阅读说明：本技术 使用空肠弯曲杆菌crispr/cas系统衍生的rna引导的工程化核酸酶的基因编辑 (Gene editing using Campylobacter jejuni CRISPR/CAS system-derived RNA-guided engineered nucleases ) 是由 金殷智 金奭中 于 2015-08-06 设计创作，主要内容包括：本文提供的公开涉及空肠弯曲杆菌(Campylobacter jejuni)CRISPR/CAS系统衍生的RNA引导的工程核酸酶(RGEN)及使用其的方法。(The disclosure provided herein relates to Campylobacter jejuni (Campylobacter jejuni) CRISPR/CAS system-derived RNA-guided engineered nucleases (RGENs) and methods of using the same.)

1. Use of a Cas protein in the preparation of a CRISPR-Cas system for editing a genome comprising a target DNA, wherein the sequence of the target DNA is adjacent to a protospacer adjacent motif sequence of NNNNRYAC (SEQ ID NO:1),

wherein the CRISPR-Cas system comprises:

a Cas protein that recognizes the sequence of NNRYAC (SEQ ID NO:1), wherein the Cas protein is Campylobacter jejuni Cas9 protein; and

a guide RNA comprising a crRNA and a tracrRNA of the Cas protein, wherein the crRNA has a sequence capable of forming a duplex with the complementary strand of the target DNA sequence adjacent to the protospacer-adjacent motif sequence of SEQ ID NO:1 and a sequence of an essential portion of the crRNA.

2. Use of a Cas protein according to claim 1, wherein said Cas protein consists of SEQ ID No. 22.

3. Use of a Cas protein according to claim 1, wherein said Cas protein has a nickase activity.

4. Use of a Cas protein according to claim 1, wherein said cell is a eukaryotic cell.

5. Use of a Cas protein according to claim 1, wherein the sequence of said target DNA is derived from the genome of a eukaryotic cell.

6. Use of a Cas protein according to claim 1, wherein said CRISPR-Cas system further comprises a Nuclear Localization Signal (NLS).

7. Use of a Cas protein according to claim 1, wherein said guide RNA is a dual guide RNA.

8. Use of a Cas protein according to claim 1, wherein said guide RNA is a single guide RNA in which said crRNA and said tracrRNA are fused to each other.

9. A method for editing a target DNA sequence adjacent to a protospacer adjacent motif sequence of NNNNRYAC (SEQ ID NO:1) in an isolated eukaryotic cell from a human or a eukaryotic cell from a non-human comprising: introducing the composition into a cell in a cell culture,

wherein the composition comprises:

a Cas protein or a nucleic acid encoding the Cas protein that recognizes a motif sequence adjacent to the protospacer sequence of NNRYAC (SEQ ID NO:1), wherein the Cas protein is a Campylobacter jejuni Cas9 protein; and

a guide RNA comprising a crRNA and a tracrRNA of a CRISPR-Cas system, or a DNA sequence encoding the guide RNA, wherein the crRNA has a sequence capable of forming a duplex with the complementary strand of the target DNA sequence and a sequence of an essential part of the crRNA.

10. The method of claim 9, wherein the first and second light sources are selected from the group consisting of,

wherein the composition comprises the Cas protein and the guide RNA,

wherein the composition is introduced into the cell in the form of ribonucleoproteins.

11. The method of claim 9, wherein the composition comprises nucleic acid encoding the Cas protein and nucleic acid encoding the guide RNA.

12. The method of claim 11, wherein the nucleic acid encoding the Cas protein and the nucleic acid encoding the guide RNA are contained in one or more vectors selected from a plasmid vector, a viral vector.

13. A method for designing a sequence of a guide RNA of a campylobacter jejuni Cas9 protein, comprising:

identifying a target DNA in a genome, wherein the target DNA sequence is adjacent to a PAM sequence of NNNNRYAC (SEQ ID NO:1) in the genome;

designing a sequence of a guide RNA comprising a crRNA and a tracrRNA, wherein the crRNA has a sequence capable of forming a duplex with the complementary strand of the target DNA sequence and a sequence of an essential part of the crRNA of the campylobacter jejuni Cas9 protein, wherein the tracrRNA is directed against the campylobacter jejuni Cas9 protein.

14. The method of claim 13, wherein the target DNA is 17 to 23bp in length.

Technical Field

The present invention relates to RNA-guided engineered nucleases (RGENs) derived from the Campylobacter jejuni (Campylobacter jejuni) CRISPR/CAS system and methods of using the same.

Background

Engineered nucleases can be used to efficiently manipulate genes in living cells or in whole organisms by generating site-specific double-strand breaks at desired locations in the genome (Nat Rev Genet, 2014.15 (5): pages 321-34). Engineered nucleases comprising DNA binding and nuclease domains tailored for type II restriction enzymes have a broad spectrum of genomic engineering applications in the biotechnology and medical fields as well as in various other industries. More recently, a more efficient RGEN platform was developed based on the CRISPR/CAS9 bacterial adaptive immune system.

The sequence targeted by RGEN is limited to a Protospacer Adjacent Motif (PAM), which is the DNA sequence immediately following the DNA sequence targeted by Cas9 nuclease. PAM sequences were not previously reprogrammable in the CRISPR bacterial adaptive immune system. The canonical PAM contains the sequence 5'-NGG-3' and is linked to RGEN derived from the CAS9 nuclease from Streptococcus pyogenes. Therefore, the GG motif is a prerequisite for DNA recognition by RGENs. In order to amplify the sequences used as PAM, attempts have been made to isolate RGENs from different bacterial species with universal PAM. In fact, it has been found that different PAMs correlate with the CAS9 protein of Streptococcus thermophilus (PAM: NNAGAAW) and Neisseria meningitidis (PAM: NNNNGATT), widening the selection range for determining RGEN target sites.

Disclosure of Invention

Technical problem

As described herein, intensive and thorough research into the development of RGENs from bacteria other than Streptococcus pyogenes (Streptococcus pyogenes) has led to the discovery that Cas proteins derived from Campylobacter jejuni (c.jejuni) specifically recognize nnnrryac sequences, which can be used as PAMs in targeting target DNA. In addition, guide RNAs can be engineered to optimize DNA, resulting in efficient genome editing, transcriptional regulation, and isolation of the DNA of interest.

