GENOME EDITING USING CAMPYLOBACTER JEJUNI CRISPR/CAS SYSTEM-DERIVED RGEN

Abstract

The disclosure provided herewith relates to a Campylobacter jejuni CRISPR/CAS system-derived RGEN and a use thereof.

Description

CROSS-REFERENCE TO RELATED APPLICATION(S)

[0001] This application is a Continuation Application of International Application No. PCT/KR2015/008269, filed Aug. 6, 2015, designating the United States of America, which claims the benefit of U.S. Provisional Application No. 62/033,852, filed Aug. 6, 2014, which applications are incorporated herein by reference in their entirety.

REFERENCE TO SEQUENCE LISTING

[0002] The contents of the text file named "52029-501C01US_ST25.txt" which was created on Jan. 31, 2017, and is 25641 bytes, are hereby incorporated by reference in their entireties.

TECHNICAL FIELD

[0003] The disclosure provided herewith relates to a Campylobacter jejuni CRISPR/CAS system-derived, RNA-guided engineered nuclease (RGEN) and methods for using the same.

BACKGROUND ART

[0004] Engineered nucleases can be used to effectively manipulate genes in living cells or whole organisms by creating site-specific double-strand breaks at desired locations in the genome (Nat Rev Genet, 2014. 15(5): p. 321-34.). Engineered nucleases, which comprise a DNA-binding domain and a nuclease domain customized for type II restriction enzymes, have a broad spectrum of genome engineering applications in the biotechnology and medical fields as well as various other industries. More recently, a more potent RGEN platform has been developed based on the CRISPR/CAS9 bacterial adaptive immune system.

[0005] The sequence that RGEN targets is limited to a protospacer adjacent motif (PAM), which is a DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease. The PAM sequence was not previously reprogrammable in the CRISPR bacterial adaptive immune system. The canonical PAM comprises the sequence 5'-NGG-3' and is associated with the RGEN derived from the CAS9 nuclease of Streptococcus pyogenes. Hence, the GG motif is a prerequisite for DNA recognition by the RGEN. To expand sequences for use as PAMs, attempts have been made to separate RGENs from different bacterial species with versatile PAMs. In fact, different PAMs have been found to be associated with the CAS9 protein of the bacteria Streptococcus thermophilus (PAM: NNAGAAW) and Neisseria meningitidis (PAM: NNNNGATT), widening the range of selection in determining RGEN target loci.

SUMMARY

[0006] As described herein, intensive and thorough research into the development of RGENs from bacteria other than Streptococcus pyogenes has resulted in the discovery that a Cas protein derived from Campylobacter jejuni (C. jejuni) specifically recognizes an NNNNRYAC sequence, which can be used as a PAM in the targeting of a DNA of interest. Further, a guide RNA can be engineered for optimal of a DNA, thereby resulting in efficient genome editing, transcription regulation, and separation of a DNA of interest.

[0007] Accordingly, in one aspect, the present invention provides a method for targeting a DNA sequence comprising a PAM sequence of SEQ ID NO: 1, the method comprising introducing a Cas protein that recognizes the PAM sequence of SEQ ID NO: 1, or a nucleic acid encoding the Cas protein into a cell.

[0008] In another aspect, the present invention provides an isolated guide RNA comprising a sequence capable of forming a duplex (forming a base pair or hybridizing) with a complementary strand of a target DNA sequence of interest adjacent to the PAM sequence of SEQ ID NO: 1, or a composition comprising the same.

[0009] In still another aspect, the disclosure provided herewith provides a CRISPR-CAS system, comprising: (i) a guide RNA comprising a sequence capable of duplexing with a target NDA sequence adjacent to the PAM sequence of NNNNRYAC (SEQ ID NO: 1), or DNA encoding the guide RNA, and (ii) a Cas protein recognizing the NNNNRYAC sequence (SEQ ID NO: 1), or a nucleic acid encoding the Cas protein.

[0010] In still another aspect, the disclosure provided herewith provides a recombinant viral vector, comprising (i) an expression cassette for a guide RNA comprising a sequence capable of forming a duplex with a target DNA sequence adjacent to the PAM sequence NNNNRYAC (SEQ ID NO: 1), and (ii) an expression cassette for a Cas protein recognizing the PAM sequence NNNNRYAC (SEQ ID NO: 1).

[0011] In still another aspect, the disclosure provides an isolated guide RNA comprising a sequence, 21-23 bp in length, capable of forming a duplex with a complementary strand of a target DNA sequence, or a composition comprising the same.

[0012] In still another aspect, the disclosure provides an isolated guide RNA, comprising: a first region comprising a sequence capable of forming a duplex with a complementary strand of a target DNA sequence, and a second region comprising a stem-loop structure characterized by a stem 13-18 bp in length, or a composition comprising the isolated guide RNA.

[0013] In still another aspect, the disclosure provides an isolated guide RNA, comprising: a first region comprising a sequence capable of forming a duplex with a complementary strand of a target DNA sequence, and a second region comprising a stem-loop structure characterized by a loop 5-10 bp in length, or a composition comprising the isolated guide RNA.

[0014] In still another aspect, the disclosure provided herewith provides a method of genome editing in a cell, comprising introducing an isolated guide RNA or a DNA encoding the isolated guide RNA, along with a Cas protein or a nucleic acid encoding the Cas protein, into the cell.

[0015] In still another aspect, the disclosure provides a method of cleaving a target DNA in a cell, comprising introducing an isolated guide RNA or a DNA encoding the isolated guide RNA, and a Cas protein or a nucleic acid encoding the Cas protein into the cell.

[0016] In still another aspect, the disclosure provides a method for preparing a target DNA-recognizing sequence of a guide RNA, comprising: (i) identifying the presence of a PAM sequence NNNNRYAC (SEQ ID NO: 1) in a given sequence; and (ii) determining a sequence located upstream of the PAM sequence NNNNRYAC (SEQ ID NO: 1) as being recognizable by a guide RNA, if the presence of the PAM sequence is identified in step (i).

[0017] In still another aspect, the disclosure provides a method for isolating a DNA of interest, comprising: (i) introducing a guide RNA or a DNA encoding the guide RNA, along with a deactivated Cas protein or a nucleic acid encoding the deactivated Cas protein, into a cell, to allow the guide RNA and the deactivated Cas protein to form a complex together with the DNA of interest comprising a target DNA sequence; and (ii) separating the complex from a sample.

[0018] In still another aspect, the disclosure provides a method for Cas-mediated gene expression regulation in a DNA of interest comprising a target DNA sequence, comprising introducing an isolated guide RNA specifically recognizing the target DNA sequence or a DNA encoding the guide RNA, and an deactivated Cas protein fused to a transcription effector domain or a nucleic acid encoding the deactivated Cas protein into a cell.

[0019] As described above, in some embodiments, the CRISPR/Cas system can be effectively used for targeting a target DNA, thereby achieving genome editing, transcription regulation, and isolation of a DNA of interest.

BRIEF DESCRIPTION OF DRAWINGS

[0020] FIG. 1 depicts a schematic diagram of a C. jejuni Cas9 expression vector. The vector is designed so that a humanized Cas9 protein is expressed under the regulation of a CMV promoter and is provided with a nuclear localization signal (NLS) and an HA tag at a C-terminal region.

[0021] FIG. 2A and FIG. 2B depict the experiments for C. jejuni RGEN-induced mutation in an endogenous human AAVS1 target locus. FIG. 2A shows that RGEN-driven chromosomal mutations were detected using a T7E1 assay. Asterisks (*) indicate DNA bands that are anticipated to be cleaved by T7E1. HEK293 wild-type (wt) gDNA was used as a negative control (-). A previously proven RGEN was used as a positive control (+). FIG. 2B shows DNA sequences of hAAVS1 mutant clones. Target sequence regions complementary to chimeric RNA are shown in bold. PAM sequences recognized by CAS9 are underlined. The WT sequence of FIG. 2B is represented by SEQ ID NO: 4, the (-2, x1) sequence by SEQ ID NO: 5, and the (-1, x1) sequence by SEQ ID NO: 6.

[0022] FIG. 3A and FIG. 3B show the experiments for C. jejuni RGEN-induced mutation in an endogenous mouse ROSA26 (mROSA) target locus. FIG. 3A shows that RGEN-driven chromosomal mutations were detected using a T7E1 assay. Asterisks (*) indicate DNA bands that are anticipated to be cleaved by T7E1. NIH3T3 wt gDNA was used as a negative control (-). A previously proven RGEN was used as a positive control (+). FIG. 3B shows DNA sequences of mROSA mutant clones. Target sequence regions complementary to chimeric RNA are shown in bold. PAM sequences recognized by C. jejuni CAS9 are underlined. The WT sequence of FIG. 3B is represented by SEQ ID NO: 7, the (-1, x1) sequence by SEQ ID NO: 8, and the (+1, x1) sequence by SEQ ID NO: 9.

[0023] FIG. 4 shows certain mutations induced in endogenous AAVS1 target loci by a mutant C. jejuni sgRNA structure. RGEN-driven chromosomal mutations were detected using a T7E1 assay. Asterisks (*) indicate DNA bands that are anticipated to be cleaved by T7E1. HEK293 wt gDNA was used as a negative control (-). A previously proven RGEN was used as a positive control (+).

[0024] FIG. 5A to FIG. 5C illustrate the optimization of the spacer length of sgRNAs. FIG. 5A shows various sgRNA structures. Additional nucleotides immediately upstream of the 5' end of the spacer of sgRNA are underlined, wherein small letters represent mismatched nucleotides with regard to the target sequence. The PAM sequence is boxed. In FIG. 5A, the target sequence is represented by SEQ ID NO: 10, GX19 by SEQ ID NO: 11, GX20 by SEQ ID NO: 12, GX21 by SEQ ID NO: 13, GX22 by SEQ ID NO: 14, GX23 by SEQ ID NO: 15, GGX20 by SEQ ID NO: 16, and GGGX20 by SEQ ID NO: 17. FIG. 5B shows target sites of sgRNA wherein sequences for hAAVS-CJ1, hAAVS-NRG1, hAAVS-NRG3, and hAAVS-NRGS are represented by SEQ ID NOs: 18, 19, 20, and 21, respectively. FIG. 5C shows the efficiency of the sgRNA constructs in inducing RGEN-mediated mutations. Briefly, sgRNAs were constructed to have various lengths of spacers (19-23 bp) and various numbers of additional G (guanine) residues present immediately upstream of the spacer. Each of the sgRNAs shown in FIG. 5A was designed for 4 target sites of the human AAVS1 locus (FIG. 5B), and was delivered to human 293-cells. Subsequently, mutations induced by NHEJ were identified in the cells. In this embodiment, the target sites were amplified by PCR, and analyzed by deep sequencing using miSEQ (Illumine) to detect the mutations. On the whole, genome editing (mutation) frequency was increased when the recognition sequence was 21-23 bp in length or was provided with 2 or 3 additional G residues at the 5' end thereof, compared to GX19 or GX20 used in C. jejuni or other species.

