C delivery, engineering and optimization of systems, methods and compositions for sequence manipulation and therapeutic applications

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 deliver 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 ceils 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, ENGINEERING AND OPTIMIZATIONOF SYSTEMS, METHODS AND COMPOSITIONS FOR SEQUENCE MANIPULATION AND THERAPEUTIC APPLICATIONSRELATED APPLICATIONS AND INC RPORATION BY REFERENCE

[0001] This application claims benefit of and priority to US provisional patent application numbers 61 / 736,527 filed December 12, 2012; 61 / 748,427 filed January 2, 2013; 61 / 758,468 filed January 30, 2013, 61 / 769,046 filed February 25, 2013; 61 / 791,409 and 61 / 802,174 filed March 15, 2013, 61 / 806,375 filed March 28, 2013; 61 / 814,263 filed April 20, 2013; 61 / 819,803 filed May 6, 2013; 61 / 828,130 filed May 28, 2013; 61 / 835,931 and 61 / 836,123 filed June 17, 2013 and 61 / 847,537 filed July 17, 2013.

[0002] Reference is also made to LIS provisional patent application numbers 61 / 799,800 filed March 15, 2013; 61 / 835,931, 61 / 835,936, 61 / 836,127, 61 / 836, 101, 61 / 836,080 and 61 / 835,973 filed June 17, 2013; 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.

[0003] The foregoing applications, and all documents cited therein or during 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

[0004] The present inventio generally relates to the delivery, engineering, optimization and therapeutic applications of systems, methods, and compositions used for the control of gene expressio involving sequence targeting, such as genome perturbatio or gene-editing, that relate to Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and components thereof.STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

[0005] This invention was made with government support under the NIL! Pioneer Award (1 DPI MH 100706) awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND OF THE INVENTION

[0006] Recent advances in genome sequencing techniques and analysis methods have significantly accelerated the ability to catalog and map ge etic 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[0ΘΘ7] 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. NA molecule to recognize a specific DNA target. Adding the CRISPR-Cas system to the repertoire of genome sequencing techniques and analysis methods may significa tly simplify the methodology and accelerate the ability to catalog and map genetic factors associated with a diverse ra ge of biological functio s a d 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 deliver}' of these genome engineering tools, which are aspects of the claimed invention.[ΟΘ08] 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. An exemplar}' CRISPR comple comprises a CRISP R enzyme complexed with a guide sequence hybridized to a target sequence within the targetpolynucleotide. 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 CR ISPR comple 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, dr g discovery, drug screening, disease diagnosis, and prognosis.

[0010] Aspects of the invention relate to Cas9 enzymes having improved targeting specificity in a CR1SPR-Cas9 system having guide RNAs having optimal activity, smal ler 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 deliver}' 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. Al so provided are uses of the same in gene or genome editin g.

[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 (D I O 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 Cas 9 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 DNAmethyltransferase, a cryptochrome, a light inducible / controllable domain or a chemically induci b 1 e / contro liable domain .

[0013] In a further embodiment, the invention provides for methods to generate mutant tracrl A 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 oiigo and added onto the guide RNA. The resulting PGR 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 ceil. In a preferred embodiment the eukaryotic cell is a human cell, hi a more preferred embodiment the human cell is a patient specific cell.

[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 a y chimaeras, mutants, homo logs or orthologs. In a preferred embodiment, the Cas9 is delivered into the ceil in the form of mRNA. This allows for the transient expression of the enzyme thereby reducing toxicity. 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. 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 organizationof 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. tbermopmTus CRISPR locus 1, S. thermoph ius CRISPR locus 3, and Franciscilla novicida type 11 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 Π CRI SPR 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 thai 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, StlCas9 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 protems. Chimeric Cas9 proteins chimeric Cas9s may be new Cas9 containing fragments from more than one naturally occurring Cas9. These methods may comprise fusing N-teraiinal 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 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 ceil,(b) a tracr mate sequence, and(c) a tracr sequence, andIL 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 to the target sequence, and (2) the tracr mate sequence that is hybridized 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 cukaryotie cell, and(b) at least one or more tracr mate sequences,II, a polynucleotide sequence encoding a CRISPR enzyme, andIII. 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 to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, and the polynucleotide sequence encoding a CRISPR. enzyme is DNA or RNA.

[0025] Any or ail 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, nanoparticies, exosomes, micro vesicles, 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 RN A 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 sequencecapable 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 encodi g a CRISPR enzyme comprising at least one or more nuclear localizatio sequences (or optionally at least one or more nuclear localization sequences as some embodiments can involve o NLS), wherei (a), (b) and (c) are arranged in a 5' to 3' orientation, wherem components I and I I 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 to the target sequence, and (2) the tracr mate sequence that is hybridized 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, a d (b) at least o e 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 (I) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate seque ce that is hybridized to the tracr sequence. In some embodiments, components 1, 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, 11 and III is located on different vectors. The invention also provides a viral or plasmid vector system as described herein.0028j 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 know (such as yeast systems, microvesic!es, gene guns / means of attaching vectors to gold nanoparticies) and areprovided. 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 metliylation 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.

[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 (if the organism is multicellular). In the case of humans, for instance, Applicants envisage, inter alia, a single ceil or a population of cells and these may preferabl be modified ex vivo and then re-introduced. In this case, a biopsy or other tissue or biological fluid sample may be necessary. Stem ceils 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 a defect in a target 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 the target 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 AAA'' 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 Π. 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 oneor 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 to the target sequence, and (2) the tracr mate sequence that is hybridized 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 ( If 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 to the target sequence, and (2) the tracr mate sequence that is hybridized 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 IK are located on the same vector, while component II is located on another vector. In other embodiments, components I I 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 111 is located on different vectors. The invention also provides a viral (e.g. AAV or lentivirus) vector system as described herein. , and can be part of a vector system as described herein.

[0032] Some methods of the invention can include inducing expression. In some methods of the invention 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. Anon-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 algae, including microaigae, or is a fungus. In some methods of the mvention 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.

[0033] The invention in some embodiments comprehends a method of delivering a CRISPR enzyme comprising delivering to a ceil mRNA encoding the CRISPR enzyme. In some of these methods the CRISPR enzyme is a Cas9.|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-mfected 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 obligator}7for replication and packaging of the AA V 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 cel ls. And in some embodiments the cells are insect ceils and the helper virus is baculovirus. In other embodiments, the virus is a lentivirus.

[0035] In plants, pathogens are often host-specific. For example, Fusarium oxysporum f. sp. lycopersici causes tomato wilt but attacks only tomato, and F. oxysporum f. dianthii Puccinia graminis f. sp. tritici attacks only wheat. Plants have existing and induced defenses to resist most pathogens. Mutations and recombination events across plant generations lead to genetic variability thai gives rise to susceptibility, especially as pathogens reproduce with more frequency than plants. In plants there can be non-host resistance, e.g., the host and pathogen are incompatible. There can also be Horizontal Resistance, e.g., partial resistance against all races of a pathogen, typically control led by many genes and Vertical Resistance, e.g., complete resistance to some races of a pathogen but not to other races, typically controlled by a few genes. In a Gene-for-Gene level, plants and pathogens evolve together, and the genetic changes in onebalance changes in other. Accordingly, using Natural Variability, breeders combine most useful genes for Yield, Quality, Uniformity, Hardiness, Resistance. The sources of resistance genes include native or foreig Varieties, Heirloom Varieties, Wild Plant Relatives, and Induced Mutations, e.g., treating plant material with mutagenic agents. Using the present invention, plant breeders are provided with a new tool to induce mutations. Accordingly, one skilled in the art can analyze the genome of sources of resistance genes, and in Varieties having desired characteristics or traits employ the present invention to induce the rise of resistance genes, with more precision than previous mutagenic agents and hence accelerate and improve plant breeding programs.

[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 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 comprehends in some embodiments a composition of the Invention or a CRISPR enzyme thereof (including or alternatively mRN A encoding the CRISPR enzyme), wherein the target sequence is flanked at its 3 ' end by a PAM (protospacer adjacent motif) sequence comprising 5 '-motif, 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.

[0037] It will be appreciated that SpCas9 or SaCas9 are those from or derived from 5. pyogenes or S. aureus Cas9.

[0038] 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 ofmodifying 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 noii-naturaily occurring or engineered composition comprising :

[0039] 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,

[0040] 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,(h) a second tracr mate sequence, and(c) a second tracr seque ce, and

[0041] 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 comple comprises the CRISPR enzyme complexed with (1) the first guide sequence that is hybridized to the first target sequence, and (2) the first tracr mate sequence that is hybridized to the first tracr sequence, wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized to the second target sequence, and (2) the second tracr mate sequence that is hybridized 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.

[0042] 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 CR ISPR 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, micro vesicles, 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 in vention 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 DI0A mutation. In preferred embodiments, the first CRISPR enzyme has one or more mutations such thai 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.1 043| 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.

[0044] 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-naturaily occurring or engineered composition comprising a vector system comprising one or more vectors comprising

[0045] I. a first regulatory element operabiy 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,

[0046] II. a second regulatory element operabiy 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,

[0047] III. a third regulatory element operabiy linked to an enzyme-coding sequence encoding a CRISPR enzyme, and

[0048] IV. a fourth regulatory element operabiy linked to a tracr sequence,

[0049] wherein components L 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 a d the second guide sequence direct sequence-specific binding of a first and a seco d 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 seque ce that is hybridized to the first target sequence, and (2) the tracr .mate sequence that is hybridized to the tracr sequence, wherein the second CRISPR complex comprises the CRISPR enzyme complexed with (1) the second guide sequence that is hybridized to the second target sequence, and (2) the tracr mate sequence that is hybridized 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.

[0050] 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, I I, I II and IV can be located on the same vector; components I, II, III and IV can each be located on different vectors; components I, ( 1, II I and I V may be located on a total of two or three different vectors, with al l combinations of locations envisaged, etc.

[0051] 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 CRJSPR enzyme comprises one or more mutations in a catalytic domain, wherein the one or more mutations are selected from the group consisting of DIOA, E762A, H840A, N854A, N863A and D986A. In a highly preferred embodiment the CRISPR enzyme has the DIOA 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, micro vesicles, or a gene-gun.

[0052] 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.

[0053] 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 CR ISPR-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 DNAmolecule 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.

[0054] In preferred methods of the invention the Cas protein nicking each of the first stra d and the second strand of the DM A molecule encoding the gene product results in a 5' overhang. I 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] Embodiments of the invention also comprehend the guide RNAs comprising a guide sequence fused to a tracr mate sequence and a tract" sequence. In an aspect of the invention the Cas protein is codon optimized for expression in a eukaryotic cel l, preferably a mammalian cell or a human ceil. In further embodiments of the invention the Cas protein is a type II CRISPR- Cas protein, e.g. a Cas 9 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.

[0056] Aspects of the in vention 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.

[0057] 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,

[0058] 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 atype II CRISPR-Cas protein. In an aspect of the invention the Cas protein is eodon 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 Cas 9 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.

[0059] Aspects of the in vention 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.

[0060] The invention also comprehends a engineered, noii-naturaily occurring vector system comprising one or more vectors com rising: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 a d the Cas protein icks 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.

[0061] 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 embodimen t 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 ceil, preferably a mammalian cell or a human cell. In further embodiments of the invention the Cas protein is a t pe I I CR ISPR-Cas protein, e.g. a Cas 9 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 groupconsisting of D10A, E762A, H840A, N854A, N863A and D986A. In a highly preferred embodiment the Cas protein has the D10A mutation.

[0062] 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, nanoparticies, exosomes, microvesicl.es, or a gene-gun.10063| 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 compiexed 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 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 tosaid modifying. In some embodiments, the method further comprises returning said eukaryotie cell and / or cells derived therefrom to said subject.

[0064] In one aspect, the invention provides a method of modifying expression of a polynucleotide in a eukaryotie cell. In some embodiments, the method comprises allowing a CRISPR complex to bind to the polynucleotide such that said binding results in i creased 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. In some embodiments, the method further comprises delivering one or more vectors to said eukaryotie 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.[CI065] i one aspect, the invention provides a method of generating a model eukaryotie cell comprising a mutated disease gene. In some embodiments, a disease gene is any gene associated with an increase in the risk of having or developing a disease. In some embodiments, the method comprises (a) introducing one or more vectors into a eukaryotie cell, wherein the one or more vectors drive expression of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, and a tracr sequence; and (b) allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target poly ucleotide within said disease ge e, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (I) the guide sequence that is hybridized to the target sequence within the target poly ucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence, thereby generating a model eukaryotie cell comprising a mutated disease gene. 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 expression from a gene comprising the target sequence.

[0066] In one aspect the invention provides for a method of selecting one or more prokaryotie cell(s) by introducing one or more mutations in a gene in the one or more prokaryotie cell (s), the method comprising: introducing one or more vectors into the prokaryotie cel l (s), wherein the one or more vectors drive expressio of one or more of: a CRISPR enzyme, a guide sequence linked to a tracr mate sequence, a tracr sequence, and an editing template; wherein the editing template comprises the one or more mutations that abolish CRISPR enzyme cleavage; allowing homologous recombination of the editing template with the target polynucleotide in the cell(s) to be selected; allowing a CRISPR complex to bind to a target polynucleotide to effect cleavage of the target polynucleotide within said gene, wherein the CRISPR complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence within the target polynucleotide, and (2) the tracr mate sequence that is hybridized to the tracr sequence, wherein binding of the CRISPR complex to the target polynucleotide induces cell death, thereby allowing one or more prokaryotie cell(s) in which one or more mutations have been introduced to be selected. In a preferred embodiment, the CRISPR enzyme is Cas9, In another aspect of the invention the cell to be selected may be a eukaryotic cell. Aspects of the invention allow for selection of specific ceils without requiring a selection marker or a two-step process thai may include a counter-selection system,

[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 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 increasing or decreasing expression of a target polynucleotide by using a CRJSPR 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 encodingsaid 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 to the target sequence, and (2) the tracr mate sequence that is hybridized 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 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 RuvCl or HNH domain of the CRISPR enzyme or is a mutatio 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.

[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 I I 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 CRISPR99system. 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 wlidtype 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, Stl Cas9 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, deliver}.' 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 nanopartic!es) 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 deliver}' 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 ceil niR A 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 CR ISPR 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] It will be appreciated that SpCas9 or SaCas9 are those from or derived from S. pyogenes or S. aureus Cas9.

[0079] Accordingly, 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. § 1 12, first paragraph) or the EPO (Article 83 of the EPC), such that Applicants reserve the right and hereby disclose a disclaimer of any previously described product, process of making the product, or method of using the product.

[0080] 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.

[0081] These and other embodiments are disclosed or are obvious from and encompassed by, the following Detailed Description.BRIEF DESCRIPTION OF THE DRAWINGS[CI082] The novel features of the invention are set forth with particularity i 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:

[0083] 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 seque ce (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 P AM (red triangle).

[0084] 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.

[0085] Figure 3A-D shows results of an evaluation of SpCas9 specificity for an example target.

[0086] Figure 4A-G show an exemplary vector system and results for its use in directing homologous recombination in eukaryotic cells.

[0087] 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 ceil 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).

[0088] Figure 6A-C shows a comparison of different tracrRNA transcripts for Cas9-mediated gene targeting.

[0089] Figure 7 shows a schematic of a surveyor nuclease assay for detection of double strand break-induced micro-insertions and -deletions.

[0090] Figure 8A-B shows exemplary bicistronic expression vectors for expression of CRISPR system elements in eukaryotic cel ls.[00 J ] 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).

[0092] Figure 10A-D shows an exemplary CRISPR. system, an example adaptation for expression in eukaryotic cells, and results of tests assessing CRISPR activity.

[0093] Figure 11 A-C shows exemplary manipulations of a CRISPR system for targeting of genomic loci in mammalian cells.

[0094] Figure 12A-B shows the results of a Northern blot analysis of crRNA processing in mammalian cells.

[0095] Figure 13A-B shows an exemplary selection of protospacers in the human PVALB and mouse Th loci.

[0096] Figure 14 shows example protospacer and corresponding PAM sequence targets of the S. thermophilus CRISPR. system in the human EMXl locus.

[0097] Figure 15 provides a table of sequences for primers and probes used for Surveyor, RFLP, genomic sequencing, and Northern blot assays.

[0098] Figure 16 A-C shows exemplary manipulation of a CRISPR system with chimeric RNAs and results of SURVEYOR assays for system activity in eukaryotic cells.

[0099] Figure 17A-B shows a graphical representation of the resul ts of SURVEYOR, assays for CRISPR system activity in eukaryotic cells.

[0100] Figure 18 shows an exemplary visualization of some S. pyogenes Cas9 target sites in the human genome using the UCSC genome browser.

[0101] Figure 19A-D shows a circular depiction of the phylo genetic analysis revealing five families of Cas9s, including three groups of large Cas9s (- 1400 amino acids) and two of small Cas9s (~1100 amino acids),

[0102] 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 (~.l .100 amino acids).

[0103] 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 (FIR.) at the human EMXl locus using either sense or amisense single stranded oligonucleotides as repair templates. Red arrow above indicates sgRNA cleavage site; PCR primers for genotyping (Tables J and ) are indicated as arrows in right panel, (c) Sequence of region modified by HR. d, SURVEYOR assay for wildtype (wt) andnickase (D10A) SpCas9-mediated indels at the EM XI target 1 locus (n~3). Arrows indicate positions of expected fragment sizes.

[0104] Figure 22A-B shows single vector designs for SpCas9.

[0105] Figure 23 shows a graph representing the length distribution of Cas9 ortho!ogs.

[0106] Figure 24A-M shows sequences where the mutation points are located within the SpCas9 gene.

[0107] Figure 25A shows the Conditional Cas9, Rosa26 targeting vector map.

[0108] Figure 25B shows the Constitutive Cas9, Rosa26 targeting vector map.

[0109] Figure 26 shows a schematic of the important elements in the Constitutive andConditional Cas9 constructs.

[0110] Figure 27 shows delivery and in vivo mouse brain Cas9 expression data.

[0111] 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. cel ls. (B) Delivery of Cas9 and chimeric RNA against the Ieam2 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.[001 12] 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, hide! mutations occurring within the coding region of a gene can result in frame-shift and a premature stop codoii, leading to gene knockout. Alternatively, a repair template in the form of a plasmid or single-stranded oligodcoxynuelcotides (ssODN) can be suppli ed to leverage the homo logy-directed repair (HDR) pathway, which allows high fidelity and precise editing.

[0113] 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, we introduced a Hindlll site (red bar) into the target locus, which was PCR-amplified with primers that anneal outside of the region of homology. Digestion of the PGR. product wit Hindlll 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 Cas977to achieve efficient BDR-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

[0114] Figure 31 A-C shows the repair strategy for Cystic Fibrosis delta F508 mutation.

[0115] 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.

[0116] Figure 33 shows a screen for efficient SpCas9 mediated targeting of Tetl-3 and Drsmtl , 3a and 3b gene loci. Surveyor assay on DMA from transfected N2A ceils demonstrates efficient DNA cleavage by using different gRNAs.[001 J T] Figure 34 shows a strategy of multiplex genome targeting using a 2 -vector system in an AAV 1 / 2 delivery system. Tetl-3 and Dnmtl, 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 AAV 1 / 2 virus in a 1 :1 ratio.

[0118] Figure 35 shows verification of multiplex DNMT targeting vector #1 functionality using Surveyor assay. N2A ceils 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.

[0119] Figure 36 shows verification of multiplex DNMT targeting vector #2 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.

[0120] Figure 37 shows schematic overview of short promoters and short polyA versions used for HA-SpCas9 expression in vivo. Sizes of the encoding region from L-ITR to R-ITR are shown on the right.

[0121] Figure 38 shows schematic overview of short promoters and short poiyA versions used for HA-SaCas9 expression in vivo. Sizes of the encoding region from L-ITR to R-ITR are shown on the right.

[0122] Figure 39 shows expression of SpCas9 and SaCas9 in N2A ceils. Representative Western blot of HA-tagged SpCas9 and SaCas9 versions under the control of different short promoters and with or short polyA (spA) sequences. Tubulin is loading control. mCherry (mCh) is a transfection control. Cells were harvested and further processed for Western blotting 48 h after transfection.

[0123] Figure 40 shows screen for efficient SaCas9 mediated targeting of Tet.3 gene locus. Surveyor assay on DNA from transfecte N2A cells demonstrates efficient DNA cleavage by using different gRNAs wit NNGGGT PUM sequence. GFP transfected cells and ceils expressing only SaCas9 are controls.

[0124] Figure 41 shows expression of A-SaCas9 in the mouse brain. Animals were injected into dentate gyri with vims driving expression of HA-SaCas9 under the control of human Synapsin promoter. Animals were sacrificed 2 weeks after surgery. HA tag was detected using rabbit monoclonal antibody C29F4 (Cell Signaling). Cell nuclei stained in blue with DAPI stain.

[0125] Figure 42 shows expression of SpCas9 and SaCas9 in cortical primary neurons in culture 7 days after transduction. Representative Western blot of HA-tagged SpCas9 and SaCas9 versions under the control of different promoters and with bgh or short polyA (spA) sequences. Tubulin is loading control.

[0126] Figure 43 shows LIVE DEAD stain of primary cortical neurons 7 days after transduction with AAV 1 particles carrying SpCas9 with different promoters and multiplex gRNAs constructs (example shown on the last panel for DNMTs). Neurons after AAV transduction were compared with control untransduced neurons. Red nuclei indicate permeabilized, dead cells (second line of panels). Live ceils are marked in green color (third line of panels).

[0127] Figure 44 shows LIVE / DEAD stain of primary cortical neurons 7 days after transduction with AAVl particles carrying SaCas9 with different promoters. Red nuclei indicate permeabilized, dead ceils (second line of panels). Live cells are marked in green color (third line of panels).

[0128] Figure 45 shows comparison of morphology of neurons after transduction with AAV1 virus carrying SpCas9 and gRNA multiplexes for TETs and DNMTs genes loci. Neurons without transduction are shown as a control.

[0129] Figure 46 shows verification of multiplex DNMT targeting vector #1 functionality using Surveyor assay in primary cortical neurons. Cells were co -transduced with the DNMT targeting vector #1 and the SpCas9 viruses with different promoters for testing SpCas9 mediated cleavage of DNMTs genes family loci.

[0130] Figure 47 shows in vivo efficiency of SpCas9 cleavage in the brain. Mice were injected with AAV 1 / 2 virus carrying gRNA multiplex targeting DNMT family genes loci together with SpCas9 viruses under control of 2 different promoters: mouse Mecp2 and rat- Map lb. Two weeks after injection brain tissue was extracted and nuclei were prepped and sorted using FACS, based on the GFP expression driven by Synapsin promoter from gRNA multiplex construct. After gDNA extraction Surveyor assay was run. + indicates GFP positive nuclei and - control, GFP -negative nuclei from the same animal. Numbers on the gel indicate assessed SpCas9 efficiency.

