WO2018097657A1 - Genome sequencing method and genome editing identification method, which use chromatin dna - Google Patents

Abstract

The present invention relates to a method for detecting cleavage sites and/or off-target sites of a target-specific nuclease in genomic DNA and, particularly, treats chromatin protein-containing genomic DNA in vitro with a target-specific nuclease so as to cleave a genome, and then subjects the same to whole genome sequencing so as to identify off-target sites through data analysis.

Description

 【Specification】

 [Name of invention]

 Genome sequencing method using chromatin DNA and genome calibration confirmation method

 The present invention relates to a method for detecting the cleavage site and / or off-target site of a target specific nuclease in genomic DNA, specifically a genome comprising chromatin protein in Un in vitro. After processing the target specific nuclease to DNA, the genome is cleaved, and then whole genome sequencing is performed to identify non-target positions through data analysis.

[Background technology]

 The development of RNA-guided engineered nucleases (RGEN) has been used to correct genomes of a wide variety of animals and plants, including human cells. .

For example, ZFN (zinc finger nuclease), TALEN (transcript ional act ivator-1 ike effector nuclease), and type 2 CRISPR I Cas (clustered regularly interspaced repeat I CRISPR-associated) prokaryotic immune system derived RNA-guided Programmable nucleases, such as engineered nucleases, are widely used for genome editing of cultured cells and / or individuals. The genome correction technology using the genetic scissors is a very useful technology that can be used for various purposes in the life sciences, biotechnology and medical fields. For example, gene / cell therapy for various genetic or acquired diseases has been made possible by causing targeted genetic modifications in stem or somatic cells. However, the genetic scissors may cut not only an on-target site but also an off-target site having homology thereto, thereby causing unwanted mutations. "A typical example, 5. pyogenes-derived ^'Cas9 protein and sgRNA (small guide RNA) PAM ( protospacer-adjacent motif) that is RGEN is recognized by a 20-bp (base pair) sequence that is common and Cas9 torch and sgRNA consists of SEQ ID NO: It recognizes a 23-bp target DNA sequence consisting of phosphorus 5 'ᅳ 00-3' but can also work if some nucleotide sequences do not match. Furthermore, RGEN can also cleave non-target DNA sequences that have additional base sequences (DNA bulge) or one or more bases (RNA bulge) compared to sgRNA sequences. Similarly, ZFN and TALEN can also cleave sequences with some bases different. This suggests that applying genetic scissors to a genome may have a significant number of nontarget positions in addition to the target position.

 Nontarget DNA cleavage causes mutations in unwanted genes, such as proto-oncogenes and tumor suppressor genes, and / or translocations, deletions, and inversions. Genomic recombination, such as can be increased, is a serious problem in the use of genetic scissors in the field of research and medicine. Various strategies have been reported to reduce the non-target effects of these gene shears, but no genetic shears have been reported that operate specifically at the target site without bar targeting effects at the overall genome level. To address this problem, there is a need for the development of a technique that can confirm the specificity of genetic scissors at the genome level.

[Detailed Description of the Invention]

 [Technical problem]

 One example is

 (a) cleaving the isolated genomic DNA with a target specific nuclease; And

 (b) performing whole genome sequencing (WGS) on the cleaved DNA

 Including

 The isolated genomic DNA is chromatin containing chromatin protein

DNA,

 It provides a genomic DNA sequencing method.

 Another example is

 (a) cleaving the isolated genomic DNA with a target specific nuclease; And

 (b) performing whole genome sequencing on the cleaved DNA; And

(c) identifying the cleaved position in the sequence read obtained by the sequencing ^' ,

 The isolated genomic DNA is chromatin containing chromatin protein

DNA,

 Provided are methods for detecting the cleavage site or non-target site of a target specific nuclease.

 Another example provides a method of selecting a target site having a low non-target position using a method of detecting the cleavage position or the non-target position of the target specific nuclease. Technical Solution

 In the present specification, part of a method for identifying a non-target cleavage site of a target specific nuclease such as a programmable nuc lease (RNA-guided engineered nuc leases, RGEN) in the whole genome. To provide a technique for digested genome sequencing (Digenome—seq; a method of identifying before and after target-specific nuclease treatment at a glance to distinguish truncated positions). Recognizing that there is a limitation that does not consider the actual state of the chromatin in the cell because it proceeds to the DNA from which all the chromatin proteins have been removed, the truncated genome sequencing method using the chromatin DNA which preserved the chromatin structure to compensate for this limitation Suggest to come.

 For example,

 (a) cleaving the isolated genomi c DNA with a target specific nuclease; And

 (b) performing whole genome sequencing on the cleaved DNA

 Including

 The isolated genomic DNA is chromatin containing chromatin proteinol

DNA,

 A method of sequencing genomic DNA is provided.

 Another example is

 (a) cleaving the isolated genomi c DNA with a target specific nuclease;

(b) performing whole genome sequencing on the cleaved DNA; And (c) identifying the cleaved position in the sequence read obtained by the sequencing

 Including

 The isolated genomic DNA is chromatin DNA containing chromatin protein,

 Methods of detecting the cleavage site or off-target site of a target specific nuclease are provided.

 The method of detecting the off-target site may include, after step (c), if the identified truncated position is not an on-target site, the off-target site. The method may further include the step (d) of determining.

 Another example is

 (a) cleaving the isolated genomic DNA with a target specific nuclease;

 (b) performing whole genome sequencing on the cleaved DNA; And

 (c) identifying the cleaved position in the sequence read obtained by the sequencing

 Including,

 The isolated genomic DNA is a chromatin protein containing chromatin protein,

 Provided are methods for confirming the calibration efficiency and / or accuracy of target specific nucleases.

The method of confirming the calibration efficiency of the target specific nuclease is performed after the step (c), when the identified cleaved position is not an on-target site, the off-target site. Determining (d) step and the degree of cut at the non-target position (the number of non-target positions and / or the frequency of cut at the non-target position) and comparing it with the degree of cut of the comparison target (d— 1 ), In which case, the lower the degree of cleavage at the non-target position, the higher the calibration efficiency and / or accuracy may be determined-the comparison target is a target for a target sequence of any target DNA. In particular, it may be a specific nuclease, and may be any one selected from among commonly used or known target specific nucleases (eg, RGEN and guide RNA combinations). Digested genome sequencing (Digenome-seq) technology herein refers to nucleases. Herein to mean a sequence analysis of the genome by cleavage, nuclease non-target Jl of the entire genome of the cell and (f of target-ef fect) i and analysis; ^7! It can be applied to in vitro nuc lease-digested who l e-genome sequencing. Generate sequence reads with identical 5 ′ ends at the cleavage site of the nuclease, which can be identified by computer by an appropriate program (eg, Digenome program). In one example, cleavage genomic sequencing is a step in a genomic DNA sequencing method as described above or in a method of detecting a non-target site of nucleases) and (b), Or may comprise steps (a), (b), and (c). In other words, the steps (a) and (b), or (a), (b), and (c) may be performed by cleavage genome sequencing.

 Genomic correction and / or genetic correction techniques allow the introduction of target-directed mutations into the genome sequences of animal and plant cells, including human cells, by knocking out or knocking in a specific gene. And introducing a mutation into a non-coding DNA sequence that does not produce a protein. The method proposed herein is to detect a non-target position of a target specific nuclease used in the genome correction and / or genetic correction technique, which target specific nuclease system operates specifically at the target position. This can be useful to develop.

 The step (a) is a step of cleaving genomic DNA isolated from a living body or cell with a target specific nuclease, wherein the genomic DNA is isolated from ny / ro in vitro to specifically target a specific target. Cleavage with nuclease. The nucleases have the potential to cleave other sites, i.e., non-target positions, depending on the specificity, even if targeted specifically. Therefore, as a result, by the step (a), a genomic DNA fragment whose specific position is cleaved by cleaving a target position or a plurality of non-target positions, which are positions where the target specific nuclease used can be active with respect to the genomic DNA ( l ead).

The isolated genomic DNA may be isolated from non-transformed cells (wild-type cells) and / or cells expressing target specific nucleases or transformed to have nuclease activity, and may be isolated from target specific nucleases. Depending on the purpose to detect the non-target position can be used without limitation in its origin.

 The isolated genomic DNA is chromatin. It is characterized by including a DNA. In the present specification, chromatin DNA refers to one or more non-DNA chromatin components, such as histones, non-histone proteins, RNA, etc., that are not removed from a cell (or nucleus). It means DNA of the containing form. The chromatin DNA may be obtained by removing the cytoplasmic layer or taking a chromatin DNA layer from the cytoplasmic layer and chromatin DNA layer obtained by centrifugation after cell membrane removal (see FIG. 1). The cell membrane removal may be performed by treating a cell with a conventional lysis buffer, but is not limited thereto. The isolated genomic DNA described herein may be meant to exclude a state in which only DNA is separated from chromatin, for example, obtained by removing cytoplasm from cell lysate or by taking a chromatin DNA layer generated by centrifugation. It may be one containing chromatin DNA or chromatin DNA and cytoplasm.

 As used herein, target specific nucleases, also called progra 隱 able (nuclease), are any type of nuclease (eg, capable of recognizing and cleaving a specific position on the desired genomic DNA). , Endonuclease).

 For example, the target specific nuclease may be one or more selected from all nucleases that recognize a specific sequence of the target gene and have nucleotide cleavage activity that may result in insertion and / or deletion (Indel) in the target gene. Can be.