[0025] FIG. 6 is a graph showing the activity of C. jejuni CRISPR/Cas9 in which the AAVS1-CJ1 locus is inserted to a surrogate reporter. Relative to the activity (100) detected for the ACAC sequence at the PAM site, activities were calculated when different nucleotides were substituted at each position. At the first position, G as well as A guaranteed high activity. T as well as C were effective at the second position. However, only A and C exhibited activity at the third and fourth positions, respectively. Therefore, NNNN-A/G-C/T-C-A (or NNNNRYAC, SEQ ID NO: 1, wherein A/G=R, C/T=Y) is inferred to be an optimal PAM sequence at least in some embodiments.

[0026] FIG. 7 shows a consensus logo for a potential off-target sequence of hAAVS1-CJ1 sgRNA, as excavated by the Digenome-Seq analysis.

[0027] FIG. 8 shows the test results for the PAM sequences of C. jejuni Cas9. Seven target sites of NNNNRYAC (SEQ ID NO: 1) were analyzed for mutation efficiency. hAAVS1-RYN1-7: ratio of mutation at each site in sgRNA/Cas9-treated cells, WT1-7: ratio of mutation at each site in genomic DNA of mock-treated cells.

[0028] FIG. 9 is a schematic diagram showing the structure of a C. jejuni CRISPR/Cas9 expression AAV vector.

[0029] FIG. 10 shows the genome editing, performed by C. jejuni CRISPR/Cas9 AAV (adeno-associated virus), in the Rosa26 locus. Briefly, C2C12 cells were infected with a recombinant AAV vector carrying both Rosa26-sgRNA and C. jejuni Cas9 at different MOI (multiplicity of infectivity). At 3, 5, 7, 10, and 14 days post-infection, genomic DNA was isolated, and analyzed for mutation ratio by deep sequencing.

DETAILED DESCRIPTION OF CERTAIN EMBODIMENTS

[0030] One embodiment of the present invention provides a method for targeting a DNA sequence of interest, comprising introducing into a cell a Cas protein or a nucleic acid encoding the same.

[0031] In detail, in accordance with one aspect, the present disclosure provides a method for targeting a DNA sequence comprising a PAM (protospacer adjacent motif) sequence of SEQ ID NO: 1, comprising introducing a Cas protein that recognizes the PAM sequence NNNNRYAC of SEQ ID NO: 1 or a nucleic acid encoding the Cas protein into a cell. In SEQ ID NO: 1, according to the IUPAC nomenclature, "N" refers to any nucleotide, for example, selected from A, C, G, and T; "R" refers to purine (AIG); and "Y" refers to pyrimidine (C/T).

[0032] In an aspect of the present disclosure, the method may further comprise introducing a guide RNA comprising a sequence capable of forming a duplex with a complementary strand of a DNA of interest (target DNA) adjacent to the PAM sequence of SEQ ID NO: 1. The guide RNA can be introduced simultaneously or sequentially with the Cas protein that recognizes the PAM sequence of SEQ ID NO: 1 or the nucleic acid encoding the Cas protein. As used herein, the term "targeting" is intended to encompass the binding of a Cas protein to a DNA sequence of interest, either with or without DNA cleavage. The terminology that will be described later is applicable to all embodiments of the present disclosure, and can be used in combination.

[0033] The Cas protein can perform its activity after forming a complex with CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). The Cas protein may exhibit endonuclease or nickase activity.

[0034] Information related to Cas proteins or genes encoding Cas proteins can be found in well-known databases, such as GenBank of the NCBI (National Center for Biotechnology Information). According to one embodiment, the Cas protein may be a Cas9 protein. In another embodiment, the Cas protein may be one originating (derived) from a Campylobacter spp. (i.e., the genus Campylobacter) and may particularly be of Campylobacter jejuni in origin. More particularly, the Cas9 protein can be derived from Campylobacter jejuni. In some embodiments of the present disclosure, the Cas protein may comprise the amino acid sequence represented by SEQ ID NO: 22, or may be homologous to the amino acid sequence of SEQ ID NO: 22, retaining the intrinsic activity thereof For example, without limitation, the Cas protein and its homologous sequences encompassed by the present disclosure may have a sequence identity at least 50%, 60%, 70%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 22.

[0035] Moreover, the Cas protein, as used in certain embodiments of the present disclosure, is intended to encompass any variant that can serve as an activated endonuclease or nickase in cooperation with a guide RNA, as well as a native protein. The activated endonuclease or nickase may cleave a target DNA, or may be able to perform genome editing with the cleavage function. As for deactivated variants, their functions may be used for regulating transcription or isolating a DNA of interest.

[0036] The Cas9 protein variant may be a derivative, variant, or mutant of Cas9 resulting from the substitution of a catalytic aspartate or histidine residue with a different amino acid. For example, the different amino acid may be alanine, but is not limited thereto.

[0037] Specifically, the Cas protein, for example, a Cas9 protein derived from C. jejuni may include a substitution of the catalytic aspartic acid (D) at position 8 or the histidine residue (H) at position 559 with an amino acid that differs from the wild type amino acid sequence. In some embodiments, the catalytic aspartic acid (D) at position 8 or the histidine residue (H) at position 559 of the sequence of SEQ ID NO. 22 is substituted with a different amino acid. For example, the different amino acid may be, without limitation, alanine. The Cas9 nuclease variant prepared by introducing a mutation to one active site of the native Cas9 nuclease can act as a nickase in association with a guide RNA. When bound to one guide RNA molecule, two nickase molecules can cleave both strands of a DNA duplex of interest, thereby creating double-strand breaks (DSB). Hence, such variants also belong to the scope of RGENs encompassed by the present disclosure.

[0038] As used herein, the term "deactivated Cas protein" refers to a Cas nuclease, the function of which is entirely or partially deactivated. The deactivated Cas protein may be abbreviated to dCas. The Cas may be a Cas9 protein. Further, it may originate from Campylobacter spp., and particularly from C. jejuni. Any method may be used in the preparation of the deactivated Cas9 nuclease, so long as it eliminates nuclease activity. For example, a dCAS9 protein can be constructed by introducing mutations into the two above-mentioned active loci of the Cas9 nuclease. The dCAS9 can then act as a DNA-bound complex with a guide DNA, while lacking a DNA cleavage function. Moreover, the dCAS9 protein may have substituents having other than the aspartic acid (D) at position 8 and the histidine (H) at position 559. For example, in some embodiments, the dCAS9 protein may have substituents other than the aspartic acid (D) at position 8 and the histidine (H) at position 559 of the sequence of SEQ ID NO. 22. The substituents may be, without limitation, alanine. As used herein, the term "cleavage" refers to the breakage of the covalent backbone of a nucleotide molecule.

[0039] In some embodiments of the present disclosure, the Cas protein can be a recombinant protein. The term "recombinant", as used in conjunction with, for example, a cell, nucleic acid, protein, or vector, refers to the cell, nucleic acid, protein or vector that is modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or which is derived from such a modified cell. Thus, for example, a recombinant Cas protein may be generated by reconstituting a Cas protein-encoding nucleic acid sequence (i.e., a sequence encoding a Cas protein), based on the human codon table.

[0040] In some embodiments of the present disclosure, the Cas protein or the nucleic acid encoding the same may be in a form that is allowed to be active within the nucleus.

[0041] In some embodiments of the present disclosure, the isolated Cas protein may be in a form that is easy to introduce into cells. For example, the Cas protein may be linked to a cell-penetrating peptide or a protein transduction domain. The protein transduction domain may be, without limitation, poly-arginine or an HIV-derived TAT protein. The present disclosure encompasses various examples of cell-penetrating peptides or protein transduction domains that are well known in the art.

[0042] In some embodiments of the present disclosure, the Cas protein or the nucleic acid encoding the same may further comprise a nuclear localization signal (NSL) for transporting the protein or nucleic acid into a nucleus in a cell by nuclear transport. In addition, the nucleic acid encoding the Cas protein may further comprise a nuclear localization signal (NLS) sequence. Thus, the Cas protein-encoding nucleic acid may be present as a component of an expression cassette that may contain, but is not limited to, an NLS sequence as well as a regulatory element, such as a promoter.

[0043] In some embodiments of the present disclosure, the Cas protein may be linked with a tag that facilitates separation and/or purification. As a non-limiting example, a small peptide tag, such as a His tag, a Hag tag, an S tag, etc., a glutathione S-transferase (GST) tag, or a maltose binding protein (MBP) tag may be used, depending on the purpose.

[0044] In some embodiments of the present disclosure, where the Cas protein is associated with target DNA-specific guide RNA, the Cas protein may be collectively termed an RGEN (RNA-Guided Engineered Nuclease). As used herein, the term "RGEN" refers to a nuclease having a target DNA-specific guide RNA and a Cas protein.

[0045] For application to cells, according to some embodiments of the present disclosure, the RGEN may have a target DNA-specific guide RNA or a DNA encoding the guide RNA; as well as an isolated Cas protein or a nucleic acid encoding the Cas protein. In this regard, the guide RNA or the DNA encoding the guide RNA may be applied to cells simultaneously or sequentially with the Cas protein or the nucleic acid encoding the Cas protein.

[0046] In an aspect of the present disclosure, the RGEN for delivery to cells include 1) a target DNA-specific guide RNA and an isolated Cas protein, or 2) a DNA encoding the guide RNA or a nucleic acid encoding the Cas protein. Delivery in the form of 1) is designated "RNP delivery." Examples of the isolated guide RNA may comprise, but are not limited to, in vitro transcribed RNAs.

[0047] In some embodiments of the present disclosure, the guide RNA-coding DNA (DNA encoding the guide RNA) and the Cas protein-coding nucleic acid may themselves be used as isolated nucleic acids. Alternatively, without limitation, they may be present in a vector having an expression cassette for expressing the guide RNA and/or the Cas protein. Examples of suitable vectors include a viral vector, a plasmid vector, and an agrobacterium vector. The viral vector may be exemplified by, but is not limited to, an AAV (adeno-associated virus).

[0048] In some embodiments of the present disclosure, without limitation, the guide RNA-coding DNA and the Cas protein-coding nucleic acid may be present separately in respective vectors or together in a single vector.

[0049] The foregoing application embodiments of the subject matter can be applied to more exemplary embodiments as described in this specification. In addition, application embodiments that will be described later may be applied in combination with other constitutional elements.

[0050] As used herein, the term "guide RNA" may refer to a RNA having specificity to a target DNA (i.e., a target DNA-specific RNA), which can be coupled with a Cas protein to guide the Cas protein to the target DNA.