[0131] Figure 48 shows purification of GFP-KASH labeled cell nuclei from hippocampal neurons. The outer nuclear membrane (ONM) of the cell nuclear membrane is tagged with, a fusion of GFP and the ASH protein transmembrane domain. Strong GFP expression in the brain after one week of stereotactic surgery and AAV 1 / 2 injection. Density gradient eentrifugation step to purify cell nuclei from intact brain. Purified nuclei are shown. Chromatin stain by Vybrant® DyeCycle™ Ruby Stain is shown in red, GFP labeled nuclei are green. Representative FACS profile of GFP+ and GFP- cell nuclei (Magenta: Vybrant® DyeCycle™ Ruby Stain, Green: GFP).[00.1.32] Figure 49 shows efficiency of SpCas9 cleavage in the mouse brain. Mice were injected with AAV 1 / 2 virus carrying gRNA multiplex targeting TET family genes loci together with SpCas9 viruses under control of 2 different promoters: mouse Mecp2 and rat Map lb. Three weeks after injection brain tissue was extracted, nuclei were prepped and sorted using FACS, based on the GFP expression driven by Synapsin promoter from gRNA multiplex construct. After gDNA extraction Surveyor assay was run. + indicates GFP positive nuclei and - control, GFP-negative nuclei from the same animal. Numbers on the gel indicate assessed SpCas9 efficiency.

[0133] Figure 50 shows GFP-KASH expression in cortical neurons in culture. Neurons were transduced with AAV1 virus carrying gRNA multiplex constructs targeting TET genes loci. The strongest signal localize around cells nuclei due to KASH domain localization.

[0134] Figure 51 shows (top) a list of spacing (as indicated by the pattern of arrangement for two PAM sequences) between pairs of guide RNAs. Only guide RNA pairs satisfying patterns 1, 2, 3, 4 exhibited indels when used with SpCas9(D10A) nickase. (bottom) GeS images showing that combination of SpCas9(Dl OA) with pairs of guide RNA satisfying patterns 1, 2, 3, 4 led to the formation of indels in the target site.

[0135] Figure 52 shows a list of U6 reverse primer sequences used to generate U6-guide RNA expression casssett.es. Each primer needs to be paired with the U6 forward primer "gcactgagggcctatttcccatgattc" to generate amplicons containing U6 and the desired guide RN A.

[0136] Figure 53 shows a Genomic sequence map from the human Emxl locus showmg the locations of the 24 patterns listed in Figure 33.

[0137] Figure 54 shows on (right) a gel image indicating the formation of indels at the target site when variable 5' overhangs are present after cleavage by the Cas9 nickase targeted by different pairs of guide RNAs. on (left) a table indicating the lane numbers of the gel on the right and various parameters including identifying the guide RNA pairs used and the length of the 5' overhang present following cleavage by the Cas9 nickase.

[0138] Figure 55 shows a Genomic sequence map from the human Emxl locus showing the locations of the different pairs of guide RNAs that result in the gel patterns of Fig. 54 (right) and which are further described in Example 35.

[0139] Figure 56 shows staining of HA-SpCas9 in dorsal and ventral hippocampus 8 weeks after injection of viruses encoding M ecp2-HA-SpCas9 and 3xgRNA-TETS with Syn-KASH- GFP.

[0140] Figure 57 shows Syn_GFP- ASH expression 8 weeks after 3xgRNA virus injection is specific for neurons (NeuN positive cells) and not for glia cells (GFAP positive).

[0141] Figure 58 shows behavior tests conducted 5 weeks after CRISPR-mediated KD of TETs and DNMTs in dentate gyrus (ventral and dorsal part) showed increased level of anxiety and learning deficits. A) time spend in the open arm during elevated plus maze test. B) open field test, time spent in the center of arena vs time in the corners was measured. C) Novel object recognition test, results were measured 3h after familiarization phase. D) Barnes maze;efficiency in finding escape within 3 days of training. E) Barnes maze results. F) Freezing behavior during contextual fear conditioning. G) Latency to first freezing episode during contextual fear conditioning. H) Trace fear conditioning results for TETs KD and DNMTs KD (1). Control - animals injected with SpCas9 virus and GFP- ASH construct without g NAs. TETs - animals injected with both SpCas9 and construct encoding gRNAs against Tetl, Tet2 and Tet3. DNMTs - animals injected wit both SpCas9 and construct encoding gRNAs against Dnmt l , Dnmt3a and Dnmt3b.

[0142] Figure 59 shows cutting efficiency of Tet loci in the brain, 8 weeks after Mecp_SpCas9 vims injection in compare to control animals mjected with Mecp2_SpCas9 virus only.[00143 J Figure 60 shows cutting efficiency of Dnmt loci in the brain, 8 weeks after Mecp SpCas9 virus injection in compare to control animals injected with Mecp2 SpCas9 virus only,

[0144] Figure 61 shows Dnmt3a staining in the brain, 8 weeks after stereotaxic injection of virus encoding Mecp2 SpCas9 and gRNAs targeting Dnmt loci. Bottom panel shows magnification of ROl indicated on the upper panel.

[0145] Figure 62 shows staining of Syn HA-SaCas9 in the dorsal hippocampus, 4 weeks after injection of virus. First column shows animal injected with Sa-Cas9 only, middle column animal injected with both SaCas9 and gRNAs against TETs loci and the right column represents animal injected with only gRNAs encoding virus. SaCas9 nuclear localization depends on the presence of gRNA.

[0146] Figure 63 shows SpCas9 in 2a cells. A) Targeting- and SpCas9 expression vector, B) Western Blot analysis of N2a cells expressing HA-tagged SpCas9 under the control of different promoters. C) Cutting efficiency of Dnmt loci. D) Western blot analysis demonstrating efficient knock down of Dnmt3a. e) Cutting efficiency of Tet loci.

[0147] Figure 64 shows SpCas9 in primary neurons. A) Schematic overview of SpCas9 cloning strategies used in this study. Short promoters and short polyA for efficient packaging into AAV delivering system. B) Schematic overview of combined multiplex targeting and nuclear envelope labeling strategy. C) Western blot analysis showing expression of HA-tagged SpCas9 under the control of rMaplb and HiMecp2 promoter and bGH and spA signal. D) Immunocytochemistry demonstrating co-expression of SpCas9 and GFP-KASH in primaryneurons, SpCas9 under the control of the mMecp2 promoter is expressed in neurons (Map lb, NeiiN) but not in astroglia cells (GFAP).

[0148] Figure 65 shows knock down of Dnmt3a in primary neurons. A) Immunocytochemistry demonstrating efficient knock down of Dnmt3a after targeting with multiplex targeting vector and mMeep2-SpCas9, B) Quantification of Dnmt3a antibody staining in control and targeted neurons. C) Western blot analysis demonstrating reduced Dnmt3a protein level. D) Quantification of W'"estcrn Blot analysis demonstrating a total knock down of Dnmt3a protein level of approx. 75% in a mixed primary neuron culture (neurons and astroglia).

[0149] Figure 66 shows knock down of Dnmt3a in vivo. A) Cutting efficiency of Dnmt loci in the brain, 8 weeks after Mecp SpCas9 virus injection in compare to control animals injected with Mecp2_SpCas9 vims only. B) Western blot analysis showing reduced Dnmt3a protein level in targeted neuronal nuclei (KASH-GFP positive) compared to control nuclei (RubyDye positive) after sorting cell nuclei using FACS.

[0150] Figure 67 shows expression of SaCas9 in primary neurons. A) Size of SaCas9 expression vector using hSynapsin promoter and bGH signal. B) Expression of SaCas9 in primary neurons (NeuN) but not in astroglia (GFAP). C) Extranuclear localization of SaCas9 in absence of gRNA. C Higher magnification of SaCas9 positive neurons shown in C). D) Nuclear localization of SaCas9 in presence of gRNA . D') Higher magnification of SaCas9 positive neurons shown in D). E) Western blot analysis demonstrating expression of HA-tagged SaCas9 and GFP- ASH. F) Cutting efficie cy of Dnmt foci 1 week after AAV infection.

[0151] Figure 68 shows gRNA dependent nuclear localization of SaCas9. A) Confocal imaging analysis demonstrating extranuclear localization of SaCas9 in absence of gRNA in primary neurons. B) Nuclear localization of SaCas9 in presence of gRNA. C) Line Scan analysis of confocal picture A) showing extranuclear localization of SaCas9 in absence of gRNA (red, SaCas9 signal; blue, DAPI signal; green, GFP-KASH signal . D) Line Scan analysis of confocal picture B) showing nuclear localization of SaCas9 in presence of gRNA (red, SaCas9 signal; blue, DAPI signal; green, GFP-KASH signal). E) Subcellular localization of SaCas9 and SpCas9 under conditions without (-) and with (+) gRNA in N2a cells. SaCas9 signal at 250 kDa in the cytoplasm fraction (Tubulin positive) indicating dirnerization of SaCas9 in the cytoplasm. In the presence of gRNA a shift of SaCas9 protein into the nuclear fraction (Sun2 positive) is visible. SaCas9 signal at 100 kDa indicates a gRNA dependent formation of SaCas9 homomers andtransport into the cell nucleus. In contrast, SpCas9 is mainly present as homomer and its nuclear localization is independent of gRNA.

[0152] Figure 69 shows an AAV-Sa-Cas9 vector, a liver-specific AAV-Sa-Cas9 vector and an alternate AAV-Sa-Cas9 vector,

[0153] Figure 70 shows data on optimized CMV-SaCas9-NLS-U6-sgRNA vector (submitted vector design last time); new data compares N'-term vs C'-term tagged SaCas9 and shows enhanced cleavage efficiency using C'-term NLS tagging.

[0154] Figure 71 shows SURVEYOR image showing indels generated by new Pcsk9 targets.

[0155] Figure 72 shows SaCas9 specificity: genome -wide off target sites (GWOTs) are predicted based on 2 criteria: they contain 4 or fewer mismatched bases to intended SaCas9 target and bear the least restrictive PAM for SaCas9, W'GRR. H i-. 293FT cells are transfected with either SpCas9 or SaCas9 with their corresponding sgRNAs at a target site (EMX1 : T AGGGTT A GG GG CC C C AGGC) that has CGGGGT as a PAM so that it can be cut by either SpCas9 (CGG) or SaCas9 (CGGGGT). DNAs from ceils are harvested and analyzed for indels by Illumina sequencing at on-target and 41 predicted off-target loci (following protocols from Hsu et al. Nature Biotech 2013 and data analysis pipeline developed by David Scott and Josh Wei stein).

[0156] Figure 73 shows that that SaCas9 may have a higher level of off-target activity than SpCas9 at certain loci,|00157| The figures herein are for illustrative purposes only and are not necessarily drawn to scale.DETAILED DESCRIPTION OF THE INVENTION

[0158] 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 -C as system and components thereof. In advantageous embodiments, the Cas enzyme is Cas9.

[0159] An advantage of the present methods is that the CRISPR system 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

[0160] Cas9 optimization may be used to enhance function or to develop new functions, one can generate chimeric Cas9 proteins. Examples that the Applicants have generated are provided in Example 6. Chimeric Cas9 proteins can be made by combining fragments from different Cas9 homoSogs. For example, two example chimeric Cas9 proteins from the Cas9s described herein. For example. Applicants fused the N-term of StlCas9 (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.

[0161] The Cas9 may be used as a generic DNA binding protein . For example, and as shown in Example 7, Applicants used Cas9 as a generic DNA binding protein by mutating the two catalytic domains (DIG and H840) responsible for cleaving both strands of the DNA target. In order to upreguSate gene transcription at a target locus Applicants fused a transcriptional activation domain (VP64) to Cas9. Other transcriptional activation domains are known. As shown in Example 17, transcriptional activation is possible. As also shown in Example 1 7, gene repression (in this case of the beta-eatenm gene) is possible using a Cas9 repressor (DNA- binding domain) that binds to the target gene sequence, thus repressing its activity.

[0162] Cas9 and one or more guide RNA can be delivered using adeno associated vims (AAV), ientivirus, 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 Ientivirus, 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, 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.

[0163] 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 might use the Synapsin I promoter.Transgenic animals and plants

[0164] 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 knoekin 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 os. 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 presen t invention .

[0165] 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 Rl mESCs can have active Cas9 when Cre is expressed. Because Cas9 is fol lowed 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 deliver}'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.

[0166] As mentioned above, transgenic animals are also provided, as are transgenic plants, especially crops and algae. The transgenic plants may be useful in applications outside of providing a disease model. These may include food or feed production through expression of, for instance, higher protein, carbohydrate, nutrient or vitamin levels than would normally be seen in the wildtypc. In this regard, transgenic plants, especially pulses and tubers, and animals, especially mammals such as livestock (cows, sheep, goats and pigs), but also poultry and edible insects, are preferred.

[0167] Transgenic algae or other plants such as rape may be particularly useful in the production of vegetable oils or biofuels such as alcohols (especially methanol and ethanol), for instance. These may be engineered to express or overexpress high levels of oil or alcohols for use in the oil or biofuel industries.Adeno associated vims (AAV)

[0168] In terms of in vivo delivery, AAV is advantageous over other viral vectors for a couple of reasons:

[0169] Low toxicity (this may be due to the purification method not requiring ultra eentrifugation of cel l particles that can activate the immune response)

[0170] Low probability of causing insertional mutagenesis because it doesn't integrate into the host genome.

[0171] 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 vims 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 SizeCorynebacter diphtheriae 3252 Eubacterium ventriosumStreptococcus pasteurianus 3390Lactobacillus farciminis 3378 Sphaerochaeta globus 3537350431503246342031113159339630093396

[0172] These species are therefore, in general, preferred Cas9 species. Applicants have shown delivery and in vivo mouse brai Cas9 expression data.

[0173] Two ways to package Cas9 coding nucleic acid molecules, e.g., DNA, into viral vectors to mediate genome modification in vivo are preferred:

[0174] To achieve NHEJ-mediated gene knockout:

[0175] Single virus vector:Vector containing two or more expression cassettes:Promoter-Cas9 coding nucleic acid molecule -terminatorPromoter-gRNA 1 -terminatorPromoter~gRNA2-terminatorPromoter- gl^NA( N) -terminator (up to size limit of vector)

[0176] Double virus vector;Vector 1 containing one expression cassette for driving the expression of Cas9 Promoter~Cas9 coding nucleic acid molecule-temiinatorVector 2 containing one more expression cassettes for driving the expression of one or more guideRNAsPromoter-gR A 1 -terminatorPromoter-gRNA(N)-terminator (up to size limit of vector)

[0177] To mediate homology-directed repair. In addition to the single and double vims vector approaches described above, an additional vector is used to deliver a homology-direct repair template.

[0178] Promoter used to drive Cas9 coding nucleic acid molecule expression can include:

[0179] 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 spacefreed 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 toxicity due to over expression of Cas9.

[0180] For ubiquitous expression, can use promoters: CMV, CAG, CBh, PGK, SV40, Ferritin heavy or light chains, etc.

[0181] For brain expression, can use promoters: Synapsinl for all neurons, CaMKIIalpha for excitatory neurons, GAD67 or GAD65 or VGAT for G ABAergic neurons, etc.

[0182] For liver expression, can use Albumin promoter.

[0183] For lung expression, can use SP~B.

[0184] For endothelial cells, can use ICAM.

[0185] For hematopoietic cells can use IFNbeta or CD45.

[0186] For Osteoblasts can use OG-2.

[0187] Promoter used to drive guide RNA can include:Pol 10 promoters such as U6 or HIUse of Pol II promoter and mtronic cassettes to express gRNA

[0188] 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 or capsid AAV J , AAV2, AAV5 or any combination thereof for targeting brain or neuronal cells; and one can select AAV4 for targeting cardiac tissue. AAA'S is useful for delivery to the liver. The above promoters and vectors are preferred individually.

[0189] RN A delivery is also a useful method of in vivo delivery. Fig, 27 shows delivery and in vivo mouse brain Cas9 expression data. 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.

[0190] 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 u86. HR efficiency canalso be increased by co-expressing prokaryotic or eukaryotic homologous recombination enzymes such as RecBCD, RecA.

[0191] Various means of delivery are described herein, and further discussed in this section.

[0012] Viral deliver)?: The CRISPR enzyme, for instance a Cas9, and / or any of the present RNAs, for instance a guide RNA, can be delivered using 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 viral vectors. In some embodiments, the viral vector is delivered to the tissue of interest by, for example, an intramuscular injection, while other times the viral delivery is via intravenous, transdermal, intranasal, oral, mucosal, or other delivery methods. Such deliver}' 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 chose, the target cell, organism, or tissue, the general condition of the subject to be treated, the degree of transformation^nodification sought, the administration route, the administration mode, the type of transformation / modification sought, etc,

[0193] 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 pharmaceuticaily-acceptable carrier (e.g., phosphate-buffered saline), a pharmaceuticaily-acceptabie excipient, an adjuvant to enhance antigenicity, an immunostimulatory compound or molecule, and / or other compounds known in the art. The adjuvant herein may contain a suspension of minerals (alum, aluminum hydroxide, aluminum phosphate) on which antigen is adsorbed; or water-in-oil emulsion in which antigen solution is emulsified in oil (MF-59, Freund's incomplete adjuvant), sometimes with the inclusion of killed mycobacteria (Freund's complete adjuvant) to further enhance antigenicity (inhibits degradation of antigen and / or causes influx of macrophages). Adjuvants also include immunostimulatory molecules, such as cyiokincs. costimulatory molecules, and for example, immunostimulatory DNA or RNA molecules, such as CpG oligonucleotides. Such a dosage formulation is readily ascertainable by one skilled 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 aswetting 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, antieaking agents, fillers, chelatmg agents, coating agents, chemical stabilizers, etc. may also be present, especially if the dosage form is a reeonstitutable form. Suitable exemplary ingredients include macrocrystalline cellulose, earboxymethyicelhilose 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 excipienis is available in REMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991) which is incorporated by reference herein.

[0194] In an embodiment herein the delivery is via an adenovirus, which may be at a single booster dose containing at least 1 x 105particles (also referred to as particle units, pu) of adenoviral vector. In an embodiment herein, the dose preferably is at least about 1 x I06particles (for example, about 1 x 10°- 1 x 1012particles), more preferably at least about 1 x 10'' particles, more preferably at least about 1 x 108particles (e.g., about 1 x 10δ-1 x 10nparticles or about 1 x Ι Ο'-Ι x 10" particles), and most preferably at least about 1 10' particles (e.g., about 1 * x 109-1 x 1010particles or about 1 x 109-1 x l Ql / ~ particles), or even at least about 1 x 10f 0particles (e.g., about 1 x IQlU-l x 10 particles) of the adenoviral vector. Alternatively, the dose comprises no more than about 1 x 10f 4particles, preferably no more than about 1 x lO1' particles, even more preferably no more than about 1 x 101particles, even more preferably no more than about 1 x 10Hparticles, and most preferably no more than about 1 x 10'° particles (e.g., no more than about 1 x 109articles). Thus, the dose may contain a single dose of adenoviral vector with, for example, about 1 x 10° particle units (pu), about 2 x 106pu, about 4 x 106pu, about 1 x 10 ' pu, about 2 x 10 ' pu, about 4 x 10' pu, about 1 x 10Bpu, about 2 x 108pu, about 4 x 108pu, about 1 x 109pu, about 2 x 1Q9pu, about 4 x 109pu, about 1 x 1010pu, about 2 X 1()ϊ ύpu, about 4 x 101pu, about 1 x 10f 1pu, about 2 x ! Q5 ipu, about 4 x 10npu, about 1 x 10'2pu, about 2 x 10f / pu, or about 4 x 1012pu 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.

[0195] 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 I x IQ1Jto about I x I01Jfunctional 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 105to 1 x 1050genomes AAV, from about 1 x 108to 1 x 10"° genomes AAV, from about 1 x 10i0to about 1 x 10i6genomes, or about 1 x 10f 1to about 1 x 10f 6genomes AAV. A human dosage may be about 1 x 10ljgenomes 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.

[0196] 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 I μ¾ to about 10 ug.

[0197] 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.Lenti virus

[0018] 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 lcntivirus is the human immunodeficiency virus (H IV), which uses the envelope glycoproteins of other viruses to target a broad range of cell types.

[0199] Lentiviruses may be prepared as follows. After cloning pCasES!O (which contains a lenti viral 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. Ceils were transfected with 10 iig of lentiviral transfer plasmid (pCasESlO) and the following packaging plasmids: 5 iig of pMD2.G (VSV-gpseudotype), and 7,5ug of psPAX2 (gag / pol / rev / tat). Transfeetion was done in 4mL OptiMEM with a eationie lipid delivery agent (50uL Lipofectamine 2000 and lOOui Plus reagent). After 6 hours, the media was changed to antibiotic-free DMEM with 10% fetal bovine serum.[0020Θ] Lenti virus may be purified as follows. Viral supematants were harvested after 48 hours. Supematants 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. Viraf pellets were resuspended in 50ul of DMEM overnight at 4C. They were then aliquotted and immediately frozen at -80C.

[0201] 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, Published online 21 November 2005 in Wiley InterScieiice (www.interscience.wiley.com). DOI: 10.1002 / jgm.845). In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostain 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)) may be modified for the CRISPR-Cas system of the present invention.

[0202] 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 Trans! Med 2:36ra43) may be used / and or adapted to the CRISPR-Cas system of the present invention. A minimum of 2.5 x 106CD34+ cells per kilogram patient weight may be collected and prestimulated for 16 to 20 hours in X- VIVO 15 medium (Lonza) containing 2mML-glutamine, stem cell factor ( 100 ng / ml), Fit- 3 ligand (Flt-3L) (100 ng / ml), and thrombopoietin (10 ng / ml) (CellGemx) at a density of 2 x 106cells / ml. Prestimulated cells may be transduced with lentiviral at a multiplicity of infection of 5 for 16 to 24 hours in 75-cnr tissue culture flasks coated with fibronectin (25 mg / cm") (Retro Neetm,Takara Bio Inc.).

[0203] 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. 20060281 180, 20090007284, US201101 17189; US20090017543;US2G07005496J , US20100317J 09. Lenti viral vectors have also been disclosed for deliveiy to the train, see, e.g., US Patent Publication Nos. US201 10293571; US201 10293571 , US20040013648, US2007G025970, US20090111106 and US Patent No. US7259015.RNA delivery

[0204] RNA delivery: The CRISPR e zyme, for instance a Cas9, a d / 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 PGR 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 tra scription from a cassette containing T7_promoter-GG-guide RNA sequence.[0Θ205] To enhance expression and reduce toxicity, the CRISPR enzyme and / or guide RNA can be modified using pseudo-U or 5-Methyl-C.

[0206] mRNA deliveiy methods are especially promising for liver delivery currently. In particular, for AA.V8 is particularly preferred for delivery to the liver.Nartoparticles[00207J CRISPR enzyme mRNA and guide RNA may be delivered simultaneously using nanoparticles or lipid envelopes.

[0208] For example, Su X, Fricke J, Kavanagh DG, Irvine DJ ("In vitro and in vivo mRNA delivery using Hpid-enveloped pH-responsive polymer nanoparticles" Mof Pharm. 2011 Jun 6;8(3):774-87. doi: 10.1021 / mpl00390w. Epub 2011 Apr I) describes biodegradable core-shell structured nanoparticles with a poly(p-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.

[0209] In one embodiment, nanoparticles based on self assembling bioadhesive polymers are contemplated, which may be applied to oral deliveiy of peptides, intravenous deliver}.' of peptides and nasal delivery of peptides, all to the brain. Other embodiments, such as oral absorption and ocular deliver of hydrophobic drags are also contemplated. The molecular envelope technology involves an engineered polymer envelope which is protected and deliveredto the site of the disease (see, e.g., Mazza, M. et al. ACSNano, 2013. 7(2): 1016-1026; Siew, A., et al. Moi 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, .L., et al. J Biophotomcs, 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. 7:8423-33; Uchegbu, IF. Expert Opin Drug Deliv, 2006. 3(5):629-40; Qu, X.,et al. Biomacromoiecul.es, 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 contem lated, with single or multiple doses, depending on the target tissue.