 For example, the target specific nuclease may be

 Transcription activator-like effector nuclease (TALEN) in which a TAL activator-like effector (TAL) activator domain and a cleavage domain are derived from a plant pathogenic gene, a domain that recognizes a specific target sequence on the genome;

 Zinc— finger nuclease;

 Meganucleases;

 RGEN (RNA-guided engineered nuclease; derived from the microbial immune system CRISPR; eg, Cas protein (eg, Cas9, etc.), Cpfl, etc.);

Ago homolog (Ago homo 1 og, DNA ᅳ guided endonuc lease) It may be one or more selected from the group consisting of, but is not limited thereto.

 The target specific nuclease may cause a double strand break (DSB) by recognizing specific sequences in the genomes of animal and plant cells (eg, eukaryotic cells) including prokaryotic cells and / or human cells. The double helix cutting may cut a double helix of 纖 to create a blunt end or a cohesive end. DSBs can be efficiently repaired by homologous recombination or non-homologous end-joining (NHEJ) mechanisms in cells, in which desired mutations can be introduced at target sites.

 Said meganucleases can be naturally-occurring meganucleases, including but not limited to, they recognize 15-40 base pair cleavage sites, which are generally classified into four families: the LAGLIDADG family, the GIY-YIG family, His-Cyst box family, and HNH family. Exemplary meganucleases include I-Scel, I-Ceul, PI-PspI, PI-See I, I—SceIV, I-Csml, I- Panl, I-SceII, I-Ppol, I— Scelll, I- Crel, I-Tevl, I-Tevl I and I-TevIII.

 Site-specific genomic modifications have been promoted in plants, yeasts, Drosophila, mammalian cells and mice using naturally-occurring meganucleases, primarily DNA binding domains derived from the LAGLIDADG family, but this approach has Modification of homologous genes in which the clease target sequence is conserved (Monet et al. (1999) Biochem. Biophysics. Res. Common.255: 88-93), which is limited to modification of the pre-engineered genome into which the target sequence is introduced. There was. Thus, attempts have been made to engineer meganucleases to exhibit novel binding specificities at medically and biotechnologically relevant sites. In addition, naturally-occurring or engineered DNA binding domains derived from meganucleases are operably linked to cleavage domains derived from heterologous nucleases (eg, Fokl).

The ZFN comprises a selected gene and a zinc-finger protein engineered to bind to the target site of the cleavage domain or cleavage half-domain. The ZFN may be an artificial restriction enzyme comprising a zinc-finger DNA binding domain and a DNA cleavage domain. Here, the zinc-finger DNA binding domain can be engineered to bind to the selected sequence. For example, Beerli et al. (2002) Nature Biotechnol. 20: 135—141; Pabo et al. (2001) Ann. Rev. Biochem. 70: 313—340; Isalan et al, (2001) Nature Biotechnol. 19: 656-660; Segal et al. (2001) Curr. Opin. Biotechnol. 12: 632—637; Choo et al. (2000) Curr. Opin. Struct. Biol. 10: 411-416 may be incorporated herein by reference. Compared to naturally occurring zinc finger proteins, engineered zinc finger binding domains can have novel binding specificities. Manipulation methods include, but are not limited to, rational design and various types of selection. Rational design includes, for example, the use of a database comprising triplet (or quadruple) nucleotide sequences, and individual zinc finger amino acid sequences, wherein each triplet or quadruple nucleotide sequence is associated with a zinc finger that binds to a particular triplet or quadruple sequence. Is associated with one or more sequences.

 Selection of target sequences, design and construction of fusion proteins (and polynucleotides encoding them) are known to those of skill in the art and are described in detail in the entirety of US Patent Application Publications 2005/0064474 and 2006/0188987, which are incorporated by reference. The entirety of the patent is incorporated herein by reference of the invention. In addition, as disclosed in these references and other references in the art, zinc finger domains and / or multi-finger zinc finger proteins are provided by linkers comprising any suitable linker sequence, eg, a linker of 5 or more amino acids in length. Can be linked together. Examples of linker sequences of six or more amino acids in length are described in US Pat. No. 6,479,626; 6,903,185; 7,153,949. The proteins described herein may comprise any combination of linkers that are appropriate between each zinc finger of the protein.

 In addition, nucleases, such as ZFNs, contain nuclease active moieties (cleavage domain cleavage half-domains). As noted, the cleavage domain can be heterologous to the DNA binding domain, such as, for example, a cleavage domain from a nuclease different from the zinc finger DNA binding domain. Heterologous cleavage domains can be obtained from any endonuclease or exonuclease. Exemplary endonucleases from which a cleavage domain can be derived include, but are not limited to, restriction endonucleases and meganucleases.

Similarly, cleaved half-domains can be derived from any nuclease or portion thereof that requires dimerization for cleavage activity, as shown above. If the fusion protein comprises a cleavage half-domain, two fusion proteins are generally required for cleavage. Alternatively, a single protein comprising two truncated half-domains may be used. Two cleaved hafnium domains are from the same endonuclease (or functional fragments thereof) Endonucleases that may be derived or that each cleaved half-domain is different

(Or functional fragments thereof). In addition, the target sites of the two fusion proteins are cleaved by the binding of the two fusion proteins and their respective target sites—the half domains are spatially oriented with respect to each other so that the cleavage half-domains are, for example, divalent. It is preferably arranged in a relationship that allows formation of a functional cleavage domain by sieving. Thus, in one embodiment, the neighboring edges of the target site are separated by 3-8 nucleotides or 14-18 nucleotides. However, the nucleotide or nucleotides at any integer pairs may be interposed between _{the two} target sites (e.g., 2 to 50 nucleotide pairs or more). In general, the cleavage site lies between the target sites.

 Restriction endonucleases (limiting enzymes) are present in many species and can sequence-specifically bind (at the target site) to DNA, thereby cleaving the DNA at or near the binding site. Some restriction enzymes (eg, Type I IS) cleave DNA at sites removed from the recognition site and have separable bonds and cleavable domains. For example, the Type I IS enzyme Fokl catalyzes double strand cleavage of DNA at 9 nucleotides from the recognition site on one strand and 13 nucleotides from the recognition site on the other strand. Thus, in one embodiment, the fusion protein comprises a cleavage domain (or cleavage half-domain) from at least one Type I IS restriction enzyme and one or more zinc-finger binding domains (which may or may not be engineered). .

^' TALEN ^' refers to a nuclease capable of recognizing and cleaving target regions of DNA. TALEN refers to a fusion protein comprising a TALE domain and a nucleotide cleavage domain. In the present invention, the terms "TAL effector nuclease" and "TALEN" are compatible. TAL effectors are known to be proteins that are secreted through their type IE secretion system when Xanthomonas bacteria are infected with various plant species. The protein may bind to a promoter sequence in the host plant to activate expression of plant genes to aid bacterial infection. The protein recognizes plant DNA sequences through a central repeat domain consisting of up to 34 different numbers of amino acid repeats. Thus, it is believed that TALE could be a new platform for tools of genome engineering. However, in order to produce a functional TALEN with genome-correcting activity, a few key parameters that have not been known to date should be defined. i) minimal DNA-binding domain of TALE, ii) one The length of the spacer between the two half-sites that make up the target region, and iii) a linker or fusion junction connecting the Fokl nuclease domain to dTALE.

 TALE domains of the invention refer to protein domains that bind nucleotides in a sequence-specific manner through one or more TALE-repeat parents. The TALE domain includes, but is not limited to, at least one TALE 'repeating modulus, more specifically 1 to 30 TALE repeating moduli. In the present invention, the terms “TAL effector domain” and “TALE domain” are interchangeable.The TALE domain may comprise half of the TALE-repeat modalities. The entire contents disclosed in US Pat. No. 093833 or US Patent Publication No. 2013-0217131 are incorporated herein by reference.

 In one embodiment, the target specific nuclease is a Cas protein (e.g., a Cas9 protein (CRISPR (Clustered regularly interspaced short pal indromic repeats) associated protein 9), a Cpf 1 protein (CRISPR from Prevotel la and Franci sella 1)). And one or more selected from the group consisting of nucleases (eg, endonucleases) and the like involved in the CRISPR system of type Π and / or type V, and the like. In this case, the target specific nuclease may further comprise a target DNA specific guide RNA for guiding to the target site of the genomic DNA. The guide R A may be transcribed in vitro, for example, but may be transcribed from an oligonucleotide double strand or plasmid template, but is not limited thereto. The target specific nuclease can act in the form of ribonucleic acid protein (RNP) by forming a ribonucleic acid-protein complex bound to guide RNA (RNA-Guided Engineered Nuclease).

 Cas protein is a major protein component of the CRISPR / Cas system, a protein capable of forming activated endonucleases or nickases.

 Cas protein or genetic information can be obtained from known databases such as GenBank of the National Center for Biotechnology Informat ion (NCBI). For example, the Cas protein,

 Streptococcus sp. {Streptococcus sp.), Such as Cas protein from Streptococcus pyogenes, such as Cas9 protein (eg SwissProt Accession number Q99ZW2 (NP — 269215.1));

Cas proteins, such as Cas9 protein, of the genus Campylobacter, such as, for example, Campylobacter jejuni; Genus Straptococcus, for example, Stemtococcus thermophilus

Cas proteins, such as Cas9 proteins from Streptococcus thermophiles or Streptocuccus aureus;

 Cas proteins from Neisseria meningitidis), such as Cas9 protein;

 Cas proteins such as the Cas9 protein from Pasteurella iPasteurel d, such as Pasteurella multocida;

 Cas proteins, such as Cas9 proteins, from the genus Francis i sell a, such as Francisella novicida

 It may be one or more selected from the group consisting of, but is not limited thereto.