[0051] Moreover, at least in some embodiments, the guide RNA may be designed to be specific for a certain target to be cleaved.

[0052] In some embodiments of the present disclosure, the guide RNA may be a dual RNA consisting of two RNAs, that is, a crRNA and a tracrRNA. In other embodiments, the guide RNA may be a sgRNA comprising or consisting of a first region containing a sequence complementary to the target DNA capable of forming a duplex with a complementary strand of the target DNA, and a second region containing a sequence responsible for interacting with the Cas protein. More particularly, the guide RNA may be a sgRNA (single guide RNA or single-stranded guide RNA) synthesized by fusing respective essential portions of a crRNA and a tracrRNA.

[0053] In some embodiments of the present disclosure, the sequence capable of forming a duplex with a complementary strand of a target DNA sequence in the guide RNA may range, without limitation, in length from 17 to 23 bp, from 18 to 23 bp, from 19 to 23 bp, particularly from 20 to 23 bp, and more particularly from 21 to 23 bp. The length may be applied to both the dual RNA and the sgRNA, and more particularly to the sgRNA.

[0054] In some embodiments of the present disclosure, the guide RNA may comprise one to three, more particularly two or three additional nucleotides just prior to the 5' end of the sequence capable of forming a duplex with a complementary strand of a target DNA sequence. The nucleotides are selected from among A, T, G, C, and combinations thereof The guide RNA may comprise, as additional nucleotides, one to three consecutive guanine (G) residues, more preferably, two or three consecutive G residues. This is applied, without limitation, to both dualRNAs and sgRNAs, and more preferably to sgRNAs.

[0055] In some embodiments of the present disclosure, the sgRNA may comprise a region complementary to a target DNA sequence (termed "Spacer region", "Target DNA recognition sequence", "base pairing region", etc.), and a hairpin structure for binding to the Cas protein.

[0056] In some embodiments of the present disclosure, the sgRNA may comprise a region complementary to a target DNA sequence, a hairpin structure for binding to the Cas protein, and a terminator sequence. These elements may be, without limitation, sequentially arranged in the 5' to 3' direction.

[0057] In some embodiments of the present disclosure, any form of guide RNA can be used, as long as it contains respective essential portions of a crRNA and a tracrRNA and a region complementary to a target DNA.

[0058] In some embodiments of the present disclosure, the crRNA may hybridize with a target DNA.

[0059] In some embodiments of the present disclosure, the RGEN may consist of a Cas protein and a dualRNA, or a Cas protein and an sgRNA. Alternatively, the RGEN may comprise respective nucleic acids encoding a Cas protein and an sgRNA as constitutional elements, but is not limited thereto.

[0060] In some embodiments of the present disclosure, the guide RNA, e.g., crRNA or sgRNA, may contain a sequence complementary to a target DNA sequence, and may comprise one or more additional nucleotides located upstream of the crRNA or sgRNA, particularly at the 5' end of the crRNA of sgRNA or dualRNA. The additional nucleotides may be, but are not limited to, guanine (G) residues.

[0061] In some embodiments of the present disclosure, the guide RNA may comprise a sequence capable of forming a duplex with (i.e., forming a base pair with or hybridizing to) a complementary strand of a target DNA sequence adjacent to the PAM (proto-spacer-adjacent motif) sequence NNNNRYAC (SEQ ID NO: 1).

[0062] In some embodiments of the present disclosure, the guide RNA may comprise a first region, capable of forming a duplex with a complementary stand of a target DNA sequence, and a second region, comprising a stem-loop structure characterized by a stem 13-18 bp in length. In certain embodiments, the stem may comprise the nucleotide sequence of SEQ ID NO: 2 (5'-GUUUUAGUCCCUUGUG-3') and a complementary sequence thereof.

[0063] In some embodiments of the present disclosure, the guide RNA may comprise a first region, capable of forming a duplex with a complementary stand of a target DNA sequence, and a second region comprising a stem-loop structure characterized by a loop 5-10 bp in length. The loop may comprise the nucleotide sequence of SEQ ID NO: 3 (5'-AUAUUCAA-3').

[0064] In some embodiments of the present disclosure, the Cas proteins and the guide RNAs, especially sgRNAs, which are described above or later, may be those that are not naturally occurring or are engineered. In addition, the factors described for each matter may be combined together for application.

[0065] In some embodiments of the present disclosure, the intracellular introduction of RGEN can be achieved by, but is not limited to, (1) delivering the Cas9 protein, purified after bacterial overexpression, and the sgRNA (single guided RNA), that recognizes a specific HLA target sequence, which is prepared after in vitro transcription in cells, or (2) delivering a plasmid carrying the Cas9 gene and the sgRNA into cells for expression or transcription.

[0066] In addition, proteins, RNAs or plasmid DNAs encompassed within the scope of the present disclosure can be introduced into cells through various methods known in the art, such as, without limitation, electroporation, or techniques using liposomes, viral vectors, nanoparticles, or PTD (protein translocation domain) fusion proteins.

[0067] In some embodiments, a method of the present disclosure may be used to cleave a target DNA comprising the PAM sequence of SEQ ID NO: 1, and more particularly to edit a genome. In this context, the Cas protein may be in an active form with a nuclease or nickase activity.

[0068] In certain embodiments, the Cas protein may be in a deactivated (inactivated) form. In this case, the method of the present disclosure is conducted in such a way that a target DNA sequence comprising the PAM sequence of SEQ ID NO: 1 is not cleaved, but is associated with the Cas protein.

[0069] Moreover, in some other embodiments, the Cas protein, more particularly, the deactivated Cas protein, may further comprise a transcription effector domain. In detail, the deactivated Cas protein may be linked to, without limitation, an activator, a repressor, or so on.

[0070] Given the transcription effector domain, the method, at least in some embodiments, may be applied to Cas-mediated gene expression regulation comprising transcriptional regulation or epigenetic regulation.

[0071] In accordance with another aspect, the present disclosure provides an isolated guide RNA comprising a sequence capable of forming a duplex with a complementary strand of a target DNA sequence adjacent to the PAM (proto-spacer-adjacent motif) NNNNRYAC (SEQ ID NO: 1). The isolated guide RNA may be one that is not naturally occurring or is artificially engineered. The individual elements are as described above.

[0072] In some embodiments of the present disclosure, the guide RNA may be single guide RNA in which the sequence capable of forming a duplex with a complementary strand of a target DNA may range in length from 17 to 23 bp, from 18 to 23 bp, from 19 to 23 bp, particularly from 20 to 23 bp, and more particularly from 21 to 23 bp, without being limited thereto.

[0073] Further, the guide RNA, at least in some embodiments, may comprise one to three consecutive guanine (G) residues just upstream of the 5' end of the complementary strand of the target DNA, but is not limited thereto. Additionally, the foregoing description of the additional nucleotides can also be applicable to this embodiment.

[0074] Also, provided in accordance with a another aspect of the present disclosure is a composition comprising a guide RNA comprising a sequence capable of forming a duplex with a complementary strand of a target DNA sequence adjacent to the PAM (proto-spacer-adjacent motif) sequence NNNNRYAC (SEQ ID NO: 1), or a DNA encoding the guide RNA. Each of the components, in at least some embodiments, is as described above.

[0075] In some embodiments of the present disclosure, the composition may further comprise a Cas protein recognizing the sequence NNNNRYAC (SEQ ID NO: 1) or a nucleic acid encoding the Cas protein.

[0076] In addition, in certain embodiments, the composition may be used for genome editing.

[0077] Further, in some embodiments, the composition may comprise: (i) a guide RNA comprising a sequence capable of forming a duplex with a complementary strand of a target DNA sequence adjacent to the PAM (proto-spacer-adjacent motif) NNNNRYAC (SEQ ID NO: 1), or a DNA encoding the guide RNA; and (ii) an deactivated Cas protein (dCas) or a nucleic acid encoding the dCas.

[0078] In an embodiment, the deactivated Cas protein may further comprise a transcription effector domain.

[0079] In some embodiments of the present disclosure, the composition may be used to isolate a DNA of interest comprising a target DNA sequence. In this regard, the deactivated Cas protein may be labeled with a tag useful for separation and purification, but is not limited thereto. The tag may be as described above.

[0080] In some embodiments of the present disclosure, the composition may be used for Cas-mediated gene expression regulation, comprising transcriptional regulation or epigenetic regulation.

[0081] In some embodiments of the present disclosure, the target DNA may be present in isolated cells, for example, eukaryotic cells. Examples of the eukaryotic cells include yeasts, fungi, protozoa, cells from plants, higher plants, insects or amphibians, and mammalian cells such as CHO, HeLa, HEK293, and COS-1 cells. Without limitation, cultured cells (in vitro), graft cells, primary cell culture (in vitro and ex vivo), in vivo cells, and mammalian cells including human cells are commonly used in the art.

[0082] In accordance with a still further aspect, the present disclosure provides a CRISPR-CAS system, comprising (i) a guide RNA comprising a sequence capable of forming a duplex with a target DNA sequence adjacent to the PAM (proto-spacer-adjacent motif) NNNNRYAC (SEQ ID NO: 1), or a DNA encoding the guide RNA; and (ii) a Cas protein recognizing the PAM sequence NNNNRYAC (SEQ ID NO: 1) or a nucleic acid encoding the Cas protein. The individual factors are as described above. These factors may be non-naturally occurring or engineered.

[0083] Still another aspect of the present disclosure pertains to a recombinant viral vector, comprising (i) an expression cassette for a guide RNA comprising a sequence capable of forming a duplex with a target DNA sequence adjacent to the PAM (proto-spacer-adjacent Motif) sequence of NNNNRYAC (SEQ ID NO: 1), and (ii) an expression cassette for a Cas protein recognizing the PAM sequence of NNNNRYAC (SEQ ID NO: 1). The individual factors are as described above. These factors may be non-naturally occurring or engineered. The viral vector, at least in some embodiments, may be of AAV (Adeno-associated virus) origin.

[0084] Yet another aspect of the present disclosure pertains to an isolated guide RNA comprising a sequence of 21-23 bp in length, capable of forming a duplex with a complementary strand of a target DNA sequence. The guide RNA is as defined above. The guide RNA may be non-naturally occurring or engineered.

[0085] Yet still another aspect of the present disclosure pertains to a composition comprising the guide RNA or a DNA encoding the guide RNA. The individual factors are as described above. These factors may be non-naturally occurring or engineered.

[0086] The composition, at least in some embodiments, may comprise a Cas protein that recognizes the PAM sequence NNNNRYAC (SEQ ID NO: 1), or a nucleic acid that encodes the Cas protein.

[0087] In addition, the composition, in some embodiments, may comprise a deactivated Cas recognizing the NNNNRYAC sequence (SEQ ID NO: 1), or a nucleic acid encoding the deactivated Cas protein. The deactivated Cas protein, in some embodiments, may further comprise a transcription effector domain.

