US20200172912A1

US20200172912A1 - Genome editing using campylobacter jejuni crispr/cas system-derived rgen

Info

Publication number: US20200172912A1
Application number: US16/700,942
Authority: US
Inventors: Eun Ji Kim; Seok Joong Kim
Original assignee: Toolgen Inc
Current assignee: Toolgen Inc
Priority date: 2014-08-06
Filing date: 2019-12-02
Publication date: 2020-06-04
Also published as: CN113789317B; CN106922154B; AU2020267249A1; AU2015299850B2; CA2957441A1; US20170145425A1; US10519454B2; JP2017526387A; KR101817482B1; CN113789317A; KR20180015731A; EP3178935A4; EP4194557A1; KR20170020535A; CN106922154A; EP3178935B1; AU2020267249B2; WO2016021973A1; EP3178935A1; JP6715419B2

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)

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

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

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

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.
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

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.
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.
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.
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.
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).
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.
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.
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.
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.
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.
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).
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.
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.
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

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.

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.

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.

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 (+).

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.

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.

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

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.

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

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

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.
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).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
Moreover, at least in some embodiments, the guide RNA may be designed to be specific for a certain target to be cleaved.
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.
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.
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.
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.
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.
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.
In some embodiments of the present disclosure, the crRNA may hybridize with a target DNA.
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.
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.
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).
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.
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′).
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
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.
In addition, in certain embodiments, the composition may be used for genome editing.
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.
In an embodiment, the deactivated Cas protein may further comprise a transcription effector domain.
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.
In some embodiments of the present disclosure, the composition may be used for Cas-mediated gene expression regulation, comprising transcriptional regulation or epigenetic regulation.
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.
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.
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.
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.
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.
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.
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.
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.
In certain embodiments, the stem may comprise the nucleotide sequence of SEQ ID NO: 2 (5′-GUUUUAGUCCCUUGUG-3′) and a complementary sequence thereof.
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.
In certain embodiments, the loop may comprise the nucleotide sequence of SEQ ID NO: 3 (5′-AUAUUCAA-3′).
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.
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.
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.
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.
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.
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.
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).
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.
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.
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.
In some embodiments, isolation of a DNA of interest from the complex may be conducted using RNase and protease.
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.
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.
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.
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

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

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 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-

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 2

		SEQ
		ID
sgRNAs	sgRNA sequence	NO

C. jejuni_	NNNNNNNNNNNNNNNNNNNN GTTTTAGTCCCT	23

sgRNA	GAAA AGGGACTAAAAT AAAGAGTTTGCGGGAC

	TCTGCGGGGTTACAATCCCCTAAAACCGCTTT

	TTTT

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 3

		SEQ
		ID
sgRNAs	Target Sequence	NO

Human AAVS1_	ATATAAGGTGGTCCCAGCTCGGGGACA	24
C. Jejuni

Mouse Rosa26_	ATTCCCCTGCAGGACAACGCCCACACA		25
C. Jejuni

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 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

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).
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

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 5

		SEQ
		ID
sgRNAs	sgRNA Sequence	NO

C. jejuni_	NNNNNNNNNNNNNNNNNNNN GTTTTAGTCCC	23
sgRNA	T GAAA AGGGACTAAAAT AAAGAGTTTGCGGG
	ACTCTGCGGGGTTACAATCCCCTAAAACCGC
	TTTTTTT

C. jejuni _	NNNNNNNNNNNNNNNNNNNN GTTTTAGTCCC		30
sgRNA_stem	T TGTGGAAATATA AGGGACTAAAAT AAAGAG
modified	TTTGCGGGACTCTGCGGGGTTACAATCCCCT
	AAAACCGCTTTTTTT

C. jejuni_	NNNNNNNNNNNNNNNNNNNN GTTTTAGTCCC	31
sgRNA_loop	T ATATTCAA AGGGACTAAAAT AAAGAGTTTG
modified	CGGGACTCTGCGGGGTTACAATCCCCTAAAA
	CCGCTTTTTTT

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

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 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

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 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

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 8

		Activ-
		ity		SEQ
		(T7E1		ID
	sgRNA	assay)	Sequence	NO

Human

AAVS1	hAAVS1-	O	ATATAAGGTGGTCCCAGCTCGGG	42
	CJ1		GACA C
	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*		ACA C
	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		GACA C
	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

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

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.
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.
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 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

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

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 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

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

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.
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.
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

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.
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.
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.
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 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.

A total of 200 μl of a mixture solution containing DNA: dCas9 protein: sgRNA at a molar ratio of 1:20:100 was incubated at 37° C. for 1.5 hrs. Then, the solution was mixed with 50 μl of Ni-NTA magnetic beads binding specifically to His-tag, and washed twice with 200 μl of a wash buffer, followed by purifying a dCas9-sgRNA-target DNA complex with 200 μl of an eluting buffer (Bioneer, K-7200).
Thereafter, the eluate was incubated at 37° C. for 2 hrs with 0.2 mg/ml RNase A (Amresco, E866) and then at 55° 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.
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.
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.
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.

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).