Cpf l protein is the CRISPR / Cas system is ^old, as the endonuclease of the new CRISPR system be specific, not relatively require a smaller size as compared to t racrRNA Cas9, may function by a single guide RNA. It also recognizes thymine-rich PAM (protospacer-adj acent mot if) sequences and cuts the double chains of DNA to create a cohesive end (cohesive doubl e-st rand break).

 For example, the Cpf l protein may be used in the genus Candidatus, genus Lachinospira, Genus Livibrib Butyrivibrio, Peregrinibacteria, and Aximinococcus.

(Aci ominococcus), Porphyromonas genus, Prevotella genus, Franci sel la genus, Candidatus Methanoplasma, or Eubacterium genus Parcubacteria bacterium (GWC2011—GWC2 ′ 44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasi icus, Peregr in ibact ia ia bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevi or i cam 's, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp. (SC_K08D17), Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum, Candidatus Paceibacter, Eubacterium eligens, etc., but are not limited thereto. The target specific endonucleases are isolated from microorganisms or artificially or unnaturally produced, such as recombinant or synthetic methods. It can be (non-natura l ly occurr i ng). In one example, the target specific endonucleases (eg Cas9, Cpf l, etc.) may be recombinant proteins made by recombinant DNA. Recombinant DNA (rDNA) refers to a DNA molecule artificially produced by genetic recombination methods such as molecular cloning to include heterologous or homologous genetic material obtained from various organisms. For example, when the recombinant DNA is expressed in an appropriate organism to produce a target specific endonuclease, either in vivo or in

The DNA may be one having a nucleotide sequence reconstituted by selecting a codon optimized for expression in the organism among codons encoding a protein to be prepared.

 The inactivated target specific endonuclease inactivated target specific endonuclease refers to a target specific endonuclease that has lost endonuclease activity that cleaves DNA double strands, eg, endonuclease. It may be at least one selected from an inactivated target specific endonuclease that has lost its activity and has a Nikase activity and an inactivated target specific endonuclease that has lost both endonuclease activity and Nikase activity. . When the inactivated target specific endonuclease is of Nikase activity, the strand in which the cytosine is converted to uracil or vice versa simultaneously or sequentially with or without the cytosine being converted to uracil (eg, Ni ck is introduced (e.g., ni ck is introduced between the third and fourth nucleotides in the 5 'terminal direction of the PAM sequence). Such modification of the surface specific endonuclease (mutation) is at least as catalytic acid activity (cat alytic aspartate res i due; for example, aspartic acid at position 10 in the case of Streptococcus pyogenes-derived Cas9 protein) (D10) residues, etc.) may include a mutation of Cas9 substituted with any other amino acid, and the other amino acid may be al anine, but is not limited thereto.

As used herein, the 'other amino acids' are alanine, isoleucine, leucine, methionine, phenylalanine, plin, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine serine, threonine, tyrosine, aspartic acid, glutamic acid , Arginine, histidine, lysine, among all known variants of the amino acid, means an amino acid selected from among amino acids except the amino acid has the original mutation position. In one embodiment, where the inactivated target specific endonuclease is a modified Cas9 protein, the modified Cas9 protein is a Cas9 protein derived from Streptococcus pyogenes (eg, Swi ssProt Access i on number Q99ZW2 (NP_269215. 1) )) Introduced a mutation at the D10 position (e.g., substitution with another amino acid) resulting in a loss of endonuclease activity and a Cas9 protein from a modified Cas9, Streptococcus pyogenes, having Nikase activity. A group consisting of a modified Cas9 protein or the like which has introduced both a mutation at the position (e.g., substitution with another amino acid) and a mutation (e.g., substitution with another amino acid) at the H840 position, thereby losing both endonuclease activity and Nikase activity. It may be one or more selected from. For example, the mutation at the D10 position of the CAs9 protein means a D10A mutation (mutation in which D, the tenth amino acid of the amino acids of the Cas9 protein, is substituted with A; hereinafter, a mutation introduced into Cas9 is represented by the same method). And the mutation at the H840 position may be a H840A mutation.

 As used herein, "nuclease", unless otherwise indicated, means "target specific nuclease (endonuclease)" as described above, eg, Cas9, Cpf l, and the like.

The nuclease may be isolated from a microorganism or artificially or non-naturally produced, such as a recombinant method or a synthetic method. In one example, the nuclease (eg Cas9, Cpf l, etc.) may be a recombinant protein made by recombinant DNA. Recombinant DAN (Recombinant DNA; rDNA) refers to a DNA molecule artificially made by genetic recombination methods such as molecular cloning to include heterologous or homologous genetic material obtained from various organisms. For example, when recombinant DNA is expressed in an appropriate organism to produce a protein (endonuclease) in vivo or in w ^' ro), the recombinant DNA is optimized for expression in the organism among the protein-coding codons to be prepared. The selected codon may be one having a nucleotide sequence reconstituted.

 The nuclease is a protein, a nucleic acid molecule encoding the same, a guide

A ribonucleic acid protein coupled to RNA, a nucleic acid molecule encoding the ribonucleic acid protein, or a recombinant vector comprising the nucleic acid molecule may be used. The nuclease or nucleic acid molecule encoding the same may be in a form that can be delivered, acted, and / or expressed into the nucleus.

 The nuclease may be in a form that is easy to introduce into the cell. In one example, the nuclease may be linked to a cell penetrating peptide and / or a protein transduction domain. The protein delivery domain may be, but is not limited to, poly-arginine or HIV derived TAT protein. Cell penetrating peptides or protein delivery domains are known in the art in addition to the examples described above, so those skilled in the art can apply various examples without being limited to these examples.

 In addition, the nuclease or nucleic acid molecule encoding a nuclear position signal

(nuclear localization signal, NLS) sequence or may further comprise a sequence encoding it. _. Thus, an expression cassette comprising a nucleic acid molecule encoding said nuclease may comprise a regulatory sequence, such as a promoter sequence for expressing said nuclease, or in addition, an NLS sequence. Such NLS sequences are well known in the art.

 The nuclease or nucleic acid molecule encoding the same may be linked to a tag for isolation and / or purification or to a nucleic acid sequence encoding the tag. For example, the tag may be appropriately selected from the group consisting of a small peptide tag such as His tag, Flag tag, S tag, GST (Glutathione S-transferase) tag, MBP (Maltose binding protein) tag, but is not limited thereto. Do not.

 In the present invention, the term "guide RNA" refers to a target DNA specific NA (eg, RNA that can be localized with a target site of DNA), and binds to a nucleotide such as Cas protein, Cpfl, etc., and leads to the target DNA. It plays a role.

The guide RNA may be appropriately selected depending on the type of nuclease and / or the microorganism derived from the nuclease. For example, the guide NA may be selected from the group consisting of CRISPR RNA (crRNA) comprising a DNA target site and a site capable of hybridization; / ^ Y? 5 ^to activating crRNA (tracrRNA), including a site that interacts with endonnucleotides such as Cas protein, Cpfl, etc .; And

 Single guide RNA (sgRNA) in a fused form of the major sites of the crRNA and tracrRNA (eg, the localization site of the crRNA and the interaction site of the tracrRNA)

At least one selected from the group consisting of, Specifically, it may be a dual RNA comprising CRISPR RNA (crRNA) and raJS-act ivat ing crR A (tracrRNA), or a single guide RNA (sgRNA) comprising the major sites of crRNA and tracrRNA.

 The sgRNA may include a portion having a sequence complementary to a sequence in the target DNA (also referred to as a spacer region, a target DNA recognition sequence, a base pairing region, etc.) and a hairpin structure for Cas protein binding. More specifically, it may include a moiety having a sequence complementary to a sequence in the target DNA, a hairpin structure for Cas protein binding, and a terminator sequence. The structure described above may be present in order from 5 'to 3', but is not limited thereto. Any form of guide R A may be used in the present invention, provided that the guide RNA comprises a major portion of crRNA and tracrRNA and complementary portions of the target DNA.

 For example, a Cas9 protein can be used for two types of guide RNA for correcting the target gene, namely CRISPR RNA (crRNA) having a nucleotide sequence that is capable of hybridizing with a target sequence region of the target gene, and a trans—activating crRNA (tracrRNA; Cas9) that interacts with the Cas9 protein. Interacting with proteins) and these crRNA and tracrRNA can be used in the form of a double .stranded crRNA: tracrRNA complex bound to each other, or linked through a linker to form a single guide RNA (sgRNA). In one embodiment, when using a Cas9 protein derived from Streptococcus pyogenes, the sgRNA comprises at least a portion of all or part of the crRNA comprising the localizable nucleotide sequence of the crRNA of the Cas9 and a site that interacts with the Cas9 protein of the tracrRNA of the Cas9. Some or all of the tracrRNA may be to form a hairpin structure (stem- loop structure) through the nucleotide linker (the nucleotide linker may correspond to the loop structure).

The guide RNA, specifically or crRNA sgRNA is within the target DNA sequence and a sequence complementary to and including ^■, crRNA or upstream portion of the sgRNA, specifically, one or more of the 5 'end of the sgRNA crRNA dualR or A, for example, 1 It may contain 10, 1-5, or 1-3 additional nucleotides. The additional nucleotide may be guanine (G), but is not limited thereto.

In another example, when the nuclease is Cpfl, the guide RNA may include crRNA, and may be appropriately selected depending on the type of Cpfl protein and / or the microorganism derived therefrom. The specific sequence of the guide RNA can be appropriately selected according to the type of nuclease (Cas9 protein or Cpf l) (ie, the derived microorganism), which can be easily understood by those skilled in the art. It is possible.