Technical solution

Thus, in one aspect, the invention provides a method for targeting a polypeptide comprising SEQ ID NO:1, the method comprising converting a DNA sequence that recognizes the PAM sequence of SEQ ID NO:1, or a nucleic acid encoding the Cas protein.

In another aspect, the invention provides an isolated guide RNA comprising a sequence capable of forming a duplex (forming base pairs 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 another aspect, the disclosure provided herein 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 sequence of NNNNRYAC (SEQ ID NO:1), or a DNA encoding a guide RNA, and (ii) a Cas protein, or a nucleic acid encoding a Cas protein, that recognizes the nnryac sequence (SEQ ID NO: 1).

In another aspect, the disclosure provided herein 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 that recognizes the PAM sequence nnryac (SEQ ID NO: 1).

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

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

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

In another aspect, the present disclosure provides a method of genome editing in a cell, comprising introducing into the cell an isolated guide RNA or DNA encoding an isolated guide RNA, and a Cas protein or a nucleic acid encoding a Cas protein.

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

In another aspect, the present disclosure provides a method of preparing a target DNA recognition sequence for a guide RNA, comprising: (i) identifying the presence of the 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 recognizable by the guide RNA if the presence of the PAM sequence is identified in step (i).

In another aspect, the present disclosure provides a method of isolating a target DNA, comprising: (i) introducing a guide RNA or DNA encoding a guide RNA together with an inactivated Cas protein or a nucleic acid encoding an inactivated Cas protein into a cell to allow the guide RNA and the inactivated Cas protein to form a complex with a target DNA comprising a target DNA sequence; and (ii) isolating the complex from the sample.

In another aspect, the present disclosure provides a method for Cas-mediated regulation of gene expression in a target DNA comprising a target DNA sequence, comprising introducing into a cell an isolated guide RNA or DNA encoding a guide RNA that specifically recognizes the target DNA sequence, and an inactive Cas protein or a nucleic acid encoding an inactive Cas protein fused to a transcription effector (transcription effector) domain.

Advantageous effects

As described above, in some embodiments, the CRISPR/Cas system can be effectively used to target DNA, thereby enabling genome editing, transcriptional regulation, and isolation of target DNA.

Drawings

Fig. 1 depicts a schematic of a campylobacter jejuni Cas9 expression vector. The vector was designed such that the humanized Cas9 protein was expressed under the control of the CMV promoter and had a Nuclear Localization Signal (NLS) and an HA tag in the C-terminal region.

FIGS. 2A and 2B depict experiments on Campylobacter jejuni RGEN-induced mutations at endogenous human AAVS1 target sites. FIG. 2A shows the detection of RGEN-driven chromosomal mutations using the T7E1 assay. Asterisks indicate DNA bands expected to be cut by T7E 1. HEK293 wild type (wt) gDNA was used as a negative control (-). Previously confirmed RGEN was used as positive control (+). FIG. 2B shows the DNA sequence of the hAAVS1 mutant clone. The regions of the target sequence complementary to the chimeric RNA are shown in bold. The PAM sequence identified by CAS9 is underlined. The WT sequence of fig. 2B consists of SEQ ID NO: 4, (-2, x1) sequence represented by SEQ ID NO: 5, (-1, x1) sequence represented by SEQ ID NO: and 6, representation.

FIGS. 3A and 3B show experiments with mutations induced by Campylobacter jejuni RGEN in the endogenous mouse ROSA26(mROSA) target site. FIG. 3A shows the detection of RGEN-driven chromosomal mutations using the T7E1 assay. Asterisks indicate DNA bands expected to be cut by T7E 1. NIH3T3wt gDNA was used as a negative control (-). Previously confirmed RGEN was used as positive control (+). FIG. 3B shows the DNA sequence of a clone of a mutant of mROSA. The regions of the target sequence complementary to the chimeric RNA are shown in bold. The PAM sequence recognized by campylobacter jejuni CAS9 is underlined. The WT sequence of fig. 3B consists of SEQ ID NO: 7, (-1, x1) sequence represented by SEQ ID NO: 8, and the (+1, x1) sequence is represented by SEQ ID NO: and 9, the specification.

Figure 4 shows certain mutations induced in the endogenous AAVS1 target site by the mutant campylobacter jejuni sgRNA structure. The T7E1 assay was used to detect RGEN-driven chromosomal mutations. Asterisks indicate DNA bands expected to be cut by T7E 1. HEK293wt gDNA was used as negative control (-). Previously confirmed RGEN was used as positive control (+).

Fig. 5A to 5C illustrate optimization of spacer length of sgrnas. Fig. 5A shows various sgRNA structures. The additional nucleotides immediately upstream of the 5' end of the spacer sequence of the sgRNA are underlined, with lower case letters indicating mismatched nucleotides with respect to the target sequence. And adding frames to the PAM sequence. In fig. 5A, the target sequence consists of SEQ ID NO: 10, GX19 is represented by SEQ ID NO: 11, GX20 is represented by SEQ ID NO: 12, GX21 is represented by SEQ ID NO: 13, GX22 consisting of SEQ ID NO: 14, GX23 is represented by SEQ ID NO: 15, GGX20 is represented by SEQ ID NO: 16, GGGX20 is represented by SEQ ID NO: and 17, are shown. FIG. 5B shows the target site for sgRNA in which the sequences of hAVS-CJ 1, hAVS-NRG 1, hAVS-NRG 3 and hAVS-NRG 5 are encoded by SEQ ID NO: 18. 19, 20 and 21. Fig. 5C shows the efficiency of the sgRNA construct to induce RGEN-mediated mutations. Briefly, sgrnas were constructed to have spacer sequences of different lengths (19-23bp) and different numbers of additional G (guanine) residues present immediately upstream of the spacer sequence. Each sgRNA 5A shown in fig. 1 was designed for 4 target sites of the human AAVS1 site (fig. 5B) and delivered to human 293 cells. Subsequently, mutations induced by NHEJ were identified in the cells. In this embodiment, the target site is amplified by PCR and analyzed by deep sequencing using miseq (illumine) to detect the mutation. In general, the frequency of genome editing (mutation) is increased when the recognition sequence is 21-23bp in length or when 2 or 3 additional G residues are provided at the 5' end compared to GX19 or GX20 used in campylobacter jejuni or other species.

