves an engineered polymer envelope which is protected and delivered to the site of the disease (see, e.g., Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. Mol Pharm, 2012. 9(1):14-28; Lalatsa, A., et al. J Contr Rel, 2012. 161(2):523-36; Lalatsa, A., et al., Mol Pharm, 2012. 9(6):1665-80; Lalatsa, A., et al. Mol Pharm, 2012. 9(6):1764-74; Garrett, N.L., et al. J Biophotonics, 2012. 5(5-6):458-68; Garrett, N.L., et al. J Raman Spect, 2012. 43(5):681-688; Ahmad, S., et al. J Royal Soc Interface 2010. 7:S423-33; Uchegbu, I.F. Expert Opin Drug Deliv, 2006. 3(5):629-40; Qu, X.,et al. Biomacromolecules, 2006. 7(12):3452- 9 and Uchegbu, I.F., et al. Int J Pharm, 2001. 224:185-199). Doses of about 5 mg/kg are contemplated, with single or multiple doses, depending on the target tissue.
[0293] In one embodiment, nanoparticles that can deliver RNA to a cancer cell to stop tumor growth developed by Dan Anderson’s lab at MIT may be used/and or adapted to the CRISPR Cas system of the present invention. In particular, the Anderson lab developed fully automated, combinatorial systems for the synthesis, purification, characterization, and formulation of new biomaterials and nanoformulations. See, e.g., Alabi et al., Proc Natl Acad Sci U S A. 2013 Aug 6;110(32):12881-6; Zhang et al., Adv Mater. 2013 Sep 6;25(33):4641-5; Jiang et al., Nano Lett. 2013 Mar 13;13(3):1059-64; Karagiannis et al., ACS Nano. 2012 Oct 23;6(10):8484-7; Whitehead et al., ACS Nano. 2012 Aug 28;6(8):6922-9 and Lee et al., Nat Nanotechnol. 2012 Jun 3;7(6):389-93. US patent application 20110293703 relates to lipidoid compounds are also particularly useful in the administration of polynucleotides, which may be applied to deliver the CRISPR Cas system of the present invention. In one aspect, the aminoalcohol lipidoid compounds are combined with an agent to be delivered to a cell or a subject to form microparticles, nanoparticles, liposomes, or micelles. The agent to be delivered by the particles, liposomes, or micelles may be in the form of a gas, liquid, or solid, and the agent may be a polynucleotide, protein, peptide, or small molecule. The minoalcohol lipidoid compounds may be combined with other aminoalcohol lipidoid compounds, polymers (synthetic or natural), surfactants, cholesterol, carbohydrates, proteins, lipids, etc. to form the particles. These particles may then optionally be combined with a pharmaceutical excipient to form a pharmaceutical composition. US Patent Publication No. 20110293703 also provides methods of preparing the aminoalcohol lipidoid compounds. One or more equivalents of an amine are allowed to react with one or more equivalents of an epoxide-terminated compound under suitable conditions to form an aminoalcohol lipidoid compound of the present invention. In certain embodiments, all the amino groups of the amine are fully reacted with the epoxide-terminated compound to form tertiary amines. In other embodiments, all the amino groups of the amine are not fully reacted with the epoxide-terminated compound to form tertiary amines thereby resulting in primary or secondary amines in the aminoalcohol lipidoid compound. These primary or secondary amines are left as is or may be reacted with another electrophile such as a different epoxide-terminated compound. As will be appreciated by one skilled in the art, reacting an amine with less than excess of epoxide-terminated compound will result in a plurality of different aminoalcohol lipidoid compounds with various numbers of tails. Certain amines may be fully functionalized with two epoxide-derived compound tails while other molecules will not be completely functionalized with epoxide-derived compound tails. For example, a diamine or polyamine may include one, two, three, or four epoxide-derived compound tails off the various amino moieties of the molecule resulting in primary, secondary, and tertiary amines. In certain embodiments, all the amino groups are not fully functionalized. In certain embodiments, two of the same types of epoxide-terminated compounds are used. In other embodiments, two or more different epoxide- terminated compounds are used. The synthesis of the aminoalcohol lipidoid compounds is performed with or without solvent, and the synthesis may be performed at higher temperatures ranging from 30.-100 C., preferably at approximately 50.-90 C. The prepared aminoalcohol lipidoid compounds may be optionally purified. For example, the mixture of aminoalcohol lipidoid compounds may be purified to yield an aminoalcohol lipidoid compound with a particular number of epoxide-derived compound tails. Or the mixture may be purified to yield a particular stereo- or regioisomer. The aminoalcohol lipidoid compounds may also be alkylated using an alkyl halide (e.g., methyl iodide) or other alkylating agent, and/or they may be acylated. US Patent Publication No. 20110293703 also provides libraries of aminoalcohol lipidoid compounds prepared by the inventive methods. These aminoalcohol lipidoid compounds may be prepared and/or screened using high-throughput techniques involving liquid handlers, robots, microtiter plates, computers, etc. In certain embodiments, the aminoalcohol lipidoid compounds are screened for their ability to transfect polynucleotides or other agents (e.g., proteins, peptides, small molecules) into the cell. US Patent Publication No. 20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) has been prepared using combinatorial polymerization. The inventive PBAAs may be used in biotechnology and biomedical applications as coatings (such as coatings of films or multilayer films for medical devices or implants), additives, materials, excipients, non-biofouling agents, micropatterning agents, and cellular encapsulation agents. When used as surface coatings, these PBAAs elicited different levels of inflammation, both in vitro and in vivo, depending on their chemical structures. The large chemical diversity of this class of materials allowed us to identify polymer coatings that inhibit macrophage activation in vitro. Furthermore, these coatings reduce the recruitment of inflammatory cells, and reduce fibrosis, following the subcutaneous implantation of carboxylated polystyrene microparticles. These polymers may be used to form polyelectrolyte complex capsules for cell encapsulation. The invention may also have many other biological applications such as antimicrobial coatings, DNA or siRNA delivery, and stem cell tissue engineering. The teachings of US Patent Publication No. 20130302401 may be applied to the CRISPR Cas system of the present invention.
[0294] In another embodiment, lipid nanoparticles (LNPs) are contemplated. In particular, an antitransthyretin small interfering RNA encapsulated in lipid nanoparticles (see, e.g., Coelho et al., N Engl J Med 2013;369:819-29) may be applied to the CRISPR Cas system of the present invention. Doses of about 0.01 to about 1 mg per kg of body weight administered intravenously are contemplated. Medications to reduce the risk of infusion-related reactions are contemplated, such as dexamethasone, acetampinophen, diphenhydramine or cetirizine, and ranitidine are contemplated. Multiple doses of about 0.3 mg per kilogram every 4 weeks for five doses are also contemplated. LNPs have been shown to be highly effective in delivering siRNAs to the liver (see, e.g., Tabernero et al., Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470) and are therefore contemplated for delivering RNA encoding CRISPR Cas to the liver. A dosage of about four doses of 6 mg/kg of the LNP every two weeks may be contemplated. Tabernero et al. demonstrated that tumor regression was observed after the first 2 cycles of LNPs dosed at 0.7 mg/kg, and by the end of 6 cycles the patient had achieved a partial response with complete regression of the lymph node metastasis and substantial shrinkage of the liver tumors. A complete response was obtained after 40 doses in this patient, who has remained in remission and completed treatment after receiving doses over 26 months. Two patients with RCC and extrahepatic sites of disease including kidney, lung, and lymph nodes that were progressing following prior therapy with VEGF pathway inhibitors had stable disease at all sites for approximately 8 to 12 months, and a patient with PNET and liver metastases continued on the extension study for 18 months (36 doses) with stable disease. However, the charge of the LNP must be taken into consideration. As cationic lipids combined with negatively charged lipids to induce nonbilayer structures that facilitate intracellular delivery. Because charged LNPs are rapidly cleared from circulation following intravenous injection, ionizable cationic lipids with pKa values below 7 were developed (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011). Negatively charged polymers such asiRNA may be loaded into LNPs at low pH values (e.g., pH 4) where the ionizable lipids display a positive charge. However, at physiological pH values, the LNPs exhibit a low surface charge compatible with longer circulation times. Four species of ionizable cationic lipids have been focused upon, namely 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-keto-N,N-dimethyl-3-aminopropane (DLinKDMA), and 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA). It has been shown that LNP siRNA systems containing these lipids exhibit remarkably different gene silencing properties in hepatocytes in vivo, with potencies varying according to the series DLinKC2-DMA>DLinKDMA>DLinDMA>>DLinDAP employing a Factor VII gene silencing model (see, e.g., Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011). A dosage of 1 g/ml levels may be contemplated, especially for a formulation containing DLinKC2-DMA.
[0295] Preparation of LNPs and CRISPR Cas encapsulation may be used/and or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011). The cationic lipids 1,2-dilineoyl-3-dimethylammonium-propane (DLinDAP), 1,2-dilinoleyloxy-3-N,N- dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxyketo-N,N-dimethyl-3-aminopropane (DLinK-DMA), 1,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLinKC2-DMA), (3- o-[2Ǝ-(methoxypolyethyleneglycol 2000) succinoyl]-1,2-dimyristoyl-sn-glycol (PEG-S-DMG), and R-3-[( -methoxy-poly(ethylene glycol)2000) carbamoyl]-1,2-dimyristyloxlpropyl-3-amine (PEG-C-DOMG) may be provided by Tekmira Pharmaceuticals (Vancouver, Canada) or synthesized. Cholesterol may be purchased from Sigma (St Louis, MO). The specific CRISPR Cas RNA may be encapsulated in LNPs containing DLinDAP, DLinDMA, DLinK-DMA, and DLinKC2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40:10:40:10 molar ratios). When required, 0.2% SP-DiOC18 (Invitrogen, Burlington, Canada) may be incorporated to assess cellular uptake, intracellular delivery, and biodistribution. Encapsulation may be performed by dissolving lipid mixtures comprised of cationic lipid:DSPC:cholesterol:PEG-c-DOMG (40:10:40:10 molar ratio) in ethanol to a final lipid concentration of 10 mmol/l. This ethanol solution of lipid may be added drop-wise to 50 mmol/l citrate, pH 4.0 to form multilamellar vesicles to produce a final concentration of 30% ethanol vol/vol. Large unilamellar vesicles may be formed following extrusion of multilamellar vesicles through two stacked 80 nm Nuclepore polycarbonate filters using the Extruder (Northern Lipids, Vancouver, Canada). Encapsulation may be achieved by adding RNA dissolved at 2 mg/ml in 50 mmol/l citrate, pH 4.0 containing 30% ethanol vol/vol drop-wise to extruded preformed large unilamellar vesicles and incubation at 31 °C for 30 minutes with constant mixing to a final RNA/lipid weight ratio of 0.06/1 wt/wt. Removal of ethanol and neutralization of formulation buffer were performed by dialysis against phosphate-buffered saline (PBS), pH 7.4 for 16 hours using Spectra/Por 2 regenerated cellulose dialysis membranes. Nanoparticle size distribution may be determined by dynamic light scattering using a NICOMP 370 particle sizer, the vesicle/intensity modes, and Gaussian fitting (Nicomp Particle Sizing, Santa Barbara, CA). The particle size for all three LNP systems may be ~70 nm in diameter. siRNA encapsulation efficiency may be determined by removal of free siRNA using VivaPureD MiniH columns (Sartorius Stedim Biotech) from samples collected before and after dialysis. The encapsulated RNA may be extracted from the eluted nanoparticles and quantified at 260 nm. siRNA to lipid ratio was determined by measurement of cholesterol content in vesicles using the Cholesterol E enzymatic assay from Wako Chemicals USA (Richmond, VA). Preparation of large LNPs may be used/and or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 2011. A lipid premix solution (20.4 mg/ml total lipid concentration) may be prepared in ethanol containing DLinKC2-DMA, DSPC, and cholesterol at 50:10:38.5 molar ratios. Sodium acetate may be added to the lipid premix at a molar ratio of 0.75:1 (sodium acetate:DLinKC2- DMA). The lipids may be subsequently hydrated by combining the mixture with 1.85 volumes of citrate buffer (10 mmol/l, pH 3.0) with vigorous stirring, resulting in spontaneous liposome formation in aqueous buffer containing 35% ethanol. The liposome solution may be incubated at 37 °C to allow for time-dependent increase in particle size. Aliquots may be removed at various times during incubation to investigate changes in liposome size by dynamic light scattering (Zetasizer Nano ZS, Malvern Instruments, Worcestershire, UK). Once the desired particle size is achieved, an aqueous PEG lipid solution (stock = 10 mg/ml PEG-DMG in 35% (vol/vol) ethanol) may be added to the liposome mixture to yield a final PEG molar concentration of 3.5% of total lipid. Upon addition of PEG-lipids, the liposomes should their size, effectively quenching further growth. RNA may then be added to the empty liposomes at an siRNA to total lipid ratio of approximately 1:10 (wt:wt), followed by incubation for 30 minutes at 37 °C to form loaded LNPs. The mixture may be subsequently dialyzed overnight in PBS and filtered with a 0.45- m syringe filter.
[0296] Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) are also contemplate as a means to delivery CRISPR/Cas system to intended targets. Significant data show that AuraSense Therapeutics' Spherical Nucleic Acid (SNA™) constructs, based upon nucleic acid-functionalized gold nanoparticles, are superior to alternative platforms based on multiple key success factors, such as: High in vivo stability: Due to their dense loading, a majority of cargo (DNA or siRNA) remains bound to the constructs inside cells, conferring nucleic acid stability and resistance to enzymatic degradation. Deliverability: For all cell types studied (e.g., neurons, tumor cell lines, etc.) the constructs demonstrate a transfection efficiency of 99% with no need for carriers or transfection agents. Therapeutic targeting: The unique target binding affinity and specificity of the constructs allow exquisite specificity for matched target sequences (i.e., limited off-target effects). Superior efficacy: The constructs significantly outperform leading conventional transfection reagents (Lipofectamine 2000 and Cytofectin). Low toxicity: The constructs can enter a variety of cultured cells, primary cells, and tissues with no apparent toxicity. No significant immune response: The constructs elicit minimal changes in global gene expression as measured by whole-genome microarray studies and cytokine-specific protein assays. Chemical tailorability: Any number of single or combinatorial agents (e.g., proteins, peptides, small molecules) can be used to tailor the surface of the constructs. This platform for nucleic acid-based therapeutics may be applicable to numerous disease states, including inflammation and infectious disease, cancer, skin disorders and cardiovascular disease. Citable literature includes: Cutler et al.,. J. Am. Chem. Soc. 2011 133:9254-9257, Hao et al., Small. 2011 7:3158-3162, Zhang et al., ACS Nano. 2011 5:6962- 6970, Cutler et al., J. Am. Chem. Soc. 2012 134:1376-1391, Young et al.,. Nano Lett. 2012 12:3867-71, Zheng et al., Proc. Natl. Acad. Sci. USA. 2012 109:11975-80, Mirkin, Nanomedicine 2012 7:635-638 Zhang et al., J. Am. Chem. Soc. 2012 134:16488-1691, Weintraub, Nature 2013 495:S14-S16, Choi et al., Proc. Natl. Acad. Sci. USA. 2013 110(19):7625-7630, Jensen et al., Sci. Transl. Med. 5, 209ra152 (2013) and Mirkin, et al., Small, 10:186-192.
[0297] Self-assembling nanoparticles with siRNA may be constructed with polyethyleneimine (PEI) that is PEGylated with an Arg-Gly-Asp (RGD) peptide ligand attached at the distal end of the polyethylene glycol (PEG), for example, as a means to target tumor neovasculature expressing integrins and used to deliver siRNA inhibiting vascular endothelial growth factor receptor-2 (VEGF R2) expression and thereby tumor angiogenesis (see, e.g., Schiffelers et al., Nucleic Acids Research, 2004, Vol. 32, No. 19). Nanoplexes may be prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid to give a net molar excess of ionizable nitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6. The electrostatic interactions between cationic polymers and nucleic acid resulted in the formation of polyplexes with average particle size distribution of about 100 nm, hence referred to here as nanoplexes. A dosage of about 100 to 200 mg of CRISPR Cas is envisioned for delivery in the self-assembling nanoparticles of Schiffelers et al.
[0298] The nanoplexes of Bartlett et al. (PNAS, September 25, 2007,vol. 104, no. 39) may also be applied to the present invention. The nanoplexes of Bartlett et al. are prepared by mixing equal volumes of aqueous solutions of cationic polymer and nucleic acid to give a net molar excess of ionizable nitrogen (polymer) to phosphate (nucleic acid) over the range of 2 to 6. The electrostatic interactions between cationic polymers and nucleic acid resulted in the formation of polyplexes with average particle size distribution of about 100 nm, hence referred to here as nanoplexes. The DOTA-siRNA of Bartlett et al. was synthesized as follows: 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid mono(N-hydroxysuccinimide ester) (DOTA- NHSester) was ordered from Macrocyclics (Dallas, TX). The amine modified RNA sense strand with a 100-fold molar excess of DOTA-NHS-ester in carbonate buffer (pH 9) was added to a microcentrifuge tube. The contents were reacted by stirring for 4 h at room temperature. The DOTA-RNAsense conjugate was ethanol-precipitated, resuspended in water, and annealed to the unmodified antisense strand to yield DOTA-siRNA. All liquids were pretreated with Chelex-100 (Bio-Rad, Hercules, CA) to remove trace metal contaminants. Tf-targeted and nontargeted siRNA nanoparticles may be formed by using cyclodextrin-containing polycations. Typically, nanoparticles were formed in water at a charge ratio of 3 (+/-) and an siRNA concentration of 0.5 g/liter. One percent of the adamantane-PEG molecules on the surface of the targeted nanoparticles were modified with Tf (adamantane-PEG-Tf). The nanoparticles were suspended in a 5% (wt/vol) glucose carrier solution for injection.
[0299] Davis et al. (Nature, Vol 464, 15 April 2010) conducts a siRNA clinical trial that uses a targeted nanoparticle-delivery system (clinical trial registration number NCT00689065). Patients with solid cancers refractory to standard-of-care therapies are administered doses of targeted nanoparticles on days 1, 3, 8 and 10 of a 21 -day cycle by a 30-min intravenous infusion. The nanoparticles consist of a synthetic delivery system containing: (1) a linear, cyclodextrin- based polymer (CDP), (2) a human transferrin protein (TF) targeting ligand displayed on the exterior of the nanoparticle to engage TF receptors (TFR) on the surface of the cancer cells, (3) a hydrophilic polymer (polyethylene glycol (PEG) used to promote nanoparticle stability in biological fluids), and (4) siRNA designed to reduce the expression of the RRM2 (sequence used in the clinic was previously denoted siR2B+5). The TFR has long been known to be upregulated in malignant cells, and RRM2 is an established anti-cancer target. These nanoparticles (clinical version denoted as CALAA-01) have been shown to be well tolerated in multi-dosing studies in non-human primates. Although a single patient with chronic myeloid leukaemia has been administered siRNAby liposomal delivery, Davis et al.’s clinical trial is the initial human trial to systemically deliver siRNA with a targeted delivery system and to treat patients with solid cancer. To ascertain whether the targeted delivery system can provide effective delivery of functional siRNA to human tumours, Davis et al. investigated biopsies from three patients from three different dosing cohorts; patients A, B and C, all of whom had metastatic melanoma and received CALAA-01 doses of 18, 24 and 30 mg m-2 siRNA, respectively. Similar doses may also be contemplated for the CRISPR Cas system of the present invention. The delivery of the invention may be achieved with nanoparticles containing a linear, cyclodextrin-based polymer (CDP), a human transferrin protein (TF) targeting ligand displayed on the exterior of the nanoparticle to engage TF receptors (TFR) on the surface of the cancer cells and/or a hydrophilic polymer (for example, polyethylene glycol (PEG) used to promote nanoparticle stability in biological fluids). Exosomes
[0300] Exosomes are endogenous nano-vesicles that transport RNAs and proteins which can deliver short interfering (si)RNA to the brain and other target organs. To reduce immunogenicity, Alvarez-Erviti et al. (2011, Nat Biotechnol 29: 341) used self-derived dendritic cells for exosome production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptide3. Purified exosomes were loaded with exogenous siRNA by electroporation. Intravenously injected RVG-targeted exosomes delivered GAPDH siRNA specifically to neurons, microglia, oligodendrocytes in the brain, resulting in a specific gene knockdown. Pre-exposure to RVG exosomes did not attenuate knockdown, and non-specific uptake in other tissues was not observed. The therapeutic potential of exosome-mediated siRNA delivery was demonstrated by the strong mRNA (60%) and protein (62%) knockdown of BACE1, a therapeutic target in Alzheimer's disease. To obtain a pool of immunologically inert exosomes, Alvarez-Erviti et al. harvested bone marrow from inbred C57BL/6 mice with a homogenous major histocompatibility complex (MHC) haplotype. As immature dendritic cells produce large quantities of exosomes devoid of T-cell activators such as MHC-II and CD86, Alvarez-Erviti et al. selected for dendritic cells with granulocyte/macrophage-colony stimulating factor (GM-CSF) for 7 d. Exosomes were purified from the culture supernatant the following day using well-established ultracentrifugation protocols. The exosomes produced were physically homogenous, with a size distribution peaking at 80 nm in diameter as determined by nanoparticle tracking analysis (NTA) and electron microscopy. Alvarez-Erviti et al. obtained 6-12 g of exosomes (measured based on protein concentration) per 106 cells. Next, Alvarez-Erviti et al. investigated the possibility of loading modified exosomes with exogenous cargoes using electroporation protocols adapted for nanoscale applications. As electroporation for membrane particles at the nanometer scale is not well-characterized, nonspecific Cy5-labeled siRNA was used for the empirical optimization of the electroporation protocol. The amount of encapsulated siRNA was assayed after ultracentrifugation and lysis of exosomes. Electroporation at 400 V and 125 F resulted in the greatest retention of siRNA and was used for all subsequent experiments. Alvarez-Erviti et al. administered 150 g of each BACE1 siRNA encapsulated in 150 g of RVG exosomes to normal C57BL/6 mice and compared the knockdown efficiency to four controls: untreated mice, mice injected with RVG exosomes only, mice injected with BACE1 siRNA complexed to an in vivo cationic liposome reagent and mice injected with BACE1 siRNA complexed to RVG-9R, the RVG pep tide conjugated to 9 D-arginines that electrostatically binds to the siRNA Cortical tissue samples were analyzed 3 d after administration and a significant protein knockdown (45%, P < 0.05, versus 62%, P < 0.01) in both siRNA-RVG-9R-treated and siRNARVG exosome- treated mice was observed, resulting from a significant decrease in BACE1 mRNA levels (66% [+ or -] 15%, P < 0.001 and 61% [+ or -] 13% respectively, P < 0.01). Moreover, Applicants demonstrated a significant decrease (55%, P < 0.05) in the total [beta]-amyloid 1-42 levels, a main component of the amyloid plaques in Alzheimer's pathology, in the RVG-exosome-treated animals. The decrease observed was greater than the ȕ-amyloid 1-40 decrease demonstrated in normal mice after intraventricular injection of BACE1 inhibitors. Alvarez-Erviti et al. carried out 5'-rapid amplification of cDNA ends (RACE) on BACE1 cleavage product, which provided evidence of RNAi-mediated knockdown by the siRNA. Finally, Alvarez-Erviti et al. investigated whether siRNA-RVG exosomes induced immune responses in vivo by assessing IL-6, IP-10, TNFĮ and IFN-Į serum concentrations. Following siRNA-RVG exosome treatment, nonsignificant changes in all cytokines were registered similar to siRNA-transfection reagent treatment in contrast to siRNA-RVG-9R, which potently stimulated IL-6 secretion, confirming the immunologically inert profile of the exosome treatment. Given that exosomes encapsulate only 20% of siRNA, delivery with RVG-exosome appears to be more efficient than RVG-9R delivery as comparable mRNA knockdown and greater protein knockdown was achieved with fivefold less siRNA without the corresponding level of immune stimulation. This experiment demonstrated the therapeutic potential of RVG-exosome technology, which is potentially suited for long-term silencing of genes related to neurodegenerative diseases. The exosome delivery system of Alvarez-Erviti et al. may be applied to deliver the CRISPR-Cas system of the present invention to therapeutic targets, especially neurodegenerative diseases. A dosage of about 100 to 1000 mg of CRISPR Cas encapsulated in about 100 to 1000 mg of RVG exosomes may be contemplated for the present invention.
[0301] El-Andaloussi et al. (Nature Protocols 7,2112–2126(2012)) discloses how exosomes derived from cultured cells can be harnessed for delivery of siRNA in vitro and in vivo. This protocol first describes the generation of targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. Next, El- Andaloussi et al. explain how to purify and characterize exosomes from transfected cell supernatant. Next, El-Andaloussi et al. detail crucial steps for loading siRNA into exosomes. Finally, El-Andaloussi et al. outline how to use exosomes to efficiently deliver siRNA in vitro and in vivo in mouse brain. Examples of anticipated results in which exosome-mediated siRNA delivery is evaluated by functional assays and imaging are also provided. The entire protocol takes 3 weeks. Delivery or administration according to the invention may be performed using exosomes produced from self-derived dendritic cells.
[0302] In another embodiment, the plasma exosomes of Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) are contemplated. Exosomes are nano-sized vesicles (30– 90nm in size) produced by many cell types, including dendritic cells (DC), B cells, T cells, mast cells, epithelial cells and tumor cells. These vesicles are formed by inward budding of late endosomes and are then released to the extracellular environment upon fusion with the plasma membrane. Because exosomes naturally carry RNA between cells, this property might be useful in gene therapy.
[0303] Exosomes from plasma are prepared by centrifugation of buffy coat at 900g for 20 min to isolate the plasma followed by harvesting cell supernatants, centrifuging at 300g for 10 min to eliminate cells and at 16 500g for 30 min followed by filtration through a 0.22 mm filter. Exosomes are pelleted by ultracentrifugation at 120 000g for70 min. Chemical transfection of siRNA into exosomes is carried out according to the manufacturer’s instructions in RNAi Human/Mouse Starter Kit (Quiagen, Hilden, Germany). siRNA is added to 100 ml PBS at a final concentration of 2 mmol/ml. After adding HiPerFect transfection reagent, the mixture is incubated for 10 min at RT. In order to remove the excess of micelles, the exosomes are re- isolated using aldehyde/sulfate latex beads. The chemical transfection of CRISPR Cas into exosomes may be conducted similarly to siRNA. The exosomes may be co-cultured with monocytes and lymphocytes isolated from the peripheral blood of healthy donors. Therefore, it may be contemplated that exosomes containing CRISPR Cas may be introduced to monocytes and lymphocytes of and autologously reintroduced into a human. Accordingly, delivery or administration according to the invention may beperformed using plasma exosomes.
Liposomes’
[0304] Delivery or administration according to the invention can be performed with liposomes. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bilayer surrounding internal aqueous compartments and a relatively impermeable outer lipophilic phospholipid bilayer. Liposomes have gained considerable attention as drug delivery carriers because they are biocompatible, nontoxic, can deliver both hydrophilic and lipophilic drug molecules, protect their cargo from degradation by plasma enzymes, and transport their load across biological membranes and the blood brain barrier (BBB) (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). Liposomes can be made from several different types of lipids; however, phospholipids are most commonly used to generate liposomes as drug carriers. Although liposome formation is spontaneous when a lipid film is mixed with an aqueous solution, it can also be expedited by applying force in the form of shaking by using a homogenizer, sonicator, or an extrusion apparatus (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). Several other additives may be added to liposomes in order to modify their structure and properties. For instance, either cholesterol or sphingomyelin may be added to the liposomal mixture in order to help stabilize the liposomal structure and to prevent the leakage of the liposomal inner cargo. Further, liposomes are prepared from hydrogenated egg phosphatidylcholine or egg phosphatidylcholine, cholesterol, and dicetyl phosphate, and their mean vesicle sizes were adjusted to about 50 and 100ௗnm. (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). A liposome formulation may be mainly comprised of natural phospholipids and lipids such as 1,2-distearoryl-sn-glycero-3-phosphatidyl choline (DSPC), sphingomyelin, egg phosphatidylcholines and monosialoganglioside. Since this formulation is made up of phospholipids only, liposomal formulations have encountered many challenges, one of the ones being the instability in plasma. Several attempts to overcome these challenges have been made, specifically in the manipulation of the lipid membrane. One of these attempts focused on the manipulation of cholesterol. Addition of cholesterol to conventional formulations reduces rapid release of the encapsulated bioactive compound into the plasma or 1,2-dioleoyl-sn-glycero-3- phosphoethanolamine (DOPE) increases the stability (see, e.g., Spuch and Navarro, Journal of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doi:10.1155/2011/469679 for review). In a particularly advantageous embodiment, Trojan Horse liposomes (also known as Molecular Trojan Horses) are desirable and protocols may be found at http://cshprotocols.cshlp.org/content/2010/4/pdb.prot5407.long. These particles allow delivery of a transgene to the entire brain after an intravascular injection. Without being bound by limitation, it is believed that neutral lipid particles with specific antibodies conjugated to surface allow crossing of the blood brain barrier via endocytosis. Utilizing Trojan Horse Liposomes to deliver the CRISPR family of nucleases to the brain via an intravascular injection, can allow whole brain transgenic animals without the need for embryonic manipulation. About 1 -5 g of DNA or RNA may be contemplated for in vivo administration in liposomes. [0305] In another embodiment, the CRISPR Cas system may be administered in liposomes, such as a stable nucleic-acid-lipid particle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005). Daily intravenous injections of about 1, 3 or 5 mg/kg/day of a specific CRISPR Cas targeted in a SNALP are contemplated. The daily treatment may be over about three days and then weekly for about five weeks. In another embodiment, a specific CRISPR Cas encapsulated SNALP) administered by intravenous injection to at doses of about 1 or 2.5 mg/kg are also contemplated (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006). The SNALP formulation may contain the lipids 3-N- [(wmethoxypoly(ethylene glycol) 2000) carbamoyl] -1 ,2-dimyristyloxy-propylamine (PEG-C- DMA), 1,2-dilinoleyloxy-N,N-dimethyl-3-aminopropane (DLinDMA), 1,2-distearoyl-sn- glycero-3-phosphocholine (DSPC) and cholesterol, in a 2:40:10:48 molar per cent ratio (see, e.g., Zimmerman et al., Nature Letters, Vol. 441, 4 May 2006).
[0306] In another embodiment, stable nucleic-acid-lipid particles (SNALPs) have proven to be effective delivery molecules to highly vascularized HepG2-derived liver tumors but not in poorly vascularized HCT-116 derived liver tumors (see, e.g., Li, Gene Therapy (2012) 19, 775– 780). The SNALP liposomes may be prepared by formulating D-Lin-DMA and PEG-C-DMA with distearoylphosphatidylcholine (DSPC), Cholesterol and siRNA using a 25:1 lipid/siRNA ratio and a 48/40/10/2 molar ratio of Cholesterol/D-Lin-DMA/DSPC/PEG-C-DMA. The resulted SNALP liposomes are about 80–100 nm in size.
[0307] In yet another embodiment, a SNALP may comprise synthetic cholesterol (Sigma- Aldrich, St Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), 3-N-[(w-methoxy poly(ethylene glycol)2000)carbamoyl]-1,2- dimyrestyloxypropylamine, and cationic 1,2-dilinoleyloxy-3-N,Ndimethylaminopropane (see, e.g., Geisbert et al., Lancet 2010; 375: 1896-905). A dosage of about 2 mg/kg total CRISPR Cas per dose administered as, for example, a bolus intravenous infusion may be contemplated.
[0308] In yet another embodiment, a SNALP may comprise synthetic cholesterol (Sigma- Aldrich), 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG- cDMA, and 1,2-dilinoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g., Judge, J. Clin. Invest. 119:661-673 (2009)). Formulations used for in vivo studies may comprise a final lipid/RNA mass ratio of about 9:1.
[0309] The safety profile of RNAi nanomedicines has been reviewed by Barros and Gollob of Alnylam Pharmaceuticals (see, e.g., Advanced Drug Delivery Reviews 64 (2012) 1730– 1737). The stable nucleic acid lipid particle (SNALP) is comprised of four different lipids— an ionizable lipid (DLinDMA) that is cationic at low pH, a neutral helper lipid, cholesterol, and a diffusible polyethylene glycol (PEG)-lipid. The particle is approximately 80 nm in diameter and is charge-neutral at physiologic pH. During formulation, the ionizable lipid serves to condense lipid with the anionic siRNA during particle formation. When positively charged under increasingly acidic endosomal conditions, the ionizable lipid also mediates the fusion of SNALP with the endosomal membrane enabling release of siRNA into the cytoplasm. The PEG-lipid stabilizes the particle and reduces aggregation during formulation, and subsequently provides a neutral hydrophilic exterior that improves pharmacokinetic properties.
[0310] To date, two clinical programs have been initiated using SNALPsiRNA formulations. Tekmira Pharmaceuticals recently completed a phase I single-dose study of SNALP-ApoB in adult volunteers with elevated LDL cholesterol. ApoB is predominantly expressed in the liver and jejunum and is essential for the assembly and secretion of VLDL and LDL. Seventeen subjects received a single dose of SNALP-ApoB (dose escalation across 7 dose levels). There was no evidence of liver toxicity (anticipated as the potential dose-limiting toxicity based on preclinical studies). One (of two) subjects at the highest dose experienced flu-like symptoms consistent with immune system stimulation, and the decision was made to conclude the trial. Alnylam Pharmaceuticals has similarly advanced ALN-TTR01, which employs the SNALP technology described above and targets hepatocyte production of both mutant and wild-type TTR to treat TTR amyloidosis (ATTR). Three ATTR syndromes have been described: familial amyloidotic polyneuropathy (FAP) and familial amyloidotic cardiomyopathy (FAC)— both caused by autosomal dominant mutations in TTR; and senile systemic amyloidosis (SSA) cause by wildtype TTR. A placebo-controlled, single dose-escalation phase I trial of ALN-TTR01 was recently completed in patients with ATTR. ALN-TTR01 was administered as a 15-minute IV infusion to 31 patients (23 with study drug and 8 with placebo) within a dose range of 0.01 to 1.0 mg/kg (based on siRNA). Treatmentwaswell tolerated with no significant increases in liver function tests. Infusion-related reactions were noted in 3 of 23 patients at 0.4 mg/kg; all responded to slowing of the infusion rate and all continued on study. Minimal and transient elevations of serum cytokines IL-6, IP-10 and IL-1ra were noted in two patients at the highest dose of 1 mg/kg (as anticipated from preclinical and NHP studies). Lowering of serum TTR, the expected pharmacodynamics effect of ALN-TTR01, was observed at 1 mg/kg.
[0311] In yet another embodiment, a SNALP may be made by solubilizing a cationic lipid, DSPC, cholesterol and PEG-lipid were solubilized in ethanol at a molar ratio of 40:10:40:10, respectively (see, Semple et al., Nature Niotechnology, Volume 28 Number 2 February 2010, pp. 172-177). The lipid mixture was added to an aqueous buffer (50 mM citrate, pH 4) with mixing to a final ethanol and lipid concentration of 30% (vol/vol) and 6.1 mg/ml, respectively, and allowed to equilibrate at 22 °C for 2 min before extrusion. The hydrated lipids were extruded through two stacked 80 nm pore-sized filters (Nuclepore) at 22 °C using a Lipex Extruder (Northern Lipids) until a vesicle diameter of 70–90 nm, as determined by dynamic light scattering analysis, was obtained. This generally required 1–3 passes. The siRNA (solubilized in a 50 mM citrate, pH 4 aqueous solution containing 30% ethanol) was added to the pre- equilibrated (35 °C) vesicles at a rate of ~5 ml/min with mixing. After a final target siRNA/lipid ratio of 0.06 (wt/wt) was reached, the mixture was incubated for a further 30 min at 35 °C to allow vesicle reorganization and encapsulation of the siRNA. The ethanol was then removed and the external buffer replaced with PBS (155 mM NaCl, 3 mM Na2HPO4, 1 mM KH2PO4, pH 7.5) by either dialysis or tangential flow diafiltration. siRNA were encapsulated in SNALP using a controlled step-wise dilution method process. The lipid constituents of KC2-SNALP were DLin-KC2-DMA (cationic lipid), dipalmitoylphosphatidylcholine (DPPC; Avanti Polar Lipids), synthetic cholesterol (Sigma) and PEG-C-DMA used at a molar ratio of 57.1:7.1:34.3:1.4. Upon formation of the loaded particles, SNALP were dialyzed against PBS and filter sterilized through a 0.2 m filter before use. Mean particle sizes were 75–85 nm and 90–95% of the siRNA was encapsulated within the lipid particles. The final siRNA/lipid ratio in formulations used for in vivo testing was ~0.15 (wt/wt). LNP-siRNA systems containing Factor VII siRNA were diluted to the appropriate concentrations in sterile PBS immediately before use and the formulations were administered intravenously through the lateral tail vein in a total volume of 10 ml/kg. This method may be extrapolated to the CRISPR Cas system of the present invention.
Other Lipids
[0312] Other cationic lipids, such as amino lipid 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]- dioxolane (DLin-KC2-DMA) may be utilized to encapsulate CRISPR Cas similar to SiRNA (see, e.g., Jayaraman, Angew. Chem. Int. Ed. 2012, 51, 8529–8533). A preformed vesicle with the following lipid composition may be contemplated: amino lipid, distearoylphosphatidylcholine (DSPC), cholesterol and (R)-2,3-bis(octadecyloxy) propyl-1- (methoxy poly(ethylene glycol)2000)propylcarbamate (PEG-lipid) in the molar ratio 40/10/40/10, respectively, and a FVII siRNA/total lipid ratio of approximately 0.05 (w/w). To ensure a narrow particle size distribution in the range of 70–90 nm and a low polydispersity index of 0.11_0.04 (n=56), the particles may be extruded up to three times through 80 nm membranes prior to adding the CRISPR Cas RNA. Particles containing the highly potent amino lipid 16 may be used, in which the molar ratio of the four lipid components 16, DSPC, cholesterol and PEG-lipid (50/10/38.5/1.5) which may be further optimized to enhance in vivo activity. Michael S D Kormann et al. ("Expression of therapeutic proteins after delivery of chemically modified mRNA in mice: Nature Biotechnology, Volume:29, Pages: 154–157 (2011)) describes the use of lipid envelopes to deliver RNA. Use of lipid envelopes is also preferred in the present invention.
[0313] In another embodiment, lipids may be formulated with the CRISPR Cas system of the present invention to form lipid nanoparticles (LNPs). Lipids include, but are not limited to, DLin-KC2-DMA4, C12-200 and colipids disteroylphosphatidyl choline, cholesterol, and PEG- DMG may be formulated with CRISPR Cas instead of siRNA (see, e.g., Novobrantseva, Molecular Therapy–Nucleic Acids (2012) 1, e4; doi:10.1038/mtna.2011.3) using a spontaneous vesicle formation procedure. The component molar ratio may be about 50/10/38.5/1.5 (DLin- KC2-DMA or C12-200/disteroylphosphatidyl choline/cholesterol/PEG-DMG). The final lipid:siRNA weight ratio may be ~12:1 and 9:1 in the case of DLin-KC2-DMA and C12-200 lipid nanoparticles (LNPs), respectively. The formulations may have mean particle diameters of ~80 nm with >90% entrapment efficiency. A 3 mg/kg dose may be contemplated.
[0314] Tekmira has a portfolio of approximately 95 patent families, in the U.S. and abroad, that are directed to various aspects of LNPs and LNP formulations (see, e.g., U.S. Pat. Nos. 7,982,027; 7,799,565; 8,058,069; 8,283,333; 7,901,708; 7,745,651; 7,803,397; 8,101,741; 8,188,263; 7,915,399; 8,236,943 and 7,838,658 and European Pat. Nos .1766035; 1519714; 1781593 and 1664316), all of which may be used and/or adapted to the present invention.
[0315] The CRISPR Cas system may be delivered encapsulated in PLGA Microspheres such as that further described in US published applications 20130252281 and 20130245107 and 20130244279 (assigned to Moderna Therapeutics) which relate to aspects of formulation of compositions comprising modified nucleic acid molecules which may encode a protein, a protein precursor, or a partially or fully processed form of the protein or a protein precursor. The formulation may have a molar ratio 50:10:38.5:1.5-3.0 (cationic lipid:fusogenic lipid:cholesterol:PEG lipid). The PEG lipid may be selected from, but is not limited to PEG-c- DOMG, PEG-DMG. The fusogenic lipid may be DSPC. See also, Schrum et al., Delivery and Formulation of Engineered Nucleic Acids, US published application 20120251618.
[0316] Nanomerics’ technology addresses bioavailability challenges for a broad range of therapeutics, including low molecular weight hydrophobic drugs, peptides, and nucleic acid based therapeutics (plasmid, siRNA, miRNA). Specific administration routes for which the technology has demonstrated clear advantages include the oral route, transport across the blood- brain-barrier, delivery to solid tumours, as well as to the eye. See, e.g., Mazza et al., 2013, ACS Nano. 2013 Feb 26;7(2):1016-26; Uchegbu and Siew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al., 2012, J Control Release. 2012 Jul 20;161(2):523-36. US Patent Publication No. 20050019923 describes cationic dendrimers for delivering bioactive molecules, such as polynucleotide molecules, peptides and polypeptides and/or pharmaceutical agents, to a mammalian body. The dendrimers are suitable for targeting the delivery of the bioactive molecules to, for example, the liver, spleen, lung, kidney or heart. Dendrimers are synthetic 3- dimensional macromolecules that are prepared in a step-wise fashion from simple branched monomer units, the nature and functionality of which can be easily controlled and varied. Dendrimers are synthesised from the repeated addition of building blocks to a multifunctional core (divergent approach to synthesis), or towards a multifunctional core (convergent approach to synthesis) and each addition of a 3-dimensional shell of building blocks leads to the formation of a higher generation of the dendrimers. Polypropylenimine dendrimers start from a diaminobutane core to which is added twice the number of amino groups by a double Michael addition of acrylonitrile to the primary amines followed by the hydrogenation of the nitriles. This results in a doubling of the amino groups. Polypropylenimine dendrimers contain 100% protonable nitrogens and up to 64 terminal amino groups (generation 5, DAB 64). Protonable groups are usually amine groups which are able to accept protons at neutral pH. The use of dendrimers as gene delivery agents has largely focused on the use of the polyamidoamine. and phosphorous containing compounds with a mixture of amine/amide or N--P(O2)S as the conjugating units respectively with no work being reported on the use of the lower generation polypropylenimine dendrimers for gene delivery. Polypropylenimine dendrimers have also been studied as pH sensitive controlled release systems for drug delivery and for their encapsulation of guest molecules when chemically modified by peripheral amino acid groups. The cytotoxicity and interaction of polypropylenimine dendrimers with DNA as well as the transfection efficacy of DAB 64 has also been studied. US Patent Publication No. 20050019923 is based upon the observation that, contrary to earlier reports, cationic dendrimers, such as polypropylenimine dendrimers, display suitable properties, such as specific targeting and low toxicity, for use in the targeted delivery of bioactive molecules, such as genetic material. In addition, derivatives of the cationic dendrimer also display suitable properties for the targeted delivery of bioactive molecules. See also, Bioactive Polymers, US published application 20080267903, which discloses "Various polymers, including cationic polyamine polymers and dendrimeric polymers, are shown to possess anti-proliferative activity, and may therefore be useful for treatment of disorders characterised by undesirable cellular proliferation such as neoplasms and tumours, inflammatory disorders (including autoimmune disorders), psoriasis and atherosclerosis. The polymers may be used alone as active agents, or as delivery vehicles for other therapeutic agents, such as drug molecules or nucleic acids for gene therapy. In such cases, the polymers' own intrinsic anti-tumour activity may complement the activity of the agent to be delivered."
Supercharged proteins
[0317] Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supernegatively and superpositively charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositively charged proteins are also able to penetrate mammalian cells. Associating cargo with these proteins, such as plasmid DNA, siRNA, or other proteins, can enable the functional delivery of these macromolecules into mammalian cells both in vitro and in vivo. David Liu’s lab reported the creation and characterization of supercharged proteins in 2007 (Lawrence et al., 2007, Journal of the American Chemical Society 129, 10110– 10112). The nonviral delivery of siRNA and plasmid DNA into mammalian cells are valuable both for research and therapeutic applications (Akinc et al., 2010, Nat. Biotech. 26, 561–569). Purified +36 GFP protein (or other superpositively charged protein) is mixed with siRNAs in the appropriate serum-free media and allowed to complex prior addition to cells. Inclusion of serum at this stage inhibits formation of the supercharged protein-siRNA complexes and reduces the effectiveness of the treatment. The following protocol has been found to be effective for a variety of cell lines (McNaughton et al., 2009, Proc. Natl. Acad. Sci. USA 106, 6111–6116). However, pilot experiments varying the dose of protein and RNA, e.g., siRNA or CRISPR RNA can be performed to optimize the procedure for specific cell lines.
(1) One day before treatment, plate 1 x 105 cells per well in a 48-well plate.
(2) On the day of treatment, dilute purified +36 GFP protein in serumfree media to a final concentration 200nM. Add siRNA or CRISPR RNA to a final concentration of 50nM. Vortex to mix and incubate at room temperature for 10min.
(3) During incubation, aspirate media from cells and wash once with PBS. (4) Following incubation of +36 GFP and siRNA or CRISPR RNA, add the protein-siRNA or CRISPR RNA complexes to cells.
(5) Incubate cells with complexes at 37 C for 4h.
(6) Following incubation, aspirate the media and wash three times with 20 U/mL heparin PBS. Incubate cells with serum-containing media for a further 48h or longer depending upon the assay for knockdown.
(7) Analyze cells by immunoblot, qPCR, phenotypic assay, or other appropriate method.
[0318] David Liu’s lab has further found +36 GFP to be an effective plasmid delivery reagent in a range of cells. As plasmid DNA is a larger cargo than siRNA, proportionately more +36 GFP protein is required to effectively complex plasmids. For effective plasmid delivery Applicants have developed a variant of +36 GFP bearing a C-terminal HA2 peptide tag, a known endosome-disrupting peptide derived from the influenza virus hemagglutinin protein. The following protocol has been effective in a variety of cells, but as above it is advised that plasmid DNA and supercharged protein doses be optimized for specific cell lines and delivery applications.
(1) One day before treatment, plate 1 x 105 per well in a 48-well plate. (2) On the day of treatment, dilute purified þ36 GFP protein in serumfree media to a final concentration 2 mM. Add 1mg of plasmid DNA. Vortex to mix and incubate at room temperature for 10min.
(3) During incubation, aspirate media from cells and wash once with PBS.
(4) Following incubation of þ36 GFP and plasmid DNA, gently add the protein- DNA complexes to cells.
(5) Incubate cells with complexes at 37 C for 4h.
(6) Following incubation, aspirate the media and wash with PBS. Incubate cells in serum-containing media and incubate for a further 24–48h.
(7) Analyze plasmid delivery (e.g., by plasmid-driven gene expression) as appropriate.
[0319] See also, e.g., McNaughton et al., Proc. Natl. Acad. Sci. USA 106, 6111-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752 (2010); Cronican et al., Chemistry & Biology 18, 833-838 (2011); Thompson et al., Methods in Enzymology 503, 293-319 (2012); Thompson, D.B., et al., Chemistry & Biology 19 (7), 831-843 (2012). The methods of the super charged proteins may be used and/or adapted for delivery of the CRISPR Cas system of the present invention.