[0210] In one embodiment, nanoparticles that can deliver RNA to a cancer ceil to stop tumor growth developed by Dan Anderson's lab at MIT may be used / and or adapted to the CR1SPR 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., Alahi et ah, 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, Na o Lett. 2013 Mar 13;13(3):1059-64; aragiannis et al, ACS Nano. 2012 Oct 23;6(30):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.

[0211] US patent application 201 10293703 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 aminoalcohoi 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 aminoalcohoi 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.[00212 j US Patent Publication No. 01 10293703 also provides methods of preparing the aminoalcohoi lipidoid compounds. One or more equivalents of an amine are allowed to react with one or more equivalents of an epoxide-termmated compound under suitable conditions toform an aminoalcohol lipidoid compoimd 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, ail 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 funetionalizcd 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 alky! halide (e.g., methyl iodide) or other alkylating agent, and / or they may be acylated.

[0213] US Patent Publication No. 0110293703 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, microliter 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.

[0214] US Patent Publication No. 20130302401 relates to a class of poly(beta-amino alcohols) (PBAAs) has been prepared using combinatorial polymerization. The inventive PBAAsmay 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, micropatteming age ts, and cellular encapsulation agents. When used as surface coatings, these PES A As 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, foi lowing 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 cel l tissue engineering. The teachings of US Patent Publication No. 20130302401 may be applied to the CRISPR Cas system of the present invention.[002 J 5] in another embodiment, lipid nanoparticles (LNPs) are contemplated. In particular, an antitransthyretin small interfering RNA encapsulated in lipid nanoparticles (see, e.g., Coeiho et a!., 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.

[0216] LNPs have been shown to be highly effective in delivering siRNAs to the liver (see, e.g., Tabemero et a!,, Cancer Discovery, April 2013, Vol. 3, No. 4, pages 363-470) and are therefore contemplated for delivering CRISPR Cas to the liver. A dosage of about four doses of 6 mg / kg of the LNP every two weeks may be contemplated. Tabemero 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 apatient with PNET and liver metastases continued on the extension study for 18 months (36 doses) with stable disease.

[0217] However, the charge of the LNP must be taken into consideration. As cationic lipids combined wit negatively charged lipids to induce rsonbi Saver 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 ai, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 201 1). Negatively charged polymers such as siRNA oligonucleotides 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), l,2-dilinoleyloxy-3-N,N-dimethylaminopropane (DLinDMA), 1 ,2-diliiioleyloxy ceto-N,N-dimethyi-3-aminopropaiie (DLinKDMA), and 1 ,2- dilinoleyl-4-(2-dimethylaminoethyl)-[l ,3]-dioxolane (DLinKC2-DMA). It has been shown that LNP siRNA systems containing these lipids exhibit remarkably different gene silencing properties in iiepatocytes in vivo, with potencies varying according to the series DLinKC2- DMA>DLm DMA>DLinDMA>>DLinDAP employing a Factor VI] gene silencing model (see, e.g., Rosin et ai, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 201 1). A dosage of 1 μ^'Ίη! levels may be contemplated, especially for a formulation containing DLin C2-DMA.

[0218] Preparation of LN Ps and CRISPR Cas encapsulation may be used / and or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 201 1). The cationic lipids 1 ,2-di lineoyl-3-dimethylammonium-propaiie (DLinDAP), 1 ,2-dilinoley]oxy~3~N,N~ dimethylaminopropane (DLinDMA), 1 ,2-dilinoleyioxyketo-N,N-dimethyi-3-aminopropane (DLinK-DMA), l ,2-dilinoleyl-4-(2-dimethylaminoethyl)-[l ,3]-dioxolane (DLinKC2-DMA), (3- o-[2 " -(methoxypolyethyleneglycol 2000) succinoyl]-] ,2-dimyristoyl-sii-glycol (PEG-S-DMG), and R-3-[(o')-methoxy-poly(ethyiene glycol )2000) carbamoyl]- l ,2-dimyristyloxlpropyl-3-amme (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 DLin .C2-DMA (cationic lipid:DSPC:CHOL: PEGS-DMG or PEG-C-DOMG at 40: 10:40: 10 molar ratios). When required, 0.2% SP-DiOC1 8 (Invitrogen, Burlington, Canada) may beincorporated 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 concentratioii 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 agai st 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 scatteri g usi g 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 iniH 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 LISA (Richmond, VA).[002 J 9] Preparation of large LNPs may be used / and or adapted from Rosin et al, Molecular Therapy, vol. 19, no. 12, pages 1286-2200, Dec. 201 1. A lipid prcmix 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 al low 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 (Zeiasizer 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-μη syringe filter.

[0220] Spherical Nucleic Acid (SNA™) constructs and other nanoparticles (particularly gold nanoparticles) are also contemplate as a means to delivery CRJSPR / 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:

[0221] 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.

[0222] 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.

[0223] 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).

[0224] Superior efficacy. The constructs significantly outperform leading conventional transfection reagents (Lipofectamine 2000 and Cytofectin).

[0225] Low toxicity. The constructs can enter a variety of cultured cells, primary cells, and tissues with no apparent toxicity.

[0226] No significant immune response. The constructs elicit minimal changes in global gene expression as measured by whole-genome microarray studies and cytokine-specifi.c protein assays.[00227J 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.

[0228] 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.

[0229] 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 1 10< 19):7625-7630, Jensen et al., Sci. Transi. Med. 5, 209ral 52 (2013) and Mirkin, et al, Small, doi.org / 10.1002 / smll.201302143.

[0230] Self-assembling nanoparticles with siRNA may be constructed with polyethyieneimine (PEI) that is PEGvlated 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 iieovasculature expressing integrins and used to deliver siRNA inhibiting vascular endothelial growth factor receptor-2 (VEGF 2) 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 polyp lex es 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 deliver)? in the self-assembling nanoparticles of Schiffelers et al.

[0231] 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 poly lexes 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 strandwith 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 ethanoi-preeipitated, resuspended in water, and annealed to the unmodified antisense strand to yield DGTA -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 cydodextrin-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.[00232J Davis et al. (Nature, Vol 464, 15 April 2010) conducts a siRNA clinical trial that uses a targeted naiioparticle-deiivery system (clinical trial registration number NCT00689065). Patients with solid cancers refractory to standard-of-care therapies are administered doses of targeted nanoparticles on days L 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, cyciodextrin- 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 sl R.2B · 5 ). The TFR has long been known to be upregulated in malignant ceils, 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"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

[0233] Exosomes are endogenous nano-vesicles that transport R'NAs and proteins which can deliver short interfering (si)RNA to the brain in mice. To reduce immimogcnieity, Alvarez -Erviti et ai. (2011, Nat Biotecimol 29: 341) used self-derived dendritic cells for exosorne production. Targeting was achieved by engineering the dendritic cells to express Lamp2b, an exosomal membrane protein, fused to the neuron-specific RVG peptides. 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 BACE l, a therapeutic target in Alzheimer's disease.

[0234] To obtain a pool of immunologically inert exosomes, Alvarez-Erviti et ai. harvested bone marrow from inbred C57BL / 6 mice with a homogenous major histocompatibility complex (MHC) haplotype. As immature dendritic ceils produce large quantities of exosomes devoid of T-celi activators such as MHC-I I and CD86, Alvarez-Erviti et al, selected for dendritic cel ls 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 iig of exosomes (measured based on protein concentration) per 106cel ls.

[0235] 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 ofexosomes. Electroporation at 400 V and 125 μΕ resulted in the greatest retention of siRNA and was used for all subsequent experiments.

[0236] Alvarez-Erviti et ai. administered 150 μ§ of each BACEl siRNA encapsulated in 150 p.g of VG 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 BACEl siRNA complexed to an in vivo eationie liposome reagent and mice injected with BACEl siRNA comple ed 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 BACEl 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 [betaj-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 β-amyloid 1- 40 decrease demonstrated in normal mice after intraventricular injection of BACEl inhibitors. Alvarez-Erviti et al. carried out 5 -rapid amplification of cDNA ends (RACE) on BACEl cleavage product, which provided evidence of RNAi-mediated knockdown by the siRNA..

[0237] Finally, Alvarez-Erviti et al. investigated whether siRNA- RVG exosomes induced immune responses in vivo by assessing IL-6, IP- 10, TNFa and IFN-a serum concentrations. Following si NA-RVG exosome treatment, nonsignificant changes in all cytokines were registered similar to siRNA-transfection reagent treatment in contrast to siRNA-RVG-9R, which potentl stimulated I L-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 deliver}' 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.

[0238] 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 expressio vector, comprising an exosomal protein fused with a peptide ligand. Next, El-Andaloussi et al, explain how to purify and characterize exosomes from traiisfected ceil supernata t. 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.

[0239] In another embodiment, the plasma exosomes of Wahlgren et al. (Nucleic Acids Research, 2012, Vol. 40, No. 17 el30) 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 cany RNA between cells, this property might be useful in gene therapy.

[0240] Exosomes from plasma are prepared by centrifugation of buffy coat at 900g for 20 min to isolate the plasma followed by harvesting ceil supernata ts, 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 OOOg 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 transfectio 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 monocytesand lymphocytes of and autologously reintroduced into a human. Accordingly, delivery or administration according to the invention may beperformed using plasma exosomes.Liposomes

[0241] Delivery or administration according to the invention can be performed with liposomes. Liposomes are spherical vesicle structures composed of a uni- or multilamellar lipid bi!ayer 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. 201 1 , Article ID 469679, 12 pages, 2011. doi: 10.1155 / 2011 / 469679 for review).

[0242] 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 Deliver}', vol. 2011. Article ID 469679, 12 pages, 2011. doi: 10.1155 / 201 1 / 469679 for review).

[0243] 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 100 nm. (see, e.g., Spuch and Navarro, Journal of Drag Delivery, vol. 201 1 , Article ID 469679, 12 pages, 2011. doi: 10.1 155 / 2011 / 469679 for review).

[0244] Conventional liposome formulation is main!)? comprised of natural phospholipids and lipids such as 1, 2-distearoryl-sn-giycero-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 l,2-dioleoyl-sn-giyeero-3- phosphoethanolamine (DOPE) increases the stability (see, e.g., Spuch and Navarro, Journal, of Drug Delivery, vol. 2011, Article ID 469679, 12 pages, 2011. doL IG. l 155 / 2011 / 469679 for review).0Θ245] In a particularly advantageous embodiment, Trojan Horse liposomes (also known as Molecular Trojan Horses) are desirable and protocols may be found at bi{p: / / cshpro ocois sh)p.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. Applicant postulates utilizing Trojan Horse Liposomes to deliver the C ISPR family of nucleases to the brain via an intravascular injection, which would allow whole brain transgenic animals without the need for embryonic manipulation. About 1 -5 g of DNA may be contemplated for in vivo administration in liposomes.

[0246] 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 abpit 1 or 2.5 rng / '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-propyiamine (PEG-CDMA), 1 ,2-dilinoIeyloxy-N,N-dimethyI-3-aminopropane (DLinDMA), 1 ,2-distearoy -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. 44.1 , 4 May 2006).

[0247] 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-DMAwith distearoylphosphatidylcholme (DSPC), Cholesterol and siRNA using a 25: 1 lipid / siRNA ratio and a 48 / 40 / 10 / 2 molar ratio of Cholestero! / D-Lin-DMA DSPC / PEG-C-DMA. The resulted SNALP liposomes are about 80-100 nm in size.

[0248] in yet another embodiment, a SNALP may comprise synthetic cholesterol (Sigma- Aldric , St Louis, MO, USA), dipalmitoylphosphatidylcholine (Avanti Polar Lipids, Alabaster, AL, USA), 3-N-[(w-methoxy polyethylene glycol )2000)carbamo l]- 1 ,2- dimyrestyloxypropylamine, and cationic l,2-dilinoleyioxy-3-N,Ndimethylammopropane (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.

[0249] In yet another embodiment, a SN ALP may comprise synthetic cholesterol (Sigma- Aidrich), l,2-distearoyl-sn-glycero-3-phosphocholine (DSPC; Avanti Polar Lipids Inc.), PEG- cDMA, and l,2-dilmoleyloxy-3-(N;N-dimethyl)aminopropane (DLinDMA) (see, e.g., Judge, J. Clin. Invest. 1 19:661-673 (2009)). Formulations used for in vivo studies may comprise a final lipid / RNA mass ratio of about 9: 1.

[0250] The safety profile of RNAi nanomedicin.es 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 H, a neutral helper lipid, cholesterol, and a diffusible polyethylene glycol (PEG)-iipid. 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 hydrophiiic exterior that improves pharmacokinetic properties.

[0251] 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 onpreclinical 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 .

[0252] Alnyiam 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 (I AC) both caused by autosomal dominant mutations in TTR; and senile systemic amyloidosis (SSA) cause by wild type TTR. A placebo-controlled, single dose-escalation phase I trial of ALN- TTR01 was recently completed in patients with ATTR. ALN-TTROl was administered as a 15- minute IV infusion to 31 patients (23 with study drag and 8 with placebo) within a dose range of 0.01 to 1.0 mg / kg (based on siRNA). Treatmentwaswel! 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-lra 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-TTROl, was observed at 1 mg / kg.

[0253] In yet another embodiment, a SNALP may be made by solubilizing a cationic lipid, DSPC, cholesterol and PEG-iipid 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 raM 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 allo w vesicle reorganization and encapsulation of the siRNA . The ethanol was then removed and the external buffer replaced with PBS (155 mM NaCl, 3 mM Na2HP04, 1 mM KH2P04, pH 7.5) by either dialysis or tangential flow diafiltration. siRNA were encapsulated in SNALP usinga controlled step-wise dilution method process. The lipid constituents of KC2-SNALP were DLm-KC2-DMA (eatiome lipid), dipalmitoylphosphatidyleholine (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 agamst PBS and filter steri lized through a 0.2 μηι 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

[0254] Other cationic lipids, such as amino lipid 2,2-dilinoleyl-4~dimethylaminoethyI-[l,3]- dioxolane (DLin- C2-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, distearoylphosphatidyl ioline (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 (u 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,

[0255] Michael S D ormann et al. ("Expression of therapeutic proteins after delivery of chemically modified mRNA. in mice: Nature Biotechnology, Volume:29, Pages: 154-157 (2011) Published online 09 January 201 1 ) describes the use of lipid envelopes to deliver RNA. Use of lipid envelopes is also preferred in the present invention.

[0256] In another embodiment, lipids may e formulated with the CRISPR Cas system of the present invention to form lipid nanoparticles (LNPs). Lipids include, but are not limited to, DLin- C2~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, c4; doi: 10.1038 / mtna.201 1.3) using a spontaneous vesicle formation procedure. The component molar ratio may be about 50 / 10 / 38.5 / 1.5 (DLin- KC2-DMA or C 12-200 / disteroyf phosphatidyl cholme / cholesterol PEG-DMG). The final lipid :siRNA weight ratio may be -12: 1 and 9: 1 in the case of DLin-KC2-DMA and C 12-200 lipid nanopartieles (LNPs), respectively. The formulations may have mean particle diameters of ~80 urn with >90% entrapment efficiency. A 3 mg / kg dose may be contemplated.

[0257] 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.

[0258] 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 Modema 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 !ipid:fusogemc iipidxholesterobPEG 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 a!., Delivery and Formulation of Engineered Nucleic Acids, US published application 20120251618.

[0259] Nanomerics' technology addresses bioavailability challenges for a broad range of therapeutics, including low molecular weight hydrophobic drags, peptides, and nucleic acid based therapeutics (piasmid, 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 ): 101 -26; Uchegbu and Slew, 2013, J Pharm Sci. 102(2):305-10 and Lalatsa et al, 2012, J Control Release. 2012 Jul 20;161(2):523-36.00260J 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 thedelivery of the bioactive molecules to, for example, the liver, spleen, lung, kidney or heart. Dendrimers are synthetic 3-dimensional maeromolecules 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 aerylonitrile to the primary amines followed by the hydrogenation of the nitriies. This results in a doubling of the amino groups. Polypropylenimine dendrimers contai 100% protonable nitrogens and up to 64 terminal amino groups (generation 5, DAB 64). Protoiiable groups are usually amine groups which are able to accept protons at neutral pH. The use of dendrimers as gene deli very agents has l argely focused on the use of the polyamidoamine. and phosphorous containing compounds with a mixture of amine / amide or N--P(02)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.

[0261] 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 drugmolecules or nucleic acids for gene therapy. In such cases, the polymers' own intrinsic anti- tumour activity may complement the activity of the agent to he delivered."Supercharged proteins

[0262] Supercharged proteins are a class of engineered or naturally occurring proteins with unusually high positive or negative net theoretical charge. Both supe negatively and superpositiveiy charged proteins exhibit a remarkable ability to withstand thermally or chemically induced aggregation. Superpositiveiy 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 macromoiecules 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 ah, 2007, Journal of the American Chemical Society 129, 101 10- 10112).

[0263] The nonviral delivery of siRNA and plasmid DNA into mammalian cel ls are valuable both for research and therapeutic applications (Akiiic et al, 2010, Nat. Biotech. 26, 561-569). Purified +36 GFP protein (or other superpositiveiy 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 siRN A should be performed to optimize the procedure for specific ceil lines.(1) One day before treatment, plate I x 10Jcel ls 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 to a final concentration of 50nM. Vortex to mix and incubate at room temperature for lOmin.(3) During incubation, aspirate media from cells and wash once with PBS.(4) Following incubation of +36 GFP and siRN A, add the protein-siRNA complexes to ceils.(5) Incubate cells with complexes at 37 C for 4h.(6) Following incubation, aspirate the media and wash three times with 20 U / raL heparin PBS. Incubate cells with serum -containing media for a further 48h or longer depending upon the assay for knockdown.(7) Analyze cells by miniunoblot, qPCR, phenotypic assay, or other appropriate method.ΘΘ264] David Lars 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 p!asmids. 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 10'per well in a 48- well plate.(2) On the day of treatment, dilute purified p36 GFP protein in serumfree media to a final concentration 2 niM. Add Img of plasmid DNA. Vortex to mix and incubate at room temperature for lOmin.(3) During incubation, aspirate media from ceils and wash once with PBS.(4) Following incubation of b36 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 cel ls in seami-containing media and incubate for a further 24-48h.(7) Analyze plasmid delivery (e.g., by plasmid-driven gene expression) as appropriate.

[0265] See also, e.g., McNaughton et al, Proc. Natl. Acad. Sci. USA 106, 61 11-6116 (2009); Cronican et al., ACS Chemical Biology 5, 747-752 (2010); Cronican et ah, 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.

[0266] In another embodiment, implantable devices are also contemplated for delivery of the CRISPR Cas system. For example, US Patent Publication 201 10195123 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 polymeri c substrate, such as a matrix for exampl e, that is used as the device body, and dmgs, 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 RJN A, 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 i 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. Typical ly a few devices are implanted during the same treatment procedure.

[0267] As described in US Patent Publication 201 10195123, 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 201 10195 123.

[0268] 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 nr to 1000 mm", as required by the volume for the agent load. The Loder may optionallybe larger, for example when incorporated with a device whose size is determined by functionality, for example and without limitation, a knee joint, an infra-uterine or cervical ring and the like,

[0269] The drug delivery system (for delivering the composition) is designed in some embodiments to preferably employ degradable polymers, wherei 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, a d 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.

[0270] 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,

[0271] The drug delivery system as described in US Patent Publication 201 10195123 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.

[0272] According to some embodiments of US Patent Publication 20110195123, the site for local deliver}'may optionally include target sites characterized by high abnormal proliferation of cells, and suppressed apoptosis, including tumors, active and or chronic inflammation and infectio including autoimmune diseases states, degenerating tissue including muscle andnervous 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 local deliver}.' also may optionally include sites enabling performing preventive activities including pregnancy, prevention of infection and aging.

[0273] The site for implantatio 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.

[0274] 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.

[0275] 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.

[0276] 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 i 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 vagi al; 10. Inner ear— auditory system, labyrinth of the inner ear, vestibular system; 11. Intra tracheal; 12. Intra-cardiac; coronary, epicardiac; 13. urmary 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. !ntra-ocuiar; 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.

[0277] 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.

[0278] 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.[0Θ279] According to other embodiments of US Patent Publication 201 10195123, 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. Moreover, 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. Such drugs include approved drugs that are delivered today by methods other than of this invention, including Amphotericin B for fungal infection; antibiotics such as in osteomyelitis; pain killers such as narcotics; anti degenerative such as in Alzheimer or Parkinson diseases in a Loder implanted in the vicinity of the spine in the case of back pain. Such a system may be used and / or adapted to deliver the CRI SPR Cas system of the present invention .

[0280] For example, for specific applications such as prevention of growth or regrowth of smooth muscle cells (that are injured during a stenting procedure and as a result tend to proliferate), the drug may optionally be siRNA that silence smooth muscle cells, including HI 9 silencing, or a drug selected from the group consisting of taxoi, rapamycin and rapamyein- analogs. In such cases the Loder is preferably either a Drug Eluting Stent (DES), with prolonged release at constant rate, or a dedicated device that is implanted separately, in association to the stent. Al l of this may be used / and or adapted to the CRISPR Cas system of the present invention.

[0281] As another example of a specific application, neuro and muscular degenerative diseases develop due to abnormal gene expression. Local delivery of silencing R As may have therapeutic properties for interfering with such abnormal gene expression. Local delivery of anti apoptotic, anti inflammatory and anti degenerative drugs including smal l drugs and macromolecuies may also optionally be therapeutic. In such cases the Loder is applied forprolonged release at constant rate and / or through a dedicated device that is implanted separately. All of this may be used and / or adapted to the CRISPR Cas system of the present invention.

[0282] As yet another example of a specific application, psychiatric and cognitive disorders are treated with gene modifiers. Gene knockdown with silencing R A is a treatment option. Loders locally delivering nucleotide based agents to central nervous system sites are therapeutic options for psychiatric and cognitive disorders including but not limited to psychosis, bi -polar diseases, neurotic disorders and behavioral maladies. The Loders could also deliver locally drags including small drugs and macromolecules upon implantation at specific brain sites. All of this may be used and / or adapted to the CRISPR Cas system of the present invention.

[0283] As another example of a specific application, silencing of innate and / or adaptive immune mediators at local sites enables the prevention of organ transplant rejection. Local delivery of silencing R As and immunomodulating reagents with the Loder implanted into the transplanted organ and / or the implanted site renders local immune suppression by repelling immune ceils such as CDS activated against the transplanted organ. Ail of this may be used / and or adapted to the CRISPR Cas system of the present invention.

[0284] As another example of a specific application, vascular growth factors including VEGFs and angiogenin and others are essential for neovascularization . Local delivery of the factors, peptides, peptidomimetics, or suppressing their repressors is an important therapeutic modality; silencing the repressors and local delivery of the factors, peptides, macromolecules and small drugs stimulating angiogenesis with the Loder is therapeutic for peripheral, systemic and cardiac vascular disease.[CI0285] 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 hrachymerapy 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.RISj^R e z me m

[0286] CRISPR enzyme rnRNA 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 beexpressed, CRISPR enzyme mRNA might be administered 1 -12 hours (preferably around 2-6 hours) prior to the administration of guide RNA.