Fig. 6 is a graph showing the activity of campylobacter jejuni CRISPR/CAS9, in which AAVS1-CJ1 site was inserted into alternative reporter (reporter). Relative to the activity of the ACAC sequence detected at the PAM site (100), the activity was calculated when a different nucleotide was substituted at each position. In the first position, G and A ensure high activity. T and C are valid in the second position. However, only a and C showed activity at the third and fourth positions, respectively. Thus, at least in some embodiments, NNNN-A/G-C/T-C-A (or NNNNRYAC, SEQ ID NO:1, where A/G ═ R, C/T ═ Y) is inferred to be the optimal PAM sequence.

FIG. 7 shows the consensus markers of potential off-target sequences of hAAVS1-CJ1sgRNA developed by Digenome-Seq analysis.

Fig. 8 shows the test results of the PAM sequence of campylobacter jejuni Cas 9. The seven target sites of NNNNRYAC (SEQ ID NO:1) were analyzed for mutation efficiency. hAVS 1-RYN 1-7: mutation rate per site in sgRNA/Cas9 treated cells, WT 1-7: the mutation rate at each site in the genomic DNA of the treated cells was simulated.

Fig. 9 is a schematic diagram showing the structure of a campylobacter jejuni CRISPR/CAS9 expression AAV vector.

Fig. 10 shows genome editing by campylobacter jejuni CRISPR/CAS9AAV (adeno-associated virus) in Rosa26 site. Briefly, C2C12 cells were infected with recombinant AAV vectors carrying Rosa26-sgRNA and campylobacter jejuni Cas9 at different MOIs (multiplicity of infection). At 3, 5, 7, 10 and 14 days post infection, genomic DNA was isolated and mutation rates were analyzed by deep sequencing.

Detailed Description

One embodiment of the invention provides a method of targeting a target DNA sequence comprising introducing a Cas protein or a nucleic acid encoding it into a cell.

Specifically, according to one aspect, the present disclosure provides a method for targeting a polypeptide comprising SEQ ID NO:1, comprising a DNA sequence that recognizes the PAM (protomer spacer adjacent motif) sequence of SEQ ID NO:1 or a nucleic acid encoding the Cas protein of the PAM sequence NNNNRYAC. In SEQ ID NO:1, "N" refers to any nucleotide, e.g. selected from A, C, G and T; "R" refers to purine (A/G); "Y" refers to pyrimidine (C/T).

In one aspect of the disclosure, the method may further comprise introducing a nucleic acid comprising a sequence capable of hybridizing to a sequence adjacent to SEQ ID NO:1 (target DNA) of a sequence in which complementary strands of target DNA (target DNA) of the PAM sequence form a duplex: the guide RNA can hybridize to a sequence that recognizes SEQ ID NO:1 or a nucleic acid encoding the Cas protein, simultaneously or sequentially.

As used herein, the term "targeting" is intended to include binding of the Cas protein to the target DNA sequence, with or without DNA cleavage.

Terms to be described later are applicable to all embodiments of the present disclosure, and may be used in combination.

The Cas protein may exhibit its activity after forming a complex with CRISPR RNA (crRNA) and trans-activated 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 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 a protein derived from (derived from) campylobacter, and may in particular be derived from campylobacter jejuni. More specifically, the Cas9 protein may be derived from campylobacter jejuni. In some embodiments of the disclosure, the Cas protein may comprise a protein consisting of SEQ ID NO:22, or may be identical to the amino acid sequence represented by SEQ ID NO:22, and retains its intrinsic activity. For example, but not limited to, Cas proteins and their homologous sequences encompassed by the present disclosure may be homologous to SEQ ID NOs: 22 has 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% sequence identity.

Furthermore, Cas proteins as used in certain embodiments of the present disclosure are intended to include any variant of an endonuclease or nickase that can act as an activator in cooperation with a guide RNA as well as a native protein. The activated endonuclease or nickase can cleave the target DNA, or can use the cleavage function for genome editing. For inactive variants, their function may be used to regulate transcription or to isolate the target DNA.

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

In particular, a Cas protein, such as the catalytic aspartic acid (D) at position 8 or the histidine residue (H) at position 559 of a Cas9 protein derived from campylobacter jejuni, may be substituted with an amino acid different from the wild-type amino acid sequence. In some embodiments, the catalytic aspartic acid at position 8 (D) or the histidine residue at position 559 (H) of the sequence of SEQ ID No.22 is substituted with a different amino acid. For example, the different amino acid may be, but is not limited to, alanine. The Cas9 nuclease variant, prepared by introducing mutations into one active site of the native Cas9 nuclease, can act as a nickase that binds to guide RNA. When bound to one guide RNA molecule, two nickase molecules can cleave both strands of the target DNA duplex, thereby creating a Double Strand Break (DSB). Accordingly, such variants are also within the scope of RGENs encompassed by the present disclosure.

As used herein, the term "inactivated Cas protein" refers to a Cas nuclease, the function of which is completely or partially inactivated. An inactivated Cas protein may be abbreviated dCas. The Cas may be a Cas9 protein. Furthermore, it may originate from Campylobacter, in particular from Campylobacter jejuni. Any method can be used to prepare an inactivated Cas9 nuclease, as long as it eliminates nuclease activity. For example, dCAS9 protein can be constructed by introducing mutations into the two above-mentioned active sites of Cas9 nuclease. dCAS9 can then act as a DNA binding complex with the guide DNA, lacking DNA cleavage function. In addition, the dCAS9 protein may have substituents other than aspartic acid (D) at position 8 and histidine (H) at position 559. For example, in some embodiments, the dCAS9 protein may have an amino acid sequence other than SEQ ID NO:22 sequence except aspartic acid (D) at position 8 and histidine (H) at position 559. The substituent may be, but is not limited to, alanine.