Implantable devices
[0320] In another embodiment, implantable devices are also contemplated for delivery of the CRISPR Cas system. For example, US Patent Publication 20110195123 discloses an implantable medical device which elutes a drug locally and in prolonged period is provided, including several types of such a device, the treatment modes of implementation and methods of implantation. The device comprising of polymeric substrate, such as a matrix for example, that is used as the device body, and drugs, and in some cases additional scaffolding materials, such as metals or additional polymers, and materials to enhance visibility and imaging. The selection of drug is based on the advantageous of releasing drug locally and in prolonged period, where drug is released directly to the extracellular matrix (ECM) of the diseased area such as tumor, inflammation, degeneration or for symptomatic objectives, or to injured smooth muscle cells, or for prevention. One kind of drug is the gene silencing drugs based on RNA interference (RNAi), including but not limited to si RNA, sh RNA, or antisense RNA/DNA, ribozyme and nucleoside analogs. Therefore, this system may be used/and or adapted to the CRISPR Cas system of the present invention. The modes of implantation in some embodiments are existing implantation procedures that are developed and used today for other treatments, including brachytherapy and needle biopsy. In such cases the dimensions of the new implant described in this invention are similar to the original implant. Typically a few devices are implanted during the same treatment procedure. As described in US Patent Publication 20110195123, there is provided a drug delivery implantable or insertable system, including systems applicable to a cavity such as the abdominal cavity and/or any other type of administration in which the drug delivery system is not anchored or attached, comprising a biostable and/or degradable and/or bioabsorbable polymeric substrate, which may for example optionally be a matrix. It should be noted that the term "insertion" also includes implantation. The drug delivery system is preferably implemented as a "Loder" as described in US Patent Publication 20110195123. The polymer or plurality of polymers are biocompatible, incorporating an agent and/or plurality of agents, enabling the release of agent at a controlled rate, wherein the total volume of the polymeric substrate, such as a matrix for example, in some embodiments is optionally and preferably no greater than a maximum volume that permits a therapeutic level of the agent to be reached. As a non-limiting example, such a volume is preferably within the range of 0.1 m3 to 1000 mm3, as required by the volume for the agent load. The Loder may optionally be larger, for example when incorporated with a device whose size is determined by functionality, for example and without limitation, a knee joint, an intra-uterine or cervical ring and the like. The drug delivery system (for delivering the composition) is designed in some embodiments to preferably employ degradable polymers, wherein the main release mechanism is bulk erosion; or in some embodiments, non degradable, or slowly degraded polymers are used, wherein the main release mechanism is diffusion rather than bulk erosion, so that the outer part functions as membrane, and its internal part functions as a drug reservoir, which practically is not affected by the surroundings for an extended period (for example from about a week to about a few months). Combinations of different polymers with different release mechanisms may also optionally be used. The concentration gradient at the surface is preferably maintained effectively constant during a significant period of the total drug releasing period, and therefore the diffusion rate is effectively constant (termed "zero mode" diffusion). By the term "constant" it is meant a diffusion rate that is preferably maintained above the lower threshold of therapeutic effectiveness, but which may still optionally feature an initial burst and/or fluctuate, for example increasing and decreasing to a certain degree. The diffusion rate is preferably so maintained for a prolonged period, and it can be considered constant to a certain level to optimize the therapeutically effective period, for example the effective silencing period. The drug delivery system optionally and preferably is designed to shield the nucleotide based therapeutic agent from degradation, whether chemical in nature or due to attack from enzymes and other factors in the body of the subject. The drug delivery system as described in US Patent Publication 20110195123 is optionally associated with sensing and/or activation appliances that are operated at and/or after implantation of the device, by non and/or minimally invasive methods of activation and/or acceleration/deceleration, for example optionally including but not limited to thermal heating and cooling, laser beams, and ultrasonic, including focused ultrasound and/or RF (radiofrequency) methods or devices. According to some embodiments of US Patent Publication 20110195123, the site for local delivery may optionally include target sites characterized by high abnormal proliferation of cells, and suppressed apoptosis, including tumors, active and or chronic inflammation and infection including autoimmune diseases states, degenerating tissue including muscle and nervous tissue, chronic pain, degenerative sites, and location of bone fractures and other wound locations for enhancement of regeneration of tissue, and injured cardiac, smooth and striated muscle. The site for implantation of the composition, or target site, preferably features a radius, area and/or volume that is sufficiently small for targeted local delivery. For example, the target site optionally has a diameter in a range of from about 0.1 mm to about 5 cm. The location of the target site is preferably selected for maximum therapeutic efficacy. For example, the composition of the drug delivery system (optionally with a device for implantation as described above) is optionally and preferably implanted within or in the proximity of a tumor environment, or the blood supply associated thereof. For example the composition (optionally with the device) is optionally implanted within or in the proximity to pancreas, prostate, breast, liver, via the nipple, within the vascular system and so forth. The target location is optionally selected from the group consisting of (as non-limiting examples only, as optionally any site within the body may be suitable for implanting a Loder): 1. brain at degenerative sites like in Parkinson or Alzheimer disease at the basal ganglia, white and gray matter; 2. spine as in the case of amyotrophic lateral sclerosis (ALS); 3. uterine cervix to prevent HPV infection; 4. active and chronic inflammatory joints; 5. dermis as in the case of psoriasis; 6. sympathetic and sensoric nervous sites for analgesic effect; 7. Intra osseous implantation; 8. acute and chronic infection sites; 9. Intra vaginal; 10. Inner ear--auditory system, labyrinth of the inner ear, vestibular system; 11. Intra tracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urinary bladder; 14. biliary system; 15. parenchymal tissue including and not limited to the kidney, liver, spleen; 16. lymph nodes; 17. salivary glands; 18. dental gums; 19. Intra-articular (into joints); 20. Intra-ocular; 21. Brain tissue; 22. Brain ventricles; 23. Cavities, including abdominal cavity (for example but without limitation, for ovary cancer); 24. Intra esophageal and 25. Intra rectal. Optionally insertion of the system (for example a device containing the composition) is associated with injection of material to the ECM at the target site and the vicinity of that site to affect local pH and/or temperature and/or other biological factors affecting the diffusion of the drug and/or drug kinetics in the ECM, of the target site and the vicinity of such a site. Optionally, according to some embodiments, the release of said agent could be associated with sensing and/or activation appliances that are operated prior and/or at and/or after insertion, by non and/or minimally invasive and/or else methods of activation and/or acceleration/deceleration, including laser beam, radiation, thermal heating and cooling, and ultrasonic, including focused ultrasound and/or RF (radiofrequency) methods or devices, and chemical activators. According to other embodiments of US Patent Publication 20110195123, the drug preferably comprises a gene silencing biological RNAi drug, for example for localized cancer cases in breast, pancreas, brain, kidney, bladder, lung, and prostate as described below. Although examplifieid with RNAi, many drugs other than siRNA are applicable to be encapsulated in Loder, and can be used in association with this invention, as long as such drugs can be encapsulated with the Loder substrate, such as a matrix for example, and this system may be used and/or adapted to deliver the CRISPR Cas system of the present invention.
[0321] The method of insertion, such as implantation, may optionally already be used for other types of tissue implantation and/or for insertions and/or for sampling tissues, optionally without modifications, or alternatively optionally only with non-major modifications in such methods. Such methods optionally include but are not limited to brachytherapy methods, biopsy, endoscopy with and/or without ultrasound, such as ERCP, stereotactic methods into the brain tissue, Laparoscopy, including implantation with a laparoscope into joints, abdominal organs, the bladder wall and body cavities.
CRISPR enzyme mRNA and guide RNA
[0322] CRISPR enzyme mRNA and guide RNA might also be delivered separately. CRISPR enzyme mRNA can be delivered prior to the guide RNA to give time for CRISPR enzyme to be expressed. CRISPR enzyme mRNA might be administered 1-12 hours (preferably around 2-6 hours) prior to the administration of guide RNA. Alternatively, CRISPR enzyme mRNA and guide RNA can be administered together. Advantageously, a second booster dose of guide RNA can be administered 1-12 hours (preferably around 2-6 hours) after the initial administration of CRISPR enzyme mRNA + guide RNA. Additional administrations of CRISPR enzyme mRNA and/or guide RNA might be useful to achieve the most efficient levels of genome modification. For minimization of toxicity and off-target effect, it will be important to control the concentration of CRISPR enzyme mRNA and guide RNA delivered. Optimal concentrations of CRISPR enzyme mRNA and guide RNA can be determined by testing different concentrations in a cellular or animal model and using deep sequencing the analyze the extent of modification at potential off-target genomic loci. For example, for the guide sequence targeting 5’- GAGTCCGAGCAGAAGAAGAA-3’ in the EMX1 gene of the human genome, deep sequencing can be used to assess the level of modification at the following two off-target loci, 1: 5’-GAGTCCTAGCAGGAGAAGAA-3’ and 2: 5’-GAGTCTAAGCAGAAGAAGAA-3’. The concentration that gives the highest level of on-target modification while minimizing the level of off-target modification should be chosen for in vivo delivery. Alternatively, to minimize the level of toxicity and off-target effect, CRISPR enzyme nickase mRNA (for example S. pyogenes Cas9 with the D10A mutation) can be delivered with a pair of guide RNAs targeting a site of interest. The two guide RNAs need to be spaced as follows. Guide sequences in red (single underline) and blue (double underline) respectively (these examples are based on the PAM requirement for Streptococcus pyogenes Cas9).
Figure imgf000108_0001
Figure imgf000109_0001
Figure imgf000110_0001
Figure imgf000111_0001
Figure imgf000112_0001
[0323] Further interrogation of the system have given Applicants evidence of the 5’ overhang (see, e.g., Ran et al., Cell. 2013 Sep 12;154(6):1380-9 and US Provisional Patent Application Serial No. 61/871,301 filed August 28, 2013). Applicants have further identified parameters that relate to efficient cleavage by the Cas9 nickase mutant when combined with two guide RNAs and these parameters include but are not limited to the length of the 5’ overhang. In embodiments of the invention the 5’ overhang is at most 200 base pairs, preferably at most 100 base pairs, or more preferably at most 50 base pairs. In embodiments of the invention the 5’ overhang is at least 26 base pairs, preferably at least 30 base pairs or more preferably 34-50 base pairs or 1-34 base pairs. In other preferred methods of the invention the first guide sequence directing cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directing cleavage of other strand near the second target sequence results in a blunt cut or a 3’ overhang. In embodiments of the invention the 3’ overhang is at most 150, 100 or 25 base pairs or at least 15, 10 or 1 base pairs. In preferred embodiments the 3’ overhang is 1- 100 basepairs.
[0324] Aspects of the invention relate to the expression of the gene product being decreased or a template polynucleotide being further introduced into the DNA molecule encoding the gene product or an intervening sequence being excised precisely by allowing the two 5’ overhangs to reanneal and ligate or the activity or function of the gene product being altered or the expression of the gene product being increased. In an embodiment of the invention, the gene product is a protein. sgRNA pairs creating 5’ overhangs with less than 8bp overlap between the guide sequences (offset greater than -8 bp) were able to mediate detectable indel formation. Importantly, each guide used in these assays is able to efficiently induce indels when paired with wildtype Cas9, indicating that the relative positions of the guide pairs are the most important parameters in predicting double nicking activity. Since Cas9n and Cas9H840A nick opposite strands of DNA, substitution of Cas9n with Cas9H840A with a given sgRNA pair should result in the inversion of the overhang type. For example, a pair of sgRNAs that will generate a 5’ overhang with Cas9n should in principle generate the corresponding 3’ overhang instead. Therefore, sgRNA pairs that lead to the generation of a 3’ overhang with Cas9n might be used with Cas9H840A to generate a 5’ overhang. Applicants tested Cas9H840A with a set of sgRNA pairs designed to generate both 5’ and 3’ overhangs (offset range from–278 to +58 bp), but were unable to observe indel formation, indicating that mutation Cas9H840A may substantially reduce Cas9 nuclease activity, thereby making sgRNA pairing as to mutant Cas9H840A as to double nicking of interest.
Liver, proprotein convertase subtilisin kexin 9 (PCSK9)
[0325] Bailey et al. (J Mol Med (Berl). 1999 Jan;77(1):244-9) discloses insulin delivery by ex-vivo somatic cell gene therapy involves the removal of non-B-cell somatic cells (e.g. fibroblasts) from a diabetic patient, and genetically altering them in vitro to produce and secrete insulin. The cells can be grown in culture and returned to the donor as a source of insulin replacement. Cells modified in this way could be evaluated before implantation, and reserve stocks could be cryopreserved. By using the patient's own cells, the procedure should obviate the need for immunosuppression and overcome the problem of tissue supply, while avoiding a recurrence of cell destruction. Ex-vivo somatic cell gene therapy requires an accessible and robust cell type that is amenable to multiple transfections and subject to controlled proliferation. Special problems associated with the use of non-B-cell somatic cells include the processing of proinsulin to insulin, and the conferment of sensitivity to glucose-stimulated proinsulin biosynthesis and regulated insulin release. Preliminary studies using fibroblasts, pituitary cells, kidney (COS) cells and ovarian (CHO) cells suggest that these challenges could be met, and that ex-vivo somatic cell gene therapy offers a feasible approach to insulin replacement therapy. The system of Bailey et al. may be used/and or adapted to the CRISPR Cas system of the present invention for delivery to the liver.
[0326] The methods of Sato et al. (Nature Biotechnology Volume 26 Number 4 April 2008, Pages 431-442) may be applied to the CRISPR Cas system of the present invention for delivery to the liver. Sato et al. found that treatments with the siRNA-bearing vitamin A–coupled liposomes almost completely resolved liver fibrosis and prolonged survival in rats with otherwise lethal dimethylnitrosamine-induced liver cirrhosis in a dose- and duration-dependent manner. Cationic liposomes (Lipotrust) containing O,O’-ditetradecanoyl-N-(a- trimethylammonioacetyl) diethanolamine chloride (DC-6-14) as a cationic lipid, cholesterol and dioleoylphosphatidylethanolamine at a molar ratio of 4:3:3 (which has shown high transfection efficiency under serumcontaining conditions for in vitro and in vivo gene delivery) were purchased from Hokkaido System Science. The liposomes were manufactured using a freeze- dried empty liposomes method and prepared at a concentration of 1 mM (DC-16-4) by addition of double-distilled water (DDW) to the lyophilized lipid mixture under vortexing before use. To prepare VA-coupled liposomes, 200 nmol of vitamin A (retinol, Sigma) dissolved in DMSO was mixed with the liposome suspensions (100 nmol as DC-16-4) by vortexing in a 1.5 ml tube at 25 1C. To prepare VA-coupled liposomes carrying siRNAgp46 (VA-lip-siRNAgp46), a solution of siRNAgp46 (580 pmol/ml in DDW) was added to the retinol-coupled liposome solution with stirring at 25 C. The ratio of siRNA to DC-16-4 was 1:11.5 (mol/mol) and the siRNA to liposome ratio (wt/wt) was 1:1. Any free vitamin A or siRNA that was not taken up by liposomes were separated from liposomal preparations using a micropartition system (VIVASPIN 2 concentrator 30,000 MWCO PES, VIVASCIENCE). The liposomal suspension was added to the filters and centrifuged at 1,500g for 5 min 3 times at 25 1C. Fractions were collected and the material trapped in the filter was reconstituted with PBS to achieve the desired dose for in vitro or in vivo use. Three injections of 0.75 mg/kg siRNA were given every other day to rats. The system of Sato et al. may be used/and or adapted to the CRISPR Cas system of the present invention for delivery to the liver by delivering about 0.5 to 1 mg/kg of CRISPR Cas RNA in the liposomes as described by Sato et al. to humans.
[0327] The methods of Rozema et al. (PNAS, August 7, 2007, vol. 104, no. 32) for a vehicle for the delivery of siRNA to hepatocytes both in vitro and in vivo, which Rozema et al. have named siRNA Dynamic PolyConjugates may also be applied to the present invention. Key features of the Dynamic Poly-Conjugate technology include a membrane-active polymer, the ability to reversibly mask the activity of this polymer until it reaches the acidic environment of endosomes, and the ability to target this modified polymer and its siRNA cargo specifically to hepatocytes in vivo after simple, low-pressure i.v. injection. SATA-modified siRNAs are synthesized by reaction of 5’ aminemodified siRNA with 1 weight equivalents (wt eq) of Nsuccinimidyl-S-acetylthioacetate (SATA) reagent (Pierce) and 0.36 wt eq of NaHCO3 in water at 4°C for 16 h. The modified siRNAs are then precipitated by the addition of 9 vol of ethanol and incubation at 80°C for 2 h. The precipitate is resuspended in 1X siRNA buffer (Dharmacon) and quantified by measuring absorbance at the 260-nm wavelength. PBAVE (30 mg/ml in 5mMTAPS, pH 9) is modified by addition of 1.5 wt % SMPT (Pierce). After a 1-h incubation, 0.8 mg of SMPT-PBAVE was added to 400 l of isotonic glucose solution containing 5 mM TAPS (pH 9). To this solution was added 50 g of SATA-modified siRNA. For the dose– response experiments where [PBAVE] was constant, different amounts of siRNA are added. The mixture is then incubated for 16 h. To the solution is then added 5.6 mg of Hepes free base followed by a mixture of 3.7 mg ofCDM-NAGand 1.9mg of CDM-PEG. The solution is then incubated for at least 1 h at room temperature before injection. CDM-PEG and CDM-NAG are synthesized from the acid chloride generated by using oxalyl chloride. To the acid chloride is added 1.1 molar equivalents polyethylene glycol monomethyl ether (molecular weight average of 450) to generate CDM-PEG or (aminoethoxy)ethoxy-2-(acetylamino)-2-deoxy-ȕ-D- glucopyranoside to generate CDM-NAG. The final product is purified by using reverse-phase HPLC with a 0.1% TFA water/acetonitrile gradient. About 25 to 50 g of siRNA was delivered to mice. The system of Rozema et al. may be applied to the CRISPR Cas system of the present invention for delivery to the liver, for example by envisioning a dosage of about 50 to about 200 mg of CRISPR Cas for delivery to a human.
Brain
[0328] Delivery options for the brain include encapsulation of CRISPR enzyme and guide RNA in the form of either DNA or RNA into liposomes and conjugating to molecular Trojan horses for trans-blood brain barrier (BBB) delivery. Molecular Trojan horses have been shown to be effective for delivery of B-gal expression vectors into the brain of non-human primates. The same approach can be used to delivery vectors containing CRISPR enzyme and guide RNA. For instance, Xia CF and Boado RJ, Pardridge WM ("Antibody-mediated targeting of siRNA via the human insulin receptor using avidin-biotin technology." Mol Pharm. 2009 May- Jun;6(3):747-51. doi: 10.1021/mp800194) describes how delivery of short interfering RNA (siRNA) to cells in culture, and in vivo, is possible with combined use of a receptor-specific monoclonal antibody (mAb) and avidin-biotin technology. The authors also report that because the bond between the targeting mAb and the siRNA is stable with avidin-biotin technology, and RNAi effects at distant sites such as brain are observed in vivo following an intravenous administration of the targeted siRNA.
[0329] Zhang et al. (Mol Ther. 2003 Jan;7(1):11-8.)) describe how expression plasmids encoding reporters such as luciferase were encapsulated in the interior of an "artificial virus" comprised of an 85 nm pegylated immunoliposome, which was targeted to the rhesus monkey brain in vivo with a monoclonal antibody (MAb) to the human insulin receptor (HIR). The HIRMAb enables the liposome carrying the exogenous gene to undergo transcytosis across the blood-brain barrier and endocytosis across the neuronal plasma membrane following intravenous injection. The level of luciferase gene expression in the brain was 50-fold higher in the rhesus monkey as compared to the rat. Widespread neuronal expression of the beta-galactosidase gene in primate brain was demonstrated by both histochemistry and confocal microscopy. The authors indicate that this approach makes feasible reversible adult transgenics in 24 hours. Accordingly, the use of immunoliposome is preferred. These may be used in conjunction with antibodies to target specific tissues or cell surface proteins. Other means of delivery or RNA are also preferred, such as via nanoparticles (Cho, S., Goldberg, M., Son, S., Xu, Q., Yang, F., Mei, Y., Bogatyrev, S., Langer, R. and Anderson, D., Lipid-like nanoparticles for small interfering RNA delivery to endothelial cells, Advanced Functional Materials, 19: 3112-3118, 2010) or exosomes (Schroeder, A., Levins, C., Cortez, C., Langer, R., and Anderson, D., Lipid-based nanotherapeutics for siRNA delivery, Journal of Internal Medicine, 267: 9-21, 2010, PMID: 20059641).
[0330] Indeed, exosomes have been shown to be particularly useful in delivery siRNA, a system with some parallels to the CRISPR system. For instance, El-Andaloussi S, et al. (“Exosome-mediated delivery of siRNA in vitro and in vivo.” Nat Protoc. 2012 Dec;7(12):2112- 26. doi: 10.1038/nprot.2012.131. Epub 2012 Nov 15.) describe how exosomes are promising tools for drug delivery across different biological barriers and can be harnessed for delivery of siRNA in vitro and in vivo. Their approach is to generate targeted exosomes through transfection of an expression vector, comprising an exosomal protein fused with a peptide ligand. The exosomes are then purify and characterized from transfected cell supernatant, then siRNA is loaded into the exosomes. Delivery or administration according to the invention can be performed with exosomes, in particular but not limited to the brain. Vitamin E (Į -tocopherol) may be conjugated with CRISPR Cas and delivered to the brain along with high density lipoprotein (HDL), for example in a similar manner as was done by Uno et al. (HUMAN GENE THERAPY 22:711–719 (June 2011)) for delivering short-interfering RNA (siRNA) to the brain. Mice were infused via Osmotic minipumps (model 1007D; Alzet, Cupertino, CA) filled with phosphate-buffered saline (PBS) or free TocsiBACE or Toc-siBACE/HDL and connected with Brain Infusion Kit 3 (Alzet). A brain-infusion cannula was placed about 0.5mm posterior to the bregma at midline for infusion into the dorsal third ventricle. Uno et al. found that as little as 3 nmol of Toc-siRNA with HDL could induce a target reduction in comparable degree by the same ICV infusion method. A similar dosage of CRISPR Cas conjugated to Į -tocopherol and co- administered with HDL targeted to the brain may be contemplated for humans in the present invention, for example, about 3 nmol to about 3 mol of CRISPR Cas targeted to the brain may becontemplated.
[0331] Zou et al. ((HUMAN GENE THERAPY 22:465-475 (April 2011)) describes a method of lentiviral-mediated delivery of short-hairpin RNAs targeting PKCȖ for in vivo gene silencing in the spinal cord of rats. Zou et al. administered about 10 l of a recombinant lentivirus having a titer of 1 x 109 transducing units (TU)/ml by an intrathecal catheter. A similar dosage of CRISPR Cas expressed in a lentiviral vector targeted to the brain may be contemplated for humans in the present invention, for example, about 10-50 ml of CRISPR Cas targeted to the brain in a lentivirus having a titer of 1 x 109 transducing units (TU)/ml may be contemplated.
[0332] In terms of local delivery to the brain, this can be achieved in various ways. For instance, material can be delivered intrastriatally e.g. by injection. Injection can be performed stereotactically via a craniotomy.
Targeted deletion, therapeutic applications
[0333] Targeted deletion of viral genes or other viral elements is preferred. Examples are exemplified in Example 18. Preferred are, therefore, latent viral genes. As exemplified here, Applicants prefer gene delivery of a CRISPR-Cas system to the liver, brain, ocular, epithelial, hematopoetic, or another tissue of a subject or a patient in need thereof, suffering from latent viral infections, using either viral or nonviral (e.g. nanoparticle) delivery system.
[0334] Therapeutic applications of the CRISPR-Cas system include treatment of viral infections, such as HBV infection.
[0335] Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. The immunogenicity of protein drugs can be ascribed to a few immunodominant helper T lymphocyte (HTL) epitopes. Reducing the MHC binding affinity of these HTL epitopes contained within these proteins can generate drugs with lower immunogenicity (Tangri S, et al. (“Rationally engineered therapeutic proteins with reduced immunogenicity” J Immunol. 2005 Mar 15;174(6):3187-96.) In the present invention, the immunogenicity of the CRISPR enzyme in particular may be reduced following the approach first set out in Tangri et al with respect to erythropoietin and subsequently developed. Accordingly, directed evolution or rational design may be used to reduce the immunogenicity of the CRISPR enzyme (for instance a Cas9) in the host species (human or other species). [0336] Applicants used 3 guideRNAs of interest and able to visualize efficient DNA cleavage in vivo occurring only in a small subset of cells. Essentially, what Applicants have shown here is targeted in vivo cleavage. In particular, this provides proof of concept that specific targeting in higher organisms such as mammals can also be achieved. It also highlights multiplex aspect in that multiple guide sequences (i.e. separate targets) can be used simultaneously (in the sense of co-delivery). In other words, Applicants used a multiple approach, with several different sequences targeted at the same time, but independently.
[0337] A suitable example of a protocol for producing AAV, a preferred vector of the invention is provided in the Examples.
Blood
[0338] The present invention also contemplates delivering the CRISPR-Cas system to the blood. The plasma exosomes of Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 e130) were previously described and may be utilized to deliver the CRISPR Cas system to the blood.
Heart
[0339] The present invention also contemplates delivering the CRISPR-Cas system to the heart. For the heart, a myocardium tropic adena-associated virus (AAVM) is preferred, in particular AAVM41 which showed preferential gene transfer in the heart (see, e.g., Lin-Yanga et al., PNAS, March 10, 2009, vol. 106, no. 10). Administration may be systemic or local. A dosage of about 1-10 x 1014 vector genomes are contemplated for systemic administration. See also, e.g., Eulalio et al. (2012) Nature 492: 376 and Somasuntharam et al. (2013) Biomaterials 34: 7790.
Kidneys
[0340] The present invention also contemplates delivering the CRISPR-Cas system to the kidney. Delivery strategies to induce cellular uptake of the therapeutic nucleic acid include physical force or vector systems such as viral-, lipid- or complex- based delivery, or nanocarriers. From the initial applications with less possible clinical relevance, when nucleic acids were addressed to renal cells with hydrodynamic high pressure injection systemically, a wide range of gene therapeutic viral and non-viral carriers have been applied already to target posttranscriptional events in different animal kidney disease models in vivo (Csaba Révész and Péter Hamar (2011). Delivery Methods to Target RNAs in the Kidney, Gene Therapy Applications, Prof. Chunsheng Kang (Ed.), ISBN: 978-953-307-541-9, InTech, Available from: http://www.intechopen.com/books/gene-therapy-applications/delivery-methods-to-target-rnas-in- the-kidney). Delivery methods to the kidney are summarized as follows:
Figure imgf000119_0001
Figure imgf000120_0001
Figure imgf000121_0001
Lungs
[0341] The present invention also contemplates delivering the CRISPR-Cas system to oneor both lungs. Although AAV-2-based vectors were originally proposed for CFTR delivery to CF airways, other serotypes such as AAV-1, AAV-5, AAV-6, and AAV-9 exhibit improved gene transfer efficiency in a variety of models of the lung epithelium (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009). AAV-1 was demonstrated to be ~100-fold more efficient than AAV-2 and AAV-5 at transducing human airway epithelial cells in vitro,5 although AAV-1 transduced murine tracheal airway epithelia in vivo with an efficiency equal to that of AAV-5. Other studies have shown that AAV-5 is 50-fold more efficient than AAV-2 at gene delivery to human airway epithelium (HAE) in vitro and significantly more efficient in the mouse lung airway epithelium in vivo. AAV-6 has also been shown to be more efficient than AAV-2 in human airway epithelial cells in vitro and murine airways in vivo.8 The more recent isolate, AAV-9, was shown to display greater gene transfer efficiency than AAV-5 in murine nasal and alveolar epithelia in vivo with gene expression detected for over 9 months suggesting AAV may enable long-term gene expression in vivo, a desirable property for a CFTR gene delivery vector. Furthermore, it was demonstrated that AAV-9 could be readministered to the murine lung with no loss of CFTR expression and minimal immune consequences. CF and non- CF HAE cultures may be inoculated on the apical surface with 100 l of AAV vectors for hours (see, e.g., Li et al., Molecular Therapy, vol. 17 no. 12, 2067-2077 Dec 2009). The MOI may vary from 1 × 103 to 4 × 105 vector genomes/cell, depending on virus concentration and purposes of the experiments. The above cited vectors are contemplated for the delivery and/or administration of the invention.
[0342] Zamora et al. (Am J Respir Crit Care Med Vol 183. pp 531–538, 2011) reported an example of the application of an RNA interference therapeutic to the treatment of human infectious disease and also a randomized trial of an antiviral drug in respiratory syncytial virus (RSV)-infected lung transplant recipients. Zamora et al. performed a randomized, double-blind, placebocontrolled trial in LTX recipients with RSV respiratory tract infection. Patients were permitted to receive standard of care for RSV. Aerosolized ALN-RSV01 (0.6 mg/kg) or placebo was administered daily for 3 days. This study demonstrates that an RNAi therapeutic targeting RSV can be safely administered to LTX recipients with RSV infection. Three daily doses of ALN-RSV01 did not result in any exacerbation of respiratory tract symptoms or impairment of lung function and did not exhibit any systemic proinflammatory effects, such as induction of cytokines or CRP. Pharmacokinetics showed only low, transient systemic exposure after inhalation, consistent with preclinical animal data showing that ALN-RSV01, administered intravenously or by inhalation, is rapidly cleared from the circulation through exonucleasemediated digestion and renal excretion. The method of Zamora et al. may be applied to the CRISPR Cas system of the present invention and an aerosolized CRISPR Cas, for example with a dosage of 0.6 mg/kg, may be contemplated for the present invention.
Muscles
[0343] The present invention also contemplates delivering the CRISPR-Cas system to muscle(s).
Skin
[0344] The present invention also contemplates delivering the CRISPR-Cas system to the skin. Hickerson et al. (Molecular Therapy—Nucleic Acids (2013) 2, e129) relates to a motorized microneedle array skin delivery device for delivering self-delivery (sd)-siRNA to human and murine skin. The primary challenge to translating siRNA-based skin therapeutics to the clinic is the development of effective delivery systems. Substantial effort has been invested in a variety of skin delivery technologies with limited success. In a clinical study in which skin was treated with siRNA, the exquisite pain associated with the hypodermic needle injection precluded enrollment of additional patients in the trial, highlighting the need for improved, more“patient-friendly” (i.e., little or no pain) delivery approaches. Microneedles represent an efficient way to deliver large charged cargos including siRNAs across the primary barrier, the stratum corneum, and are generally regarded as less painful than conventional hypodermic needles. Motorized“stamp type” microneedle devices, including the motorized microneedle array (MMNA) device used by Hickerson et al., have been shown to be safe in hairless mice studies and cause little or no pain as evidenced by (i) widespread use in the cosmetic industry and (ii) limited testing in which nearly all volunteers found use of the device to be much less painful than a flushot, suggesting siRNA delivery using this device will result in much less pain than was experienced in the previous clinical trial using hypodermic needle injections. The MMNA device (marketed as Triple-M or Tri-M by Bomtech Electronic Co, Seoul, South Korea) was adapted for delivery of siRNA to mouse and human skin. sd-siRNA solution (up to 300 l of 0.1 mg/ml RNA) was introduced into the chamber of the disposable Tri-M needle cartridge (Bomtech), which was set to a depth of 0.1 mm. For treating human skin, deidentified skin (obtained immediately following surgical procedures) was manually stretched and pinned to a cork platform before treatment. All intradermal injections were performed using an insulin syringe with a 28-gauge 0.5-inch needle. The MMNA device and method of Hickerson et al. could be used and/or adapted to deliver the CRISPR Cas of the present invention, for example, at a dosage of up to 300 l of 0.1 mg/ml CRISPR Cas to the skin.
Latent and chronic viral infections
[0345] The present invention may also be applied to treat latent or chronic viral infections. Viral latency is the abiity of a pathogenic virus to remain latent or dormant within a cell during the lysogenic part of its life cycle. Latent infection is distinct from chronic infection in which a virus continues to replicate and proliferate. Instead, proliferation of the virus ceases but the viral genome is not eradicated, and so it can reactivate and again result in production of viral progeny (lytic part of the life cycle) without requiring reinfection of the host. Thus the invention provides the use of a CRISPR-Cas system to inactivate a virus within a eukaryotic cell, and in particular a latent form of the virus. For instance, the CRISPR-Cas system can be used to excise an integrated provirus from a cell’s genome and/or to inactivate a latent virus which is present in episomal form (e.g. to cleave a cccDNA form).
[0346] Thus the invention provides a CRISPR-Cas system comprising one or more vectors for delivery to a eukaryotic cell, wherein the vector(s) encode(s): (i) a CRISPR enzyme; (ii) a guide RNA capable of hybridizing to a target sequence in a viral genome within the cell; and (iii); a tracr mate sequence; and (iv) a tracr sequence, wherein, when expressed within the cell, the guide RNA directs sequence-specific binding of a CRISPR complex to the target sequence, and the CRISPR complex comprises (a) the tracr mate sequence hybridised to the tracr sequence and (b) a CRISPR enzyme bound to the guide RNA, such that the guide RNA can hybridise to its target sequence in the viral genome.
[0347] Further characteristics of the vector(s), the encoded enzyme, the guide sequences, etc. are disclosed elsewhere herein. For instance, the guide sequences can be part of a chiRNA sequence which provides the guide, tracr mate and tracr sequences within a single RNA, such that the system can encode (i) a CRISPR enzyme; (ii) a chiRNA comprising a sequence capable of hybridizing to a target sequence in a viral genome, a tracr mate sequence, and a tracr sequence. Similarly, the enzyme can include one or more NLS, etc.
[0348] Latent viruses can exist in episomal form or integrated proviral form, and the invention can be used to treat both types. The invention is particularly useful with DNA viruses, and especially viruses with a double-stranded DNA genome. Examples of pathogenic DNA viruses which have an episomal latent form herpes simplex virus (HSV) 1 and 2, human papillomavirus (HPV) of any type, Epstein Barr virus (EBV), and varicella zoster virus (VZV). The invention can be used to treat infections with any of these viruses. Some plant viruses also exhibit a latent form and the invention can be used to eliminate these as well. CRISPR systems of the invention can be targeted to cell types which maintain a latent virus e.g. B cells or epithelial cells for EBV, neurons for HSV and VZV, epithelial cells for HPV, liver cells for HBV, etc. Genomic sequences for viruses are widely available, and guide sequences for targeting the viral genome can thus be designed without difficulty. Where a virus has various sequence variants (e.g. different subtypes of HBV) it is useful to design guide sequences to target regions of the genome which are conserved, thereby providing broad activity. It is preferred to use guide RNAs against more than one site in the viral genome. A key advantage of CRISPR technology when compared to ZFNs or TALENs is the relative ease by which multiple sequences can be targeted. Targeting multiple sites in a viral genome offers two main advantages. Firstly, it reduces the possibility that a viral strain might escape (e.g. by mutation), and helps to ensure that at least one target is present in any subject’s unique ensemble of genomes and variants or quasispecies. Secondly, because episomeal forms are generally small and circular (~3-4kb), targeting multiple sites with different guides may allow fragmentation of the episome into multiple pieces that cannot easily be repaired by NHEJ. Thus, for example, a CRISPR system can target multiple genes or ORFs within a viral genome. As well as targeting latent viruses, CRISPR systems can be used to target chronic viral infections by viruses whose life cycles include a dsDNA form which can be bound by CRISPR complexes. In these embodiments the CRISPR system can be used in conjunction with an antiviral compound, such as tenofovir (HBV), entecavir (HBV), aciclovir (HSV, VZV), etc.
Hepatitis viruses
[0349] The present invention may also be applied to treat hepatitis B virus (HBV). Thus the invention provides the use of a CRISPR-Cas system to inactivate HBV within a mammalian cell, and in particular a latent form of HBV. For instance, the CRISPR-Cas system can be used to excise integrated HBV provirus from a cell’s genome (a rare occurrence) and/or to inactivate latent HBV which is present in covalently closed circular DNA (cccDNA) form. HBV genomic sequences are widely available, and guide sequences for targeting the HBV genome can thus be designed without difficulty. HBV exists in several serological subtypes (e.g. adw, ayw, ady, adr), which differ by >8% in primary sequence, and it is useful to design guide sequences to target regions of the genome which are conserved between multiple subtypes. Applicants have designed 24 guide RNAs to target the HBV genome. These include targets which are highly conserved within the HBV genome; and the location of 9 of these guide sequences is mapped against the HBV genome (See Figures 36, 57). It is preferred to use guide RNAs against more than one site in the HBV genome. For instance, it is useful to provide guide sequences which recognise two or more of: ORF S, which encodes the surface antigen; ORF C, which encodes core protein; ORF P, which encodes the polymerase; ORF X, which encodes HBX protein; the EnhI enhancer regulatory element; and/or the EnhII enhancer regulatory element. CRISPR systems for treating HBV are ideally delivered to liver cells, and in particular to hepatocytes. Thus an AAV8 vector may be useful.. Similarly, expression of the components of the CRISPR system are ideally under the transcriptional control of a liver-specific or hepatocyte-specific promoter.
[0350] The CRISPR system can be used in conjunction with an anti-HBV compound such as tenofovir or entecavir. As Cas9 targeting to cccDNA is likely at least partially dependent on the cccDNA structure, co-treatment with epigenetic modifiers (for example Class I and Class III HDAC inhibitors trichostatin A (TSA), valproate, and nicotinamide (NAM), and Type I interferons) may be useful for increasing Cas9 occupancy on HBV cccDNA. For treating HBV in practice, the CRISPR Cas system must avoid the shortcomings of RNAi, such as the risk of oversaturating endogenous small RNA pathways, by for example, optimizing dose and sequence (see, e.g., Grimm et al., Nature vol. 441, 26 May 2006). For example, low doses, such as about 1-10 x 1014 particles per human are contemplated. In another embodiment, the CRISPR Cas system directed against HBV may be administered in liposomes, such as a stable nucleic-acid- lipid particle (SNALP) (see, e.g., Morrissey et al., Nature Biotechnology, Vol. 23, No. 8, August 2005). Daily intravenous injections of about 1, 3 or 5 mg/kg/day of CRISPR Cas targeted to HBV RNA in a SNALP are contemplated. The daily treatment may be over about three days and then weekly for about five weeks. In another embodiment, the system of Chen et al. (Gene Therapy (2007) 14, 11–19) may be used/and or adapted for the CRISPR Cas system of the present invention. Chen et al. use a double-stranded adenoassociated virus 8-pseudotyped vector (dsAAV2/8) to deliver shRNA. A single administration of dsAAV2/8 vector (1 x 1012 vector genomes per mouse), carrying HBV-specific shRNA, effectively suppressed the steady level of HBV protein, mRNA and replicative DNA in liver of HBV transgenic mice, leading to up to 2–3 log10 decrease in HBV load in the circulation. Significant HBV suppression sustained for at least 120 days after vector administration. The therapeutic effect of shRNA was target sequence dependent and did not involve activation of interferon. For the present invention, a CRISPR Cas system directed to HBV may be cloned into an AAV vector, such as a dsAAV2/8 vector and administered to a human, for example, at a dosage of about 1 x 1015 vector genomes to about 1 x 1016 vector genomes per human. In another embodiment, the method of Wooddell et al. (Molecular Therapy vol. 21 no. 5, 973–985 May 2013) may be used/and or adapted to the CRISPR Cas system of the present invention. Woodell et al. show that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, Wooddell et al. show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. Intraveinous coinjections, for example, of about 6 mg/kg of NAG-MLP and 6 mg/kg of HBV specific CRISPR Cas may be envisioned for the present invention. In the alternative, about 3 mg/kg of NAG-MLP and 3 mg/kg of HBV specific CRISPR Cas may be delivered on day one, followed by administration of about about 2-3 mg/kg of NAG- MLP and 2-3 mg/kg of HBV specific CRISPR Cas two weeks later.
[0351] The present invention may also be applied to treat hepatitis C virus (HCV). The methods of Roelvinki et al. (Molecular Therapy vol. 20 no. 9, 1737-1749 Sep 2012) may be applied to the CRISPR Cas system. For example, an AAV vector such as AAV8 may be a contemplated vector and for example a dosage of about 1.25 × 1011 to 1.25 × 1013 vector genomes per kilogram body weight (vg/kg) may be contemplated.
[0352] It will be readily apparent that a host of other viruses can be treated in a similar fashion.
Nucleic acids, amino acids and proteins
[0353] The invention uses nucleic acids to bind target DNA sequences. This is advantageous as nucleic acids are much easier and cheaper to produce than proteins, and the specificity can be varied according to the length of the stretch where homology is sought. Complex 3-D positioning of multiple fingers, for example is not required. The terms“polynucleotide”, “nucleotide”, “nucleotide sequence”, “nucleic acid” and “oligonucleotide” are used interchangeably. They refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides, or analogs thereof. Polynucleotides may have any three dimensional structure, and may perform any function, known or unknown. The following are non-limiting examples of polynucleotides: coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, short interfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA), ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. The term also encompasses nucleic-acid-like structures with synthetic backbones, see, e.g., Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO 96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. A polynucleotide may comprise one or more modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be imparted before or after assembly of the polymer. The sequence of nucleotides may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component.
[0354] As used herein the term“wild type” is a term of the art understood by skilled persons and means the typical form of an organism, strain, gene or characteristic as it occurs in nature as distinguished from mutant or variant forms.
[0355] As used herein the term“variant” should be taken to mean the exhibition of qualities that have a pattern that deviates from what occurs in nature. [0356] The terms“non-naturally occurring” or“engineered” are used interchangeably and indicate the involvement of the hand of man. The terms, when referring to nucleic acid molecules or polypeptides mean that the nucleic acid molecule or the polypeptide is at least substantially free from at least one other component with which they are naturally associated in nature and as found in nature.
[0357] “Complementarity” refers to the ability of a nucleic acid to form hydrogen bond(s) with another nucleic acid sequence by either traditional Watson-Crick base pairing or other non- traditional types. A percent complementarity indicates the percentage of residues in a nucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crick base pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9, 10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” means that all the contiguous residues of a nucleic acid sequence will hydrogen bond with the same number of contiguous residues in a second nucleic acid sequence. “Substantially complementary” as used herein refers to a degree of complementarity that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refers to two nucleic acids that hybridize under stringent conditions.
[0358] As used herein,“stringent conditions” for hybridization refer to conditions under which a nucleic acid having complementarity to a target sequence predominantly hybridizes with the target sequence, and substantially does not hybridize to non-target sequences. Stringent conditions are generally sequence-dependent, and vary depending on a number of factors. In general, the longer the sequence, the higher the temperature at which the sequence specifically hybridizes to its target sequence. Non-limiting examples of stringent conditions are described in detail in Tijssen (1993), Laboratory Techniques In Biochemistry And Molecular Biology- Hybridization With Nucleic Acid Probes Part I, Second Chapter“Overview of principles of hybridization and the strategy of nucleic acid probe assay”, Elsevier, N.Y. Where reference is made to a polynucleotide sequence, then complementary or partially complementary sequences are also envisaged. These are preferably capable of hybridising to the reference sequence under highly stringent conditions. Generally, in order to maximize the hybridization rate, relatively low-stringency hybridization conditions are selected: about 20 to 25° C lower than the thermal melting point (Tm ). The Tm is the temperature at which 50% of specific target sequence hybridizes to a perfectly complementary probe in solution at a defined ionic strength and pH. Generally, in order to require at least about 85% nucleotide complementarity of hybridized or hybridizable sequences, highly stringent washing conditions are selected to be about 5 to 15° C lower than the Tm . In order to require at least about 70% nucleotide complementarity of hybridized or hybridizable sequences, moderately-stringent washing conditions are selected to be about 15 to 30° C lower than the Tm . Highly permissive (very low stringency) washing conditions may be as low as 50° C below the Tm , allowing a high level of mis-matching between hybridized or hybridizable sequences. Those skilled in the art will recognize that other physical and chemical parameters in the hybridization and wash stages can also be altered to affect the outcome of a detectable hybridization signal from a specific level of homology between target and probe sequences. Preferred highly stringent conditions comprise incubation in 50% formamide, 5×SSC, and 1% SDS at 42° C, or incubation in 5×SSC and 1% SDS at 65° C, with wash in 0.2×SSC and 0.1% SDS at 65° C.
[0359] “Hybridization” refers to a reaction in which one or more polynucleotides react to form a complex that is stabilized via hydrogen bonding between the bases of the nucleotide residues. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner. The complex may comprise two strands forming a duplex structure, three or more strands forming a multi stranded complex, a single self- hybridizing strand, or any combination of these. A hybridization reaction may constitute a step in a more extensive process, such as the initiation of PCR, or the cleavage of a polynucleotide by an enzyme. A sequence capable of hybridizing with a given sequence is referred to as the “complement” of the given sequence.
[0360] As used herein, the term“genomic locus” or“locus” (plural loci) is the specific location of a gene or DNA sequence on a chromosome. A“gene” refers to stretches of DNA or RNA that encode a polypeptide or an RNA chain that has functional role to play in an organism and hence is the molecular unit of heredity in living organisms. For the purpose of this invention it may be considered that genes include regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites and locus control regions.
[0361] As used herein,“expression of a genomic locus” or“gene expression” is the process by which information from a gene is used in the synthesis of a functional gene product. The products of gene expression are often proteins, but in non-protein coding genes such as rRNA genes or tRNA genes, the product is functional RNA. The process of gene expression is used by all known life - eukaryotes (including multicellular organisms), prokaryotes (bacteria and archaea) and viruses to generate functional products to survive. As used herein "expression" of a gene or nucleic acid encompasses not only cellular gene expression, but also the transcription and translation of nucleic acid(s) in cloning systems and in any other context. As used herein, “expression” also refers to the process by which a polynucleotide is transcribed from a DNA template (such as into and mRNA or other RNA transcript) and/or the process by which a transcribed mRNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as“gene product.” If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in a eukaryotic cell.
[0362] The terms“polypeptide”,“peptide” and“protein” are used interchangeably herein to refer to polymers of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non amino acids. The terms also encompass an amino acid polymer that has been modified; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component. As used herein the term“amino acid” includes natural and/or unnatural or synthetic amino acids, including glycine and both the D or L optical isomers, and amino acid analogs and peptidomimetics.
[0363] As used herein, the term“domain” or“protein domain” refers to a part of a protein sequence that may exist and function independently of the rest of the protein chain.
[0364] As described in aspects of the invention, sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences. In some preferred embodiments, the capping region of the dTALEs described herein have sequences that are at least 95% identical or share identity to the capping region amino acid sequences provided herein.
[0365] Sequence homologies may be generated by any of a number of computer programs known in the art, for example BLAST or FASTA, etc. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12:387). Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid– Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60). However it is preferred to use the GCG Bestfit program.
[0366] Percentage (%) sequence homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an“ungapped” alignment. Typically, such ungapped alignments are performed only over a relatively short number of residues.
[0367] Although this is a very simple and consistent method, it fails to take into consideration that, for example, in an otherwise identical pair of sequences, one insertion or deletion may cause the following amino acid residues to be put out of alignment, thus potentially resulting in a large reduction in % homology when a global alignment is performed. Consequently, most sequence comparison methods are designed to produce optimal alignments that take into consideration possible insertions and deletions without unduly penalizing the overall homology or identity score. This is achieved by inserting“gaps” in the sequence alignment to try to maximize local homology or identity.
[0368] However, these more complex methods assign“gap penalties” to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting higher relatedness between the two compared sequences - may achieve a higher score than one with many gaps.“Affinity gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties may, of course, produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
[0369] Calculation of maximum % homology therefore first requires the production of an optimal alignment, taking into consideration gap penalties. A suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (Devereux et al., 1984 Nuc. Acids Research 12 p387). Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 Short Protocols in Molecular Biology, 4th Ed.– Chapter 18), FASTA (Altschul et al., 1990 J. Mol. Biol. 403-410) and the GENEWORKS suite of comparison tools. Both BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999, Short Protocols in Molecular Biology, pages 7-58 to 7-60). However, for some applications, it is preferred to use the GCG Bestfit program. A new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174(2): 247-50; FEMS Microbiol Lett. 1999177(1): 187-8 and the website of the National Center for Biotechnology information at the website of the National Institutes for Health).
[0370] Although the final % homology may be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison. Instead, a scaled similarity score matrix is generally used that assigns scores to each pair-wise comparison based on chemical similarity or evolutionary distance. An example of such a matrix commonly used is the BLOSUM62 matrix - the default matrix for the BLAST suite of programs. GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table, if supplied (see user manual for further details). For some applications, it is preferred to use the public default values for the GCG package, or in the case of other software, the default matrix, such as BLOSUM62.
[0371] Alternatively, percentage homologies may be calculated using the multiple alignment feature in DNASISTM (Hitachi Software), based on an algorithm, analogous to CLUSTAL (Higgins DG & Sharp PM (1988), Gene 73(1), 237-244). Once the software has produced an optimal alignment, it is possible to calculate % homology, preferably % sequence identity. The software typically does this as part of the sequence comparison and generates a numerical result.
[0372] The sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance. Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups. Amino acids may be grouped together based on the properties of their side chains alone. However, it is more useful to include mutation data as well. The sets of amino acids thus derived are likely to be conserved for structural reasons. These sets may be described in the form of a Venn diagram (Livingstone C.D. and Barton G.J. (1993)“Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput. Appl. Biosci. 9: 745-756) (Taylor W.R. (1986)“The classification of amino acid conservation” J. Theor. Biol. 119; 205-218). Conservative substitutions may be made, for example according to the table below which describes a generally accepted Venn diagram grouping of amino acids.
Figure imgf000133_0001
[0373] Embodiments of the invention include sequences (both polynucleotide or polypeptide) which may comprise homologous substitution (substitution and replacement are both used herein to mean the interchange of an existing amino acid residue or nucleotide, with an alternative residue or nucleotide) that may occur i.e., like-for-like substitution in the case of amino acids such as basic for basic, acidic for acidic, polar for polar, etc. Non-homologous substitution may also occur i.e., from one class of residue to another or alternatively involving the inclusion of unnatural amino acids such as ornithine (hereinafter referred to as Z), diaminobutyric acid ornithine (hereinafter referred to as B), norleucine ornithine (hereinafter referred to as O), pyriylalanine, thienylalanine, naphthylalanine and phenylglycine.
[0374] Variant amino acid sequences may include suitable spacer groups that may be inserted between any two amino acid residues of the sequence including alkyl groups such as methyl, ethyl or propyl groups in addition to amino acid spacers such as glycine or E-alanine residues. A further form of variation, which involves the presence of one or more amino acid residues in peptoid form, may be well understood by those skilled in the art. For the avoidance of doubt,“the peptoid form” is used to refer to variant amino acid residues wherein the D-carbon substituent group is on the residue’s nitrogen atom rather than the D-carbon. Processes for preparing peptides in the peptoid form are known in the art, for example Simon RJ et al., PNAS (1992) 89(20), 9367-9371 and Horwell DC, Trends Biotechnol. (1995) 13(4), 132-134. [0375] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and Maniatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc.): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).
Vectors
[0376] In one aspect, the invention provides for vectors that are used in the engineering and optimization of CRISPR-Cas systems. A used herein, a“vector” is a tool that allows or facilitates the transfer of an entity from one environment to another. It is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a vector is capable of replication when associated with the proper control elements. In general, the term“vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double-stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a“plasmid,” which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally- derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno-associated viruses (AAVs)). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively-linked. Such vectors are referred to herein as“expression vectors.” Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
[0377] Recombinant expression vectors can comprise a nucleic acid of the invention in a form suitable for expression of the nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory elements, which may be selected on the basis of the host cells to be used for expression, that is operatively-linked to the nucleic acid sequence to be expressed. Within a recombinant expression vector,“operably linked” is intended to mean that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows for expression of the nucleotide sequence (e.g. in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell). With regards to recombination and cloning methods, mention is made of U.S. patent application 10/815,730, published September 2, 2004 as US 2004-0171156 A1, the contents of which are herein incorporated by reference in their entirety.