In one example, when using a Cas9 protein from Streptococcus pyogenes as a target specific endonuclease, the crRNA can be ^' expressed in Formula 1 below:

5 '-(N _cas9 ) (GUUUUAGAGCUA)-(X _cas9 ) _m -3' (Formula 1)

 In the general formula 1,

N _cas9 is a targeting sequence, i.e., a site determined according to the sequence of a target site of a target gene (i.e., a sequence that is capable of hybridizing with the sequence of the target site), 1 is the targeting sequence Representing the number of nucleotides contained in and may be an integer from 17 to 23 or 18 to 22, such as 20;

 The site comprising 12 consecutive nucleotides (GUUUUAGAGCUA) (SEQ ID NO: 1) located adjacent to the 3 'direction of the target sequence is an essential part of crR,

X _C as9 is a site comprising m nucleotides located at the 3 ′ .terminal side of the crRNA (ie, located adjacent to the 3 ′ direction of an essential part of the crRNA), where m is an integer from 8 to 12, such as 11 M and the nucleotides may be the same as or different from each other, and may be independently selected from the group consisting of A, U, C, and G.

In one example, the X _cas9 may comprise a UGCUGUUUUG (SEQ ID NO: 2), but is not limited thereto.

 In addition, the t racrRNA may be represented by the following general formula (2):

(Formula 2)

 In the general formula 2,

 60 nucleotides

(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC)

The site marked (SEQ ID NO: 3) is an essential part of t racrRNA,

Y _{c aS} 9 is a site comprising p nucleotides located adjacent to the 5 'end of the essential part of the t racrRNA, p is an integer of 6 to 20 For example, it may be an integer of 8 to 19, the p nucleotides may be the same or different from each other, and may be independently selected from the group consisting of A, U, C and G.

 In addition, the sgRNA is a crRNA portion comprising the targeting sequence and the essential portion of the crRNA and a tracrRNA portion including the essential portion (60 nucleotides) of the tracrRNA form a hairpin structure (stem-loop structure) through the oligonucleotide linker. Where the oligonucleotide linker corresponds to the loop structure. More specifically, the sgRNA is a double-stranded RA molecule in which a crRNA portion including a targeting sequence and an essential portion of a crRNA and a tracrRNA portion including an essential portion of a tracrRNA are bonded to each other. ′ May have a hairpin structure linked via an oligonucleotide linker.

 In one example, the sgRNA can be represented by the following general formula 3:

5 '-(N _cas9 ) 厂 (GUIMJAGAGCUA)-(oligonucleotide linker)-

(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC) -3 *

(Formula 3)

 In Formula 3, (Ν ^)! Is the same as described above in Formula 1 as the targeting sequence.

 The oligonucleotide linker included in the sgRNA may be three to five, for example four nucleotides, the nucleotides may be the same or different from each other, each independently selected from the group consisting of A, U, C and G Can be.

 The crRNA or sgRNA may further comprise 1-3 guanine (G) at the 5 'end (ie, the 5' end of the targeting sequence region of the crRNA).

 The tracrRNA or sgRNA may further comprise a termination comprising 5 to 7 uracils (U) at the 3 ′ end of the—essential portion (60nt) of the tracrRNA.

If the target sequence of the RNA are targeted PAM guide (Protospacer Adjacent on the DNA sequence Motif (6 ^". Pyogenes Cas9, the 5'-NGG-3 '(N is A, T, G, or C Im)) of the 5 ' And from about 17 to about 23 or about 18 to about 22, such as 20 contiguous nucleic acid sequences located contiguously.

The target sequence of the guide RNA capable of hybridizing with the target sequence of the guide RNA is a DNA strand (ie, PAM sequence (5'-NGG-3 '(N is At least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, at least 99% with the nucleotide sequence of the complementary strand of the DNA strand) where A, T, G, or C) is located Or nucleotide sequence having 100% sequence complementarity, and complementary binding to the nucleotide sequence of the complementary strand is possible.

In this specification, the nucleic acid sequence of the target site is represented by the nucleic acid sequence of the strand where the PAM sequence is located among the two DNA strands of the corresponding gene site of the target gene. At this time, since the DNA strand to which the guide RNA actually binds is the complementary strand of the strand where the PAM sequence is located, the targeting sequence included in the guide RNA is a sequence of the target site, except that T is changed to U due to RNA characteristics. It will have the same nucleic acid sequence as. Thus, in this specification, the targeting sequence of the guide RNA and the sequence of the target site (or the sequence of the cleavage site) are identical nucleic acids except that T and U are mutually altered _. It is represented by the sequence.

 The guide RNA may be used (or included in the composition) in the form of RNA (or included in the composition), or in the form of a plasmid containing 腿 encoding it.

Terminology in the present invention _. “On—target site” means the position at which a mutation (cutting, insertion, and / or deletion) is to be introduced using the target specific nuclease, and may be arbitrarily selected according to the purpose. As well as being present within the coding sequence of a particular gene, but may also be present in non-coding DNA sequences that do not produce a protein.

 Since the target specific nuclease has sequence specificity, the target specific nuclease acts at the target position, but side effects may occur at the off-target site depending on the target sequence.

As used herein, an off-target site refers to a position having a sequence that is not identical to a target sequence of a target specific nuclease, but wherein the target specific nuclease is active. That is, it refers to a position cleaved by a target specific nuclease other than the target position. In one example, the non-target position may be used as a concept including not only the actual non-target position for a specific target specific nuclease but also a position that is likely to be a non-target position. The non-target position may be any position other than the target position cleaved by the target specific nuclease in Un / o) in vitro, but not limited thereto. Genetic shearing activity at a position other than the target position can be caused by a variety of causes. For example, there is a possibility that genetic scissors work for non-target sequences that have high sequence homology with the target position, which has a nucleotide mismatch with the target sequence designed for the target position. The non-target position may be a position having one or more nucleotide mismatches with a target sequence, but not limited thereto.

 This can cause mutations in unwanted genes in the genome, which can be a serious problem in using such target specific nucleases. Therefore, the process of accurately detecting and analyzing non-target positions as well as the activity at target positions of target specific nucleases can also be very important, which is a target specific nucleus that works specifically at target positions without non-target effects. It may be useful to develop clease.

 For the purposes of the present invention, since the nuclease may have nuclease activity in vivo in vivo and in vitro, it may be used to detect non-target positions of genomic DNA in vitro, which may be used in vivo. When applied within it can be expected to have activity at the same position as the detected non-target position.

 Step (b) is a step of performing whole genome sequence (WGS) using the DNA cut through the step (a), and finds a sequence having homology with the sequence of the target position Unlike indirect methods of predicting position, it is performed to detect non-target positions substantially cleaved by target specific nucleases at the entire genome level.

 In the present invention, the term "whole genome sequencing" (WGS) '' reads the full-length genome sequencing by next gene sequencing in multiple 10x, 20x, 40x format in multiple multiples "Next-Generation Sequencing" fragments the full-length genome in chip-based and PCR-based pai red end formats, and then fragments are ultra-fast based on chemical hybr idi zat ions. This refers to a technique for performing sequencing.

Step (c) is a step of determining the position where the DNA is cleaved from the sequential read obtained from the whole genome sequencing, by analyzing the sequencing data to target and non-target position of the target specific nuclease Can be detected easily. DNA from the sequence data Determining a particular location cut may be performed in a variety of approaches, and the present invention provides a variety of reasonable ways to determine the location. However, this is only an example included in the technical idea of the present invention, and the scope of the present invention is not limited by these methods.

For example, determining the cut position _. As an example, the 5 'terminal is vertically aligned when sequence data obtained through whole genome sequencing is aligned according to the position on the genome using an analysis program (eg, BWA / GATK or ISAAC). May refer to a position where the DNA is cleaved. As used herein, the term "vertical alignment" refers to adjacent Watson strands and Crick strands, respectively, when analyzing whole genome sequencing results with a program such as BWA / GATK or ISAAC. For, refers to an arrangement where the 5 'end of two or more nucleotide sequences data starts at the same position on the genome (nucleotide posit ion). This results in the sequencing of each of the DNA fragments that are cut by the target specific nuclease and have the same 5 'end.

 That is, when a target specific nuclease exhibits nuclease activity at a target position and a non-target position and cleaves the position, when sequencing the sequence data, the commonly cleaved sites start at 5 'ends. Therefore, the vertical alignment is performed, but since the 5 'end does not exist in the uncut portion, the alignment may be arranged in a staggered manner. Thus, the vertically aligned position can be viewed as the site cleaved by the target specific nuclease, which may mean the target position or non-target position of the target specific nuclease.

 The term "alignment" means mapping base sequence data to a reference genome, and arranging bases having the same position in the genome according to each position. Any computer program may be used as long as it can be sorted by a computer program, which may be a known program known in the art, or may be selected from among programs designed for the purpose. However, the present invention is not limited thereto.

As a result of the alignment, the position where the DNA is cleaved by the target specific nuclease can be determined by a method such as finding a position where the 5 'end is vertically aligned as described above, and the cleaved position is the target position (on- If it is not a target site, it can be determined as an off target site. In other words, The same sequence as the base sequence designed as the target position of the target specific nuclease is the target position, and sequences not identical to the base sequence can be regarded as non-target positions. This is obvious in the definition of the non-target positions described above. The non-target positions may in particular be composed of sequences having homology with the sequence of the target position, and in particular one or more nucleotide mismatches with the target sequence (mi smat ch). ), More specifically 1 to 6, 1 to 5, 1 to 4, 1 to 3, 1 to 2, or 1 nucleotide mismatch with the target position It is not particularly limited thereto, and may be included in the scope of the present invention as long as the target specific nuclease is cleavable. In this case, the target position may be a 15 to 30 nucleotide sequence complementary to the guide RNA, and may further include a sequence recognized by the target specific nuclease (eg, Cas9 recognizes Cas9 PAM sequence).