As used herein, the term "cleavage" refers to the breaking of the covalent backbone of a nucleotide molecule.

In some embodiments of the disclosure, the Cas protein may be a recombinant protein.

The term "recombinant" as used in connection with, for example, a cell, nucleic acid, protein or vector, refers to a cell, nucleic acid, protein or vector modified by the introduction of a heterologous nucleic acid or protein or by alteration of a native nucleic acid or protein, or derived from a cell so modified. Thus, for example, a recombinant Cas protein can be produced by reconstructing a nucleic acid sequence encoding the Cas protein (i.e., a sequence encoding the Cas protein) based on the human codon table.

In some embodiments of the disclosure, the Cas protein or the nucleic acid encoding it may be in a form that allows activity within the nucleus.

In some embodiments of the disclosure, the isolated Cas protein may be in a form that is readily introduced into a cell. For example, the Cas protein may be linked to a cell penetrating peptide or protein transduction domain. The protein transduction domain may be, but is not limited to, polyarginine or HIV-derived TAT protein. The present disclosure includes various examples of cell penetrating peptide or protein transduction domains well known in the art.

In some embodiments of the 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 the nucleus of the cell by nuclear transport. In addition, the nucleic acid encoding the Cas protein may further comprise a Nuclear Localization Signal (NLS) sequence. Thus, the nucleic acid encoding the Cas protein may be present as a component of an expression cassette, which may include, but is not limited to, an NLS sequence, as well as regulatory elements, such as a promoter.

In some embodiments of the disclosure, the Cas protein may be linked to a tag that facilitates isolation and/or purification. As non-limiting examples, depending on the purpose, small peptide tags such as His-tag, Flag-tag, S-tag, etc., glutathione S-transferase (GST) tag or Maltose Binding Protein (MBP) tag may be used.

In some embodiments of the disclosure, when the Cas protein is associated with a target DNA-specific guide RNA, the Cas protein may be collectively referred to as RGEN (RNA-guided engineered nuclease). As used herein, the term "RGEN" refers to a nuclease with a target DNA-specific guide RNA and a Cas protein.

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

In one aspect of the disclosure, an RGEN for delivery to a cell includes 1) a target-DNA-specific guide RNA and an isolated Cas protein, or 2) a guide-RNA-encoding DNA or Cas-protein-encoding nucleic acid. Delivery in the form of 1) is designated as "RNP delivery".

Examples of isolated guide RNAs may include, but are not limited to, in vitro transcribed RNAs.

In some embodiments of the present disclosure, guide RNA-encoding DNA (guide RNA-encoding DNA) and the Cas protein-encoding nucleic acid itself can be used as the isolated nucleic acid. Alternatively, but not limited to, they may be present in a vector having expression cassettes for expression of the guide RNA and/or Cas protein.

Examples of suitable vectors include viral vectors, plasmid vectors, and agrobacterium vectors. The viral vector may be exemplified by, but not limited to, AAV (adeno-associated virus).

In some embodiments of the present disclosure, without limitation, the guide RNA-encoding DNA and Cas protein-encoding nucleic acid may be present separately in each vector or together in a single vector.

The foregoing application embodiments of the present subject matter can be applied to more exemplary embodiments as described in this specification. In addition, application embodiments to be described later may be applied in combination with other constituent elements.

As used herein, the term "guide RNA" can refer to an RNA specific for target DNA (i.e., a target-DNA specific RNA) that can be coupled to a Cas protein to guide the Cas protein to the target DNA.

Furthermore, at least in some embodiments, the guide RNA can 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 double RNA composed of two RNAs, i.e., a crRNA and a tracrRNA. In other embodiments, the guide RNA can be a sgRNA that comprises or consists of a first region comprising a sequence complementary to a target DNA capable of forming a duplex with a complementary strand of the target DNA and a second region comprising a sequence responsible for interaction with the Cas protein. More specifically, the guide RNA may be sgRNA (single guide RNA or single-stranded guide RNA) synthesized by fusing each necessary part of crRNA and tracrRNA.

In some embodiments of the present disclosure, the sequence capable of forming a duplex with the complementary strand of the target DNA sequence in the guide RNA may be not limited in length to 17 to 23bp, 18 to 23bp, 19 to 23bp, particularly 20 to 23bp, more particularly 21 to 23 bp. The length can be applied to both double RNAs and sgrnas, more specifically to sgrnas.

In some embodiments of the present disclosure, the guide RNA may comprise one to three, more particularly two or three additional nucleotide sequences prior to the 5' end of the sequence capable of forming a duplex with the complementary strand of the target DNA. The nucleotide is selected from A, T, G, C and combinations thereof. The guide RNA may comprise one to three consecutive guanine (G) residues, more preferably, two or three consecutive G residues as additional nucleotides. This is not limited to application to double RNAs and sgrnas, and more preferably to sgrnas.

In some embodiments of the disclosure, the sgRNA can comprise a region complementary to the target DNA sequence (referred to as a "spacer sequence", "target DNA recognition sequence", "base-pairing region", etc.) and a hairpin structure for binding the Cas protein.

In some embodiments of the disclosure, the sgRNA can comprise a region complementary to the target DNA sequence for binding to the hairpin structure of the Cas protein and the terminator sequence. These elements may be, but are not limited to being, arranged sequentially in the 5 'to 3' direction.

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

In some embodiments of the disclosure, the crRNA may hybridize to the target DNA.

In some embodiments of the disclosure, the RGEN may consist of a Cas protein and a double RNA, or a Cas protein and a sgRNA. Alternatively, RGEN may comprise the respective nucleic acid encoding the Cas protein and sgRNA as constituent elements, but is not limited thereto.

In some embodiments of the disclosure, the guide RNA (e.g., crRNA or sgRNA) may contain a sequence complementary to the target DNA sequence, and may comprise one or more additional nucleotides upstream of the crRNA or sgRNA, particularly at the 5' end of the crRNA of the sgRNA or dirna. The additional nucleotide may be, but is not limited to, a guanine (G) residue.