[0378] Aspects of the invention relate to bicistronic vectors for chimeric RNA and Cas9. Bicistronic expression vectors for chimeric RNA and Cas9 are preferred. In general and particularly in this embodiment Cas9 is preferably driven by the CBh promoter. The chimeric RNA may preferably be driven by a U6 promoter. Ideally the two are combined. The chimeric guide RNA typically consists of a 20bp guide sequence (Ns) and this may be joined to the tracr sequence (running from the first“U” of the lower strand to the end of the transcript). The tracr sequence may be truncated at various positions as indicated. The guide and tracr sequences are separated by the tracr-mate sequence, which may be GUUUUAGAGCUA. This may be followed by the loop sequence GAAA as shown. Both of these are preferred examples. Applicants have demonstrated Cas9-mediated indels at the human EMX1 and PVALB loci by SURVEYOR assays. ChiRNAs are indicated by their“+n” designation, and crRNA refers to a hybrid RNA where guide and tracr sequences are expressed as separate transcripts. Throughout this application, chimeric RNA may also be called single guide, or synthetic guide RNA (sgRNA). The loop is preferably GAAA, but it is not limited to this sequence or indeed to being only 4bp in length. Indeed, preferred loop forming sequences for use in hairpin structures are four nucleotides in length, and most preferably have the sequence GAAA. However, longer or shorter loop sequences may be used, as may alternative sequences. The sequences preferably include a nucleotide triplet (for example, AAA), and an additional nucleotide (for example C or G). Examples of loop forming sequences include CAAA and AAAG. [0379] The term“regulatory element” is intended to include promoters, enhancers, internal ribosomal entry sites (IRES), and other expression control elements (e.g. transcription termination signals, such as polyadenylation signals and poly-U sequences). Such regulatory elements are described, for example, in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Regulatory elements include those that direct constitutive expression of a nucleotide sequence in many types of host cell and those that direct expression of the nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). A tissue-specific promoter may direct expression primarily in a desired tissue of interest, such as muscle, neuron, bone, skin, blood, specific organs (e.g. liver, pancreas), or particular cell types (e.g. lymphocytes). Regulatory elements may also direct expression in a temporal-dependent manner, such as in a cell-cycle dependent or developmental stage-dependent manner, which may or may not also be tissue or cell-type specific. In some embodiments, a vector comprises one or more pol III promoter (e.g. 1, 2, 3, 4, 5, or more pol III promoters), one or more pol II promoters (e.g. 1, 2, 3, 4, 5, or more pol II promoters), one or more pol I promoters (e.g. 1, 2, 3, 4, 5, or more pol I promoters), or combinations thereof. Examples of pol III promoters include, but are not limited to, U6 and H1 promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521 -530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the ȕ-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1Į promoter. Also encompassed by the term“regulatory element” are enhancer elements, such as WPRE; CMV enhancers; the R-U5’ segment in LTR of HTLV-I (Mol. Cell. Biol., Vol. 8(1), p. 466-472, 1988); SV40 enhancer; and the intron sequence between exons 2 and 3 of rabbit ȕ-globin (Proc. Natl. Acad. Sci. USA., Vol. 78(3), p. 1527-31, 1981). It will be appreciated by those skilled in the art that the design of the expression vector can depend on such factors as the choice of the host cell to be transformed, the level of expression desired, etc. A vector can be introduced into host cells to thereby produce transcripts, proteins, or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., clustered regularly interspersed short palindromic repeats (CRISPR) transcripts, proteins, enzymes, mutant forms thereof, fusion proteins thereof, etc.). With regards to regulatory sequences, mention is made of U.S. patent application 10/491,026, the contents of which are incorporated by reference herein in their entirety. With regards to promoters, mention is made of PCT publication WO 2011/028929 and U.S. application 12/511,940, the contents of which are incorporated by reference herein in their entirety.
[0380] Vectors can be designed for expression of CRISPR transcripts (e.g. nucleic acid transcripts, proteins, or enzymes) in prokaryotic or eukaryotic cells. For example, CRISPR transcripts can be expressed in bacterial cells such as Escherichia coli, insect cells (using baculovirus expression vectors), yeast cells, or mammalian cells. Suitable host cells are discussed further in Goeddel, GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990). Alternatively, the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase (although any suitable DNA-dependent RNA polymerase can be used, such as SP6, T3 or T7). Amplification by T7 RNA polymerase requires the presence of suitable promoters in the RNA-coding DNA. Sequence requirements for polymerase binding sites are well known in the art. Various T7 RNA polymerase promoter sequences are known, including natural sequences and artificial ones. Different T7 RNA polymerases can have different promoter sequence preferences, and mutant T7 RNA polymerases have been produced to match specific promoters (e.g. see US patents 5,122,457 and 5,385,834), but the skilled person can routinely obtain both T7 RNA polymerases and promoter sequences, and can easily match any particular T7 RNA polymerase to its preferred promoter sequence. The consensus 23 base-pair T7 DNA promoter is classically divided into two domains, an upstream binding domain (-17 to -5, numbered relative to the start of transcription), and a downstream initiation domain (-4 to +6). One strand of this 23mer is 5'-TAATACGACTCACTATAGGGAGA-3'. The minimum sequence required for efficient transcription is the first 19mer of this 23mer.
[0381] Vectors may be introduced and propagated in a prokaryote or prokaryotic cell. In some embodiments, a prokaryote is used to amplify copies of a vector to be introduced into a eukaryotic cell or as an intermediate vector in the production of a vector to be introduced into a eukaryotic cell (e.g. amplifying a plasmid as part of a viral vector packaging system). In some embodiments, a prokaryote is used to amplify copies of a vector and express one or more nucleic acids, such as to provide a source of one or more proteins for delivery to a host cell or host organism. Expression of proteins in prokaryotes is most often carried out in Escherichia coli with vectors containing constitutive or inducible promoters directing the expression of either fusion or non-fusion proteins. Fusion vectors add a number of amino acids to a protein encoded therein, such as to the amino terminus of the recombinant protein. Such fusion vectors may serve one or more purposes, such as: (i) to increase expression of recombinant protein; (ii) to increase the solubility of the recombinant protein; and (iii) to aid in the purification of the recombinant protein by acting as a ligand in affinity purification. Often, in fusion expression vectors, a proteolytic cleavage site is introduced at the junction of the fusion moiety and the recombinant protein to enable separation of the recombinant protein from the fusion moiety subsequent to purification of the fusion protein. Such enzymes, and their cognate recognition sequences, include Factor Xa, thrombin and enterokinase. Example fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith and Johnson, 1988. Gene 67: 31-40), pMAL (New England Biolabs, Beverly, Mass.) and pRIT5 (Pharmacia, Piscataway, N.J.) that fuse glutathione S-transferase (GST), maltose E binding protein, or protein A, respectively, to the target recombinant protein.
[0382] Examples of suitable inducible non-fusion E. coli expression vectors include pTrc (Amrann et al., (1988) Gene 69:301-315) and pET 11d (Studier et al., GENE EXPRESSION TECHNOLOGY: METHODS IN ENZYMOLOGY 185, Academic Press, San Diego, Calif. (1990) 60-89).
[0383] In some embodiments, a vector is a yeast expression vector. Examples of vectors for expression in yeast Saccharomyces cerivisae include pYepSec1 (Baldari, et al., 1987. EMBO J. 6: 229-234), pMFa (Kuijan and Herskowitz, 1982. Cell 30: 933-943), pJRY88 (Schultz et al., 1987. Gene 54: 113-123), pYES2 (Invitrogen Corporation, San Diego, Calif.), and picZ (InVitrogen Corp, San Diego, Calif.).
[0384] In some embodiments, a vector drives protein expression in insect cells using baculovirus expression vectors. Baculovirus vectors available for expression of proteins in cultured insect cells (e.g., SF9 cells) include the pAc series (Smith, et al., 1983. Mol. Cell. Biol. 3: 2156-2165) and the pVL series (Lucklow and Summers, 1989. Virology 170: 31-39).
[0385] In some embodiments, a vector is capable of driving expression of one or more sequences in mammalian cells using a mammalian expression vector. Examples of mammalian expression vectors include pCDM8 (Seed, 1987. Nature 329: 840) and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195). When used in mammalian cells, the expression vector’s control functions are typically provided by one or more regulatory elements. For example, commonly used promoters are derived from polyoma, adenovirus 2, cytomegalovirus, simian virus 40, and others disclosed herein and known in the art. For other suitable expression systems for both prokaryotic and eukaryotic cells see, e.g., Chapters 16 and 17 of Sambrook, et al., MOLECULAR CLONING: A LABORATORY MANUAL. 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989.
[0386] In some embodiments, the recombinant mammalian expression vector is capable of directing expression of the nucleic acid preferentially in a particular cell type (e.g., tissue- specific regulatory elements are used to express the nucleic acid). Tissue-specific regulatory elements are known in the art. Non-limiting examples of suitable tissue-specific promoters include the albumin promoter (liver-specific; Pinkert, et al., 1987. Genes Dev. 1: 268-277), lymphoid-specific promoters (Calame and Eaton, 1988. Adv. Immunol. 43: 235-275), in particular promoters of T cell receptors (Winoto and Baltimore, 1989. EMBO J. 8: 729-733) and immunoglobulins (Baneiji, et al., 1983. Cell 33: 729-740; Queen and Baltimore, 1983. Cell 33: 741-748), neuron-specific promoters (e.g., the neurofilament promoter; Byrne and Ruddle, 1989. Proc. Natl. Acad. Sci. USA 86: 5473-5477), pancreas-specific promoters (Edlund, et al., 1985. Science 230: 912-916), and mammary gland-specific promoters (e.g., milk whey promoter; U.S. Pat. No. 4,873,316 and European Application Publication No. 264,166). Developmentally- regulated promoters are also encompassed, e.g., the murine hox promoters (Kessel and Gruss, 1990. Science 249: 374-379) and the Į -fetoprotein promoter (Campes and Tilghman, 1989. Genes Dev. 3: 537-546). With regards to these prokaryotic and eukaryotic vectors, mention is made of U.S. Patent 6,750,059, the contents of which are incorporated by reference herein in their entirety. Other embodiments of the invention may relate to the use of viral vectors, with regards to which mention is made of U.S. Patent application 13/092,085, the contents of which are incorporated by reference herein in their entirety. Tissue-specific regulatory elements are known in the art and in this regard, mention is made of U.S. Patent 7,776,321, the contents of which are incorporated by reference herein in their entirety.
Regulatory elements
[0387] In some embodiments, a regulatory element is operably linked to one or more elements of a CRISPR system so as to drive expression of the one or more elements of the CRISPR system. In general, CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats), also known as SPIDRs (SPacer Interspersed Direct Repeats), constitute a family of DNA loci that are usually specific to a particular bacterial species. The CRISPR locus comprises a distinct class of interspersed short sequence repeats (SSRs) that were recognized in E. coli (Ishino et al., J. Bacteriol., 169:5429-5433 [1987]; and Nakata et al., J. Bacteriol., 171:3553- 3556 [1989]), and associated genes. Similar interspersed SSRs have been identified in Haloferax mediterranei, Streptococcus pyogenes, Anabaena, and Mycobacterium tuberculosis (See, Groenen et al., Mol. Microbiol., 10:1057-1065 [1993]; Hoe et al., Emerg. Infect. Dis., 5:254-263 [1999]; Masepohl et al., Biochim. Biophys. Acta 1307:26-30 [1996]; and Mojica et al., Mol. Microbiol., 17:85-93 [1995]). The CRISPR loci typically differ from other SSRs by the structure of the repeats, which have been termed short regularly spaced repeats (SRSRs) (Janssen et al., OMICS J. Integ. Biol., 6:23-33 [2002]; and Mojica et al., Mol. Microbiol., 36:244-246 [2000]). In general, the repeats are short elements that occur in clusters that are regularly spaced by unique intervening sequences with a substantially constant length (Mojica et al., [2000], supra). Although the repeat sequences are highly conserved between strains, the number of interspersed repeats and the sequences of the spacer regions typically differ from strain to strain (van Embden et al., J. Bacteriol., 182:2393-2401 [2000]). CRISPR loci have been identified in more than 40 prokaryotes (See e.g., Jansen et al., Mol. Microbiol., 43:1565-1575 [2002]; and Mojica et al., [2005]) including, but not limited to Aeropyrum, Pyrobaculum, Sulfolobus, Archaeoglobus, Halocarcula, Methanobacterium, Methanococcus, Methanosarcina, Methanopyrus, Pyrococcus, Picrophilus, Thermoplasma, Corynebacterium, Mycobacterium, Streptomyces, Aquifex, Porphyromonas, Chlorobium, Thermus, Bacillus, Listeria, Staphylococcus, Clostridium, Thermoanaerobacter, Mycoplasma, Fusobacterium, Azarcus, Chromobacterium, Neisseria, Nitrosomonas, Desulfovibrio, Geobacter, Myxococcus, Campylobacter, Wolinella, Acinetobacter, Erwinia, Escherichia, Legionella, Methylococcus, Pasteurella, Photobacterium, Salmonella, Xanthomonas, Yersinia, Treponema, and Thermotoga.
[0388] In general,“CRISPR system” refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence (e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a“direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a“spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In embodiments of the invention the terms guide sequence and guide RNA are used interchangeably. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex,“target sequence” refers to a sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.
[0389] In some embodiments, direct repeats may be identified in silico by searching for repetitive motifs that fulfill any or all of the following criteria: 1. found in a 2Kb window of genomic sequence flanking the type II CRISPR locus; 2. span from 20 to 50 bp; and 3. interspaced by 20 to 50 bp. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used. In some embodiments, candidate tracrRNA may be subsequently predicted by sequences that fulfill any or all of the following criteria: 1. sequence homology to direct repeats (motif search in Geneious with up to 18-bp mismatches); 2. presence of a predicted Rho-independent transcriptional terminator in direction of transcription; and 3. stable hairpin secondary structure between tracrRNA and direct repeat. In some embodiments, 2 of these criteria may be used, for instance 1 and 2, 2 and 3, or 1 and 3. In some embodiments, all 3 criteria may be used. In some embodiments, chimeric synthetic guide RNAs (sgRNAs) designs may incorporate at least 12 bp of duplex structure between the direct repeat and tracrRNA. In preferred embodiments of the invention, the CRISPR system is a type II CRISPR system and the Cas enzyme is Cas9, which catalyzes DNA cleavage. Enzymatic action by Cas9 derived from Streptococcus pyogenes or any closely related Cas9 generates double stranded breaks at target site sequences which hybridize to 20 nucleotides of the guide sequence and that have a protospacer-adjacent motif (PAM) sequence (examples include NGG/NRG or a PAM that can be determined as described herein) following the 20 nucleotides of the target sequence. CRISPR activity through Cas9 for site- specific DNA recognition and cleavage is defined by the guide sequence, the tracr sequence that hybridizes in part to the guide sequence and the PAM sequence. More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA-guided defence in bacteria and archaea, Mole Cell 2010, January 15; 37(1): 7.
[0390] The type II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30bp each). In this system, targeted DNA double-strand break (DSB) is generated in four sequential steps (Fig. 2A). First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats of pre-crRNA, which is then processed into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer (Fig. 2A). Fig. 2B demonstrates the nuclear localization of the codon optimized Cas9. To promote precise transcriptional initiation, the RNA polymerase III-based U6 promoter was selected to drive the expression of tracrRNA (Fig. 2C). Similarly, a U6 promoter-based construct was developed to express a pre-crRNA array consisting of a single spacer flanked by two direct repeats (DRs, also encompassed by the term“tracr-mate sequences”; Fig. 2C). The initial spacer was designed to target a 33-base-pair (bp) target site (30-bp protospacer plus a 3-bp CRISPR motif (PAM) sequence satisfying the NGG recognition motif of Cas9) in the human EMX1 locus (Fig. 2C), a key gene in the development of the cerebral cortex.
[0391] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized or hybridizable to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 50, or more base pairs from) the target sequence. Without wishing to be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5’ with respect to (“upstream” of) or 3’ with respect to (“downstream” of) a second element. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more of the guide sequence, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter.
[0392] In some embodiments, a vector comprises one or more insertion sites, such as a restriction endonuclease recognition sequence (also referred to as a“cloning site”). In some embodiments, one or more insertion sites (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more insertion sites) are located upstream and/or downstream of one or more sequence elements of one or more vectors. In some embodiments, a vector comprises an insertion site upstream of a tracr mate sequence, and optionally downstream of a regulatory element operably linked to the tracr mate sequence, such that following insertion of a guide sequence into the insertion site and upon expression the guide sequence directs sequence-specific binding of a CRISPR complex to a target sequence in a eukaryotic cell. In some embodiments, a vector comprises two or more insertion sites, each insertion site being located between two tracr mate sequences so as to allow insertion of a guide sequence at each site. In such an arrangement, the two or more guide sequences may comprise two or more copies of a single guide sequence, two or more different guide sequences, or combinations of these. When multiple different guide sequences are used, a single expression construct may be used to target CRISPR activity to multiple different, corresponding target sequences within a cell. For example, a single vector may comprise about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, or more guide sequences. In some embodiments, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more such guide-sequence-containing vectors may be provided, and optionally delivered to a cell.
[0393] In some embodiments, a vector comprises a regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Cas protein. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, homologues thereof, or modified versions thereof. In some embodiments, the unmodified CRISPR enzyme has DNA cleavage activity, such as Cas9. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands at the location of a target sequence, such as within the target sequence and/or within the complement of the target sequence. In some embodiments, the CRISPR enzyme directs cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of a target sequence. In some embodiments, a vector encodes a CRISPR enzyme that is mutated to with respect to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence. For example, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of Cas9 from S. pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (cleaves a single strand). Other examples of mutations that render Cas9 a nickase include, without limitation, H840A, N854A, and N863A. As a further example, two or more catalytic domains of Cas9 (RuvC I, RuvC II, and RuvC III or the HNH domain) may be mutated to produce a mutated Cas9 substantially lacking all DNA cleavage activity. In some embodiments, a D10A mutation is combined with one or more of H840A, N854A, or N863A mutations to produce a Cas9 enzyme substantially lacking all DNA cleavage activity. In some embodiments, a CRISPR enzyme is considered to substantially lack all DNA cleavage activity when the DNA cleavage activity of the mutated enzyme is less than about 25%, 10%, 5%, 1%, 0.1%, 0.01%, or lower with respect to its non-mutated form. Where the enzyme is not SpCas9, mutations may be made at any or all residues corresponding to positions 10, 762, 840, 854, 863 and/or 986 of SpCas9 (which may be ascertained for instance by standard sequence comparison tools . In particular, any or all of the following mutations are preferred in SpCas9: D10A, E762A, H840A, N854A, N863A and/or D986A; as well as conservative substitution for any of the replacement amino acids is also envisaged. The same (or conservative substitutions of these mutations) at corresponding positions in other Cas9s are also preferred. Particularly preferred are D10 and H840 in SpCas9 . However, in other Cas9s, residues corresponding to SpCas9 D10 and H840 are also preferred.
[0394] An aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of SpCas9 was engineered to convert the nuclease into a nickase (SpCas9n) (see e.g. Sapranauskas et al., 2011, Nucleic Acis Research, 39: 9275; Gasiunas et al., 2012, Proc. Natl. Acad. Sci. USA, 109:E2579), such that nicked genomic DNA undergoes the high-fidelity homology-directed repair (HDR). Surveyor assay confirmed that SpCas9n does not generate indels at the EMX1 protospacer target. Co-expression of EMX1-targeting chimeric crRNA (having the tracrRNA component as well) with SpCas9 produced indels in the target site, whereas co-expression with SpCas9n did not (n=3). Moreover, sequencing of 327 amplicons did not detect any indels induced by SpCas9n. The same locus was selected to test CRISPR-mediated HR by co- transfecting HEK 293FT cells with the chimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, as well as a HR template to introduce a pair of restriction sites (HindIII and NheI) near the protospacer.
[0395] Preferred orthologs are described herein. A Cas enzyme may be identified Cas9 as this can refer to the general class of enzymes that share homology to the biggest nuclease with multiple nuclease domains from the type II CRISPR system. Most preferably, the Cas9 enzyme is from, or is derived from, spCas9 or saCas9. By derived, Applicants mean that the derived enzyme is largely based, in the sense of having a high degree of sequence homology with, a wildtype enzyme, but that it has been mutated (modified) in some way as described herein.
[0396] It will be appreciated that the terms Cas and CRISPR enzyme are generally used herein interchangeably, unless otherwise apparent. As mentioned above, many of the residue numberings used herein refer to the Cas9 enzyme from the type II CRISPR locus in Streptococcus pyogenes. However, it will be appreciated that this invention includes many more Cas9s from other species of microbes, such as SpCas9, SaCa9, St1Cas9 and so forth.
Codon optimization
[0397] An example of a codon optimized sequence, in this instance optimized for humans (i.e. being optimized for expression in humans) is provided herein, see the SaCas9 human codon optimized sequence. Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species is known. In some embodiments, an enzyme coding sequence encoding a CRISPR enzyme is codon optimized for expression in particular cells, such as eukaryotic cells. The eukaryotic cells may be those of or derived from a particular organism, such as a mammal, including but not limited to human, mouse, rat, rabbit, dog, or non- human mammal or primate. In some embodiments, processes for modifying the germ line genetic identity of human beings and/or processes for modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal, and also animals resulting from such processes, may be excluded.
[0398] In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www.kazusa.orjp/codon/ (visited Jul. 9, 2002), and these tables can be adapted in a number of ways. See Nakamura, Y., et al.“Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28:292 (2000). Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available. In some embodiments, one or more codons (e.g. 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a CRISPR enzyme correspond to the most frequently used codon for a particular amino acid.
[0399] Codon usage can even be optimized for expression in particular cell types e.g. for brain cells. For instance, Plotkin et al. (2004) PNAS USA 101:12588-91 reports on tissue-specific codon usage and notes, for instance, that brain-specific genes show a characteristically different codon usage than liver-specific genes. Thus a protein-coding sequence can be codon-optimised for expression in a target cell type of interest e.g. for expression in the liver.
Nuclear localization sequences (NLSs)
[0400] In some embodiments, a vector encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs), such as about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In some embodiments, the CRISPR enzyme comprises about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the amino-terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs at or near the carboxy-terminus, or a combination of these (e.g. one or more NLS at the amino-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each may be selected independently of the others, such that a single NLS may be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies. In a preferred embodiment of the invention, the CRISPR enzyme comprises at most 6 NLSs. In some embodiments, an NLS is considered near the N- or C-terminus when the nearest amino acid of the NLS is within about 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50, or more amino acids along the polypeptide chain from the N- or C-terminus. Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV; the NLS from nucleoplasmin (e.g. the nucleoplasmin bipartite NLS with the sequence KRPAATKKAGQAKKKK); the c-myc NLS having the amino acid sequence PAAKRVKLD or RQRRNELKRSP; the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY; the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV of the IBB domain from importin-alpha; the sequences VSRKRPRP and PPKKARED of the myoma T protein; the sequence POPKKKPL of human p53; the sequence SALIKKKKKMAP of mouse c-abl IV; the sequences DRLRR and PKQKKRK of the influenza virus NS1; the sequence RKLKKKIKKL of the Hepatitis virus delta antigen; the sequence REKKKFLKRR of the mouse Mx1 protein; the sequence KRKGDEVDGVDEVAKKKSKK of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK of the steroid hormone receptors (human) glucocorticoid.
[0401] In general, the one or more NLSs are of sufficient strength to drive accumulation of the CRISPR enzyme in a detectable amount in the nucleus of a eukaryotic cell. In general, strength of nuclear localization activity may derive from the number of NLSs in the CRISPR enzyme, the particular NLS(s) used, or a combination of these factors. Detection of accumulation in the nucleus may be performed by any suitable technique. For example, a detectable marker may be fused to the CRISPR enzyme, such that location within a cell may be visualized, such as in combination with a means for detecting the location of the nucleus (e.g. a stain specific for the nucleus such as DAPI). Cell nuclei may also be isolated from cells, the contents of which may then be analyzed by any suitable process for detecting protein, such as immunohistochemistry, Western blot, or enzyme activity assay. Accumulation in the nucleus may also be determined indirectly, such as by an assay for the effect of CRISPR complex formation (e.g. assay for DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity affected by CRISPR complex formation and/or CRISPR enzyme activity), as compared to a control no exposed to the CRISPR enzyme or complex, or exposed to a CRISPR enzyme lacking the one or more NLSs. Guide sequence
[0402] In general, a guide sequence is any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence. In some embodiments, the degree of complementarity between a guide sequence and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting example of which include the Smith-Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows-Wheeler Transform (e.g. the Burrows Wheeler Aligner), ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies; available at www.novocraft.com), ELAND (Illumina, San Diego, CA), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net). In some embodiments, a guide sequence is about or more than about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In some embodiments, a guide sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length. The ability of a guide sequence to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay. For example, the components of a CRISPR system sufficient to form a CRISPR complex, including the guide sequence to be tested, may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence, such as by Surveyor assay as described herein. Similarly, cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide sequence to be tested and a control guide sequence different from the test guide sequence, and comparing binding or rate of cleavage at the target sequence between the test and control guide sequence reactions. Other assays are possible, and will occur to those skilled in the art.
[0403] A guide sequence may be selected to target any target sequence. In some embodiments, the target sequence is a sequence within a genome of a cell. Exemplary target sequences include those that are unique in the target genome. For example, for the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGG where NNNNNNNNNNNNXGG (N is A G T or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGG where NNNNNNNNNNNXGG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. For the S. thermophilus CRISPR1 Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. A unique target sequence in a genome may include an S. thermophilus CRISPR1 Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXXAGAAW where NNNNNNNNNNNXXAGAAW (N is A, G, T, or C; X can be anything; and W is A or T) has a single occurrence in the genome. For the S. pyogenes Cas9, a unique target sequence in a genome may include a Cas9 target site of the form MMMMMMMMNNNNNNNNNNNNXGGXG where NNNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. A unique target sequence in a genome may include an S. pyogenes Cas9 target site of the form MMMMMMMMMNNNNNNNNNNNXGGXG where NNNNNNNNNNNXGGXG (N is A, G, T, or C; and X can be anything) has a single occurrence in the genome. In each of these sequences“M” may be A, G, T, or C, and need not be considered in identifying a sequence as unique. In some embodiments, a guide sequence is selected to reduce the degree secondary structure within the guide sequence. In some embodiments, about or less than about 75%, 50%, 40%, 30%, 25%, 20%, 15%, 10%, 5%, 1%, or fewer of the nucleotides of the guide sequence participate in self-complementary base pairing when optimally folded. Optimal folding may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy. An example of one such algorithm is mFold, as described by Zuker and Stiegler (Nucleic Acids Res. 9 (1981), 133-148). Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
Tracr mate sequence
[0404] In general, a tracr mate sequence includes any sequence that has sufficient complementarity with a tracr sequence to promote one or more of: (1) excision of a guide sequence flanked by tracr mate sequences in a cell containing the corresponding tracr sequence; and (2) formation of a CRISPR complex at a target sequence, wherein the CRISPR complex comprises the tracr mate sequence hybridized or hybridizable to the tracr sequence. In general, degree of complementarity is with reference to the optimal alignment of the tracr mate sequence and tracr sequence, along the length of the shorter of the two sequences. Optimal alignment may be determined by any suitable alignment algorithm, and may further account for secondary structures, such as self-complementarity within either the tracr sequence or tracr mate sequence. In some embodiments, the degree of complementarity between the tracr sequence and tracr mate sequence along the length of the shorter of the two when optimally aligned is about or more than about 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97.5%, 99%, or higher. In some embodiments, the tracr sequence is about or more than about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or more nucleotides in length. In some embodiments, the tracr sequence and tracr mate sequence are contained within a single transcript, such that hybridization between the two produces a transcript having a secondary structure, such as a hairpin. In an embodiment of the invention, the transcript or transcribed polynucleotide sequence has at least two or more hairpins. In preferred embodiments, the transcript has two, three, four or five hairpins. In a further embodiment of the invention, the transcript has at most five hairpins. In a hairpin structure the portion of the sequence 5’ of the final“N” and upstream of the loop corresponds to the tracr mate sequence, and the portion of the sequence 3’ of the loop corresponds to the tracr sequence Further non-limiting examples of single polynucleotides comprising a guide sequence, a tracr mate sequence, and a tracr sequence are as follows (listed 5’ to 3’), where“N” represents a base of a guide sequence, the first block of lower case letters represent the tracr mate sequence, and the second block of lower case letters represent the tracr sequence, and the final poly-T sequence represents the transcription terminator: (1) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaagatttaGAAAtaaatcttgcagaagctacaaagataa ggcttcatgccgaaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (2) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgttttcgttatttaaTTTTTT; (3) NNNNNNNNNNNNNNNNNNNNgtttttgtactctcaGAAAtgcagaagctacaaagataaggcttcatgccg aaatcaacaccctgtcattttatggcagggtgtTTTTTT; (4) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaactt gaaaaagtggcaccgagtcggtgcTTTTTT; (5) NNNNNNNNNNNNNNNNNNNNgttttagagctaGAAATAGcaagttaaaataaggctagtccgttatcaac ttgaaaaagtgTTTTTTT; and (6) NNNNNNNNNNNNNNNNNNNNgttttagagctagAAATAGcaagttaaaataaggctagtccgttatcaTT TTTTTT. In some embodiments, sequences (1) to (3) are used in combination with Cas9 from S. thermophilus CRISPR1. In some embodiments, sequences (4) to (6) are used in combination with Cas9 from S. pyogenes. In some embodiments, the tracr sequence is a separate transcript from a transcript comprising the tracr mate sequence.
Recombination template
[0405] In some embodiments, a recombination template is also provided. A recombination template may be a component of another vector as described herein, contained in a separate vector, or provided as a separate polynucleotide. In some embodiments, a recombination template is designed to serve as a template in homologous recombination, such as within or near a target sequence nicked or cleaved by a CRISPR enzyme as a part of a CRISPR complex. A template polynucleotide may be of any suitable length, such as about or more than about 10, 15, 20, 25, 50, 75, 100, 150, 200, 500, 1000, or more nucleotides in length. In some embodiments, the template polynucleotide is complementary to a portion of a polynucleotide comprising the target sequence. When optimally aligned, a template polynucleotide might overlap with one or more nucleotides of a target sequences (e.g. about or more than about 1, 5, 10, 15, 20, or more nucleotides). In some embodiments, when a template sequence and a polynucleotide comprising a target sequence are optimally aligned, the nearest nucleotide of the template polynucleotide is within about 1, 5, 10, 15, 20, 25, 50, 75, 100, 200, 300, 400, 500, 1000, 5000, 10000, or more nucleotides from the target sequence.
Fusion protein
[0406] In some embodiments, the CRISPR enzyme is part of a fusion protein comprising one or more heterologous protein domains (e.g. about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more domains in addition to the CRISPR enzyme). A CRISPR enzyme fusion protein may comprise any additional protein sequence, and optionally a linker sequence between any two domains. Examples of protein domains that may be fused to a CRISPR enzyme include, without limitation, epitope tags, reporter gene sequences, and protein domains having one or more of the following activities: methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity and nucleic acid binding activity. Non-limiting examples of epitope tags include histidine (His) tags, V5 tags, FLAG tags, influenza hemagglutinin (HA) tags, Myc tags, VSV-G tags, and thioredoxin (Trx) tags. Examples of reporter genes include, but are not limited to, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) beta-galactosidase, beta-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and autofluorescent proteins including blue fluorescent protein (BFP). A CRISPR enzyme may be fused to a gene sequence encoding a protein or a fragment of a protein that bind DNA molecules or bind other cellular molecules, including but not limited to maltose binding protein (MBP), S-tag, Lex A DNA binding domain (DBD) fusions, GAL4 DNA binding domain fusions, and herpes simplex virus (HSV) BP16 protein fusions. Additional domains that may form part of a fusion protein comprising a CRISPR enzyme are described in US20110059502, incorporated herein by reference. In some embodiments, a tagged CRISPR enzyme is used to identify the location of a target sequence.
Inducible system
[0407] In some embodiments, a CRISPR enzyme may form a component of an inducible system. The inducible nature of the system would allow for spatiotemporal control of gene editing or gene expression using a form of energy. The form of energy may include but is not limited to electromagnetic radiation, sound energy, chemical energy and thermal energy. Examples of inducible system include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc), or light inducible systems (Phytochrome, LOV domains, or cryptochrome).In one embodiment, the CRISPR enzyme may be a part of a Light Inducible Transcriptional Effector (LITE) to direct changes in transcriptional activity in a sequence-specific manner. The components of a light may include a CRISPR enzyme, a light-responsive cytochrome heterodimer (e.g. from Arabidopsis thaliana), and a transcriptional activation/repression domain. Further examples of inducible DNA binding proteins and methods for their use are provided in US 61/736465 and US 61/721,283, which is hereby incorporated by reference in its entirety (see also Konerman et al. (2013) Nature doi:10.1038/nature12466).
Delivery
[0408] In some aspects, the invention provides methods comprising delivering one or more polynucleotides, such as or one or more vectors as described herein, one or more transcripts thereof, and/or one or proteins transcribed therefrom, to a host cell. In some aspects, the invention further provides cells produced by such methods, and animals comprising or produced from such cells. In some embodiments, a CRISPR enzyme in combination with (and optionally complexed with) a guide sequence is delivered to a cell. Conventional viral and non-viral based gene transfer methods can be used to introduce nucleic acids in mammalian cells or target tissues. Such methods can be used to administer nucleic acids encoding components of a CRISPR system to cells in culture, or in a host organism. Non-viral vector delivery systems include DNA plasmids, RNA (e.g. a transcript of a vector described herein), naked nucleic acid, and nucleic acid complexed with a delivery vehicle, such as a liposome. Viral vector delivery systems include DNA and RNA viruses, which have either episomal or integrated genomes after delivery to the cell. For a review of gene therapy procedures, see Anderson, Science 256:808- 813 (1992); Nabel & Felgner, TIBTECH 11:211-217 (1993); Mitani & Caskey, TIBTECH 11:162-166 (1993); Dillon, TIBTECH 11:167-175 (1993); Miller, Nature 357:455-460 (1992); Van Brunt, Biotechnology 6(10):1149-1154 (1988); Vigne, Restorative Neurology and Neuroscience 8:35-36 (1995); Kremer & Perricaudet, British Medical Bulletin 51(1):31 -44 (1995); Haddada et al., in Current Topics in Microbiology and Immunology Doerfler and Böhm (eds) (1995); and Yu et al., Gene Therapy 1:13-26 (1994).
[0409] Methods of non-viral delivery of nucleic acids include lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, polycation or lipid:nucleic acid conjugates, naked DNA, artificial virions, and agent-enhanced uptake of DNA. Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those of Felgner, WO 91/17424; WO 91/16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).
[0410] The preparation of lipid:nucleic acid complexes, including targeted liposomes such as immunolipid complexes, is well known to one of skill in the art (see, e.g., Crystal, Science 270:404-410 (1995); Blaese et al., Cancer Gene Ther. 2:291-297 (1995); Behr et al., Bioconjugate Chem. 5:382-389 (1994); Remy et al., Bioconjugate Chem. 5:647-654 (1994); Gao et al., Gene Therapy 2:710-722 (1995); Ahmad et al., Cancer Res. 52:4817-4820 (1992); U.S. Pat. Nos. 4,186,183, 4,217,344, 4,235,871, 4,261,975, 4,485,054, 4,501,728, 4,774,085, 4,837,028, and 4,946,787).
[0411] The use of RNA or DNA viral based systems for the delivery of nucleic acids take advantage of highly evolved processes for targeting a virus to specific cells in the body and trafficking the viral payload to the nucleus. Viral vectors can be administered directly to patients (in vivo) or they can be used to treat cells in vitro, and the modified cells may optionally be administered to patients (ex vivo). Conventional viral based systems could include retroviral, lentivirus, adenoviral, adeno-associated and herpes simplex virus vectors for gene transfer. Integration in the host genome is possible with the retrovirus, lentivirus, and adeno-associated virus gene transfer methods, often resulting in long term expression of the inserted transgene. Additionally, high transduction efficiencies have been observed in many different cell types and target tissues.
[0412] The tropism of a retrovirus can be altered by incorporating foreign envelope proteins, expanding the potential target population of target cells. Lentiviral vectors are retroviral vectors that are able to transduce or infect non-dividing cells and typically produce high viral titers. Selection of a retroviral gene transfer system would therefore depend on the target tissue. Retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 kb of foreign sequence. The minimum cis-acting LTRs are sufficient for replication and packaging of the vectors, which are then used to integrate the therapeutic gene into the target cell to provide permanent transgene expression. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al., J. Virol. 66:2731-2739 (1992); Johann et al., J. Virol. 66:1635-1640 (1992); Sommnerfelt et al., Virol. 176:58-59 (1990); Wilson et al., J. Virol. 63:2374-2378 (1989); Miller et al., J. Virol. 65:2220-2224 (1991); WO94/26877).
[0413] In another embodiment, Cocal vesiculovirus envelope pseudotyped retroviral vector particles are contemplated (see, e.g., US Patent Publication No. 20120164118 assigned to the Fred Hutchinson Cancer Research Center). Cocal virus is in the Vesiculovirus genus, and is a causative agent of vesicular stomatitis in mammals. Cocal virus was originally isolated from mites in Trinidad (Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964)), and infections have been identified in Trinidad, Brazil, and Argentina from insects, cattle, and horses. Many of the vesiculoviruses that infect mammals have been isolated from naturally infected arthropods, suggesting that they are vector-borne. Antibodies to vesiculoviruses are common among people living in rural areas where the viruses are endemic and laboratory-acquired; infections in humans usually result in influenza-like symptoms. The Cocal virus envelope glycoprotein shares 71.5% identity at the amino acid level with VSV-G Indiana, and phylogenetic comparison of the envelope gene of vesiculoviruses shows that Cocal virus is serologically distinct from, but most closely related to, VSV-G Indiana strains among the vesiculoviruses. Jonkers et al., Am. J. Vet. Res. 25:236-242 (1964) and Travassos da Rosa et al., Am. J. Tropical Med. & Hygiene 33:999- 1006 (1984). The Cocal vesiculovirus envelope pseudotyped retroviral vector particles may include for example, lentiviral, alpharetroviral, betaretroviral, gammaretroviral, deltaretroviral, and epsilonretroviral vector particles that may comprise retroviral Gag, Pol, and/or one or more accessory protein(s) and a Cocal vesiculovirus envelope protein. Within certain aspects of these embodiments, the Gag, Pol, and accessory proteins are lentiviral and/or gammaretroviral.
[0414] In applications where transient expression is preferred, adenoviral based systems may be used. Adenoviral based vectors are capable of very high transduction efficiency in many cell types and do not require cell division. With such vectors, high titer and levels of expression have been obtained. This vector can be produced in large quantities in a relatively simple system.
[0415] Adeno-associated virus (“AAV”) vectors may also be used to transduce cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and for in vivo and ex vivo gene therapy procedures (see, e.g., West et al., Virology 160:38-47 (1987); U.S. Pat. No. 4,797,368; WO 93/24641; Kotin, Human Gene Therapy 5:793-801 (1994); Muzyczka, J. Clin. Invest. 94:1351 (1994). Construction of recombinant AAV vectors are described in a number of publications, including U.S. Pat. No. 5,173,414; Tratschin et al., Mol. Cell. Biol. 5:3251-3260 (1985); Tratschin, et al., Mol. Cell. Biol. 4:2072-2081 (1984); Hermonat & Muzyczka, PNAS 81:6466-6470 (1984); and Samulski et al., J. Virol. 63:03822-3828 (1989).
[0416] Packaging cells are typically used to form virus particles that are capable of infecting a host cell. Such cells include 293 or PER.C6 cells, which package adenovirus, and 2 cells or PA317 cells, which package retrovirus. Viral vectors used in gene therapy are usually generated by producer a cell line that packages a nucleic acid vector into a viral particle. The vectors typically contain the minimal viral sequences required for packaging and subsequent integration into a host, other viral sequences being replaced by an expression cassette for the polynucleotide(s) to be expressed. The missing viral functions are typically supplied in trans by the packaging cell line. For example, AAV vectors used in gene therapy typically only possess ITR sequences from the AAV genome which are required for packaging and integration into the host genome. Viral DNA is packaged in a cell line, which contains a helper plasmid encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The cell line may also infected with adenovirus as a helper. The helper virus promotes replication of the AAV vector and expression of AAV genes from the helper plasmid. The helper plasmid is not packaged in significant amounts due to a lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV. Accordingly, AAV is considered an ideal candidate for use as a transducing vector. Such AAV transducing vectors can comprise sufficient cis-acting functions to replicate in the presence of adenovirus or herpesvirus or poxvirus (e.g., vaccinia virus) helper functions provided in trans. Recombinant AAV (rAAV) can be used to carry exogenous genes into cells of a variety of lineages. In these vectors, the AAV cap and/or rep genes are deleted from the viral genome and replaced with a DNA segment of choice. Current AAV vectors may accommodate up to 4300 bases of inserted DNA.
[0417] There are a number of ways to produce rAAV, and the invention provides rAAV and methods for preparing rAAV. For example, plasmid(s) containing or consisting essentially of the desired viral construct are transfected into AAV-infected cells or into a packaging cell. In addition, a second or additional helper plasmid is cotransfected into these cells to provide the AAV rep and/or cap genes which are obligatory for replication and packaging of the recombinant viral construct. Under these conditions, the rep and/or cap proteins of AAV act in trans to stimulate replication and packaging of the rAAV construct. Two to Three days after transfection, rAAV is harvested. Traditionally rAAV is harvested from the cells along with adenovirus. The contaminating adenovirus is then inactivated by heat treatment. In the instant invention, rAAV is advantageously harvested not from the cells themselves, but from cell supernatant. Accordingly, in an initial aspect the invention provides for preparing rAAV, and in addition to the foregoing, rAAV can be prepared by a method that comprises or consists essentially of: infecting susceptible cells with a rAAV containing exogenous DNA including DNA for expression, and helper virus (e.g., adenovirus, herpesvirus, poxvirus such as vaccinia virus) wherein the rAAV lacks functioning cap and/or rep (and the helper virus (e.g., adenovirus, herpesvirus, poxvirus such as vaccinia virus) provides the cap and/or rev function that the rAAV lacks); or infecting susceptible cells with a rAAV containing exogenous DNA including DNA for expression, wherein the recombinant lacks functioning cap and/or rep, and transfecting said cells with a plasmid supplying cap and/or rep function that the rAAV lacks; or infecting susceptible cells with a rAAV containing exogenous DNA including DNA for expression, wherein the recombinant lacks functioning cap and/or rep, wherein said cells supply cap and/or rep function that the recombinant lacks; or transfecting the susceptible cells with an AAV lacking functioning cap and/or rep and plasmids for inserting exogenous DNA into the recombinant so that the exogenous DNA is expressed by the recombinant and for supplying rep and/or cap functions whereby transfection results in an rAAV containing the exogenous DNA including DNA for expression that lacks functioning cap and/or rep. [0418] The rAAV can be from an AAV as herein described, and advantageously can be an rAAV1, rAAV2, AAV5 or rAAV having hybrid or capsid which may comprise AAV1, AAV2, AAV5 or any combination thereof. One can select the AAV of the rAAV with regard to the cells to be targeted by the rAAV; e.g., one can select AAV serotypes 1, 2, 5 or a hybrid or capsid AAV1, AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. In addition to 293 cells, other cells that can be used in the practice of the invention and the relative infectivity of certain AAV serotypes in vitro as to these cells (see Grimm, D. et al, J. Virol. 82: 5887-5911 (2008)) are as follows:
Figure imgf000157_0001
[0419] The invention provides rAAV that contains or consists essentially of an exogenous nucleic acid molecule encoding a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system, e.g., a plurality of cassettes comprising or consisting a first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding a CRISPR-associated (Cas) protein (putative nuclease or helicase proteins), e.g., Cas9 and a terminator, and a two, or more, advantageously up to the packaging size limit of the vector, e.g., in total (including the first cassette) five, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator ... Promoter- gRNA(N)-terminator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector), or two or more individual rAAVs, each containing one or more than one cassette of a CRISPR system, e.g., a first rAAV containing the first cassette comprising or consisting essentially of a promoter, a nucleic acid molecule encoding Cas, e.g., Cas9 and a terminator, and a second rAAV containing a plurality, four, cassettes comprising or consisting essentially of a promoter, nucleic acid molecule encoding guide RNA (gRNA) and a terminator (e.g., each cassette schematically represented as Promoter-gRNA1-terminator, Promoter-gRNA2-terminator ... Promoter-gRNA(N)-terminator (where N is a number that can be inserted that is at an upper limit of the packaging size limit of the vector). As rAAV is a DNA virus, the nucleic acid molecules in the herein discussion concerning AAV or rAAV are advantageously DNA. The promoter is in some embodiments advantageously human Synapsin I promoter (hSyn).
[0420] Additional methods for the delivery of nucleic acids to cells are known to those skilled in the art. See, for example, US20030087817, incorporated herein by reference. In some embodiments, a host cell is transiently or non-transiently transfected with one or more vectors described herein. In some embodiments, a cell is transfected as it naturally occurs in a subject. In some embodiments, a cell that is transfected is taken from a subject. In some embodiments, the cell is derived from cells taken from a subject, such as a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, C8161, CCRF-CEM, MOLT, mIMCD-3, NHDF, HeLa-S3, Huh1, Huh4, Huh7, HUVEC, HASMC, HEKn, HEKa, MiaPaCell, Panc1, PC-3, TF1, CTLL-2, C1R, Rat6, CV1, RPTE, A10, T24, J82, A375, ARH-77, Calu1, SW480, SW620, SKOV3, SK-UT, CaCo2, P388D1, SEM-K2, WEHI-231, HB56, TIB55, Jurkat, J45.01, LRMB, Bcl-1, BC-3, IC21, DLD2, Raw264.7, NRK, NRK-52E, MRC5, MEF, Hep G2, HeLa B, HeLa T4, COS, COS-1, COS-6, COS-M6A, BS-C-1 monkey kidney epithelial, BALB/ 3T3 mouse embryo fibroblast, 3T3 Swiss, 3T3-L1, 132-d5 human fetal fibroblasts; 10.1 mouse fibroblasts, 293-T, 3T3, 721, 9L, A2780, A2780ADR, A2780cis, A172, A20, A253, A431, A-549, ALC, B16, B35, BCP-1 cells, BEAS-2B, bEnd.3, BHK-21, BR 293, BxPC3, C3H-10T1/2, C6/36, Cal-27, CHO, CHO-7, CHO-IR, CHO-K1, CHO-K2, CHO-T, CHO Dhfr -/-, COR-L23, COR-L23/CPR, COR-L23/5010, COR-L23/R23, COS-7, COV-434, CML T1, CMT, CT26, D17, DH82, DU145, DuCaP, EL4, EM2, EM3, EMT6/AR1, EMT6/AR10.0, FM3, H1299, H69, HB54, HB55, HCA2, HEK-293, HeLa, Hepa1c1c7, HL-60, HMEC, HT-29, Jurkat, JY cells, K562 cells, Ku812, KCL22, KG1, KYO1, LNCap, Ma-Mel 1-48, MC-38, MCF-7, MCF-10A, MDA-MB-231, MDA-MB-468, MDA-MB- 435, MDCK II, MDCK II, MOR/0.2R, MONO-MAC 6, MTD-1A, MyEnd, NCI-H69/CPR, NCI-H69/LX10, NCI-H69/LX20, NCI-H69/LX4, NIH-3T3, NALM-1, NW-145, OPCN / OPCT cell lines, Peer, PNT-1A / PNT 2, RenCa, RIN-5F, RMA/RMAS, Saos-2 cells, Sf-9, SkBr3, T2, T-47D, T84, THP1 cell line, U373, U87, U937, VCaP, Vero cells, WM39, WT-49, X63, YAC-1, YAR, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassus, Va.)). In some embodiments, a cell transfected with one or more vectors described herein is used to establish a new cell line comprising one or more vector-derived sequences. In some embodiments, a cell transiently transfected with the components of a CRISPR system as described herein (such as by transient transfection of one or more vectors, or transfection with RNA), and modified through the activity of a CRISPR complex, is used to establish a new cell line comprising cells containing the modification but lacking any other exogenous sequence. In some embodiments, cells transiently or non-transiently transfected with one or more vectors described herein, or cell lines derived from such cells are used in assessing one or more test compounds.
[0421] In another embodiment, a fluid delivery device with an array of needles (see, e.g., US Patent Publication No. 20110230839 assigned to the Fred Hutchinson Cancer Research Center) may be contemplated for delivery of CRISPR Cas to solid tissue. A device of US Patent Publication No. 20110230839 for delivery of a fluid to a solid tissue may comprise a plurality of needles arranged in an array; a plurality of reservoirs, each in fluid communication with a respective one of the plurality of needles; and a plurality of actuators operatively coupled to respective ones of the plurality of reservoirs and configured to control a fluid pressure within the reservoir. In certain embodiments each of the plurality of actuators may comprise one of a plurality of plungers, a first end of each of the plurality of plungers being received in a respective one of the plurality of reservoirs, and in certain further embodiments the plungers of the plurality of plungers are operatively coupled together at respective second ends so as to be simultaneously depressable. Certain still further embodiments may comprise a plunger driver configured to depress all of the plurality of plungers at a selectively variable rate. In other embodiments each of the plurality of actuators may comprise one of a plurality of fluid transmission lines having first and second ends, a first end of each of the plurality of fluid transmission lines being coupled to a respective one of the plurality of reservoirs. In other embodiments the device may comprise a fluid pressure source, and each of the plurality of actuators comprises a fluid coupling between the fluid pressure source and a respective one of the plurality of reservoirs. In further embodiments the fluid pressure source may comprise at least one of a compressor, a vacuum accumulator, a peristaltic pump, a master cylinder, a microfluidic pump, and a valve. In another embodiment, each of the plurality of needles may comprise a plurality of ports distributed along its length.
Modifying a target
[0422] In one aspect, the invention provides for methods of modifying a target polynucleotide in a eukaryotic cell, which may be in vivo, ex vivo or in vitro. In some embodiments, the method comprises sampling a cell or population of cells from a human or non- human animal, or a plant, and modifying the cell or cells. Culturing may occur at any stage ex vivo. The cell or cells may even be re-introduced into the non-human animal or plant. For re- introduced cells it is particularly preferred that the cells are stem cells. In some embodiments, the method comprises allowing a CRISPR complex to bind to the target polynucleotide to effect cleavage of said target polynucleotide thereby modifying the target polynucleotide, wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said target polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence.
[0423] In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in increased or decreased expression of said polynucleotide; wherein the CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within said polynucleotide, wherein said guide sequence is linked to a tracr mate sequence which in turn hybridizes to a tracr sequence. Similar considerations and conditions apply as above for methods of modifying a target polynucleotide. In fact, these sampling, culturing and re-introduction options apply across the aspects of the present invention. Indeed, in any aspect of the invention, the CRISPR complex may comprise a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence, wherein said guide sequence may be linked to a tracr mate sequence which in turn may hybridize to a tracr sequence. Similar considerations and conditions apply as above for methods of modifying a target polynucleotide.
Kits
[0424] In one aspect, the invention provides kits containing any one or more of the elements disclosed in the above methods and compositions. Elements may be provided individually or in combinations, and may be provided in any suitable container, such as a vial, a bottle, or a tube. In some embodiments, the kit includes instructions in one or more languages, for example in more than one language. In some embodiments, a kit comprises one or more reagents for use in a process utilizing one or more of the elements described herein. Reagents may be provided in any suitable container. For example, a kit may provide one or more reaction or storage buffers. Reagents may be provided in a form that is usable in a particular assay, or in a form that requires addition of one or more other components before use (e.g. in concentrate or lyophilized form). A buffer can be any buffer, including but not limited to a sodium carbonate buffer, a sodium bicarbonate buffer, a borate buffer, a Tris buffer, a MOPS buffer, a HEPES buffer, and combinations thereof. In some embodiments, the buffer is alkaline. In some embodiments, the buffer has a pH from about 7 to about 10. In some embodiments, the kit comprises one or more oligonucleotides corresponding to a guide sequence for insertion into a vector so as to operably link the guide sequence and a regulatory element. In some embodiments, the kit comprises a homologous recombination template polynucleotide. In some embodiments, the kit comprises one or more of the vectors and/or one or more of the polynucleotides described herein. The kit may advantageously allows to provide all elements of the systems of the invention.