[0088] According to an additional aspect, the present disclosure provides an isolated guide RNA, comprising a first region comprising a sequence capable of forming a duplex with a complementary strand of a target DNA sequence, and a second region comprising a stem-loop structure characterized by a stem 13-18 bp in length. The individual factors are as defined above. These factors may be non-naturally occurring or engineered.

[0089] In certain embodiments, the stem may comprise the nucleotide sequence of SEQ ID NO: 2 (5'-GUUUUAGUCCCUUGUG-3') and a complementary sequence thereof.

[0090] According to a further additional aspect, the present disclosure provides an isolated guide RNA, comprising a first region comprising a sequence capable of forming a duplex with a complementary strand of a target DNA sequence, and a second region comprising a stem-loop structure characterized by a loop 5-10 bp in length. The individual factors are as defined above. These factors may be non-naturally occurring or engineered.

[0091] In certain embodiments, the loop may comprise the nucleotide sequence of SEQ ID NO: 3 (5'-AUAUUCAA-3').

[0092] According to yet an additional aspect, the present disclosure provides a composition comprising a guide RNA, along with a Cas protein or a nucleic acid encoding the Cas protein. The individual factors are as defined above. These factors may be non-naturally occurring or engineered.

[0093] Yet still another aspect of the present disclosure provides a method for genome editing in a cell, comprising introducing into the cell an isolated guide RNA or a DNA encoding the isolated guide RNA, together with a Cas protein or a nucleic acid encoding the Cas protein. The individual factors are as defined above. These factors may be non-naturally occurring or engineered.

[0094] Yet a further aspect of the present disclosure provides a method for cleaving a target DNA in a cell, comprising introducing into the cell an isolated guide RNA or a DNA encoding the isolated guide RNA, along with a Cas protein or a nucleic acid encoding the Cas protein. The individual factors are as defined above. These factors may be non-naturally occurring or engineered.

[0095] In certain embodiments, the guide RNA or the DNA encoding the guide RNA may be introduced into a cell simultaneously or sequentially with the Cas protein or the nucleic acid encoding the Cas protein.

[0096] A still further aspect of the present disclosure provides a method for preparing a target DNA-recognizing sequence of a guide RNA (i.e., a sequence in a guide RNA that is responsible for recognizing a target DNA), comprising: (i) identifying the presence of a PAM sequence NNNNRYAC (SEQ ID NO: 1) in a given sequence; and (ii) determining a sequence located just upstream of the PAM sequence NNNNRYAC (SEQ ID NO: 1) as being recognizable by a guide RNA, if the presence of the PAM sequence is identified in step (i). The individual factors are as defined above. These factors may be non-naturally occurring or engineered.

[0097] In some embodiments of the disclosure, the sequence located upstream of the PAM sequence may range, without limitation, in length from 17 to 23 bp, from 18 to 23 bp, from 19 to 23 bp, more particularly from 20 to 23 bp, and even more particularly from 21 to 23 bp.

[0098] Yet another aspect of the present disclosure provides a method for isolating a DNA of interest, comprising: (i) introducing into a cell a guide RNA or a DNA encoding the guide RNA, along with a deactivated Cas protein or a nucleic acid encoding the deactivated Cas protein, thereby permitting the guide RNA and the deactivated Cas protein to form a complex together with the DNA of interest comprising a target DNA sequence; and (ii) separating the complex from a sample. The individual factors are as defined above. These factors may be non-naturally occurring or engineered. The deactivated Cas protein, at least in some embodiments, may recognize the PAM (protospacer-adjacent Motif) sequence NNNNRYAC (SEQ ID NO: 1).

[0099] In certain embodiments, the method for isolating a DNA of interest may be performed by allowing a guide RNA (gRNA), binding specifically to the DNA of interest, and a deactivated Cas protein (dCas) to form a dCas-gRNA-DNA of interest complex with the DNA of interest; and separating the complex from a sample. The DNA of interest, in some embodiments, may be identified using a well-known detection method, such as PCR amplification, etc. The isolation method, in some embodiments, may be adapted for cell-free DNA in vitro without forming crosslinks via covalent bonds between the DNA, the gRNA, and the dCas. In addition, the isolation method may further comprise isolating the DNA of interest from the complex in some embodiments.

[0100] The deactivated Cas protein, in some embodiments, may be linked with an affinity tag for use in isolating the DNA of interest. The affinity tag may be selected from the group consisting of a His tag, a Flag tag, an S tag, a GST (Glutathione S-transferase) tag, an MBP (Maltose binding protein) tag, a CBP (chitin binding protein) tag, an Avi tag, a calmodulin tag, a polyglutamate tag, an E tag, an HA tag, a myc tag, an SBP tag, softag 1, softag 3, a strep tag, a TC tag, an Xpress tag, a BCCP (biotin carboxyl carrier protein) tag, and a GFP (green fluorescent protein) tag, but are not limited thereto. The deactivated Cas protein, in some embodiments, may be a Cas protein that lacks DNA cleavage activity.

[0101] Isolation of a DNA of interest, in some embodiments, may be achieved using an affinity column or magnetic beads capable of binding the tag used. For example, when a His tag is used to isolate the DNA of interest, a metal affinity column or magnetic beads capable of binding the His tag may be employed. The magnetic beads may comprise, but are not limited to, Ni-NTA magnetic beads.

[0102] In some embodiments, isolation of a DNA of interest from the complex may be conducted using RNase and protease.

[0103] In some embodiments in the method for isolating a DNA of interest, a certain genotype DNA, or two or more different DNAs of interest can be isolated from an isolated sample containing a mixture of two or more different genotype DNAs. When the method involves isolating two or more different DNAs of interest, guide RNAs respectively specific for the two or more different DNAs of interest may be employed to isolate two or more DNAs of interest.

[0104] In certain embodiments, the guide RNA may be single guide RNA (sgRNA), or dualRNA comprising crRNA and tracrRNA. The guide RNA may be an isolated RNA, or may be encoded in a plasmid.

[0105] The isolation method, in certain embodiments, may be performed by binding a guide RNA (gRNA) specifically to 1) a DNA of interest and 2) a deactivated Cas protein (dCas) to form a dCas-gRNA-DNA complex with the DNA of interest; and separating the complex from the sample.

[0106] Yet an additional aspect of the present disclosure provides a method for Cas-mediated gene expression regulation in a DNA of interest comprising a target DNA sequence, the method comprising introducing an isolated guide RNA, specifically recognizing the target DNA or a DNA encoding the guide RNA, along with a deactivated Cas protein fused to a transcription effector domain or a nucleic acid encoding the deactivated Cas protein, into a cell. The individual factors are as defined above. These factors may be non-naturally occurring or engineered.

EXAMPLES

[0107] The following examples are provided for the purpose of illustrating some aspects of the disclosure provided herewith and they should not be construed as limiting the scope of the present disclosure in any manner.

C. jejuni CRISPR/Cas9 System

Example 1: Genome Editing using C. jejuni CRISPR/Cas9

[0108] The present inventors succeeded in isolating RGEN from C. jejuni. To identify the characteristics of the C. jejuni CRISPR/CAS 9-derived RGEN with regard to genome editing, a C. jejuni CAS9 gene optimized for human codons was synthesized (TABLE 1) and then inserted into a mammalian expression vector to construct a C. jejuni CAS9 expression cassette in which the HA-tagged, NLS-linked Cas gene was under the regulation of a CMV promoter (FIG. 1).

TABLE-US-00001 TABLE 1 Amino Acid Sequence of C. jejuni Cas9 Protein SEQ ID Amino acid sequence size NO MARILAFDIGISSIGWAFSENDELKDCGVRIFTKVE 1003a.a 22 NPKTGESLALPRRLARSARKRLARRKARLNHLKHLI ANEFKLNYEDYQSFDESLAKAYKGSLISPYELRFRA LNELLSKQDFARVILHIAKRRGYDDIKNSDDKEKGA ILKAIKQNEEKLANYQSVGEYLYKEYFQKFKENSKE FTNVRNKKESYERCIAQSFLKDELKLIFKKQREFGF SFSKKFEEEVLSVAFYKRALKDFSHLVGNCSFFTDE KRAPKNSPLAFMFVALTRIINLLNNLKNTEGILYTK DDLNALLNEVLKNGTLTYKQTKKLLGLSDDYEFKGE KGTYFIEFKKYKEFIKALGEHNLSQDDLNEIAKDIT LIKDEIKLKKALAKYDLNQNQIDSLSKLEFKDHLNI SFKALKLVTPLMLEGKKYDEACNELNLKVAINEDKK DFLPAFNETYYKDEVTNPVVLRAIKEYRKVLNALLK KYGKVHKINIELAREVGKNHSQRAKIEKEQNENYKA KKDAELECEKLGLKINSKNILKLRLFKEQKEFCAYS GEKIKISDLQDEKMLEIDHIYPYSRSFDDSYMNKVL VFTKQNQEKLNQTPFEAFGNDSAKWQKIEVLAKNLP TKKQKRILDKNYKDKEQKNFKDRNLNDTRYIARLVL NYTKDYLDFLPLSDDENTKLNDTQKGSKVHVEAKSG MLTSALRHTWGFSAKDRNNHLHHAIDAVIIAYANNS IVKAFSDFKKEQESNSAELYAKKISELDYKNKRKFF EPFSGFRQKVLDKIDEIFVSKPERKKPSGALHEETF RKEEEFYQSYGGKEGVLKALELGKIRKVNGKIVKNG DMFRVDIFKHKKTNKFYAVPIYTMDFALKVLPNKAV ARSKKGEIKDWILMDENYEFCFSLYKDSLILIQTKD MQEPEFVYYNAFTSSTVSLIVSKHDNKFETLSKNQK ILFKNANEKEVIAKSIGIQNLKVFEKYIVSALGEVT KAEFRQREDFKKSGPPKKKRKVYPYDVPDYA-

[0109] The native guide RNA of the C. jejuni CRISPR/CAS9 system consists of tracrRNA and target-specific crRNA. In view of the notion that the guide RNA is used as the two RNA molecules in themselves or as a single guide RNA (sgRNA) in which crRNA and tracrRNA are fused to each other, the present inventors designed and constructed an expression plasmid for C. jejuni sgRNA (TABLE 2).

TABLE-US-00002 TABLE 2 SEQ ID sgRNAs sgRNA sequence NO C. jejuni_ NNNNNNNNNNNNNNNNNNNNGTTTTAGTCCCT 23 sgRNA GAAAAGGGACTAAAATAAAGAGTTTGCGGGAC TCTGCGGGGTTACAATCCCCTAAAACCGCTTT TTTT

[0110] Then, potential target loci for human AAVS1 and mouse Rosa-26 were selected based on the PAM sequence (NNNACA) of the C. jejuni CRISPR/CAS9 system (TABLE 3).