In some embodiments of the present disclosure, the guide RNA may comprise a sequence capable of forming a duplex (i.e., forming base pairs or hybridizing) with a complementary strand of a target DNA sequence adjacent to the PAM (protospacer 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 the complementary framework of the target DNA sequence and a second region comprising a stem-loop structure characterized by a stem of 13-18bp in length. In certain embodiments, the stem may comprise SEQ ID NO: 2(5'-GUUUUAGUCCCUUGUG-3') and the complement thereof.

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

In some embodiments of the disclosure, the Cas protein and the guide RNA, particularly the sgrnas, described above or later, may be non-naturally occurring or engineered. In addition, the factors described for each subject may be combined together for application.

In some embodiments of the disclosure, intracellular introduction of RGENs can be achieved by, but is not limited to, (1) delivery of Cas9 protein purified after bacterial overexpression and sgrnas (single-guide RNAs) that recognize specific HLA target sequences prepared after in vitro transcription in cells, or (2) delivery of plasmids carrying Cas9 gene and sgrnas into cells for expression or transcription.

In addition, proteins, RNA or plasmid DNA encompassed within the scope of the present disclosure may be introduced into cells by various methods known in the art, such as, but not limited to, electroporation or techniques using liposomes, viral vectors, nanoparticles or PTD (protein transport domain) fusion proteins.

In some embodiments, the methods of the present disclosure can be used to cleave a nucleic acid comprising SEQ ID NO:1, and more particularly, for editing a genome. Herein, the Cas protein may be in an active form with nuclease or nickase activity.

In certain embodiments, the Cas protein may be in an inactivated (inactivated) form. In this case, the methods of the present disclosure are performed with a nucleic acid comprising SEQ ID NO:1 is not cleaved but proceeds in a manner associated with the Cas protein.

Moreover, in some other embodiments, the Cas protein, more particularly, the inactivated Cas protein, may further comprise a transcriptional effector domain. In detail, the inactivated Cas protein may be linked to (but not limited to) an activator, repressor, etc.

Given a transcriptional effector domain, the methods can be applied, at least in some embodiments, to Cas-mediated regulation of gene expression comprising transcriptional regulation or epigenetic regulation.

According to 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 a PAM (protospacer adjacent motif) NNNNRYAC (SEQ ID NO: 1). The isolated guide RNA may be a non-naturally occurring or artificially engineered RNA.

The individual elements are as described above.

In some embodiments of the present disclosure, the guide RNA may be a single guide RNA, wherein the length of the sequence capable of forming a duplex with the complementary strand of the target DNA may be 17 to 23bp, 18 to 23bp, 19 to 23bp, particularly 20 to 23bp, more particularly 21 to 23bp, but is not limited thereto.

Furthermore, the guide RNA, at least in some embodiments, can 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 so limited. In addition, the above description of additional nucleotides may also apply to this embodiment.

Further, according to another aspect of the present disclosure, there is provided 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 a PAM (protospacer adjacent motif) sequence NNNNRYAC (SEQ ID NO:1), or a DNA encoding the guide RNA.

In at least some embodiments, the components are as described above.

In some embodiments of the disclosure, the composition can further comprise a Cas protein or a nucleic acid encoding a Cas protein that recognizes the sequence NNRYAC (SEQ ID NO: 1).

Furthermore, in certain embodiments, the compositions are useful 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 adjacent to a target DNA sequence of PAM (protospacer adjacent motif) NNRYAC (SEQ ID NO:1) or a DNA encoding a guide RNA; and (ii) inactivating the Cas protein (dCas) or a nucleic acid encoding dCas.

In one embodiment, the inactivated Cas protein may further comprise a transcription effector domain.

In some embodiments of the present disclosure, the compositions can be used to isolate a DNA of interest comprising a target DNA sequence. In this regard, the inactivated Cas protein may be labeled with a tag that can be used for isolation and purification, but is not limited thereto. The label may be as described above.

In some embodiments of the present disclosure, the compositions are useful for Cas-mediated gene expression regulation, including transcriptional regulation or epigenetic regulation.

In some embodiments of the present disclosure, the target DNA may be present in an isolated cell, such as a eukaryotic cell. Examples of eukaryotic cells include yeast, 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), transplanted cells, primary cell cultures (in vitro and ex vivo), in vivo cells, and mammalian cells including human cells are commonly used in the art.

According to another 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 a PAM (protospacer-proximity motif) NNNNRYAC (SEQ ID NO:1), or a DNA encoding the guide RNA; and (ii) a Cas protein or nucleic acid encoding a Cas protein that recognizes the PAM sequence NNRYAC (SEQ ID NO: 1).

The respective factors are as described above. These factors may be non-naturally occurring or engineered.

Another aspect of the present disclosure relates 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 a PAM (protospacer-adjacent motif) NNNNRYAC (SEQ ID NO:1), and (ii) an expression cassette for a Cas protein that recognizes the PAM sequence of nnryac (SEQ ID NO: 1).

The respective factors are as described above. These factors may be non-naturally occurring or engineered.

The viral vector, at least in some embodiments, can be of AAV (adeno-associated virus) origin.

Another aspect of the disclosure relates to an isolated guide RNA comprising a sequence of 21-23bp in length, which is 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.

Another aspect of the present disclosure relates to a composition comprising a guide RNA or DNA encoding a guide RNA.

The respective factors are as described above. These factors may be non-naturally occurring or engineered.

Compositions, at least in some embodiments, can comprise a Cas protein or a nucleic acid encoding a Cas protein that recognizes the PAM sequence NNNNRYAC (SEQ ID NO: 1).

Furthermore, in some embodiments, the compositions can comprise an inactive Cas or a nucleic acid encoding an inactive Cas protein that recognizes the NNRYAC sequence (SEQ ID NO: 1).

In some embodiments, the inactivated Cas protein may further comprise a transcriptional effector domain.

According to another 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 of 13-18bp in length.

The respective factors are as defined above. These factors may be non-naturally occurring or engineered.

In certain embodiments, the stem may comprise SEQ ID NO: 2(5'-GUUUUAGUCCCUUGUG-3') and the complement thereof.

According to a further 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 of 5-10bp in length.

The respective factors are as defined above. These factors may be non-naturally occurring or engineered.

In certain embodiments, the loop may comprise SEQ ID NO: 3(5 '-AUAUUCAA-3').