CRISPR complex
[0425] In one aspect, the invention provides methods for using one or more elements of a CRISPR system. The CRISPR complex of the invention provides an effective means for modifying a target polynucleotide. The CRISPR complex of the invention has a wide variety of utility including modifying (e.g., deleting, inserting, translocating, inactivating) a target polynucleotide in a multiplicity of cell types. An exemplary CRISPR complex comprises a CRISPR enzyme complexed with a guide sequence hybridized or hybridizable to a target sequence within the target polynucleotide. The guide sequence is linked to a tracr mate sequence, which in turn hybridizes to a tracr sequence. In one embodiment, this invention provides a method of cleaving a target polynucleotide. The method comprises modifying a target polynucleotide using a CRISPR complex that binds to the target polynucleotide and effect cleavage of said target polynucleotide. Typically, the CRISPR complex of the invention, when introduced into a cell, creates a break (e.g., a single or a double strand break) in the genome sequence. For example, the method can be used to cleave an integrated viral gene in a cell. The break created by the CRISPR complex can be repaired by a repair processes such as the error prone non-homologous end joining (NHEJ) pathway or the high fidelity homology-directed repair (HDR) (Fig. 29). During these repair process, an exogenous polynucleotide template can be introduced into the genome sequence. In some methods, the HDR process is used modify genome sequence. For example, an exogenous polynucleotide template comprising a sequence to be integrated flanked by an upstream sequence and a downstream sequence is introduced into a cell. The upstream and downstream sequences share sequence similarity with either side of the site of integration in the chromosome. Where desired, a donor polynucleotide can be DNA, e.g., a DNA plasmid, a bacterial artificial chromosome (BAC), a yeast artificial chromosome (YAC), a viral vector, a linear piece of DNA, a PCR fragment, a naked nucleic acid, or a nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer. The exogenous polynucleotide template comprises a sequence to be integrated (e.g., a mutated gene). The sequence for integration may be a sequence endogenous or exogenous to the cell. Examples of a sequence to be integrated include polynucleotides encoding a protein or a non-coding RNA (e.g., a microRNA). Thus, the sequence for integration may be operably linked to an appropriate control sequence or sequences. Alternatively, the sequence to be integrated may provide a regulatory function. The upstream and downstream sequences in the exogenous polynucleotide template are selected to promote recombination between the chromosomal sequence of interest and the donor polynucleotide. The upstream sequence is a nucleic acid sequence that shares sequence similarity with the genome sequence upstream of the targeted site for integration. Similarly, the downstream sequence is a nucleic acid sequence that shares sequence similarity with the chromosomal sequence downstream of the targeted site of integration. The upstream and downstream sequences in the exogenous polynucleotide template can have 75%, 80%, 85%, 90%, 95%, or 100% sequence identity with the targeted genome sequence. Preferably, the upstream and downstream sequences in the exogenous polynucleotide template have about 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with the targeted genome sequence. In some methods, the upstream and downstream sequences in the exogenous polynucleotide template have about 99% or 100% sequence identity with the targeted genome sequence. An upstream or downstream sequence may comprise from about 20 bp to about 2500 bp, for example, about 50, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, 2400, or 2500 bp. In some methods, the exemplary upstream or downstream sequence have about 200 bp to about 2000 bp, about 600 bp to about 1000 bp, or more particularly about 700 bp to about 1000 bp. In some methods, the exogenous polynucleotide template may further comprise a marker. Such a marker may make it easy to screen for targeted integrations. Examples of suitable markers include restriction sites, fluorescent proteins, or selectable markers. The exogenous polynucleotide template of the invention can be constructed using recombinant techniques (see, for example, Sambrook et al., 2001 and Ausubel et al., 1996). In an exemplary method for modifying a target polynucleotide by integrating an exogenous polynucleotide template, a double stranded break is introduced into the genome sequence by the CRISPR complex, the break is repaired via homologous recombination an exogenous polynucleotide template such that the template is integrated into the genome. The presence of a double-stranded break facilitates integration of the template.
[0426] In other embodiments, this invention provides a method of modifying expression of a polynucleotide in a eukaryotic cell. The method comprises decreasing expression of a target polynucleotide by using a CRISPR complex that binds to the polynucleotide. In some methods, a target polynucleotide can be inactivated to effect the modification of the expression in a cell. For example, upon the binding of a CRISPR complex to a target sequence in a cell, the target polynucleotide is inactivated such that the sequence is not transcribed, the coded protein or RNA is not produced, or the sequence does not function as the wild-type sequence does. For example, a protein or microRNA coding sequence may be inactivated such that the protein or microRNA is not produced. In some methods, a control sequence can be inactivated such that it no longer functions as a control sequence. As used herein,“control sequence” refers to any nucleic acid sequence that effects the transcription, translation, or accessibility of a nucleic acid sequence. Examples of a control sequence include, a promoter, a transcription terminator, and an enhancer are control sequences. The inactivated target sequence may include a deletion mutation (i.e., deletion of one or more nucleotides), an insertion mutation (i.e., insertion of one or more nucleotides), or a nonsense mutation (i.e., substitution of a single nucleotide for another nucleotide such that a stop codon is introduced). In some methods, the inactivation of a target sequence results in“knock-out” of the target sequence.
[0427] The term“wild type StCas9” refers to wild type Cas9 from S thermophilus, the protein sequence of which is given in the SwissProt database under accession number G3ECR1. Similarly, S pyogenes Cas9 is included in SwissProt under accession number Q99ZW2.
Preclinical translation
[0428] To switch to a CRISPR/Cas system compatible with clinically-tested liver-directed gene delivery, Applicants may switch from using SpCas9 to the smaller SaCas9.
[0429] A first step is to redesign and produce sgRNA sequences compatible with the SaCas9 PAM. Then, the sgRNAs are cloned into a viral vector (likely lentiviral for initial in vitro cell line studies) and these new guides are screened for their ability to cleave cccDNA in de novo infections. These experiments use the HepG2-hNTCP knockin cell lines and HBV virions purified from HepG2.2.15 cells. The screen is performed either with guides in individual wells side-by-side, or using a pooled format in which lentivirus is produced with a pool of all possible sgRNA sequences against HBV, and then deep sequencing identifes regions of the HBV genome most susceptible to cleavage by SaCas9.
[0430] Once a smaller list of 3-10 guides is chosen, more targeted experiments are performed in both in vitro and in vivo models of HBV infection in primary human hepatocytes (see e.g., Schlomai et al., (2014) Proceedings of the National Academy of Sciences, 111(33): 12193-12198; Bissig et al., (2014) Journal of Clinical Investigation, 120(3): 924-930; Legrand N et al. (2009) Cell Host & Microbe, 6(1): 5-9). These experiments can also utilize targeting with multiple sgRNAs simultaneously to induce cccDNA fragmentation). Additional possibilities include using other delivery systems (perhaps nonviral) as herein described.
[0431] Rationale for choosing appropriate sgRNAs for anti-HBV CRISPR/Cas9 systems: The process is a multistep one in which several parameters should be optimized: efficacy of the sgRNA sequence, targeting to an accessible part of cccDNA, conservation of the target sequence across viral genotypes, and minimization of target sequence homology to the human genome. These criteria should be general criteria across all episomal viruses, although the specifics may differ (for example, HBV cccDNA seems to be most accessible to cleavage in the ORF for Core, and it is possible that latent HSV may be most accessible in the region encoding latency- associated transcript LAT). The general workflow is as follows:
• Use CRISPR design tool to identify all possible sgRNAs for the virus of interest (for targeting dsDNA forms, such as HBV, look at targets on both positive and negative strands), based on the PAM for the Cas9 of interest (e.g. SpCas0, SaCas9)
• Use available efficacy prediction tools (e.g. Doench et al. 2014. Nat Biotech for SpCas9) to predict on-target efficacy of the sgRNAs for initial prioritization
• Perform some combination of literature search and pilot experiments using sgRNAs targeting diverse regions of the viral genome, followed by assessing cleavage efficiency at these sites, to determine which parts of viral genome are most efficiently cleaved by Cas9. Prioritize sgRNAs that hit these regions. • From this downselected list of sgRNAs, further prioritize by how strongly conserved the target sequences are across viral genotypes. This can be done by performing primer BLAST against the full set of viral whole genome sequences deposited in GenBank. For example, for HBV there are a little over 5000 whole genome sequences from different strains and patient isolates. Ideal conservation across these sequences is 90+%.
• Finally, further downselect by choosing sgRNAs that have low homology to the human genome- typically this will not be a problem for viral genomes, since they are generally divergent from human genomic sequences (perhaps endogenous retroviruse regions are an exception, but these are predominantly nonfunctional)
• Efficacy in vitro and in vivo, as well as selectivity and minimizatrion of off- target effects, can then be determined experimentally in the proper model systems.
[0432] Below exhaustive list of possible sgRNAs for both SaCas9 and SpCas9. In these tables, the columns correspond to the sgRNA target DNA sequence (target sequence + PAM), the strand on which the target sequence and PAM are found (cccDNA is double-stranded), the nucleotide of the circular HBV genome at which the target sequence starts (HBV cccDNA is 3182 bp total), whether the sgRNA targets the Core ORF (which Applicants have found to be most capable of cleavage by Cas9), and finally whether or not the sgRNA is very close (+/- 50nt) on the HBV genome to where Applicants’ most effective sgRNA (g17) targets.
[0433] SaCas9 sgRNAs
Figure imgf000165_0001
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Figure imgf000196_0001
[0435] Preliminary next-gen sequencing was performed to identify the level of indel formation at top predicted off-target sites (using the Hsu 2013 algorithm), and Applicants’ current data shows no indel formation at 3 separate off-target sites for each of 3 separate sgRNAs (g6, g17, g21) in the HepG2.2.15 model system.
[0436] In the plot as depicted in Figure 73, the columns labeled‘D’ signify where Applicants used a nuclease-deficient Cas9 as an internal control. The 29 dpt corresponds to 29 days post transduction, where a single lentiviral vector encoding U6-sgRNA and EFS-hSpCas9-2A-Puro was transduced into HepG2.2.15 cells followed by selection with puromycin. The caveat is that the read depth was lower than ideal (~200-1000+ reads per target site), and that the on-target indels determined in this experiment are somewhat lower than what Applicants saw by Surveyor assay—however, it is promising that after stringent puromycin selection and constitutive expression of Cas9 and sgRNA for 29 days, still no indels are detected at possible off-target sites.
[0437] Additional anti-HBV CRISPR strategies: Previous studies have shown that CRISPR/Cas9 systems can be used for transcriptional activation when nuclease-deficient Cas9 is fused to activator domains. Specifically regarding HBV, it was recently shown that specific perturbations which upregulate APOBEC3A or APOBEC3B activity in HBV-infected hepatocytes can lead to HBV cccDNA clearance by specific CÆUÆT editing of the HBV genome leading to cccDNA degradation (Lucifora et al. Science 14 March 2014: Vol. 343 no. 6176 pp. 1221-1228). Since the CRISPR-based activation system can specifically upregulate targeted genes, it is possible to target APOBEC3A, APOBEC3B, and/or other antiviral interferon-stimulated genes (ISGs) using this system in order to target HBV cccDNA for degradation. While this is an indirect approach, the advantage here may be that the use of a nuclease-competent Cas9 is not required, potentially reducing the chance of deleterious off- target effects.
EXAMPLES
[0438] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art. Example 1: CRISPR Complex Activity in the Nucleus of a Eukaryotic Cell
[0439] An example type II CRISPR system is the type II CRISPR locus from Streptococcus pyogenes SF370, which contains a cluster of four genes Cas9, Cas1, Cas2, and Csn1, as well as two non-coding RNA elements, tracrRNA and a characteristic array of repetitive sequences (direct repeats) interspaced by short stretches of non-repetitive sequences (spacers, about 30bp each). In this system, targeted DNA double-strand break (DSB) is generated in four sequential steps (Fig. 2A). First, two non-coding RNAs, the pre-crRNA array and tracrRNA, are transcribed from the CRISPR locus. Second, tracrRNA hybridizes to the direct repeats of pre- crRNA, which is then processed into mature crRNAs containing individual spacer sequences. Third, the mature crRNA:tracrRNA complex directs Cas9 to the DNA target consisting of the protospacer and the corresponding PAM via heteroduplex formation between the spacer region of the crRNA and the protospacer DNA. Finally, Cas9 mediates cleavage of target DNA upstream of PAM to create a DSB within the protospacer (Fig. 2A). This example describes an example process for adapting this RNA-programmable nuclease system to direct CRISPR complex activity in the nuclei of eukaryotic cells.
[0440] Cell culture and transfection
[0441] Human embryonic kidney (HEK) cell line HEK 293FT (Life Technologies) was maintained in Dulbecco’s modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum (HyClone), 2mM GlutaMAX (Life Technologies), 100U/mL penicillin, and 100 g/mL streptomycin at 37ºC with 5% CO2 incubation. Mouse neuro2A (N2A) cell line (ATCC) was maintained with DMEM supplemented with 5% fetal bovine serum (HyClone), 2mM GlutaMAX (Life Technologies), 100U/mL penicillin, and 100 g/mL streptomycin at 37ºC with 5% CO2.
[0442] HEK 293FT or N2A cells were seeded into 24-well plates (Corning) one day prior to transfection at a density of 200,000 cells per well. Cells were transfected using Lipofectamine 2000 (Life Technologies) following the manufacturer’s recommended protocol. For each well of a 24-well plate a total of 800ng of plasmids were used.
[0443] Surveyor assay and sequencing analysis for genome modification
[0444] HEK 293FT or N2A cells were transfected with plasmid DNA as described above. After transfection, the cells were incubated at 37ºC for 72 hours before genomic DNA extraction. Genomic DNA was extracted using the QuickExtract DNA extraction kit (Epicentre) following the manufacturer’s protocol. Briefly, cells were resuspended in QuickExtract solution and incubated at 65ºC for 15 minutes and 98ºC for 10 minutes. Extracted genomic DNA was immediately processed or stored at–20ºC.
[0445] The genomic region surrounding a CRISPR target site for each gene was PCR amplified, and products were purified using QiaQuick Spin Column (Qiagen) following manufacturer’s protocol. A total of 400ng of the purified PCR products were mixed with 2 l 10X Taq polymerase PCR buffer (Enzymatics) and ultrapure water to a final volume of 20 l, and subjected to a re-annealing process to enable heteroduplex formation: 95ºC for 10min, 95ºC to 85ºC ramping at– 2ºC/s, 85ºC to 25ºC at– 0.25ºC/s, and 25ºC hold for 1 minute. After re- annealing, products were treated with Surveyor nuclease and Surveyor enhancer S (Transgenomics) following the manufacturer’s recommended protocol, and analyzed on 4-20% Novex TBE poly-acrylamide gels (Life Technologies). Gels were stained with SYBR Gold DNA stain (Life Technologies) for 30 minutes and imaged with a Gel Doc gel imaging system (Bio-rad). Quantification was based on relative band intensities, as a measure of the fraction of cleaved DNA. Fig. 7 provides a schematic illustration of this Surveyor assay.
[0446] Restriction fragment length polymorphism assay for detection of homologous recombination.
[0447] HEK 293FT and N2A cells were transfected with plasmid DNA, and incubated at 37°C for 72 hours before genomic DNA extraction as described above. The target genomic region was PCR amplified using primers outside the homology arms of the homologous recombination (HR) template. PCR products were separated on a 1% agarose gel and extracted with MinElute GelExtraction Kit (Qiagen). Purified products were digested with HindIII (Fermentas) and analyzed on a 6% Novex TBE poly-acrylamide gel (Life Technologies).
[0448] RNA secondary structure prediction and analysis
[0449] RNA secondary structure prediction was performed using the online webserver RNAfold developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. A.R. Gruber et al., 2008, Cell 106(1): 23-24; and PA Carr and GM Church, 2009, Nature Biotechnology 27(12): 1151-62).
[0450] RNA purification
[0451] HEK 293FT cells were maintained and transfected as stated above. Cells were harvested by trypsinization followed by washing in phosphate buffered saline (PBS). Total cell RNA was extracted with TRI reagent (Sigma) following manufacturer’s protocol. Extracted total RNA was quantified using Naonodrop (Thermo Scientific) and normalized to same concentration. [0452] Northern blot analysis of crRNA and tracrRNA expression in mammalian cells
[0453] RNAs were mixed with equal volumes of 2X loading buffer (Ambion), heated to 95qC for 5 min, chilled on ice for 1 min, and then loaded onto 8% denaturing polyacrylamide gels (SequaGel, National Diagnostics) after pre-running the gel for at least 30 minutes. The samples were electrophoresed for 1.5 hours at 40W limit. Afterwards, the RNA was transferred to Hybond N+ membrane (GE Healthcare) at 300 mA in a semi-dry transfer apparatus (Bio-rad) at room temperature for 1.5 hours. The RNA was crosslinked to the membrane using autocrosslink button on Stratagene UV Crosslinker the Stratalinker (Stratagene). The membrane was pre-hybridized in ULTRAhyb-Oligo Hybridization Buffer (Ambion) for 30 min with rotation at 42qC, and probes were then added and hybridized overnight. Probes were ordered from IDT and labeled with [gamma-32P] ATP (Perkin Elmer) with T4 polynucleotide kinase (New England Biolabs). The membrane was washed once with pre-warmed (42qC) 2xSSC, 0.5% SDS for 1 min followed by two 30 minute washes at 42qC. The membrane was exposed to a phosphor screen for one hour or overnight at room temperature and then scanned with a phosphorimager (Typhoon).
[0454] Bacterial CRISPR system construction and evaluation
[0455] CRISPR locus elements, including tracrRNA, Cas9, and leader were PCR amplified from Streptococcus pyogenes SF370 genomic DNA with flanking homology arms for Gibson Assembly. Two BsaI type IIS sites were introduced in between two direct repeats to facilitate easy insertion of spacers (Fig. 8). PCR products were cloned into EcoRV-digested pACYC184 downstream of the tet promoter using Gibson Assembly Master Mix (NEB). Other endogenous CRISPR system elements were omitted, with the exception of the last 50bp of Csn2. Oligos (Integrated DNA Technology) encoding spacers with complimentary overhangs were cloned into the BsaI-digested vector pDC000 (NEB) and then ligated with T7 ligase (Enzymatics) to generate pCRISPR plasmids. Challenge plasmids containing spacers with PAM expression in mammalian cells (expression constructs illustrated in Fig. 6A, with functionality as determined by results of the Surveyor assay shown in Fig. 6B). Transcription start sites are marked as +1, and transcription terminator and the sequence probed by northern blot are also indicated. Expression of processed tracrRNA was also confirmed by Northern blot. Fig. 6C shows results of a Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying long or short tracrRNA, as well as SpCas9 and DR-EMX1(1)-DR. Left and right panels are from 293FT cells transfected without or with SpRNase III, respectively. U6 indicate loading control blotted with a probe targeting human U6 snRNA Transfection of the short tracrRNA expression construct led to abundant levels of the processed form of tracrRNA (~75bp). Very low amounts of long tracrRNA are detected on the Northern blot.
[0456] To promote precise transcriptional initiation, the RNA polymerase III-based U6 promoter was selected to drive the expression of tracrRNA (Fig. 2C). Similarly, a U6 promoter- based construct was developed to express a pre-crRNA array consisting of a single spacer flanked by two direct repeats (DRs, also encompassed by the term“tracr-mate sequences”; Fig. 2C). The initial spacer was designed to target a 33-base-pair (bp) target site (30-bp protospacer plus a 3-bp CRISPR motif (PAM) sequence satisfying the NGG recognition motif of Cas9) in the human EMX1 locus (Fig. 2C), a key gene in the development of the cerebral cortex.
[0457] To test whether heterologous expression of the CRISPR system (SpCas9, SpRNase III, tracrRNA, and pre-crRNA) in mammalian cells can achieve targeted cleavage of mammalian chromosomes, HEK 293FT cells were transfected with combinations of CRISPR components. Since DSBs in mammalian nuclei are partially repaired by the non-homologous end joining (NHEJ) pathway, which leads to the formation of indels, the Surveyor assay was used to detect potential cleavage activity at the target EMX1 locus (Fig. 7) (see e.g. Guschin et al., 2010, Methods Mol Biol 649: 247). Co-transfection of all four CRISPR components was able to induce up to 5.0% cleavage in the protospacer (see Fig. 2D). Co-transfection of all CRISPR components minus SpRNase III also induced up to 4.7% indel in the protospacer, suggesting that there may be endogenous mammalian RNases that are capable of assisting with crRNA maturation, such as for example the related Dicer and Drosha enzymes. Removing any of the remaining three components abolished the genome cleavage activity of the CRISPR system (Fig. 2D). Sanger sequencing of amplicons containing the target locus verified the cleavage activity: in 43 sequenced clones, 5 mutated alleles (11.6%) were found. Similar experiments using a variety of guide sequences produced indel percentages as high as 29% (see Figs. 3-6, 10, and 11). These results define a three-component system for efficient CRISPR-mediated genome modification in mammalian cells. To optimize the cleavage efficiency, Applicants also tested whether different isoforms of tracrRNA affected the cleavage efficiency and found that, in this example system, only the short (89-bp) transcript form was able to mediate cleavage of the human EMX1 genomic locus (Fig. 6B).
[0458] Fig. 12 provides an additional Northern blot analysis of crRNA processing in mammalian cells. Fig. 12A illustrates a schematic showing the expression vector for a single spacer flanked by two direct repeats (DR-EMX1(1)-DR). The 30bp spacer targeting the human EMX1 locus protospacer 1 (see Fig. 6) and the direct repeat sequences are shown in the sequence beneath Fig. 12A. The line indicates the region whose reverse-complement sequence was used to generate Northern blot probes for EMX1(1) crRNA detection. Fig. 12B shows a Northern blot analysis of total RNA extracted from 293FT cells transfected with U6 expression constructs carrying DR-EMX1(1)-DR. Left and right panels are from 293FT cells transfected without or with SpRNase III respectively. DR-EMX1(1)-DR was processed into mature crRNAs only in the presence of SpCas9 and short tracrRNA and was not dependent on the presence of SpRNase III. The mature crRNA detected from transfected 293FT total RNA is ~33bp and is shorter than the 39-42bp mature crRNA from S. pyogenes. These results demonstrate that a CRISPR system can be transplanted into eukaryotic cells and reprogrammed to facilitate cleavage of endogenous mammalian target polynucleotides.
[0459] Fig. 2 illustrates the bacterial CRISPR system described in this example. Fig. 2A illustrates a schematic showing the CRISPR locus 1 from Streptococcus pyogenes SF370 and a proposed mechanism of CRISPR-mediated DNA cleavage by this system. Mature crRNA processed from the direct repeat-spacer array directs Cas9 to genomic targets consisting of complimentary protospacers and a protospacer-adjacent motif (PAM). Upon target-spacer base pairing, Cas9 mediates a double-strand break in the target DNA. Fig. 2B illustrates engineering of S. pyogenes Cas9 (SpCas9) and RNase III (SpRNase III) with nuclear localization signals (NLSs) to enable import into the mammalian nucleus. Fig. 2C illustrates mammalian expression of SpCas9 and SpRNase III driven by the constitutive EF1a promoter and tracrRNA and pre- crRNA array (DR-Spacer-DR) driven by the RNA Pol3 promoter U6 to promote precise transcription initiation and termination. A protospacer from the human EMX1 locus with a satisfactory PAM sequence is used as the spacer in the pre-crRNA array. Fig. 2D illustrates surveyor nuclease assay for SpCas9-mediated minor insertions and deletions. SpCas9 was expressed with and without SpRNase III, tracrRNA, and a pre-crRNA array carrying the EMX1- target spacer. Fig. 2E illustrates a schematic representation of base pairing between target locus and EMX1-targeting crRNA, as well as an example chromatogram showing a micro deletion adjacent to the SpCas9 cleavage site. Fig. 2F illustrates mutated alleles identified from sequencing analysis of 43 clonal amplicons showing a variety of micro insertions and deletions. Dashes indicate deleted bases, and non-aligned or mismatched bases indicate insertions or mutations. Scale bar = 10 m.
[0460] To further simplify the three-component system, a chimeric crRNA-tracrRNA hybrid design was adapted, where a mature crRNA (comprising a guide sequence) may be fused to a partial tracrRNA via a stem-loop to mimic the natural crRNA:tracrRNA duplex. To increase co- delivery efficiency, a bicistronic expression vector was created to drive co-expression of a chimeric RNA and SpCas9 in transfected cells. In parallel, the bicistronic vectors were used to express a pre-crRNA (DR-guide sequence-DR) with SpCas9, to induce processing into crRNA with a separately expressed tracrRNA (compare Fig. 11B top and bottom). Fig. 8 provides schematic illustrations of bicistronic expression vectors for pre-crRNA array (Figure 8A) or chimeric crRNA (represented by the short line downstream of the guide sequence insertion site and upstream of the EF1Į promoter in Fig. 8B) with hSpCas9, showing location of various elements and the point of guide sequence insertion. The expanded sequence around the location of the guide sequence insertion site in Fig. 8B also shows a partial DR sequence (GTTTAGAGCTA) and a partial tracrRNA sequence (TAGCAAGTTAAAATAAGGCTAGTCCGTTTTT). Guide sequences can be inserted between BbsI sites using annealed oligonucleotides. Sequence design for the oligonucleotides are shown below the schematic illustrations in Fig. 8, with appropriate ligation adapters indicated. WPRE represents the Woodchuck hepatitis virus post-transcriptional regulatory element. The efficiency of chimeric RNA-mediated cleavage was tested by targeting the same EMX1 locus described above. Using both Surveyor assay and Sanger sequencing of amplicons, Applicants confirmed that the chimeric RNA design facilitates cleavage of human EMX1 locus with approximately a 4.7% modification rate (Fig. 3).
[0461] Generalizability of CRISPR-mediated cleavage in eukaryotic cells was tested by targeting additional genomic loci in both human and mouse cells by designing chimeric RNA targeting multiple sites in the human EMX1 and PVALB, as well as the mouse Th loci. Fig. 13 illustrates the selection of some additional targeted protospacers in human PVALB (Fig. 13A) and mouse Th (Fig. 13B) loci. Schematics of the gene loci and the location of three protospacers within the last exon of each are provided. The underlined sequences include 30bp of protospacer sequence and 3bp at the 3’ end corresponding to the PAM sequences. Protospacers on the sense and anti-sense strands are indicated above and below the DNA sequences, respectively. A modification rate of 6.3% and 0.75% was achieved for the human PVALB and mouse Th loci respectively, demonstrating the broad applicability of the CRISPR system in modifying different loci across multiple organisms (Fig. 5). While cleavage was only detected with one out of three spacers for each locus using the chimeric constructs, all target sequences were cleaved with efficiency of indel production reaching 27% when using the co-expressed pre-crRNA arrangement (Figs. 6 and 13). [0462] Fig. 11 provides a further illustration that SpCas9 can be reprogrammed to target multiple genomic loci in mammalian cells. Fig. 11A provides a schematic of the human EMX1 locus showing the location of five protospacers, indicated by the underlined sequences. Fig. 11B provides a schematic of the pre-crRNA/trcrRNA complex showing hybridization between the direct repeat region of the pre-crRNA and tracrRNA (top), and a schematic of a chimeric RNA design comprising a 20bp guide sequence, and tracr mate and tracr sequences consisting of partial direct repeat and tracrRNA sequences hybridized in a hairpin structure (bottom). Results of a Surveyor assay comparing the efficacy of Cas9-mediated cleavage at five protospacers in the human EMX1 locus is illustrated in Fig. 11C. Each protospacer is targeted using either processed pre-crRNA/tracrRNA complex (crRNA) or chimeric RNA (chiRNA).
[0463] Since the secondary structure of RNA can be crucial for intermolecular interactions, a structure prediction algorithm based on minimum free energy and Boltzmann-weighted structure ensemble was used to compare the putative secondary structure of all guide sequences used in the genome targeting experiment (see e.g. Gruber et al., 2008, Nucleic Acids Research, 36: W70). Analysis revealed that in most cases, the effective guide sequences in the chimeric crRNA context were substantially free of secondary structure motifs, whereas the ineffective guide sequences were more likely to form internal secondary structures that could prevent base pairing with the target protospacer DNA. It is thus possible that variability in the spacer secondary structure might impact the efficiency of CRISPR-mediated interference when using a chimeric crRNA.
[0464] Further vector designs for SpCas9 are shown in Fig. 22, which illustrates single expression vectors incorporating a U6 promoter linked to an insertion site for a guide oligo, and a Cbh promoter linked to SpCas9 coding sequence. The vector shown in Fig. 22b includes a tracrRNA coding sequence linked to an H1 promoter.
[0465] In the bacterial assay, all spacers facilitated efficient CRISPR interference (Fig. 3C). These results suggest that there may be additional factors affecting the efficiency of CRISPR activity in mammalian cells.
[0466] To investigate the specificity of CRISPR-mediated cleavage, the effect of single- nucleotide mutations in the guide sequence on protospacer cleavage in the mammalian genome was analyzed using a series of EMX1-targeting chimeric crRNAs with single point mutations (Fig. 3A). Fig. 3B illustrates results of a Surveyor nuclease assay comparing the cleavage efficiency of Cas9 when paired with different mutant chimeric RNAs. Single-base mismatch up to 12-bp 5’ of the PAM substantially abrogated genomic cleavage by SpCas9, whereas spacers with mutations at farther upstream positions retained activity against the original protospacer target (Fig. 3B). In addition to the PAM, SpCas9 has single-base specificity within the last 12- bp of the spacer. Furthermore, CRISPR is able to mediate genomic cleavage as efficiently as a pair of TALE nucleases (TALEN) targeting the same EMX1 protospacer. Fig. 3C provides a schematic showing the design of TALENs targeting EMX1, and Fig. 3D shows a Surveyor gel comparing the efficiency of TALEN and Cas9 (n=3).
[0467] Having established a set of components for achieving CRISPR-mediated gene editing in mammalian cells through the error-prone NHEJ mechanism, the ability of CRISPR to stimulate homologous recombination (HR), a high fidelity gene repair pathway for making precise edits in the genome, was tested. The wild type SpCas9 is able to mediate site-specific DSBs, which can be repaired through both NHEJ and HR. In addition, an aspartate-to-alanine substitution (D10A) in the RuvC I catalytic domain of SpCas9 was engineered to convert the nuclease into a nickase (SpCas9n; illustrated in Fig. 4A) (see e.g. Sapranausaks et al., 2011, Nucleic Acids Resch, 39: 9275; Gasiunas et al., 2012, Proc. Natl. Acad. Sci. USA, 109:E2579), such that nicked genomic DNA undergoes the high-fidelity homology-directed repair (HDR). Surveyor assay confirmed that SpCas9n does not generate indels at the EMX1 protospacer target. As illustrated in Fig. 4B, co-expression of EMX1-targeting chimeric crRNA with SpCas9 produced indels in the target site, whereas co-expression with SpCas9n did not (n=3). Moreover, sequencing of 327 amplicons did not detect any indels induced by SpCas9n. The same locus was selected to test CRISPR-mediated HR by co-transfecting HEK 293FT cells with the chimeric RNA targeting EMX1, hSpCas9 or hSpCas9n, as well as a HR template to introduce a pair of restriction sites (HindIII and NheI) near the protospacer. Fig. 4C provides a schematic illustration of the HR strategy, with relative locations of recombination points and primer annealing sequences (arrows). SpCas9 and SpCas9n indeed catalyzed integration of the HR template into the EMX1 locus. PCR amplification of the target region followed by restriction digest with HindIII revealed cleavage products corresponding to expected fragment sizes (arrows in restriction fragment length polymorphism gel analysis shown in Fig. 4D), with SpCas9 and SpCas9n mediating similar levels of HR efficiencies. Applicants further verified HR using Sanger sequencing of genomic amplicons (Fig. 4E). These results demonstrate the utility of CRISPR for facilitating targeted gene insertion in the mammalian genome. Given the 14-bp (12- bp from the spacer and 2-bp from the PAM) target specificity of the wild type SpCas9, the availability of a nickase can significantly reduce the likelihood of off-target modifications, since single strand breaks are not substrates for the error-prone NHEJ pathway. [0468] Expression constructs mimicking the natural architecture of CRISPR loci with arrayed spacers (Fig. 2A) were constructed to test the possibility of multiplexed sequence targeting. Using a single CRISPR array encoding a pair of EMX1- and PVALB-targeting spacers, efficient cleavage at both loci was detected (Fig. 4F, showing both a schematic design of the crRNA array and a Surveyor blot showing efficient mediation of cleavage). Targeted deletion of larger genomic regions through concurrent DSBs using spacers against two targets within EMX1 spaced by 119bp was also tested, and a 1.6% deletion efficacy (3 out of 182 amplicons; Fig. 4G) was detected. This demonstrates that the CRISPR system can mediate multiplexed editing within a single genome.
[0469] Example 2: CRISPR system modifications and alternatives
[0470] The ability to use RNA to program sequence-specific DNA cleavage defines a new class of genome engineering tools for a variety of research and industrial applications. Several aspects of the CRISPR system can be further improved to increase the efficiency and versatility of CRISPR targeting. Optimal Cas9 activity may depend on the availability of free Mg2+ at levels higher than that present in the mammalian nucleus (see e.g. Jinek et al., 2012, Science, 337:816), and the preference for an NGG motif immediately downstream of the protospacer restricts the ability to target on average every 12-bp in the human genome (Fig. 9, evaluating both plus and minus strands of human chromosomal sequences). Some of these constraints can be overcome by exploring the diversity of CRISPR loci across the microbial metagenome (see e.g. Makarova et al., 2011, Nat Rev Microbiol, 9:467). Other CRISPR loci may be transplanted into the mammalian cellular milieu by a process similar to that described in Example 1. For example, Fig. 10 illustrates adaptation of the Type II CRISPR system from CRISPR 1 of Streptococcus thermophilus LMD-9 for heterologous expression in mammalian cells to achieve CRISPR-mediated genome editing. Fig. 10A provides a Schematic illustration of CRISPR 1 from S. thermophilus LMD-9. Figure 10B illustrates the design of an expression system for the S. thermophilus CRISPR system. Human codon-optimized hStCas9 is expressed using a constitutive EF1Į promoter. Mature versions of tracrRNA and crRNA are expressed using the U6 promoter to promote precise transcription initiation. Sequences from the mature crRNA and tracrRNA are illustrated. A single base indicated by the lower case“a” in the crRNA sequence is used to remove the polyU sequence, which serves as a RNA polIII transcriptional terminator. Fig. 10C provides a schematic showing guide sequences targeting the human EMX1 locus. Fig. 10D shows the results of hStCas9-mediated cleavage in the target locus using the Surveyor assay. RNA guide spacers 1 and 2 induced 14% and 6.4%, respectively. Statistical analysis of cleavage activity across biological replica at these two protospacer sites is also provided in Fig. 5. Fig. 14 provides a schematic of additional protospacer and corresponding PAM sequence targets of the S. thermophilus CRISPR system in the human EMX1 locus. Two protospacer sequences are highlighted and their corresponding PAM sequences satisfying NNAGAAW motif are indicated by underlining 3’ with respect to the corresponding highlighted sequence. Both protospacers target the anti-sense strand.
[0471] Example 3: Sample target sequence selection algorithm
[0472] A software program is designed to identify candidate CRISPR target sequences on both strands of an input DNA sequence based on desired guide sequence length and a CRISPR motif sequence (PAM) for a specified CRISPR enzyme. For example, target sites for Cas9 from S. pyogenes, with PAM sequences NGG, may be identified by searching for 5’-Nx-NGG-3’ both on the input sequence and on the reverse-complement of the input. Likewise, target sites for Cas9 of S. thermophilus CRISPR1, with PAM sequence NNAGAAW, may be identified by searching for 5’-Nx-NNAGAAW-3’ both on the input sequence and on the reverse-complement of the input. Likewise, target sites for Cas9 of S. thermophilus CRISPR3, with PAM sequence NGGNG, may be identified by searching for 5’-Nx-NGGNG-3’ both on the input sequence and on the reverse-complement of the input. The value“x” in Nx may be fixed by the program or specified by the user, such as 20.
[0473] Since multiple occurrences in the genome of the DNA target site may lead to nonspecific genome editing, after identifying all potential sites, the program filters out sequences based on the number of times they appear in the relevant reference genome. For those CRISPR enzymes for which sequence specificity is determined by a‘seed’ sequence, such as the 11-12bp 5’ from the PAM sequence, including the PAM sequence itself, the filtering step may be based on the seed sequence. Thus, to avoid editing at additional genomic loci, results are filtered based on the number of occurrences of the seed:PAM sequence in the relevant genome. The user may be allowed to choose the length of the seed sequence. The user may also be allowed to specify the number of occurrences of the seed:PAM sequence in a genome for purposes of passing the filter. The default is to screen for unique sequences. Filtration level is altered by changing both the length of the seed sequence and the number of occurrences of the sequence in the genome. The program may in addition or alternatively provide the sequence of a guide sequence complementary to the reported target sequence(s) by providing the reverse complement of the identified target sequence(s). An example visualization of some target sites in the human genome is provided in Fig. 18. [0474] Further details of methods and algorithms to optimize sequence selection can be found in U.S. application Serial No. 61/064,798 (Attorney docket 44790.11.2022; Broad Reference BI-2012/084); incorporated herein by reference.
[0475] Example 4: Evaluation of multiple chimeric crRNA-tracrRNA hybrids
[0476] This example describes results obtained for chimeric RNAs (chiRNAs; comprising a guide sequence, a tracr mate sequence, and a tracr sequence in a single transcript) having tracr sequences that incorporate different lengths of wild-type tracrRNA sequence. Fig.16a illustrates a schematic of a bicistronic expression vector for chimeric RNA and Cas9. Cas9 is driven by the CBh promoter and the chimeric RNA is driven by a U6 promoter. The chimeric guide RNA consists of a 20bp guide sequence (Ns) joined to the tracr sequence (running from the first“U” of the lower strand to the end of the transcript), which is truncated at various positions as indicated. The guide and tracr sequences are separated by the tracr-mate sequence GUUUUAGAGCUA followed by the loop sequence GAAA. Results of SURVEYOR assays for Cas9-mediated indels at the human EMX1 and PVALB loci are illustrated in Figs. 16b and 16c, respectively. Arrows indicate the expected SURVEYOR fragments. ChiRNAs are indicated by their“+n” designation, and crRNA refers to a hybrid RNA where guide and tracr sequences are expressed as separate transcripts. Quantification of these results, performed in triplicate, are illustrated by histogram in Figs. 17a and 17b, corresponding to Figs. 16b and 16c, respectively (“N.D.” indicates no indels detected). Protospacer IDs and their corresponding genomic target, protospacer sequence, PAM sequence, and strand location are provided in the following Table. Guide sequences were designed to be complementary to the entire protospacer sequence in the case of separate transcripts in the hybrid system, or only to the underlined portion in the case of chimeric RNAs.
Figure imgf000208_0001
[0477] Further details to optimize guide sequences can be found in U.S. application Serial No. 61/836,127 (Attorney docket 44790.08.2022; Broad Reference BI-2013/004G); incorporated herein by reference. [0478] Initially, three sites within the EMX1 locus in human HEK 293FT cells were targeted. Genome modification efficiency of each chiRNA was assessed using the SURVEYOR nuclease assay, which detects mutations resulting from DNA double-strand breaks (DSBs) and their subsequent repair by the non-homologous end joining (NHEJ) DNA damage repair pathway. Constructs designated chiRNA(+n) indicate that up to the +n nucleotide of wild-type tracrRNA is included in the chimeric RNA construct, with values of 48, 54, 67, and 85 used for n. Chimeric RNAs containing longer fragments of wild-type tracrRNA (chiRNA(+67) and chiRNA(+85)) mediated DNA cleavage at all three EMX1 target sites, with chiRNA(+85) in particular demonstrating significantly higher levels of DNA cleavage than the corresponding crRNA/tracrRNA hybrids that expressed guide and tracr sequences in separate transcripts (Figs. 16b and 17a). Two sites in the PVALB locus that yielded no detectable cleavage using the hybrid system (guide sequence and tracr sequence expressed as separate transcripts) were also targeted using chiRNAs. chiRNA(+67) and chiRNA(+85) were able to mediate significant cleavage at the two PVALB protospacers (Figs. 16c and 17b).
[0479] For all five targets in the EMX1 and PVALB loci, a consistent increase in genome modification efficiency with increasing tracr sequence length was observed. Without wishing to be bound by any theory, the secondary structure formed by the 3’ end of the tracrRNA may play a role in enhancing the rate of CRISPR complex formation.
[0480] Example 5: Cas9 diversity
[0481] The CRISPR-Cas system is an adaptive immune mechanism against invading exogenous DNA employed by diverse species across bacteria and archaea. The type II CRISPR- Cas9 system consists of a set of genes encoding proteins responsible for the“acquisition” of foreign DNA into the CRISPR locus, as well as a set of genes encoding the“execution” of the DNA cleavage mechanism; these include the DNA nuclease (Cas9), a non-coding transactivating cr-RNA (tracrRNA), and an array of foreign DNA-derived spacers flanked by direct repeats (crRNAs). Upon maturation by Cas9, the tracRNA and crRNA duplex guide the Cas9 nuclease to a target DNA sequence specified by the spacer guide sequences, and mediates double-stranded breaks in the DNA near a short sequence motif in the target DNA that is required for cleavage and specific to each CRISPR-Cas system. The type II CRISPR-Cas systems are found throughout the bacterial kingdom and highly diverse in in Cas9 protein sequence and size, tracrRNA and crRNA direct repeat sequence, genome organization of these elements, and the motif requirement for target cleavage. One species may have multiple distinct CRISPR-Cas systems. [0482] Applicants evaluated 207 putative Cas9s from bacterial species identified based on sequence homology to known Cas9s and structures orthologous to known subdomains, including the HNH endonuclease domain and the RuvC endonuclease domains [information from the Eugene Koonin and Kira Makarova]. Phylogenetic analysis based on the protein sequence conservation of this set revealed five families of Cas9s, including three groups of large Cas9s (~1400 amino acids) and two of small Cas9s (~1100 amino acids) (see Figs. 19 and 20A-F).
[0483] Further details of Cas9s and mutations of the Cas9 enzyme to convert into a nickase or DNA binding protein and use of same with altered functionality can be found in U.S. application Serial Nos 61/836,101 and 61/835,936 (Attorney docket 44790.09.2022 and 4790.07.2022 and Broad Reference BI-2013/004E and BI-2013/004F respectively) incorporated herein by reference.
[0484] Example 6: Cas9 orthologs
[0485] Applicants analyzed Cas9 orthologs to identify the relevant PAM sequences and the corresponding chimeric guide RNA. Having an expanded set of PAMs provides broader targeting across the genome and also significantly increases the number of unique target sites and provides potential for identifying novel Cas9s with increased levels of specificity in the genome.
[0486] The specificity of Cas9 orthologs can be evaluated by testing the ability of each Cas9 to tolerate mismatches between the guide RNA and its DNA target. For example, the specificity of SpCas9 has been characterized by testing the effect of mutations in the guide RNA on cleavage efficiency. Libraries of guide RNAs were made with single or multiple mismatches between the guide sequence and the target DNA. Based on these findings, target sites for SpCas9 can be selected based on the following guidelines:
[0487] To maximize SpCas9 specificity for editing a particular gene, one should choose a target site within the locus of interest such that potential‘off-target’ genomic sequences abide by the following four constraints: First and foremost, they should not be followed by a PAM with either 5’-NGG or NAG sequences. Second, their global sequence similarity to the target sequence should be minimized. Third, a maximal number of mismatches should lie within the PAM-proximal region of the off-target site. Finally, a maximal number of mismatches should be consecutive or spaced less than four bases apart.
[0488] Similar methods can be used to evaluate the specificity of other Cas9 orthologs and to establish criteria for the selection of specific target sites within the genomes of target species. As mentioned previously phylogenetic analysis based on the protein sequence conservation of this set revealed five families of Cas9s, including three groups of large Cas9s (~1400 amino acids) and two of small Cas9s (~1100 amino acids) (see Figs. 19 and 20A-F). Further details on Cas orthologs can be found in U.S. application Serial Nos 61/836,101 and 61/835,936 (Attorney docket 44790.09.2022 and 4790.07.2022 and Broad Reference BI-2013/004E and BI-2013/004F respectively) incorporated herein by reference.
[0489] Example 7: Methodological improvement to simplify cloning and delivery.
[0490] Rather than encoding the U6-promoter and guide RNA on a plasmid, Applicants amplified the U6 promoter with a DNA oligo to add on the guide RNA. The resulting PCR product may be transfected into cells to drive expression of the guide RNA.
[0491] Example primer pair that allows the generation a PCR product consisting of U6- promoter::guideRNA targeting human Emx1 locus:
[0492] Forward Primer: AAACTCTAGAgagggcctatttcccatgattc
[0493] Reverse Primer (carrying the guide RNA, which is underlined): acctctagAAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGC CTTATTTTAACTTGCTATGCTGTTTTGTTTCCAAAACAGCATAGCTCTAAAACCCC TAGTCATTGGAGGTGACGGTGTTTCGTCCTTTCCACaag
[0494] Example 8: Methodological improvement to improve activity:
[0495] Rather than use pol3 promoters, in particular RNA polymerase III (e.g. U6 or H1 promoters), to express guide RNAs in eukaryotic cells, Applicants express the T7 polymerase in eukaryotic cells to drive expression of guide RNAs using the T7 promoter.
[0496] One example of this system may involve introduction of three pieces of DNA:
[0497] 1. expression vector for Cas9
[0498] 2. expression vector for T7 polymerase
[0499] 3. expression vector containing guideRNA fused to the T7 promoter
[0500] Example 9: Methodological improvement to reduce toxicity of Cas9: Delivery of Cas9 in the form of mRNA.
[0501] Delivery of Cas9 in the form of mRNA enables transient expression of Cas9 in cells, to reduce toxicity. For example, humanized SpCas9 may be amplified using the following primer pair:
[0502] Forward Primer (to add on T7 promoter for in vitro transcription): TAATACGACTCACTATAGGAAGTGCGCCACCATGGCCCCAAAGAAGAAGCGG
[0503] Reverse Primer (to add on polyA tail): GGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTttcttaCTTTTTCTTTTTTGCCTGGCCG [0504] Applicants transfect the Cas9 mRNA into cells with either guide RNA in the form of RNA or DNA cassettes to drive guide RNA expression in eukaryotic cells.
[0505] Example 10: Methodological improvement to reduce toxicity of Cas9: Use of an inducible promoter
[0506] Applicants transiently turn on Cas9 expression only when it is needed for carrying out genome modification. Examples of inducible system include tetracycline inducible promoters (Tet-On or Tet-Off), small molecule two-hybrid transcription activations systems (FKBP, ABA, etc), or light inducible systems (Phytochrome, LOV domains, or cryptochrome).
[0507] Example 11: Improvement of the Cas9 system for in vivo application
[0508] Applicants conducted a Metagenomic search for a Cas9 with small molecular weight. Most Cas9 homologs are fairly large. For example the SpCas9 is around 1368aa long, which is too large to be easily packaged into viral vectors for delivery. A graph representing the length distribution of Cas9 homologs is generated from sequences deposited in GenBank (Fig. 23). Some of the sequences may have been mis-annotated and therefore the exact frequency for each length may not necessarily be accurate. Nevertheless it provides a glimpse at distribution of Cas9 proteins and suggest that there are shorter Cas9 homologs.
[0509] Through computational analysis, Applicants found that in the bacterial strain Campylobacter, there are two Cas9 proteins with less than 1000 amino acids. The sequence for one Cas9 from Campylobacter jejuni is presented below. At this length, CjCas9 can be easily packaged into AAV, lentiviruses, Adenoviruses, and other viral vectors for robust delivery into primary cells and in vivo in animal models. In a preferred embodiment of the invention, the Cas9 protein from S. aureus is used.
[0510] >Campylobacter jejuni Cas9 (CjCas9)
[0511] MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVENPKTGESLALPRRLAR SARKRLARRKARLNHLKHLIANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRALN ELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGAILKAIKQNEEKLANYQSVGEYLYK EYFQKFKENSKEFTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGFSFSKKFEEEVL SVAFYKRALKDFSHLVGNCSFFTDEKRAPKNSPLAFMFVALTRIINLLNNLKNTEGIL YTKDDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGEKGTYFIEFKKYKEFIKAL GEHNLSQDDLNEIAKDITLIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNISFKAL KLVTPLMLEGKKYDEACNELNLKVAINEDKKDFLPAFNETYYKDEVTNPVVLRAIKE YRKVLNALLKKYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKAKKDAELECEK LGLKINSKNILKLRLFKEQKEFCAYSGEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMN KVLVFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLPTKKQKRILDKNYKDKEQ KNFKDRNLNDTRYIARLVLNYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSGM LTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNSIVKAFSDFKKEQESNSAELYAK KISELDYKNKRKFFEPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETFRKEEEFYQS YGGKEGVLKALELGKIRKVNGKIVKNGDMFRVDIFKHKKTNKFYAVPIYTMDFALK VLPNKAVARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKDMQEPEFVYYNAFTSS TVSLIVSKHDNKFETLSKNQKILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVTKA EFRQREDFKK.
[0512] The putative tracrRNA element for this CjCas9 is:
[0513] TATAATCTCATAAGAAATTTAAAAAGGGACTAAAATAAAGAGTTTGCG GGACTCTGCGGGGTTACAATCCCCTAAAACCGCTTTTAAAATT
[0514] The Direct Repeat sequence is:
[0515] ATTTTACCATAAAGAAATTTAAAAAGGGACTAAAAC
[0516] An example of a chimeric guideRNA for CjCas9 is:
[0517] NNNNNNNNNNNNNNNNNNNNGUUUUAGUCCCGAAAGGGACUAAAAU AAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU
[0518] Example 12: Cas9 optimization
[0519] For enhanced function or to develop new functions, Applicants generate chimeric Cas9 proteins by combining fragments from different Cas9 homologs. For example, two example chimeric Cas9 proteins:
[0520] For example, Applicants fused the N-term of St1Cas9 (fragment from this protein is in bold) with C-term of SpCas9 (fragment from this protein is underlined).