 In another example, in addition to finding a position where the 5 'end is vertically aligned, when viewing the double peak pattern in the 5' end plot, it may be determined as a non-target position if the position is not the target position. When counting the number of nucleotides constituting the 5 'end of the same base for each position in genomic DNA, a double peak pattern appears at a specific position, which is cleaved by a target specific nuclease. It is represented by each strand of the double strand.

 In one embodiment, the genomic DNA is cleaved with a target specific nuclease (eg, RGEN), followed by whole genome analysis and then aligned with ISAAC, in a vertical alignment at the cut position and in a staggered position at the uncut position. The alignment pattern was confirmed, and when it is represented by the 5 'terminal plot, it was confirmed that a unique pattern of double peaks appeared at the cleavage position.

 Further, but not limited to, Watson strand as a specific example

The position where the sequence data corresponding to (Wat son st rand) and the creek strand (Cr ck st rand) are vertically aligned by two or more, respectively, may be determined as a non-target position. It can be determined that at least 10 nucleotide sequence data is vertically aligned, and the position at which the number of nucleotide sequence data having the same 5 'end in each of the Watson strand and the creek strand is 10 or more is a non-target position, that is, a cleavage position.

Make the non-target position (detection) it can be carried out by treating the target-specific nucleases in in vitro w ^'o) in the dielectric DNA. Above this Through the method, it is possible to confirm whether the non-target effect is substantially observed in vivo (/ ra) for the non-target position identified (detected). However, since this is only an additional verification process, it is not an essential step in the scope of the present invention, but is only a step that may be additionally performed as necessary. As used herein, the term “off-target effect” may be a concept that is distinct from off—target sites. That is, as described above, in the present invention, the concept of non-target position means a position other than the target position among the positions where the target specific nuclease can operate, and indicates a position that is cleaved by the target specific nuclease. As said, non-target effects refer to the effects of insertion and / or deletion by target specific nucleases at non-target locations in the cell.

The term “indel” refers to a variation in which some bases are inserted or deleted in the base sequence of DNA. In addition, the non-target position where said indel was caused by a target specific nuclease is called a non-target indel position. In conclusion, the non-target position of the present specification can be viewed as a concept including a non-target indel position, and the target specific nuclease may be a position capable of having activity, and the indel may be identified by genetic scissors. It does not have to be. On the other hand, ^"non-target position in the present invention is a non-target position candidates (candidate off-target site), non-target indel position may be referred to as a proven non-target position (validated off-target site).

 Specifically, the verification process is not limited thereto, but the genomic DNA is isolated from the cells expressing the target specific nucleases for the non-target positions, and the indels are identified at the non-target positions of the DNA. It may be to confirm the non-target effect of. This is the T7E1 analysis, Cel-

It may be to confirm the non-target effect by performing indel identification methods known in the art such as mutation detection analysis or targeted deep sequencing using the I enzyme. The step of identifying the non-target effect may be to directly check whether the indel occurred at the non-target position. However, even though indel did not occur in the in vivo verification process, this is not confirmed until indel occurs at a frequency below the detectable level and should be regarded as an auxiliary means to the last. Identifying vertically aligned positions as described above, or identifying double peaks in the 5 'end plots, is sufficient to detect non-target positions, which is highly sensitive. There is a problem of having reproducibility, but a non-uniform cutting pattern or a low sequencing depth may cause missing tooth locations. In this specification, based on the alignment pattern of the sequence data, the position of each nucleotide i. (I.e., nucleotide positions on genomic DNA) provides the formula for calculating DNA cleavage scores as follows (see Figure 4): score at position =

ι위치에서 시작하는정방향염기서열 데이터의 수

Number of forward base sequence data starting at position

 Number of reverse base sequence data starting at position I

 Sequencing Depth at i Position

 Any constant

 In the above formula, the number of nucleotide sequence data means the number of nucleotide reads, and the sequencing depth means the number of sequencing reads at a specific position.

In addition, the above formula can detect a number of additional positions that were not detected in the existing Digenome-seq, and thus can easily filter out false-positive positions. The C value in the above formula is not limited by the embodiment of the present invention as one skilled in the art can apply any constant. In one example, C may be 1 to 1000, 1 to 500, 1 to 100, .1 to 50, 1 to 10, 1 to 5, or 1 to 3, but is not limited thereto. In particular, but not limited to, for example, in the nucleotide sequence of any position (cutting position), the calculated score with a C value of 1 is 2.5 or more, or 0.1 or more and On-target sequence and homology In the case of (eg, containing PAM (5'-NGN-3 'or 5'-NNG-3') with Οη-target sequence and no more than 10 mismatches) Position) can be determined as a non-target position. However, the criteria of the score may be appropriately adjusted and changed by those skilled in the art according to the purpose.

 In one example, the Di genotne-seQ method provided herein may be performed using a plurality of target specific nucleases (eg, target specific nucleases comprising multiple guide RAs with different target sites) In the present specification, this is referred to as “complex Di genome—seq '” In this case, the target specific nuclease may be a combination of target specific nucleases for two or more, specifically 2 to 100 targets. It may be, but is not limited thereto.

 In the case of the complex Di genome seq, genomic DNA is cleaved by each target specific nuclease, so it is important to identify which gene was cut by the cleavage site. This can be achieved by classifying non-target positions according to the edit distance from the target position (ed i t st ance), and it is assumed that the base sequence of the non-target position has homology with the target position. This makes it possible to clearly distinguish between target and non-target positions for each gene shear.

 In a specific embodiment of the present invention, a nucleotide mismatch with a target position among non-target positions detected by Digenome-seq in the whole genome for RGEN (R A-gu i ded engineered nuclei ease) targeting a specific position. If there are no more than 13, 000 homology positions with no more than 6, and no homology positions with no more than 2 nucleotide matches, selecting the specific position as the target position of the RGEN can minimize non-target effects. It was confirmed. This is an example showing the process of establishing a desirable criterion for selecting a target position using the Digenome-seq of the present invention, Di genome-seq is expected to minimize the non-target effect of the genetic scissors.

On the other hand, it was confirmed that the number of positions having homology with the sequence of the target position is detected at a small rate through Di genome-seq as the level of nucleotide mismatch increases. This is because, in selecting the target position of RGEN, the more nucleotide sequences having homology in the target sequence and the genome, and in particular, the more nucleotide sequences having high homology, are relatively more specific. The target location of the selected RGEN may be a non-target effect is minimized. Another example is a target site having a low non-target position and / or a target specific target for the target site using a method of detecting the cleavage site or off-target site of the target specific nuclease. Nuclease * Provides a method for screening.

 The screening method,

 (a) cleaving the isolated genomic DNA with a target specific nuclease;

 (b) performing whole genome sequencing on the cleaved DNA;

 (c) identifying the cleaved position in sequence reads obtained by the sequencing;

 (d) if the identified truncated position is not an on-target site, determining it as an off-target site; And

 (e) analyzing the determined non-target position data to select a target site to which the target specific nuclease targets and / or a target specific nuclease as a target when the non-target position appears less than the comparison target Steps to

 It may include.

 In one embodiment, where the target specific nuclease is targeted to a target site by a guide NA, the selected target site and / or target specific nuclease may be linked to a target DNA sequence other than the target non-target of the guide RNA. It may be characterized as comprising a sequence that can be localized.

 The comparison subject may be a target specific nuclease for the target sequence of any target DNA, and in one embodiment is selected from among the commonly used or known target specific nucleases (eg, RGEN and guide RNA combinations) It can be either.

【Effects of the Invention】

 Digenome-seq of the present invention can detect the non-target position of the gene scissors at the genome level with high reproducibility, and can be used for the construction and research for the gene scissors with high target specificity.

[Brief Description of Drawings]

Figure 1 shows the cleavage genomic sequencing method using chromatin DNA It is shown schematically.

 Figures 2a to 2c shows the comparison of the cleavage genome sequencing results using chromatin DNA and chromatin-free DNA,

 Figure 2a shows that when sequencing the whole genome without processing RA gene shears on DNA without chromatin protein (isolated DNA) (top), RNA gene shears on DNA without chromatin protein (isolated DNA) DNA cl eavage score for post genome sequencing (middle) and for sequencing the genome (bottom) after RNA genotyping to chromatin DNA (including chromatin proteins) Dark colored bars represent the total number of positions beyond the cut DNA sequencing results measured DNA cleavage score (0.01 000-10), and light colored bars represent the sequences (NNG or NGN) is present and indicates the number of positions where a sequence of 20 bp of target position coincides with a sequence of 10 bp or more;

 Figure 2b is one with DNA without chromatin protein. A van diagram showing the result of comparing the cleavage genome sequencing with the number of non-target positions resulting from cleavage genomic sequencing with DNA containing chromatin protein;

 FIG. 2C shows the correlation between the truncation score of a nontarget location from a truncated genomic sequencing with DNA without chromatin protein and the truncation score of a nontarget location from a truncated genomic sequence with DNA containing chromatin protein. This is a graph comparing.