TABLE-US-00003 TABLE 3 SEQ ID sgRNAs Target Sequence NO Human AAVS1_ ATATAAGGTGGTCCCAGCTCGGGGACA 24 C. Jejuni Mouse Rosa26_ ATTCCCCTGCAGGACAACGCCCACACA 25 C. Jejuni

[0111] To examine whether the C. jejuni RGEN can be used for the targeted disruption of endogenous genes in mammalian cells, genomic DNA, isolated from transfected cells using T7 endonuclease I (T7E1), a mismatch-sensitive endonuclease that specifically recognizes and cleaves heteroduplexes formed by the hybridization of wild-type and mutant DNA sequences, was analyzed. The primer sequences used are as follows (TABLE 4).

TABLE-US-00004 TABLE 4 SEQ ID Primer Sequence NO Human AAVS1-F TGCTTCTCCTCTTGGGAAGT 26 Human AAVS1-R CCCCGTTCTCCTGTGGATTC 27 Mouse Rosa26-F ACGTTTCCGACTTGAGTTGC 28 Mouse Rosa26-R CCCAGCTACAGCCTCGATTT 29

[0112] As a result, mutations (interchangeably substitution or variation) were detected only in the cells into which the CAS9 protein and the guide RNA were introduced together. The mutation frequency was found to be RNA-dose dependent, as measured based on relative DNA band intensities (FIG. 2A). In addition, DNA sequencing analysis of the PCR amplicons corroborated the induction of RGEN-mediated mutations at the endogenous sites. Indels and microhomologies, which are characteristic of error-prone nonhomologous end joining (NHEJ) repair, were observed at the target sites (FIG. 2B). The mutation frequency was 16.7% as measured by direct sequencing (=2 mutant clones/12 clones).

[0113] Likewise, when mouse Rosa26 C. jejuni RGEN was delivered into mouse NHI3T3 cells, mutations were effectively induced at the mouse Rosa26 site, as measured by a T7E1 assay (FIG. 3A). In addition, DNA sequencing analysis of the PCR amplicons revealed the induction of C. jejuni RGEN-mediated mutation at the endogenous gene sites (FIG. 3B). The mutation frequency was found to be 22.2% as measured by direct sequencing (2 mutant clones/9 clones).

Example 2: Structural Modification of sgRNA

[0114] With the anticipation that the C. jejuni crRNA: tracrRNA complex would comprise a shorter loop structure than those from other bacterial species, a modified stem or loop structure was designed to structurally stabilize the C. jejuni RGEN sgRNA constructed in Example 1 (TABLE 5).

TABLE-US-00005 TABLE 5 SEQ ID sgRNAs sgRNA Sequence NO C. jejuni_ NNNNNNNNNNNNNNNNNNNNGTTTTAGTCCC 23 sgRNA TGAAAAGGGACTAAAATAAAGAGTTTGCGGG ACTCTGCGGGGTTACAATCCCCTAAAACCGC TTTTTTT C. jejuni_ NNNNNNNNNNNNNNNNNNNNGTTTTAGTCCC 30 sgRNA_stem TTGTGGAAATATAAGGGACTAAAATAAAGAG modified TTTGCGGGACTCTGCGGGGTTACAATCCCCT AAAACCGCTTTTTTT C. jejuni_ NNNNNNNNNNNNNNNNNNNNGTTTTAGTCCC 31 sgRNA_loop TATATTCAAAGGGACTAAAATAAAGAGTTTG modified CGGGACTCTGCGGGGTTACAATCCCCTAAAA CCGCTTTTTTT

[0115] In TABLE 5, norm stem parts are shown in bold and underlined.

[0116] When the modified sgRNA was introduced to target the target site of the human AAVS1 C. jejuni RGEN into which mutations were successfully induced through the normal sgRNA structure, similar mutation frequencies were observed (FIG. 4). In this regard, the primer sequences used are as shown in TABLE 4.

Example 3: Optimization of Length of sgRNA Spacer

[0117] The spacer sequence of C. jejuni crRNA, recognizing a target sequence, was reported to be 20 bp in length in the literature. To determine which spacer length is optimal, a genome editing test was performed for 4 target sites of Cj Cas9 on human AAVS1 loci, as shown in TABLE 6, using spacers with various lengths, and sgRNA mutant structures with additional nucleotides at 5' terminus (FIGS. 5A to 5C). For the method used in this experiment, reference was made to Genome Res. 2014 January; 24(1):132-41.

TABLE-US-00006 TABLE 6 Target Site SEQ Sequence (20bp- ID sgRNA SPACERnnnnACA) NO Human AAVS1- ATATAAGGTGGTCCCAGCTCggggACA 32 CJ1 Human AAVS1- GTAGAGGCGGCCACGACCTGgtgaACA 33 NRG1 Human AAVS1- TCACAAAGGGAGTTTTCCACacggACA 34 NRG3 Human AAVS1- TAGGCAGATTCCTTATCTGGtgacACA 35 NRG5

[0118] Three days after an sgRNA expression vector was delivered into 293-cells, genomic DNA was isolated and analyzed for mutation efficiency by deep sequencing. The results are depicted in FIG. 5C. As can be seen, high efficiency was detected when the spacers ranged in length from 21 to 23 bp. In addition, even when 2-3 additional G residues were added to the 5' end of sgRNA of a 20 bp-long spacer, an improvement in genome editing was observed.

TABLE-US-00007 TABLE 7 NGS- NGS- primer- primer- Target F* Sequences R** Sequences sgRNA Human AS-AV- ACACTCTTTC AS-AV- GTGACTGGAG CJ1 AAVS1 F1 CCTACACGAC R1 TTCAGACGTG GCTCTTCCGA TGCTCTTCCG TCTAGGAGGA ATCTTGTCAT GGCCTAAGGA GGCATCTTCC TGG AGGG (SEQ ID (SEQ ID NO: 36) NO: 39) AS-AV- ACACTCTTTC AS-AV- GTGACTGGAG NRG1, F2 CCTACACGAC R2 TTCAGACGTG NRG3 GCTCTTCCGA TGCTCTTCCG TCTGCTCTGG ATCTTCCGTG GCGGAGGAAT CGTCAGTTTT ATG ACCT (SEQ ID (SEQ ID NO: 37) NO: 40) AS-AV- ACACTCTTTC AS-AV- GTGACTGGAG NRG5 F4 CCTACACGAC R4 TTCAGACGTG GCTCTTCCGA TGCTCTTCCG TCTATCCTCT ATCTCCGGTT CTGGCTCCAT AATGTGGCTC CGT TGGT (SEQ ID (SEQ ID NO: 38) NO: 41) Here, F* indicates a forward primer and R** indicates a reverse primer.

Example 4: C. jejuni Cas9 PAM Sequence Analysis

[0119] In the present disclosure, the PAM sequence of C. jejuni Cas9 was inferred to comprise "NNNNACA", based on data in the existing literature, and experiments were conducted. Of 34 C. jejuni CRISPR/Cas9 systems constructed for five genome sites, only three exhibited activity. Particularly, additional analysis of the sequences covering the sites in the three active systems showed that the nucleotide "C" was identified immediately after the PAM sequence (NNNNACA) in all three sites (TABLE 8).

TABLE-US-00008 TABLE 8 Activ- ity SEQ (T7E1 ID sgRNA assay) Sequence NO Human AAVS1 hAAVS1- O ATATAAGGTGGTCCCAGCTCGGG 42 CJ1 GACAC hAAVS1- X TGGCCCCACTGTGGGGTGGAGGGGA 43 CJ2 CAG hAAVS1- X CACCCCACAGTGGGGCCACTAGGGA 44 CJ3 CAG CCR5 CCR5- X CTAGCAGCAAACCTTCCCTTCACTAC 45 CJ1 AA CCR5- X CTCCATGAATGCAAACTGTTTTATAC 46 CJ2 AT CCR5- X TGCATTCATGGAGGGCAACTAAATA 47 CJ3 CAT CCR5- X ATCAAGTGTCAAGTCCAATCTATGA 48 CJ4 CAT CCR5- X CCAATCTATGACATCAATTATTATAC 49 CJ5 AT CCR5- X GCAAAAGGCTGAAGAGCATGACTG 50 CJ6 ACAT CCR5- X GCAGCATAGTGAGCCCAGAAGGGG 51 CJ7 ACAG CCR5- X GCCGCCCAGTGGGACTTTGGAAATA 52 CJ8 CAA Mouse Rosa26 ROSA26- X TCCACTGCAGCTCCCTTACTGATAAC 53 CJ1 AA ROSA26- O ATTCCCCTGCAGGACAACGCCCAC 54 CJ2* ACAC ROSA26- X ACACCTGTTCAATTCCCCTGCAGGA 55 CJ3 CAA ROSA26- X TTGAACAGGTGTAAAATTGGAGGGA 56 CJ4 CAA ROSA26- X TTGCCCCTATTAAAAAACTTCCCGAC 57 CJ5 AA ROSA26- X AGATCCTTACTACAGTATGAAATTA 58 CJ6 CAG ROSA26- X AGCCTTATCAAAAGGTATTTTAGAA 59 CJ7 CAC TP53 TP53- X CGGGGCCCACTCACCGTGCACATAA 60 CJ1 CAG TP53- X GCCGTGTCCGCGCCATGGCCATCTA 61 CJ2 CAA TP53- X TGGCCATCTACAAGAAGTCACAGCA 62 CJ3 CAT TP53- X CCGAGTGTCAGGAGCTCCTGCAGCA 63 CJ4 CAG TP53- X CTCCCCGGGGCCCACTCACCGTGCA 64 CJ5 CAT TP53- X CCTGTGCAGTTGTGGGTCAGCGCCA 65 CJ6 CAC TP53- X GGTGTGGCGCTGACCCACAACTGCA 66 CJ7 CAG TP53- O TTCTTGTAGATGGCCATGGCGCG 67 CJ8 GACAC TP53- X CGCCATGGCCATCTACAAGAAGTCA 68 CJ9 CAG PTEN mPTEN- X ACATCATCAATATTGTTCCTGTATAC 69 CJ1 AC mPTEN- X TGAATCCAAAAACCTTAAAACAAAA 70 CJ2 CAA mPTEN- X TGCTTTGAATCCAAAAACCTTAAAA 71 CJ3 CAA mPTEN- X AGCATAAAAACCATTACAAGATATA 72 CJ4 CAA mPTEN- X GTAGATGTGCTGAGAGACATTATGA 73 CJ5 CAC mPTEN- X GGCGGTGTCATAATGTCTCTCAGCA 74 CJ6 CAT mPTEN- X ATTTAACTGCAGAGGTATGTATAAA 75 CJ7 CAT

[0120] Based on this result, the PAM sequence was inferred to contain "NNNNACAC". While the nucleotide at each site of "ACAC" were substituted by A/T/G/C, the activity of C. jejuni Cas9 was analyzed to identify the PAM sequence of C. jejuni RGEN. For this, a surrogate reporter assay was utilized. As a result, C. jejuni was identified to comprise the PAM sequence of "NNNNRYAC (SEQ ID NO: 1)" (FIG. 6, wherein R is a purine residue (A or G) and Y is a pyrimidine residue (CIF)). This experiment was carried out using the surrogate reporter assay described in Nat Methods. 2011 Oct. 9; 8(11):941-3.