[0521] >St1(N)Sp(C)Cas9
[0522] MSDLVLGLDIGIGSVGVGILNKVTGEIIHKNSRIFPAAQAENNLVRRTN RQGRRLARRKKHRRVRLNRLFEESGLITDFTKISINLNPYQLRVKGLTDELSNEE LFIALKNMVKHRGISYLDDASDDGNSSVGDYAQIVKENSKQLETKTPGQIQLERY QTYGQLRGDFTVEKDGKKHRLINVFPTSAYRSEALRILQTQQEFNPQITDEFINR YLEILTGKRKYYHGPGNEKSRTDYGRYRTSGETLDNIFGILIGKCTFYPDEFRAAK ASYTAQEFNLLNDLNNLTVPTETKKLSKEQKNQIINYVKNEKAMGPAKLFKYIAK LLSCDVADIKGYRIDKSGKAEIHTFEAYRKMKTLETLDIEQMDRETLDKLAYVLT LNTEREGIQEALEHEFADGSFSQKQVDELVQFRKANSSIFGKGWHNFSVKLMME LIPELYETSEEQMTILTRLGKQKTTSSSNKTKYIDEKLLTEEIYNPVVAKSVRQAIK IVNAAIKEYGDFDNIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVE NTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLT RSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDK AGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQ FYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQ EIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKV LSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSV LVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYS LFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLF VEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNL GAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
[0523] >Sp(N)St1(C)Cas9
[0524] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGA LLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVE EDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRG HFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENL IAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIG DQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQ QLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLL RKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGN SRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYE YFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIE CFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIE ERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFAN RNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELV KVMGRHKPENIVIEMARETNEDDEKKAIQKIQKANKDEKDAAMLKAANQYNGKA ELPHSVFHGHKQLATKIRLWHQQGERCLYTGKTISIHDLINNSNQFEVDHILPLSI TFDDSLANKVLVYATANQEKGQRTPYQALDSMDDAWSFRELKAFVRESKTLSNK KKEYLLTEEDISKFDVRKKFIERNLVDTRYASRVVLNALQEHFRAHKIDTKVSVVR GQFTSQLRRHWGIEKTRDTYHHHAVDALIIAASSQLNLWKKQKNTLVSYSEDQL LDIETGELISDDEYKESVFKAPYQHFVDTLKSKEFEDSILFSYQVDSKFNRKISDATI YATRQAKVGKDKADETYVLGKIKDIYTQDGYDAFMKIYKKDKSKFLMYRHDPQT FEKVIEPILENYPNKQINEKGKEVPCNPFLKYKEEHGYIRKYSKKGNGPEIKSLKYY DSKLGNHIDITPKDSNNKVVLQSVSPWRADVYFNKTTGKYEILGLKYADLQFEKG TGTYKISQEKYNDIKKKEGVDSDSEFKFTLYKNDLLLVKDTETKEQQLFRFLSRT MPKQKHYVELKPYDKQKFEGGEALIKVLGNVANSGQCKKGLGKSNISIYKVRTD VLGNQHIIKNEGDKPKLDF
[0525] The benefit of making chimeric Cas9 include:
[0526] reduce toxicity
[0527] improve expression in eukaryotic cells
[0528] enhance specificity
[0529] reduce molecular weight of protein, make protein smaller by combining the smallest domains from different Cas9 homologs.
[0530] Altering the PAM sequence requirement
[0531] Example 13: Utilization of Cas9 as a generic DNA binding protein
[0532] Applicants used Cas9 as a generic DNA binding protein by mutating the two catalytic domains (D10 and H840) responsible for cleaving both strands of the DNA target. In order to upregulate gene transcription at a target locus Applicants fused the transcriptional activation domain (VP64) to Cas9. Applicants hypothesized that it would be important to see strong nuclear localization of the Cas9-VP64 fusion protein because transcription factor activation strength is a function of time spent at the target. Therefore, Applicants cloned a set of Cas9-VP64-GFP constructs, transfected them into 293 cells and assessed their localization under a fluorescent microscope 12 hours post-transfection.
[0533] The same constructs were cloned as a 2A-GFP rather than a direct fusion in order to functionally test the constructs without a bulky GFP present to interfere. Applicants elected to target the Sox2 locus with the Cas9 transactivator because it could be useful for cellular reprogram and the locus has already been validated as a target for TALE-TF mediated transcriptional activation. For the Sox2 locus Applicants chose eight targets near the transcriptional start site (TSS). Each target was 20bp long with a neighboring NGG protospacer adjacent motif (PAM). Each Cas9-VP64 construct was co-transfected with each PCR generated chimeric crispr RNA (chiRNA) in 293 cells. 72 hours post transfection the transcriptional activation was assessed using RT-qPCR.
[0534] To further optimize the transcriptional activator, Applicants titrated the ratio of chiRNA (Sox2.1 and Sox2.5) to Cas9 (NLS-VP64-NLS-hSpCas9-NLS-VP64-NLS), transfected into 293 cells, and quantified using RT-qPCR. These results indicate that Cas9 can be used as a generic DNA binding domain to upregulate gene transcription at a target locus.
[0535] Applicants designed a second generation of constructs. (Table below).
Figure imgf000216_0001
[0536] Applicants use these constructs to assess transcriptional activation (VP64 fused constructs) and repression (Cas9 only) by RT-qPCR. Applicants assess the cellular localization of each construct using anti-His antibody, nuclease activity using a Surveyor nuclease assay, and DNA binding affinity using a gel shift assay. In a preferred embodiment of the invention, the gel shift assay is an EMSA gel shift assay.
[0537] Example 14: Cas9 transgenic and knock in mice
[0538] To generate a mouse that expresses the Cas9 nuclease Applicants submit two general strategies, transgenic and knock in. These strategies may be applied to generate any other model organism of interest, for e.g. Rat. For each of the general strategies Applicants made a constitutively active Cas9 and a Cas9 that is conditionally expressed (Cre recombinase dependent). The constitutively active Cas9 nuclease is expressed in the following context: pCAG-NLS-Cas9-NLS-P2A-EGFP-WPRE-bGHpolyA. pCAG is the promoter, NLS is a nuclear localization signal, P2A is the peptide cleavage sequence, EGFP is enhanced green fluorescent protein, WPRE is the woodchuck hepatitis virus posttranscriptional regulatory element, and bGHpolyA is the bovine growth hormone poly-A signal sequence (Figs. 25A-B). The conditional version has one additional stop cassette element, loxP-SV40 polyA x3-loxP, after the promoter and before NLS-Cas9-NLS (i.e. pCAG-loxP-SV40polyAx3-loxP-NLS-Cas9-NLS- P2A-EGFP-WPRE-bGHpolyA). The important expression elements can be visualized as in Fig. 26. The constitutive construct should be expressed in all cell types throughout development, whereas, the conditional construct will only allow Cas9 expression when the same cell is expressing the Cre recombinase. This latter version will allow for tissue specific expression of Cas9 when Cre is under the expression of a tissue specific promoter. Moreover, Cas9 expression could be induced in adult mice by putting Cre under the expression of an inducible promoter such as the TET on or off system.
[0539] Validation of Cas9 constructs: Each plasmid was functionally validated in three ways: 1) transient transfection in 293 cells followed by confirmation of GFP expression; 2) transient transfection in 293 cells followed by immunofluorescence using an antibody recognizing the P2A sequence; and 3) transient transfection followed by Surveyor nuclease assay. The 293 cells may be 293FT or 293 T cells depending on the cells that are of interest. In a preferred embodiment the cells are 293FT cells. The results of the Surveyor were run out on the top and bottom row of the gel for the conditional and constitutive constructs, respectively. Each was tested in the presence and absence of chimeric RNA targeted to the hEMX1 locus (chimeric RNA hEMX1.1). The results indicate that the construct can successfully target the hEMX1 locus only in the presence of chimeric RNA (and Cre in the conditional case). The gel was quantified and the results are presented as average cutting efficiency and standard deviation for three samples.
[0540] Transgenic Cas9 mouse: To generate transgenic mice with constructs, Applicants inject pure, linear DNA into the pronucleus of a zygote from a pseudo pregnant CB56 female. Founders are identified, genotyped, and backcrossed to CB57 mice. The constructs were successfully cloned and verified by Sanger sequencing.
[0541] Knock in Cas9 mouse: To generate Cas9 knock in mice Applicants target the same constitutive and conditional constructs to the Rosa26 locus. Applicants did this by cloning each into a Rosa26 targeting vector with the following elements: Rosa26 short homology arm– constitutive/conditional Cas9 expression cassette– pPGK-Neo-Rosa26 long homology arm– pPGK-DTA. pPGK is the promoter for the positive selection marker Neo, which confers resistance to neomycin, a 1 kb short arm, a 4.3 kb long arm, and a negative selection diphtheria toxin (DTA) driven by PGK.
[0542] The two constructs were electroporated into R1 mESCs and allowed to grow for 2 days before neomycin selection was applied. Individual colonies that had survived by days 5-7 were picked and grown in individual wells. 5-7 days later the colonies were harvested, half were frozen and the other half were used for genotyping. Genotyping was done by genomic PCR, where one primer annealed within the donor plasmid (AttpF) and the other outside of the short homology arm (Rosa26-R) Of the 22 colonies harvested for the conditional case, 7 were positive (Left). Of the 27 colonies harvested for the constitutive case, zero were positive (Right). It is likely that Cas9 causes some level of toxicity in the mESC and for this reason there were no positive clones. To test this Applicants introduced a Cre expression plasmid into correctly targeted conditional Cas9 cells and found very low toxicity after many days in culture. The reduced copy number of Cas9 in correctly targeted conditional Cas9 cells (1 -2 copies per cell) is enough to allow stable expression and relatively no cytotoxicity. Moreover, this data indicates that the Cas9 copy number determines toxicity. After electroporation each cell should get several copies of Cas9 and this is likely why no positive colonies were found in the case of the constitutive Cas9 construct. This provides strong evidence that utilizing a conditional, Cre- dependent strategy should show reduced toxicity. Applicants inject correctly targeted cells into a blastocyst and implant into a female mouse. Chimerics are identified and backcrossed. Founders are identified and genotyped.
[0543] Utility of the conditional Cas9 mouse: Applicants have shown in 293 cells that the Cas9 conditional expression construct can be activated by co-expression with Cre. Applicants also show that the correctly targeted R1 mESCs can have active Cas9 when Cre is expressed. Because Cas9 is followed by the P2A peptide cleavage sequence and then EGFP Applicants identify successful expression by observing EGFP. This same concept is what makes the conditional Cas9 mouse so useful. Applicants may cross their conditional Cas9 mouse with a mouse that ubiquitously expresses Cre (ACTB-Cre line) and may arrive at a mouse that expresses Cas9 in every cell. It should only take the delivery of chimeric RNA to induce genome editing in embryonic or adult mice. Interestingly, if the conditional Cas9 mouse is crossed with a mouse expressing Cre under a tissue specific promoter, there should only be Cas9 in the tissues that also express Cre. This approach may be used to edit the genome in only precise tissues by delivering chimeric RNA to the same tissue.
[0544] Example 15: Cas9 diversity and chimeric RNAs
[0545] The CRISPR-Cas system is an adaptive immune mechanism against invading exogenous DNA employed by diverse species across bacteria and archaea. The type II CRISPR- Cas system consists of a set of genes encoding proteins responsible for the“acquisition” of foreign DNA into the CRISPR locus, as well as a set of genes encoding the“execution” of the DNA cleavage mechanism; these include the DNA nuclease (Cas9), a non-coding transactivating cr-RNA (tracrRNA), and an array of foreign DNA-derived spacers flanked by direct repeats (crRNAs). Upon maturation by Cas9, the tracrRNA and crRNA duplex guide the Cas9 nuclease to a target DNA sequence specified by the spacer guide sequences, and mediates double-stranded breaks in the DNA near a short sequence motif in the target DNA that is required for cleavage and specific to each CRISPR-Cas system. The type II CRISPR-Cas systems are found throughout the bacterial kingdom and highly diverse in in Cas9 protein sequence and size, tracrRNA and crRNA direct repeat sequence, genome organization of these elements, and the motif requirement for target cleavage. One species may have multiple distinct CRISPR-Cas systems. [0546] Applicants evaluated 207 putative Cas9s from bacterial species identified based on sequence homology to known Cas9s and structures orthologous to known subdomains, including the HNH endonuclease domain and the RuvC endonuclease domains [information from the Eugene Koonin and Kira Makarova]. Phylogenetic analysis based on the protein sequence conservation of this set revealed five families of Cas9s, including three groups of large Cas9s (~1400 amino acids) and two of small Cas9s (~1100 amino acids) (Figs.19A-D and 20A-F).
[0547] Applicants have also optimized Cas9 guide RNA using in vitro methods.
[0548] Example 16: Cas9 mutations
[0549] In this example, Applicants show that the following mutations can convert SpCas9 into a nicking enzyme: D10A, E762A, H840A, N854A, N863A, D986A.
[0550] Applicants provide sequences showing where the mutation points are located within the SpCas9 gene (Fig. 24A-M). Applicants also show that the nickases are still able to mediate homologous recombination. Furthermore, Applicants show that SpCas9 with these mutations (individually) do not induce double strand break.
[0551] Cas9 orthologs all share the general organization of 3-4 RuvC domains and a HNH domain. The 5' most RuvC domain cleaves the non-complementary strand, and the HNH domain cleaves the complementary strand. All notations are in reference to the guide sequence.
[0552] The catalytic residue in the 5' RuvC domain is identified through homology comparison of the Cas9 of interest with other Cas9 orthologs (from S. pyogenes type II CRISPR locus, S. thermophilus CRISPR locus 1, S. thermophilus CRISPR locus 3, and Franciscilla novicida type II CRISPR locus), and the conserved Asp residue is mutated to alanine to convert Cas9 into a complementary-strand nicking enzyme. Similarly, the conserved His and Asn residues in the HNH domains are mutated to Alanine to convert Cas9 into a non-complementary- strand nicking enzyme.
[0553] Example 17: Cas9 Transcriptional Activation and Cas9 Repressor
[0554] Cas9 Transcriptional Activation
[0555] A second generation of constructs were designed and tested (Table 1). These constructs are used to assess transcriptional activation (VP64 fused constructs) and repression (Cas9 only) by RT-qPCR. Applicants assess the cellular localization of each construct using anti- His antibody, nuclease activity using a Surveyor nuclease assay, and DNA binding affinity using a gel shift assay.
[0556] Cas Repressor [0557] It has been shown previously that dCas9 can be used as a generic DNA binding domain to repress gene expression. Applicants report an improved dCas9 design as well as dCas9 fusions to the repressor domains KRAB and SID4x. From the plasmid library created for modulating transcription using Cas9 in the following Table, the following repressor plasmids were functionally characterized by qPCR: pXRP27, pXRP28, pXRP29, pXRP48, pXRP49, pXRP50, pXRP51, pXRP52, pXRP53, pXRP56, pXRP58, pXRP59, pXRP61, and pXRP62.
[0558] Each dCas9 repressor plasmid was co-transfected with two guide RNAs targeted to the coding strand of the beta-catenin gene. RNA was isolated 72 hours after transfection and gene expression was quantified by RT-qPCR. The endogenous control gene was GAPDH. Two validated shRNAs were used as positive controls. Negative controls were certain plasmids transfected without gRNA, these are denoted as“pXRP## control”. The plasmids pXRP28, pXRP29, pXRP48, and pXRP49 could repress the beta-catenin gene when using the specified targeting strategy. These plasmids correspond to dCas9 without a functional domain (pXRP28 and pXRP28) and dCas9 fused to SID4x (pXRP48 and pXRP49).
[0559] Further work investigates: repeating the above experiment, targeting different genes, utilizing other gRNAs to determine the optimal targeting position, and multiplexed repression.
Figure imgf000220_0001
Figure imgf000221_0001
Example 18: Targeted deletion of genes involved in cholesterol biosynthesis, fatty acid biosynthesis and other metabolic disorders genes encoding mis folded proteins involved in amyloid and other diseases, oncogenes leading to cellular transformation, latent viral genes, and genes leading to dominant-negative disorders, amongst other disorders.
[0560] Applicants demonstrate gene delivery of a CRISPR-Cas system in the liver, brain, ocular, epithelial, hematopoetic, or another tissue of a subject or a patient in need thereof, suffering from metabolic disorders, amyloidosis and protein-aggregation related diseases, cellular transformation arising from genetic mutations and translocations, dominant negative effects of gene mutations, latent viral infections, and other related symptoms, using either viral or nanoparticle delivery system.
[0561] Study Design: Subjects or patients in need thereof suffering from metabolic disorders, amyloidosis and protein aggregation related disease which include but are not limited to human, non-primate human, canine, feline, bovine, equine, other domestic animals and related mammals. The CRISPR-Cas system is guided by a chimeric guide RNA and targets a specific site of the human genomic loci to be cleaved. After cleavage and non-homologous end-joining mediated repair, frame-shift mutation results in knock out of genes.
[0562] Applicants select guide-RNAs targeting genes involved in above-mentioned disorders to be specific to endogenous loci with minimal off-target activity. Two or more guide RNAs may be encoded into a single CRISPR array to induce simultaneous double-stranded breaks in DNA leading to micro-deletions of affected genes or chromosomal regions.
[0563] Identification and design of gene targets
[0564] For each candidate disease gene, Applicants select DNA sequences of interest include protein-coding exons, sequences including and flanking known dominant negative mutation sites, sequences including and flanking pathological repetitive sequences. For gene-knockout approaches, early coding exons closest to the start codon offer best options for achieving complete knockout and minimize possibility of truncated protein products retaining partial function.
[0565] Applicants analyze sequences of interest for all possible targetable 20-bp sequences immediately 5’ to a NGG motif (for SpCas9 system) or a NNAGAAW (for St1Cas9 system). Applicants choose sequences for unique, single RNA-guided Cas9 recognition in the genome to minimize off-target effects based on computational algorithm to determine specificity.
[0566] Cloning of guide sequences into a delivery system
[0567] Guide sequences are synthesized as double-stranded 20-24 bp oligonucleotides. After 5’-phosphorylation treatment of oligos and annealing to form duplexes, oligos are ligated into suitable vector depending on the delivery method: [0568] Virus-based delivery methods
[0569] AAV-based vectors (PX260, 330, 334, 335) have been described elsewhere
[0570] Lentiviral-based vectors use a similar cloning strategy of directly ligating guide sequences into a single vector carrying a U6 promoter-driven chimeric RNA scaffold and a EF1a promoter-driven Cas9 or Cas9 nickase.
[0571] Virus production is described elsewhere.
[0572] Nanoparticle-based RNA delivery methods
[0573] 1. Guide sequences are synthesized as an oligonucleotide duplex encoding T7 promoter—guide sequence—chimeric RNA. A T7 promoter is added 5’ of Cas9 by PCR method.
[0574] 2. T7-driven Cas9 and guide-chimeric RNAs are transcribed in vitro, and Cas9 mRNA is further capped and A-tailed using commercial kits. RNA products are purified per kit instructions.
[0575] Hydrodynamic tail vein delivery methods (for mouse)
[0576] Guide sequences are cloned into AAV plasmids as described above and elsewhere in this application.
[0577] In vitro validation on cell lines
[0578] Transfection
[0579] 1. DNA plasmid transfection
[0580] Plasmids carrying guide sequences are transfected into human embryonic kidney (HEK293T) or human embryonic stem (hES) cells, other relevant cell types using lipid-, chemical-, or electroporation-based methods. For a 24-well transfection of HEK293T cells (~260,000 cells), 500ng of total DNA is transfected into each single well using Lipofectamine 2000. For a 12-well transfection of hES cells, 1ug of total DNA is transfected into a single well using Fugene HD.
[0581] 2. RNA transfection
[0582] Purified RNA described above is used for transfection into HEK293T cells. 1-2ug of RNA may be transfected into ~260,000 using Lipofectamine 2000 per manufacturer’s instruction. RNA delivery of Cas9 and chimeric RNA is shown in Fig. 28.
[0583] Assay of indel formation in vitro
[0584] Cells are harvested 72-hours post-transfection and assayed for indel formation as an indication of double-stranded breaks. [0585] Briefly, genomic region around target sequence is PCR amplified (~400-600 bp amplicon size) using high-fidelity polymerase. Products are purified, normalized to equal concentration, and slowly annealed from 95°C to 4°C to allow formation of DNA heteroduplexes. Post annealing, the Cel-I enzyme is used to cleave heteroduplexes, and resulting products are separated on a polyacrylamide gel and indel efficiency calculated.
[0586] In vivo proof of principle in animal
[0587] Delivery mechanisms
[0588] AAV or Lentivirus production is described elsewhere.
[0589] Nanoparticle formulation: RNA mixed into nanoparticle formulation
[0590] Hydrodynamic tail vein injections with DNA plasmids in mice are conducted using a commercial kit
[0591] Cas9 and guide sequences are delivered as virus, nanoparticle-coated RNA mixture, or DNA plasmids, and injected into subject animals. A parallel set of control animals is injected with sterile saline, Cas9 and GFP, or guide sequence and GFP alone.
[0592] Three weeks after injection, animals are tested for amelioration of symptoms and sacrificed. Relevant organ systems analyzed for indel formation. Phenotypic assays include blood levels of HDL, LDL, lipids,
[0593] Assay for indel formation
[0594] DNA is extracted from tissue using commercial kits; indel assay will be performed as described for in vitro demonstration.
[0595] Therapeutic applications of the CRISPR-Cas system are amenable for achieving tissue-specific and temporally controlled targeted deletion of candidate disease genes. Examples include genes involved in cholesterol and fatty acid metabolism, amyloid diseases, dominant negative diseases, latent viral infections, among other disorders.
[0596] Examples of a single guide-RNA to introduce targeted indels at a gene locus
Figure imgf000224_0001
Figure imgf000225_0001
Example 19: Targeted correction of deltaF508 or other mutations in cystic fibrosis using AAV
[0598] An aspect of the invention provides for a pharmaceutical composition that may comprise an CRISPR-Cas gene therapy particle and a biocompatible pharmaceutical carrier. According to another aspect, a method of gene therapy for the treatment of a subject having a mutation in the CFTR gene comprises administering a therapeutically effective amount of a CRISPR-Cas gene therapy particle to the cells of a subject.
[0599] This Example demonstrates gene transfer or gene delivery of a CRISPR-Cas system in airways of subject or a patient in need thereof, suffering from cystic fibrosis or from cystic fibrosis related symptoms, using adeno-associated virus (AAV) particles.
[0600] Study Design: Subjects or patients in need there of: Human, non-primate human, canine, feline, bovine, equine and other domestic animals, related. This study tests efficacy of gene transfer of a CRISPR-Cas system by a AAV vector. Applicants determine transgene levels sufficient for gene expression and utilize a CRISPR-Cas system comprising a Cas9 enzyme to target deltaF508 or other CFTR-inducing mutations.
[0601] The treated subjects receive pharmaceutically effective amount of aerosolized AAV vector system per lung endobronchially delivered while spontaneously breathing. The control subjects receive equivalent amount of a pseudotyped AAV vector system with an internal control gene. The vector system may be delivered along with a pharmaceutically acceptable or biocompatible pharmaceutical carrier. Three weeks or an appropriate time interval following vector administration, treated subjects are tested for amelioration of cystic fibrosis related symptoms.
[0602] Applicants use an adenovirus or an AAV particle.
[0603] Applicants clone the following gene constructs, each operably linked to one or more regulatory sequences (Cbh or EF1a promoter for Cas9, U6 or H1 promoter for chimeric guide RNA), into one or more adenovirus or AAV vectors or any other compatible vector: A CFTRdelta508 targeting chimeric guide RNA (Fig. 31B), a repair template for deltaF508 mutation (Fig. 31C) and a codon optimized Cas9 enzyme with optionally one or more nuclear localization signal or sequence(s) (NLS(s)), e.g., two (2) NLSs.
[0604] Identification of Cas9 target site
[0605] Applicants analyzed the human CFTR genomic locus and identified the Cas9 target site (Fig. 31A). (PAM may contain a NGG or a NNAGAAW motif).
[0606] Gene Repair Strategy
[0607] Applicants introduce an adenovirus/AAV vector system comprising a Cas9 (or Cas9 nickase) and the guide RNA along with a adenovirus/AAV vector system comprising the homology repair template containing the F508 residue into the subject via one of the methods of delivery discussed earlier. The CRISPR-Cas system is guided by the CFTRdelta 508 chimeric guide RNA and targets a specific site of the CFTR genomic locus to be nicked or cleaved. After cleavage, the repair template is inserted into the cleavage site via homologous recombination correcting the deletion that results in cystic fibrosis or causes cystic fibrosis related symptoms. This strategy to direct delivery and provide systemic introduction of CRISPR systems with appropriate guide RNAs can be employed to target genetic mutations to edit or otherwise manipulate genes that cause metabolic, liver, kidney and protein diseases and other disorders.
[0608] Example 20: Delivery of CRISPR System
[0609] Cas9 and its chimeric guide RNA, or combination of tracrRNA and crRNA, can be delivered either as DNA or RNA. Delivery of Cas9 and guide RNA both as RNA (normal or containing base or backbone modifications) molecules can be used to reduce the amount of time that Cas9 protein persist in the cell. This may reduce the level of off-target cleavage activity in the target cell. Since delivery of Cas9 as mRNA takes time to be translated into protein, it might be advantageous to deliver the guide RNA several hours following the delivery of Cas9 mRNA, to maximize the level of guide RNA available for interaction with Cas9 protein.
[0610] In situations where guide RNA amount is limiting, it may be desirable to introduce Cas9 as mRNA and guide RNA in the form of a DNA expression cassette with a promoter driving the expression of the guide RNA. This way the amount of guide RNA available will be amplified via transcription.
[0611] A variety of delivery systems can be introduced to introduce Cas9 (DNA or RNA) and guide RNA (DNA or RNA) into the host cell. These include the use of liposomes, viral vectors, electroporation, nanoparticles , nanowires (Shalek et al., Nano Letters, 2012), exosomes. Molecular trojan horses liposomes (Pardridge et al., Cold Spring Harb Protoc; 2010; doi:10.1101/pdb.prot5407) may be used to deliver Cas9 and guide RNA across the blood brain barrier.
[0612] Example 21: Therapeutic strategies for Trinucleotide repeat disorders; guide design
[0613] As previously mentioned in the application, the target polynucleotide of a CRISPR complex may include a number of disease-associated genes and polynucleotides and some of these disease associated gene may belong to a set of genetic disorders referred to as Trinucleotide repeat disorders (referred to as also trinucleotide repeat expansion disorders, triplet repeat expansion disorders or codon reiteration disorders).
[0614] These diseases are caused by mutations in which the trinucleotide repeats of certain genes exceed the normal, stable threshold which may usually differ in a gene. The discovery of more repeat expansion disorders has allowed for the classification of these disorders into a number of categories based on underlying similar characteristics. Huntington’s disease (HD) and the spinocerebellar ataxias that are caused by a CAG repeat expansion in protein-coding portions of specific genes are included in Category I. Diseases or disorders with expansions that tend to make them phenotypically diverse and include expansions are usually small in magnitude and also found in exons of genes are included in Category II. Category III includes disorders or diseases which are characterized by much larger repeat expansions than either Category I or II and are generally located outside protein coding regions. Examples of Category III diseases or disorders include but are not limited to Fragile X syndrome, myotonic dystrophy, two of the spinocerebellar ataxias, juvenile myoclonic epilepsy, and Friedreich's ataxia.
[0615] Similar therapeutic strategies, like the one mentioned for Friedreich's ataxia below may be adopted to address other trinucleotide repeat or expansion disorders as well. For example, another triple repeat disease that can be treated using almost identical strategy is dystrophia myotonica 1 (DM1), where there is an expanded CTG motif in the 3' UTR. In Friedreich's ataxia, the disease results from expansion of GAA trinucleotides in the first intron of frataxin (FXN). One therapeutic strategy using CRISPR is to excise the GAA repeat from the first intron. The expanded GAA repeat is thought to affect the DNA structure and leads to recruit the formation of heterochromatin which turn off the frataxin gene (Fig. 32A).
[0616] Competitive Advantage over other therapeutic strategies are listed below:
[0617] siRNA knockdown is not applicable in this case, as disease is due to reduced expression of frataxin. Viral gene therapy is currently being explored. HSV-1 based vectors were used to deliver the frataxin gene in animal models and have shown therapeutic effect. However, long term efficacy of virus-based frataxin delivery suffer from several problems: First, it is difficult to regulate the expression of frataxin to match natural levels in health individuals, and second, long term over expression of frataxin leads to cell death.
[0618] Nucleases may be used to excise the GAA repeat to restore healthy genotype, but Zinc Finger Nuclease and TALEN strategies require delivery of two pairs of high efficacy nucleases, which is difficult for both delivery as well as nuclease engineering (efficient excision of genomic DNA by ZFN or TALEN is difficult to achieve).
[0619] In contrast to above strategies, the CRISPR-Cas system has clear advantages. The Cas9 enzyme is more efficient and more multiplexible, by which it is meant that one or more targets can be set at the same time. So far, efficient excision of genomic DNA > 30% by Cas9 in human cells and may be as high as 30%, and may be improved in the future. Furthermore, with regard to certain trinucleotide repeat disorders like Huntington’s disease (HD), trinucleotide repeats in the coding region may be addressed if there are differences between the two alleles. Specifically, if a HD patient is heterozygous for mutant HTT and there are nucleotide differences such as SNPs between the wt and mutant HTT alleles, then Cas9 may be used to specifically target the mutant HTT allele. ZFN or TALENs will not have the ability to distinguish two alleles based on single base differences.
[0620] In adopting a strategy using the CRISPR-Cas9 enzyme to address Friedreich’s ataxia, Applicants design a number of guide RNAs targeting sites flanking the GAA expansion and the most efficient and specific ones are chosen (Fig. 32B).
[0621] Applicants deliver a combination of guide RNAs targeting the intron 1 of FXN along with Cas9 to mediate excision of the GAA expansion region. AAV9 may be used to mediate efficient delivery of Cas9 and in the spinal cord.
[0622] If the Alu element adjacent to the GAA expansion is considered important, there may be constraints to the number of sites that can be targeted but Applicants may adopt strategies to avoid disrupting it.
[0623] Alternative Strategies:
[0624] Rather than modifying the genome using Cas9, Applicants may also directly activate the FXN gene using Cas9 (nuclease activity deficient)-based DNA binding domain to target a transcription activation domain to the FXN gene.
[0625] Example 22: Strategies for minimizing off-target cleavage using Cas9 nickase
[0626] As previously mentioned in the application, Cas9 may be mutated to mediate single strand cleavage via one or more of the following mutations: D10A, E762A, and H840A.
[0627] To mediate gene knockout via NHEJ, Applicants use a nickase version of Cas9 along with two guide RNAs. Off-target nicking by each individual guide RNA may be primarily repaired without mutation, double strand breaks (which can lead to mutations via NHEJ) only occur when the target sites are adjacent to each other. Since double strand breaks introduced by double nicking are not blunt, co-expression of end-processing enzymes such as TREX1 will increase the level of NHEJ activity.
[0628] The following list of targets in tabular form are for genes involved in the following diseases:
[0629] Lafora’s Disease– target GSY1 or PPP1R3C (PTG) to reduce glycogen in neurons.
[0630] Hypercholesterolemia– target PCSK9
[0631] Target sequences are listed in pairs (L and R) with different number of nucleotides in the spacer (0 to 3bp). Each spacer may also be used by itself with the wild type Cas9 to introduce double strand break at the target locus.
Figure imgf000230_0001
Figure imgf000231_0001
[0632] Alternative strategies for improving stability of guide RNA and increasing specificity
1. Nucleotides in the 5’ of the guide RNA may be linked via thiolester linkages rather than phosphoester linkage like in natural RNA. Thiolester linkage may prevent the guide RNA from being digested by endogenous RNA degradation machinery.
2. Nucleotides in the guide sequence (5’ 20bp) of the guide RNA can use bridged nucleic acids (BNA) as the bases to improve the binding specificity. Example 23: CRISPR-Cas for rapid, multiplex genome editing
[0633] Aspects of the invention relate to protocols and methods by which efficiency and specificity of gene modification may be tested within 3-4 days after target design, and modified clonal cell lines may be derived within 2-3 weeks.
[0634] Programmable nucleases are powerful technologies for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial CRISPR adaptive immune system can be used to facilitate efficient genome editing in eukaryotic cells by simply specifying a 20-nt targeting sequence in its guide RNA. Applicants describe a set of protocols for applying Cas9 to facilitate efficient genome editing in mammalian cells and generate cell lines for downstream functional studies. Beginning with target design, efficient and specific gene modification can be achieved within 3-4 days, and modified clonal cell lines can be derived within 2-3 weeks.
[0635] The ability to engineer biological systems and organisms holds enormous potential for applications across basic science, medicine, and biotechnology. Programmable sequence- specific endonucleases that facilitate precise editing of endogenous genomic loci are now enabling systematic interrogation of genetic elements and causal genetic variations in a broad range of species, including those that have not been genetically tractable previously. A number of genome editing technologies have emerged in recent years, including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the RNA-guided CRISPR- Cas nuclease system. The first two technologies use a common strategy of tethering endonuclease catalytic domains to modular DNA-binding proteins for inducing targeted DNA double stranded breaks (DSB) at specific genomic loci. By contrast, Cas9 is a nuclease guided by small RNAs through Watson-Crick base-pairing with target DNA, presenting a system that is easy to design, efficient, and well-suited for high-throughput and multiplexed gene editing for a variety of cell types and organisms. Here Applicants describe a set of protocols for applying the recently developed Cas9 nuclease to facilitate efficient genome editing in mammalian cells and generate cell lines for downstream functional studies.
[0636] Like ZFNs and TALENs, Cas9 promotes genome editing by stimulating DSB at the target genomic loci. Upon cleavage by Cas9, the target locus undergoes one of two major pathways for DNA damage repair, the error-prone non-homologous end joining (NHEJ) or the high-fidelity homology directed repair (HDR) pathway. Both pathways may be utilized to achieve the desired editing outcome.
[0637] NHEJ: In the absence of a repair template, the NHEJ process re-ligates DSBs, which may leave a scar in the form of indel mutations. This process can be harnessed to achieve gene knockouts, as indels occurring within a coding exon may lead to frameshift mutations and a premature stop codon. Multiple DSBs may also be exploited to mediate larger deletions in the genome.
[0638] HDR: Homology directed repair is an alternate major DNA repair pathway to NHEJ. Although HDR typically occurs at lower frequencies than NHEJ, it may be harnessed to generate precise, defined modifications at a target locus in the presence of an exogenously introduced repair template. The repair template may be either in the form of double stranded DNA, designed similarly to conventional DNA targeting constructs with homology arms flanking the insertion sequence, or single-stranded DNA oligonucleotides (ssODNs). The latter provides an effective and simple method for making small edits in the genome, such as the introduction of single nucleotide mutations for probing causal genetic variations. Unlike NHEJ, HDR is generally active only in dividing cells and its efficiency varies depending on the cell type and state.
[0639] Overview of CRISPR: The CRISPR-Cas system, by contrast, is at minimum a two- component system consisting of the Cas9 nuclease and a short guide RNA. Re-targeting of Cas9 to different loci or simultaneous editing of multiple genes simply requires cloning a different 20- bp oligonucleotide. Although specificity of the Cas9 nuclease has yet to be thoroughly elucidated, the simple Watson-Crick base-pairing of the CRISPR-Cas system is likely more predictable than that of ZFN or TALEN domains.
[0640] The type II CRISPR-Cas (clustered regularly interspaced short palindromic repeats) is a bacterial adaptive immune system that uses Cas9, to cleave foreign genetic elements. Cas9 is guided by a pair of non-coding RNAs, a variable crRNA and a required auxiliary tracrRNA. The crRNA contains a 20-nt guide sequence determines specificity by locating the target DNA via Watson-Crick base-pairing. In the native bacterial system, multiple crRNAs are co-transcribed to direct Cas9 against various targets. In the CRISPR-Cas system derived from Streptococcus pyogenes, the target DNA must immediately precede a 5’-NGG/NRG protospacer adjacent motif (PAM), which can vary for other CRISPR systems.
[0641] CRISPR-Cas is reconstituted in mammalian cells through the heterologous expression of human codon-optimized Cas9 and the requisite RNA components. Furthermore, the crRNA and tracrRNA can be fused to create a chimeric, synthetic guide RNA (sgRNA). Cas9 can thus be re-directed toward any target of interest by altering the 20-nt guide sequence within the sgRNA.
[0642] Given its ease of implementation and multiplex capability, Cas9 has been used to generate engineered eukaryotic cells carrying specific mutations via both NHEJ and HDR. In addition, direct injection of sgRNA and mRNA encoding Cas9 into embryos has enabled the rapid generation of transgenic mice with multiple modified alleles; these results hold promise for editing organisms that are otherwise genetically intractable.
[0643] A mutant Cas9 carrying a disruption in one of its catalytic domains has been engineered to nick rather than cleave DNA, allowing for single-stranded breaks and preferential repair through HDR, potentially ameliorating unwanted indel mutations from off-target DSBs. Additionally, a Cas9 mutant with both DNA-cleaving catalytic residues mutated has been adapted to enable transcriptional regulation in E. coli, demonstrating the potential of functionalizing Cas9 for diverse applications. Certain aspects of the invention relate to the construction and application of Cas9 for multiplexed editing of human cells.
[0644] Applicants have provided a human codon-optimized, nuclear localization sequence- flanked Cas9 to facilitate eukaryotic gene editing. Applicants describe considerations for designing the 20-nt guide sequence, protocols for rapid construction and functional validation of sgRNAs, and finally use of the Cas9 nuclease to mediate both NHEJ- and HDR-based genome modifications in human embryonic kidney (HEK-293FT) and human stem cell (HUES9) lines. This protocol can likewise be applied to other cell types and organisms.
[0645] Target selection for sgRNA: There are two main considerations in the selection of the 20-nt guide sequence for gene targeting: 1) the target sequence should precede the 5’-NGG PAM for S. pyogenes Cas9, and 2) guide sequences should be chosen to minimize off-target activity. Applicants provided an online Cas9 targeting design tool that takes an input sequence of interest and identifies suitable target sites. To experimentally assess off-target modifications for each sgRNA, Applicants also provide computationally predicted off-target sites for each intended target, ranked according to Applicants’ quantitative specificity analysis on the effects of base- pairing mismatch identity, position, and distribution.
[0646] The detailed information on computationally predicted off-target sites is as follows:
[0647] Considerations for Off-target Cleavage Activities: Similar to other nucleases, Cas9 can cleave off-target DNA targets in the genome at reduced frequencies. The extent to which a given guide sequence exhibit off-target activity depends on a combination of factors including enzyme concentration, thermodynamics of the specific guide sequence employed, and the abundance of similar sequences in the target genome. For routine application of Cas9, it is important to consider ways to minimize the degree of off-target cleavage and also to be able to detect the presence of off-target cleavage.
[0648] Minimizing off-target activity: For application in cell lines, Applicants recommend following two steps to reduce the degree of off-target genome modification. First, using Applicants’ online CRISPR target selection tool, it is possible to computationally assess the likelihood of a given guide sequence to have off-target sites. These analyses are performed through an exhaustive search in the genome for off-target sequences that are similar sequences as the guide sequence. Comprehensive experimental investigation of the effect of mismatching bases between the sgRNA and its target DNA revealed that mismatch tolerance is 1) position dependent– the 8-14 bp on the 3’ end of the guide sequence are less tolerant of mismatches than the 5’ bases, 2) quantity dependent– in general more than 3 mismatches are not tolerated, 3) guide sequence dependent– some guide sequences are less tolerant of mismatches than others, and 4) concentration dependent– off-target cleavage is highly sensitive to the amount of transfected DNA. The Applicants’ target site analysis web tool (available at the website genome- engineering.org/tools) integrates these criteria to provide predictions for likely off-target sites in the target genome. Second, Applicants recommend titrating the amount of Cas9 and sgRNA expression plasmid to minimize off-target activity.
[0649] Detection of off-target activities: Using Applicants’ CRISPR targeting web tool, it is possible to generate a list of most likely off-target sites as well as primers performing SURVEYOR or sequencing analysis of those sites. For isogenic clones generated using Cas9, Applicants strongly recommend sequencing these candidate off-target sites to check for any undesired mutations. It is worth noting that there may be off target modifications in sites that are not included in the predicted candidate list and full genome sequence should be performed to completely verify the absence of off-target sites. Furthermore, in multiplex assays where several DSBs are induced within the same genome, there may be low rates of translocation events and can be evaluated using a variety of techniques such as deep sequencing.
[0650] The online tool provides the sequences for all oligos and primers necessary for 1) preparing the sgRNA constructs, 2) assaying target modification efficiency, and 3) assessing cleavage at potential off-target sites. It is worth noting that because the U6 RNA polymerase III promoter used to express the sgRNA prefers a guanine (G) nucleotide as the first base of its transcript, an extra G is appended at the 5’ of the sgRNA where the 20-nt guide sequence does not begin with G.
[0651] Approaches for sgRNA construction and delivery: Depending on the desired application, sgRNAs may be delivered as either 1) PCR amplicons containing an expression cassette or 2) sgRNA-expressing plasmids. PCR-based sgRNA delivery appends the custom sgRNA sequence onto the reverse PCR primer used to amplify a U6 promoter template. The resulting amplicon may be co-transfected with a plasmid containing Cas9 (PX165). This method is optimal for rapid screening of multiple candidate sgRNAs, as cell transfections for functional testing can be performed mere hours after obtaining the sgRNA-encoding primers. Because this simple method obviates the need for plasmid-based cloning and sequence verification, it is well suited for testing or co-transfecting a large number of sgRNAs for generating large knockout libraries or other scale-sensitive applications. Note that the sgRNA-encoding primers are over 100-bp, compared to the ~20-bp oligos required for plasmid-based sgRNA delivery.
[0652] Construction of an expression plasmid for sgRNA is also simple and rapid, involving a single cloning step with a pair of partially complementary oligonucleotides. After annealing the oligo pairs, the resulting guide sequences may be inserted into a plasmid bearing both Cas9 and an invariant scaffold bearing the remainder of the sgRNA sequence (PX330). The transfection plasmids may also be modified to enable virus production for in vivo delivery.
[0653] In addition to PCR and plasmid-based delivery methods, both Cas9 and sgRNA can be introduced into cells as RNA.
[0654] Design of repair template: Traditionally, targeted DNA modifications have required use of plasmid-based donor repair templates that contain homology arms flanking the site of alteration. The homology arms on each side can vary in length, but are typically longer than 500 bp. This method can be used to generate large modifications, including insertion of reporter genes such as fluorescent proteins or antibiotic resistance markers. The design and construction of targeting plasmids has been described elsewhere. [0655] More recently, single-stranded DNA oligonucleotides (ssODNs) have been used in place of targeting plasmids for short modifications within a defined locus without cloning. To achieve high HDR efficiencies, ssODNs contain flanking sequences of at least 40 bp on each side that are homologous to the target region, and can be oriented in either the sense or antisense direction relative to the target locus.
[0656] Functional testing
[0657] SURVEYOR nuclease assay: Applicants detected indel mutations either by the SURVEYOR nuclease assay (or PCR amplicon sequencing. Applicants online CRISPR target design tool provides recommended primers for both approaches. However, SURVEYOR or sequencing primers may also be designed manually to amplify the region of interest from genomic DNA and to avoid non-specific amplicons using NCBI Primer-BLAST. SURVEYOR primers should be designed to amplify 300-400 bp (for a 600-800 bp total amplicon) on either side of the Cas9 target for allowing clear visualization of cleavage bands by gel electrophoresis. To prevent excessive primer dimer formation, SURVEYOR primers should be designed to be typically under 25-nt long with melting temperatures of ~60°C. Applicants recommend testing each pair of candidate primers for specific PCR amplicons as well as for the absence of non- specific cleavage during the SURVEYOR nuclease digestion process.
[0658] Plasmid- or ssODN-mediated HDR: HDR can be detected via PCR-amplification and sequencing of the modified region. PCR primers for this purpose should anneal outside the region spanned by the homology arms to avoid false detection of residual repair template (HDR Fwd and Rev, Fig. 30). For ssODN-mediated HDR, SURVEYOR PCR primers can be used.
[0659] Detection of indels or HDR by sequencing: Applicants detected targeted genome modifications by either Sanger or next-generation deep sequencing (NGS). For the former, genomic DNA from modified region can be amplified using either SURVEYOR or HDR primers. Amplicons should be subcloned into a plasmid such as pUC19 for transformation; individual colonies can be sequenced to reveal clonal genotype.
[0660] Applicants designed next-generation sequencing (NGS) primers for shorter amplicons, typically in the 100-200 bp size range. For detecting NHEJ mutations, it is important to design primers with at least 10-20 bp between the priming regions and the Cas9 target site to allow detection of longer indels. Applicants provide guidelines for a two-step PCR method to attach barcoded adapters for multiplex deep sequencing. Applicants recommend the Illumina platform, due to its generally low levels of false positive indels. Off-target analysis (described previously) can then be performed through read alignment programs such as ClustalW, Geneious, or simple sequence analysis scripts.
Materials and Reagents
[0661] sgRNA preparation:
UltraPure DNaseRNase-free distilled water (Life Technologies, cat. no. 10977-023) Herculase II fusion polymerase (Agilent Technologies, cat. no. 600679)
CRITICAL. Standard Taq polymerase, which lacks 3’-5’ exonuclease proofreading activity, has lower fidelity and can lead to amplification errors. Herculase II is a high-fidelity polymerase (equivalent fidelity to Pfu) that produces high yields of PCR product with minimal optimization. Other high-fidelity polymerases may be substituted.
Herculase II reaction buffer (5x; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L)
MgCl2 (25mM; ThermoScientific, cat. no. R0971)
QIAquick gel extraction kit (Qiagen, cat. no. 28704)
QIAprep spin miniprep kit (Qiagen, cat. no. 27106)
UltraPure TBE buffer (10X; Life Technologies, cat. no. 15581 -028)
SeaKem LE agarose (Lonza, cat. no. 50004)
SYBR Safe DNA stain (10,000x; Life Technologies, cat. no. S33102)
1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018)
TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest BbsI (BpiI) (Fermentas/ThermoScientific, cat. no. FD1014)
Fermentas Tango Buffer (Fermentas/ThermoScientific, cat. no. BY5)
DL-dithiothreitol (DTT; Fermentas/ThermoScientific, cat. no. R0862)
T7 DNA ligase (Enzymatics, cat. no. L602L)
Critical:Do not substitute the more commonly used T4 ligase. T7 ligase has 1,000- fold higher activity on the sticky ends than on the blunt ends and higher overall activity than commercially available concentrated T4 ligases.
T7 2X Rapid Ligation Buffer (included with T7 DNA ligase, Enzymatics, cat. no.
L602L)
T4 Polynucleotide Kinase (New England Biolabs, cat. no M0201S)
T4 DNA Ligase Reaction Buffer (10X; New England Biolabs, cat. no B0202S) Adenosine 5’-triphosphate (10 mM; New England Biolabs, cat. no. P0756S)
PlasmidSafe ATP-dependent DNase (Epicentre, cat. no. E3101K)
One Shot Stbl3 chemically competent Escherichia coli (E. coli) (Life Technologies, cat. no. C7373-03)
SOC medium (New England Biolabs, cat. no. B9020S)
LB medium (Sigma, cat. no. L3022)
LB agar medium (Sigma, cat. no. L2897)
Ampicillin, sterile filtered (100 mg mlí 1; Sigma, cat. no. A5354)
[0662] Mammalian cell culture:
HEK293FT cells (Life Technologies, cat. no. R700-07)
Dulbecco's minimum Eagle’s medium (DMEM, 1X, high glucose; Life Technologies, cat. no. 10313-039)
Dulbecco’s minimum Eagle’s medium (DMEM, 1X, high glucose, no phenol red;
Life Technologies, cat. no. 31053-028)
Dulbecco’s phosphate-buffered saline (DPBS, 1X; Life Technologies, cat. no. 14190- 250)
Fetal bovine serum, qualified and heat inactivated (Life Technologies, cat. no. 10438- 034)
Opti-MEM I reduced-serum medium (FBS; Life Technologies, cat. no. 11058-021) Penicillin-streptomycin (100x; Life Technologies, cat. no. 15140-163)
TrypLE™ Express (1X, no Phenol Red; Life Technologies, cat. no. 12604-013) Lipofectamine 2000 transfection reagent (Life Technologies, cat. no. 11668027) Amaxa SF Cell Line 4D-Nucleofector® X Kit S (32 RCT; Lonza, cat. no V4XC- 2032)
HUES 9 cell line (HARVARD STEM CELL SCIENCE)
Geltrex LDEV-Free Reduced Growth Factor Basement Membrane Matrix (Life Technologies, cat. no. A1413201)
mTeSR1 medium (Stemcell Technologies, cat. no. 05850)
Accutase cell detachment solution (Stemcell Technologies, cat. no. 07920) ROCK Inhibitor (Y-27632; Millipore, cat. no. SCM075)
Amaxa P3 Primary Cell 4D-Nucleofector® X Kit S (32 RCT; Lonza cat. no. V4XP- 3032)
[0663] Genotyping analysis: QuickExtract DNA extraction solution (Epicentre, cat. no. QE09050) PCR primers for SURVEYOR, RFLP analysis, or sequencing (see Primer table) Herculase II fusion polymerase (Agilent Technologies, cat. no. 600679)
CRITICAL. As Surveyor assay is sensitive to single-base mismatches, it is particularly important to use a high-fidelity polymerase. Other high-fidelity polymerases may be substituted.