[0287] 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.

[0288] Additional administrations of CRISPR enzyme mRNA and / or guide RNA might be useful to achieve the most efficient levels of genome modification.

[0289] 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.

[0290] Alternatively, to minimize the level of toxicity and off-target effect, CRISPR enzyme niekase mRN A (for example S. pyogenes Cas9 with the DIOA 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).O srhang Guide RNA design (guide sequence and PAM color coded) length (bp)14 N N NCC >^U^:s;i?;NN>^U^:s;i?;NN>^U^[sN G NN-3'3' -13 NNNNNNNNNNNNNNNNNNNNG^NNN ^-5'12 5' -NNNNNNNNNNNNNNNNNNNNCC^r^N-3'11 3' -NNNNNNNNNNNNNNNNNNNNG^NNNN ^N-5'105' -NNNNNNNNNNNNNNNNNNNNCC :>;NG ; ΐί ί ;i;NG N N i ;i;NGNGGNNNNNNNNNNNNNNNN-3'3' -N-5'5' -NNNNNNNNNNNNNNNNNNNNCC:>VNGii NN>VSG;i NN>GNGGNNNNNNNNNNNNNNNNN-3'3' -NNNNNNNNNNNNNNNNNNNNG^^N-5'N-3'3' -NNNNNNNNNNNNNNNNNNNNGGN NNN ¾^N-5fNN NNNN NN NN^N-3'' _NNNNNNNNNNNNNNNNN^N-5'blunt NNNNNNNNNNNNN^N-3'3 ' --NNNNNNNNNNNNNNNNN^N-5'NNNNNNNNNNNNNN^N-3'3 ' -- NNN NN NNN NN M^N-5'NNNN NN NNNNK^N-3'3' -NNNNNNNNNNNNNNNNNNNNGGNNNN^^N-5'NNNNNNNNNNNNNNNNNN :i NCCNNNN :ί N?¾NNNNN¾NNNNNNNNNNNNNNNNNNNNNNN-3'3' -NNNNNNNNNNNNNNNNNNNNGGNNN^N-5'NNNNNNN NNNN!^ :ί :i?;NN>;CC :ί :ί :i?;NGGNNNN NNNNNNN NNNNNNN NN -3'3 ' -NNNN NN NNNNN NN^N-5' NNNNNNN NNN^U^ :i :i?;NN>;CC :ί :i ii GGNNNNN NNNNNNN NNNNNNN NNN-3'3 ' -NNNNNNNNNNNNNNNNNNNNGGNNNNN ^N-5'NNNNNNNNNNNNNN :i :ii¾i;NCC :ί :ί?¾ί;ΝΝΝ iNGGNNNNNNNNNNNNNNNNNNNNNNNNNN-3'3' -NNNNNNNNNNNNNNNNNNNNGGNNNJxi^N-5'5' - NN ir^ ¾\U ^N-3'3' -NNNNNNNNNNNNNNNNNNNNGjGN^N-5'5' -ΝΝΝΝΝΝΝΝΝΝίϊΝΝ ;i Ni;N( c Ni;NNNGGNNNNNNNNNNNNNNNNNNNNNNNNNNNN-3'' _NNNNNNNNNNNNNNNNNNNNG^^N-5'5' -Ν ΝΝΝΝΝ ΝΝ>νΚ^;¾ ΝΝ>ν;> "0;¾N-3fNN N NNy¾K;\¾?;>;^Nf N N NNy¾K;\¾?;^NNNNNNNNNNNNNNNNNNNNNN ;χΤNNNN ^ ^N ^ ^V^NN-5'5 ' -N-3'3' --NNNNNNNNNNNNNNNNNNNNGGNCC^N-5'5 ' - :i :i :i :i CGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-3'3' -NNNNNNNNNNNNNNNNNNNNNGGC^N-5'5' - :i :i NN>^U^ :i :i NN>;N!^ :i ii NGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-3'3' -NNNNNNNNNNNNNNNNNNNNNNNCCG^N-5'5' - :i :i NN>^U^ :i :i NN>;N!^ :i ii CGGNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-3'3' -NNNNNNNNNNNNNNNNNNNNNNNCCNGGNl^^N-5'5' -ΝΝ Ν>;ΝΝ : Ν>;ΝΝ : Οΰ(;ΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝΝN-3'3' -NNNNNNNNNNNNNNNNNNNNNNNCCNNGjSN^N-5'5' - Ν>^Ν; Ν ΝΝ>^Ν^N-3'3' -NNNN NNNNNNN NNNNNNN NNCCNNNGGNNNNN NNNN NNNNN NNNNNNN-5'5' - Ν>^Ν; Ν ΝΝ>^Ν^N-3'' _NNNNNNNNNNNNNNNNNNNNNNNCCNNNNGGNN N N NN N N NN NNNNNNNNN-5'5' -Ν >ν^;¾ ΝΝ>ν^ί¾N-3'' _NNNNNNNNNNNNNNNNNNNNNNNCCNNNNNGGN N NNNN N NNNN N NNNNNNN-5'

[0291] 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.

[0292] 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 reannea! and ligate or the activity or function of the gene product being al tered or the expression of the gene product being increased. In an embodiment of the invention, the gene product is a protein.[002931 Only sgRNA pairs creating 5' overhangs with less than 8 bp overlap between the guide sequences (offset greater than -8 bp) were able to mediate delectable 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.

[0294] 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. Unexpectedly, 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. Further work may be needed to identify the necessary design rules for sgRNA pairing to allow double nicking by Cas9H840A.Liver, proprotein convertase subtiiisin kexin 9 (PCSK9)[CI 295] Proprotein convertase subtiiisin kexin 9 (PCSK9) is a member of the subtiiisin serine protease family. PCSK9 is primarily expressed by the liver and is critical for the down regulation of hepatocyte LDL receptor expression. LDL-C levels in plasma are highly elevated in humans with gain of function mutations in PCSK9, classifying them as having severe hypercholesterolemia. Therefore, PCSK9 is an attractive target for CRISPR. PCS9K-targeted CRISPR may be formulated in a lipid particle and for example administered at about 15, 45, 90, 150, 250 and 400 .ug / kg intraveneously (see, e.g., http: / / www.ataylam.com / capella / wp~ coriteot mioad / 'iO 13 / 08 / AI ."N~PCS0?.~001 -Proto ol-l .ancet odf)[002961 Bailey et al. (J Mol Med (Berl). 1999 Jan;77(l):244-9) discloses insulin delivery by ex-vivo somatic cell gene therapy involves the removal of non-B-ce l 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 eryopreserved. 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 ceil 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.

[0297] 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 dimethyinitrosamine-induced liver cirrhosis in a dose- and duration-dependent manner. Cationic liposomes (Li otrust) containing 0,0'-ditetradecanoyl-N-(a rimethylammonioacetyl) diethanoianiine chloride (DC-6-14) as a cationic lipid, cholesterol and dioleoylphosphatidyiethanolamine at a molar ratio of 4:3:3 (which has shown high transfection efficiency under semmcontaining 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 ratvi (DC -16-4) by addition of double-distilled, water (DDW) to the lyophilized lipid mixture under vortexing before use. To prepare V A-coupled liposomes, 200 nmoi of vitamin A (retinoi, 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 1 C. To prepare V A-coupled liposomes carrying siRNAgp46 (VA-lip~siRNAgp46), a solution of siRNAgp46 (580 pmol / ml in DDW) was added to the retinoi-coupled liposome solution with stirring at 25 C. The ratio of siRNA to DC- 16-4 was .1 : 1 .1.5 (mol / mol) and the siRNA toliposome ratio (w† / t) was 1 :1. Any free vitamin A or siRNA that was not taken up by liposomes were separated from liposomal preparations using a mieropartition system (VIVASPIN 2 concentrator 30,000 MWCO PES, VIVASCIENCE). The liposomal suspension was added to the filters and centrifuged at 1 ,500g for 5 mm 3 times at 25 1 C. 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 si RNA 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.

[0298] 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 PoiyConjugates may also be applied to the present invention. Key features of the Dynamic Poly-Conjugate technology include a membrane-active polymer, the ability to reversibiy 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' ammemodified siRNA with 1 weight equivalents (wt eq) of Nsuccmiinidyi-S-acctylthioacetate (SAT A) reagent (Pierce) and 0.36 wt eq of NaHC03in 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 IX siRNA buffer (Dharmacon) and quantified by measuring absorbanee 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 μΐ of isotonic glucose solution containing 5 mM TAPS (pH 9). To this solution was added 50 ug 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 L9mg 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-P-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 μ§ 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 deiiver to the liver, for example by envisioning a dosage of about 50 to about 200 mg of CRISPR Cas for delivery to a human.Bong[002991 Oakes and Lieberman (Clin Orthop Reiat Res. 2000 Oct;(379 Suppi):S101 -12) discusses delivery of genes to the bone. By transferring genes into cells at a specific anatomic site, the osteoinductive properties of growth factors can be used at physiologic doses for a sustained period to facilitate a more significant healing response. The specific anatomic site, the quality of the bone, and the soft-tissue envelope, influences the selection of the target cells for regional gene therapy. Gene therapy vectors delivered to a treatment site in osteoinductive carriers have yielded promising results. Several investigators have shown exciting results using ex vivo and in vivo regional gene therapy in animal models. Such a system may be used / and or adapted to the CRISPR Cas system for delivery to the bone.Brain

[0300] Delivery options for the brain include encapsulation of CRISPR enzyme and guide RNA in the form of either D'NA or RNA into liposomes and conjugating to molecular Trojan horses for trans-blood brain barrier (BBB) deliver}7. 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.

[0301] Zhang et al. (Mol Ther. 2003 Jan;7(l):l l-8.)) describe how expression piasmids encoding reporters such as luciferase were encapsulated in the interior of an "artificial virus"comprised of an 85 ran pegylated immunoiiposome, 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 transeytosis across the blood-brain barrier and endocyiosis 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 histochemistiy and confocal microscopy. The authors indicate that this approach makes feasible reversible adult transgenics in 24 hours. Accordingly, the use of immunoiiposome is preferred. These may be used in conjunction with antibodies to target specific tissues or cell surface proteins.[00302 J Other means of deliver}' 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-! ike 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, PM1D: 20059641).

[0303] Indeed, exosomes have been shown to be particularly useful in delivery siRNA, a system with some parallels to the CRISPR system. For instance, Ei-Andaloussi S, et al. ("Exosome-mediated delivery of siRNA in vitro and in vivo." Nat Protoc. 2012 Dec;7(12):2112- 26. doi: 10.1()38 / 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 ceil 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.

[0304] Vitamin E (a-tocopherol) may be conjugated with CRISPR Cas and delivered to the brain alo g with high density lipoprotein (HDL), for example in a similar ma ner as was do e by Uno et al. (HUMAN GENE THERAPY 22:71 1-719 (June 201 1 )) 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 Toe-siBA.CE / 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 a-tocopheroi 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 μηιοΐ of CRISPR Cas targeted to the brain may becontempiated.

[0305] Zou et al. ({HUMAN GENE THERAPY 22:465-475 (April 2011}) describes a method of lentiviral-mediated delivery of short-hairpin RNAs targeting PKCy for in vivo gene silencing in the spinal cord of rats. Zou et al. administered about 10 μΐ of a recombinant lentivirus having a titer of 1 x 109transducing units (TU) / ml by an intrathecal catheter. A similar dosage of CRISPR Cas expressed in a lenti iral 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 109transducing units (TU) / ml may becontempiated.Targeted deletion, therapeutic applications[003061 Targeted deletion of genes is preferred. Examples are exemplified in Example 18. Preferred are, therefore, 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. 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 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.

[0307] Therapeutic applications of the CRISPR-Cas system include Glaucoma, Amyloidosis, and Huntington's disease. These are exemplified in Example 20 and the features described therein are preferred alone or in combination.

[0308] Another example of a polyglutamine expansion disease that may be treated by the present invention includes spinocerebellar ataxia type 1 (SCAl). Upon intracerebellar injection, recombinant adenoassociated virus (AAV) vectors expressing short hairpin RNAs profoundlyimprove motor coordination., restored cerebellar morphology and resolved characteristic ataxin-1 inclusions in Purkinje cells of SCA1 mice (see, e.g., Xia et al., Nature Medicine, Vol. 10, No. 8, Aug. 2004). In particular, AAV1 and AAV5 vectors are preferred a d AAV titers of about 1 x 10" vector genomes / ml are desirable.

[0309] As an example, chronic infectio by HIV-1 may be treated or prevented. In order to accomplish this, one may generate CRISPR-Cas guide R'NAs that target the vast majority of the HIV-1 genome while taking into account HIV- 1 strain variants for maximal coverage and effectiveness. One may accomplish delivery of the CRISPR-Cas system by conventional adenoviral or lentiviral-mediated infection of the host immune system. Depending on approach, host immune cells could be a) isolated, transduced with CRISPR-Cas, selected, and reintroduced in to the host or b) transduced in vivo by systemic delivery of the CRISPR-Cas system. The first approach allows for generation of a resistant immune population whereas the second is more likely to target latent viral reservoirs within the host. This is discussed in more detail in the Examples section.

[0310] In another example, US Patent Publication No. 20130171732 assigned to Sangamo Biosciences, Inc. relates to insertion of an anti-HJV transgene into the genome, methods of which may be applied to the CRISPR Cas system of the present invention. In another embodiment, the CXCR4 gene may be targeted and the TALE system of US Patent Publication No. 20100291048 assig ed to Sangamo Biosciences, Inc. may be modified to the CRISPR Cas system of the present invention. The method of US Patent Publication Nos. 20130137104 and 20130122591 assigned to Sangamo Biosciences, Inc. and US Patent Publication No. 20100146651 assigned to Cel Sectis may be more generally applicable for transgene expression as it involves modifying a hypoxanthine-guanine phosphoribosyltransferase (HPRT) locus for increasing the frequency of gene modification.

[0311] It is also envisaged that the present invention generates a gene knockout cell library. Each cel l may have a single gene knocked out. This is exemplified in Example 23.

[0312] One may make a library of ES cells where each cell has a single gene knocked out, and the entire library of ES ceils will have every single gene knocked out. This library is useful for the screening of gene function in cellular processes as well as diseases. To make this cell library, one may integrate Cas9 driven by an inducible promoter (e.g. doxycycline inducible promoter) into the ES cell. In addition, one .may integrate a single guide RNA targeting a specificgene in the ES cell. To make the ES cell library, one may simply mix ES cells with a library of genes encoding guide NAs targeting each gene in the human genome. One may first introduce a single BxBl attB site into the AAVS1 locus of the human ES cell. Then one may use the BxBl mtegrase to facilitate the integration of individual guide RNA genes into the BxBl attB site in AAVS1 locus. To facilitate integration, each guide RNA gene may be contained on a plasmid that carries of a single attP site. This way BxBl will recombine the attB site in the genome with the attP site on the guide RN A containing plasmid. To generate the ceil library, one may take the library of cells thai have single guide RNAs integrated and induce Cas9 expression. After induction, Cas9 mediates double strand break at sites specified by the guide RNA.

[0313] Chronic administration of protein therapeutics may elicit unacceptable immune responses to the specific protein. The immunogenic! ty 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 i 5; 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).

[0314] In Example 28, Applicants used 3 guideR As 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 multiple 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.

[0315] A suitable example of a protocol for producing AAV, a preferred vector of the invention is provided in Example 34.

[0316] Trinucleotide repeat disorders are preferred conditions to be treated. These are also exemplified herein.

[0317] For example, US Patent Publication No. 20110016540, describes use of zinc finger nucleases to genetically modify ceils, animals and proteins associated with trinucleotide repeat expansion disorders. Trinucleotide repeat expansion disorders are complex, progressive disorders that involve developmental neurobiology and often affect cognition as well as sensori-motor functions.

[0318] Trinucleotide repeat expansion proteins are a diverse set of proteins associated with susceptibility for developing a trinucleotide repeat expansion disorder, the presence of a trinucleotide repeat expansion disorder, the severity of a trinucleotide repeat expansion disorder or any combination thereof. Trinucleotide repeat expansion disorders are divided into two categories determined by the type of repeat. The most common repeat is the triplet CAG, which, when present in the coding region of a gene, codes for the amino acid gfutamine (Q). Therefore, these disorders are referred to as the polyglutamine (polyQ) disorders and comprise the following diseases: Huntington Disease (HD); Spinobulbar Muscular Atrophy (SBMA); Spinocerebellar Ataxias (SCA types 1, 2, 3, 6, 7, and 17); and Dentatorubro-Pallidoluysian Atrophy (DRPLA). The remaining trinucleotide repeat expansion disorders either do not involve the CAG triplet or the CAG triplet is not in the coding region of the gene and are, therefore, referred to as the non-polyglutamine disorders. The non-polyglutarmne disorders comprise Fragile X Syndrome (FRAXA); Fragile XE Mental Retardation (FRAXE); Friedreich Ataxia (FRDA); Myotonic Dystrophy (DM); and Spinocerebellar Ataxias (SCA types 8, and 12).

[0319] The proteins associated with trinucleotide repeat expansion disorders are typical ly selected based on an experimental association of the protein associated with a trinucleotide repeat expansion disorder to a trinucleotide repeat expansion disorder. For example, the production rate or circulating concentration of a protein associated with a trinucleotide repeat expansion disorder may be elevated or depressed in a population having a trinucleotide repeat- expansion disorder relative to a population lacking the trinucleotide repeat expansion disorder. Differences in protein levels may be assessed using proteomic techniques including but not limited to Western blot, immunohistochemicaf staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the proteins associated with trinucleotide repeat expansion disorders may be identified by obtaining gene expression profiles of the genes encoding the proteins using genomic techniques including but not limited to DNA microarrayanalysis, serial analysis of gene expression (S AGE), and quantitative real-time polymerase chai reaction (Q-PCR).

[0320] Non-limiti g examples of proteins associated with trinucleotide repeat expansion disorders include AR (androgen receptor), FMR1 (fragile X mental retardation 1), HTT (huntmgtin). DMPK (dystrophia myotonica-protein kinase), FXN (frataxin), ATXN2 (ataxin 2), ATN1 (atrophin 1), FEN1 (flap structure-specific endonuc lease 1), TNRC6A (trinucleotide repeat containing 6 A), PABPN 1 (poly(A) binding protein, nuclear 1), JPH3 (junctophilin 3), MED 15 (mediator complex subunit 15), ATXNl (ataxin 1), ATXN3 (ataxin 3), TBP (TATA box binding protein), CACNA 1A (calcium channel, voltage-dependent, P / Q type, alpha 1A subunit), ATXN8QS (ATXN8 opposite strand (non-protein coding)), PPP2R2B (protein phosphatase 2, regulatory subunit B, beta), ATXN7 (ataxin 7), TNRC6B (trinucleotide repeat containing 6B), TNRC6C (trinucleotide repeat containing 6C), CELF3 (CUGBP, Elav-like family member 3), MAB21 L1 (mab-21 -like I (C. elegans)), MSH2 (mutS homolog 2, colon cancer, nonpolyposis type 1 (E. coii)), TMEM185A (transmembrane protein 185 A), SIX5 (SIX homeobox 5), CNPY3 (canopy 3 homolog (zebrafish)), FRAXE (fragile site, folic acid type, rare, fra(X)(q28) E), GNB2 (guanine nucleotide binding protein (G protein), beta polypeptide 2), RPL14 (ribosomal protein L14), ATXN8 (ataxin 8), INSR (insulin receptor), TTR (transthyretin), EP400 (El A binding protein p400), GIGYF2 (GRB10 interacting GYF protein 2), OGG1 (8-oxoguanine DNA glycosylase), STCl (stanniocalcin 1), CNDPl (carnosine dipeptidase 1 (metallopeptidase M20 family)), ClOorfZ (chromosome 10 open reading frame 2), MAML3 mastermind -like 3 (Drosophiia), D C1 (dyskeratosis congenita 1, dyskerin), PAXIP1 (PAX interacting (with transcription-activation domain) protein 1 ), CAS (calcium / calmodulin-dependent serine protein kinase (MAGUK family)), MAPT (microtubule-associated protein tau), S l (S l transcription factor), POLG (polymerase (DNA directed), gamma), AFF2 (AF4 / FMR2 family, member 2), THBS1 (thrombospondin 1 ), TP53 (tumor protein p53), ESR l (estrogen receptor 1 ), CGGBP1 (CGG triplet repeat binding protein 1), ABT1 (activator of basal transcription 1), KLK3 (kailikrein-reiated peptidase 3), PRNP (prion protein), JUN (jun oncogene), KCXN (potassium intermediate / small conductance calcium-activated channel, subfamily N, member 3), BAX (BCL2-associated X protein), FRAXA (fragile site, folic acid type, rare, fra(X)(q27.3) A (macroorchidism, mental retardation)), KBTBDIQ (kelch repeat and BTB (POZ) domain containing 10), MBNLi (muscleblind-iike (Drosophiia)), RAD51 (RAD51 homolog (RecAhomolog, E, coli) (S. cerevisiae)), NCOA3 (nuclear receptor coactivator 3), ERDA1 (expanded repeat domain, CAG / CTG 1), TSC1 (tuberous sclerosis 1), COMP (cartilage oligomeric matrix protein), GCLC (glutamate-cysteine ligase, catalytic subunit), RRAD (Ras-related associated with diabetes), SH3 (mutS homolog 3 (E. coli)), DRD2 (dopamine receptor D2), CD44 (CD44 molecule (Indian blood group)), CTCF (CCCTC-binding factor (zinc finger protein)), CCND1 (eye fin Dl ), CLSPN (claspin homolog (Xenopus laevis)), MEF2A (myocyte enhancer factor 2A), PTPRU (protein tyrosine phosphatase, receptor type, U), GAPDH (glyceraidchyde-3- phosphate dehydrogenase), TRXM22 (tripartite motif-containing 22), WT1 (Wilms tumor 1). AHR (aryl hydrocarbon receptor), GPX1 (glutathione peroxidase 1), TPMT (thiopurine S- methyltransferase), NDP (Norrie disease (pseudogiioma)), ARX (aristaless related homeobox), M l S I (MUS81 endonuclease homolog (S. cerevisiae)), TYR (tyrosinase (oculocutaneous albinism IA)), EGR1 (early growth response 1), UNG (uracil-DNA glycosylate), NUMBL (numb homolog (Drosophila)-like), FABP2 (fatty acid binding protein 2, intestinal), EN2 (engrailed homeobox 2), CRYGC (crystaliin, gamma C), SR 14 (signal recognition particle 14 kDa (homologous Alu RNA binding protein)), CRYGB (crystaliin, gamma B), PDCD1 (programmed cell death 1), eOXAl (homeobox Al ), ATXN2L (ataxin 2-like), PMS2 (PMS2 postmeiotie segregation increased 2 (S. cerevisiae)), GLA (galactosidase, alpha), CBL (Cas-Br- M (murine) ecotropic retroviral transforming sequence), FT H I (ferritin, heavy polypeptide 1), IL12RB2 (interleukin 12 receptor, beta 2), OTX2 (orthodenticle homeobox 2), HOXA5 (homeobox A5), POLG2 (polymerase (D A directed), gamma 2, accessory subunit), DLX2 (distal-less homeobox 2), SIRPA (signal-regulatory protem alpha), OTXI (orthodenticle homeobox 1 ), AHRR (aryl -hydrocarbon receptor repressor), MANF (mesencephalic astrocyte- derived neurotrophic factor), TMEM 158 (transmembrane protein 158 (gene / pseudogene)), and ENSG00000078687.