According to another aspect, the present disclosure provides a composition comprising a guide RNA and a Cas protein or a nucleic acid encoding a Cas protein.

The respective factors are as defined above. These factors may be non-naturally occurring or engineered.

Yet 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 DNA encoding an isolated guide RNA and a Cas protein or a nucleic acid encoding a Cas protein.

The respective factors are as defined above. These factors may be non-naturally occurring or engineered.

Another aspect of the disclosure provides a method for cleaving a target DNA in a cell, comprising introducing into the cell an isolated guide RNA or DNA encoding an isolated guide RNA together with a Cas protein or a nucleic acid encoding a Cas protein.

The respective factors are as defined above. These factors may be non-naturally occurring or engineered.

In certain embodiments, the guide RNA or DNA encoding the guide RNA can be introduced into the cell simultaneously or sequentially with the Cas protein or nucleic acid encoding the Cas protein.

Yet another aspect of the present disclosure provides a method for preparing a target DNA recognition sequence of a guide RNA (i.e., a sequence in a guide RNA responsible for recognizing a target DNA), comprising: (i) identifying the presence of the PAM sequence NNNNRYAC (SEQ ID NO:1) in a given sequence; and (ii) determining a sequence located just upstream of the PAM sequence NNRYAC (SEQ ID NO:1) as recognizable by the guide RNA if the presence of the PAM sequence is identified in step (i).

The respective factors are as defined above. These factors may be non-naturally occurring or engineered.

In some embodiments of the present disclosure, the sequence located upstream of the PAM sequence may be, but is not limited to, within a length range from 17 to 23bp, from 18 to 23bp, from 19 to 23bp, more particularly from 20 to 23bp, even more particularly 21 to 23 bp.

Another aspect of the present disclosure provides a method of isolating a target DNA, comprising: (i) introducing a guide RNA or DNA encoding a guide RNA into a cell together with an inactivated Cas protein or a nucleic acid encoding an inactivated Cas protein, thereby allowing the guide RNA and the inactivated Cas protein to form a complex with a target DNA comprising a target DNA sequence; and (ii) isolating the complex from the sample.

The respective factors are as defined above. These factors may be non-naturally occurring or engineered.

In at least some embodiments, an inactivated Cas protein can recognize the PAM (protomer spacer adjacent motif) sequence NNRYAC (SEQ ID NO: 1).

In certain embodiments, the methods for isolating a target DNA can form a dCas-gRNA-target DNA complex by allowing a guide rna (gRNA) that specifically binds the target DNA and an inactivated Cas protein (dCas) and the target DNA; and separating the complex from the sample.

In some embodiments, the target DNA may be identified using well known detection methods such as PCR amplification and the like.

In some embodiments, the isolation method may be suitable for in vitro cell-free DNA that does not form cross-links through covalent bonds between the DNA, gRNA, and dCas.

Furthermore, in some embodiments, the isolation method may further comprise isolating the target DNA from the complex.

In some embodiments, the inactivated Cas protein may be linked to an affinity tag for use in isolating the target DNA. 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 polyglutamic acid tag, an E-tag, an HA-tag, a myc-tag, an SBP-tag, softag 1, softag 3, strep-tag, a TC-tag, an Xpress-tag, a BCCP (biotin carboxyl carrier protein) tag, and a GFP (green fluorescent protein) tag, but is not limited thereto.

In some embodiments, the inactivated Cas protein may be a Cas protein that lacks DNA cleavage activity.

In some embodiments, the separation of target DNA may be achieved using affinity columns or magnetic beads that are capable of binding the label used. For example, when a His-tag is used for isolating target DNA, a metal affinity column or magnetic beads capable of binding the His-tag may be used. The magnetic beads may include, but are not limited to, Ni-NTA magnetic beads.

In some embodiments, ribonuclease and protease can be used to separate the target DNA from the complex.

In some embodiments of the methods for isolating target DNA, a certain genotype DNA or two or more different target DNAs may be isolated from an isolated sample containing a mixture of two or more different genotype DNAs. When the method comprises isolating two or more different target DNAs, the two or more target DNAs may be isolated using guide RNAs specific to the two or more different target DNAs, respectively.

In certain embodiments, the guide RNA may be a single guide RNA (sgrna), or a double RNA comprising crRNA and tracrRNA. The guide RNA may be an isolated RNA, or may be encoded in a plasmid.

In certain embodiments, the separation method may be performed by: specifically binding a guide rna (gRNA) to 1) a target DNA and 2) an inactivated Cas protein (dCas) to form a dCas-gRNA-DNA complex with the target DNA; and separating the complex from the sample.

Another aspect of the present disclosure provides a method for Cas-mediated regulation of gene expression in a target DNA comprising a target DNA sequence, the method comprising: an isolated guide RNA or DNA encoding a guide RNA that specifically recognizes the target DNA is introduced into the cell along with an inactivated Cas protein or a nucleic acid encoding an inactivated Cas protein fused to a transcription effector domain.

The respective factors are as defined above. These factors may be non-naturally occurring or engineered.

Examples

The following examples are provided to illustrate some aspects of the present disclosure, and they should not be construed as limiting the scope of the present disclosure in any way.

Campylobacter jejuni CRISPR/CAS9 system

Example 1: genome editing using campylobacter jejuni CRISPR/CAS9

The present inventors have successfully isolated RGEN from Campylobacter jejuni. To identify features of campylobacter jejuni CRISPR/CAS 9-derived RGENs for genome editing, a campylobacter jejuni CAS9 gene optimized for human codons was synthesized (table 1) and then inserted into a mammalian expression vector to construct a campylobacter jejuni CAS9 expression cassette in which the HA-labeled NLS-linked CAS gene is under the regulation of the CMV promoter (fig. 1).

TABLE 1 amino acid sequence of Campylobacter jejuni Cas9 protein

The natural guide RNA of the campylobacter jejuni CRISPR/CAS9 system consists of tracrRNA and target-specific crRNA. Considering the concept that the guide RNA itself is used as two RNA molecules 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 campylobacter jejuni sgRNA (c.jejuni sgRNA) (table 2).