Example 5: Assay of Specificity and PAM Sequence of C. jejuni CRISPR/Cas9

[0121] The cleavage sites of C. jejuni CRISPR/CAS9 in the AAVS1-CJ1 loci were analyzed at the genomic level using Digenome-seq, a CRISPR/Cas9 off-target assay developed and submitted for patent protection by the present inventors. The experiment was carried out using a method described in Nat Methods. 2015 March; 12(3):237-43.

[0122] Through Digenome-Seq, 41 loci at which AAVS1-CJ1 CRISPR/Cas9 seemed to be cleaved were determined (Genomic locations in TABLE 9). Consensus sequences were obtained from alignments of cleavage site sequences of the 41 loci, and PAM consistent with that identified in Example 4 was verified.

[0123] Further, to examine whether an off-target mutation is actually introduced into the potential off-targets acquired by Digenome-Seq, genomic DNA from 293-cells to which AAVS1-CJ1 CRISPR engineered nuclease was delivered was subjected to deep sequencing for 40 potential off-target sites. As shown in TABLE 9, no significant mutations were observed.

TABLE-US-00009 TABLE 9 Indel Frequency Genomic Location Mock C. Jejuni CRISPR On-target chr19 55627221 0.02 5.123 CJ_AAVS1_1 chr1 24521012 0.019 0.034 CJ_AAVS1_2 chr1 29848565 0.157 0.136 CJ_AAVS1_3 chr1 30381084 0.041 0.035 CJ_AAVS1_4 chr1 37283269 0.016 0.016 CJ_AAVS1_5 chr2 55333369 0.079 0.091 CJ_AAVS1_6 chr4 153532801 0.003 0.003 CJ_AAVS1_7 chr4 153926891 0 0 CJ_AAVS1_8 chr4 183304101 0.033 0.046 CJ_AAVS1_9 chr6 51746466 0.41 0.43 CJ_AAVS1_10 chr7 11346020 0.02 0.038 CJ_AAVS1_11 chr7 128481430 0.024 0.036 CJ_AAVS1_12 chr7 142878579 0.024 0.028 CJ_AAVS1_13 chr8 25979587 0.138 0.155 CJ_AAVS1_14 chr8 80240626 0.043 0.049 CJ_AAVS1_15 chr8 141347249 0.028 0.024 CJ_AAVS1_16 chr8 141688584 0.088 0.092 CJ_AAVS1_17 chr8 143120119 0.016 0.013 CJ_AAVS1_18 chr9 83960768 0.032 0.037 CJ_AAVS1_19 chr9 102650644 0.029 0.034 CJ_AAVS1_20 chr9 129141695 0.014 0.009 CJ_AAVS1_21 chr10 103862556 0.053 0.073 CJ_AAVS1_22 chr12 9085293 0.21 0.277 CJ_AAVS1_23 chr14 70581187 0.013 0.025 CJ_AAVS1_24 chr14 95327446 0.046 0.041 CJ_AAVS1_25 chr14 102331176 0.015 0.028 CJ_AAVS1_26 chr14 104753692 0.035 0.041 CJ_AAVS1_27 chr15 67686972 0.061 0.096 CJ_AAVS1_28 chr16 85565862 0.028 0.028 CJ_AAVS1_29 chr17 17270109 0.003 0 CJ_AAVS1_30 chr17 79782954 0.03 0.043 CJ_AAVS1_31 chr18 42305670 0.035 0.043 CJ_AAVS1_32 chr19 12826405 0.024 0.039 CJ_AAVS1_33 chr19 32268337 0.043 0.042 CJ_AAVS1_35 chr20 40758976 0 0 CJ_AAVS1_36 chr21 41295936 0.011 0.007 CJ_AAVS1_37 chr22 20990738 0.004 0.004 CJ_AAVS1_38 chr22 46402289 0.006 0.011 CJ_AAVS1_39 chr22 46426607 0.003 0 CJ_AAVS1_40 chrX 27472673 0.279 0.318

[0124] Further, consensus sequences were obtained from the entire alignment of the sequences of 41 loci that showed cleavages in vitro. Consistent with previous results, PAM was actually observed as NNNNRYAC (SEQ ID NO: 1).

Example 6: Degeneracy at First Two Nucleotides of PAM

[0125] The PAM sequence of C. jejuni was found to be NNNNRYAC'' as well as "NNNNACAC" in Example 5, showing degeneracy at the first two positions. In order to corroborate the degeneracy, sgRNAs were constructed respectively for the 7 PAM target sequences of C. jejuni of human AAVS1 loci, which carried G or T residues at the first two positions (TABLE 10), and analyzed for mutation efficiency in HEK293 cells.

TABLE-US-00010 TABLE 10 SEQ Di- ID sgRNA rection PAM Target Sequence NO hAAVS1- + NNNNRYAC gCCACGACCTGGTGA 76 RYN1 ACACCTAGGACGCAC hAAVS1- + gGCCTTATCTCACAG 77 RYN2 GTAAAACTGACGCAC hAAVS1- + cTCTTGGGAAGTGTA 78 RYN3 AGGAAGCTGCAGCAC hAAVS1- + aGCTGCAGCACCAGG 79 RYN4 ATCAGTGAAACGCAC hAAVS1- + cTGTGGGGTGGAGGG 80 RYN5 GACAGATAAAAGTAC hAAVS1- - gCCGGTTAATGTGGC 81 RYN6 TCTGGTTCTGGGTAC hAAVS1- + gCCATGACAGGGGGC 82 RYN7 TGGAAGAGCTAGCAC

[0126] Of the seven constructed sgRNAs, six were found to induce mutations, demonstrating degeneracy at the first two positions of the PAM sequences (FIG. 8). Accordingly, this degeneracy increases the frequency of the PAM sequences, allowing improved accuracy of the genome editing of C. jejuni.

Example 7: Genome Editing through C. jejuni CRISPR/CAS9 Delivery Using AAV

[0127] Representative among promising fields in which genome editing finds application are genome editing technologies for gene and cell therapy. The practical application of genome editing to therapy needs a clinically applicable vector for effectively delivering an engineered nuclease and a donor DNA to target cells in vitro or in vivo. The two most widely used engineered nuclease platforms, TALENs and RGEN, are limited to application to established gene therapy vectors due to their large sizes. In contrast, the C. jejuni RGEN of the present disclosure consists of the smallest CAS9 protein and sgRNA among the RGENs developed so far. Thanks to its small size, the C. jejuni RGEN can allow large-sized gene therapy vectors to be used in genome manipulation. For example, AAV (adeno-associated virus), serving as one of the most important vectors for gene therapy, imposes strict limitations on the size of the DNA to be carried thereby, and thus is difficult to apply to the RGEN derived from S. pyogenes, S. thermophilus, or N. meningitidis, or to the currently used engineered nuclease platform TALEN. In contrast, the C. jejuni RGEN can be applied to an AAV vector.

[0128] In the present disclosure, examination was made of the operation of the C. jejuni Cas9 through practical AAV delivery. To this end, an AAV vector carrying both a C. jejuni Cas9 expression cassette and an sgRNA expression cassette was constructed (FIG. 9) and used to produce AAV. After infection with the AAV, mouse C2C12 cells were quantitatively analyzed for mutations (FIG. 10). As can be seen, mutations were induced in target sites in an AAV dose- and time-dependent manner. Particularly, 4 weeks after infection at high MOI (100), mutations were induced at an efficiency of 90% or higher in the target sites.

[0129] Consequently, the C. jejuni RGEN was proven to effectively perform genome editing in cultured cells. In addition, the PAM sequence of the C. jejuni CRISPR/Cas9 system was actually determined, as the sequence proposed in previous studies was found not to be perfect. Further, the C. jejuni RGEN can be loaded into a single virus thanks to the small sizes of its elements, and thus can be used for effective genome editing.

Enrichment of Target DNA Using dCAS9: gRNA Complex

[0130] Moreover, a target DNA was isolated and enriched using the RGEN (dCas9:gRNA complex) composed of a Streptococcus pyogenes-derived, deactivated Cas9 protein and a guide RNA.

[0131] In this regard, the dCas9 protein was tagged with six consecutive His residues, so that it could be purified using Ni-NTA magnetic beads for selectively binding to the His tag. In addition, the dCas protein-sgRNA complex can be used for the selective purification of a target DNA because the complex can bind specifically to a certain DNA sequence, but lacks nuclease activity.

[0132] The RGEN (dCas9: gRNA complex) composed of a guide RNA and a deactivated Cas nuclease was tested for ability to isolate a target DNA. For this, first, the plasmid pUC19 was digested with restriction enzymes (SpeI, XmaI, XhoI) to yield plasmid DNA fragments 4134 bp, 2570 bp, and 1263 bp in length, respectively.

[0133] For each of the plasmid DNA fragments digested with the restriction enzymes, two different sgRNAs were synthesized (4134bp_sg #1, 4134bp_sg #2, 2570bp_sg #1, 2570bp_sg #2, 1263bp_sg #1, and 1263bp_sg #2). A purification procedure was carried out using the sgRNAs corresponding to target DNAs, singularly or in combination (4134bp_sg #1+2, 2570bp_sg #1+2, and 1263bp_sg #1+2). The nucleotide sequences of the sgRNAs are listed in TABLE 11 below.

TABLE-US-00011 TABLE 11 PAM sgRNA Target sequence Sequence 4134bp_ GAGAACCAGACCACCCAGAA GGG sg#1 (SEQ ID NO: 83) 4134bp_ GGCAGCCCCGCCATCAAGAA GGG sg#2 (SEQ ID NO: 84) 2570bp_ GTAAGATGCTTTTCTGTGAC TGG sg#1 (SEQ ID NO: 85) 2570bp_ GATCCTTTGATCTTTTCTAC GGG sg#2 (SEQ ID NO: 86) 1270bp_ GCCTCCAAAAAAGAAGAGAA AGG sg#1 (SEQ ID NO: 87) 1270bp_ TGACATCAATTATTATACAT CGG sg#2 (SEQ ID NO: 88) *nucleotide sequences of the sgRNAs are identical to those of the target DNA, except for U in place of T.