 3a to 3c show the results of cleavage genome sequencing analysis using DNA containing chromatin,

 FIG. 3A is a graph showing DNA cleavage scores when sequencing cleavage genomes using mutation rates in cells and DNA containing chromatin proteins, FIG.

 Figure 3b is a graph showing the DNA cleavage score when the mutant ratio in the cell and cleavage genome sequencing using DNA without chromatin protein,

 FIG. 3C is a graph showing the correlation between the mutation rate (Inde l frequency) and DNA cleavage scores shown in FIGS. 3A and 3B.

 4 shows an in vitro DNA cleavage scoring system for Di genome-seq analysis.

Figure 5a shows Digenome 1.0 in Hel a ce ll using HBB specific Cas9. Venn diagram showing non-target location results identified.

Figure 5b is a graph showing the relationship between the cutting points of the cutting points of the nuclei pellet in a non-target location by Digenotne 1.0 using HBB specific Cas9 from Hela cell and _nat i _V e chromatin.

 FIG. 6A is a venn diagram showing non-target location results identified by Di genome 2.0 using HBB specific Cas9 in Hela cells.

 6B is a graph showing the correlation between cleavage scores of nuclear targets identified by Di genome 2.0 and non-target positions in native chromatin using HBB specific Cas9 in Hela cells.

 Figure 7a shows Digenome using HBB specific Cas9 in HEK293T cells

Venn diagram showing non-target location results identified by 2.0.

 FIG. 7B is a graph showing the correlation between the cleavage score of the nuclear pellet and the cleavage score of native chromatin at the non-target position identified by Digenome 2.0 using HBB specific Cas9 in HEK293T cells.

 FIG. 8A is a band diagram showing in vitro cleavage sites (non-target positions) identified by performing Digenome 1.0 using HBB specific Cas9 on nuclear pellets and chromatin-free DNA of Hela cells. . FIG. 8B is a graph showing the correlation between the nuclear Peltiff identified by Digenome 1.0 using HBB specific Cas9 in Hela cells and the cleavage scores of non-target sites in chromatin-removed DNA. FIG. 9A is a diagram of Hela cells. This is a band diagram showing in vitro cleavage sites (non-target positions) identified by performing Digenome 2.0 using HBB specific Cas9 on nuclear pellet and chromatin-depleted DNA.

 9B shows the sequence logo (DNA cleavage score> 0.1) obtained via WebLogo using DNA sequences at Digenome-capture sites.

 9C is a graph showing the correlation between the nuclear pellets identified by Digenome 2.0 using HBB specific Cas9 in Hela cells and the cleavage scores of non-target positions in chromatin-removed DNA.

 9D is a graph showing the correlation between Indel frequency and DNA cleavage scores obtained by performing Digenome-seq using HBB target sequences on chromatin DNA and chromatin-removed DNA.

FIG. 9E is a graph showing R ² values obtained by performing Digenome-seq on chromatin DNA and chromatin-removed DNA using HBB target sequences. [Form to carry out invention]

 Through the following examples will be described in more detail the present invention. These examples are only for illustrating the present invention in more detail, it will be apparent to those of ordinary skill in the art that the scope of the present invention is not limited by these examples. Example 1: Chromatin DNA cleavage using endonucleases Un vitro cleavage and sequence analysis of cleaved DNA

 For use in genomic DNA cleavage and sequencing, He la (ATCC, CCL-

2), HEK293T (ATCC, CRL-11268), K562 (ATCC, CCL-243), and T cells (extracted from donor blood) were prepared. Lysis buf fer (lxPBS, 0.4% NP-40 (CAS 9016-45-9), and 3 mM MgCl ₂ ) were treated with each of the cells (5xl0 ⁵ each) to remove the cell membranes, and then, at 500 X g. Centrifugation was performed for a minute to separate the cytoplasmic layer (supernatant) and the chromatin DNA layer.

After removal of the cellular layer, 300 nM of Cas9 protein Streptococcus pyogenes, SwissProt Accession number Q99ZW2; SEQ ID NO: 4) and 900 nM of guide RNA were added to the reaction buffer (100 mM NaCl, 50 mM Tris-HCl, 10 mM MgCl ₂ , 100 μg). / ml BSA, and pH 7.9) and incubated for 8 hours at 37 ^° C. The RNA was cleaved by Cas9 and guide RNA treatment, and then treated with R ase A (50 ug / mL) to remove the guide RNA and purified whole genome DNA using DNeasy Tissue kit (Qiagen).

 The separated whole genomic DNA was taken in an amount of lug, cut about 400-500 bp using a Covaris system (Life Technologies), and the DNA protrusion generated by the cleavage was removed using an End Repair Mix. In order to make a library for sequencing, the obtained cut DNA is combined with an adapter (a piece of DNA that binds to the cut DNA, using a kit provided by Illumina), and then using HiSeq X Ten Sequencer (Macrogen). Whole genome sequencing was performed.

 The procedure described in this example is schematically shown in FIG.

In addition, the guide NA used above has the following nucleotide sequence: 5'- (target sequence)-(GUUUUAGAGCUA; SEQ ID NO: 1)-(nucleotide linker)-(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAMGUGGCACCGAGUCGGUGC;

SEQ ID NO: 3) -3 '

 (The target sequence is the "NGG" at the 3 'end in the DNA sequence at target site (sequenced by a square box or on-target) or a target sequence (on-target sequence) described in Tables 1 to 8 below. (N is any nucleotide, a nucleotide sequence having the base of A, T, G, or C), except for the nucleotide sequence converted to "T",

 The nucleotide linker has a nucleotide sequence of GAAA. In the examples below, the complex of Cas9 protein and guide RNA is called genetic scissors.

 For comparison, genome cleavage and sequencing were performed on the chromatin free DNA obtained by purifying DNA from the chromatin DNA obtained above. The chromatin-removed DNA was prepared by purifying genomic DNA from each of the prepared cells using a DNeasy tissue kit (Qiagen) according to the manufacturer's instructions. Example 2 Comparison of Cleavage Sequence Analysis Using Chromatin DNA and Chromatin Removal DNA

 Using the whole genome DM sequencing results obtained for four cells of Hela, HE 293T, K562, and T cells in Example 1, non-target positions were determined according to the DNA Cleavage Scoring system. target site) (cut genome sequencing).

The DNA Cleavage Scoring System was obtained according to the following equation, for more details, see "Kim, 0., Kim, S., Kim, S., Park, J. & Kim, JS Genome-wide". target specificities of CRISPR ᅳ Cas9 nucleases revealed by multiplex Digenome-seq. Genome research 26, 406-415 (2016) ": Score at position =

F _t : Forward sequence data starting at position i

 R ,： i Starting of reverse base sequence data

 Di ： Positivity at / position ο

 C όΐ Q] ol ΛΤ- τ

 In the above formula, the number of nucleotide sequence data means the number of nucleotide reads, and the sequencing depth means (the number of sequencing reads at a specific position). In addition, the said C value was set to 1.

 First, the obtained dielectric without any treatment to the cell lysate

DNA cleavage score calculated by the above method using the whole genome sequencing of (cytoplasm + chromatin), and the result of the whole genome sequencing of the resultant half-cutting result obtained by processing the scissors with DNA that contained or removed the chromatin. Were compared with each other. Among these, the results corresponding to the conditions of Table 1 (He l ce l l and the t arget sequence (HBB target sequence) of Table 1) are shown in FIGS. 2A to 2C.

First, the number of total positions over a specific DNA cleavage score (0.0001-10) and the sequence to which the Cas9 protein binds among the positions over a specific DNA cleavage score (NNG or NGN; N is any nucleotide, A, T (U) Nucleotide having a base of, G, or C), and the number of positions where the sequence of the target position of 20 bp and the sequence of 10 bp or more coincides with each other, and the result is shown in FIG. 2A. FIG. 2A shows a case in which the genome sequencing is performed without processing the genetic scissors in the chromatin-removed DNA (denoted as chromat in f ree DNA) (upper), and the genome sequencing is performed after the genetic shearing is performed on the chromatin-removed DNA. (Middle), and the number of sit es according to the DNA cleavage score in the (same bottom) when the genetic scissors were treated with chromatin DNA (bottom). In FIG. 2A, the result of cleavage genome sequencing shows that the total number of positions (dark color bars) above the measured DNA cleavage score (0.0001-10) and the sequence to which cas9 binds (NNG or NGN) The number of positions (light color bars) present and where the sequence of the target position of 20 bp and the sequence of 10 bp or more coincident is shown.

 When the measured DNA cleavage score is 0.1 or higher, there is a sequence (NNG or NGN) to which the positional Cas9 binds from the genome sequence of the genome without Cas9 treatment, and the target position of 20 bp matches the sequence of 10 bp or more The cutoff value of the DNA cleavage score was set to 0.1 because there was no place to.

 Based on these criteria, there were 44 non-target cleavage sites for cleavage genomic sequencing on chromatin DNA, which were included in all 119 non-target positions resulting from cleavage genome sequencing on chromatin free DNA. This became (see Figure 2b). 2B shows the number of non-target positions obtained as a result of cleavage genomic sequencing for chromatin-removed DNA and cleavage genomic sequencing for chromatin DNA through a van diagram. 2B shows that by performing cleavage genome sequencing using chromatin DNA, the number of non-target positions is significantly reduced as compared with the case of using chromatin-free DNA, thereby enabling more accurate cleavage genome sequencing.