[0321] Preferred proteins associated with trinucleotide repeat expansion disorders include HTT (Huntingtin), AR (androgen receptor), FXN (frataxin), Atxn3 (ataxin), Atxnl (ataxin), Atxn2 (ataxin), Atxn7 (ataxin), AtxnlO (ataxin), DM (dystrophia myotonica-protein kinase), Atnl (atrophin 1), CBP (creb binding protein), VLDLR (very low density lipoprotein receptor), and any combination thereof.

[0322] According to another aspect, a method of gene therapy for the treatment of a subject having a mutation in the CFTR gene is provided and comprises administering a therapeuticallyeffective amount of a CRISPR-Cas gene therapy particle, optionally via a biocompatible pharmaceutical carrier, to the cells of a subject. Preferably, the target DNA comprises the mutation cIeltaF5G8. In general, it is of preferred that the mutation is repaired to the wildtype. In this case, the mutation is a deletion of the three nucleotides that comprise the codon for phenylalanine (F) at position 508. Accordi gly, repair in this instance requires reiiitroduction of the missing codon into the mutant.0Θ323] To implement this Gene Repair Strategy, it is preferred that an adenovirus / AAV vector system is introduced into the host cell, cells or patient. Preferably, the system comprises a Cas9 (or Cas9 nickase) and the guide RNA along with a adenovirus / AAV vector system comprising the homology repair template containing the F5Q8 residue. This may be introduced into the subject via one of the methods of deliver}' discussed earlier. The CRISPR-Cas system may be guided by the CFTRdelta 508 chimeric guide RNA. It 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 disorders such as those in 1 able B.Genome editing

[0324] The CR ISPR / Cas9 sy.si.cnr··> of the present invention can be used to correct genetic mutations that were previously attempted with limited success using TALEN and ZFN, For example, WQ2013163628 A2, Genetic Correction of Mutated Genes, published application of Duke University describes efforts to correct, for example, a frameshift mutation which causes a premature stop codon and a truncated gene product that can be corrected via nuclease .mediated non-homologous end joining such as those responsible for Duchenne Muscular Dystrophy, ("DMD") a recessive, fatal, X-linked disorder that results in .muscle degeneration due to mutations in the dystrophin gene. The majority of dystrophin mutations that cause DMD are deletions of exons that disrupt the reading frame and cause premature translation termination in the dystrophin gene. Dystrophin is a cytoplasmic protein that provides structural stability to the dystroglycan complex of the cell membrane that is responsible for regulating muscle cell integrity and function. The dystrophin gene or "DMD gene" as used interchangeably herein is 2.2megabases at locus Xp21. The primary transcription measures about 2,400 kb with the mature mR'NA being about 14 kb. 79 exons code for the protein which is over 3500 amino acids. Exon 51 is frequently adjacent to frame-disrupting deletions in DMD patients and has been targeted in clinical trials for oligonucSeotide-based exon skipping. A clinical trial for the exon 51 skipping compound eteplirsen recently reported a significa t functional benefit across 48 weeks, with an average of 47% dystrophin positive fibers compared to baseline. Mutations in exon 51 are ideal ly suited for permanent correction by NHEJ-based genome editing.

[0325] The methods of US Patent Publication No. 20130145487 assigned to Ceilectis, which relates to meganuclease variants to cleave a target sequence from the human dystrophin gene (DMD), may also be modified to for the CRISPR Cas system of the present invention.Blood

[0326] The present invention also contemplates delivering the CRISPR-Cas system to the blood.

[0327] The plasma exosomes of Wahlgren et ai, (Nucleic Acids Research, 2012, Vol. 40, No. 17 el30) were previously described and may be utilized to deliver the CRISPR Cas system to the blood.

[0328] The CRISPR Cas system of the present invention is also contemplated to treat hemoglobinopathies, such as thalassemias and sickle cell disease. See, e.g., International Patent Publication No. WO 2013 / 126794 for potential targets that may be targeted by the CRISPR Cas system of the present invention.

[0329] US Patent Publication Nos. 20110225664, 20110091441, 20100229252, 20090271881 and 20090222937 assigned to Ceilectis, relates to CREI variants , wherein at least one of the two I-Crel monomers has at least two substitutions, one in each of the two functional subdomains of the LAGLIDADG core domain situated respectively from positions 26 to 40 and 44 to 77 of I-Crel, said variant being able to cleave a DNA target sequence from the human interleukin-2 receptor gamma chain (IL2RG) gene also named common cytokine receptor gamma chain gene or gamma C gene. The target sequences identified in US Patent Publication Nos. 20110225664, 20110091441, 20100229252, 20090271881 and 20090222937 may be utilized for the CRJSPR Cas system of the present invention.

[0330] Severe Combined Immune Deficiency (SCID) results from a defect in lymphocytes T maturation, always associated with a functional defect in lymphocytes B (Cavazzana-Calvo etal, Annu. Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109). Overall incidence is estimated to 1 in 75 000 births. Patients with untreated SCID are subject to multiple opportunist micro-organism infections, and do generally not live beyond one year, SCID can be treated by al logenic hematopoietic stem cell transfer, from a fami lial donor. Histocompatibility with the donor can vary widely. In the case of Adenosine Deaminase (ADA) deficiency, one of the SCID forms, patients can be treated by injection of recombinant Adenosine Deaminase enzyme.

[0331] Since the ADA gene has been shown to be mutated in SCID patients (Giblett et al, Lancet, 1972, 2, 1067-1069), several other genes involved in SCID have been identified (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al., Immunol. Rev., 2005, 203, 98-109). There are four major causes for SCID: (i) the most frequent form of SCID, SCID-X1 (X-linked SCID or X-SCID), is caused by mutation in the IL2RG gene, resulting in the absence of mature T lymphocytes and NK. cells. IL2RG encodes the gamma C protein (Noguchi, et al., Cell, 1993, 73, 147-157), a common component of at least five interleukiii receptor complexes. These receptors activate several targets through the J AO kinase (Macchi et al., Nature, 1995, 377, 65-68), which inactivation results in the same syndrome as gamma C inactivation; (ii) mutation in the ADA gene results in a defect in purine metabolism that is lethal for lymphocyte precursors, which in turn results in the quasi absence of B, T and NK cells; (iii) V(D)J recombination is an essential step in the maturation of immunoglobulins and T lymphocytes receptors (TCl s). Mutations in Recombination Activating Gene 1 and 2 (RAG 1 and RAG2) and Artemis, three genes involved in this process, result in the absence of mature T and B lymphocytes; and (iv) Mutations in other genes such as CD45, involved in T cell specific signaling have also been reported, although they represent a minority of cases (Cavazzana-Calvo et al., Annu. Rev. Med., 2005, 56, 585-602; Fischer et al, Immunol . Rev., 2005, 203, 98-109).[003321 Since when their genetic bases have been identified, the different SCID forms have become a paradigm for gene therapy approaches (Fischer et al., Immunol. Rev., 2005, 203, 98- 109) for two major reasons. First, as in all blood diseases, an ex vivo treatment can be envisioned. Hematopoietic Stem Ceils (HSCs) can be recovered from bone marrow, and keep their piuripotent properties for a few cell divisions. Therefore, they can be treated in vitro, and then reinjected into the patient, where they repopulate the bone marrow. Second, since the maturation of lymphocytes is impaired in SCID patients, corrected cells have a selectiveadvantage. Therefore, a small number of corrected cells can restore a functional immune system. This hypothesis was validated several times by (i) the partial restoration of immune functions associated with the reversion of mutations in SCID patients (Hirschhorn et al., Nat. Genet., 1996, 13, 290-295; Stephan et al, N, Engl. J. Med., 1996, 335, 1563-1567; Bousso et al, Proc. Natl, Acad. Sci. USA, 2000, 97, 274-278; Wada et al., Proc. Natl Acad. Sci. USA, 2001, 98, 8697- 8702; Nishikomori et al., Blood, 2004, 103, 4565-4572), (ii) the correction of SCID-X 1 deficiencies in vitro in hematopoietic ceils (Candotti et al, Blood, 1996, 87, 3097-3102; Cavazzana-Caivo et al, Blood, 1996, Blood, 88, 3901-3909; Taylor et al, Blood, 1996, 87, 3103-3 107; Hacein-Bey et al, Blood, 1998, 92, 4090-4097), (iii) the correction of SCID-X1 (Soudais et al., Blood, 2000, 95, 3071-3077; Tsai et al., Blood, 2002, 100, 72-79), JA -3 (Bunting et al, Nat. Med., 1998, 4, 58-64; Bunting et al., Hum. Gene Ther., 2000, 1 1 , 2353- 2364) and RAG2 (Yates et al., Blood, 2002, 100, 3942-3949) deficiencies in vivo in animal models and (iv) by the result of gene therapy clinical trials (Cavazzana-Caivo et al., Science, 2000, 288, 669-672; Aiuti et al., Nat. Med., 2002; 8, 423-425; Gaspar et al, Lancet, 2004, 364, 2181-2187).[00333 S US Patent Publication No. 20110182867 assigned to the Children's Medical Center Corporation and the President and Fellows of Harvard College relates to methods and uses of modulating fetal hemoglobin expression (HbF) in a hematopoietic progenitor cells via inhibitors of BCL11A expression or activity, such as R Ai and a tibodies. The targets disclosed in US Patent Publication No. 201 101 82867, such as BCL11A, may be targeted by the CRJSPR Cas system of the present invention for modulating fetal hemoglobin expression. See also Bauer et al (Science 11 October 2013: Vol 342 no. 6155 pp. 253-257) and Xu et al. (Science 18 November 2011 : Vol. 334 no. 6058 pp. 993-996) for additional BCL11A targets.Ears

[0334] The present invention also contemplates delivering the CRISPR-Cas system to one or both ears.

[0335] Researchers are looking into whether gene therapy could be used to aid current deafness treatments— namely, cochlear implants. Deafness is often caused by lost or damaged hair cells that cannot relay signals to auditor}' neurons. In such cases, cochlear implants may be used to respond to sound and transmit electrical signals to the nerve cells. But these neuronsoften degenerate and retract from the cochlea as fewer growth factors are released by impaired hair cells.

[0336] US patent application 20120328580 describes injection of a pharmaceutical composition into the ear (e.g., auricular administration), such as into the luminae of the cochlea (e.g., the Scala media, Sc vestibulae, and Sc tympani), e.g., using a syringe, e.g., a single-dose syringe. For example, one or more of the compounds described herein can be administered by intratympanic injection (e.g., into the middle ear), and / or injections into the outer, middle, and / or inner ear. Such methods are routinely used in the art, for example, for the administration of steroids and antibiotics into human ears. Injection can be, for example, through the round window of the ear or through the cochlear capsule. Other inner ear administration methods are known in the art (see, e.g., Salt and Plontke, Drug Discovery Today, 10: 1299-1306, 2005).

[0337] In another mode of administratio , the pharmaceutical composition can be administered in situ,"via a catheter or pump. A catheter or pump can, for example, direct a pharmaceutical composition into the cochlear luminae or the round window of the ear and / or the lumen of the colon. Exemplary drug delivery apparatus and methods suitable for administering one or more of the compounds described herein into an ear, e.g., a human ear, are described by McKenna et al., (U.S. Publication No. 2006 / 0030837) and Jacobsen et al., (U.S. Pat. No. 7,206,639). In some embodiments, a catheter or pump can be positioned, e.g., in the ear (e.g., the outer, middle, and / or i ner ear) of a patient during a surgical procedure. In some embodiments, a catheter or pump can be positioned, e.g., in the ear (e.g., the outer, middle, and / or inner ear) of a patient without the need for a surgical procedure.

[0338] Alternatively or in addition, one or more of the compounds described herein can be administered in combination with a mechanical device such as a cochlear implant or a hearing aid, which is worn in the outer ear. An exemplary cochlear implant that is suitable for use with the present invention is described by Edge et al., (U.S. Publication No. 2007 / 0093878).

[0339] In some embodiments, the modes of administration described above may be combined in any order and can be simultaneous or interspersed.

[0340] Alternatively or in addition, the present invention may be administered according to any of the Food and Drug Administration approved methods, for example, as described in CDER Data Standards Manual, version number 004 (which is available at fda.give / cder / dsm DRG / drg00301.htm).

[0341] In general, the cell therapy methods described in US patent application 20120328580 can be used to promote complete or partial d fferentiation of a cell to or towards a mature cell type of the inner ear (e.g., a hair cell) in vitro. Cells resulting from such methods can then be transplanted or implanted into a patient in need of such treatment. The cell culture methods required to practice these methods, including methods for identifying and selecting suitable cell types, methods for promoting complete or partial differentiation of selected cel ls, methods for identifying complete or partially differentiated cell types, and methods for implanting complete or partially differentiated cells are described below.

[0342] Cells suitable for use in the present invention include, but are not limited to, cells that are capable of differentiating completely or partially into a mature cell of the inner ear, e.g., a hair cel l (e.g., an inner and / or outer hair cel l), when contacted, e.g., in vitro, with one or more of the compounds described herein. Exemplary cells that are capable of differentiating into a hair cel l include, but are not limited to stem cells (e.g., inner ear stem cells, adult stem cells, bone marrow derived stem cells, embryonic stem cells, mesenchymal stem cells, skin stem cells, iPS cells, and fat derived stem cells), progenitor cells (e.g., inner ear progenitor cells), support cells (e.g., Deiters' cells, pillar ceils, inner phalangeal cells, tectal cells and Hansen's cells), and / or germ cells. The use of stem cells for the replacement of inner ear sensory cells is described in Li et al, (U.S. Publication No. 2005 / 0287127) and Li et al, (U.S. patent Ser. No. 11 / 953,797). The use of bone marrow derived stem cells for the replacement of inner ear sensor}.' cells is described in Edge et al, PCT / US2007 / 084654. IPS cells are described, e.g., at Takahashi et al., Ceil, Volume 131, Issue 5, Pages 861-872 (2007); Takahashi and Yamanaka, Cell 126, 663-76 (2006); Okita et al, Nature 448, 260-262 (2007); Yu, J. et al, Science 318(5858):! 917-1920 (2007); Nakagawa et al, Nat. Biotechnol. 26:101-106 (2008); and Zaehres and Scholer, Cell 131(5):834- 835 (2007).[003431 Such suitable cells can be identified by analyzing (e.g., qualitatively or quantitatively) the presence of one or more tissue specific genes. For example, gene expression can be detected by detecting the protein product of one or more tissue-specific genes. Protein detection techniques involve staining proteins (e.g., using cell extracts or whole cells) using antibodies against the appropriate antigen. In this case, the appropriate antigen is the protein product of the tissue-specific gene expression. Although, in principle, a first antibody (i.e., the antibody that binds the antigen) can be labeled, it is more common (and improves the visualization) to use asecond antibody directed against the first (e.g., an anti-IgG). This second antibody is conjugated either with fluorochromes, or appropriate enzymes for coiorimetric reactions, or gold beads (for electron microscopy), or with the biotin-avidin system, so that the location of the primary antibody, and thus the antigen, can be recognized.

[0344] The CR1SPR Cas molecules of the present invention may be delivered to the ear by direct application of pharmaceutical composition to the outer ear, with compositions modified from US Published application, 201 10142917. In some embodiments the pharmaceutical composition is applied to the ear canal. Delivery to the ear may also be refered to as aural or otic delivery.

[0345] In some embodiments the RNA molecules of the invention are delivered in liposome or !ipofectirs 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.

[0346] 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: 1 1 1-114; 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, 3 1 , 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.

[0347] Qi et al. discloses methods for efficient siRNA transfeetion to the inner ear through the intact round window by a novel proteidic delivery technology which may be applied to the CRISPR Cas system of the present invention (see, e.g., Qi et al, Gene Therapy (2013), 1-9). In particular, a TAT double stranded RNA-binding domains (TAT-DRBDs), which can transfect Cy3-!abeled siRNA. into cells of the inner ear, including the inner and outer hair cells, crista ampullaris, macula utriculi and macula saeculi, through intact round-window permeation was successful for delivering double stranded siRNAs in vivo for treating various inner ear ailments and preservation of hearing function. About 40 μΐ of lOmM RNA may be contemplated as the dosage for administration to the ear.

[0348] According to Rejaii et al. (Hear Res, 2007 Jun;228(l -2): 180-7), cochlear implant function can be improved by good preservation of the spiral ganglion neurons, which are the target of electrical stimulation by the implant and brain derived neurotrophic factor (BDNF) has previously been shown to enhance spiral ganglion survival in experimentally deafened ears. Rejaii et al. tested a modified design of the cochlear implant electrode that includes a coating of"fi broblast cel ls transduced by a viral vector with a BDNF gene insert. To accomplish this type of ex vivo gene transfer, Rejaii et al. transduced guinea pig fibroblasts with an adenovirus with a BDNF gene cassette insert, and determined that these cells secreted BDNF and then attached BDNF-secreting cells to the cochlear implant electrode via an agarose gel, and implanted the electrode in the scala tympani. Rejaii et al. determined that the BDNF expressing electrodes were able to preserve significantl more spiral ganglion neurons in the basal turns of the cochlea after 48 days of implantation when compared to control electrodes and demonstrated the feasibility of combining cochlear implant therapy with ex vivo gene transfer for enhancing spiral ganglion neuron survival. Such a system may be applied to the CRISPR Cas system of the present invention for delivery to the ear.[003491 Mukherjea et al. (Antioxidants & Redox Signaling, Volume 13, Number 5, 2010) document that knockdown of NQX3 using short interfering (si) RNA abrogated cisplatin ototoxicity, as evidenced by protection of OHCs from damage and reduced threshold shifts in auditory brainstem responses (ABRs). Different doses of siNOX3 (0.3, 0.6, and 0.9 fig) were administered to rats and NOX3 expression was evaluated by real time RT-PCR, The lowest dose of NOX3 siRNA used (0.3 iig) did not show any inhibition of NOX3 mRNA when compared to transtympanic administration of scrambled siRNA or untreated cochleae. However, administration of the higher doses of NOX3 siRNA (0.6 and 0.9 μg) reduced NOX3 expression compared to control scrambled siRNA. Such a system may be applied to the CRISPR Cas system of the present invention for transtympanic administration with a dosage of about 2 mg to about 4 mg of CRISPR Cas for administratio to a human.

[0350] Jung et al. ( Molecular Therapy, vol 21 no. 4, 834-841 apr. 2013) demonstrate that Hes5 levels in the utricle decreased after the application of siRNA and that the number of hair cells in these utricles was significantly larger than following control treatment. The data suggest that siRNA technology may be useful for inducing repair and regeneration in the inner ear and that the Notch signaling pathway is a potentially useful target for specific gene expressioninhibition. Jung et al. injected 8 μ§ of Hes5 siRNA in 2 μ! volume, prepared by adding sterile normal saline to the lyophilized siRNA to a vestibular epithelium of the ear. Such a system may be applied to the CRISPR Cas system of the present invention for administration to the vestibular epithelium of the ear with a dosage of about 1 to about 30 mg of CRISPR Cas for administration to a human.Eyes[003511 The present invention also contemplates delivering the CRISPR-Cas system to one or both eyes.

[0352] In yet another aspect of the invention, the CRISPR-Cas system may be used to correct ocular defects that arise from several genetic mutations further described in Genetic Diseases of the Eye, Second Edition, edited by Elias I. Traboulsi, Oxford University Press, 2012.

[0353] For administration to the eye, lentiviral vectors, in particular equine infectious anemia viruses ( EJAV) are particularly preferred.

[0354] In another embodiment, minimal non-primate lentiviral vectors based on the equine infectious anemia virus (E1AV) are also contemplated, especially for ocular gene therapy (see, e.g., Balagaan, J Gene Med 2006; 8: 275 - 285, Published online 21 November 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / jgm.845). The vectors are contemplated to have cytomegalovirus (CMV) promoter driving expression of the target gene. Intracameral, subretinal, intraocular and intravitreal injections are all contemplated (see, e.g., Balagaan, .1 Gene Med 2006; 8: 275 - 285, Published online 21 November 2005 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002 / jgm.845). Intraocular injections may be performed with the aid of an operating microscope. For subretinal and intravitreal injections, eyes may be prolapsed by gentle digital pressure and fundi visualised using a contact lens system consisting of a drop of a coupling medium solution on the cornea covered with a glass microscope slide coverslip. For subretinal injections, the tip of a I Q-mm 34-gauge needle, mounted on a 5-μ1 Hamilton syringe may be advanced under direct visualisation through the superior equatorial sclera tangential ly towards the posterior pole until the aperture of the needle was visible in the subretinal space. Then, 2 μΐ of vector suspension may be injected to produce a superior bullous retinal detachment, thus confirming subretinal vector admin stration. This approach creates a self-sealing sclerotomy allowing the vector suspension to be retained in the subretinal space until it is absorbed by the RPE, usually within 48 h of the procedure. Thisprocedure may be repeated in the interior hemisphere to produce an inferior retinal detachment. This technique results in the exposure of approximately 70% of neurosensory retina and RPE to the vector suspension. For intr vitreal injections, the needle tip may be advanced through the sclera 1 mm posterior to the corneoscleral limbus and 2 μΐ of vector suspension injected into the vitreous cavity. For intracameral injections, the needle tip may be advanced through a corneoscleral limbal paracentesis, directed towards the central cornea, and 2 μΐ of vector suspension may be injected. For intracameral injections, the needle tip may be advanced through a corneoscleral limbal paracentesis, directed towards the central cornea, and 2 μΐ of vector suspension may be injected. These vectors may be injected at titres of either 1.0-1.4 x 10'° or 1.0-1.4 x 109transducing units (TU) / ml.

[0355] In another embodiment, RetinoStat®, an equine infectious anemia virus-based lentiviral gene therapy vector that expresses angiostatic proteins endostain 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)). Such a vector may be modified for the C ISPR-Cas system of the present invention. Each eye may be treated with either RctinoStat® at a dose of 1.1 x 103transducing units per eye (TU / eye) in a total volume of 100 μΐ.

[0356] In another embodiment, an E 1 -, partial E3-, E4-deIeted adenoviral vector may be contemplated for delivery to the eye. Twenty-eight patients with advanced neovaseular age- related macular degeneration (AMD) were given a single intravitreous injection of an E1 -, partial E3-, E4-deleted adenoviral vector expressing human pigment ep- itheiium-derived factor (AdPEDF.ll) (see, e.g., Campochiaro et al,, Human Gene Therapy 17: 167-176 (Februan' 2006)). Doses ranging from 106to 109'5particle units (PU) were investigated and there were no serious adverse events related to AdPEDF.ll and no dose-limiting toxicities (see, e.g., Campochiaro et al., Human Gene Therapy 17: 167-176 (February 2006)). Adenoviral vector- mediated ocular gene transfer appears to be a viable approach for the treatment of ocular disorders and could be applied to the CRISPR Cas system.

[0357] In another embodiment, the sd-rxRNA® system of RXi Pharmaceuticals may be used / and or adapted for delivering CRISPR Cas to the eye. in this system, a single intravitreal administration of 3 μg of sd-rxRNA results in sequence-specific reduction of PPIB mRNA levels for 14 days. The the sd-rxRNA® system may be applied to the CRISPR. Cas system of thepresent invention, contemplating a dose of about 3 to 20 mg of CRISPR administered to a human.