TABLE 2

Potential target sites for human AAVS1 and mouse Rosa-26 were then selected based on the PAM sequence of the campylobacter jejuni CRISPR/CAS9 system (NNNACA) (table 3).

TABLE 3

sgRNAs	Target sequence	Sequence ID number
			Human AAVS1_ c	ATATAAGGTGGTCCCAGCTCGGGGACA	24
Mouse Rosa26_ c. jejuni	ATTCCCCTGCAGGACAACGCCCACACA	25

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

TABLE 4

Primer and method for producing the same	Sequence of	Sequence ID number
			Human AAVS1-F	TGCTTCTCCTCTTGGGAAGT	26
Human AAVS1-R	CCCCGTTCTCCTGTGGATTC	27
			Mouse Rosa26-F	ACGTTTCCGACTTGAGTTGC	28
Mouse Rosa26-R	CCCAGCTACAGCCTCGATTT	29

As a result, a mutation (interchangeably, substitution or variation) was detected only in the cells into which the CAS9 protein and guide RNA were introduced together. The mutation frequency was found to be RNA-dose dependent based on relative DNA band intensity measurements (fig. 2A). In addition, DNA sequencing analysis of the PCR amplification products confirmed that RGEN-mediated mutations were induced at the endogenous site. Insertions/deletions (indels) and micro-homologies (microhomologies) were observed at the target site, characterized by error-prone non-homologous end joining (NHEJ) repair (fig. 2B). The mutation frequency measured by direct sequencing (═ 2 mutant clones/12 clones) was 16.7%.

Likewise, when mouse Rosa26 campylobacter jejuni RGEN was delivered into mouse NHI3T3 cells, mutations were efficiently induced at mouse Rosa26 site as measured by T7E1 assay (fig. 3A). In addition, DNA sequencing analysis of the PCR amplification products revealed induction of Campylobacter jejuni RGEN-mediated mutations 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 sgrnas

Campylobacter jejuni crRNA: the tracrRNA complex will contain a shorter loop structure than loop structures from other bacterial species, and the modified stem or loop structure is designed to structurally stabilize the campylobacter jejuni RGEN sgRNA constructed in example 1 (table 5).

TABLE 5

In table 5, the standard stem portions are shown in bold and underlined.

Similar mutation frequencies were observed when a modified sgRNA was introduced to target the target site of human AAVS1 campylobacter jejuni RGEN, which successfully induced mutation by the normal sgRNA structure (fig. 4). In this regard, the primer sequences used are shown in Table 4.

Example 3: optimization of sgRNA gap length

It is reported in the literature that the spacer sequence of Campylobacter jejuni crRNA recognizing the target sequence is 20bp in length. To determine which spacer length is optimal, genome editing tests were performed on 4 target sites of campylobacter jejuni Cas9 at the human AAVS1 site using spacer sequences of various lengths and sgRNA mutant structures with additional nucleotides at the 5' end as shown in table 6 (fig. 5A to 5C). For the method used in this experiment, reference is made to Genome res.2014jan; 24(1): 132-41.

TABLE 6

Target site

sgRNA	Sequence (20 bp-SPACERnnnACA)	Sequence ID number
			Human AAVS1-CJ1	ATATAAGGTGGTCCCAGCTCggggACA	32
Human AAVS1-NRG1	GTAGAGGCGGCCACGACCTGgtgaACA	33
			Human AAVS1-NRG3	TCACAAAGGGAGTTTTCCACacggACA	34
Human AAVS1-NRG5	TAGGCAGATTCCTTATCTGGtgacACA	35

Three days after delivery of the sgRNA expression vector to 293-cells, genomic DNA was isolated and mutation efficiency was analyzed by deep sequencing. The results are shown in fig. 5C. It can be seen that high efficiency is detected when the length of the spacer sequence is 21-23 bp. Furthermore, even when 2-3 additional G residues were added at the 5' end of the sgRNA of the 20bp long spacer, an improvement in genome editing was observed.

TABLE 7

Here, F denotes a forward primer, and R denotes a reverse primer.

Example 4: campylobacter jejuni Cas9 PAM sequence analysis

In the present disclosure, based on data in the existing literature, the PAM sequence of campylobacter jejuni Cas9 was inferred to comprise "NNNNACA", and experiments were performed. For the 34 campylobacter jejuni CRISPR/CAS9 system constructed for five genomic loci, only three showed activity. In particular, additional analysis of the sequences covering sites in the three active systems showed that in all three sites, nucleotide "C" was identified immediately after the PAM sequence (NNNNACA) (table 8).

TABLE 8

Based on this result, it was concluded that the PAM sequence comprises "NNNNACAC". When the nucleotides at each site of "ACAC" were substituted with a/T/G/C, the activity of campylobacter jejuni Cas9 was analyzed to identify the PAM sequence of campylobacter jejuni RGEN. To this end, alternative reporter vectors are utilized. As a result, Campylobacter jejuni was identified as containing the PAM sequence of "NNRYAC (SEQ ID NO: 1)" (FIG. 6, wherein R is a purine residue (A or G) and Y is a pyrimidine residue (C/T)). Nat methods.2011oct 9 was used for this experiment; 8(11) 941-3.

Example 5: determination of specificity and PAM sequence of Campylobacter jejuni CRISPR/CAS9

The cleavage site of Campylobacter jejuni CRISPR/CAS9 in the AAVS1-CJ1 site was analyzed at the genome level using digomere-seq (the CRISPR/CAS9 off-target assay developed and patented by the inventors). Nat methods.2015mar; 12(3) 237-43.

Through digomer-Seq, 41 sites where AAVS1-CJ1 CRISPR/CAS9 appeared to be cleaved were identified (genomic positions in table 9). The consensus sequence was obtained from an alignment of the cleavage site sequences of 41 sites and verified for PAM identity as identified in example 4.

Furthermore, to examine whether off-target mutations were actually introduced into potential off-targets obtained by Digenome-Seq, genomic DNA from 293-cells in which AAVS1-CJ1 CRISPR engineered nuclease was delivered was deep sequenced for 40 potential off-target sites. As shown in table 9, no significant mutations were observed.