In addition, the correlation between the cleavage score of the non-target position obtained as a result of cleavage genome sequencing for chromatin-removed DNMchromatin free DNA) and the cleavage score of the non-target position as a result of cleavage genome sequencing for chromatin DNA is shown in FIG. 2C. As shown in FIG. 2C, when the correlation between the DNA cleavage scores obtained when the cleavage genome sequencing was performed for each of the chromatin DNA and the chromatin-removed DNA, was obtained. It can be seen that there is little correlation. In addition, cleavage genomic sequencing using chromatin DNA was performed on various cells, such as HEK293T, 562, and T cells, in addition to Hela cells, demonstrating their applicability to these cells (Table 1 (target sequences in order). Nos. 5 to 48, wherein SEQ ID No. 5 is the HBB on—target sequence and the rest is off-target sequence and Table 2 (target sequences are in sequence order except for the on-target sequence indicated by square boxes). 49-84), the nucleotides represented by lowercase letters in Tables 1 and 2 are mismatch nucleotides).

TABLE 2

 HB 93T

 Chr Location DNA C! Eavaoe Score DNA seauence at taraet sites

1 chr 11 5248215 14.63 CTTGCCCCACAGGGCAGTAACGG | chr9 104595883 8.17 tcaGCCCCACAGGGCAGTAAGGG

Chir17 8370253 3.65 tTgctCC CACAGGG CAGTAAACG c r12 124803834 2.96 gcTGCCCCACAGGGCAGcAAAGG chr14 94585327 t.82 a Tg GCCC CACAa GGCAiGa AATG G

Chir12 93549202 1.01 aTT & CCCCACgGGGCAGTgACGG chr2 121715240 0 88 gTgtCCC CACAGGG CAGg AAAG G c r12 27234755 070 gaTGCCtCACAGGa CAGgAAGGG chrX 75006257 0.46 gTgGCCC CACAGGG CAGgaATG G chr 14 36889538 0.40 gTTatCC CACAGGa CAGTgAGGG chr6 23709579 0.29 ga a GCCC tACAGGG CAGcAATC G chr7 97874171 0.28 CcctCCC CACAGGG CAG cATGG chr 10 72286 450 0.23 Caa GCCC CACAGGG CAGa c AG GG chr8 24931381 0.19 agTGCC a CACAcaG CA.GTAAGGG chr9 134994964 0.12 C TGCCCCtCAGGGCAGctAAGG

 K562

 Chr Location DNA C! Eavaae Score DNA seauence at taraet sites

1 chr 1 1 5248215 16,29 CTT & CC CCACAGGG C AGTAAC GG | chr9 104595883 9. 1 t-c a GCCC CAC AGGG CAGTAA.GGG chr 17 8370 253 3.80 tTgctCC CACAGGG CAGTAAACG

. chr 12 124803834 2,65 gcTGCCCCAC GGGCAGcAAAGe chrX 75006257 2.62 gTgGCCC CACAGGG CAGgaATGG chr 14 94585327 1.04 aTgGCCCCACAaGGCAGaAATGG chr2 12171 5240 0.56 gTgtCCC CACAGGGCAGgAAAGG chr22 17230623 0.30 tg GCC CCACAGaGCAcTAAGGG chr 12 93549202 0,28 aTTGCCCCACgGGGCAGTgACGG chfi 17346702 0.15 '. gglc CCC a cagGGt C AGTAAG GG chr? 9787417 t 0.1 Ccc CCC CACAGGG CAGTcATGG chr6 23709579 0.12 ga a GCCC tACAGGG CAGcAATGG

 T cell

 Chr Location DMA CIeavaqe Score OIMA seciuence at taraet sites

1 c r1 1 5248215 15.29 CTTGCG CCACAGGG GAGTAACGG | chr9 104595883 5.64 tea GCCC CACAGGG CAG AAG GG chr 12 124803834 3,78 gcTGCC CCACAGGGCAGCAAAG G chr17 8370253 3.27 CTgc tCC CACAGGG CAGTAAACG chr14 94585327 3.03 a ¾ GCCC CACAOGG CAGaAATG G chr12 93549202 t 94 aTTGCCCCACgGGGCAGTgACGG chr22 17230623 1.52 tg GCCCCACAGaGCAcTAAGGG chrX 75006257 1.48 gTgGCCC CACAGGGCAGgAATGG chr6 23709579 1 23 gaaGCCCtACAGGGCAGcaATGG chr2: 121715240 1,06 gTgtCCC CACAGGG CAGgAAAG (3 chr 19 5001 0010 0.50 aT GCCCCcCAGG CAGTAgGGG chr5 131423385 0.23 tcTGCCCCACAGGc CAGgAAGG G Analysis DNA Example 3 DNA Analysis: Cleavage genome sequencing using chromatin DNA using seven different gene shears (including guide RNAs containing different target sequences) confirmed the indel frequency in the Hela cell. Tables 3 (target sequences are SEQ ID NOs: 85 to 115), Table 4 (target sequences are SEQ ID NOs: 116 to 151), Table 5 (target sequences are SEQ ID NOs: 152 to 164) and Table 6 (targets The sequences are shown in sequence in SEQ ID NOs: 165 to 181) (the nucleotides shown in lowercase in Tables 3 to 6 are mismatch nucleotides).

FA.NCF

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In cells, the ratio of mutations to non-target positions on the truncated genome sequencing results shown in Tables 3 to 6 was determined, and the DNA at that position was measured. I compared it with the score of ^' dan. The comparison result is shown in FIG. 3A. For comparison, the same test was performed on chromatin-removed DNA, and the results are shown in FIG. 3B, and the correlations shown in FIGS. 3A and 3B (the closer the mean is, the higher the correlation) are shown in FIG. 3C. . As shown in FIGS. 3A to 3C, when cleavage genomics was performed using chromatin DNA, the correlation between the DNA cleavage score and the mutation rate at a specific position (mean ^ = 0.62) was used for the chromatin removal DNA. It can be seen that significantly higher than the correlation (mean ^ = 0.20) when the cleavage dielectric sequencing. Example 4: Cleavage test of chromatin DNA (nuclei pellets and native chromatin DNA)

 In the previous examples, it was confirmed that chromatin plays an important role in Cas9 nuclease activity, and therefore, it was originally intended to reflect the chromatin state in the profile of the Genome 'wide Cas9 off-target. The following tests were performed to show that the Digenome-seq (cleavage genome sequencing) method using native chromatin DNA had an advantage.

 To identify Cas9 target sites throughout the genome, Di genome—seq was performed using chromatin DNA. Referring to Example 1 and the target sequence of Table 7 below (sequence in box (HBB target sequence)), nuclear pellets and native chromatin isolated from Hela cells or HEK293T cells were pre-assembled Cas9 protein (300 nM). ) And incubated with ribonucleic acid protein including sgRNA (900nM), followed by whole-genome sequencing (WGS) after DNA purification (see FIG. 1). The nuclear pellet is in the state of the nuclear membrane after removing the cytoplasm from the cells, centrifuged for 5 minutes at 500 xg after lysis buffer (lx PBS, 0.4% NP-40, 1 mM EDTA) to the cells and the cytoplasm layer (supernatant) Prepared by removal (same as the chromatin DNA used in Examples 1 to 3), native chromatin was removed from the nuclear membrane, cells lysis buffer (lx PBS, 0.4% NP-40, 1 mM EDTA) After centrifugation at 500 xg for 5 minutes after treatment, the cytoplasm layer (supernatant) was removed, and then treated with a Nucleation solution (10 mM EDTA, 0.5 mM EGTA, 0.1% Triton X100) to remove Nucleoplasm (supernatant). It was.

After sequence alignment to the human reference genome (hgl9), a linear alignment induced by Cas9—mediated DNA digestion at the target site was observed using an integrative genomics viewer (IGV). Previous studies involving Digenome-seq (Kim, D., Kim, S., Kim, S., Park, J. & Kim, J.S. Genome-wide target specificities of CRISPR ᅳ Cas9 nucleases revealed by multiplex Digenome-seq Cas9 non-target location was confirmed by DNA cleavage score used in Genome research 26, 406-415 (2016). The Cas9 non-target location was determined by Digenome 1.0 (determining sites having a DNA cleavage score of 2.5 or more as a non-target location candidate group) and Digenome 2.0 (DNA cleavage score of 0.1 or more and having a mismatch of 10 or less PAM (5'-). Sites with NGN-3 'or 5'-NNG-3') were determined as non-target locations.

 The obtained results are shown in FIG. 5A (vendiagram showing the non-target position result obtained by Digenonie 1.0 using HBB specific Cas9 in He la cell), 5b (Digenome 1.0 obtained using HBB specific Cas9 in Hela cell). A graph showing the correlation between the cleavage score of the nuclear pel¾ at the non-target position and the cleavage score of the native chromatin), 6a (vena diagram showing the non-target position result obtained by Digenome 2.0 using HBB-specific Cas9 in Hela cells). ), 6b (graph showing the correlation between cleavage scores of nuclear pellets obtained by Digenome 2.0 and non-target positions in native chromatin using HBB specific Cas9 in Hela cells), 7a (HBB specific Cas9 in HEK293T cells) Venn diagram showing non-target position results obtained by Digenome 2.0 using the method and 7b (HBB specific Cas9 in HEK293T cells) Graph showing the correlation between the cleavage score of the nuclear pellet at the non-target position obtained by Digenome 2.0 and the cleavage score of the native chromatin, respectively.