Herculase II reaction buffer (5x; Agilent Technologies, included with polymerase) dNTP solution mix (25 mM each; Enzymatics, cat. no. N205L)
QIAquick gel extraction kit (Qiagen, cat. no. 28704)
Taq Buffer (10x; Genscript, cat. no. B0005)
SURVEYOR mutation detection kit for standard gel electrophoresis (Transgenomic, cat. no. 706025)
UltraPure TBE buffer (10x; Life Technologies, cat. no. 15581-028)
SeaKem LE agarose (Lonza, cat. no. 50004)
4-20% TBE Gels 1.0 mm, 15 Well (Life Technologies, cat. no. EC62255BOX) Novex® Hi-Density TBE Sample Buffer (5X; Life Technologies, cat. no. LC6678) SYBR Gold Nucleic Acid Gel Stain (10,000X; Life Technologies, cat. no. S-11494) 1-kb Plus DNA ladder (Life Technologies, cat. no. 10787-018)
TrackIt CyanOrange loading buffer (Life Technologies, cat. no. 10482-028) FastDigest HindIII (Fermentas/ThermoScientific, cat. no. FD0504)
[0664] Equipment
Filtered sterile pipette tips (Corning)
Standard 1.5ml microcentrifuge tubes (Eppendorf, cat. no. 0030 125.150) Axygen 96-well PCR plates (VWR, cat. no. PCR-96M2-HSC)
Axygen 8-Strip PCR tubes (Fischer Scientific, cat. no. 14-222-250)
Falcon tubes, polypropylene, 15 ml (BD Falcon, cat. no. 352097)
Falcon tubes, polypropylene, 50 ml (BD Falcon, cat. no. 352070)
Round-bottom Tube with cell strainer cap, 5ml (BD Falcon, cat. no. 352235) Petri dishes (60 mm × 15 mm; BD Biosciences, cat. no. 351007)
Tissue culture plate (24 well; BD Falcon, cat. no. 353047)
Tissue culture plate (96 well, flat bottom; BD Falcon, cat. no. 353075)
Tissue culture dish (100 mm; BD Falcon, 353003) 96-well thermocycler with programmable temperature stepping functionality (Applied Biosystems Veriti, cat. no. 4375786).
Desktop microcentrifuges 5424, 5804 (Eppendorf)
Gel electrophoresis system (PowerPac basic power supply, Bio-Rad, cat. no. 164- 5050, and Sub-Cell GT System gel tray, Bio-Rad, cat. no. 170-4401)
Novex XCell SureLock Mini-Cell (Life Technologies, cat. no. EI0001)
Digital gel imaging system (GelDoc EZ, Bio-Rad, cat. no. 170-8270, and blue sample tray, Bio-Rad, cat. no. 170-8273)
Blue light transilluminator and orange filter goggles (SafeImager 2.0; Invitrogen, cat. no. G6600)
Gel quantification software (Bio-Rad, ImageLab, included with GelDoc EZ,or open- source ImageJ from the National Institutes of Health, available at the website rsbweb.nih.gov/ij/) UV spectrophotometer (NanoDrop 2000c, Thermo Scientific)
Reagent Setup
[0665] Tris-borate EDTA (TBE) electrophoresis solution Dilute TBE buffer in distilled water to 1X working solution for casting agarose gels and for use as a buffer for gel electrophoresis. Buffer may be stored at room temperature (18 í 22 °C) for at least 1 year.
• ATP, 10 mM Divide 10 mM ATP into 50- l aliquots and store at í 20 °C for up to 1 year; avoid repeated freeze-thaw cycles.
• DTT, 10 mM Prepare 10 mM DTT solution in distilled water and store in 20- l aliquots at í 70 °C for up to 2 years; for each reaction, use a new aliquot, as DTT is easily oxidized.
• D10 culture medium For culture of HEK293FT cells, prepare D10 culture medium by supplementing DMEM with 1X GlutaMAX and 10% (vol/vol) fetal bovine serum. As indicated in the protocol, this medium can also be supplemented with 1X penicillin-streptomycin . D10 medium can be made in advance and stored at 4 °C for up to 1 month.
• mTeSR1 culture medium For culture of human embryonic stem cells, prepare mTeSR1 medium by supplementing the 5X supplement (included with mTeSR1 basal medium), and 100 ug/ml Normocin. Procedure
[0666] Design of targeting components and use of the online tool• Timing 1 d
[0667] 1| Input target genomic DNA sequence. Applicants provide an online Cas9 targeting design tool that takes an input sequence of interest, identifies and ranks suitable target sites, and computationally predicts off-target sites for each intended target. Alternatively, one can manually select guide sequence by identifying the 20-bp sequence directly upstream of any 5’- NGG.
[0668] 2| Order necessary oligos and primers as specified by the online tool. If the site is chosen manually, the oligos and primers should be designed.
[0669] Preparation of sgRNA expression construct
[0670] 3| To generate the sgRNA expression construct, either the PCR- or plasmid-based protocol can be used.
(A) via PCR amplification• Timing 2 h
(i) Applicants prepare diluted U6 PCR template. Applicants recommend using PX330 as a PCR template, but any U6-containing plasmid may likewise be used as the PCR template. Applicants diluted template with ddH2O to a concentration of 10 ng/ul. Note that if a plasmid or cassette already containing an U6-driven sgRNA is used as a template, a gel extraction needs to be performed to ensure that the product contains only the intended sgRNA and no trace sgRNA carryover from template.
(ii) Applicants prepared diluted PCR oligos. U6-Fwd and U6-sgRNA-Rev primers are diluted to a final concentration of 10 uM in ddH2O (add 10 ul of 100 uM primer to 90 ul ddH2O).
(iii) U6-sgRNA PCR reaction. Applicants set up the following reaction for each U6-sgRNA-Rev primer and mastermix as needed:
Component: Amount (ul) Final concentration Herculase II PCR buffer, 5X 10 1X dNTP, 100mM (25mM each) 0.5 1 mM
U6 template (PX330) 1 0.2 ng/ul
U6-Fwd primer 1 0.2 uM
U6-sgRNA-Rev primer (variable) 1 0.2 uM
Herculase II Fusion polymerase 0.5
Distilled water 36
Total 50 (iv) Applicants performed PCR reaction on the reactions from step (iii) using the following cycling conditions:
Cycle number Denature Anneal Extend
1 95°C, 2 m
2-31 95°C, 20 s 60°C, 20 s 72°C, 20 s 32 72°C, 3 m (v) After the reaction is completed, Applicants ran the product on a gel to verify successful, single-band amplification. Cast a 2% (wt/vol) agarose gel in 1X TBE buffer with 1X SYBR Safe dye. Run 5 ul of the PCR product in the gel at 15 V cm-1 for 20-30 min. Successful amplicons should yield one single 370-bp product and the template should be invisible. It should not be necessary to gel extract the PCR amplicon.
(vi) Applicants purified the PCR product using the QIAquick PCR purification kit according to the manufacturer’s directions. Elute the DNA in 35 ul of Buffer EB or water. Purified PCR products may be stored at 4°C or -20°C.
(B) Cloning sgRNA into Cas9-containing bicistronic expression vector• Timing 3 d (i) Prepare the sgRNA oligo inserts. Applicants resuspended the top and bottom strands of oligos for each sgRNA design to a final concentration of 100 uM. Phosphorylate and anneal the oligo as follows:
Oligo 1 (100 uM) 1 ul Oligo 2 (100 uM) 1 ul T4 Ligation Buffer, 10X 1 ul T4 PNK 1 ul ddH2O 6 ul Total 10 ul (ii) Anneal in a thermocycler using the following parameters:
37°C for 30 m
95°C for 5 m
Ramp down to 25°C at 5°C per m
(iii) Applicants diluted phosphorylated and annealed oligos 1:200 by add 1ul of oligo to 199 ul room temperature ddH2O. (iv) Clone sgRNA oligo into PX330. Applicants set up Golden Gate reaction for each sgRNA. Applicants recommend also setting up a no-insert, PX330 only negative control.
PX330 (100 ng) x ul
Diluted oligo duplex from step (iii) 2 ul
Tango Buffer, 10X 2 ul
DTT, 10mM 1 ul
ATP, 10mM 1 ul
FastDigest BbsI 1 ul
T7 Ligase 0.5 ul ddH2O x ul
Total 20 ul
(v) Incubate the Golden Gate reaction for a total of 1 h:
Cycle number Condition
1-6 37°C for 5 m, 21°C for 5 m (vi) Applicants treated Golden Gate reaction with PlasmidSafe exonuclease to digest any residual linearized DNA. This step is optional but highly recommended.
Golden Gate reaction from step 4 11 ul
10X PlasmidSafe Buffer 1.5 ul
ATP, 10 mM 1.5 ul
PlasmidSafe exonuclease 1 ul
Total 15 ul
(vii) Applicants incubated the PlasmidSafe reaction at 37°C for 30 min, followed by inactivation at 70°C for 30 min. Pause point: after completion, the reaction may be frozen and continued later. The circular DNA should be stable for at least 1 week.
(viii) Transformation. Applicants transformed the PlasmidSafe-treated plasmid into a competent E. coli strain, according to the protocol supplied with the cells. Applicants recommend Stbl3 for quick transformation. Briefly, Applicants added 5ul of the product from step (vii) into 20ul of ice-cold chemically competent Stbl3 cells. This is then incubated on ice for 10 m, heat shocked at 42°C for 30 s, returned immediately to ice for 2 m, 100 ul of SOC medium is added, and this is plated onto an LB plate containing 100 ug/ml ampicillin with incubation overnight at 37°C. (ix) Day 2: Applicants inspected plates for colony growth. Typically, there are no colonies on the negative control plates (ligation of BbsI-digested PX330 only, no annealed sgRNA oligo), and tens to hundreds of colonies on the PX330-sgRNA cloning plates.
(x) From each plate, Applicants picked 2-3 colonies to check correct insertion of sgRNA. Applicants used a sterile pipette tip to inoculate a single colony into a 3 ml culture of LB medium with 100 ug/ml ampicillin. Incubate and shake at 37°C overnight.
(xi) Day 3: Applicants isolated plasmid DNA from overnight cultures using a QiAprep Spin miniprep kit according to the manufacturer’s instructions.
(xii) Sequence validate CRISPR plasmid. Applicants verified the sequence of each colony by sequencing from the U6 promoter using the U6-Fwd primer. Optional: sequence the Cas9 gene using primers listed in the following Primer table.
Figure imgf000244_0001
Figure imgf000245_0001
[0671] Applicants referenced the sequencing results against the PX330 cloning vector sequence to check that the 20 bp guide sequence was inserted between the U6 promoter and the remainder of the sgRNA scaffold. Details and sequence of the PX330 map in GenBank vector map format (*.gb file) can be found at the website crispr.genome-engineering.org.
(Optional) Design of ssODN template• Timing 3 d planning ahead
[0672] 3| Design and order ssODN. Either the sense or antisense ssODN can be purchased directly from supplier. Applicants recommend designing homology arms of at least 40 bp on either side and 90 bp for optimal HDR efficiency. In Applicants’ experience, antisense oligos have slightly higher modification efficiencies.
[0673] 4| Applicants resuspended and diluted ssODN ultramers to a final concentration of 10 uM. Do not combine or anneal the sense and antisense ssODNs. Store at -20°C. [0674] 5| Note for HDR applications, Applicants recommend cloning sgRNA into the PX330 plasmid.
Functional validation of sgRNAs: cell culture and transfections• Timing 3-4 d
[0675] The CRISPR-Cas system has been used in a number of mammalian cell lines. Conditions may vary for each cell line. The protocols below details transfection conditions for HEK239FT cells. Note for ssODN-mediated HDR transfections, the Amaxa SF Cell Line Nucleofector Kit is used for optimal delivery of ssODNs. This is described in the next section.
[0676] 7| HEK293FT maintenance. Cells are maintained according to the manufacturer’s recommendations. Briefly, Applicants cultured cells in D10 medium (GlutaMax DMEM supplemented with 10% Fetal Bovine Serum), at 37°C and 5% CO2.
[0677] 8| To passage, Applicants removed medium and rinsed once by gently adding DPBS to side of vessel, so as not to dislodge cells. Applicants added 2 ml of TrypLE to a T75 flask and incubated for 5 m at 37°C. 10 ml of warm D10 medium is added to inactivate and transferred to a 50 ml Falcon tube. Applicants dissociated cells by triturating gently, and re-seeded new flasks as necessary. Applicants typically passage cells every 2-3 d at a split ratio of 1:4 or 1:8, never allowing cells to reach more than 70% confluency. Cell lines are restarted upon reaching passage number 15.
[0678] 9| Prepare cells for transfection. Applicants plated well-dissociated cells onto 24-well plates in D10 medium without antibiotics 16-24 h before transfection at a seeding density of 1.3 x 105 cells per well and a seeding volume of 500 ul. Scale up or down according to the manufacturer’s manual as needed. It is suggested to not plate more cells than recommended density as doing so may reduce transfection efficiency.
[0679] 10| On the day of transfection, cells are optimal at 70-90% confluency. Cells may be transfected with Lipofectamine 2000 or Amaxa SF Cell Line Nucleofector Kit according to the manufacturers’ protocols.
(A) For sgRNAs cloned into PX330, Applicants transfected 500 ng of sequence- verified CRISPR plasmid; if transfecting more than one plasmid, mix at equimolar ratio and no more than 500 ng total.
(B) For sgRNA amplified by PCR, Applicants mixed the following:
PX165 (Cas9 only) 200 ng
sgRNA amplicon (each) 40 ng
pUC19 fill up total DNA to 500 ng [0680] Applicants recommend transfecting in technical triplicates for reliable quantification and including transfection controls (e.g. GFP plasmid) to monitor transfection efficiency. In addition, PX330 cloning plasmid and/or sgRNA amplicon may be transfected alone as a negative control for downstream functional assays.
[0681] 11| Applicants added Lipofectamine complex to cells gently as HEK293FT cells may detach easily from plate easily and result in lower transfection efficiency.
[0682] 12| Applicants checked cells 24 h after transfection for efficiency by estimating the fraction of fluorescent cells in the control (e.g., GFP) transfection using a fluorescence microscope. Typically cells are more than 70% transfected.
[0683] 13| Applicants supplemented the culture medium with an additional 500 ul of warm D10 medium. Add D10 very slowly to the side of the well and do not use cold medium, as cells can detach easily.
[0684] 14| Cells are incubated for a total of 48-72 h post-transfection before harvested for indel analysis. Indel efficiency does not increase noticeably after 48 h.
(Optional) Co-transfection of CRISPR plasmids and ssODNs or targeting plasmids for HR • Timing 3-4 d
[0685] 15| Linearize targeting plasmid. Targeting vector is linearized if possible by cutting once at a restriction site in the vector backbone near one of the homology arms or at the distal end of either homology arm.
[0686] 16| Applicants ran a small amount of the linearized plasmid alongside uncut plasmid on a 0.8-1% agarose gel to check successful linearization. Linearized plasmid should run above the supercoiled plasmid.
[0687] 17| Applicants purified linearized plasmid with the QIAQuick PCR Purification kit.
[0688] 18| Prepare cells for transfection. Applicants cultured HEK293FT in T75 or T225 flasks. Sufficient cell count before day of transfection is planned for. For the Amaxa strip- cuvette format, 2 x 106 cells are used per transfection.
[0689] 19| Prepare plates for transfection. Applicants added 1 ml of warm D10 medium into each well of a 12 well plate. Plates are placed into the incubator to keep medium warm.
[0690] 20| Nucleofection. Applicants transfected HEK293FT cells according to the Amaxa SF Cell Line Nucleofector 4D Kit manufacturer’s instructions, adapted in the steps below.
a. For ssODN and CRISPR cotransfection, pre-mix the following DNA in PCR tubes: pCRISPR plasmid (Cas9 + sgRNA) 500 ng ssODN template (10uM) 1 ul b. For HDR targeting plasmid and CRISPR cotransfection, pre-mix the following DNA in PCR tubes:
CRISPR plasmid (Cas9 + sgRNA) 500 ng
Linearized targeting plasmid 500 ng [0691] For transfection controls, see previous section. In addition, Applicants recommend transfecting ssODN or targeting plasmid alone as a negative control.
[0692] 21| Dissociate to single cells. Applicants removed medium and rinsed once gently with DPBS, taking care not to dislodge cells. 2 ml of TrypLE is added to a T75 flask and incubated for 5 m at 37°C. 10 ml of warm D10 medium is added to inactivate and triturated gently in a 50 ml Falcon tube. It is recommended that cells are triturated gently and dissociated to single cells. Large clumps will reduce transfection efficiency. Applicants took a 10 ul aliquot from the suspension and diluted into 90 ul of D10 medium for counting. Applicants counted cells and calculated the number of cells and volume of suspension needed for transfection. Applicants typically transfected 2 x 105 cells per condition using the Amaxa Nucleocuvette strips, and recommend calculating for 20% more cells than required to adjust for volume loss in subsequent pipetting steps. The volume needed is transferred into a new Falcon tube.
[0693] 23| Applicants spun down the new tube at 200 x g for 5 m.
[0694] Applicants prepared the transfection solution by mixing the SF solution and S1 supplement as recommended by Amaxa. For Amaxa strip-cuvettes, a total of 20 ul of supplemented SF solution is needed per transfection. Likewise, Applicants recommend calculating for 20% more volume than required.
[0695] 25| Applicants removed medium completely from pelleted cells from step 23 and gently resuspended in appropriate volume (20 ul per 2 x 105 cells) of S1-supplemented SF solution. Do not leave cells in SF solution for extended period of time.
[0696] 26| 20 ul of resuspended cells is pipetted into each DNA pre-mix from step 20. Pipette gently to mix and transfer to Nucleocuvette strip chamber. This is repeated for each transfection condition.
[0697] Electroporate cells using the Nucleofector 4D program recommended by Amaxa, CM-130.
[0698] 28| Applicants gently and slowly pipetted 100 ul of warm D10 medium into each Nucleocuvette strip chamber, and transferred all volume into the pre-warmed plate from step 19. CRITICAL. Cells are very fragile at this stage and harsh pipetting can cause cell death. Incubate for 24 h. At this point, transfection efficiency can be estimated from fraction of fluorescent cells in positive transfection control. Nucleofection typically results in greater than 70-80% transfection efficiency. Applicants slowly added 1 ml warm D10 medium to each well without dislodging the cells. Incubate cells for a total of 72 h.
[0699] Human embryonic stem cell (HUES 9) culture and transfection• Timing 3-4 d
[0700] Maintaining hESC (HUES9) line. Applicants routinely maintain HUES9 cell line in feeder-free conditions with mTesR1 medium. Applicants prepared mTeSR1 medium by adding the 5X supplement included with basal medium and 100 ug/ml Normocin. Applicants prepared a 10 ml aliquot of mTeSR1 medium supplemented further with 10 uM Rock Inhibitor. Coat tissue culture plate. Dilute cold GelTrex 1:100 in cold DMEM and coat the entire surface of a 100 mm tissue culture plate.
[0701] Place plate in incubator for at least 30 m at 37°C. Thaw out a vial of cells at 37°C in a 15 ml Falcon tube, add 5 ml of mTeSR1 medium, and pellet at 200 x g for 5 m. Aspirate off GelTrex coating and seed ~1 x 106 cells with 10 ml mTeSR1 medium containing Rock Inhibitor. Change to normal mTeSR1 medium 24 h after transfection and re-feed daily. Passaging cells. Re-feed cells with fresh mTeSR1 medium daily and passage before reaching 70% confluency. Aspirate off mTeSR1 medium and wash cells once with DPBS. Dissociate cells by adding 2 ml Accutase and incubating at 37°C for 3– 5 m. Add 10 ml mTeSR1 medium to detached cells, transfer to 15 ml Falcon tube and resuspend gently. Re-plate onto GelTrex-coated plates in mTeSR1 medium with 10 uM Rock Inhibitor. Change to normal mTeSR1 medium 24 h after plating.
[0702] Transfection. Applicants recommend culturing cells for at least 1 week post-thaw before transfecting using the Amaxa P3 Primary Cell 4-D Nucleofector Kit (Lonza). Re-feed log-phase growing cells with fresh medium 2 h before transfection. Dissociate to single cells or small clusters of no more than 10 cells with accutase and gentle resuspension. Count the number of cells needed for nucleofection and spin down at 200 x g for 5 m. Remove medium completely and resuspend in recommended volume of S1-supplemented P3 nucleofection solution. Gently plate electroporated cells into coated plates in presence of 1X Rock Inhibitor.
[0703] Check transfection success and re-feed daily with regular mTeSR1 medium beginning 24 h after nucleofection. Typically, Applicants observe greater than 70% transfection efficiency with Amaxa Nucleofection. Harvest DNA. 48-72 h post transfection, dissociate cells using accutase and inactivate by adding 5 x volume of mTeSR1. Spin cells down at 200 x g for 5 m. Pelleted cells can be directed processed for DNA extraction with QuickExtract solution. It is recommended to not mechanically dissociate cells without accutase. It is recommended to not spin cells down without inactivating accutase or above the recommended speed; doing so may cause cells to lyse.
[0704] Isolation of clonal cell lines by FACS. Timing• 2-3 h hands-on; 2-3 weeks expansion
[0705] Clonal isolation may be performed 24 h post-transfection by FACS or by serial dilution.
[0706] 54| Prepare FACS buffer. Cells that do not need sorting using colored fluorescence may be sorted in regular D10 medium supplemented with 1X penicillin/streptinomycin. If colored fluorescence sorting is also required, a phenol-free DMEM or DPBS is substituted for normal DMEM. Supplement with 1X penicillin/streptinomycin and filter through a .22 um Steriflip filter.
[0707] 55| Prepare 96 well plates. Applicants added 100 ul of D10 media supplemented with 1X penicillin/streptinomycin per well and prepared the number of plates as needed for the desired number of clones.
[0708] 56| Prepare cells for FACS. Applicants dissociated cells by aspirating the medium completely and adding 100 ul TrypLE per well of a 24-well plate. Incubate for 5 m and add 400 ul warm D10 media.
[0709] 57| Resuspended cells are transferred into a 15 ml Falcon tube and gently triturated 20 times. Recommended to check under the microscope to ensure dissociation to single cells.
[0710] 58| Spin down cells at 200 x g for 5 minutes.
[0711] 59| Applicants aspirated the media, and resuspended the cells in 200 ul of FACS media.
[0712] 60| Cells are filtered through a 35 um mesh filter into labeled FACS tubes. Applicants recommend using the BD Falcon 12 x 75 mm Tube with Cell Strainer cap. Place cells on ice until sorting.
[0713] 61| Applicants sorted single cells into 96-well plates prepared from step 55. Applicants recommend that in one single designated well on each plate, sort 100 cells as a positive control.
[0714] NOTE. The remainder of the cells may be kept and used for genotyping at the population level to gauge overall modification efficiency. [0715] 62| Applicants returned cells into the incubator and allowed them to expand for 2-3 weeks. 100 ul of warm D10 medium is added 5 d post sorting. Change 100 ul of medium every 3-5 d as necessary.
[0716] 63| Colonies are inspected for“clonal” appearance 1 week post sorting: rounded colonies radiating from a central point. Mark off wells that are empty or may have been seeded with doublets or multiplets.
[0717] 64| When cells are more than 60% confluent, Applicants prepared a set of replica plates for passaging. 100 ul of D10 medium is added to each well in the replica plates. Applicants dissociated cells directly by pipetting up and down vigorously 20 times. 20% of the resuspended volume was plated into the prepared replica plates to keep the clonal lines. Change the medium every 2-3 d thereafter and passage accordingly.
[0718] 65| Use the remainder 80% of cells for DNA isolation and genotyping.
Optional: Isolation of clonal cell lines by dilution. Timing• 2-3 h hands-on; 2-3 weeks expansion
[0719] 66| Applicants dissociated cells from 24-well plates as described above. Make sure to dissociate to single cells. A cell strainer can be used to prevent clumping of cells.
[0720] 67| The number of cells are counted in each condition. Serially dilute each condition in D10 medium to a final concentration of 0.5 cells per 100 ul. For each 96 well plate, Applicants recommend diluting to a final count of 60 cells in 12 ml of D10. Accurate count of cell number is recommended for appropriate clonal dilution. Cells may be recounted at an intermediate serial dilution stage to ensure accuracy.
[0721] 68| Multichannel pipette was used to pipette 100 ul of diluted cells to each well of a 96 well plate.
[0722] NOTE. The remainder of the cells may be kept and used for genotyping at the population level to gauge overall modification efficiency.
[0723] 69| Applicants inspected colonies for“clonal” appearance ~1 week post plating: rounded colonies radiating from a central point. Mark off wells that may have seeded with doublets or multiplets.
[0724] 70| Applicants returned cells to the incubator and allowed them to expand for 2-3 weeks. Re-feed cells as needed as detailed in previous section. SURVEYOR assay for CRISPR cleavage efficiency. Timing• 5-6 h
[0725] Before assaying cleavage efficiency of transfected cells, Applicants recommend testing each new SURVEYOR primer on negative (untransfected) control samples through the step of SURVEYOR nuclease digestion using the protocol described below. Occasionally, even single-band clean SURVEYOR PCR products can yield non-specific SURVEYOR nuclease cleavage bands and potentially interfere with accurate indel analysis.
[0726] 71| Harvest cells for DNA. Dissociate cells and spin down at 200 x g for 5 m. NOTE. Replica plate at this stage as needed to keep transfected cell lines.
[0727] 72| Aspirate the supernatant completely.
[0728] 73| Applicants used QuickExtract DNA extraction solution according to the manufacturer’s instructions. Applicants typically used 50 ul of the solution for each well of a 24 well plate and 10 ul for a 96 well plate.
[0729] 74| Applicants normalized extracted DNA to a final concentration of 100-200 ng/ul with ddH2O. Pause point: Extracted DNA may be stored at -20°C for several months.
[0730] 75| Set up the SURVEYOR PCR. Master mix the following using SURVEYOR primers provided by Applicants online/computer algorithm tool:
Component: Amount (ul) Final concentration Herculase II PCR buffer, 5X 10 1X dNTP, 100mM (25mM each) 1 1 mM
SURVEYOR Fwd primer (10uM) 1 0.2 uM SURVEYOR Rev primer (10uM) 1 0.2 uM Herculase II Fusion polymerase 1
MgCl2 (25mM) 2 1 mM
Distilled water 33 Total 49 (for each reaction) [0731] 76| Applicants added 100-200 ng of normalized genomic DNA template from step 74 for each reaction.
[0732] 77| PCR reaction was performed using the following cycling conditions, for no more than 30 amplification cycles:
Cycle number Denature Anneal Extend 1 95°C, 2 min
2-31 95°C, 20 s 60°C, 20 s 72°C, 30 s 32 72°C, 3 min [0733] 78| Applicants ran 2-5 ul of PCR product on a 1% gel to check for single-band product. Although these PCR conditions are designed to work with most pairs of SURVEYOR primers, some primers may need additional optimization by adjusting the template concentration, MgCl2 concentration, and/or the annealing temperature.
[0734] 79| Applicants purified the PCR reactions using the QIAQuick PCR purification kit and normalized eluant to 20 ng/ul. Pause point: Purified PCR product may be stored at -20°C.
[0735] 80| DNA heteroduplex formation. The annealing reaction was set up as follows:
Taq PCR buffer, 10X 2 ul
Normalized DNA (20 ng/ul) 18 ul
Total volume 20 ul [0736] 81| Anneal the reaction using the following conditions:
Cycle number Condition
1 95°C, 10 mn
2 95°C-85°C, -2°C/s
3 85°C, 1 min
4 85°C-75°C, -0.3°C/s
5 75°C, 1 min
6 75°C-65°C, -0.3°C/s
7 65°C, 1 min
8 65°C-55°C, -0.3°C/s
9 55°C, 1 min
10 55°C-45°C, -0.3°C/s
11 45°C, 1 min
12 45°C-35°C, -0.3°C/s
13 35°C, 1 min
14 35°C-25°C, -0.3°C/s
15 25°C, 1 min [0737] 82| SURVEYOR nuclease S digestion. Applicants prepared master-mix and added the following components on ice to annealed heteroduplexes from step 81 for a total final volume of 25 ul: Component Amount (ul) Final Concentration MgCl2 solution, 0.15M 2.5 15mM
ddH2O 0.5
SURVEYOR nuclease S 1 1X
SURVEYOR enhancer S 1 1X
Total 5 [0738] 83| Vortex well and spin down. Incubate the reaction at 42°C for 1 h.
[0739] 84| Optional: 2 ul of the Stop Solution from the SURVEYOR kit may be added. Pause point. The digested product may be stored at -20°C for analysis at a later time.
[0740] 85| Visualize the SURVEYOR reaction. SURVEYOR nuclease digestion products may be visualized on a 2% agarose gel. For better resolution, products may be run on a 4-20% gradient Polyacrylamide TBE gel. Applicants loaded 10 ul of product with the recommended loading buffer and ran the gel according to manufacturer’s instructions. Typically, Applicants run until the bromophenol blue dye has migrated to the bottom of the gel. Include DNA ladder and negative controls on the same gel.
[0741] 86| Applicants stained the gel with 1X SYBR Gold dye diluted in TBE. The gel was gently rocked for 15 m.
[0742] 87| Applicants imaged the gel using a quantitative imaging system without overexposing the bands. The negative controls should have only one band corresponding to the size of the PCR product, but may have occasionally non-specific cleavage bands of other sizes. These will not interfere with analysis if they are different in size from target cleavage bands. The sum of target cleavage band sizes, provided by Applicants online/computer algorithm tool, should be equal to the size of the PCR product.
[0743] 88| Estimate the cleavage intensity. Applicants quantified the integrated intensity of each band using ImageJ or other gel quantification software.
[0744] 89| For each lane, Applicants calculated the fraction of the PCR product cleaved (fcut) using the following formula: fcut = (b + c) / (a + b + c), where a is the integrated intensity of the undigested PCR product and b and c are the integrated intensities of each cleavage product. 90| Cleavage efficiency may be estimated using the following formula, based on the binomial probability distribution of duplex formation:
[0745] 91| Ψ ൌ ൈ െ െ ௨௧ ȁ ̴ ȁ ȁ
[0746] Sanger sequencing for assessing CRISPR cleavage efficiency. Timing• 3 d [0747] Initial steps are identical to Steps 71-79 of the SURVEYOR assay. Note: SURVEYOR primers may be used for Sanger sequencing if appropriate restriction sites are appended to the Forward and Reverse primers. For cloning into the recommended pUC19 backbone, EcoRI may be used for the Fwd primer and HindIII for the Rev primer.
[0748] 92| Amplicon digestion. Set up the digestion reaction as follows:
Component Amount (ul)
Fast Digest buffer, 10X 3
FastDigest EcoRI 1
FastDigest HindIII 1
Normalized DNA (20 ng/ul) 10
ddH2O 15
Total volume 30 [0749] 93| pUC19 backbone digestion. Set up the digestion reaction as follows:
Component Amount (ul)
Fast Digest buffer, 10X 3
FastDigest EcoRI 1
FastDigest HindIII 1
FastAP Alkaline Phosphatase 1
pUC19 vector (200 ng/ul) 5
ddH2O 20
Total volume 30 [0750] 94| Applicants purified the digestion reactions using the QIAQuick PCR purification kit. Pause point: Purified PCR product may be stored at -20°C.
[0751] 95| Applicants ligated the digested pUC19 backbone and Sanger amplicons at a 1:3 vector:insert ratio as follows:
Component Amount (ul)
Digested pUC19 x (50 ng)
Digested insert x (1:3 vector:insert molar ratio)
T7 ligase 1
2X Rapid Ligation Buffer 10
ddH2O x
Total volume 20 [0752] 96| Transformation. Applicants transformed the PlasmidSafe-treated plasmid into a competent E. coli strain, according to the protocol supplied with the cells. Applicants recommend Stbl3 for quick transformation. Briefly, 5ul of the product from step 95 is added into 20ul of ice-cold chemically competent Stbl3 cells, incubated on ice for 10 m, heat shocked at 42°C for 30 s, returned immediately to ice for 2 m, 100 ul of SOC medium is added, and plated onto an LB plate containing 100 ug/ml ampicillin. This is incubated overnight at 37°C.
[0753] 97| Day 2: Applicants inspected plates for colony growth. Typically, there are no colonies on the negative control plates (ligation of EcoRI-HindIII digested pUC19 only, no Sanger amplicon insert), and tens to hundreds of colonies on the pUC19-Sanger amplicon cloning plates.
[0754] 98| Day 3: Applicants isolated plasmid DNA from overnight cultures using a QIAprep Spin miniprep kit according to the manufacturer’s instructions.
[0755] 99| Sanger sequencing. Applicants verified the sequence of each colony by sequencing from the pUC19 backbone using the pUC19-For primer. Applicants referenced the sequencing results against the expected genomic DNA sequence to check for the presence of Cas9-induced NHEJ mutations. % editing efficiency = (# modified clones)/(# total clones). It is important to pick a reasonable number of clones (>24) to generate accurate modification efficiencies.
Genotyping for microdeletion. Timing• 2-3 d hands on; 2-3 weeks expansion
[0756] 100| Cells were transfected as described above with a pair of sgRNAs targeting the region to be deleted.
[0757] 101| 24 h post-transfection, clonal lines are isolated by FACS or serial dilution as described above.
[0758] 102| Cells are expanded for 2-3 weeks.
[0759] 103| Applicants harvested DNA from clonal lines as described above using 10 ul QuickExtract solution and normalized genomic DNA with ddH2O to a final concentration of 50- 100 ng/ul.
[0760] 104| PCR Amplify the modified region. The PCR reaction is set up as follows:
Component: Amount (ul) Final concentration Herculase II PCR buffer, 5X 10 1X dNTP, 100mM (25mM each) 1 1 mM Out Fwd primer (10uM) 1 0.2 uM Out Rev primer (10uM) 1 0.2 uM Herculase II Fusion polymerase 1 MgCl2 (25mM) 2 1 mM ddH2O 32 Total 48 (for each reaction) [0761] Note: if deletion size is more than 1 kb, set up a parallel set of PCR reactions with In- Fwd and In-Rev primers to screen for the presence of the wt allele.
[0762] 105| To screen for inversions, a PCR reaction is set up as follows:
Component: Amount (ul) Final concentration Herculase II PCR buffer, 5X 10 1X
dNTP, 100mM (25mM each) 1 1 mM
Out Fwd or Out-Rev primer 1 0.2 uM
(10uM)
In Fwd or In-Rev primer (10uM) 1 0.2 uM
Herculase II Fusion polymerase 1
MgCl2 (25mM) 2 1 mM
ddH2O 32
Total 48 (for each reaction) [0763] Note: primers are paired either as Out-Fwd + In Fwd, or Out-Rev + In-Rev.
[0764] 106| Applicants added 100-200 ng of normalized genomic DNA template from step 103 for each reaction.
[0765] 107| PCR reaction was performed using the following cycling conditions:
Cycle number Denature Anneal Extend 1 95°C, 2 min
2-31 95°C, 20 s 60°C, 20 s 72°C, 30 s 32 72°C, 3 m [0766] 108| Applicants run 2-5 ul of PCR product on a 1 -2% gel to check for product. Although these PCR conditions are designed to work with most primers, some primers may need additional optimization by adjusting the template concentration, MgCl2 concentration, and/or the annealing temperature.
Genotyping for targeted modifications via HDR. Timing• 2-3 d, 2-3 h hands on
[0767] 109| Applicants harvested DNA as described above using QuickExtract solution and normalized genomic DNA with TE to a final concentration of 100-200 ng/ul.
[0768] 110| PCR Amplify the modified region. The PCR reaction is set up as follows:
Component: Amount (ul) Final concentration Herculase II PCR buffer, 5X 10 1X
dNTP, 100 mM (25 mM each) 1 1 mM
HDR Fwd primer (10 uM) 1 0.2 uM
HDR Rev primer (10 uM) 1 0.2 uM
Herculase II Fusion polymerase 1
MgCl2 (25mM) 2 1 mM
ddH2O 33
Total 49 (for each reaction) [0769] 111| Applicants added 100-200 ng of genomic DNA template from step 109 for each reaction and run the following program.
Cycle number Denature Anneal Extend
1 95°C, 2 min
2-31 95°C, 20 s 60°C, 20 s 72°C, 30-60 s per kb 32 72°C, 3 min [0770] 112| Applicants ran 5 ul of PCR product on a 0.8-1% gel to check for single-band product. Primers may need additional optimization by adjusting the template concentration, MgCl2 concentration, and/or the annealing temperature.
[0771] 113| Applicants purified the PCR reactions using the QIAQuick PCR purification kit.
[0772] 114| In the HDR example, a HindIII restriction site is inserted into the EMX1 gene. These are detected by a restriction digest of the PCR amplicon:
Component Amount (ul)
Purified PCR amplicon (200-300ng) x F.D. buffer, Green 1
HindIII 0.5
ddH2O x
Total 10 [0773] i. The DNA is digested for 10 m at 37°C:
[0774] ii. Applicants ran 10 ul of the digested product with loading dye on a 4-20% gradient polyacrylamide TBE gel until the xylene cyanol band had migrated to the bottom of the gel.
[0775] iii. Applicants stained the gel with 1X SYBR Gold dye while rocking for 15 m.
[0776] iv. The cleavage products are imaged and quantified as described above in the SURVEYOR assay section. HDR efficiency is estimated by the formula: (b + c)/(a + b + c), where a is the integrated intensity for the undigested HDR PCR product, and b and c are the integrated intensities for the HindIII-cut fragments.
[0777] 115| Alternatively, purified PCR amplicons from step 113 may be cloned and genotyped using Sanger sequencing or NGS.
Deep sequencing and off-target analysis• Timing 1 - 2 d
[0778] The online CRISPR target design tool generates candidate genomic off-target sites for each identified target site. Off-target analysis at these sites can be performed by SURVEYOR nuclease assay, Sanger sequencing, or next-generation deep sequencing. Given the likelihood of low or undetectable modification rates at many of these sites, Applicants recommend deep sequencing with the Illumina Miseq platform for high sensitivity and accuracy. Protocols will vary with sequencing platform; here, Applicants briefly describe a fusion PCR method for attaching sequencing adapters.
[0779] 116| Design deep sequencing primers. Next-generation sequencing (NGS) primers are designed for shorter amplicons, typically in the 100-200 bp size range. Primers may be manually designed using NCBI Primer-Blast or generated with online CRISPR target design tools (website at genome-engineering.org/tools).
[0780] 117| Harvest genomic DNA from Cas9-targeted cells. Normalize QuickExtract genomic DNA to 100-200 ng/ul with ddH2O.
[0781] 118| Initial library preparation PCR. Using the NGS primers from step 116, prepare the initial library preparation PCR
Component: Amount (ul) Final concentration Herculase II PCR buffer, 5X 10 1X dNTP, 100mM (25mM each) 1 1 mM NGS Fwd primer (10uM) 1 0.2 uM
NGS Rev primer (10uM) 1 0.2 uM
Herculase II Fusion polymerase 1
MgCl2 (25mM) 2 1 mM
ddH2O 33
Total 49 (for each reaction) [0782] 119| Add 100-200 ng of normalized genomic DNA template for each reaction.
[0783] 120| Perform PCR reaction using the following cycling conditions, for no more than 20 amplification cycles:
Cycle number Denature Anneal Extend 1 95°C, 2 min
2-21 95°C, 20 s 60°C, 20 s 72°C, 15 s 22 72°C, 3 min [0784] 121| Run 2-5 ul of PCR product on a 1% gel to check for single-band product. As with all genomic DNA PCRs, NGS primers may require additional optimization by adjusting the template concentration, MgCl2 concentration, and/or the annealing temperature.
[0785] 122| Purify the PCR reactions using the QIAQuick PCR purification kit and normalize eluant to 20 ng/ul. Pause point: Purified PCR product may be stored at -20°C.
[0786] 123| Nextera XT DNA Sample Preparation Kit. Following the manufacturer’s protocol, generate Miseq sequencing-ready libraries with unique barcodes for each sample.
[0787] 124| Analyze sequencing data. Off-target analysis may be performed through read alignment programs such as ClustalW, Geneious, or simple sequence analysis scripts.
Timing
[0788] Steps 1 – 2 Design and synthesis of sgRNA oligos and ssODNs: 1 -5 d, variable depending on supplier
[0789] Steps 3– 5 Construction of CRISPR plasmid or PCR expression cassette: 2 h to 3 d
[0790] Steps 6– 53 Transfection into cell lines: 3 d (1 h hands-on time)
[0791] Steps 54– 70 Optional derivation of clonal lines: 1-3 weeks, variable depending on cell type
[0792] Steps 71– 91 Functional validation of NHEJ via SURVEYOR: 5-6 h [0793] Steps 92– 124 Genotyping via Sanger or next-gen deep sequencing: 2-3 d (3-4 h hands on time)
Addressing Situations Concerning Herein Examples
Figure imgf000261_0001
Discussion
[0794] CRISPR-Cas may be easily multiplexed to facilitate simultaneous modification of several genes and mediate chromosomal microdeletions at high efficiencies. Applicants used two sgRNAs to demonstrate simultaneous targeting of the human GRIN2B and DYRK1A loci at efficiencies of up to 68% in HEK293FT cells. Likewise, a pair of sgRNAs may be used to mediate microdeletions, such as excision of an exon, which can be genotyped by PCR on a clonal level. Note that the precise location of exon junctions can vary. Applicants also demonstrated the use of ssODNs and targeting vector to mediate HDR with both wildtype and nickase mutant of Cas9 in HEK 293FT and HUES9 cells (Fig. 30). Note that Applicants have not been able to detect HDR in HUES9 cells using the Cas9 nickase, which may be due to low efficiency or a potential difference in repair activities in HUES9 cells. Although these values are typical, there is some variability in the cleavage efficiency of a given sgRNA, and on rare occasions certain sgRNAs may not work for reasons yet unknown. Applicants recommend designing two sgRNAs for each locus, and testing their efficiencies in the intended cell type. Example 24: NLSs
[0795] Cas9 Transcriptional Modulator: Applicants set out to turn the Cas9/gRNA CRISPR system into a generalized DNA binding system in which functions beyond DNA cleavage can be executed. For instance, by fusing functional domain(s) onto a catalytically inactive Cas9 Applicants have imparted novel functions, such as transcriptional activation/repression, methylation/demethylation, or chromatin modifications. To accomplish this goal Applicants made a catalytically inactive Cas9 mutant by changing two residues essential for nuclease activity, D10 and H840, to alanine. By mutating these two residues the nuclease activity of Cas9 is abolished while maintaining the ability to bind target DNA. The functional domains Applicants decided to focus on to test Applicants’ hypothesis are the transcriptional activator VP64 and the transcriptional repressors SID and KRAB.
[0796] Cas9 Nuclear localization: Applicants hypothesized that the most effective Cas9 transcriptional modulator would be strongly localized to the nucleus where it would have its greatest influence on transcription. Moreover, any residual Cas9 in the cytoplasm could have unwanted effects. Applicants determined that wild-type Cas9 does not localize into the nucleus without including multiple nuclear localization signals (NLSs) (although a CRISPR system need not have one or more NLSs but advantageously has at least one or more NLS(s)). Because multiple NLS sequences were required it was reasoned that it is difficult to get Cas9 into the nucleus and any additional domain that is fused to Cas9 could disrupt the nuclear localization. Therefore, Applicants made four Cas9-VP64-GFP fusion constructs with different NLS sequences (pXRP02- pLenti2-EF1a-NLS-hSpCsn1(10A,840A)-NLS-VP64-EGFP, pXRP04- pLenti2-EF1a-NLS-hSpCsn1(10A,840A)-NLS-VP64-2A-EGFP-NLS, pXRP06- pLenti2-EF1a- NLS-EGFP-VP64-NLS-hSpCsn1(10A,840A)-NLS, pXRP08- pLenti2-EF1a-NLS-VP64-NLS- hSpCsn1(10A,840A)-NLS-VP64-EGFP-NLS). These constructs were cloned into a lenti backbone under the expression of the human EF1a promoter. The WPRE element was also added for more robust protein expression. Each construct was transfected into HEK 293FT cells using Lipofectame 2000 and imaged 24 hours post-transfection. The best nuclear localization is obtained when the fusion proteins have NLS sequences on both the N- and C-term of the fusion protein. The highest observed nuclear localization occurred in the construct with four NLS elements.
[0797] To more robustly understand the influence of NLS elements on Cas9 Applicants made 16 Cas9-GFP fusions by adding the same alpha importin NLS sequence on either the N- or C-term looking at zero to three tandem repeats. Each construct was transfected into HEK 293FT cells using Lipofectame 2000 and imaged 24 hours post-transfection. Notably, the number of NLS elements does not directly correlate with the extent of nuclear localization. Adding an NLS on the C-term has a greater influence on nuclear localization than adding on the N-term.
[0798] Cas9 Transcriptional Activator: Applicants functionally tested the Cas9-VP64 protein by targeting the Sox2 locus and quantifying transcriptional activation by RT-qPCR. Eight DNA target sites were chosen to span the promoter of Sox2. Each construct was transfected into HEK 293FT cells using Lipofectame 2000 and 72 hours post-transfection total RNA was extracted from the cells. 1 ug of RNA was reverse transcribed into cDNA (qScript Supermix) in a 40 ul reaction. 2 ul of reaction product was added into a single 20 ul TaqMan assay qPCR reaction. Each experiment was performed in biological and technical triplicates. No RT control and no template control reactions showed no amplification. Constructs that do not show strong nuclear localization, pXRP02 and pXRP04, result in no activation. For the construct that did show strong nuclear localization, pXRP08, moderate activation was observed. Statistically significant activation was observed in the case of guide RNAs Sox2.4 and Sox2.5.
Example 25: In Vivo Mouse Data
Material and reagents
Herculase II fusion polymerase (Agilent Technologies, cat. no. 600679)
10x NEBuffer 4 (NEB, cat. No. B7004S)
BsaI HF (NEB, cat. No. R3535S)
T7 DNA ligase (Enzymatics, cat. no. L602L) Fast Digest buffer, 10X (ThermoScientific, cat. No. B64)
FastDigest NotI (ThermoScientific, cat. No. FD0594)
FastAP Alkaline Phosphatase (ThermoScientific, cat. No. EF0651)
Lipofectamine2000 (Life Technologies, cat. No. 11668-019)
Trypsin (Life Technologies, cat. No. 15400054)
Forceps #4 (Sigma, cat. No. Z168777-1EA)
Forceps #5 (Sigma, cat. No. F6521-1EA)
10x Hank’s Balanced Salt Solution (Sigma, cat. No. H4641-500ML)
Penicillin/Streptomycin solution (Life Technologies, cat. No. P4333)
Neurobasal (Life Technologies, cat. No. 21103049)
B27 Supplement (Life Technologies, cat. No. 17504044)
L-glutamine (Life Technologies, cat. No. 25030081)
Glutamate (Sigma, cat. No. RES5063G-A7)
ȕ-mercaptoethanol (Sigma, cat. No. M6250-100ML)
HA rabbit antibody (Cell Signaling, cat. No. 3724S)
LIVE/DEAD® Cell Imaging Kit (Life Technologies, cat. No. R37601)
30G World Precision Instrument syringe (World Precision Instruments, cat. No.
NANOFIL)
Stereotaxic apparatus (Kopf Instruments)
UltraMicroPump3 (World Precision Instruments, cat. No. UMP3-4)
Sucrose (Sigma, cat. No. S7903)
Calcium chloride (Sigma, cat. No. C1016)
Magnesium acetate (Sigma, cat. No. M0631)
Tris-HCl (Sigma, cat. no T5941)
EDTA (Sigma, cat. No. E6758)
NP-40 (Sigma, cat. No. NP40)
Phenylmethanesulfonyl fluoride (Sigma, cat. No. 78830)
Magnesium chloride (Sigma, cat. No. M8266)
Potassium chloride (Sigma, cat. No. P9333)
ȕ -glycerophosphate (Sigma, cat. No. G9422)
Glycerol (Sigma, cat. No. G9012)
Vybrant® DyeCycle™ Ruby Stain (Life technologies, cat. No. S4942)
FACS Aria Flu-act-cell sorter (Koch Institute of MIT, Cambridge US) DNAeasy Blood & Tissue Kit (Qiagen, cat. No. 69504)
Procedure
Constructing gRNA multiplexes for using in vivo in the brain
[0799] Applicants designed and PCR amplified single gRNAs targeting mouse TET and DNMT family members (as described herein) Targeting efficiency was assessed in N2a cell line (Fig. 33). To obtain simultaneous modification of several genes in vivo, efficient gRNA was multiplexed in AAV-packaging vector (Fig. 34). To facilitate further analysis of system efficiency applicants added to the system expression cassette consistent of GFP-KASH domain fusion protein under control of human Synapsin I promoter (Fig. 34). This modification allows for further analysis of system efficiency in neuronal population (more detail procedure in section Sorting nuclei and in vivo results).
[0800] All 4 parts of the system were PCR amplified using Herculase II Fusion polymerase using following primers:
1st U6 Fw: gagggtctcgtccttgcggccgcgctagcgagggcctatttcccatgattc
1st gRNA Rv: ctcggtctcggtAAAAAAgcaccgactcggtgccactttttcaagttgataacggactagc cttattttaacttgctaTTTCtagctctaaaacNNNNNNNNNNNNNNNNNNNNGGTGTTTC GTCCTTTCCAC
2nd U6 Fw:
gagggtctcTTTaccggtgagggcctatttcccatgattcc
2nd gRNA Rv: ctcggtctcctcAAAAAAgcaccgactcggtgccactttttcaagttgataacggacta gc cttattttaacttgctaTTTCtagctctaaaacNNNNNNNNNNNNNNNNNNNNGGTGTTTC GTCCTTTCCAC
3rd U6 Fw:
gagggtctcTTTgagctcgagggcctatttcccatgattc
3rd gRNA Rv: ctcggtctcgcgtAAAAAAgcaccgactcggtgccactttttcaagttgataacggact ag ccttattttaacttgctaTTTCtagctctaaaacNNNNNNNNNNNNNNNNNNNNGGTGTTT CGTCCTTTCCA
hSyn_GFP-kash Fw: gagggtctcTTacgcgtgtgtctagac hSyn_GFP-kash Rv: ctcggtctcAaggaCAGGGAAGGGAGCAGTGGTTCACGCCTGTAATCCCAGCAATTTGG GA GGCCAAGGTGGGTAGATCACCTGAGATTAGGAGTTGC
(NNNNNNNNNNNNNNNNNNNN is a reverse compliment targeted genomic sequence)
[0801] Applicants used Golden Gate strategy to assemble all parts (1:1 molecular ratio) of the system in a single step reaction:
1st U6_gRNA 18 ng
2nd U6_gRNA 18 ng
3rd U6_gRNA 18 ng
Syn_GFP-kash 100 ng
10x NEBuffer 4 1.0 l
10x BSA 1.0 l
10 mM ATP 1.0 l
BsaI HF 0.75 l
T7 ligase 0.25 l
ddH2O 10 l
Cycle number Condition
1-50 37°C for 5 m, 21°C for 5 m
[0802] Golden Gate reaction product was PCR amplified using Herculase II fusion polymerase and following primers:
Fw 5’ cctgtccttgcggccgcgctagcgagggcc
Rv 5’ cacgcggccgcaaggacagggaagggagcag
[0803] PCR product was cloned into AAV backbone, between ITR sequences using NotI restriction sites:
PCR product digestion:
Fast Digest buffer, 10X 3 l
FastDigest NotI 1 l
DNA 1 g
ddH2O up to 30 l AAV backbone digestion:
Fast Digest buffer, 10X 3 l
FastDigest NotI 1 l
FastAP Alkaline Phosphatase 1 l
AAV backbone 1 g
ddH2O up to 30 l
[0804] After 20 min incubation in 37°C samples were purified using QIAQuick PCR purification kit. Standardized samples were ligated at a 1:3 vector:insert ratio as follows:
Digested pUC19 50 ng
Digested insert 1:3 vector:insert molar ratio
T7 ligase 1 l
2X Rapid Ligation Buffer 5 l
ddH2O up to 10 l
[0805] After transformation of bacteria with ligation reaction product, applicants confirmed obtained clones with Sanger sequencing.