[0134] A total of 200 .mu.l of a mixture solution containing DNA: dCas9 protein: sgRNA at a molar ratio of 1:20:100 was incubated at 37.degree. C. for 1.5 hrs. Then, the solution was mixed with 50 .mu.l of Ni-NTA magnetic beads binding specifically to His-tag, and washed twice with 200 .mu.l of a wash buffer, followed by purifying a dCas9-sgRNA-target DNA complex with 200 .mu.l of an eluting buffer (Bioneer, K-7200).

[0135] Thereafter, the eluate was incubated at 37.degree. C. for 2 hrs with 0.2 mg/ml RNase A (Amresco, E866) and then at 55.degree. C. for 45 min with 0.2 mg/ml Proteinase K to remove both the sgRNA and the dCas9 protein. The target DNA alone was precipitated in ethanol.

[0136] As a result, using the sgRNAs, whether singularly or in combinations of two thereof, for individual target DNAs, desired target DNAs could be isolated from the three DNA fragments digested by size. In addition, when multiple target DNAs were purified with combinations of sgRNA, such as a total of 4 different sgRNAs for two different target DNAs (2 sgRNAs for each target DNA), the target DNAs were associated with corresponding sgRNAs and thus purified. The results indicate that each target DNA could be isolated at a purity of 95% or higher.

[0137] Also, the purification technique is true of the Cas protein recognizing the PAM (proto-spacer-adjacent motif) sequence NNNNRYAC (SEQ ID NO: 1) of the present disclosure.

[0138] Based on the above description, it should be understood by those skilled in the art that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention without departing from the technical idea or essential features of the invention as defined in the following claims. In this regard, the above-described examples are for illustrative purposes only, and the invention is not intended to be limited by these examples. The scope of the present invention should be understood to comprise all of the modifications or modified forms derived from the meaning and scope of the following claims or equivalent concepts.

Sequence CWU 1

1

9018DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidePAM sequencemodified_base(1)..(4)Any nucleotide 1nnnnryac 8216RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidestem sequence in guideRNA 2guuuuagucc cuugug 1638RNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideloop sequence in guideRNA 3auauucaa 8460DNAHomo sapiens 4cacattaacc ggccctggga atataaggtg gtcccagctc ggggacacag gatccctgga 60558DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidehAAVS1 mutant clone (-2, x1) 5cacattaacc ggccctggga atataaggtg gtcccagcgg ggacacagga tccctgga 58659DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidehAAVS mutant clone (-1, x1) 6cacattaacc ggccctggga atataaggtg gtcccagtcg gggacacagg atccctgga 59759DNAMus musculus 7cttaaaggct aacctggtgt gtgggcgttg tcctgcaggg gaattgaaca ggtgtaaaa 59858DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideMouse Rosa26 (-1, X1) 8cttaaaggct aacctggtgt gtgggcttgt cctgcagggg aattgaacag gtgtaaaa 58960DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideMouse Rosa26 (+1, X1) 9cttaaaggct aacctggtgt gtgggcgttt gtcctgcagg ggaattgaac aggtgtaaaa 601038DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 10ggccctggga atataaggtg gtcccagctc ggggacac 381128DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideGX19 11gtataaggtg gtcccagctc ggggacac 281229DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideGX20 12gatataaggt ggtcccagct cggggacac 291330DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideGX21 13gaatataagg tggtcccagc tcggggacac 301431DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideGX22 14ggaatataag gtggtcccag ctcggggaca c 311532DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideGX23 15gggaatataa ggtggtccca gctcggggac ac 321630DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideGGX20 16ggatataagg tggtcccagc tcggggacac 301731DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideGGGX20 17gggatataag gtggtcccag ctcggggaca c 311838DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget site of hAAVS-CJ1 18ggccctggga atataaggtg gtcccagctc ggggacac 381938DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget site of hAAVS-NRG1 19gagaaaggga gtagaggcgg ccacgacctg gtgaacac 382038DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget site of hAAVS-NRG3 20cgcaccattc tcacaaaggg agttttccac acggacac 382138DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget site of hAAVS-NRG5 21cacctcctgt taggcagatt ccttatctgg tgacacac 38221003PRTCampylobacter jejuni 22Met Ala Arg Ile Leu Ala Phe Asp Ile Gly Ile Ser Ser Ile Gly Trp1 5 10 15Ala Phe Ser Glu Asn Asp Glu Leu Lys Asp Cys Gly Val Arg Ile Phe 20 25 30Thr Lys Val Glu Asn Pro Lys Thr Gly Glu Ser Leu Ala Leu Pro Arg 35 40 45Arg Leu Ala Arg Ser Ala Arg Lys Arg Leu Ala Arg Arg Lys Ala Arg 50 55 60Leu Asn His Leu Lys His Leu Ile Ala Asn Glu Phe Lys Leu Asn Tyr65 70 75 80Glu Asp Tyr Gln Ser Phe Asp Glu Ser Leu Ala Lys Ala Tyr Lys Gly 85 90 95Ser Leu Ile Ser Pro Tyr Glu Leu Arg Phe Arg Ala Leu Asn Glu Leu 100 105 110Leu Ser Lys Gln Asp Phe Ala Arg Val Ile Leu His Ile Ala Lys Arg 115 120 125Arg Gly Tyr Asp Asp Ile Lys Asn Ser Asp Asp Lys Glu Lys Gly Ala 130 135 140Ile Leu Lys Ala Ile Lys Gln Asn Glu Glu Lys Leu Ala Asn Tyr Gln145 150 155 160Ser Val Gly Glu Tyr Leu Tyr Lys Glu Tyr Phe Gln Lys Phe Lys Glu 165 170 175Asn Ser Lys Glu Phe Thr Asn Val Arg Asn Lys Lys Glu Ser Tyr Glu 180 185 190Arg Cys Ile Ala Gln Ser Phe Leu Lys Asp Glu Leu Lys Leu Ile Phe 195 200 205Lys Lys Gln Arg Glu Phe Gly Phe Ser Phe Ser Lys Lys Phe Glu Glu 210 215 220Glu Val Leu Ser Val Ala Phe Tyr Lys Arg Ala Leu Lys Asp Phe Ser225 230 235 240His Leu Val Gly Asn Cys Ser Phe Phe Thr Asp Glu Lys Arg Ala Pro 245 250 255Lys Asn Ser Pro Leu Ala Phe Met Phe Val Ala Leu Thr Arg Ile Ile 260 265 270Asn Leu Leu Asn Asn Leu Lys Asn Thr Glu Gly Ile Leu Tyr Thr Lys 275 280 285Asp Asp Leu Asn Ala Leu Leu Asn Glu Val Leu Lys Asn Gly Thr Leu 290 295 300Thr Tyr Lys Gln Thr Lys Lys Leu Leu Gly Leu Ser Asp Asp Tyr Glu305 310 315 320Phe Lys Gly Glu Lys Gly Thr Tyr Phe Ile Glu Phe Lys Lys Tyr Lys 325 330 335Glu Phe Ile Lys Ala Leu Gly Glu His Asn Leu Ser Gln Asp Asp Leu 340 345 350Asn Glu Ile Ala Lys Asp Ile Thr Leu Ile Lys Asp Glu Ile Lys Leu 355 360 365Lys Lys Ala Leu Ala Lys Tyr Asp Leu Asn Gln Asn Gln Ile Asp Ser 370 375 380Leu Ser Lys Leu Glu Phe Lys Asp His Leu Asn Ile Ser Phe Lys Ala385 390 395 400Leu Lys Leu Val Thr Pro Leu Met Leu Glu Gly Lys Lys Tyr Asp Glu 405 410 415Ala Cys Asn Glu Leu Asn Leu Lys Val Ala Ile Asn Glu Asp Lys Lys 420 425 430Asp Phe Leu Pro Ala Phe Asn Glu Thr Tyr Tyr Lys Asp Glu Val Thr 435 440 445Asn Pro Val Val Leu Arg Ala Ile Lys Glu Tyr Arg Lys Val Leu Asn 450 455 460Ala Leu Leu Lys Lys Tyr Gly Lys Val His Lys Ile Asn Ile Glu Leu465 470 475 480Ala Arg Glu Val Gly Lys Asn His Ser Gln Arg Ala Lys Ile Glu Lys 485 490 495Glu Gln Asn Glu Asn Tyr Lys Ala Lys Lys Asp Ala Glu Leu Glu Cys 500 505 510Glu Lys Leu Gly Leu Lys Ile Asn Ser Lys Asn Ile Leu Lys Leu Arg 515 520 525Leu Phe Lys Glu Gln Lys Glu Phe Cys Ala Tyr Ser Gly Glu Lys Ile 530 535 540Lys Ile Ser Asp Leu Gln Asp Glu Lys Met Leu Glu Ile Asp His Ile545 550 555 560Tyr Pro Tyr Ser Arg Ser Phe Asp Asp Ser Tyr Met Asn Lys Val Leu 565 570 575Val Phe Thr Lys Gln Asn Gln Glu Lys Leu Asn Gln Thr Pro Phe Glu 580 585 590Ala Phe Gly Asn Asp Ser Ala Lys Trp Gln Lys Ile Glu Val Leu Ala 595 600 605Lys Asn Leu Pro Thr Lys Lys Gln Lys Arg Ile Leu Asp Lys Asn Tyr 610 615 620Lys Asp Lys Glu Gln Lys Asn Phe Lys Asp Arg Asn Leu Asn Asp Thr625 630 635 640Arg Tyr Ile Ala Arg Leu Val Leu Asn Tyr Thr Lys Asp Tyr Leu Asp 645 650 655Phe Leu Pro Leu Ser Asp Asp Glu Asn Thr Lys Leu Asn Asp Thr Gln 660 665 670Lys Gly Ser Lys Val His Val Glu Ala Lys Ser Gly Met Leu Thr Ser 675 680 685Ala Leu Arg His Thr Trp Gly Phe Ser Ala Lys Asp Arg Asn Asn His 690 695 700Leu His His Ala Ile Asp Ala Val Ile Ile Ala Tyr Ala Asn Asn Ser705 710 715 720Ile Val Lys Ala Phe Ser Asp Phe Lys Lys Glu Gln Glu Ser Asn Ser 725 730 735Ala Glu Leu Tyr Ala Lys Lys Ile Ser Glu Leu Asp Tyr Lys Asn Lys 740 745 750Arg Lys Phe Phe Glu Pro Phe Ser Gly Phe Arg Gln Lys Val Leu Asp 755 760 765Lys Ile Asp Glu Ile Phe Val Ser Lys Pro Glu Arg Lys Lys Pro Ser 770 775 780Gly Ala Leu His Glu Glu Thr Phe Arg Lys Glu Glu Glu Phe Tyr Gln785 790 795 800Ser Tyr Gly Gly Lys Glu Gly Val Leu Lys Ala Leu Glu Leu Gly Lys 805 810 815Ile Arg Lys Val Asn Gly Lys Ile Val Lys Asn Gly Asp Met Phe Arg 820 825 830Val Asp Ile Phe Lys His Lys Lys Thr Asn Lys Phe Tyr Ala Val Pro 835 840 845Ile Tyr Thr Met Asp Phe Ala Leu Lys Val Leu Pro Asn Lys Ala Val 850 855 860Ala Arg Ser Lys Lys Gly Glu Ile Lys Asp Trp Ile Leu Met Asp Glu865 870 875 880Asn Tyr Glu Phe Cys Phe Ser Leu Tyr Lys Asp Ser Leu Ile Leu Ile 885 890 895Gln Thr Lys Asp Met Gln Glu Pro Glu Phe Val Tyr Tyr Asn Ala Phe 900 905 910Thr Ser Ser Thr Val Ser Leu Ile Val Ser Lys His Asp Asn Lys Phe 915 920 925Glu Thr Leu Ser Lys Asn Gln Lys Ile Leu Phe Lys Asn Ala Asn Glu 930 935 940Lys Glu Val Ile Ala Lys Ser Ile Gly Ile Gln Asn Leu Lys Val Phe945 950 955 960Glu Lys Tyr Ile Val Ser Ala Leu Gly Glu Val Thr Lys Ala Glu Phe 965 970 975Arg Gln Arg Glu Asp Phe Lys Lys Ser Gly Pro Pro Lys Lys Lys Arg 980 985 990Lys Val Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 995 100023100DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotideC.jejuni_sgRNAmodified_base(1)..(20)Any nucleotide 23nnnnnnnnnn nnnnnnnnnn gttttagtcc ctgaaaaggg actaaaataa agagtttgcg 60ggactctgcg gggttacaat cccctaaaac cgcttttttt 1002427DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence of Human AAVS1 24atataaggtg gtcccagctc ggggaca 272527DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence of Mouse Rosa26 25attcccctgc aggacaacgc ccacaca 272620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideHuman AAVS1-F 26tgcttctcct cttgggaagt 202720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideHuman AAVS1-R 27ccccgttctc ctgtggattc 202820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideMouse Rosa26-F 28acgtttccga cttgagttgc 202920DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideMouse Rosa26-R 29cccagctaca gcctcgattt 2030108DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotideC.jejuni_sgRNA_stem modifiedmodified_base(1)..(20)Any nucleotide 30nnnnnnnnnn nnnnnnnnnn gttttagtcc cttgtggaaa tataagggac taaaataaag 60agtttgcggg actctgcggg gttacaatcc cctaaaaccg cttttttt 10831104DNAArtificial SequenceDescription of Artificial Sequence Synthetic polynucleotideC.jejuni_sgRNA_loop modifiedmodified_base(1)..(20)Any nucleotide 31nnnnnnnnnn nnnnnnnnnn gttttagtcc ctatattcaa agggactaaa ataaagagtt 60tgcgggactc tgcggggtta caatccccta aaaccgcttt tttt 1043227DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideHuman AAVS1-CJ1 32atataaggtg gtcccagctc ggggaca 273327DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideHuman AAVS1-NRG1 33gtagaggcgg ccacgacctg gtgaaca 273427DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideHuman AAVS1-NRG3 34tcacaaaggg agttttccac acggaca 273527DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideHuman AAVS1-NRG5 35taggcagatt ccttatctgg tgacaca 273653DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideAS-AV-F1 36acactctttc cctacacgac gctcttccga tctaggagga ggcctaagga tgg 533753DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideAS-AV-F2 37acactctttc cctacacgac gctcttccga tctgctctgg gcggaggaat atg 533853DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideAS-AV-F4 38acactctttc cctacacgac gctcttccga tctatcctct ctggctccat cgt 533954DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideAS-AV-R1 39gtgactggag ttcagacgtg tgctcttccg atcttgtcat ggcatcttcc aggg 544054DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideAS-AV-R2 40gtgactggag ttcagacgtg tgctcttccg atcttccgtg cgtcagtttt acct 544154DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideAS-AV-R4 41gtgactggag ttcagacgtg tgctcttccg atctccggtt aatgtggctc tggt 544228DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 42atataaggtg gtcccagctc ggggacac 284328DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 43tggccccact gtggggtgga ggggacag 284428DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 44caccccacag tggggccact agggacag 284528DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 45ctagcagcaa accttccctt cactacaa 284628DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 46ctccatgaat gcaaactgtt ttatacat 284728DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 47tgcattcatg gagggcaact aaatacat 284828DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 48atcaagtgtc aagtccaatc tatgacat 284928DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 49ccaatctatg acatcaatta ttatacat 285028DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 50gcaaaaggct gaagagcatg actgacat 285128DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 51gcagcatagt gagcccagaa ggggacag 285228DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 52gccgcccagt gggactttgg aaatacaa 285328DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 53tccactgcag ctcccttact gataacaa 285428DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 54attcccctgc aggacaacgc ccacacac 285528DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 55acacctgttc aattcccctg caggacaa 285628DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 56ttgaacaggt gtaaaattgg agggacaa 285728DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 57ttgcccctat taaaaaactt cccgacaa