[0358] Miilington-Ward et al. (Molecular Therapy, vol. 19 no. 4, 642-649 apr. 2011) describes adeno-associated vims (AAV) vectors to deliver an RNA interference (RNAi)-based riiodopsin suppressor a d a codon-modified rhodopsin replacement gene resista t to suppression due to nucleotide alterations at degenerate positions over the RNAi target site. An injection of either 6.0 x 106vp or 1.8 x 10'° vp AAV were subretinaliy injected into the eyes by Millington- Ward et al. The AAV vectors of Miilington-Ward et al. may be applied to the CRISPR Cas system of the present invention, contemplating a dose of about 2 x 10f 1to about 6 x 10L' vp administered to a human.

[0359] Dalkara et al. (Sci Transl Med 5, 189ra76 (2013)) also relates to in vivo directed evolutio to fashion an AAV vector that delivers wild-type versio s of defective genes throughout the retina after noninjurious injection into the eyes' vitreous humor. Dalkara describes a a 7mer peptide display library and an AAV library constmcted by DNA shuffling of cap genes from AAVl, 2, 4, 5, 6, 8, and 9. The rcAAV libraries and r.AAV vectors expressing GFP under a CAG or Rho promoter were packaged and and deoxyribomiclease-resistant genomic titers were obtained through quantitative PGR. The libraries were pooled, and two rounds of evolution were performed, each consisting of initial library diversification followed by three in vivo selection steps. In each such step, P30 rho-GFP mice were intravitrealiy injected with 2 ml of iodixanol-purified, phosphate-buffered saline (PBS)-dialyzed library with a genomic titer of about 1 x 101"vg / ml. The AAV vectors of Dalkara et al. may be applied to the CRI SPR Cas system of the present invention, contemplating a dose of about 1 x 10° to about 1 x 10i6vg / ml administered to a human.

[0360] In another embodiment, the rhodopsin gene may be targeted for the treatment of retinitis pigmentosa (RP), wherein the system of US Patent Publication No. 20120204282 assigned to Sangamo Biosciences, Inc. may be modified in accordance of the CRISPR Cas system of the present invention.

[0361] In a other embodiment, the methods of US Patent Publication No. 20130183282 assigned to Cellectis, which is directed to methods of cleaving a target sequence from the human rhodopsin gene, may also be modified to the CRISPR Cas system of the present invention.

[0362] US Patent Publication No. 20130202678 assigned to Academia Sinica relates to methods for treating retinopathies and sight-threatening ophthalmologic disorders relating to delivering of the Puf-A gene (which is expressed in retinal ganglion and pigmented cells of eye tissues and displays a unique anti-apoptotic activity) to the sub-retinal or intravitreal space in the eye. In particular, desirable targets are zgc: 193933, prdmla, spata2, texlO, rbb4, ddx3, zp2.2, Blimp- 1 and HtrA2, all of which may be targeted by the CRISPR Cas system of the present invention.

[0363] Wu (Cell Stem Cell, 13 :659-62, 2013) designed a guide RNA that led Cas9 to a single base pair mutation that causes cataracts in mice, where it induced DNA cleavage. Then using either the other wild-type allele or oligos given to the zygotes repair mechanisms corrected the sequence of the broken al lele and corrected the cataract-causing genetic defect in mutant mouse.

[0364] US Patent Publication No. 20120159653, describes use of zinc finger nucleases to genetically modify cel ls, animals and proteins associated with macular degeration (MD). Macular degeneration (MD) is the primary cause of visual impairment in the elderly, but is also a hallmark symptom of childhood diseases such as Stargardt disease, Sorsby fundus, and fatal childhood neurodegenerative diseases, with an age of onset as young as infancy. Macular degeneration results in a loss of vision in the center of the visual field (the macula) because of damage to the retina. Currently existing animal models do not recapitulate major hallmarks of the disease as it is observed in humans. The available animal models comprising mutant genes encoding proteins associated with MD also produce highly variable phenotypes, making translations to human disease and therapy development problematic.[CI0365] One aspect of US Patent Publication No. 20120159653 relates to editing of any chromosomal sequences that encode proteins associated with MD which may be applied to the CRISPR Cas system of the present invention. The proteins associated with MD are typically selected based on an experimental association of the protein associated with MD to an MD disorder. For example, the production rate or circulating concentration of a protein associated with D may be elevated or depressed in a population having an MD disorder relative to a population lacking the MD disorder. Differences in protein levels may be assessed using proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the proteins associated with MD may be identified by obtaining gene expression profiles of thegenes encoding the proteins using genomic techniques including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

[0366] By way of non-limiting example, proteins associated with MD include but are not limited to the following proteins: (ABCA4) ATP-binding cassette, sub-family A (ABC1), member 4 ACH 1 achromatopsia (rod monochromacy) 1 ApoE Apolipoprotein E (ApoE) C1 QTNF5 (CTRP5) Clq and tumor necrosis factor related protein 5 (C 1QTNF5) C2 Complement component 2 (C2) C3 Complement components (C3) CCL2 Chemokine (C-C motif) Ligand 2 (CCL2) CCR2 Chemokine (C-C motif) receptor 2 (CCR2) CD36 Cluster of Differentiation 36 CFB Complement factor B CFH Complement factor CFH H CFHR1 complement factor H-related 1 CFHR3 complement factor H -related 3 C GB3 cyclic nucleotide gated channel beta 3 CP cemioplasmin (CP) CRP C reactive protein (CRP) CST3 cystatin C or cystatin 3 (CST3) CTSD Cathepsin D (CTSD) CX3CR1 chemoldne (C-X3-C motif) receptor 1 ELOVL4 Elongation of very long chain fatty acids 4 ERCC6 excision repair cross- complementing rodent repair deficiency, complementation group 6 FBLN5 Fibulin-5 FBLN5 Fibuiin 5 FBLN6 Fibulm 6 FSCN2 fascin (FSCN2) HMCNl Hemicentrin 1 HMCNl hemicentin I HTRA1 FItrA serine peptidase 1 (HTRA1 ) FITRA! HtrA serine peptidase 1 I F -6 Interleukin 6 IL-8 Interleukin 8 LOC387715 Hypothetical protein PLEKHA l Pleckstrin homology domain- containing family A member 1 (PLEKHAl) PROMl Prominin 1 (PROMl or CD133) PRPH2 Peripheri.n-2 RPGR retinitis pigmentosa GTPase regulator SERPINOl serpin peptidase inhibitor, clade G, member 1 (CI- inhibitor) TCOF1 Treacle TIMP3 Metalloproteinase inhibitor 3 (TTMP3) TLR3 Toil-like receptor 3

[0367] The identity of the protein associated with MD whose chromosomal sequence is edited can and will vary. In preferred embodiments, the proteins associated with MD whose chromosomal sequence is edited may be the ATP-binding cassette, sub-family A (ABC1) member 4 protein (ABCA4) encoded by the ABCR gene, the apolipoprotein E protein (APOE) encoded by the APOE gene, the chemokine (C-C motif) Ligand 2 protei (CCL2) encoded by the CCL2 gene, the chemokine (C-C motif) receptor 2 protein (CCR2) encoded by the CCR2 gene, the cemioplasmin protein (CP) encoded by the CP gene, the cathepsin D protein (CTSD) encoded by the CTSD gene, or the metalloproteinase inhibitor 3 protein (TIMP3) encoded by the T MP3 gene. In an exemplary embodiment, the genetically modified animal is a rat, and theedited chromosomal sequence encoding the protein associated with MD may be: (ABCA4) ATP- binding cassette, NM_000350 sub-family A (ABCl ), member 4 APOE Apoiipoprotein E NM 138828 (APOE) CCL2 Chemokine (C-C NM 031530 motif) Ligand 2 (CCL2) CCR2 Chemokine (C-C NM_021 S66 motif} receptor 2 (CCR2) CP ceruiopiasmin (CP) NMJ312532 CTSD Cathepsin D (CTSD) NM 134334 TIMP3 Metalloprotemase NM 012886 inhibitor 3 (TIMP3) The animal or cell may comprise 1, 2, 3, 4, 5, 6, 7 or more disrupted chromosomal sequences encoding a protein associated with MD and zero, 1 , 2, 3, 4, 5, 6, 7 or more chromosomally integrated sequences encoding the disrupted protein associated with MD.

[0368] The edited or integrated chromosomal sequence may be modified to encode an altered protein associated with MD. Several mutations in MD-related chromosomal sequences have been associated with MD. Non-limiting examples of mutations in chromosomal sequences associated with MD include those that may cause MD i cluding in the ABCR protein, E471 (i.e. glutamate at position 471 is changed to lysine), Rl 129L (i.e. arginine at position 1129 is changed to leucine), T1428M (i.e. threonine at position 1428 is changed to methionine), R1517S (i.e. arginine at position 1517 is changed to serine), I1562T (i.e. isoleucine at position 1562 is changed to threonine), and G1578R (i.e. glycine at position 1578 is changed to arginine); in the CCR2 protein, V64I (i.e. valine at position 192 is changed to isoleucine); in CP protein, G969B (i.e. glycine at position 969 is changed to asparagine or aspartate); in ΊΊΜΡ3 protein, S156C (i.e. serine at position 156 is changed to cysteine), G166C (i.e. glycine at position 166 is changed to cysteine), G167C (i.e. glycine at position 167 is changed to cysteine), Y168C (i.e. tyrosine at position 168 is cha ged to cysteine), S170C (i.e. serine at position 170 is changed to cysteine), YT72C (i.e. tyrosine at position 172 is changed to cysteine) and S1 81C (i.e. serine at position 181 is changed to cysteine). Other associations of genetic variants in MD-associated genes and disease are known in the art.Heart

[0369] The present invention also contemplates delivering the CRISPR-Cas system to the heart. For the heart, a myocardium tropic adena-assoeiated vims (AAVM) is preferred, in particular AAVM41 which showed preferential gene transfer in the heart (see, e.g., Lin-Yanga et ai., PNAS, March 10, 2009, vol. 106, no. 10). Administration may be systemic or local. A dosage of about 1-10 x 10' '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.

[0370] For example, US Patent Publication No. 20110023139, describes use of zinc finger nucleases to genetically modify cells, animals and proteins associated with cardiovascular disease. Cardiovascular diseases generally include high blood pressure, heart attacks, heart failure, and stroke and TLA.. Any chromosomal sequence involved in cardiovascular disease or the protein encoded by any chromosomal sequence involved in cardiovascular disease may be utilized in the methods described in this disclosure. The cardiovascular-re Sated proteins are typically selected based on an experimental association of the cardiovascular-related protein to the development of cardiovascular disease. For example, the production rate or circulating concentration of a cardiovascular-related protein may be elevated or depressed in a population having a cardiovascular disorder relative to a population lacking the cardiovascular disorder. Differences in protein levels may be assessed using proteomic techniques including but not limited to Western blot, immunohistochemical staining, enzyme linked immunosorbent assay (ELISA), and mass spectrometry. Alternatively, the cardiovascular-related proteins may be identified by obtaining gene expression profiles of the genes encoding the proteins using genomic techniques including but not limited to DNA microarray analysis, serial analysis of gene expression (SAGE), and quantitative real-time polymerase chain reaction (Q-PCR).