[0806] Positive DNA clones were tested in N2a cells after co-transfection with Cas9 construct (Figs. 35 and 36).
Design of new Cas9 constructs for AAV delivery
[0807] AAV delivery system despite its unique features has packing limitation – to successfully deliver expressing cassette in vivo it has to be in size < then 4.7 kb. To decrease the size of SpCas9 expressing cassette and facilitate delivery applicants tested several alteration: different promoters, shorter polyA signal and finally a smaller version of Cas9 from Staphylococcus aureus (SaCas9) (Figs. 37 and 38). All tested promoters were previously tested and published to be active in neurons, including mouse Mecp2 (Gray et al., 2011), rat Map1b and truncated rat Map1b (Liu and Fischer, 1996). Alternative synthetic polyA sequence was previously shown to be functional as well (Levitt et al., 1989; Gray et al., 2011). All cloned constructs were expressed in N2a cells after transfection with Lipofectamine 2000, and tested with Western blotting method (Fig. 39). Testing AAV multiplex system in primary neurons
[0808] To confirm functionality of developed system in neurons, Applicants use primary neuronal cultures in vitro. Mouse cortical neurons was prepared according to the protocol published previously by Banker and Goslin (Banker and Goslin, 1988).
[0809] Neuronal cells are obtained from embryonic day 16. Embryos are extracted from the euthanized pregnant female and decapitated, and the heads are placed in ice-cold HBSS. The brains are then extracted from the skulls with forceps (#4 and #5) and transferred to another change of ice-cold HBSS. Further steps are performed with the aid of a stereoscopic microscope in a Petri dish filled with ice-cold HBSS and #5 forceps. The hemispheres are separated from each other and the brainstem and cleared of meninges. The hippocampi are then very carefully dissected and placed in a 15 ml conical tube filled with ice-cold HBSS. Cortices that remain after hippocampal dissection can be used for further cell isolation using an analogous protocol after removing the brain steam residuals and olfactory bulbs. Isolated hippocampi are washed three times with 10 ml ice-cold HBSS and dissociated by 15 min incubation with trypsin in HBSS (4 ml HBSS with the addition of 10 l 2.5% trypsin per hippocampus) at 37°C. After trypsinization, the hippocampi are very carefully washed three times to remove any traces of trypsin with HBSS preheated to 37°C and dissociated in warm HBSS. Applicants usually dissociate cells obtained from 10-12 embryos in 1 ml HBSS using 1 ml pipette tips and dilute dissociated cells up to 4 ml. Cells are plated at a density of 250 cells/mm2 and cultured at 37°C and 5% CO2 for up to 3 week.
HBSS
435 ml H2O
50 ml 10x Hank’s Balanced Salt Solution
16.5 ml 0.3M HEPES pH 7.3
5 ml penicillin-streptomycin solution
Filter (0.2 m) and store 4°C
Neuron Plating Medium (100 ml)
97 ml Neurobasal
2 ml B27 Supplement
1 ml penicillin-streptomycin solution
250 l glutamine
125 l glutamate [0810] Neurons are transduced with concentrated AAV1/2 virus or AAV1 virus from filtered medium of HEK293FT cells, between 4-7 days in culture and keep for at least one week in culture after transduction to allow for delivered gene expression.
AAV-driven expression of the system
[0811] Applicants confirmed expression of SpCas9 and SaCas9 in neuronal cultures after AAV delivery using Western blot method (Fig. 42). One week after transduction neurons were collected in NuPage SDS loading buffer with ȕ-mercaptoethanol to denaturate proteins in 95°C for 5 min. Samples were separated on SDS PAGE gel and transferred on PVDF membrane for WB protein detection. Cas9 proteins were detected with HA antibody.
[0812] Expression of Syn-GFP-kash from gRNA multiplex AAV was confirmed with fluorescent microscopy (Fig. 50).
Toxicity
[0813] To assess the toxicity of AAV with CRISPR system Applicants tested overall morphology of neurons one week after virus transduction (Fig. 45). Additionally, Applicants tested potential toxicity of designed system with the LIVE/DEAD® Cell Imaging Kit, which allows to distinguish live and dead cells in culture. It is based on the presence of intracellular esterase activity (as determined by the enzymatic conversion of the non-fluorescent calcein AM to the intensely green fluorescent calcein). On the other hand, the red, cell-impermeant component of the Kit enters cells with damaged membranes only and bind to DNA generating fluorescence in dead cells. Both flourophores can be easily visualized in living cells with fluorescent microscopy. AAV-driven expression of Cas9 proteins and multiplex gRNA constructs in the primary cortical neurons was well tolerated and not toxic (Figs. 43 and 44), what indicates that designed AAV system is suitable for in vivo tests.
Virus production
[0814] Concentrated virus was produced according to the methods described in McClure et al., 2011. Supernatant virus production occurred in HEK293FT cells.
Brain surgeries
[0815] For viral vector injections 10-15 week old male C57BL/6N mice were anesthetized with a Ketamine/Xylazine cocktail (Ketamine dose of 100 mg/kg and Xylazine dose of 10 mg/kg) by intraperitoneal injection. Intraperitonial administration of Buprenex was used as a pre- emptive analgesic (1 mg/kg). Animals were immobilized in a Kopf stereotaxic apparatus using intra-aural positioning studs and tooth bar to maintain an immobile skull. Using a hand-held drill, a hole (1-2mm) at -3.0 mm posterior to Bregma and 3.5 mm lateral for injection in the CA1 region of the hippocampus was made. Using 30G World Precision Instrument syringe at a depth of 2.5 mm, the solution of AAV viral particles in a total volume of 1 ul was injected. The injection was monitored by a‘World Precision Instruments UltraMicroPump3’ injection pump at a flow rate of 0.5 ul/min to prevent tissue damage. When the injection was complete, the injection needle was removed slowly, at a rate of 0.5 mm/min. After injection, the skin was sealed with 6-0 Ethilon sutures. Animals were postoperatively hydrated with 1 mL lactated Ringer’s (subcutaneous) and housed in a temperature controlled (37°C) environment until achieving an ambulatory recovery. 3 weeks after surgery animals were euthanized by deep anesthesia followed by tissue removal for nuclei sorting or with 4% paraformaldehyde perfusion for immunochemistry.
Sorting nuclei and in vivo results
[0816] Applicants designed a method to specifically genetically tag the gRNA targeted neuronal cell nuclei with GFP for Fluorescent Activated Cell Sorting (FACS) of the labeled cell nuclei and downstream processing of DNA, RNA and nuclear proteins. To that purpose the applicants’ multiplex targeting vector was designed to express both a fusion protein between GFP and the mouse nuclear membrane protein domain KASH (Starr DA, 2011, Current biology) and the 3 gRNAs to target specific gene loci of interest (Fig. 34). GFP-KASH was expressed under the control of the human Synapsin promoter to specifically label neurons. The amino acid of the fusion protein GFP-KASH was:
MVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKFICT TGKLPVPWPTLVTTLTYGVQCFSRYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGN YKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYIMADKQKNGIK VNFKIRHNIEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSALSKDPNEKRDHMV LLEFVTAAGITLGMDELYKSGLRSREEEEETDSRMPHLDSPGSSQPRRSFLSRVIRAAL PLQLLLLLLLLLACLLPASEDDYSCTQANNFARSFYPMLRYTNGPPPT
[0817] One week after AAV1/2 mediated delivery into the brain a robust expression of GFP- KASH was observed. For FACS and downstream processing of labeled nuclei, the hippocampi were dissected 3 weeks after surgery and processed for cell nuclei purification using a gradient centrifugation step. For that purpose the tissue was homogenized in 320 mM Sucrose, 5 mM CaCl, 3 mM Mg(Ac)2, 10 mM Tris pH 7.8, 0.1 mM EDTA, 0.1% NP40, 0.1 mM Phenylmethanesulfonyl fluoride (PMSF), 1 mM ȕ-mercaptoethanol using 2ml Dounce homogenizer (Sigma) The homogenisate was centrifuged on a 25% to 29% Optiprep® gradient according to the manufacture’s protocol for 30 min at 3.500 rpm at 4 °C. The nuclear pellet was resuspended in 340 mM Sucrose, 2 mM MgCl2, 25 mM KCl, 65 mM glycerophosphate, 5% glycerol, 0.1 mM PMSF, 1 mM ȕ-mercaptoethanol and Vybrant® DyeCycle™ Ruby Stain (Life technologies) was added to label cell nuclei (offers near-infrared emission for DNA). The labeled and purified nuclei were sorted by FACS using an Aria Flu-act-cell sorter and BDFACS Diva software. The sorted GFP+ and GFP- nuclei were finally used to purify genomic DNA using DNAeasy Blood & Tissue Kit (Qiagen) for Surveyor assay analysis of the targeted genomic regions. The same approach can be easily used to purify nuclear RNA or protein from targeted cells for downstream processing. Due to the 2-vector system (Fig. 34) the applicants using in this approach efficient Cas9 mediated DNA cleavage was expected to occur only in a small subset of cells in the brain (cells which were co-infected with both the multiplex targeting vector and the Cas9 encoding vector). The method described here enables the applicants to specifically purify DNA, RNA and nuclear proteins from the cell population expressing the 3 gRNAs of interest and therefore are supposed to undergo Cas9 mediated DNA cleavage. By using this method the applicants were able to visualize efficient DNA cleavage in vivo occurring only in a small subset of cells.
[0818] Essentially, what Applicants have shown here is targeted in vivo cleavage. Furthermore, Applicants used a multiple approach, with several different sequences targeted at the same time, but independently. Presented system can be applied for studying brain pathologic conditions (gene knock out, e.g. Parkinson disease) and also open a field for further development of genome editing tools in the brain. By replacing nuclease activity with gene transcription regulators or epigenetic regulators it will be possible to answer whole spectrum of scientific question about role of gene regulation and epigenetic changes in the brain in not only in the pathologic conditions but also in physiological process as learning and memory formation. Finally, presented technology can be applied in more complex mammalian system as primates, what allows to overcome current technology limitations.
Example 26: AAV production system or protocol
[0819] An AAV production system or protocol that was developed for, and works particularly well with, high through put screening uses is provided herein, but it has broader applicability in the present invention as well. Manipulating endogenous gene expression presents various challenges, as the rate of expression depends on many factors, including regulatory elements, mRNA processing, and transcript stability. To overcome this challenge, Applicants developed an adeno-associated virus (AAV)-based vector for the delivery. AAV has an ssDNA-based genome and is therefore less susceptible to recombination.
[0820] AAV1/2 (serotype AAV1/2, i.e., hybrid or mosaic AAV1 / AAV2 capsid AAV) heparin purified concentrated virus protocol
Media: D10 + HEPES
500ml bottle DMEM high glucose + Glutamax (GIBCO)
50ml Hyclone FBS (heat-inactivated) (Thermo Fischer)
5.5ml HEPES solution (1M, GIBCO)
Cells: low passage HEK293FT (passage <10 at time of virus production, thaw new cells of passage 2-4 for virus production, grow up for 3-5 passages)
Transfection reagent: Polyethylenimine (PEI)“Max”
Dissolve 50mg PEI“Max” in 50ml sterile Ultrapure H20
Adjust pH to 7.1
Filter with 0.22um fliptop filter
Seal tube and wrap with parafilm
Freeze aliquots at -20ºC (for storage, can also be used immediately)
Cell Culture
Culture low passage HEK293FT in D10 + HEPES
Passage everyday between 1:2 and 1:2.5
Advantageously do not allow cells to reach more than 85% confluency
For T75
- Warm 10ml HBSS (-Mg2+, -Ca2+, GIBCO) + 1ml TrypLE Express (GIBCO) per flask to 37ºC (Waterbath)
Aspirate media fully
- Add 10ml warm HBSS gently (to wash out media completely)
- Add 1ml TrypLE per Flask
- Place flask in incubator (37ºC) for 1min
- Rock flask to detach cells
- Add 9ml D10 + HEPES media (37ºC)
- Pipette up and down 5 times to generate single cell suspension
- Split at 1:2– 1:2.5 (12ml media for T75) ratio (if cells are growing more slowly, discard and thaw a new batch, they are not in optimal growth) - transfer to T225 as soon as enough cells are present (for ease of handling large amounts of cells)
[0821] AAV production (5*15cm dish scale per construct):
Plate 10 million cells in 21.5 ml media into a 15cm dish
Incubate for 18-22 hours at 37ºC
Transfection is ideal at 80% confluence
[0822] Per plate
Prewarm 22ml media (D10 + HEPES)
[0823] Prepare tube with DNA mixture (use endofree maxiprep DNA):
5.2 ug vector of interest plasmid
4.35 ug AAV 1 serotype plasmid
4.35 ug AAV 2 serotype plasmid Add 434 uL DMEM (no serum!)
Add 130 ul PEI solution
Vortex 5-10 seconds
Add DNA/DMEM/PEI mixture to prewarmed media
Vortex briefly to mix
Replace media in 15cm dish with DNA/DMEM/PEI mixture
Return to 37ºC incubator
Incubate 48h before harvesting (make sure medium isn’t turning too acidic)
[0824] Virus harvest:
1. aspirate media carefully from 15cm dish dishes (advantageously do not dislodge cells)
2. Add 25 ml RT DPBS (Invitrogen) to each plate and gently remove cells with a cell scraper. Collect suspension in 50 ml tubes.
3. Pellet cells at 800x g for 10 minutes.
4. Discard supernatant
[0825] Pause point: freeze cell pellet at -80C if desired
5. resuspend pellet in 150 mM NaCl, 20 mM Tris pH 8.0, use 10 ml per tissue culture plate. 6. Prepare a fresh solution of 10% sodium deoxycholate in dH2O. Add 1.25 ml of this per tissue culture plate for a final concentration of 0.5%. Add benzonase nuclease to a final concentration of 50 units per ml. Mix tube thoroughly. 7. Incubate at 37°C for 1 hour (Waterbath).
8. Remove cellular debris by centrifuging at 3000 x g for 15 mins. Transfer to fresh 50 ml tube and ensure all cell debris has been removed to prevent blocking of heparin columns.
[0826] Heparin column purification of AAV1/2:
1. Set up HiTrap heparin columns using a peristaltic pump so that solutions flow through the column at 1 ml per minute. It is important to ensure no air bubbles are introduced into the heparin column.
2. Equilibrate the column with 10 ml 150 mM NaCl, 20 mM Tris, pH 8.0 using the peristaltic pump.
3. Binding of virus: Apply 50 ml virus solution to column and allow to flow through. 4. Wash step 1: column with 20 ml 100 mM NaCl, 20 mM Tris, pH 8.0. (using the peristaltic pump)
5. Wash step 2: Using a 3 ml or 5 ml syringe continue to wash the column with 1 ml 200 mM NaCl, 20 mM Tris, pH 8.0, followed by 1 ml 300 mM NaCl, 20 mM Tris, pH 8.0.
Discard the flow-through.
(prepare the syringes with different buffers during the 50min flow through of virus solution above)
6. Elution Using 5 ml syringes and gentle pressure (flow rate of < 1ml/min) elute the virus from the column by applying:
1.5 ml 400 mM NaCl, 20 mM Tris, pH 8.0
3.0 ml 450 mM NaCl, 20 mM Tris, pH 8.0
1.5 ml 500 mM NaCl, 20 mM Tris, pH 8.0
Collect these in a 15 ml centrifuge tube.
[0827] Concentration of AAV1/2:
1. Concentration step 1: Concentrate the eluted virus using Amicon ultra 15ml centrifugal filter units with a 100,000 molecular weight cutoff. Load column eluate into the concentrator and centrifuge at 2000x g for 2 minutes (at room temperature. Check concentrated volume– it should be approximately 500 l. If necessary, centrifuge in 1min intervals until correct volume is reached. 2. buffer exchange: Add 1ml sterile DPBS to filter unit, centrifuge in 1min intervals until correct volume (500ul) is reached.
3. Concentration step 2: Add 500ul concentrate to an Amicon Ultra 0.5ml 100K filter unit. Centrifuge at 6000g for 2min. Check concentrated volume– it should be approximately 100 l. If necessary, centrifuge in 1min intervals until correct volume is reached.
4. Recovery: Invert filter insert and insert into fresh collection tube. Centrifuge at 1000g for 2min.
Aliquot and freeze at -80ºC
1ul is typically required per injection site, small aliquots (e.g. 5ul) are therefore recommended (avoid freeze-thaw of virus).
determine DNaseI-resistant GC particle titer using qPCR (see separate protocol)
[0828] Materials
Amicon Ultra, 0.5ml, 100K; MILLIPORE; UFC510024
Amicon Ultra, 15ml, 100K; MILLIPORE; UFC910024
Benzonase nuclease; Sigma-Aldrich, E1014
HiTrap Heparin cartridge; Sigma-Aldrich; 54836
Sodium deoxycholate; Sigma-Aldrich; D5670
[0829] AAV1 supernatant production protocol
Media: D10 + HEPES
500ml bottle DMEM high glucose + Glutamax (Invitrogen)
50ml Hyclone FBS (heat-inactivated) (Thermo Fischer)
5.5ml HEPES solution (1M, GIBCO)
Cells: low passage HEK293FT (passage <10 at time of virus production) Thaw new cells of passage 2-4 for virus production, grow up for 2-5 passages Transfection reagent: Polyethylenimine (PEI)“Max”
Dissolve 50mg PEI“Max” in 50ml sterile Ultrapure H20
Adjust pH to 7.1
Filter with 0.22um fliptop filter
Seal tube and wrap with parafilm
Freeze aliquots at -20ºC (for storage, can also be used immediately) Cell Culture
Culture low passage HEK293FT in D10 + HEPES Passage everyday between 1:2 and 1:2.5
Advantageously do let cells reach more than 85% confluency
For T75
- Warm 10ml HBSS (-Mg2+, -Ca2+, GIBCO) + 1ml TrypLE Express (GIBCO) per flask to 37ºC (Waterbath)
- Aspirate media fully
- Add 10ml warm HBSS gently (to wash out media completely)
- Add 1ml TrypLE per Flask
- Place flask in incubator (37ºC) for 1min
- Rock flask to detach cells
- Add 9ml D10 + HEPES media (37ºC)
- Pipette up and down 5 times to generate single cell suspension
- Split at 1:2– 1:2.5 (12ml media for T75) ratio (if cells are growing more slowly, discard and thaw a new batch, they are not in optimal growth)
- transfer to T225 as soon as enough cells are present (for ease of handling large amounts of cells)
AAV production (single 15cm dish scale)
Plate 10 million cells in 21.5 ml media into a 15cm dish
Incubate for 18-22 hours at 37ºC
Transfection is ideal at 80% confluence per plate
Prewarm 22ml media (D10 + HEPES)
Prepare tube with DNA mixture (use endofree maxiprep DNA):
5.2 ug vector of interest plasmid
8.7 ug AAV 1 serotype plasmid
10.4 ug DF6 plasmid (adenovirus helper genes)
Vortex to mix
Add 434 uL DMEM (no serum!) Add 130 ul PEI solution
Vortex 5-10 seconds
Add DNA/DMEM/PEI mixture to prewarmed media
Vortex briefly to mix
Replace media in 15cm dish with DNA/DMEM/PEI mixture Return to 37ºC incubator
Incubate 48h before harvesting (advantageously monitor to ensure medium is not turning too acidic)
[0830] Virus harvest:
Remove supernatant from 15cm dish
Filter with 0.45 um filter (low protein binding) Aliquot and freeze at -80ºC Transduction (primary neuron cultures in 24-well format, 5DIV)
Replace complete neurobasal media in each well of neurons to be transduced with fresh neurobasal (usually 400ul out of 500ul per well is replaced)
Thaw AAV supernatant in 37ºC waterbath
Let equilibrate in incubator for 30min
Add 250 ul AAV supernatant to each well
Incubate 24h at 37ºC
Remove media/supernatant and replace with fresh complete neurobasal
Expression starts to be visible after 48h, saturates around 6-7 Days Post Infection Constructs for pAAV plasmid with GOI should not exceed 4.8kb including both ITRS.
[0831] Example of a human codon optimized sequence (i.e. being optimized for expression in humans) sequence: SaCas9 is provided below:
ACCGGTGCCACCATGTACCCATACGATGTTCCAGATTACGC TTCGCCGAAGAAAAAGCGCAAGGTCGAAGCGTCCATGAAAAGGAACTACATTCT GGGGCTGGACATCGGGATTACAAGCGTGGGGTATGGGATTATTGACTATGAAAC AAGGGACGTGATCGACGCAGGCGTCAGACTGTTCAAGGAGGCCAACGTGGAAAA CAATGAGGGACGGAGAAGCAAGAGGGGAGCCAGGCGCCTGAAACGACGGAGAA GGCACAGAATCCAGAGGGTGAAGAAACTGCTGTTCGATTACAACCTGCTGACCG ACCATTCTGAGCTGAGTGGAATTAATCCTTATGAAGCCAGGGTGAAAGGCCTGA GTCAGAAGCTGTCAGAGGAAGAGTTTTCCGCAGCTCTGCTGCACCTGGCTAAGC GCCGAGGAGTGCATAACGTCAATGAGGTGGAAGAGGACACCGGCAACGAGCTG TCTACAAAGGAACAGATCTCACGCAATAGCAAAGCTCTGGAAGAGAAGTATGTC GCAGAGCTGCAGCTGGAACGGCTGAAGAAAGATGGCGAGGTGAGAGGGTCAATT AATAGGTTCAAGACAAGCGACTACGTCAAAGAAGCCAAGCAGCTGCTGAAAGTG CAGAAGGCTTACCACCAGCTGGATCAGAGCTTCATCGATACTTATATCGACCTGC TGGAGACTCGGAGAACCTACTATGAGGGACCAGGAGAAGGGAGCCCCTTCGGAT GGAAAGACATCAAGGAATGGTACGAGATGCTGATGGGACATTGCACCTATTTTCC AGAAGAGCTGAGAAGCGTCAAGTACGCTTATAACGCAGATCTGTACAACGCCCT GAATGACCTGAACAACCTGGTCATCACCAGGGATGAAAACGAGAAACTGGAATA CTATGAGAAGTTCCAGATCATCGAAAACGTGTTTAAGCAGAAGAAAAAGCCTAC ACTGAAACAGATTGCTAAGGAGATCCTGGTCAACGAAGAGGACATCAAGGGCTA CCGGGTGACAAGCACTGGAAAACCAGAGTTCACCAATCTGAAAGTGTATCACGA TATTAAGGACATCACAGCACGGAAAGAAATCATTGAGAACGCCGAACTGCTGGA TCAGATTGCTAAGATCCTGACTATCTACCAGAGCTCCGAGGACATCCAGGAAGAG CTGACTAACCTGAACAGCGAGCTGACCCAGGAAGAGATCGAACAGATTAGTAAT CTGAAGGGGTACACCGGAACACACAACCTGTCCCTGAAAGCTATCAATCTGATTC TGGATGAGCTGTGGCATACAAACGACAATCAGATTGCAATCTTTAACCGGCTGAA GCTGGTCCCAAAAAAGGTGGACCTGAGTCAGCAGAAAGAGATCCCAACCACACT GGTGGACGATTTCATTCTGTCACCCGTGGTCAAGCGGAGCTTCATCCAGAGCATCAA AGTGATCAACGCCATCATCAAGAAGTACGGCCTGCCCAATGATATCATTATCGAGCT GGCTAGGGAGAAGAACAGCAAGGACGCACAGAAGATGATCAATGAGATGCAGAAA CGAAACCGGCAGACCAATGAACGCATTGAAGAGATTATCCGAACTACCGGGAAAGA GAACGCAAAGTACCTGATTGAAAAAATCAAGCTGCACGATATGCAGGAGGGAAAGT GTCTGTATTCTCTGGAGGCCATCCCCCTGGAGGACCTGCTGAACAATCCATTCAACT ACGAGGTCGATCATATTATCCCCAGAAGCGTGTCCTTCGACAATTCCTTTAACAACA AGGTGCTGGTCAAGCAGGAAGAGAACTCTAAAAAGGGCAATAGGACTCCTTTCCAG TACCTGTCTAGTTCAGATTCCAAGATCTCTTACGAAACCTTTAAAAAGCACATTCTG AATCTGGCCAAAGGAAAGGGCCGCATCAGCAAGACCAAAAAGGAGTACCTGCTGG AAGAGCGGGACATCAACAGATTCTCCGTCCAGAAGGATTTTATTAACCGGAATCTG GTGGACACAAGATACGCTACTCGCGGCCTGATGAATCTGCTGCGATCCTATTTCCGG GTGAACAATCTGGATGTGAAAGTCAAGTCCATCAACGGCGGGTTCACATCTTTTCTG AGGCGCAAATGGAAGTTTAAAAAGGAGCGCAACAAAGGGTACAAGCACCATGCCG AAGATGCTCTGATTATCGCAAATGCCGACTTCATCTTTAAGGAGTGGAAAAAGCTGG ACAAAGCCAAGAAAGTGATGGAGAACCAGATGTTCGAAGAGAAGCAGGCCGAATC TATGCCCGAAATCGAGACAGAACAGGAGTACAAGGAGATTTTCATCACTCCTCACC AGATCAAGCATATCAAGGATTTCAAGGACTACAAGTACTCTCACCGGGTGGATAAA AAGCCCAACAGAGAGCTGATCAATGACACCCTGTATAGTACAAGAAAAGACGATAA GGGGAATACCCTGATTGTGAACAATCTGAACGGACTGTACGACAAAGATAATGACA AGCTGAAAAAGCTGATCAACAAAAGTCCCGAGAAGCTGCTGATGTACCACCATGAT CCTCAGACATATCAGAAACTGAAGCTGATTATGGAGCAGTACGGCGACGAGAAGAA CCCACTGTATAAGTACTATGAAGAGACTGGGAACTACCTGACCAAGTATAGCAAAA AGGATAATGGCCCCGTGATCAAGAAGATCAAGTACTATGGGAACAAGCTGAATGCC CATCTGGACATCACAGACGATTACCCTAACAGTCGCAACAAGGTGGTCAAGCTGTC ACTGAAGCCATACAGATTCGATGTCTATCTGGACAACGGCGTGTATAAATTTGTGAC TGTCAAGAATCTGGATGTCATCAAAAAGGAGAACTACTATGAAGTGAATAGCAAGT GCTACGAAGAGGCTAAAAAGCTGAAAAAGATTAGCAACCAGGCAGAGTTCATCGCC TCCTTTTACAACAACGACCTGATTAAGATCAATGGCGAACTGTATAGGGTCATCGGG GTGAACAATGATCTGCTGAACCGCATTGAAGTGAATATGATTGACATCACTTACCGA GAGTATCTGGAAAACATGAATGATAAGCGCCCCCCTCGAATTATCAAAACAATTGC CTCTAAGACTCAGAGTATCAAAAAGTACTCAACCGACATTCTGGGAAACCTGTATG AGGTGAAGAGCAAAAAGCACCCTCAGATTATCAAAAAGGGCTAAGAATTC
Example 27: Minimizing off-target cleavage using Cas9 nickase and two guide RNAs
[0832] Cas9 is a RNA-guided DNA nuclease that may be targeted to specific locations in the genome with the help of a 20bp RNA guide. However the guide sequence may tolerate some mismatches between the guide sequence and the DNA-target sequence. The flexibility is undesirable due to the potential for off-target cleavage, when the guide RNA targets Cas9 to a an off-target sequence that has a few bases different from the guide sequence. For all experimental applications (gene targeting, crop engineering, therapeutic applications, etc) it is important to be able to improve the specificity of Cas9 mediated gene targeting and reduce the likelihood of off- target modification by Cas9.
[0833] Applicants developed a method of using a Cas9 nickase mutant in combination with two guide RNAs to facilitate targeted double strand breaks in the genome without off-target modifications. The Cas9 nickase mutant may be generated from a Cas9 nuclease by disabling its cleavage activity so that instead of both strands of the DNA duplex being cleaved only one strand is cleaved. The Cas9 nickase may be generated by inducing mutations in one ore more domains of the Cas9 nuclease, e.g. Ruvc1 or HNH. These mutations may include but are not limited to mutations in a Cas9 catalytic domain, e.g in SpCas9 these mutations may be at positions D10 or H840. These mutations may include but are not limited to D10A, E762A, H840A, N854A, N863A or D986A in SpCas9 but nickases may be generated by inducing mutations at corresponding positions in other CRISPR enzymes or Cas9 orthologs. In a most preferred embodiment of the invention the Cas9 nickase mutant is a SpCas9 nickase with a D10A mutation.
[0834] The way this works is that each guide RNA in combination with Cas9 nickase would induce the targeted single strand break of a duplex DNA target. Since each guide RNA nicks one strand, the net result is a double strand break. The reason this method eliminates off-target mutations is because it is very unlikely to have an off-target site that has high degrees of similarity for both guide sequences (20bp+2bp(PAM) = 22bp specificity for each guide, and two guides means any off-target site will have to have close to 44bp of homologous sequence). Although it is still likely that individual guides may have off-targets, but those off-targets will only be nicked, which is unlikely to be repaired by the mutagenic NHEJ process. Therefore the multiplexing of DNA double strand nicking provides a powerful way of introducing targeted DNA double strand breaks without off-target mutagenic effects.
[0835] Applicants carried out experiments involving the co-transfection of HEK293FT cells with a plasmid encoding Cas9(D10A) nickase as well as DNA expression cassettes for one or more guides. Applicants transfected cells using Lipofectamine 2000, and transfected cells were harvested 48 or 72 hours after transfections. Double nicking-induced NHEJ were detected using the SURVEYOR nuclease assay as described previously herein (Figs. 51, 52 and 53).
[0836] Applicants have further identified parameters that relate to efficient cleavage by the Cas9 nickase mutant when combined with two guide RNAs and these parameters include but are not limited to the length of the 5’ overhang. Efficient cleavage is reported for 5’ overhang of at least 26 base pairs. In a preferred embodiment of the invention, the 5’ overhang is at least 30 base pairs and more preferably at least 34 base pairs. Overhangs of up to 200 base pairs may be acceptable for cleavage, while 5’ overhangs less than 100 base pairs are preferred and 5’ overhangs less than 50 base pairs are most preferred (Figs. 54 and 55).
[0837] Example 28: CRISPR HBV
[0838] A CRISPR system has been designed for targeting the hepatitis B virus, as described herein and illustrated in Figures 36 to 72; and, this system has been demonstrated to be therapeutic.
[0839] The Figures associated with this Example are Figures 36 to 72, with Figures 36 and 57 demonstrating guide design. Figure 38 shows qPCR results from 1st round of HepG2.2.15 experiments. Figure 39 shows a Surveyor assay for nuclease activity. Indels form as the result of imperfect NHEJ events due to repeated DSB formation from nuclease activity. For Cas9 targeted genomic loci indel formation at rates 10-30% are often observed and can approach 50%. Figure 40 shows representative surveyor for 1st set of HepG2.2.15 experiments. Figure 41 shows HepG2.2.15 HBV quantification scheme, an experimental design motivated by noise of initial data sets. Figure 42 shows HepG2.2.15 results using sorting based normalization. Figure 43 shows low levels of indels observed with guides targeting conserved HBV sequences in 2nd round of HepG2.2.15 experiments. Figure 44 shows HepG2 co-transfection experiments. Figure 45 shows HDD data for Cohort 1. Figure 46 shows HDD data for Cohort 2. Figure 47 shows HDD data for Cohort 2. Figure 48 shows HDD data for Cohort 2. Figure 49 shows Cohort 2-liver analysis 9d post HDD. Figure 50 shows Cohort 2-liver analysis 9d post HDD. Figure 51 shows low/no indels formed during HDD experiments. Predicted band sizes for guide 21 formation: 235 + 272 + 507 bp (undigested PCR product). Figure 52 shows HDD Cohort 3 Results: HBsAg. Figure 53 shows HDD Cohort 3 Results: Viremia. Figure 54 shows HDD Cohort 3 Results: HBV in Liver. Figure 55 shows HDD Cohort 3 Results: Luciferase normalized to GAPDH. Figure 56 shows despite low/no indel formation, effects on HBV are dependent on Cas9 nuclease activity. Figure 57 shows schematic of HBV life cycle and putative anti-HBV effect of CRISPR constructs as well as HBV genome organization and location of target sequences for several tested guide RNA constructs. Figure 58 shows guide RNAs targeting conserved regions target large majority of patient-derived virus genomes, a schematic and results for HepG2 transfection experiment. Figure 59 shows Experimental schematic and results for hydrodynamic injection experiments. Figure 60 shows the HBV life cycle within HepG2.2.15 cells and schematic of lentiviral vector and experimental strategy for sustained CRISPR expression within HepG2.2.15 cells. Figure 61 shows that HBV-targeting CRISPR reduces HBV DNA and cccDNA dependent upon HBV-specific guide RNA and Cas9 activity. Figure 62 shows HBV products are reduced upon long-term CRISPR/Cas expression. Figure 63 shows that CRISPR constructs targeting HBV cause large and progressive reduction in cccDNA and total HBV DNA levels dependent on successful targeting of viral DNA. Figure 64 shows HBV DNA and cccDNA reductions upon long-term CRISPR/Cas expression are produced with multiple guides. Figure 65 shows Southern blot of HBV DNA. Figure 66 shows Southern blot of HBV DNA. Figure 67 shows Surveyor assay to detect indel formation in total HBV DNA and episomal HBV DNA. Figure 68 demonstrates large reduction in HBV Core protein staining upon targeting by g17 specifically against the Core ORF as determined by immunofluorescence. Figure 69 shows schematics for de novo infection experiments. Figure 70 shows HBsAg secretion, cccDNA copies, levels of HBV 3.5kb RNA relative to 5 bp mismatch control and titer of HBV DNA in culture medium in de novo infection context. Figure 71 shows CRISPR/Cas-mediated disruption of HBV in patient-derived virus model system. Figure 72 shows Surveyor assay results.
[0840] Construction of CRISPR Constructs. Cas9 constructs with 24 guide RNAs targeting sequences present in the HBV genome integrated into the HepG2.2.15 cell line were used for thes experiments (Sells et. al., PNAS 1987). Oligos corresponding to target sequences were ligated into BbsI (ThermoScientific #FD1014) digested plasmid PX330a or BsmBI (ThermoScientific #FD0454) digested plasmid PHBC013 using T7 ligase (Enzymatics L6020L) (Hsu et. al.,“DNA targeting specificity of RNA-guided Cas9 nucleases,” Nature Biotechnology 31,827–832(2013) (Hsu et al Nat Biotechnol 2013)). PX330a contains a U6 promoter driving expression of the +85 guide RNA described in Hsu et. al. (Nat Biotechnol 2013) and a mammalian codon optimized NLS-Cas9-NLS from S. pyogenes SF370 driven by the CBh promoter. PHBC013 contains the U6 guide RNA expression system from PX330a and NLS- Cas9-P2A-mCherry driven by the EFS promoter. PHBC013 was created from pHKO_015 by digesting pHKO_015 with NheI (ThermoScientific #FD0974) and MluI (ThermoScientific #FD0564) and then inserting mCherry using Gibson Assembly with the Gibson Assembly Master Mix (NEB, #E2611L). The red fluorescent mCherry marker makes it easy to see which cells have been transfected with the vector. See Figure 57B for an illustration of the constructs. Two sets of control constructs were generated. Mismatched guide RNA control constructs for promising guide RNA molecules were created by ligating in oligos to PX330a or PHBC013 that contained 5 basepair mismatches at the 3’ end of the spacer, but were otherwise identical to constructs designed to target HBV. Cas9 D10A/H840A nuclease dead control constructs were generated by digesting pHBC013 guide RNA containing constructs with BamHI and NheI and then inserting a PCR amplified D10A/H840A Cas9 using Gibson Assembly. D10A and H840A are mutations that are sufficient to abolish the nuclease activity of S. pyogenes SF370 Cas9 (Cong et. al.“Multiplex genome engineering using CRISPR/Cas systems,” Science 339, 819– 823, Sapranauskas et. al. “The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli,” Nucleic Acids Res Nov 2011; 39(21): 9275–9282.). The control vectors still express the sgRNA.
[0841] Guide RNAs: Available genome sequences reveal that various genomic regions are well conserved across multiple strains. Numbered according to the ayw serotype the following genomic regions are well conserved:
Figure imgf000282_0001
[0842] 24 guide RNAs have been designed to target the HBV genome (Figures 36, 57). These include targets which are highly conserved within the HBV genome. Moreover, guide sequences can be designed to minimize possible off-target sites within the human genome e.g. to include at least 2 mismatches relative to any other sequence in the published human genome sequence. For instance, the closest matches in the human genome for guide G6 differs by at least 3 nucleotides (2 hits) or 4 nucleotides (43 hits).
[0843] Furthermore, the HBV genome includes 162 NGG PAMs within genome (41 of which are in conserved tracts) and 160 NAG PAMs (32 in conserved tracts).
[0844] In addition, the target sequences can be assessed against available complete HBV genome sequences deposited in GENBANK (5052 complete sequenced genomes) to check coverage against patient-derived virus isolates. For instance, 91.2% of these sequences have a perfect match for guide G6, and 87.3% have a perfect match for guide G21.
[0845] Guide RNAs were of the form 5’-G(N19)-3’ (wherein N is any of AUC or G) with their target sequences having the form of 5’-G(N19)-NGG-3’ (wherein N is any of ATC or G). Guides 1-12 were designed to target all 4 ORFs and guides 13-24 were designed against HBV sequences that were conserved across HBV genotypes (See, e.g., US Patent No. US8350021). See also Figs 36, 57. Thus, for guides 1-12, the target motif 5’-G(N19)-NGG-3’ appears in all of the 4 ORFs of HBV, and hence the guide RNA binds accordingly to said motifs. Likewise, for guides 13-24, the target motifs 5’-G(N19)-NGG-3’ are conserved across HBV genotypes and guide RNA binds accordingly. Advantageous guides, G6, G17, and G21 have the following sequences:
Guide 6 5'-ggggcgcacctctctttacg-3'
Guide 17 5’-taaagaatttggagctactg-3’
Guide 21 5'-tcctctgccgatccatactg-3' [0846] The putative mechanism of action for the CRISPR complexes is on the cccDNA which is produced during HBV infection and can remain latent in the nucleus for years. It is postulated that cccDNA is cleaved by Cas9 after hybridization to the designed guide RNAs. Thus, Figure 37 illustrates a means for determining cccDNA of HBV.
[0847] Experiments to test the guide RNAs, as described below, used either HepG2 cells which were artificially infected with HBV, or HepG2 2.15 cells which have an overlength linear double stranded HBV DNA segment integrated into their genome and constitutively produce HBV transcripts, infectious virions and cccDNA.
[0848] Cells and Reagents. HepG2 2.15 cells were maintained in DMEM + 10% Fetal Bovine Serum/1X Penicillin/Streptomycin (maintenance medium) for long-term culture, and passaged every 5-7 days. See also Cohen et al, 2010. All cells used were between passage 4 and passage 8. Up to 1 week prior to treatment, HepG2 2.15 cells were treated with 2% DMSO in order to promote growth arrest for accurate cell quantification, and to promote hepatic differentiation and increase HBV replication and cccDNA formation.
[0849] Transient transfection Experiments HepG2 2.15 cells: HepG2 cells were plated at ~30% confluency and grown until 50% confluency, and then were co-transfected with the plasmid shown in Fig. 57B (lentiviral backbone with an mCherry reporter), and a 1.3x length WT HBV plasmid was used as previously described (Doitsh, G. et al. 2004), using the Mirus Trans-IT transfection reagent. Cells were washed and fed after 24 hours, and supernatant and cell pellets were assayed at 72 hours post transfection.
[0850] Transfection Experiments HepG2 2.15 cells. HepG2 2.15 cells were plated on standard tissue-culture plastic that had been coated with 50 ug/mL Collagen I (Rat tail collagen I, BD) at a confluency of 40-80%. 24 hours post seeding, transfection polyplexes were formed by incubating selected CRISPR-Cas or control plasmid DNA at a 1:3 ratio with TransIT 2020 (Mirus) transfection reagent in Opti-MEM reduced serum media for 30 minutes. HepG2 2.15 cells were washed, and transfection mixture was added to the cells at a dose of 250ng DNA/1cm2 growth area. Transfection was carried out for 48 hours, and then cells were washed 3 times in maintenance medium and cultured for an additional 24 hours. Finally, cells were harvested and prepared for fluorescence activated cell sorting (FACS) by standard protocols, with the addition of Sytox Blue dye to label dead cells. Cells were analyzed by FACS and a given number of and single, living cells were collected from each condition in each of two groups: mCherry+ (successfully transfected) and mCherry- (unsuccessfully transfected) (Figure 41). These cells were harvested either into lysis buffer (for DNA quantification) or Trizol reagent (for RNA quantification) and the resulting material was analyzed by Applicants’ standard assays.
[0851] Lentivirus production: 293T cells were co-transfected with the sgRNA-Cas9-2A-Puro lentiviral vectors (Figure 60B) and a 2nd-generation lentiviral packaging system (psPAX2 and pMD2.G) at a ratio of 3:2:1. Cells were washed 24h after transfection, supernatant was collected every 24h from 48-96h post transfection, and cell debris was removed by centrifugation. Lentivirus was concentrated by ultracentifugation for 1.5h at 16,600x g, incubated O/N in Optimem at 4C, then resuspended in Optimem, aliquoted and frozen at -80C the next day, prior to use.
[0852] Lentiviral transduction: HepG2 2.15 cells were plated at 50% confluency and inoculated at MOI of 1 with concentrated lentivirus (produced as described above). Transduction was performed by mixing lentivirus aliquots with standard HepG2.2.15 culture medium, washing cells and adding lentivirus-containing medium at 2.5 mL/well in a 6-well plate, centrifuging for 1h at 200 x g and then incubating for an additional 23h. 24h after addition of lentivirus, cells were washed 3x and incubated in standard medium + 2.5 ug/mL puromycin to remove untransduced cells. Puromycin selection was continued for 48h, then cells were washed 3x and maintained in standard medium. Transduced cells were then continually passaged upon reaching 80% confluence; at each passage, cells were counted, cell pellets were harvested for each condition, and a portion of the remaining cells were reseeded at 10% confluence. Cells with lentiviral constructs looked phenotypically normal and exhibited no growth defects over at least 10 weeks in culture.
[0853] In vitro Cotransfection Experiments: HepG2 cells were seeded at 60-70% confluence on collagen pre-coated plates. 24h after seeding cells were transfected with 1.3xHBV together with the corresponding CRISPR encoding constructs using Trans-IT 2020 (MIRUS) transfection reagent at a ratio of 1:4, respectively. 48-72 after transfection medium was collected and analyzed for HBsAg using a commercial ELISA kit (Bio-Rad). Cells were collected at the same time, RNA was extracted with TRIZOL and cDNA was synthesized using SuperScript (Invitrogen) kit. Q PCR was done using specific primers for HBV pgRNA. Normalization was done to human RPS11 housekeeping gene. [0854] In vivo Hydrodynamic Co-Injection Experiments. NRG mice were hydro dynamically injected (HDD) as previously described (Lewis, D. L. et al., 2005) with 1.3xHBV plasmid (15ug) together with CRIPSR encoding (20ug) and firefly luciferase (10ug) encoding plasmids (pSPORT6-Fluc) in a volume equal to 0.15 times the animal weight (in mls). Animals were injected through the tail vein in 7-9 sec and subsequentially bled every few days. At the time of bleeding animals were visualized using the IVIS machine and luciferase expression was quantified. Blood was analyzed for HBsAg using a commercial ELISA kit and for serum HBV DNA (viremia) by Q-PCR using TaqMan mastermix with HBV specific primers and probe. Quantification was done according to a standard curve with known concentrations of 2xHBV plasmid. HBsAg and DNA levels were normalized to luciferase expression at the corresponding day. 4 or 9 days after HDD animals were sacrificed, livers were collected and DNA was extracted following homogenization. The resulting DNA was subjected to QPCR for the following: total HBV DNA, cccDNA (following treatment of DNA with plasmid safe DNase and using cccDNA specific primers), GAPDH and luciferase.
[0855] Effect on pgRNA and cccDNA. The designed guide RNAs were studied for their effect on HBV pre-genomic mRNA (pgRNA) and on covalent-closed circular DNA (cccDNA), both of which are essential parts of the HBV life cycle.
[0856] Detection of secreted Hepatitis B Surface antigen in media supernatant: 100ul of medium was loaded on ELISA plates coated with mouse monoclonal anti HBsAg antibodies (Bio-Rad, GS HBsAg EIA 3.0, Cat. No. 32591). ELISA protocol was done according to the manufacturer’s instructions. Plates were read using the FLUOstar Omega machine (BMG LABTECH).
[0857] Hepatitis B e Antigen ELISA: The HBV E Antigen ELISA was performed using the Hepatitis B e Antigen (HBeAg) chemiluminescence Immunoassay kit (Autobio Diagnostics Co, Cat No.CL0312-2) according to the manufacturer’s instructions.
[0858] Immunostaining for HBV Core antigen: Cells were grown on chambered coverglasses (Lab-Tek, Rochester, NY), washed with PBS, and then fixed with 4% paraformaldehyde. Cells were washed again (3x PBS) and treated with 100 mM glycine solution in PBS. After permeabilization with 0.1 % Triton X-100 in PBS and treated with Image-iT™ FX signal enhancer (Life Technologies). Cells were blocked in PBS/10 % goat serum (Jackson Immunosearch)/1 % BSA. HBV core staining was achieved by using a polyclonal rabbit anti- HBV core antibody (Dako, CA) diluted 1:1000 in PBS/0.1 % BSA (18 h at 4 oC). As a secondary antibody a goat-anti-rabbit labeled with AlexaFluor594 (Life Technologies) diluted 1:2000 in PBS/0.1 % BSA was used. Nuclear staining was achieved using DAPI treatment. Image acquisition was performed in a Zeiss confocal microscope and image analysis was done using ImageJ (NIH, Bethesda, MD).
[0859] HBV DNA quantification: HBV genomic DNA was quantified at days 21 and 36. For cccDNA quantification DNA from cells was subjected to overnight digestion with a plasmid-safe DNase (Epicentre) as previously described (Yan H et al,“Sodium taurocholate cotransporting polypeptide is a functional receptor for human hepatitis B and D virus, eLife 2012 Nov 13;1:e00049. doi: 10.7554/eLife.00049 (2012)). Following enzyme inactivation at 70° C for 30min, DNA was subjected to real-time PCR using cccDNA specific primers previously described by Glebe et al.,“Pre-s1 antigen-dependent infection of Tupaia hepatocyte cultures with human hepatitis B virus,”. J Virol. 2003;77(17):9511–9521. The primers used for cccDNA amplification: 5’TGCACTTCGCTTCACCTF3’ (sense) 5’ AGGGGCATTTGGTGGTC3’ (anti sense). For quantification, a standard curve derived from decreasing concentrations of 2xHBV plasmid was used. Also, PCR for HBV DNA was done using the TaqMan® Universal PCR Master Mix (Applied Bio systems, Cat No 4304437) and the following primers and probe: 5’CCGTCTGTGCCTTCTCATCTG3’ (sense), 5’AGTCCAAGAGTCCTCTTATGTAAGACCTT3’ (anti sense), 5- /56-FAM/CCG TGT GCA /ZEN/CTT CGCTTC ACCTCT GC/3IABkFQ/ -3 (probe). PCR was done using the Roche LightCycler ®480 PCR machine. Quantification was done by using a standard curve composed from 2xHBV plasmid in a concentration range of 109 -101 copies.
[0860] cccDNA extraction and analysis. DNA extracted from cells was subjected to ON digestion with a plasmid-safe DNase (Epicentre) as previously described (Yan H. et al., 2012). Following enzyme inactivation at 70° C for 30min, DNA was subjected to real-time PCR using SYBR® Premix Ex Taq (TaKaRa) following a previously described protocol (Yan H. et al., 2012) and using cccDNA specific primers previously described by Glebe et al., (2012). The primers used for cccDNA amplification:
[0861] 5’TGCACTTCGCTTCACCTF3’ (sense)
[0862] 5’ AGGGGCATTTGGTGGTC3’ (anti sense).
[0863] For quantification, a standard curve derived from decreasing concentrations of 2xHBV plasmid was used. PCR was performed using the Roche LightCycler ®480 PCR machine.
[0864] HBV mRNA quantification: Total RNA was isolated via TRIZOL RNA/DNA extraction. After being subjected to DNaseI treatment, RNA was quantified using a NanoDrop and first-strand cDNA was synthesized using SuperScript® III RT kit (INVITROGEN). Quantitative PCR for 3.5kbRNA or total HBV RNA was carried out with SYBR Green PCR master Mix (Applied Biosystems) and using specific primers previously described (Yan H. et al., 2012). In each reaction an RT negative control was included to rule-out DNA carry over.
[0865] Southern blot analysis of HBV replication intermediates: Total DNA or Hirt’s extract (low MW DNA) at day 29 was run on 0.8% agarose-TAE gel, followed by denaturation and southern blotting to a Hybond N nylon membrane (Amersham). Viral DNA was detected by hybridization with a 32P random primed HBV probe, using the Prime-It II Random Primer Labeling Kit (Agilent Technologies, Cat No 300385). Following incubation and washing, membrane was visualized by phosphorImager and later exposed to film.