TABLE 9

In addition, a consensus sequence was obtained from the entire alignment of the sequences of the 41 sites showing cleavage in vitro. Consistent with previous results, PAM was actually observed as NNRYAC (SEQ ID NO: 1).

Example 6: degradation of the first two nucleotides of PAM

The PAM sequences found in example 5 for campylobacter jejuni were "NNNNRYAC" and "NNNNACAC", showing degeneration at the first two positions. To confirm the degeneracy, sgrnas of 7 PAM target sequences of campylobacter jejuni at human AAVS1 site, which carried G or T residues at the first two positions (table 10), were constructed, respectively, and the mutation efficiency in HEK293 cells was analyzed.

Watch 10

Of the seven constructed sgrnas, six induced mutations were found, demonstrating degeneration at the first two positions of the PAM sequence (fig. 8). Thus, this degeneration increases the frequency of the PAM sequence, allowing for improved accuracy of genome editing of campylobacter jejuni.

Example 7: genome editing by campylobacter jejuni CRISPR/CAS9 delivery using AAV

Representative of promising areas in which genome editing can be applied are genome editing techniques for gene and cell therapy. Practical application of genome editing to therapy requires a clinically applicable vector for efficient delivery of engineered nucleases and donor DNA to target cells in vitro or in vivo. The two most widely used engineered nuclease platforms, TALENs and RGENs, are limited in their large size to application to established gene therapy vectors. In contrast, campylobacter jejuni RGENs of the present disclosure consist of the smallest CAS9 protein and sgRNA among the RGENs developed so far. Due to its small size, Campylobacter jejuni RGEN can allow large-scale gene therapy vectors to be used for genomic manipulations. For example, AAV (adeno-associated virus), one of the most important vectors for gene therapy, imposes severe restrictions on the size of DNA carried thereby, and thus is difficult to apply to RGENs derived from streptococcus pyogenes (s.pyogenenes), streptococcus thermophilus (s.thermophilus), or neisseria meningitidis (n.meningitidis), or the engineered nuclease platform TALENs currently used. In contrast, Campylobacter jejuni RGEN can be applied to AAV vectors.

In the present disclosure, the manipulation of campylobacter jejuni Cas9 was examined by actual AAV delivery. To this end, AAV vectors carrying the campylobacter jejuni Cas9 expression cassette and sgRNA expression cassette were constructed (fig. 9) and used for production of AAV. Mouse C2C12 cells were quantitatively analyzed for mutations following infection with AAV (fig. 10). It can be seen that mutations are induced at the target site in a dose and time dependent manner of AAV. In particular, mutations were induced at the target site with an efficiency of 90% or more after 4 weeks of high MOI (100) infection.

Thus, Campylobacter jejuni RGEN was demonstrated to efficiently perform genome editing in cultured cells. Furthermore, the PAM sequence of the campylobacter jejuni CRISPR/CAS9 system was actually determined because the sequences proposed in previous studies were found to be imperfect. Furthermore, campylobacter jejuni RGEN can be loaded into a single virus due to the small size of its elements and thus can be used for efficient genome editing.

Using dCAS 9: gRNA complex enriched target DNA

In addition, RGEN (dCas 9: gRNA complex) consisting of inactivated Cas9 protein and guide RNA derived from Streptococcus pyogenes was used to isolate and enrich target DNA.

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

RGEN (dCas 9: gRNA complex) consisting of guide RNA and inactivated Cas nuclease was tested for its ability to isolate target DNA. For this, first, plasmid pUC19 was digested with restriction enzymes (SpeI, XmaI, XhoI) to give plasmid DNA fragments of 4134bp, 2570bp and 1263bp in length, respectively.

For each plasmid DNA fragment digested with 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). Purification procedures were performed using sgrnas corresponding to the target DNA, alone or in combination (4134bp _ sg #1+2, 2570bp _ sg #1+2, and 1263bp _ sg #1+ 2). The nucleotide sequence of the sgrnas is listed in table 11 below.

TABLE 11

Except for U instead of T, the nucleotide sequence of sgRNA is identical to that of the target DNA.

A total of 200. mu.l contained DNA: dCas9 protein: a mixture solution of sgRNAs (molar ratio 1:20:100) was incubated at 37 ℃ for 1.5 hours. Then, the solution was mixed with 50. mu.l of Ni-NTA magnetic beads specifically binding to His tag, and washed twice with 200. mu.l of washing buffer, followed by purification of dCas 9-sgRNA-target DNA complex buffer with 200. mu.l of elution buffer (Bioneer, K-7200).

The eluate was then incubated with 0.2mg/ml ribonuclease A (Amresco, E866) for 2 hours at 37 ℃ and then 0.2mg/ml proteinase K for 45 minutes at 55 ℃ to remove sgRNA and dCas9 protein. The target DNA alone was precipitated in ethanol.

As a result, for individual target DNA, whether sgRNAs are used alone or in combination of two, the desired target DNA can be isolated from three DNA fragments digested by size. Furthermore, when multiple target DNAs are purified with a combination of sgrnas, for example, a total of 4 different sgrnas for two different target DNAs (2 sgrnas for each target DNA), the target DNA binds to the corresponding sgRNA and is thus purified. The results show that each target DNA can be isolated with a purity of 95% or more.

In addition, purification techniques are applicable to Cas proteins that recognize the PAM (protospacer 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 present invention may be employed in practicing the invention without departing from the technical idea or essential characteristics of the invention, which are defined in the following claims. In this regard, the above examples are for illustrative purposes only, and the present invention is not intended to be limited by these examples. The scope of the present invention should be understood to include all modifications or modified forms derived from the meaning and scope of the following claims or equivalent concepts.

Sequence listing

<110> Gene tools, Inc. (TOOLGEN INCORPORATED)

BASIC SCIENCE research INSTITUTE (INSTITUTE FOR BASIC SCIENCE)

<120> Gene editing Using an RNA-guided engineered nuclease derived from the CRISPR/CAS System of Campylobacter jejuni

<130> 20211076MY

<150> US 62/033,852

<151> 2014-08-06

<160> 88

<170> SIPOSequenceListing 1.0

<210> 1

<211> 8

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<220>