[0371] By way of example, the chromosomal sequence may comprise, but is not limited to, IL1B (interieukin 1, beta), XDH (xanthine dehydrogenase), TP53 (tumor protein p53), PTG1S (prostaglandin 12 (prostacyclin) synthase), MB (myoglobin), IL4 (interieukin 4), ANGPT1 (angiopoietin 1 ), ABCG8 (ATP-bindmg cassette, sub-family G (WHITE), member 8), CTSK (cathepsiii K), PTGIR (prostaglandin 12 (prostacyclin) receptor (IP)), CNJ11 (potassium inwardly-rectifying channel, subfamily J, member 1 1), INS (insulin), CRP (C -reactive protein, pentraxm-related), PDGFRB (platelet-derived growth factor receptor, beta polypeptide), CCNA2 (cyclin A2), PDGFB (platelet -derived growth factor beta polypeptide (simian sarcoma viral (v- sis) oncogene homolog)), CNJ5 (potassium inwardly-rectifying channel, subfamily J, member 5), KCNN3 (potassium intermediate / small conductance calcium-activated channel, subfamily N, member 3), CAPN1 Q (calpam 10), PTGES (prostaglandin E synthase), ADRA2B (adrenergic, alpha-2B-, receptor), ABCG5 (ATP-binding cassette, sub-family G (WHITE), member 5), PRDX2 (peroxiredoxin 2), CAP 5 (calpain 5), PARP14 (poly (ADP-ribose) polymerase family, member 14), MEX3C (mex-3 homolog C (C. elegans)), ACE angiotensin I converting enzyme (peptidyl-dipeptidase A) I), TNF (tumor necrosis factor (TNF superfamily, member 2)), IL6(interleukin 6 (interferon, beta 2)), STN (statin), SERPINEl (serpin peptidase inhibitor, clade E (nexin, plasminogen activator mhibitor type 1), member 1), ALB (albumin), ADIPOQ (adiponectin, C1Q and collagen domain containing), APOB (apolipoprotein B (including Ag(x) antigen)), APOE (apo lipoprotein E), LEP (leptin), MTHFR (5,10-methy!enetetrahydrofolate reductase (NADPH)), APOAl (apolipoprotein A-I), EDN1 (endothelin 1), NPPB (natriuretic peptide precursor B), NOS3 (nitric oxide synthase 3 (endothelial cell)), PPARG (peroxisome proliferator-activated receptor gamma), PLAT (plasminogen activator, tissue), PTGS2 (prostagiandin-endoperoxide synthase 2 (prostaglandin G / B synthase and cyclooxygenase)), CETP (eholesteryi ester transfer protein, plasma), AGTR1 (angiotensin 11 receptor, type 1), HMGCR (3-hydroxy-3-methylglutaryl-Coenzyme A. reductase), IGF1 (insulin-like growth factor 1 (somatomedin C)), SELE (selectin E), REN (renin), PPARA (peroxisome proliferator-activated receptor alpha), PON1 (paraoxoiiase 1), KNG1 (kininogen 1), CCL2 (chemokine (C-C motif) ligand 2), LPL (lipoprotein lipase), VWF (von Willebrand factor), F2 (coagulation factor II (thrombin)), ICAM1 (intercellular adhesion molecule 1), TGFB1 (transforming growth factor, beta 1), NPPA (natriuretic peptide precursor A), ILIO (interleukin 10), EPO (erythropoietin), SOD1 (superoxide dismutase 1, soluble), VCAM 1 (vascular cell adhesion molecule 1), IFNG (interferon, gamma), LPA (lipoprotein, Lp(a)), MPO (myeloperoxidase), ESR1 (estrogen receptor 1 ), MAP 1 (mitogen-activated protein kinase 1 ), HP (haptoglobin), F3 (coagulation factor III (thromboplastin, tissue factor)), CST3 (cystatin C), COG2 (component of oligomeric golgi complex 2), MMP9 (matrix metailopeptidase 9 (gelatinase B, 92 kDa gelatinase, 92 kDa type IV eollagenase)), SERPINC1 (serpin peptidase inhibitor, clade C (antithrombin), member 1), F8 (coagulation factor VI 11, procoagulant component), HMOX1 (heme oxygenase (decycling) 1 ), APOC3 (apolipoprotein C-III), 1L8 (interleukin 8), PROK1 (prokinetiem 1), CBS (cystathionine-beta-synthase), NOS2 (nitric oxide synthase 2, inducible), TLR4 (toll-like receptor 4), SELF (selectin P (granule membrane protein 140 kDa, antigen ( 1)62 ;· ). ABCA l (ATP -binding cassette, sub-family A (ABC!), member 1), AGT (angiotensinogen (serpin peptidase inhibitor, clade A, member 8)), LDLR (low density lipoprotein receptor), GPT (glutamic-pyruvate transaminase (alanine aminotransferase)), VEGFA (vascular endothelial growth factor A), R3C2 (nuclear receptor subfamily 3, group C, member 2), IL1 8 (inter!eukirs 18 (interferon-gamma-inducing factor)), NOS1 (nitric oxide synthase 1 (neuronal)), NR3C1 (nuclear receptor subfamily 3, group C, member 1 (glucocorticoid receptor)), FGB (fibrinogenbeta chain), GF (hepatocyte growth factor (hepapoietin A; scatter factor)), ILIA (interleukin 1, alpha), RETN (resistin), Λ ΊΊ (v-akt murine thymoma viral oncogene homo log 1 ), LIPC (lipase, hepatic), HSPD1 (heat shock 60 kDa protein 1 (chaperoiiin)), MAPK14 (mitogen- activated protein kinase 14), SPP1 (secreted phosphoprotein 1), ITGB3 (integrin, beta 3 (pi ate let glycoprotein 111a, antigen CD6I)), CAT (catalase), UTS2 (urotensin 2), THBD (thrombomodulin), F1.0 (coagulation factor X), CP (ceruloplasmiti (ferroxidase)), TNFRSF1 IB (tumor necrosis factor receptor superfamily, member 1 lb), EDNRA (endothelin receptor type A), EGFR (epidermal growth factor receptor (erythroblastic leukemia viral (v-erb-b) oncogene homo log, avian)), MMP2 (matrix metailopeptidase 2 (gclatinasc A, 72 kDa geiatinase, 72 kDa type IV coilagenase)), PLG (plasminogen), NPY (neuropeptide Y), RHOD (ras homolog gene family, member D), AP 8 (mitogen-activated protein kinase 8), MYC (v-myc myelocytomatosis viral oncogene homolog (avian)), FNl (fibronectiii I), CMAl (chymase 1, mast cell), PLAU (plasminogen activator, urokinase), GNB3 (guanine nucleotide binding protein (G protein), beta polypeptide 3), ADRB2 (adrenergic, beta-2-, receptor, surface), APGA5 (apolipoprotem A-V), SOD2 (superoxide dismutase 2, mitochondrial), F5 (coagulation factor V (proacceierin, labile factor)), VDR (vitamin D (1 ,25-dihydroxyvitamin D3) receptor), ALOX5 (arachidonate 5 -lipoxygenase), HLA-D B1 (major histocompatibility complex, class II, DR beta 1), PARP1 (poly (ADP-ribose) polymerase 1 ), CD40LG (CD40 ligand), POX (paraoxonase 2), AGER (advanced glycosylation end product-specific receptor), IRS I (insulin receptor substrate 1), PTGS1 (prostaglandm-endoperoxide synthase 1 (prostaglandin G / H synthase and cyclooxygenase)), ECEl (endothelin converting enzyme 1), F7 (coagulation factor VII (semm prothrombin conversion accelerator)), URN (interleukin 1 receptor antagonist), EPHX2 (epoxide hydrolase 2, cytoplasmic), IGFBP1 (insulin-like growth factor binding protein 1 ), MAP 10 (mitogen-activated protein kinase 10), FAS (Fas (TNF receptor superfamily, member 6)), ABCB1 (ATP-binding cassette, sub-family B (MDR TAP), member 1), JUN (jun oncogene), IGFBP3 (insulin-like growth factor binding protein 3), CD 14 (CD 14 molecule), PDE5A (phosphodiesterase 5A, cGMP-specific), AGTR2 (angiotensin II receptor, type 2), CD40 (CD40 molecule, TNF receptor superfamily member 5), LCAT (lecithin-cholesterol acyitransferase), CCR5 (chemokine (C-C motif) receptor 5), MMP1 (matrix metailopeptidase 1 (interstitial coilagenase)), TIMP1 (TIMP metailopeptidase inhibitor I), ADM (adrenomedulliii), DYT10 (dystonia 10), STAT3 (signal transducer and activator of transcription 3 (acute-phase responsefactor)), MMP3 (matrix metallopeptidase 3 (stromelysin 1, progelatmase)), ELN (elastin), USF1 (upstream transcription factor 1), C'f'l 1 (complement factor H), HSPA4 (heat shock 70 kDa protein 4), MMP12 (matrix metallopeptidase 12 (macrophage elastase)), MME (membrane metallo-endopeptidase), F2R (coagulation factor ]] (thrombin) receptor), SELL (selectin L), CTSB (cathepsiii B), ANXA5 (annexin A5), ADRB1 (adrenergic, beta-1-, receptor), CYBA (cytochrome b-245, alpha polypeptide), EGA (fibrinogen alpha chain), GGT1 (gamma- glutamyitransferase 1 ), LIPG (lipase, endothelial), HIF1A (hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)), CXCR4 (chemokine (C-X-C motif) receptor 4), PROC (protem C (mactivator of coagulation factors Va and Villa)), SCARBl (scavenger receptor class B, member 1), CD79A (CD79a molecule, immunoglobulin-associated alpha), PLTP (phospholipid transfer protein), ADD! (adducin 1 (alpha)), FGG (fibrinogen gamma chain), SAA1 (serum amyloid Al), K.CNH2 (potassium voltage-gated channel, subfamily H (eag-related), member 2), DPP4 (dipeptidyl-peptidase 4), G6PD (glucose-6- phosphate dehydrogenase), NPR1 (natriuretic peptide receptor A / guanylate cyclase A (atrionatriuretic peptide receptor A)), VTN (vitronectin), KIAAOIOI (KIAAOiOl), FOS (FBJ murine osteosarcoma viral oncogene homolog), TLR2 (toll-like receptor 2), PPIG (peptidylprolyl isom erase G (cyclophilin G)), IL1R1 (mterleukin 1 receptor, type I), AR (androgen receptor), CYP1A 1 (cytochrome P450, family 1, subfamily A, polypeptide 1), SERP1NA1 (serpin peptidase inhibitor, clade A (alpha- 1 antiproteinase, antitrypsin), member 1), MTR (5- methyltetrahydrofolate-homocysteine methyltransferase), RBP4 (retmol binding protein 4, plasma), APOA4 (apolipoprotein A-IV), CDKN2A (cyclin-dependent kinase inhibitor 2A (melanoma, pi 6, inhibits CDK4)), FGF2 (fibroblast growth factor 2 (basic)), EDNRB (endothelin receptor type B), ITGA2 (integrin, alpha 2 (CD49B, alpha 2 subunit of VLA-2 receptor)), CABIN i (calcineurin binding protein 1), SHBG (sex hormone-binding globulin), HMGBl (high-mobility group box 1 ), HSP90B2P (heat shock protein 90 kDa beta (Grp94), member 2 (pseudogene)), CYP3A4 (cytochrome P450, family 3, subfamily A, polypeptide 4), GJAl (gap junction protein, alpha 1, 43 kDa), ( V I (caveolin 1, caveolae protein, 22 kDa), ESR2 (estrogen receptor 2 (ER beta)), LTA (lymphotoxin alpha (TNF superfamily, member 1)), GDF15 (growth differentiation factor 15), BDNF (brain-derived neurotrophic factor), CYP2D6 (cytochrome P450, family 2, subfamily D, polypeptide 6), NGF (nerve growth factor (beta polypeptide)), SPl (Spl transcription factor), TGIFl (TGFB-induced factor homeobox i), SRC(v-src sarcoma (Schmidt-Ruppin A-2) viral oncogene homoiog (avian)), EGF (epidermal growth factor (beta-urogastrone)), PIK3CG (phosphoinositide-3-kinase, catalytic, gamma polypeptide), HLA-A (major histocompatibiKty complex, class I, A), CNQ1 (potassium voltage-gated channel, QT-Hke subfamily, member 1), CNR1 (cannabinoid receptor 1 (brain)), FBN1 (fibrilli 1), CHELA (choline kinase alpha), BEST1 (bestrophin 1), APP (amyloid beta (A4) precursor protein), CTNNB1 (eaten in (cadherin-assoeiated protein), beta 1, 88 kDa), 1 L2 (interleukin 2), CD36 (CD36 molecule (thrombospondin receptor)), PRKAB1 (protein kinase, AMP-activated, beta 1 non-catalytic subunit), TPO (thyroid peroxidase), ALDH7A1 (aldehyde dehydrogenase 7 family, member Al), CX3CR 1 (ehemokine (C-X3-C motif) receptor 1 ), TH (tyrosine hydroxylase), F9 (coagulation factor IX), GB1 (growth hormone 1), TF (transferrin), FIFE (hemochromatosis), 1 L17A (interfeukin 17 A), PTEN (phosphatase and tensin homoiog), GSTM1 (glutathione S-transferase mu 1), DMD (dystrophin), GATA4 (GATA binding protein 4), F13A1 (coagulation factor X l f f. Al polypeptide), TTR (transthyretin), FABP4 (fatty acid binding protein 4, adipocyte), PON3 (paraoxonase 3), APOC1 (apolipoprotein C-I), INSR (insulin receptor), TNFRSF1B (tumor necrosis factor receptor superfamily, member IB), HTR2A (5-hydroxyrxyptamine (serotonin) receptor 2A), CSF3 (colony stimulating factor 3 (granulocyte)), CYP2C9 (cytochrome P450, family 2, subfamily C, polypeptide 9), TXN (thiorcdo in), CYP11 B2 (cytochrome P450, family 1 1, subfamily B, polypeptide 2), PTH (parathyroid hormone), CSF2 (colony stimulating factor 2 (granulocyte-macrophage)), KDR (kinase insert domain receptor (a type If f receptor tyrosine kinase)), PLA2G2A (phospholipase A2, group IIA (platelets, synovial fluid)), B2M (beta-2-microglobulin), THBS1 (thrombospondin 1), GCX) (glucagon), R OA (ras homoiog gene family, member A), A LDFI2 (aldehyde dehydrogenase 2 family (mitochondrial)), TCF7L2 (transcription factor 7 -like 2 (T-cell specific, HMG-box)), BDKRB2 (bradykinin receptor B2), NFE2L2 (nuclear factor (erythroid-derived 2)- like 2), NOTCH ! (Notch homoiog 1 , transiocation-associated (Drosophila)), UGTIA I (UDP giucuronosyltransferase 1 family, polypeptide Al), IFNAl (interferon, alpha 1), PPARD (peroxisome pro iiferator-activated receptor delta), SIRTl (sirtuin (silent mating type information regulation 2 homoiog) 1 (S. cerevisiae)), GNRH1 (gonadotrop in-releasing hormone 1 (luteinizing-reieasing hormone)), PAPPA (pregnancy-associated plasma protein A, pappalysin 1), ARR3 (arrestin 3, retinal (X-arrestin)), NPPC (natriuretic peptide precursor C), AHSP (alpha hemoglobin stabilizing protein), PTK2 (PTK2 protein tyrosine kinase 2), IL13 (interleukin 13),MTOR (mechanistic target of rapamycin (serine / threonine kinase)), ITGB2 (integrin, beta 2 (complement component 3 receptor 3 and 4 subunit)), GSTTl (glutathione S-transferase theta 1 ), IL6ST (interleukin 6 signal transducer (gpl30, oncostatin M receptor)), CPB2 (earboxypeptidase B2 (plasma)), CYP1A2 (cytochrome P450, family 1, subfamily A, polypeptide 2), HNF4A (hepatocyte nuclear factor 4, alpha), SLC6A4 (solute carrier family 6 (neurotransmitter transporter, serotonin), member 4), PLA2G6 (phospholipase A2, group VI (cytosoSic, calcium- independent)}, TNFSF1 1 (tumor necrosis factor (ligand) superfamily, member 11), SLC8A1 (solute carrier family 8 (sodium / calcium exchanger), member 1), F2RL3 (coagulation factor II (thrombin) receptor-like 1 ), A R1A 1 (aido-keto reductase family 1, member A l (aldehyde reductase)), ALDH9A1 (aldehyde dehydrogenase 9 family, member Al), BGLAP (bone gamma- carboxyglutamate (gla) protein), MTTP (microsomal triglyceride transfer protein), MTRR (5- methyltetrahydrofolate-homocysteine metliyltraiisferase reductase), SULT1A3 (sulfotransferase family, cytosolic, 1 A, phenol -preferring, member 3), RAG E (renal tumor antigen), C4B (complement component 4B (Chido blood group), P2RY12 (purinergic receptor P2Y, G-protein coupled, 12), RNLS (renalase, FAD-dependent amine oxidase), CREBl (cAMP responsive element binding protein 1 ), POMC (proopiomelanocortin), RAC 1 (ras-related C3 botulinum toxin substrate 1 (rho tamily, small GTP binding protein Raci)), LMNA (lamin NCj, CD59 (CD59 molecule, complement regulatory protein}, SCN5A (sodium channel, voltage-gated, type V, alpha subunit), CYP1B1 (cytochrome P450, family I, subfamily B, polypeptide 1), MIF (macrophage migration inhibitory factor (glycosylation -inhibiting factor)), MM 13 (matrix metallopeptidase 13 (collagenase 3)), TIMP2 (ΊΊΜΡ metallopeptidase inhibitor 2), CYP19A1 (cytochrome P450, family 19, subfamily A, polypeptide 1 ), CYP21 A2 (cytochrome P450, family 21, subfamily A, polypeptide 2), PTPN22 (protein tyrosine phosphatase, non-receptor type 22 (lymphoid)), MYH14 (myosin, heavy chain 14, non-muscle), MBL2 (mannose-binding lectin (protein C) 2, soluble (opsonic defect)), SELPLG (selectin P ligand), AOC3 (amine oxidase, copper containing 3 (vascular adhesion protein !)), CTSL1 (cathepsin LI), PCNA (proliferating cel l nuclear antigen), IGF2 (insulin -like growth factor 2 (somatomedin A)), ITGB1 (integrin, beta 1 (fibronectin receptor, beta polypeptide, antigen CD29 includes MDF2, MS 12)), CAST (calpastatin), CXCL12 (chemokine (C-X-C motif) ligand 12 (stromal cell-derived factor 1)), IGHE (immunoglobulin heavy constant epsilon), CNE 1 (potassium voltage-gated channel, Isk- related family, member 1), TFRC (transfemn receptor (p90. CD71)), COLIAI (collagen, type I,alpha 1), COL1A2 (collagen, type I, alpha 2), IL2RB (interleukin 2 receptor, beta), PLA2G10 (phospholipase A2, group X), ANGPT2 (angiopoietin 2), PROCR (protein C receptor, endothelial (EPCR)), NOX4 (NADPH oxidase 4), HAMP (hepcidin antimicrobial peptide), PTP 11 (protein tyrosine phosphatase, non-receptor type 1 1 ), SLC2A1 (solute carrier family 2 (facilitated glucose transporter), member 1), IL2RA (interleuki 2 receptor, alpha), CCL5 (chemokine (C-C motif) ligand 5), IRF1 (interferon regulatory factor 1), CFLAR (CASP8 and FADD-like apoptosis regulator), CALCA (caicitonin-reiated polypeptide alpha), EIF4E (eukaryotic translation initiation factor 4E), GSTP1 (glutathione S~transferase pi I), JAK2 (Janus kinase 2), CYP3A5 (cytochrome P450, family 3, subfamily A, polypeptide 5), HSPG2 (heparan sulfate proteoglycan 2), CCL3 (chemokine (C-C motif) ligand 3), MYD88 (myeloid differentiation primary response gene (88)), VIP (vasoactive intestinal peptide), SOAT1 (sterol O-acyltransferase 1), ADRB 1 (adrenergic, beta, receptor kinase 1), NR4A2 (nuclear receptor subfamily 4, group A, member 2), MMP8 (matrix metai!opeptidase 8 (neutrophil collagenase)), NPR2 (natriuretic peptide receptor B / guanylate cyclase B (atrionatriuretic peptide receptor B)), GCH3 (GTP cyclohydrolase 1), EPRS (glutamyl-prolyl-tRNA synthetase), PPARGC1A (peroxisome proliferator-activated receptor gamma, coactivator 1 alpha), F12 (coagulation factor XII (Hageman factor)), PEC AMI (platelet / endothelial cell adhesion molecule), CCL4 (chemokine (C-C motif) ligand 4), SERPINA3 (serpin peptidase inhibitor, clade A (alpha- 1 antiproteinase, antitrypsin), member 3), CASR (calcium-sensing receptor), GJA5 (gap junction protein, alpha 5, 40 kDa), FABP2 (fatty acid binding protein 2, intestinal), TTF2 (transcription termination factor, RNA polymerase II), PRGSI (protein S (alpha)), CTFl (cardiotrophm 1), SGCB (sarcoglycan, beta (43 kDa dystrophin -associated glycoprotein)), YMEI LI (YMEl-like 1 (S. ccrevisiae}}, CAMP (cathelicidin antimicrobial peptide), ZC3H 12A (zinc finger CCCH-type containing 12 A), A R1B3 (a do-keto reductase family 1 , member Bl (aldose reductase)), DBS (desmin), MMP7 (matrix metal lopeptidasc 7 (matrilysin, uterine)}, AFJR (aryl hydrocarbon receptor), CSF3 (colony stimulating factor 1 (macrophage)), HDAC9 (histone deacetylase 9), CTGF (connective tissue growth factor), KCNMA1 (potassium large conductance calcium- activated channel, subfamily M, alpha member 1), UGT1A (UDP glucuronosyltransferase I family, polypeptide A complex locus), PRKCA (protein kinase C, alpha), COMT (catechol- .beta.-methyltransferase), S100B (SI 00 calcium binding protein B), EGR1 (early growth response 1), PRL (prolactin), IL15 (interleukin 15), DRD4 (dopamine receptor D4), CAMK2G(calcium / calrnodulin-dependent protein kinase II gamma), SLC22A2 (solute carrier family 22 (organic cation transporter), member 2), CCL1 1 (ehemokine (C-C motif) Hgand 1 1), PGF (B321 placental growth factor), THPO (thrombopoietin), GP6 (glycoprotein VI (platelet)), TACR1 (tachykinin receptor 1), NTS (neurotensin), HNF A (H F.1 homeobox A), SST (somatostatin), KCND1 (potassium voltage-gated channel, Shal-reiated subfamily, member I), LOC646627 (phosphobpase inhibitor), TBXASl (thromboxane A synthase 1 (platelet)), CYP2J2 (cytochrome P450, family 2, subfamily J, polypeptide 2), TBXA2R (thromboxane A2 receptor), ADH I C (alcohol dehydrogenase 1C (class I), gamma polypeptide), ALOX12 (arachidonate 12- iipoxygenase), AHSG (alpha-2-HS-giycoprotein), BHMT (betaine- omocysteine rnethyltransferase), GJA4 (gap junction protein, alpha 4, 37 kDa), SLC25A4 (solute carrier family 25 (mitochondrial carrier; adenine nucleotide translocator), member 4), ACLY (ATP citrate lyase), ALOX5AP (arachidonate 5 -lipoxygenase-activating protein), NUMA1 (nuclear mitotic apparatus protein 1), CYP27B1 (cytochrome P450, family 27, subfamily B, polypeptide 1), CYSLTR2 (cysteinyl leukotriene receptor 2), SODS (superoxide dismutase 3, extracellular), LTC4S (leukotriene C4 synthase), UCN (urocortin), GFIRL (ghrelm / obestatin prepropeptide), APOC2 (apolipoprotein C-II), CLEC4A (C-type lectin domain family 4, member A), KBTBDIO (kelch repeat and BTB (POZ) domain containing 10), TNC (tenascin C), TYMS (thymidylate synthetase), SHCl (SHC (Src homology 2 domain containing) transforming protein 1 ), LRPl (low density lipoprotein receptor-related protein 1), SOCS3 (suppressor of cytokine signaling 3), ADFIIB (alcohol dehydrogenase I B (class I), beta polypeptide), KLK3 (kaliikrein -related peptidase 3), HSD11B1 (hydroxysteroid (11 -beta) dehydrogenase 1), VKORC1 (vitamin K epoxide reductase complex, subunit 1 ), SERP1NB2 (serpin peptidase inhibitor, clade B (ovalbumin), member 2), TNS1 (tensin 1 ), R F19A (ring finger protein 19A), EPOR (erythropoietin receptor), ITGAM (integrin, alpha M (complement component 3 receptor 3 subunit)), ΡΓΓΧ2 (paired-like homeodomain 2), MAPK7 (mitogen-activated protein kinase 7), FCGR3A (Fc fragment of IgG, low affinity H I a, receptor (CD 16a)), LEPR (leptin receptor), ENG (endoglin), GPXl (glutathione peroxidase 1), GOT2 (glutamic-oxaloacetic transaminase 2, mitochondrial (aspartate aminotransferase 2)), HRH1 (histamine receptor HI), NRI 12 (nuclear receptor subfamily 1, group I, member 2), CRH (corticotropin releasing hormone), HTR A (5- hydroxytryptamine (serotonin) receptor IA), VDAC1 (voltage-dependent anion channel 1), FIPSE (heparanase), SFTPD (surfactant protein D), TAP2 (transporter 2, ATP-binding cassette,sub-family B (MDR / TAP)), RNF123 (ring finger protein 123), PTK2B (PTK2B protein tyrosine kinase 2 beta), NTRK2 (neurotrophic tyrosine kinase, receptor, type 2), IL6R (interleukin 6 receptor), ACHE (acetylcholinesterase (Yt blood group)), GLP1R (glucagon-like peptide 1 receptor), GHR (growth hormone receptor), GSR (glutathione reductase), NQOl (NAD(P)H dehydrogenase, quinone 1), NR5A1 (nuclear receptor subfamily 5, group A, member 1), GJB2 (gap junction protein, beta 2, 26 kDa), SLC9A 1 (solute carrier family 9 (sodium / hydrogen exchanger), member 1), MAOA (monoamine oxidase A), PCSK.9 (proprotein convertase subtilisin / kexm type 9), FCGR2A (Fc fragment of IgG, low affinity Ila, receptor (CD32)), SERPINFl (serpin peptidase inhibitor, ciade F (alpha -2 antiplasmin, pigment epithelium derived factor), member 1), EDN3 (endothelin 3), DHFR (dihydro folate reductase), GAS6 (growth arrest-specific 6), SMPD1 (sphingomyeSin phosphodiesterase 1, acid lysosomal), UCP2 (uncoupling protein 2 (mitochondrial, proton carrier)), TFAP2A (transcription factor AP-2 alpha (activating enhancer binding protein 2 alpha)), C4BPA (complement component 4 binding protein, alpha), SERPINF2 (serpin peptidase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium derived factor), member 2), TYMP (thymidine phosphorylase), ALPP (alkaline phosphatase, placental (Regan isozyme)}, CXC 2 (ehemokinc (C-X-C motif) receptor 2), SLC39A3 (solute carrier family 39 (zinc transporter), member 3), ABCG2 (ATP-binding cassette, sub-family G ( WHITE), member 2), ADA (adenosine deaminase), JAK3 (Janus kinase 3), HSPA1A (heat shock 70 kDa protein 1A), FASN (fatty acid synthase), FGF1 (fibroblast growth factor 1 (acidic)), Fl ! (coagulation factor XI), ATP7A (AT ase, Cu++ transporting, alpha polypeptide), CR1 (complement component (3b / 4b) receptor 1 (Kiiops blood group)), GFAP (glial fibrillary acidic protein), ROCK1 (Rho-associated, coiled-coil containing protein kinase 1 ), MECP2 (methyl CpG binding protein 2 (Rett syndrome)), MYLK (myosin light chain kinase), BCHE (butyrylcholinesterase), LIPE (lipase, hormone-sensitive), PRDX5 (peroxiredoxin 5), ADORA1 (adenosine Al receptor), WRN (Werner syndrome, RecQ helicase- iike), CXCR3 (chemokine (C-X-C motif) receptor 3), CD81 (CD81 molecule), SMAD7 (SMAD family member 7), LAMC2 (laminin, gamma 2), MAP3K5 (mitogen -activated protein kinase kinase kinase 5), CHGA (chromogranin A (parathyroid secretory protein 1)), LAPP (islet amyloid polypeptide), RHO (rhodopsin), ENPP1 (ectonucleotide pyrophosphatase / phosphodiesterase 1 ), PTHLHhormone-like hormone), RG1 (neuregulin 1), VEGFC (vascular endothelial growth factor C), ENPEP (glutamyl aminopeptidase (aminopeptidase A)), CEBPB(CCAAT / enhancer binding protein (C / EBP), beta), NAGLU (N-acetylglucosaminidase, alpha-), F2RL3 (coagulation factor ]] (thrombin) receptor-like 3), CX3CL1 (chemokme (C-X3-C motif) ligand 1), BDKRB1 (bradykinin receptor Bl), ADAMTS13 (ADAM metaliopeptidase with thrombospondin type 1 motif, 13), ELA'NE (elastase, neutrophil expressed), ENPP2 (ectonucleotide pyrophosphatase / phosphodiesterase 2), CISH (cytokine inducible SH2- containing protein), GAST (gastrin), MYOC (myocilin, trabecular meshwork inducible glucocorticoid response), ATP1A2 (ATPase, Na+ / K+ transporting, alpha 2 polypeptide), NF1 (neurofibromin 1 ), GJB1 (gap junction protein, beta 1, 32 kDa), MEF2A (myocyte enhancer factor 2A), VCL (vinculin), BMPR2 (bone morphogenetic protein receptor, type II (serine / threonine kinase)), TUBB (tubulin, beta), CDC42 (cell division cycle 42 (GTP binding protein, 25 kDa)), KRT1 8 (keratin 18), HSFl (heat shock transcription factor 1 ), MYB (v-myb myeloblastosis viral oncogene homolog (avian)), PRKAA2 (protein kinase, AMP-activated, alpha 2 catalytic subu t), ROCK2 (Rho-associated, coiled-coil containing protein kinase 2), TFPI (tissue factor pathway inhibitor (lipoprotein-associated coagulation inhibitor)), PRKGl (protein kinase, cGMP-dependent, type I), BMP2 (bone morphogenetic protein 2), CTNND1 (catenin (cadherin-associated protein), delta 1), CTH (cystathionase (cystathionine gamma- lyase)), CTSS (cathepsin S), VAV2 (vav 2 guanine nucleotide exchange factor), NPY2R (neuropeptide Y receptor Y2), 1GFBP2 (insulin-like growth factor binding protein 2, 36 kDa), CD28 (CD28 molecule), GSTA1 (glutathione S-transferase alpha 1), PPIA (peptidylprolyl isomerase A (cyclophilin A)), APOH (apolipoprotein H (beta-2-glycoprotein 1)), S100A8 (SI.00 calcium binding protein A8), IL11 (interleukin 11), ALOX15 (arachidonate 15 -lipoxygenase), FBLNl (fibulin 1 ), NR1H3 (nuclear receptor subfamily 1 , group H, member 3), SCD (stearoyl- CoA desaturase (delta-9-desaturase)), GIP (gastric inhibitory polypeptide), CHGB (chromogranin B (secretogranin 1)), PRKCB (protein kinase C, beta), SRD5A1 (sterotd-5-alpha- reductase, alpha polypeptide 1 (3-oxo-5 alpha-steroid delta 4-dehydrogenase alpha 1)), HSD11B2 (hydroxysteroid (3 1-beta) dehydrogenase 2), CALCRL (calcitonin receptor-like), GALNT2 (UDP-N-acetyl-afpha-D-ga!actosamine:pofypeptide N-acetylgalactosaminyltransferase 2 (GalNAc-T2)), ANGPTL4 (angiopoietin-like 4), KC N4 (potassium intermediate / small conductance calcium-activated channel, subfamily N, member 4), PIK3C2A (phosphoinositide- 3-kinase, class 2, alpha polypeptide), HBEGF (heparin-binding EGF-like growth factor), CYP7A1 (cytochrome P450, family 7, subfamily A, polypeptide 1), HLA-DRB5 (majorhistocompatibility complex, class II, DR beta 5), BNIP3 (BCL2 / adeno virus E1B 19 kDa interacting protein 3), GCKR (glueokinase (hexokinase 4) regulator), S100A12 (SI 00 calcium binding protein AI2), PADI4 (peptidyl arginine deiminase, type IV), HSPAI4 (heat shock 70 kDa protein 14), CXCR l (chemokine (C-X-C motif) receptor 1 ), HI 9 (HI 9, imprinted maternally expressed transcript (non-protein coding)), KRTAP19-3 (keratin associated protem 19-3), IDDM2 (insulin-dependent diabetes mellitus 2), RAC2 (ras-related C3 botulinum toxin substrate 2 (rho family, small GTP binding protein Rac2)), RYR 1 (ryanodine receptor 1 (skeletal)), CLOCK (clock homolog (mouse)), NGFR (nerve growth factor receptor (TNFR superfamiiv, member 16)), DBH (dopamine beta-hydroxylase (dopamine beta-monooxygenase)), CHRNA4 (cholinergic receptor, nicotinic, alpha 4), CACNA1C (calcium channel, voltage- dependent, L type, alpha 1C subunit), PR AG2 (protein kinase, AMP-aetivated, gamma 2 non- catalytic subuiiit), CHAT (choline acetyltransferase), PTGDS (prostaglandin D2 synthase 21 kDa (brain)), NR1H2 (nuclear receptor subfamily 1, group H, member 2), TEK (TEK tyrosine kinase, endothelial), VEGFB (vascular endothelial growth factor B), MEF2C (myocyte enhancer factor 2C), MAPKAPK2 (mitogen-activated protein kinase-activated protein kinase 2), TNFRSF11A (tumor necrosis factor receptor superfamiiv, member 11a, NFKB activator), HSPA9 (heat shock 70 kDa protem 9 (mortalin)), CYSLTR3 (cysteinyl leukotriene receptor 1 ), MAT1A (methionine adenosyl transferase I, alpha), OPRL1 (opiate receptor-like 1), IMPA1 (inositol(myo)-l(or 4)- moiiophosphatase 1), CLCN2 (chloride channel 2), DLD (dihydrolipoamide dehydrogenase), PSMA6 (proteasome (prosome, macropain) subunit, alpha type, 6), PS B8 (proteasome (prosome, macropain) subunit, beta type, 8 (large multifunctional peptidase 7)), CHI3L1 (chitinase 3-like 1 (cartilage gSycoprotein-39)), ALDH1 B1 (aldehyde dehydrogenase 1 family, member Bl), PARP2 (poly (ADP-ribose) polymerase 2), STAR (steroidogenic acute regulatory protein), LBP (lipopolysaccharide binding protein), ABCC6 (ATP-binding cassette, sub-family C(CFTR / MRP), member 6), RGS2 (regulator of G-protein signaling 2, 24 kDa), EFNB2 (ephrin- B2), GJB6 (gap junction protein, beta 6, 30 kDa), APOA2 (apo lipoprotein A-II), AMPD1 (adenosine monophosphate deaminase 1), DYSF (dysferiin, limb girdle muscular dystrophy 2B (autosomal recessive)), FDFT1 (farnesyl-diphosphate famesyitransferase 1), EDN2 (endothelin 2), CCR6 (chemokine (C-C motif) receptor 6), GJB3 (gap junction protein, beta 3, 31 kDa), IL1RLI (interleukiii I receptor-like 1), ENTPD1 (ectonucleoside triphosphate diphosphohydrola.se 1), BBS4 (Bardet-Biedl syndrome 4), CELSR2 (eadherin, EGF LAG seven-pass G-type receptor 2 (flamingo homolog, Drosophiia)), FUR (Fl l receptor), RAPGEF3 (Rap guanine nucleotide exchange factor (GEF) 3), HYAL1 (hyaiuronoglucosaminidase 1), ZNF259 (zinc finger protein 259), ATOXI (ATX1 antioxidant protein 1 homolog (yeast)), ATF6 (activating transcription factor 6), H (ketobe okinase (fructokinase)), SAT1 (spermidine / spermine Nl-acetyltraiisferase I), GGH (gamma-glutamyl hydrolase (conjugase, folylpolygarnmaghitarnyi hydrolase)), ΊΊΜΡ4 (TIMP metallopeptidase inhibitor 4), SLC4A4 (solute carrier family 4, sodium bicarbonate cotransporter, member 4), PDE2A (phosphodiesterase 2A, cGMP-stim lated), PDE3B (phosphodiesterase 3B, cGMP-inhibited), FADS1 (fatty acid desaturase 1), FADS2 (fatty acid desaturase 2), TMSB4X (thymosin beta 4, X-linked), TXNIP (thioredoxin mteracting protein), LIMS1 (LIM and senescent cell antigen-like domains I), R OB (ras homolog gene family, member B), LY96 ( lymphocyte antigen 96), FOXOl (forkhead box 01), P PLA2 (patatin-like phospholipase domai containing 2), TRH (thyrotropin-reieasing hormone), GJC1 (gap junction protein, gamma 1 , 45 kDa), SLC1 7A5 (solute carrier family 17 (anion / sugar transporter), member 5), FTO (fat mass and obesity associated), GJD2 (gap junction protein, delta 2, 36 kDa), PSRC (proline / serine-rich coiled-coil 1), CASP12 (caspase 12 (gcne / pscudogene)), GPBAR1 (G protein-coupled bile acid receptor 1), PXK (PX domain containing serine / threonine kinase), IL33 (interleukin 33), TRIB1 (tribbles homolog 1 (Drosophiia)}, PBX4 (pre-B-cell leukemia homeobox 4), NUPR l (nuclear protein, transcriptional regulator, 1), 15-Sep(15 kDa selenoprotein), CILP2 (cartilage intermediate layer protein 2), TERC (telom erase R A component), GGT2 (gamma-glutamyltransferase 2), MT- CG1 (mitochondrialiy encoded cytochrome c oxidase I), and UOX (urate oxidase, pseudogene),