_. As confirmed in FIG. 5A, 15 and 11 in vitro cleavage sites were observed using HBB specific Cas9 through Digenome 1.0 (cutoff: 2.5) in nuclear pellets and native chromatin of Hela cells. At this time, 10 (91%; 10/11) of the in vitro cleavage sites (11%) identified in native chromatin overlap with 10 of the cleavage sites (15) identified in nuclear pellets. In addition, as shown in FIG. 5B, in Hela cells, DNA cleavage scores of in vitro cleavage sites identified in native chromatin showed a high correlation with DNA cleavage scores of in vitro cleavage sites identified in nuclear pellets (R ² = 0.97). These results show that Digenome-seq using nuclear pellets or native chromatin has high reproducibility. ^' As seen in FIGS. 6A and 6B, 44 and 37 in vitro cleavage sites were observed using HBB specific Cas9 through Digenome 2.0 (cutoff: 1.0) in nuclear pellets and native chromatin of Hela cells, among which 34 dogs were folded together and a high correlation between them was observed (R ² = 0.97).

As confirmed in FIGS. ₃ and 7B, 12 and 7 in vitro cleavage sites were observed using HBB specific Cas9 through Digenome 2.0 (cutoff: 1.0) for nuclear pellets and native chromatin of HEK293T cells, Six of them overlapped each other, and a high correlation was observed between them (R ² = 0.88).

 These results indicated that nuclear pellets and Digenome 一 seqs using native chromatin were highly correlated with each other, and that Digenome-seq using nuclear pellets had more in vitro cleavage sites than Digenome— seqs using native chromatin. In Table 7, Digenome-seq was performed using nuclear pellets. In order to determine whether Digenome-seq using chromatin DNA is generally applicable to various cell types, the method was applied to various cells such as K562 and primary T cells, and the results were compared with those of Helaell and HEK293T cells. Together, they are shown in Table 7 below (Table 7 (HBB on target sequence (square box): SEQ ID NO: 5; excluding these sequences: SEQ ID NOs: 6 to 84 in sequence) is a mismatch nucleotide in lowercase nucleotides. .

TABLE 7

_., -1111-4455

chr9 134994964 CcT GCCCCACAGGGCAa t ATGG _j chr2 43013308 CcT GCCCa g AaGGCAGTAAGGG, ¹ chr8 ^ 41296595 ca GTCCCACJ ^ G tCAGc ^ TGG__j chr22 ^J 35537395 a QCX: CX: ¾CAGGGg¾Ga HEK293T

 Chr Location DNA Cleavage Score DNA sequence at target sites

 Chr 11 5248215 14.S3 CrTGCCCCACaGGGCAGTaACGG

chr9 104595883 8.t caGCC ⁽ X¾CAGGGCAGTAAGGG

 chr 17 8370253 3. cTgctCCCACAGGGCAGT¾AACG

 chr 12 124803834 2.gcTGCCCCACAGGGCAGcAAAGG

 chr 14 94585327 1.a T gGCCCCACAaGGCAGaAATGG

 chr 12 93549202 1.aTTGCCCCACgGGGCAGT gACGG

 As shown in Table 7, it can be seen that most of the in vitro cleavage sites defined in each cell overlap each other.

Digenome-seq (Di genome l. O) using HBB-specific Cas9 was performed on nuclear pellets and chromatin-depleted DNA of Hel a cells to in situ cleavage sites (on target). all cutting positions including si te) The results are shown in Figs. 8A and 8B, measured and compared. As shown in FIG. 8A, Digenome-seq was performed on nuclear pellets and chromatin-removed DNA using Digenome 1.0 to observe 15 and 48 in vitro cleavage sites, respectively. Most of the in vitro cleavage observed in nuclear pellets was observed. The site (93%) overlapped the in vitro cleavage site observed with chromatin-removed DNA using digenome® seq.

Digenome-seq (Di enome 2.0) was performed on nuclear pellets and chromatin-free DNA of Hela cells to measure and compare in vitro cleavage sites (all cleavage sites including on target sites). The results are shown in Figures 9a to 9c. 9A and 9B, Digenome® seq was performed on nuclear pellets and chromatin-removed DNA using Digenome 2.0, and 44 and 97 in vitro cleavage sites were observed, respectively, and most of the nuclear pellets were observed. in vitro cleavage site. (98%) overlapped the in vitro cleavage site observed with digenome-seq using chromatin-removed DNA. As it is shown in Figure 8b and Figure 9c, the nucleus. For the pellets and the chromatin of DNA cut score identified in removing DNA was almost not correlated with each other (for ^{Digenome 1.0 R 2 = 0.22, Digenome} 2.0, R 2 = 0.19). Example 5: Confirmation of correlation between DNA cleavage score and Indel frequencies

Digenome-seq (using HBB target sequences from Table 7 above) was performed on chromatin DNA (see Example 1) and chromatin removal DNA (see Example 1), and the correlation between DNA cleavage score and Indel frequency The results are shown in Fig. 9 (1). There was a low correlation between DNA cleavage scores and indel frequencies of non-target candidate positions obtained from Digenome- seq using chromatin-removed DNA (R ² = 0.10; 9e), DNA cleavage scores and indel frequencies of non-target candidate positions obtained from Digenome-seq using chromatin DNA showed a relatively high correlation (R ² = 0.72) (see FIG. 9d).

Next, perform Digenome—seq using nuclear pellets against HBB target sequences (Table 7) and other target sequences (see Table 8), measure DNA cleavage scores and Indel frequencies, and calculate R ² values. Correlation between cleavage scores and Indel frequencies was measured and the results were determined in Table 7 (HBB target results), Table 8 (target sequences in SEQ ID NOs: 85-181), and FIG. 9E (HBB, VEGFA1, ΗΕΚ3, EMX1, and FANCF target results) (the lowercase nucleotides in Table 8 are mismatch nucleotides).

LP

LP

/, 59 /, 60 / 810∑; OAV

Claims

[Claim]  [Claim 1]  (a) cleaving the isolated genomic DNA with a target specific nuclease;  (b) performing whole genome sequencing on the cleaved DNA; And  (c) determining the cleaved position in sequence reads obtained by the sequencing;  The isolated genomic DNA is to include chromatin DNA,  A method of detecting off-target sites of target specific nucleases.  [Claim 2]  The method of claim 1, further comprising determining an off target site if the truncated position is not an on target site.  [Claim 3]  The method of claim 1, wherein the cleaved position is a position where the 5 ′ end is vertically aligned by aligning the obtained sequence data, or a position showing a double peak pattern in the 5 ′ end plot.  [Claim 4]  The method of claim 1, wherein the genomic DNA is isolated from cells expressing or not expressing target specific nucleases.  [Claim 5]  The method of claim 3, wherein the alignment is performed using BWA / GATK or ISAAC after mapping the sequence data to a reference genome.  [Claim 6]  The method of claim 3, wherein the determining of the non-target position as a position at which two or more sequence reads corresponding to the Watson strand and the Crick strand are vertically aligned, respectively, is performed. Further comprising.  [Claim 7] 4. The nucleotide sequence of claim 3, wherein at least 20% sequencing data is vertically aligned and has the same 5 'terminus at each Watson strand and Creek strand. And determining that the location where the number of data is at least 10 is a non-target location.  [Claim 8]  The method of claim 1, wherein the isolated genomic DNA is isolated from the target specific nuclease-expressing cells, and further comprising the step of confirming the non-target effect by checking the insertion and deletion at the non-target position of the DNA. That, including.  [Claim 9] 10. The method of claim 8, wherein validating the indel method would obtained by performing the analysis T7E1, ^"mutation detection analysis or targeting deep sequencing using the Cel-I enzyme (targeted deep sequencing) for the non-target position. [Claim 10]  The method of claim 1, wherein the nontarget position has at least one nucleotide mismatch with the target position.  [Claim 11]  The method of claim 1, wherein the nontarget position has 1-6 nucleotide mismatches with the target position.  [Claim 12]  According to claim 1, wherein the step (c) is performed by calculating the cleavage score by applying the following formula to each cleaved position, the off-target site of the target specific nuclease How to detect. Score at position i a = 1 ^LJ li-i ÷ a + Σ ^{^} W ^{1 "eu} XD ^{^} x sswe _{+ - 3 + fl - 2)} a = 1 i- 1 - 3 + ft ■ F _l : Number of forward base sequence data starting at position i . _t : Number of reverse base sequence data starting at position I Di: Detective depth at position i (de ≠ h)  C ： Arbitrary constant [Claim 13] The method according to claim 12, wherein when the constant C is 1 in the above formula, the score calculated is 2.5 or more, or the score is 0.1 or more and includes the PAM sequence with 10 or less mismatches with the target sequence. Determining the truncated position as a non-target position.  [Claim 14]  The method of claim 1, wherein the target specific nuclease combines target specific nucleases for two or more target sequences.  [Claim 15]  The method of claim 1, wherein the target specific nuclease combines target specific nucleases for 2 to 100 target sequences.  [Claim 16]  The method of claim 14, further comprising classifying the non-target position according to the edit distance to the target position (edi t di stance).  [Claim 17]  The method of claim 1, wherein the genetic scissors are meganuclease (meganuc lease), ZFN (zinc f inger nuc lease), TALEN (t ranscr ipt ion act ivator-1 ike ef fector nuc lease), and RGEN (RNA 一 guided engineered nuc l ease) , Way.  [Claim 18]  The method of claim 17, wherein the RGEN is a Cas9 protein or a Cpf l protein.  [Blue ^ term 19]  The method of claim 18, wherein the RGEN further comprises a guide RNA that is capable of localizing to the target sequence of the target gene.  [Claim 20].  20. The method of any one of claims 1 to 19, wherein the chromatin DNA is in the form of DNA comprising one or more selected from non-DNA chromatin components consisting of histones, nonhistone proteins, and RNA.