[0866] Surveyor assay: Targeted loci were amplified by PCR using Phusion Flash (NEB) or Heruclase II (Agilent) polymerases and primers listed below. PCR products were gel or PCR- purified using Qiagen kits and subject to the Surveyor assay (Transgenomics) according to the manufacturer’s instructions. Indel rate for surveyor was calculated as described in Cong, L. et al. (2013). Surveyor Primers:
Guide6-F: TATCCATGGCTGCTAGGCTG
Guide6-R: AGTCAGAAGGCAAAAACGAGAG
Guide17-F1: TATCCATGGCTGCTAGGCTG
Guide17-R1: AGGGGCATTTGGTGGTC
Guide17-F2: AAATTGGTCTGCGCACCAGC
Guide17-R2: AGGTCTCTAGATGCTGGATCTTCC
Guide 21-F1: GGTTATCCTGCGTTAATGCCC
Guide21-R1: GTCCGCGTAAAGAGAGGTG
Guide 21-F2: TGAACCTTTACCCCGTTGCCC
Guide21-R2: AGAGAGTCCCAAGCGACCCC [0867] Concerning Figure 57A, the diagram depicts the life cycle of HBV, showing where the Cas9/sgRNA targeting the virus putatively acts. cccDNA produced from HBV infection is cleaved by Cas9 after sgRNA binding to conserved HBV target site. Figure 57A also illustrates the plasmid encodes both the HBV-targeting sgRNA and the Cas9 protein along with an mCherry fluorescent protein for subsequent fluorescent sorting of successfully transfected cells. Concerning Figure 57B, targets were chosen based on regions of extremely high sequence conservation among different HBV serotypes and on their low homology to the human genome (see Figure 58A). The guide RNAs target several different regions of the genome, hitting different ORFs and transcriptional control elements.
[0868] HepG2 cells were co-transfected with 1.3x WT HBV and Cas9/sgRNA/mCherry plasmids and HBV replication was allowed to proceed for 72 hours (see schematic depicted at Figure 58B). Then, supernatants were collected and cells lysed for RNA extraction. (Figure 58C) ELISA for secreted HBsAg with either control guide RNA (untargeted guide RNA) or various HBV-targeting guide RNAs. (Figure 58D) Fold change in 3.5kb HBV pregenomic RNA (pgRNA) levels between untargeted and HBV-targeting guide RNAs. The Figures show levels of HBsAg quantified using ELISA, and guides 17 and 21 showed the best reductions. The Figures also show levels of pgRNA and, while these were lower using all of guides 13-21, the most impressive results were again seen with 17 and 21. Based on the results of these two assays, along with earlier preliminary data showing the strong cutting behavior of guide 6, the guides 6, 17, and 21 were chosen for further study.
[0869] Applicants next sought to evaluate the antiviral effect of Cas9 in vivo, to ensure that anti-HBV constructs functioned appropriately in primary hepatocytes. To do this, a mouse model of HBV was used, where HBV and Cas9/gRNA plasmids were introduced to the liver of immunodeficient mice (NRG) by hydrodynamic injection (HDI) (Lewis, D. L. et al., 2005) (Figure 59A). Animals expressing Cas9 and g21 in this model showed a progressive suppression of HBV expression as compared to controls expressing Cas9 and a mutated gRNA (g21M; 3’ 5 bp mismatch), reflected by a decrease in HBsAg secretion and a 4-fold decrease in viremia at day 4 post injection (Figures 59B and 59C).
[0870] Applicants evaluated the efficacy of sustained Cas9/gRNA expression in inhibiting HBV using a model that more reliably recapitulates the HBV life cycle. For these studies, the HepG2.2.15 hepatoblastoma cell line was used which harbors both a functional HBV integrated form and cccDNA, and constitutively produces infectious virions (see Figure 60A). Figure 60 shows (a) the HBV life cycle within HepG2.2.15 cells. HepG2.2.15 cells contain genomically integrated linear 1.3x WT HBV sequences, from which viral proteins and cccDNA are constitutively produced via transcription followed by translation (proteins) or reverse transcription and nuclear re-import (cccDNA). The persistent HBV production in this system enables assay of the long-term anti-HBV effects of CRISPR/Cas systems targeting viral DNA; and (b) schematic of lentiviral vector and experimental strategy for sustained CRISPR expression within HepG2.2.15 cells. Concentrated lentiviral stocks encoding HBV-targeting sgRNA, Cas9, and a puromycin resistance element to allow for lentiviral transduction and subsequent selection of stable lines incorporating Cas9 and each guide RNA were produced. HepG2 2.15 cells were transduced with concentrated lentivirus encoding Cas9-2A-Puro or nuclease deficient (D10A/H840A“dead”) Cas9-2A-Puro and either guide 6, 17, or 21, or one of three different untargeted sequences, followed by puromycin selection to yield HepG2 2.15 lines stably expressing the CRISPR/Cas system. Figure 61 shows that Guide 6, 17, and 21 drastically decreased total HBV DNA and cccDNA in a manner dependent on correct targeting to HBV, and on Cas9 nuclease activity. Comparing the results from 29 and 36 days post transduction, Applicants see that the levels of HBV DNA and cccDNA continue to decrease over time, consistent with results from lentiviral transduction of CRISPR/Cas in other cell systems. The Figures also show that the‘dead’ Cas9 had little impact on the amount of HBV genomic DNA at day 21 or 36 (about 50 copies per cell), but that guides 6, 17 and 21 in combination with active Cas9 reduced DNA to fewer than 10 copies per cell at day 21, and even fewer copies at day 36. Thus the Cas9 system drastically decreased total HBV DNA and cccDNA in a manner dependent on correct targeting to HBV, and on Cas9 nuclease activity.Moreover, a similar effect was seen on cccDNA, which was almost undetectable using guide 21 (much less than 1 copy per cell on average) at day 36.
[0871] In separate control experiments, cells were also transduced with constructs containing gRNAs and a nuclease deficient Cas9 (D10A/H840A; dead Cas9) to control for nuclease- independent effects of Cas9 on viral fitness, or WT Cas9 with mutated gRNAs (gXM) to control for guide sequence-independent effects. Cas9/gRNAs induced robust suppression of HBV DNA release (77-95% decrease across different gRNAs), HBeAg secretion, and viral mRNA production (greater than 50%) (Figure 62). MORE
[0872] Applicants next analyzed the effect of Cas9-mediated cleavage on the abundance of non-integrated viral forms, composed mainly of cccDNA. qPCR showed a robust reduction in total HBV DNA and in cccDNA, with the latter progressing from 71 +/- 7% reduction at day 21 to 92 +/- 4% at day 36 post transduction (Figure 63 and 64).
[0873] These results were confirmed by directly analyzing low molecular weight DNA from transduced cells by Southern blot (Figure 65). cccDNA and its deproteinated relaxed circular form (dpRC DNA) precursor were greatly depleted in Cas9/gRNA transduced cells. In contrast, when total HBV DNA was analyzed, no substantial reduction in the levels of integrated HBV DNA was detected (Figure 66). The Southern blot of HBV DNA at 29 days post transduction of Figure 65 is the results of a Southern blot that was performed on the DNA harvested from the HepG2 2.15 cell lines at 29 dpt, either using standard DNA extraction for total DNA, or using a modified Hirt’s extraction to specifically enrich for low molecular weight, non-chromosomal DNA. For Hirt’s extraction, both total DNA and mtDNA are shown as loading controls since mtDNA should persist through the extraction procedure. Note the almost complete absence of HBV DNA in the on-target, nuclease-competent lanes. Figure 65 shows that, with either high or low exposure, the rcDNA, cccDNA and ssDNA forms of HBV are essentially undetectable using guide 6 or 21, and (except for rcDNA) are barely detectable using 17. Then at day 36 linear dsDNA, cccDNA and ssDNA are essentially undetectable using 17 and 21, whereas levels of integrated HBV DNA remain unaffected (since Cas9-mediated cleavage of integrated DNA should result in NHEJ-mediated DNA repair and maintenance of the integrated HBV DNA although potentially in mutated form). This degree of reduction in HBV cccDNA (even in a cell line system) compares very favorably to the available literature on cccDNA-targeting HBV therapeutics.
[0874] The Southern blot of HBV DNA at 36 days post transduction of Figure 66 is the results of a Southern blot was performed at 36 days post transduction, and specifically looks at differential effects on integrated HBV DNA vs. intermediate HBV forms and cccDNA. Note that on-target, nuclease-competent guides drastically reduce the levels of linear dsDNA, cccDNA, and ssDNA while levels of integrated HBV DNA remain unaffected (since Cas9-mediated cleavage of integrated DNA should result in NHEJ-mediated DNA repair and maintenance of the integrated HBV DNA, although potentially in mutated form). This degree of reduction in HBV cccDNA demonstrates that the CRISPR-Cas system can be a cccDNA-targeting HBV therapeutic.
[0875] Surveyor assay was performed on DNA extracts from CRISPR-transduced HepG2 2.15 cells to directly determine whether the viral DNA was cleaved and repaired via error-prone NHEJ similar to genomic targets of CRISPR/Cas9 and the results thereof are illustrated in Figure 67. The Surveyor T7E1 endonuclease assay was performed to assess indel formation in HBV DNA in this system. Analysis of total HBV DNA forms for indel formation, an indirect measure of Cas9-mediated cleavage, revealed high levels of cutting (Figure 67, top panel). Lower levels were seen in cccDNA that was amplified from the cells after DNAse treatment to destroy non- circular integrated HBV DNA (0% vs 32%, 62% vs 88% and 21% vs 66% for guides 21, 17 and 6, respectively) (Figure 67, bottom panel).
[0876] Consistent with high levels of indel formation in the core ORF targeted by g17, immunostaining for HBV core protein (HBc) revealed a robust reduction in HBc levels in g17- expressing cells as compared to controls (Figure 68).
[0877] To evaluate Cas9 treatment in a setting of de novo infection, Applicants used HepG2 cells overexpressing the HBV receptor NTCP (Hep-NTCP) (Yan H. et al. 2012), which are permissive to infection with HBV. Because g17 showed the highest levels of indel formation in cccDNA in the HepG2.2.15 experiments, these cells were transduced with Cas9/g17, Cas9/g17M, or dead Cas9/g17 lentiviruses, co-cultured with HBV producing HepG2.2.15 cells, and selected with puromycin to eliminate non-transduced Hep-NTCP and contaminating HepG2.2.15 cells (Figure 69, left). Alternatively, Hep-NTCP cells were selected with puromycin following transduction and subsequently infected with HBV-positive patient serum (Figure 69, right). When the transduced Hep-NTCP were infected with cell culture-produced virus, Cas9/g17 greatly abrogated productive HBV infection, as reflected by reduction in HBsAg and HBV DNA secretion, as well as 3.5kbRNA and cccDNA levels, compared to controls (Figure 70); this finding was confirmed following infection with patient-derived virus (Figure 71). Surveyor assay performed using DNA from cells infected de novo with HepG2.2.15-derived virus confirmed direct cleavage of HBV episomal DNA (Figure 72). Although minor cleavage was also detected in the mutated g17M, this most likely was due to low-level cleavage with DNA bulge-containing guide RNAs (Lin Y. et al. 2014). This finding provides direct evidence that Cas9 is capable of targeting episomal forms of the virus, and exerting anti-HBV effects by directly targeting cccDNA.
[0878] These experiments show that clearance of HBV cccDNA can be mediated by the expression of CRISPR-Cas constructs in cells either constitutively expressing or de novo transfected with HBV DNA. Given the high percentage of cccDNA+ hepatocytes in chronically HBV infected livers, it would be suitable to package the CRISPR-Cas system encompassing optimal HBV target sequences into a highly efficient, liver-specific vector such as adeno- associated virus, subtype 8 (AAV8) (Sands MS,“AAV-mediated liver-directed gene therapy,” Methods Mol Biol. 2011;807:141-57. doi: 10.1007/978-1-61779-370-7_6). Additionally, to enforce the specificity or targeting and improve in vivo Cas9 expression, it will be desirable to include hepatocyte-specific transcriptional control regions (Miao CH et al,“Inclusion of the hepatic locus control region, an intron, and untranslated region increases and stabilizes hepatic factor IX gene expression in vivo but not in vitro,” Mol Ther 2000 Jun;1(6):522-32). The dosing regimens would be optimized for both in vivo expression and prevention of an anti-AAV immune response, using information taken from the 100+ clinical trials that have been undertaken using AAV vectors (http://www.abedia.com/wiley/vectors.php). While incorporation of anti-HBV CRISPR-Cas systems into AAV vectors is still ongoing, the recent gene therapeutic success of other vector types, such as Ȗ-retroviruses (Aiuti A et al,“Lentiviral Hematopoietic Stem Cell Gene Therapy in Patients with Wiskott-Aldrich Syndrome,” Science 341 no. 6148, 2013; Biffi A et al,“Lentiviral Hematopoietic Stem Cell Gene Therapy Benefits Metachromatic Leukodystrophy,” Science Vol. 341 no. 6148, 2013) opens up even more vector possibilities for human dosing, and provides a heightened level of comfort with using CRISPR-based gene therapy to excise HBV from infected hepatocytes.
[0879] Moreover, these experiments show that the CRISPR-Cas system can be used therapeutically to target and reduce the presence of HBV, especially cccDNA of HBV.
[0880] De novo HBV infection of in patient-derived hepatocytes (either primary human hepatocytes or iPS-derived hepatocytes) using the CRISPR-Cas system and constructs as herein described and illustrated obtain similar therapeutically significant results, especially because infection in these systems is performed with patient plasma-derived virus.
[0881] Cas9 cleavage of HBV cccDNA generates y-H2AX and Cas9 cleavage of HBV cccDNA linearizes the cccDNA which may be left unrepaired and potentially degraded after being linearized by Cas9.
[0882] Yan et al, eLife (2012) identified a specific 2-48aa sequence in the PreS1 protein of HBV responsible for binding to the sodium taurocholate cotransporting polypeptide (NTCP), the recently discovered receptor for HBV. Targeting sgRNAs against this region may induce indel formation and mutation of this region may result in the production of virions incapable of binding and entering new hepatocytes, leading to another mechanism of viral clearance even if all cccDNA cannot be eliminated.
[0883] Moreover, the results herein invite co-administration of CRISPR/Cas system and epigenetic modifier drugs: The HBV cccDNA‘minichromosome” is a very densely-packed, nucleosome-associated structure with HBV DNA tightly coiled around histone proteins and HBV Core protein (HBc), leading to nucleosomal spacing even shorter than standard chromosomal spacing. HBV transcription are regulated by epigenetic marks on associated histones, which lead to the recruitment of several host cellular transcription factors, and epigenetic modification of cccDNA minichromosomes has shown some promise in reducing the transcription of HBV RNAs and also in leading to cccDNA degradation (for example, IFN- alpha’s cccDNA degrading ability may be related to induction of epigenetic changes on the cccDNA). Since Cas9 targeting to cccDNA is likely at least partially dependent on the cccDNA structure, co-treatment with epigenetic modifiers (for example Class I and Class III HDAC inhibitors trichostatin A (TSA), valproate, and nicotinamide (NAM), and Type I interferons) may be a valuable strategy for increasing Cas9 occupancy on HBV cccDNA. The invention comprehends administration of the CRISPR-Cas system with such epigenetic modifier drugs; or other drugs presently used to treat HBV. The skilled person can use doses and formulations of such drugs presently used, in combination with the CRISPR-Cas system in doses and formulations as herein described to treat HBV.
[0884] Example 29: Other viruses
[0885] Other viruses can also be targeted using the same approach, and in particular DNA viruses (typically dsDNA viruses). For example, herpes simplex virus (HSV), human papillomavirus (HPV), Epstein Barr virus (EBV), varicella zoster virus (VZV), and any other virus which integrates into a host mammal’s genome and/or has a latent circular episomsal form. CRISPR systems of the invention can be targeted to cell types which maintain the latent virus e.g. B cells or epithelial cells for EBV, neurons for HSV and VZV, epithelial cells for HPV, etc.
[0886] HSV1/2 forms stable latent infection phase in neurons in episomal form, and the latent DNA is (similar to HBV) complexed with nucleosomes and other transcription-regulating machinery. There are AAV subtypes (AAV2) which efficiently target neurons, and this is a good target. HPV has a low copy-number, episomal DNA form in basal keratinocytes, activation of replication as differentiation occurs into the squamous layer; targeting these cells is relatively easy since mucosal sites are accessible, and if done early after detection, viral dissemination could be halted by using a CRISPR-Cas system as herein discussed. Plant viruses are also suitable targets. Accordingly, the herein studies as to HBV are readily extendable to other mammalian or human viruses and plant viruses.
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* * *
[0887] While preferred embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention.

Claims

WHAT IS CLAIMED IS:
1. A method of modifying an organism or a non-human organism by manipulation of a target hepatitis B virus (HBV) sequence in a genomic locus of interest comprising delivering a non-naturally occurring or engineered composition comprising:
A) - I. a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises:
(a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell,
(b) a tracr mate sequence, and
(c) a tracr sequence, and
II. a polynucleotide sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,
wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation,
wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target HBV sequence, and
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,
or
(B) I. polynucleotides comprising:
(a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell, and
(b) at least one or more tracr mate sequences,
II. a polynucleotide sequence encoding a CRISPR enzyme, and
III. a polynucleotide sequence comprising a tracr sequence,
wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target HBV sequence, and
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA.
2. The method of claim 1, wherein any or all of the polynucleotide sequence encoding a CRISPR enzyme, guide sequence, tracr mate sequence or tracr sequence, is/are RNA.
3. The method of claim 1 or 2, wherein the polynucleotides encoding the sequence encoding a CRISPR enzyme, the guide sequence, tracr mate sequence or tracr sequence is/are RNA and are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
4. The method of any of claims 1 to 3, wherein the polynucleotides are comprised within a vector system comprising one or more vectors.
5. A method of modifying an organism or a non-human organism by manipulation of a target HBV sequence in a genomic locus of interest comprising delivering a non-naturally occurring or engineered composition comprising a viral vector system comprising one or more viral vectors operably encoding a composition for expression thereof, wherein the composition comprises:
(A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
I. a first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises
(a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell,
(b) a tracr mate sequence, and
(c) a tracr sequence, and
II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,
wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation,
wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target HBV sequence, and
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, or
(B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
I. a first regulatory element operably linked to
(a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell, and
(b) at least one or more tracr mate sequences,
II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and
III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are located on the same or different vectors of the system,
wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target HBV sequence, and
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence.
6. The method of claim 5, wherein one or more of the viral vectors are delivered via liposomes, nanoparticles, exosomes, microvesicles, or a gene-gun.
7. A method of treating or inhibiting a condition caused by a defect in a target HBV sequence in a genomic locus of interest in a subject or a non-human subject in need thereof comprising modifying the subject or a non-human subject by manipulation of the target HBV sequence and wherein the condition is susceptible to treatment or inhibition by manipulation of the target HBV sequence comprising providing treatment comprising:
delivering a non-naturally occurring or engineered composition comprising an AAV or lentivirus vector system, comprising one or more AAV or lentivirus vectors operably encoding a composition for expression thereof, wherein the target HBV sequence is manipulated by the composition when expressed, wherein the composition comprises:
(A) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
I. a first regulatory element operably linked to a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises (a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell,
(b) a tracr mate sequence, and
(c) a tracr sequence, and
II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences, wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation,
wherein components I and II are located on the same or different vectors of the system, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target HBV sequence, and
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence,
or
(B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprising
I. a first regulatory element operably linked to
(a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell, and
(b) at least one or more tracr mate sequences,
II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and
III. a third regulatory element operably linked to a tracr sequence, wherein components I, II and III are located on the same or different vectors of the system,
wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target HBV sequence, and
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence.
8. The method of any preceding claim, wherein the method is carried out in vitro, and/or ex vivo.
9. The method of any preceding claim including inducing expression.
10. The method of any preceding claim wherein the organism or subject is a eukaryote, preferably a non-human eukaryote.
11. The method of claim 10 wherein the organism or subject is a non-human eukaryote.
12. The method of any of claims 1 to 11 wherein the organism or subject is a mammal or a non-human mammal.
13. The method of any of claims 4 to 8 wherein the viral vector is an AAV or lentiviral vector.
14. The method according to any preceding claim wherein the CRISPR enzyme is a Cas9.
15. The method according to any preceding claim wherein expression of the guide sequence is under the control of the T7 promoter and is driven by the expression of T7 polymerase.
16. A method of delivering a CRISPR enzyme of any preceding claim, comprising delivering to a cell mRNA encoding the CRISPR enzyme.
17. The method of any one of claims 1 to 16, wherein the polynucleotide or enzyme coding sequence encoding the CRISPR enzyme is delivered to the cell by delivering mRNA encoding the CRISPR enzyme to the cell.
18. A method of preparing the AAV or lentivirus vector of claim 7 comprising transfecting plasmid(s) containing or consisting essentially of nucleic acid molecule(s) coding for the AAV or lentivirus into AAV-infected or lentivirus-infected cells, and supplying AAV AAV or lentivirus rep and/or cap and/or helper nucleic acid molecules obligatory for replication and packaging of the AAV or lentivirus.
19. A method of preparing an AAV or lentivirus vector for use in the method of claim 7, comprising transfecting plasmid(s) containing or consisting essentially of nucleic acid molecule(s) coding for the AAV or lentivirus into AAV-infected or lentivirus-infected cells, and supplying AAV AAV or lentivirus rep and/or cap and/or helper nucleic acid molecules obligatory for replication and packaging of the AAV or lentivirus.
20. The method of claim 18 or 19 wherein the AAV or lentivirus rep and/or cap obligatory for replication and packaging of the AAV or lentivirus are supplied by transfecting the cells with helper plasmid(s) or helper virus(es).
21. The method of claim 20 wherein the helper virus is a poxvirus, adenovirus, lentivirus, herpesvirus or baculovirus.
22. The method of claim 21 wherein the poxvirus is a vaccinia virus.
23. The method of any of claims 18 to 22 wherein the cells are mammalian cells.
24. The method of any of claims 18 to 22 wherein the cells are insect cells and the helper virus (where present) is baculovirus.
25. The method of any of claims 1 to 15 wherein the target HBV sequence is flanked at its 3’ end or followed by 5’-NRG (where N is any Nucleotide), and where the CRISPR enzyme is (or is derived from) S. pyogenes or S. aureus Cas9.
26. A composition as defined in any of claims 1-25 for use in medicine or in therapy.
27. A composition as defined in any of claims 1-25 for use in a method of modifying an organism or a non-human organism by manipulation of a target HBV sequence in a genomic locus of interest or in a method of treating or inhibiting a condition caused by a defect in a target HBV sequence in a genomic locus of interest.
28. Use of a composition as defined in any of claims 1-25 in ex vivo gene or genome editing.
29. Use of a composition as defined in any of claims 1 -25 in the manufacture of a medicament for ex vivo gene or genome editing or for use in a method of modifying an organism or a non-human organism by manipulation of a target HBV sequence in a genomic locus of interest or in a method of treating or inhibiting a condition caused by a defect in a target HBV sequence in a genomic locus of interest.
30. A composition comprising:
A) - I. a CRISPR-Cas system RNA polynucleotide sequence, wherein the polynucleotide sequence comprises:
(a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell,
(b) a tracr mate sequence, and
(c) a tracr sequence, and
II. a polynucleotide sequence encoding a CRISPR enzyme, optionally comprising at least one or more nuclear localization sequences,
wherein (a), (b) and (c) are arranged in a 5’ to 3’ orientation, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target HBV sequence, and
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,
or
(B) I. polynucleotides comprising:
(a) a guide sequence capable of hybridizing to a target HBV sequence in a eukaryotic cell, and
(b) at least one or more tracr mate sequences,
II. a polynucleotide sequence encoding a CRISPR enzyme, and
III. a polynucleotide sequence comprising a tracr sequence,
wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence-specific binding of a CRISPR complex to the target hepatitis B virus (HBV) sequence, and
wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized or hybridizable to the target HBV sequence, and (2) the tracr mate sequence that is hybridized or hybridizable to the tracr sequence, and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA;
for use in medicine or therapy; or for use in a method of modifying an organism or a non- human organism by manipulation of a target HBV sequence in a genomic locus of interest; or for use in a method of treating or inhibiting a condition caused by a defect in a target HBV sequence in a genomic locus of interest; or for use in ex vivo gene or genome editing.
31. The composition of claim 30, wherein the polynucleotides are comprised within a vector system comprising one or more vectors.
32. The method, use or composition of any of the preceding claims, wherein the CRISPR-Cas system RNA is a chimeric RNA (chiRNA).
33. The method, use or composition of any of the preceding claims, wherein the CRISPR-Cas system is a multiplexed CRISPR enzyme system further comprising multiple chimeras and/or multiple multiguide sequences and a single tracr sequence.
34. The method, use or composition according any of the preceding claims, wherein the CRISPR enzyme is a nuclease directing cleavage of one or both strands at the location of the target sequence.
35. The method, use or composition according to any of the preceding claims, wherein the CRISPR enzyme comprises one or more mutations.
36. The method, use or composition according to claim 35, wherein the CRISPR enzyme comprises one or more mutations D10A, E762A, H840A, N854A, N863A or D986A.
37. The method, use or composition according to claim 35 wherein the one or more mutations is in a RuvC1 domain of the CRISPR enzyme.
38. The method, use or composition according to claim 34, wherein the CRISPR enzyme is a nickase directing cleavage at the location of the target sequence.
39. The method, use or composition according to claim 38, wherein the nickase is a double nickase.
40. The method, use or composition according to any preceding claim further comprising at least two or more NLS.
41. The method, use or composition according to any preceding claim, wherein the CRISPR enzyme has one or more mutations in a catalytic domain, wherein when transcribed, the tracr mate sequence hybridizes to the tracr sequence and the guide sequence directs sequence- specific binding of a CRISPR complex to the target sequence, and wherein the enzyme further comprises a functional domain.
42. The method, use or composition according to claim 41, wherein the functional domain is a transcriptional activation domain.
43. The method, use or composition according to claim 42, wherein the transcriptional activation domain is VP64.
44. The method of any one of claims 1-25 or 32-43 further comprising minimizing off- target modifications by manipulation of a first and a second target sequence on opposite strands of a DNA duplex in a genomic locus of interest in a cell comprising
delivering a non-naturally occurring or engineered composition comprising:
I. a CRISPR-Cas system chimeric RNA (chiRNA) polynucleotide sequence, wherein the polynucleotide sequence comprises:
(a) a first guide sequence capable of hybridizing to the first target sequence,
(b) a first tracr mate sequence, (c) a first tracr sequence,
(d) a second guide sequence capable of hybridizing to the second target sequence,
(e) a second tracr mate sequence, and
(f) a second tracr sequence, and
optionally, wherein a linker sequence is present between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem; and
II. a polynucleotide sequence encoding a CRISPR enzyme comprising at least one or more nuclear localization sequences, wherein (a), (b), (c), (d), (e) and (f) are arranged in a 5’ to 3’ orientation, wherein the polynucleotide sequence comprises a linker sequence between the first tracr sequence and the second guide sequence, whereby the first guide sequence and the second guide sequence are in tandem, and wherein when transcribed, the first and the second tracr mate sequence hybridize to the first and second tracr sequence respectively and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively,
or
II. a second regulatory element operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, and wherein components I and II are located on the same or different vectors of the system, and when transcribed, a first tracr mate sequence hybridizes to a first tracr sequence and the first and the second guide sequence directs sequence-specific binding of a first and a second CRISPR complex to the first and second target sequences respectively; wherein the first CRISPR complex comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized or hybridizable to the first target sequence, and (2) the first tracr mate sequence that is hybridized or hybridizable to the first tracr sequence,
wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized or hybridizable to the second target sequence, and (2) the second tracr mate sequence that is hybridized or hybridizable to the second tracr sequence,
wherein the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA, and wherein the first guide sequence directs cleavage of one strand of the DNA duplex near the first target sequence and the second guide sequence directs cleavage of other strand near the second target sequence inducing a double strand break, thereby modifying the organism or the non-human organism by minimizing off-target modifications.
45. A CRISPR-Cas system comprising one or more vectors for delivery to a eukaryotic cell, wherein the vector(s) encode(s): (i) a CRISPR enzyme; (ii) a guide RNA capable of hybridizing to a target sequence in a viral genome within the cell; and (iii); a tracr mate sequence; and (iv) a tracr sequence,
wherein, when expressed within the cell, the guide RNA directs sequence-specific binding of a CRISPR complex to the target sequence, and the CRISPR complex comprises (a) the tracr mate sequence hybridised to the tracr sequence and (b) a CRISPR enzyme bound to the guide RNA, such that the guide RNA can hybridise to its target sequence in the viral genome.
46. The system of claim 45 wherein the viral genome is hepatitis B virus (HBV), herpes simplex virus (HSV), human papillomavirus (HPV), Epstein Barr virus (EBV), varicella zoster virus (VZV) or a plant virus.
47. The system of claim 46 wherein the viral genome is HBV.
48. A method of treating a viral infection in an individual in need thereof comprising administering an effective amount of the system of claim 45.
49. The method of claim 48 wherein the viral infection is HBV.
50. The method of claim 49 including administering an additional HBV treatment.
51. The method of claim 50 wherein the additional treatment comprises an epigenetic modifier.
52. Use of the system of any one of claims 45-47 in treating a viral infection of an individual or in formulating a medicament or pharmaceutical composition or treatment regimen for viral infection treatment.
53. A method of modifying a cell of a eukaryotic organism by manipulating at least one target viral nucleic acid within the cell, the method comprising introducing into the cell an exogenous composition capable of forming a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) (CRISPR-Cas) complex, the composition comprising:
(A) CRISPR-Cas system polynucleotide sequences comprising:
(i) a guide sequence, which when transcribed is capable of hybridizing to a sequence of the at least one target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and (iii) a tracr sequence, wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing to the tracr mate sequence; and
(B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme,
wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
(i) the tracr mate sequence is hybridized to the tracr sequence or portion thereof;
(ii) the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and
(iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
54. An exogenous composition which, when introduced into a cell of a eukaryotic organism, is capable of forming at least one Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) complex, wherein the complex is capable of modifying the cell by manipulating at least one target viral nucleic acid within the cell, the composition comprising:
(A) Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR associated (Cas) (CRISPR-Cas) system polynucleotide sequences comprising:
(i) a guide sequence, which when transcribed is capable of hybridizing to a sequence of the at least one target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein when transcribed all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
(B) a CRISPR/Cas enzyme or a polynucleotide encoding a CRISPR/Cas enzyme,
wherein when the CRISPR/Cas system polynucleotide sequences are present as RNA within the cell and the CRISPR/Cas enzyme is present as a protein within the cell:
(i) the tracr mate sequence is hybridized to the tracr sequence or portion thereof; (ii) the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and
(iii) the guide sequence hybridizes to a sequence of the at least one target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the at least one sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
55. A Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR- associated (Cas) (CRISPR-Cas) complex which, when introduced into a cell of a eukaryotic organism, is capable of modifying the cell by manipulating a target viral nucleic acid within the cell, the complex comprising:
(A) CRISPR-Cas system RNA polynucleotide sequences comprising:
(i) a guide sequence, which is capable of hybridizing to a sequence of the target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
(B) a CRISPR/Cas enzyme,
wherein when the CRISPR/Cas system RNA polynucleotide sequences and the CRISPR/Cas enzyme are present within the cell:
(i) the tracr mate sequence is hybridized to the tracr sequence or portion thereof;
(ii) the CRISPR/Cas system polynucleotide sequences are associated with the CRISPR/Cas enzyme, so forming a CRISPR/Cas complex; and
(iii) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid, whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
56. A Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR- associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA) which, when introduced into a cell of a eukaryotic organism, is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex, wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell; the chiRNA comprising:
(i) a guide sequence, which is capable of hybridizing to a sequence of the target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
wherein when the chiRNA and the CRISPR/Cas enzyme are present within the cell:
a) the tracr mate sequence hybridizes to the tracr sequence or portion thereof;
b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex; and
c) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
57. A DNA polynucleotide molecule comprising sequences encoding a Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR)- CRISPR-associated (Cas) (CRISPR-Cas) system chimeric RNA polynucleotide molecule (chiRNA), wherein upon introduction of said chiRNA into a cell of a eukaryotic organism said chiRNA is capable of associating with a CRISPR/Cas enzyme so forming a CRISPR-Cas complex, wherein the CRISPR-Cas complex is capable of modifying the cell by manipulating a target viral nucleic acid within the cell; the chiRNA comprising:
(i) a guide sequence, which is capable of hybridizing to a sequence of the target viral nucleic acid to be manipulated;
(ii) a trans-activating CRISPR RNA (tracr) mate sequence, which is linked to the guide sequence; and
(iii) a tracr sequence, wherein all or a portion of the tracr sequence is capable of hybridizing with the tracr mate sequence; and
wherein when the chiRNA and the CRISPR/Cas enzyme are present within the cell: a) the tracr mate sequence hybridizes to the tracr sequence or portion thereof; b) the chiRNA associates with the CRISPR/Cas enzyme, so forming the CRISPR/Cas complex; and
c) the guide sequence hybridizes to a sequence of the target viral nucleic acid thereby directing sequence-specific binding of the CRISPR/Cas complex to the sequence of the target viral nucleic acid whereupon said sequence of said target viral nucleic acid is manipulated by the CRISPR/Cas enzyme of the complex.
58. The method of claim 53 or composition of claim 54, wherein the CRISPR/Cas enzyme of the exogenous composition is provided as a polynucleotide sequence which comprises either (a) RNA or (b) DNA wherein the polynucleotide sequence is operably linked to a regulatory element capable of directing expression of RNA encoding the CRISPR/Cas enzyme.
59. The method of claim 53 or composition of claim 54, wherein any of the CRISPR/Cas system polynucleotide sequences of the exogenous composition comprise either (a) RNA or (b) DNA wherein the polynucleotide sequences are operably linked to one or more regulatory elements capable of directing expression of CRISPR/Cas system RNA polynucleotide sequences.
60. The method or composition of claim 59, wherein each of the CRISPR/Cas system polynucleotide sequences of the exogenous composition consists of RNA and wherein the CRISPR/Cas system polynucleotide sequences comprise a chimeric RNA polynucleotide molecule comprising the guide sequence, the tracr mate sequence and the tracr sequence.
61. The method or composition of claim 59, wherein each of the CRISPR/Cas system polynucleotide sequences of the exogenous composition are provided as DNA polynucleotide sequences further comprising at least one regulatory element operably linked to polynucleotide sequences encoding CRISPR/Cas system RNA polynucleotide sequences and capable of directing expression thereof, and wherein the CRISPR/Cas system RNA polynucleotide sequences comprise a chimeric RNA polynucleotide molecule (chiRNA) comprising the guide sequence, the tracr mate sequence and the tracr sequence.
62. The method or composition of any of claims 59 to 61, the complex of claim 55, the chiRNA of claim 56 or the DNA polynucleotide molecule of claim 57; wherein each of the guide sequence, the tracr mate sequences and the tracr sequence are arranged in a 5’ to 3’ orientation; or wherein each of the guide sequence, the tracr mate sequences and the tracr sequence are arranged in a 3’ to 5’ orientation.
63. The method or composition of any of claims 58 to 62, wherein (a) the CRISPR/Cas system polynucleotide sequences or polynucleotide sequences encoding the CRISPR/Cas system polynucleotide sequences and/or (b) polynucleotide sequences encoding the CRISPR/Cas enzyme are comprised in one or more recombinant viral vectors
64. The method or composition of claim 69, wherein polynucleotide sequences of (a) are located on the same or different recombinant viral vector as polynucleotide sequences of (b).
65. The chiRNA of claim 56 or the DNA polynucleotide molecule of claim 57 wherein the chiRNA or the DNA polynucleotide molecule is comprised in a recombinant viral vector.
66. The method, composition, chiRNA or DNA polynucleotide of any of claims 63 to 65, wherein the viral vector is a retroviral vector, optionally a lentiviral vector, a baculoviral vector, a herpes simplex virus vector, an adenoviral vector, an adenoassociated viral (AAV) vector such as AAV8 vector, or a poxvirus such as a vaccinia virus.
67. The method of any of claims 58 to 62 wherein (a) the CRISPR/Cas system polynucleotide sequences or polynucleotide sequences encoding the CRISPR/Cas system polynucleotide sequences and/or (b) polynucleotide sequences encoding the CRISPR/Cas enzyme are delivered to the cell of the organism via liposomes, nanoparticles, exosomes, microvesicles or a gene-gun.
68. The method, composition, complex, chiRNA or DNA polynucleotide molecule of any of claims 53 to 67, wherein the tracr sequence is 30 or more nucleotides in length, 40 or more nucleotides in length, or 50 or more nucleotides in length.
69. The method, composition, complex, chiRNA or DNA polynucleotide molecule of any of claims 53 to 68, wherein hybridization between the tracr sequence and the tracr mate sequence produces a transcript having secondary structure, preferably a hairpin.
70. The method or composition of claim 69, wherein the tracr sequence comprises one or more regions capable of forming secondary structure, preferably a hairpin.
71. The method or composition of claim 70, wherein the tracr sequence comprises one or more hairpins, two or more hairpins, three or more hairpins, four or more hairpins, five or more hairpins, or at most five hairpins.
72. The method, composition, complex, chiRNA or DNA polynucleotide molecule of any of claims 53 to 71, wherein the CRISPR/Cas enzyme is a Cas9 enzyme or a biologically active fragment or derivative thereof, such as a Streptococcus pyogenes Cas9 enzyme or a biologically active fragment or derivative thereof or a Streptococcus aureus Cas9 enzyme or a biologically active fragment or derivative thereof.
73. The method, composition, complex, chiRNA or DNA polynucleotide molecule of any of claims 53 to 72, wherein the CRISPR/Cas enzyme further comprises one or more nuclear localization sequences (NLSs) capable of driving the accumulation of the CRISPR/Cas enzyme to a detectible amount in the nucleus of the cell of the organism.
74. The method, composition, complex, chiRNA or DNA polynucleotide molecule of claim 73, wherein the CRISPR/Cas enzyme comprises two or more NLSs, three or more NLSs, four or more NLSs, five or more NLSs, six or more NLSs, seven or more NLSs, eight or more NLSs, nine or more NLSs, or ten or more NLSs.
75. The method, composition, complex, chiRNA or DNA polynucleotide molecule of claim 73 or 74, wherein the CRISPR/Cas enzyme comprises at least one NLS at or near the amino-terminus of the CRISPR/Cas enzyme and/or at least one NLS at or near the carboxy- terminus the CRISPR/Cas enzyme.
76. The method, composition, complex, chiRNA or DNA polynucleotide molecule of any of claims 53 to 75, wherein when present as RNA within the cell the guide sequence is capable of hybridizing to a sequence of the target viral nucleic acid which is comprised in an episomal nucleic acid molecule which is not integrated into the genome of the organism and wherein said manipulation is a manipulation of the episomal viral nucleic acid molecule, preferably wherein the episomal nucleic acid molecule is a double-stranded DNA polynucleotide molecule.
77. The method, composition, complex, chiRNA or DNA polynucleotide molecule of claim 76, wherein the episomal viral nucleic acid molecule is a covalently closed circular DNA (cccDNA).
78. The method, composition, complex, chiRNA or DNA polynucleotide molecule of claim 76 or claim 77, wherein the CRISPR/Cas complex is capable of reducing the amount of episomal viral nucleic acid molecule in a cell of the organism compared to the amount of episomal viral nucleic acid molecule in a cell of the organism in the absence of providing the complex.
79. The method, composition, complex, chiRNA or DNA polynucleotide molecule of any of claims 76 to 78, wherein the CRISPR/Cas complex is capable of manipulating the episomal nucleic acid molecule to promote degradation of the episomal nucleic acid molecule.
80. The method, composition, complex, chiRNA or DNA polynucleotide molecule of any of claims 1 to 75, wherein when present as RNA within in the cell the guide sequence is capable of hybridizing to a sequence of the target viral nucleic acid which is integrated into the genome of the organism and wherein said manipulation is a manipulation of the integrated target nucleic acid.
81. The method, composition, complex, chiRNA or DNA polynucleotide molecule of claim 80, wherein when formed within the cell the CRISPR/Cas complex is capable of manipulating the integrated nucleic acid to promote excision of all or part of the target viral nucleic acid from the genome of the organism.
82. The use of a composition, complex, chiRNA or DNA polynucleotide molecule of any of claims 54 to 66 or any of claims 68 to 81 in the manipulation of at least one target viral nucleic acid within the cell of a eukaryotic organism.
83. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 53 to 82, wherein said at least one target viral nucleic acid is comprised in a double-stranded DNA polynucleotide cccDNA molecule and/or viral DNA integrated into the genome of the organism and wherein said manipulation of the at least one target viral nucleic acid by the CRISPR-Cas complex comprises cleavage of viral cccDNA and/or integrated viral DNA.
84. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of claim 83, wherein said cleavage comprises one or more double-strand break(s) introduced into the viral cccDNA and/or integrated viral DNA, optionally at least two double-strand break(s).
85. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of claim 83, wherein said cleavage is via one or more single-strand break(s) introduced into the viral cccDNA and/or integrated viral DNA, optionally at least two single-strand break(s).
86. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of claim 84 or 85, wherein said one or more double-strand break(s) or said one or more single- strand break(s) leads to the formation of one or more insertion and deletion mutations (INDELs) in the viral cccDNA sequences and/or integrated viral DNA sequences.
87. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of claims 83 to 86, wherein cleavage of the viral cccDNA sequences or viral DNA sequences integrated in the genome of the organism leads to excision of viral polynucleotide sequences from the cccDNA thereby reducing viral infection or excision of viral DNA sequences from the genome of the organism thereby reducing viral infection.
88. The method or composition of claim 87, wherein said composition comprises components of at least two types of CRISPR/Cas complex, wherein each type of complex comprises a guide sequence capable of hybridizing to different sequences of the target nucleic acid, wherein said cleavage is cleavage of first and second strands of the viral DNA via at least two double-strand breaks introduced into the viral cccDNA and/or into or adjacent viral DNA integrated into the genome of the organism;
wherein a first double-strand break is introduced at a first position of the DNA by manipulating a first target sequence and a second double-strand break is introduced at a second position of the DNA by manipulating a second target sequence;
wherein upon introduction of first and second double-strand breaks viral sequences between first and second double-strand breaks are excised from cccDNA and/or from the genomic DNA of the organism.
89. The method or composition of claim 87, wherein said composition comprises components of at least four types of CRISPR/Cas complex, wherein each type of complex comprises a guide sequence capable of hybridizing to different sequences of the target nucleic acid, wherein said cleavage is via at least two pairs of single-strand breaks introduced into the viral cccDNA and/or introduced into or adjacent viral DNA integrated into the genome of the organism;
wherein to introduce a first pair of single-strand breaks a first single-strand break is introduced into a first strand of DNA by manipulating a first target sequence to create a first nick and a second single-strand break is introduced into the opposite strand of DNA by manipulating a second target sequence to create a second nick;
wherein to introduce a second pair of single-strand breaks a third single-strand break is introduced into said first strand of DNA by manipulating a third target sequence to create a third nick and a fourth single-strand break is introduced into said opposite strand of DNA by manipulating a fourth target sequence to create a fourth nick;
wherein upon introduction of first and second pairs of single-strand breaks viral sequences between first and second pairs of single-strand breaks are excised from cccDNA and/or from the genomic DNA of the organism.
90. The method or composition of claim 89, wherein first and second nicks are offset relative to each other by at least one base pair so creating a first overhang, and wherein third and fourth nicks are offset relative to each other by at least one base pair so creating a second overhang.
91. The method of composition of claim 88, 89 or 90, wherein following excision of viral sequences the ends of the cleaved first strand of DNA are ligated together and the ends of the cleaved second strand of DNA are ligated together thus reforming unbroken first and second strands.
92. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 85 to 87, 89, 90 or 91, wherein said single-strand break(s) is introduced into DNA by a nickase enzyme which is a modified Cas9 enzyme comprising a substitution leading to catalytic inactivation of the HNH nuclease domain or the RuvC nuclease domain of Cas9; optionally wherein the substitution is at position D10 of SpCas9, preferably a D10A substitution or substitution of a residue corresponding to position D10 in a SpCas9-related enzyme, or wherein the substitution is at position H840 of SpCas9, preferably a H840A substitution or substitution of a residue corresponding to position H840 in a SpCas9-related enzyme.
93. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 53 to 82, wherein said target viral nucleic acid is cccDNA and/or viral DNA integrated into the genome of the organism and wherein said manipulation comprises insertion of one or more nucleotides into or adjacent viral cccDNA sequences or integrated viral DNA sequences, deletion of one or more nucleotides of viral cccDNA or integrated viral DNA, translocation of viral cccDNA sequences or integrated viral DNA sequences, repression of transcription of viral cccDNA sequences or integrated viral DNA sequences, and/or inactivation of viral cccDNA sequences or integrated viral DNA sequences.
94. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of claim 92, wherein repression of transcription of viral cccDNA sequences and/or integrated viral DNA sequences is effected by a CRISPR-Cas system comprising a CRISPR enzyme fused to one or more transcriptional repressor domains, optionally wherein the one or more transcriptional repressor domains comprises KRAB, SID and/or SID4X, preferably wherein the CRISPR enzyme is a Cas9 enzyme.
95. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 83 to 93, wherein said manipulation of nucleotide sequences of viral cccDNA or integrated viral DNA leads to disruption of one or more viral open reading frames, disruption of viral mRNA expression and/or inhibition of the production of functional virions.
96. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 83 to 93, wherein manipulation of said viral cccDNA leads to a reduction in the level of one or more of viral rcDNA, viral cccDNA and viral ssDNA compared to the level in the absence of the CRISPR/Cas complex.
97. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 83 to 95, wherein the effect of said manipulation comprises inhibiting the production of new virions.
98. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 83 to 96, wherein the effect of said modifying comprises removing viral sequences from said organism thereby reducing viral infection.
99. The method or composition of any of claims 53, 54, 58 to 87 and 93 to 98, wherein said composition further comprises components of one or more additional CRISPR/Cas complexes, wherein each type of complex comprises a different guide sequence capable hybridizing to a different sequence of the target nucleic acid within the cell.
100. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 53 to 99 wherein the target viral nucleic acid is a hepatitis B virus (HBV) nucleic acid, preferably wherein the cell expresses the sodium taurocholate cotransporting polypeptide (NTCP) or wherein the cell is a hepatocyte, preferably a primary hepatocyte, more preferably a human hepatocyte or a human primary hepatocyte, a HepG2.2.15 or a HepG2- hNTCP cell.
101. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of claim 100, wherein the guide sequence is capable of hybridizing with target viral nucleic acids of HBV ORF S, ORF C, ORF P, or ORF X, preferably ORF C.
102. The method, composition, complex, chiRNA or DNA polynucleotide molecule of claim 100 or claim 101, wherein the sequence of the guide sequence comprises 5'- gggcgcacctctctttacg-3', 5'-cctctgccgatccatactg-3' or 5’-taaagaatttggagctactg-3’.
103. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 53 to 99 wherein the target viral nucleic acid is a human papillomavirus (HPV) nucleic acid, an Epstein Barr virus (EBV) nucleic acid or a varicella zoster virus (VZV) nucleic acid.
104. The method, composition, complex, chiRNA, DNA polynucleotide molecule or use of any of claims 53 to 103 wherein said manipulation is performed in vitro or ex vivo.
105. The composition, complex, chiRNA or DNA polynucleotide molecule according to any of claims 54 to 66 and 68-99 for use as a medicament.
106. The composition, complex or chiRNA or DNA polynucleotide molecule according to any of claims 54 to 66 and 68-99 for use in the treatment of a viral infection.
107. The composition, complex or chiRNA or DNA polynucleotide molecule for use according to claim 106, wherein the viral infection is caused by hepatitis B virus (HBV).
108. The composition, complex or chiRNA or DNA polynucleotide molecule for use according to claim 106, wherein the viral infection is caused by human papillomavirus (HPV), Epstein Barr virus (EBV) or varicella zoster virus (VZV).
109. The composition, complex or chiRNA or DNA polynucleotide molecule for use according to any of claims 104 to 108 wherein said organism is a mammal.
110. The composition, complex or chiRNA or DNA polynucleotide molecule for use according to claim 109 wherein said mammal is a human.
111. The use of a composition, complex, chiRNA or DNA according to any of claims 54 to 66 or 68-99 in the manufacture of a medicament.
112. The use of a composition, complex, chiRNA or DNA according to any of claims 54 to 66 or 68-99 in the manufacture of a medicament for the treatment of a viral infection.
113. The use according to claim 112, wherein the viral infection is caused by hepatitis B virus (HBV).
114. The use according to claim 112, wherein the viral infection is caused by human papillomavirus (HPV), Epstein Barr virus (EBV) or varicella zoster virus (VZV).
115. The use according to any of claims 111 to 114 wherein said organism is a mammal.
116. The use according to claim 115 wherein said mammal is a human.
PCT/US2014/070135 2013-12-12 2014-12-12 Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders WO2015089465A1 (en)

Priority Applications (14)

Application Number Priority Date Filing Date Title
EP19168543.7A EP3540051B1 (en) 2013-12-12 2014-12-12 Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hsv and viral diseases and disorders.
EP14830911.5A EP3080261B1 (en) 2013-12-12 2014-12-12 Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders
CA2932479A CA2932479A1 (en) 2013-12-12 2014-12-12 Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders
RU2016128077A RU2016128077A (en) 2013-12-12 2014-12-12 DELIVERY, APPLICATION AND APPLICATIONS IN THE THERAPY OF CRISPR-CAS SYSTEMS AND COMPOSITIONS FOR TREATMENT OF CONDITIONED HBV AND VIRAL DISEASES AND DISORDERS
EP22182408.9A EP4183876A1 (en) 2013-12-12 2014-12-12 Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders
KR1020167018647A KR20160089530A (en) 2013-12-12 2014-12-12 Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders
CN201480072878.4A CN105899658B (en) 2013-12-12 2014-12-12 Delivery, Use and Therapeutic Applications of CRISPR-CAS Systems and Compositions for HBV and Viral Diseases and Disorders
MX2016007324A MX2016007324A (en) 2013-12-12 2014-12-12 Delivery, use and therapeutic applications of the crispr-cas systems and compositions for hbv and viral diseases and disorders.
BR112016013207A BR112016013207A2 (en) 2013-12-12 2014-12-12 administration, use and therapeutic applications of crisp systems and compositions for hbv and viral disorders and diseases
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