[0372] in an additional embodiment, the chromosomal sequence may further be selected from Ponl (paraoxonase 1 ), LDLR (LDL receptor), ApoE (Apolipoprotein E), Apo B-100 (Apoiipoprotein B-100), ApoA (Apolipoprotein(a)), ApoA l (Apolipoprotein Al), CBS (Cystathione B-synthase), Glycoprotein Ilb / l lb, MTHRF (5, 10-methyienetetrahydrofolate reductase (NADPH), and combinations thereof. In one iteration, the chromosomal sequences and proteins encoded by chromosomal sequences involved in cardiovascular disease may be chosen from CacnalC, Sodl, Pten, Ppar(alpha), Apo E, Leptin, and combinations thereof.Kidneys

[0373] The present invention also contemplates delivering the CRISPR-Cas system to the kidney. Delivery strategies to induce cellular uptake of the therapeutic nucleic acid includephysical 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 ceils with hydrodynamic high pressure injection systemicaily, a wide range of gene therapeutic"viral, and non-viral carriers have been applied already to target posttraiiscriptional events in different animal kidney disease models in vivo (Csaba Revesz and Peter Hamar (201 1). Delivery Methods to Target RNAs in the Kidney, Gene Therapy Applications, Prof. Chtinsheng Kang (Ed.), ISBN: 978-953-307-541 -9, InTech, Available from: p: / / .nHecbope^the-kidney). Delivery methods to the kidney are summarized as follows:DeliveryCarrier Target RNA Disease Model Functional assays Author methodblood pressure,serum albumin,HyperWang et al, GeneMineral serum ureaAdeno-associated tension Cold-induced Therapy, (JulViral cortieoid nitrogen, serumvirus -2 caused renal hypertension 2006), Vol 13, No.receptor creatinine, kidneydamage 14, pp. (1097-1 103) weight, urinarysodiumobayashi et al.Journal of Pharmacology andHydrodynamic Experimental pU6 vector Lucif erase n.a. n.a. uptake / Viral Therapeutics, (Feb2004), Vol. 308, No. 2, pp. (688- 693)Wolfram et al,.Uptake, binding NatureLipoproteins, affinity to Biotechnology,Lipid apoB 1 , apoM ii.a. n.a.albumin lipoproteins and (Sep 2007), Vol.albumin 25, No. 10, pp.(1 149-1 157)Molitoris et al., J ischemic andAm Soc Nephrol,Acute renal cisplatin- HistologicalLipid Lipo lecianiine2000 p53 (Aug 2009), Vol.injury induced acute scoring, apoptosis20, No. 8, injurypp. (1754-1764)MDA-MB-DOTAP / DOPE, 231 breast Mikhaylova el al., DOTAP / DO Breast adenocancer Cell viability, dancer Gene TherapyLipid COX-2PE / DOPE- carcinoma xenograft- uptake (Mar 201 i ), Vol. 16, PEG2000 bearing No. 3, pp. (217-226) mouseAlbuminuria,urinary creatinine,histology, type Ϊ Yuan et ai.. Am JStreptozotoci and IV collagen, Physiol Renal12 / 15- DiabeticLipid Choieslerol n -induced TGF-β,"Physiol, (Jun lipoxygenase ncpkro- pathydiabetes iibronectin, 2008), Vol. 295, plasminogen pp. (F605-F617) activator inhibitor1Cell proliferationand apoptosis,histology, ROS,Y. Zhang et al., i mitochondrialMitochondrial Streptozotoci Arts Soc Nephrol,Lipotectamme Diabetic import of Mn-Lipid membrane 44 n -induced (Apr 2006 Vol.2000 nephro- patby SOD and(ΤΪΜ44) diabetes 17, No. 4, pp.glutathione(1090-1101) peroxidase,cellular membranepolarizationCaki-2 kidneySinghal et al,. cancerHydrodynamic Renal Cancer Res, (MayProteolipe-some RLIP76 xenograft- uptake / Lipid carcinoina 2009), Vol. 69, No.bearing10, pp. (4244-4251) mouseDeKveryCarrier Target RNA Disease Model Functional assays Author methodMalek eJ al., Toxicology andUptake,AppliedLuciferase biodistribution,Polymer PEGylated PEI n.a. n.a. Pharmacology, pGL3 erythrocyte(Apr 2009), Vol. aggregation236, No. 1 , pp. (97- 108)Proteinuria,glomerulosclerosis Shimizu et al., JLupus , TGF- β, Am SocPEGylated Glomerulo¬Polymer MAPK.1 glomerulo fibronectin, Nephrology, (Apr poly-L-lysine nephritisnephritis plasminogen 2010), Vol 21, No.activator inhibitor 4, pp. (622-633) 1Jiang et al ,,B16F1Biodistribution, MolecularKidney melanomaPolymer / Nano Hyaluronic acid / citotoxicity, tumor Pharmaceutics,VEGF cancer / tumor- particle Quantum dot / PEI volume, (May-Jun 2009), melanoma bearingendocytosis Vol, 6, No. 3, pp. mouse(727-737)Cao et al, JPEGylated Controlled Release,Polymer / Nano cell viability,polycapro- lactone GAPDH n.a. n.a. (Jun 2010), Vol. particle uptakenanofiber 144, No. 2, pp.(203-212) urinary albumin,urinary creatinine,Ninichuk et al.. Am histopathology,Uninephreeto J Pathol, (MarSpiegelmer CC chetnokine Glomerulo glomerularAptamer - mized 2008), Vol. 172, mNOX-E36 iigand 2 sclerosis filtration rate,mouse No, 3, pp. (628- macrophage count,6.37) serum Ccl2, Mac- 2+, i-67+Binding affinity toPurschke et al., D-AVP, inhibitionProc Natl Acad Sci, vasopressin Congestive of AVP Signaling,Aptamer Aptamer OX-F37 n.a. (Mar 2006), Vol.(AVP) heart: failure Urine osmolality103, No. 13, pp. and sodium(5173-5178) concentration,

[0374] Yuan et al. (Am J Physiol Renal Physiol 295: F605--F617, 2008) investigated whether in vivo delivery of small interfering RNAs (siRNAs) targeting the 12 / 15 -lipoxygenase (12 / 15- LO) pathway of arachidonate acid metabolism can ameliorate renal injury and diabetic nephropathy (DN) in a streptozotocininjecied mouse model of type 1 diabetes. To achieve greater in vivo access and siRNA expression in the kidney, Yuan et al. used double-stranded 12 / 15-LO siRNA oligonucleotides conjugated with cholesterol. About 400 μg of siRNA was injected subcutaneouslv into mice. The method of Yuang et. al. may be applied to the CRISPR Cas system of the present invention contemplating a 1-2 g subcutaneous injection of CRISPR Cas conjugated with cholesterol to a human for delivery to the kidneys.

[0375] Molitoris et al. (J Am Soc Nephrol 20: 1754-1764, 2009) exploited proximal tubule cells (PTCs), as the site of oligonucleotide reabsorption within the kidney to test the efficacy of siRNA targeted to p53, a pivotal protein in the apoptotic pathway, to prevent kid ey injury. Naked synthetic siRNA to p53 injected intravenously 4 h after ischemic injur}'maximally protected both PTCs a d kidney functio . Molitoris et al.'s data indicates that rapid delivery of siRN A to proximal tubule cells follows intravenous administration. For dose-response analysis, rats were injected with doses of siP53, 0.33; 1, 3, or 5mg / kg, given at the same four time points, resulting in cumulative doses of 1.32; 4, 12, and 20 mg / kg, respectively. All siRNA doses tested produced a SCr reducing effect on day one with higher doses being effective over approximately five days compared with PBS-treated ischemic control rats. The 12 and 20 mg kg cumulative doses provided the best protective effect. The method of Molitoris et al. may be applied to the CRISPR Cas system of the present invention contemplating 12 a d 20 mg kg cumulative doses to a human for deliver}' to the kidneys.

[0376] Thompson et al. (Nucleic Acid Therapeutics, Volume 22, Number 4, 2012) reports the toxicoiogicai and pharmacokinetic properties of the synthetic, small interfering RNA I5NP following intravenous administration in rodents and nonhuman primates. Ϊ5ΝΡ is designed to act via the RNA interference (RNAi) pathway to temporarily inhibit expression of the pro-apoptotic protein p53 and is being developed to protect cells from acute ischemia / reperfusion injuries such as acute kid ey injury that can occur during major cardiac surgery and delayed graft function that can occur fol lowing renal transplantation. Doses of 800mg / kg 15 NP in rodents, and 1,000 mg / kg I5NP in nonhuman primates, were required to elicit adverse effects, which in the monkey were isolated to direct effects on the blood that included a sub-clinical activation of complement and slightly increased clotting times. In the rat, no additional adverse effects were observed with a rat analogue of I5NP, indicating that the effects likely represent class effects of synthetic RNA duplexes rather than toxicity related to the intended pharmacologic activity of Ϊ5ΝΡ. Taken together, these data support clinical testing of intravenous administration of I5NP for the preservation of renal function following acute isehemia / reperftision injury. The no observed adverse effect level (NOAEL) in the monkey was 500 mg / kg. No effects on cardiovascular, respirator}', and neurologic parameters were observed in monkeys following i.v. administration at dose levels up to 25 mg kg. Therefore, a similar dosage may be contemplated for i travenous administration of CRISPR Cas to the kidneys of a human.

[0377] Shimizu et al. (J Am Soc Nephrol 21 : 622-633, 2010) developed a system to target delivery of siR As to glomeruli via polyiethylene glycol)-poly(L-lysine)-based vehicles. The siRNA / nanocarrier complex was approximately 10 to 20 nm in diameter, a size that would allow it to move across the fenestrated endothelium to access to the mesangium. After intraperitoneal injection of fluorescence-labeled siRNA'naiio carrier complexes, Shimizu et al. detected siRNAs in the blood circulation for a prolonged time. Repeated intraperitoneal administration of a mitogen-activated protein kinase 1 (MAPKl) siRNA / nanocarrier complex suppressed glomerular MAPKl mRNA and protein expression in a mouse model of glomerulonephritis. For the investigation of siRNA accumulation, Cy5 -labeled siRNAs complexed with PIC nanocarriers (0.5 ml, 5 nmol of siRNA content), naked Cy5-labeled siRNAs (0.5 ml, 5 nmol), or Cy5-labeled siRN As encapsulated in HVJ-E (0.5 ml, 5 nmol of siRN A content) were administrated to BALB- c mice. The method of Shimizu et al. may be applied to the CRISPR Cas system of the present invention contemplating a dose of about of 10-20 μ / mo! CRISPR Cas complexed with nanocarriers in about 1-2 liters to a human for intraperitoneal administration and delivery to the kidneys.Lungs

[0378] The present invention also contemplates delivering the CRISPR -Cas system to oneor both lungs.

[0379] 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- 3 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 genedelivery vector. Furthermore, it was demonstrated that AAV-9 could be readmimstered 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 μΐ 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 x 103to 4 x 105vector genomes / cell, depending on virus concentration and purposes of the experiments. The above cited vectors are contemplated for the deliver}' and / or admin stration of the invention .

[0380] Zamora et ai. (Am J Respir Grit Care Med Vol 183. pp 531-538, 201 1) 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-RS V01 (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-RSVG 1 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 GRP. Pharmacokinetics showed only low, transient systemic exposure after inhalation, consistent with preclinical animal data showing that ALN-RSV01, administered intravenous])' or by inhalation, is rapidly cleared from the circulation through exonucleasemediated digestion a d renal excretion. The method of Zamora et al. may be applied to the CR ISPR 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.

[0381] For an example of CFTRdelta508 chimeric guide RNA, see Example 22 which 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 (CF) related symptoms, using adeno-associated virus (AAV) particles. In particular, they exemplify a repair strategy for Cystic Fibrosis delta F508 mutation. This type of strategy should apply across ail organisms. With particular reference to CF, suitable patients may include: Human, non-primate human, canine, feline, bovine, equine and other domestic animals. I this instance, Applica tsutilized a CRISPR-Cas system comprising a Cas9 enzyme to target deltaF508 or other CFTR- inducing mutations.

[0382] The treated subjects in this instance receive pharmaceutically effective amount of aerosolized AAV vector system per lung endobronchial^ delivered while spontaneously breathing. As such, aerosolized delivery is preferred for AAV delivery in general. An adenovirus or an AAV particle may be used for delivery. Suitable gene constructs, each operably linked to one or more regulatory sequences, may be cloned into the delivery vector. In this instance, the following constructs are provided as examples: Cbh or EFla promoter for Cas9, U6 or HI promoter for chimeric guide RNA),: A preferred arrangement is to use a CFTRdelta508 targeting chimeric guide, a repair template for deltaF508 mutation and a codon optimized Cas9 enzyme (preferred Cas9s are those with nuclease or nickase activity) with optionally one or more nuclear localization signal or sequence(s) (NLS(s)), e.g., two (2) NLSs. Constructs without NLS are also envisaged.

[0383] In order to identify the Cas9 target site, Applicants analyzed the human CFTR genomic locus and identified the Cas9 target site. Preferably, in general and in this CF case, the PAM may contain a GG or a NNAGAAW motif.

[0384] Accordingly, in the case of CF, the present method comprises manipulation of a target sequence in a genomic locus of interest comprisingdelivering 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 non-natural ly occurring or engineered composition comprising a vector system comprising one or more vectors comprisingI. a first regulator}' 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 the CF target sequence in a suitable mammalian cel l,(b) a tracr mate seque ce, and(c) a tracr sequence, andII. 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 tra scribed, 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 to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence. In respect of CF, preferred target DNA sequences comprise the CFTRdelta508 mutation. A preferred PAM is described above. A preferred CRISPR enzyme is any Cas (described herein, but particularly that described in Example 22).

[0385] Alternatives to CF include any genetic disorder and examples of these are well kno wn. Another preferred method or use of the in vention is for correcting defects in the E P2A and EMP2B genes that have been identified to be associated with Lafora disease.

[0386] hi some embodiments, a "guide sequence" may be distinct from "guide RNA". A guide sequence may refer to an approx. 20bp sequence, within the guide RNA, that specifies the target site.

[0387] In some embodiments, the Cas9 is (or is derived from) SpCas9. In such embodiments, preferred mutations are at any or all or positions 10, 762, 840, 854, 863 and / or 986 of SpCas9 or corresponding positions in other Cas9s (which may be ascertained for instance by standard sequence comparison tools. In particular, any or ail 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. These are advantageous as they provide nickase activity. Such mutations may be applied to all aspects of the present invention, not only treat ment of CF.

[0388] Schwank et al. (Ceil Stem Cell, 13:653-58, 2013) used CRISPR / Cas9 to correct a defect associated with cystic fibrosis in human stem ceils. The team's target was the gene for an ion channel, cystic"fibrosis transmembrane conductor receptor (CFTR). A deletion in CFTR causes the protein to misfold in cystic fibrosis patients. Using cultured intestinal stem cells developed from cell samples from two children with cystic fibrosis, Schwank et al. were able tocorrect the defect using CR1SPR along with a donor plasmid containing the reparative sequence to be inserted. The researchers then grew the cells into intestinal "organoids," or miniature guts, and showed that they functio ed normally. In this case, about half of clonal organoids underwent the proper genet c correction.Muscles

[0389] The present invention also contemplates delivering the CRISPR-Cas system to muscle(s).

[0390] Bortolanza et al. (Molecular Therapy vol. 19 no. 11, 2055-2064 Nov. 201 1 ) shows that systemic delivery of RNA interference expression cassettes in the FRG1 mouse, after the onset of facioscapulohumeral muscular dystrophy (FSHD), led to a dose-dependent long-term FR.G1 knockdown without signs of toxicity. Bortolanza et al, found that a single intravenous injection of 5 x 10"vg of rAAV6-shlFRGl rescues muscle histopathology and muscle function of FRG1 mice, hi detail, 200 μΐ containing 2 x 101" or 5 x 10fvg of vector in physiological solution were injected into the tail vein using a 25 -gauge Terumo syringe. The method of Bortolanza et al. may be applied to an AAV expressing CRJSPR Cas and injected into humans at a dosage of about 2χlO13or 2 x 10i 6vg of vector.

[0391] Dumonceaux et al. (Molecular Therapy vol. 18 no. 5, 881-887 May 2010) inhibit the myostatin pathway using the technique of RNA interference directed against the myostatin receptor AcvRIIb mRNA (sh-AcvRIIb). The restoration of a quasi-dystrophin was mediated by ...

Claims

WHAT IS CLAIMED IS:I . A method of modifying an organism or a non-human organism by manipulation of a target sequence in a genomic locus of interest comprisingdelivering a non-natural ly occurring or engineered composition comprising :A) - L 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 cel l,(b) a tracr mate sequence, and(c) a tracr sequence, andII. 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 comple to the target sequence, andwherein the CRISPR complex comprises the CRISPR enzyme complexed with ( 1 ) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized 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, andIII. 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, andwherein the CRISPR. complex comprises the CRISPR enzyme complexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized 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 seque ce encoding a CRISPR enzyme, the guide sequence, tracr mate sequence or tracr sequence is / are RNA and are delivered via liposomes, nanoparticies, 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 sequence in a genomic locus of interest comprisingdelivering a non-naturally occurri g or engineered compositio 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 comprisingI. a first regulatory element operably linked to a CRISP R-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, andII. 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 sequence, andwherein the CR ISPR complex comprises the CRISPR enzyme complexed with (1 ) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence, or(B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprisingI. a first regulator}? 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, andΙΪΪ. a third regulatory element operably linked to a tracr sequence,wherein components L II and I 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, andwherein the CRISPR. complex comprises the CRISPR enzyme complexed with (1 ) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized 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 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 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 lenti virus 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-natural Sy occurring or engineered composition comprising a vector system comprising one or more vectors comprising ]. 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 ceil,(b) a tracr mate sequence, and(c) a tracr sequence, andΠ. 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 comple to the target sequence, andwherein the CRISPR complex comprises the CRISPR enzyme complexed with ( 1 ) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence,or(B) a non-naturally occurring or engineered composition comprising a vector system comprising one or more vectors comprisingI. 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, andHI. a third regulatory element operably linked to a tracr sequence,wherein components 1, 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 eomplexed with (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized 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.1 1. The method of claim 10 wherein the organism or subject is a non-human eukaryote.

12. The method of any of claims 1 to 1 1 wherein the organism or subject is a mammal or a non~hurnan mammal.

13. The method of any of claims 4 to 8 wherein the viral vector is an AAV or lenti viral 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 ceil by delivering mRNA encoding the CRISPR enzyme to the cell.1 8. A method of preparing the AAV or lentivirus vector of claim 7 comprising transfecting piasmid(s) containing or consisting essentially of nucleic acid moleeulc(s) coding for the AA.V or lenti virus into AAV~mfected or lentivirus-infected cells, and supplying AAV AAV or lentiviras 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 ceils with helper plasmid(s) or helper virus(es).

21. The method of claim 20 wherein the helper vims is a poxvirus, adenovirus, lentivirus, herpesvirus or bacuio virus.

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 ceils.

24. The method of any of claims 18 to 22 wherein the cells are insect cells and the helper virus (where present) is baeulovirus.

25. The method of any of claims 1 to 15 wherein the target sequence is flanked at its 3' end or followed by 5' -NRG (where N is any Nucleotide), or where the CR iSPR enzyme is (or is derived from) a genus belonging to the group consisting of Corynebacter, Sutterella, Legionella, Treponema, Filifactor, Eubacterium, Streptococcus, Lactobacillus, Mycoplasma, Bacteroides, Flaviivola, Flavobacterium, Sphaerochaeta, Azospirillum, Gluconacetobacter, Neisseria, Roseburia, Parvibaculum, Staphylococcus, Nitratifractor, Mycoplasma and Campylobacter. ,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 sequence in a genomic locus of interest or in a method of treating or inhibiting a condition caused by a defect in a target 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 medicamen t 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 sequence in a genomic locus of interest or in a method of treating or inhibiting a condition caused by a defect in a target sequence in a genomic locus of interest.

30. A 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, andII. 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 sequence, andwherein the CRISPR complex comprises the CRISPR enzyme complexed with ( 1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized to the tracr sequence and the polynucleotide sequence encoding a CRISPR enzyme is DNA or RNA,or(B) I. poly ucleotides comprisi g:(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, andIII. 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, andwherein the CR ISPR complex comprises the CRISPR enzyme complexed wit (1) the guide sequence that is hybridized to the target sequence, and (2) the tracr mate sequence that is hybridized 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 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 sequence in a genomic locus of mterest; 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.