285828DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 58agatccttac tacagtatga aattacag 285928DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 59agccttatca aaaggtattt tagaacac 286028DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 60cggggcccac tcaccgtgca cataacag 286128DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 61gccgtgtccg cgccatggcc atctacaa 286228DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 62tggccatcta caagaagtca cagcacat 286328DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 63ccgagtgtca ggagctcctg cagcacag 286428DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 64ctccccgggg cccactcacc gtgcacat 286528DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 65cctgtgcagt tgtgggtcag cgccacac 286628DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 66ggtgtggcgc tgacccacaa ctgcacag 286728DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 67ttcttgtaga tggccatggc gcggacac 286828DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 68cgccatggcc atctacaaga agtcacag 286928DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 69acatcatcaa tattgttcct gtatacac 287028DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 70tgaatccaaa aaccttaaaa caaaacaa 287128DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 71tgctttgaat ccaaaaacct taaaacaa 287228DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 72agcataaaaa ccattacaag atatacaa 287328DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 73gtagatgtgc tgagagacat tatgacac 287428DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 74ggcggtgtca taatgtctct cagcacat 287528DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTest sequence 75atttaactgc agaggtatgt ataaacat 287630DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 76gccacgacct ggtgaacacc taggacgcac 307730DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 77ggccttatct cacaggtaaa actgacgcac 307830DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 78ctcttgggaa gtgtaaggaa gctgcagcac 307930DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 79agctgcagca ccaggatcag tgaaacgcac 308030DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 80ctgtggggtg gaggggacag ataaaagtac 308130DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 81gccggttaat gtggctctgg ttctgggtac 308230DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 82gccatgacag ggggctggaa gagctagcac 308320DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 83gagaaccaga ccacccagaa 208420DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 84ggcagccccg ccatcaagaa 208520DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 85gtaagatgct tttctgtgac 208620DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 86gatcctttga tcttttctac 208720DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 87gcctccaaaa aagaagagaa 208820DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotideTarget sequence 88tgacatcaat tattatacat 208928DNAArtificial SequenceDescription of Artificial Sequence Synthetic oligonucleotidemodified_base(21)..(24)a, c, t, g, unknown or other 89atataaggtg gtcccagctc nnnnacac 28906PRTArtificial SequenceDescription of Artificial Sequence Synthetic 6xHis tag 90His His His His His His1 5

Claims

1-67 (canceled)

68. A method of altering a genome via targeting a DNA sequence comprising a protospacer adjacent motif (PAM) sequence of NNNNRYAC (SEQ ID NO:1), the method comprising: introducing a Cas protein that recognizes the PAM sequence of SEQ ID NO:1, or a nucleic acid encoding the Cas protein, and; an isolated guide RNA, comprising a sequence capable of forming a duplex with a complementary strand of a target DNA sequence adjacent to a proto-spacer-adjacent motif (PAM) sequence of NNNNRYAC (SEQ ID NO: 1), or a nucleic acid encoding the isolated guide RNA into a cell comprising the genome, thereby altering the genome.

69. The method of claim 68, wherein the Cas protein originates from a microorganism belonging to Campylobacter.

70. The method of claim 69, wherein the microorganism is Campylobacter jejuni.

71. The method of claim 68, wherein the Cas protein is a Cas9 protein.

72. The method of claim 68, introducing a guide RNA is carried out simultaneously or sequentially with the Cas protein that recognizes the PAM sequence of SEQ ID NO: 1.

73. The method of claim 68, wherein the guide RNA is a dual RNA comprising CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA).

74. The method of claim 68, wherein the guide RNA is a single guide RNA (sgRNA).

75. The method of claim 74, wherein the sgRNA comprises a first region containing a sequence capable of forming a duplex with a complementary strand of the target DNA sequence, and a second region containing a sequence that interacts with the Cas protein.

76. The method of claim 74, wherein the sgRNA comprises a portion of a crRNA which contains a sequence capable of forming a duplex with a complementary strand of the target DNA sequence, and a portion of a tracrRNA which contains a sequence interacting with the Cas protein.

77. The method of claim 68, wherein the Cas protein having the nickase activity has a catalytic aspartic acid (D) at position 8 and histidine (H) at position 559 substituted with another amino acid.

78. The method of claim 77, wherein the substituted amino acid is alanine.

79. The method of claim 68, wherein the Cas protein binds to the target DNA sequence comprising the PAM sequence of SEQ ID NO: 1, without cleaving the target DNA.

80. A composition for altering a genome with a target DNA sequence, comprising: an isolated guide RNA or a DNA encoding the isolated guide RNA, and a Cas9 protein that recognizes a PAM sequence of NNNNRYAC (SEQ ID NO:1) or a nucleic acid encoding the Cas9 protein, wherein the target DNA sequence is adjacent to the PAM sequence of NNNNRYAC (SEQ ID NO:1).

81. The composition of claim 80, wherein the Cas protein is a deactivated Cas protein (dCas) or a nucleic acid that encodes the dCas.

82. The composition of claim 80, wherein the isolated guide RNA is a dual RNA comprising a crRNA (CRISPR RNA) and a tracrRNA (trans-activating crRNA).

83. The composition of claim 80, wherein the isolated guide RNA is a single-stranded guide RNA (sgRNA).

84. The composition of claim 83, wherein the sgRNA comprises a first region containing a sequence capable of forming a duplex with a complementary strand of the target DNA sequence, and a second region containing a sequence interacting with the Cas protein.

85. The composition of claim 83, wherein the length of the sequence capable of forming a duplex with a complementary strand of a target DNA sequence is 17 to 23 bp.

86. The composition of claim 83 wherein the guide RNA further comprises one to three additional nucleotides prior to a 5' end of the sequence capable of forming a duplex with a complementary strand of the target DNA.

87. The composition of claim 86, wherein the additional nucleotides comprise guanine (G).