KR102210700B1 - Method of identifying base editing using adenosine deaminase - Google Patents

Abstract

A composition for correcting a base comprising a deaminase and a target-specific nuclease, and a method for correcting a base using the composition for correcting a base and a method for producing a genetically modified animal are provided.

Description

Method of identifying base editing using adenosine deaminase {Method of identifying base editing using adenosine deaminase}

It relates to a base calibration verification technology using adenosine deaminase, a composition for base calibration verification comprising adenosine deaminase, and a base calibration verification method comprising the step of treating DNA with adenosine deaminase do.

The CRISPR/Cas9 system has been used for genome editing of various animals and plants including human cells, but in the case of Cas9, DNA insertion or deletion occurs at the target site due to DNA cleavage. To compensate for these shortcomings, C to T(U) (or at a lower frequency, C to G or A) without DNA cleavage by linking cytosine deaminase to Cas9 nickase recently. Transformation) transformation method was developed (Komor, AC, Kim, YB, Packer, MS, Zuris, JA & Liu, DR Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage.Nature 533, 420-424 (2016)).

However, using cytosine deaminase, C to T (or at a lower frequency, C to G or A), or G at the opposite strand corresponding to C to A (or at a lower frequency). , Converting G to C or T) is possible, but a method of converting A or T, which is a purine base to another base, has not been known.

In the present specification, adenosine deaminase is targeted-specific using digested genome sequencing (Digenome-seq; a method of distinguishing the cut position by identifying at a glance before and after target-specific nuclease treatment). Provides a method for confirming the base correction of an adenosine nucleotide correction gene scissors made by binding to an enemy nuclease (e.g., Cas9 nickase or catalytically deficient Cas9 (dCas9), etc.), through which the adenosine nucleotide correction gene scissors The specificity of the base correction by can be evaluated.

Adenine Base Editors (ABEs) consisting of S. pyogenes- derived Cas9 nickase and E. coli- derived engineered adenine deaminase enable efficient adenine/guanine single nucleotide conversion in a guide RNA targeting method, allowing animals (e.g., humans, etc. Of mammals, rodents such as mice, etc.) and/or inducing or correcting point mutations in the cells of plants. Since ABE recognizes other off-target sites such as Cas9 and BE (cytosine base editor), which are other gene correction means, it is necessary to develop an independent method to evaluate the efficiency. In the present specification, a method of evaluating the target specificity of the entire genome of ABE through whole genome sequencing (WGS) is presented. Specifically, the whole genome sequencing may be Digenome-seq. The Digenome-seq based method provided herein is sensitive enough to capture ABE non-target locations with a substitution frequency of 0.1%. In addition, it is proposed that the use of pre-assembled ABE ribonucleic acid protein, modified guide RNA, and Sniper-ABE can reduce ABE off-target activity in animal cells such as humans.

One example is

(a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid containing the nucleic acid molecule, and (2) a target-specific nuclease, a target-specific nuclease Introducing a nucleic acid molecule or a plasmid containing the nucleic acid molecule into an isolated cell or treating the isolated DNA to cut one of the DNA double strands; And

(b) a single-strand specific endonuclease (e.g., endonuclease V) or Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII (endonuclease) to the DNA from which one of the DNA double strands is cut VIII) of the combination) to cut the remaining DNA strands.

It provides a method of generating a double strand break in DNA using adenosine deaminase comprising a.

Another example is,

(a) (1) adenosine deaminase, a nucleic acid molecule encoding the same, or a plasmid containing the nucleic acid molecule, and (2) a target-specific nuclease, a nucleic acid molecule encoding the same, or a nucleic acid molecule comprising the Introducing the plasmid into the isolated cells or treating the isolated DNA to cut one of the DNA double strands;

(b) a single-strand specific endonuclease (e.g., endonuclease V) or Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII (endonuclease) to the DNA from which one of the DNA double strands is cut A combination of VIII)) to cleave the remaining DNA strands to form a double-strand break; And

(c) performing whole genome sequencing on the double-stranded DNA

It provides a DNA sequence analysis method comprising a.

Another example is,

(a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid containing the nucleic acid molecule, and (2) a target-specific nuclease, a target-specific nuclease Introducing a nucleic acid molecule or a plasmid containing the nucleic acid molecule into an isolated cell or treating the isolated DNA to cut one of the DNA double strands;

(b) a single-strand specific endonuclease (e.g., endonuclease V) or Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII (endonuclease) to the DNA from which one of the DNA double strands is cut A combination of VIII)) to cleave the remaining DNA strands to form a double-strand break;

(c) performing whole genome sequencing on the double-stranded DNA; And

(d) confirming the cut position in the sequence read obtained by the sequencing

Containing,

A method of detecting a cleavage site or an off-target site of a target-specific nuclease is provided.

By directing evolution of adenosine deaminase, A (adenosine) among the bases of DNA was deaminated and replaced with I (inosine). More specifically, Cas9 nickase (nCas9) or catalytically deficient Cas9 (dCas9) is bound to adenosine deaminase that has been directed to work on DNA, and then when introduced into cells, A of DNA is G ( I) (or at a lower frequency, A to C or T).

Based on these matters, a technique for applying adenosine deaminase to DNA sequencing is provided.

One example is

(a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid containing the nucleic acid molecule, and (2) a target-specific nuclease, a target-specific nuclease Introducing a nucleic acid molecule or a plasmid containing the nucleic acid molecule into an isolated cell or treating the isolated DNA to cut one of the DNA double strands; And

(b) a single-strand specific endonuclease (e.g., endonuclease V) or Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII (endonuclease) to the DNA from which one of the DNA double strands is cut VIII) of the combination) to cut the remaining DNA strands.

It provides a method of generating a double strand break in DNA using adenosine deaminase comprising a.

Another example is,

(a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid containing the nucleic acid molecule, and (2) a target-specific nuclease, a target-specific nuclease Introducing a nucleic acid molecule or a plasmid containing the nucleic acid molecule into an isolated cell or treating the isolated DNA to cut one of the DNA double strands;

(b) a single-strand specific endonuclease (e.g., endonuclease V) or Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII (endonuclease) to the DNA from which one of the DNA double strands is cut A combination of VIII)) to cleave the remaining DNA strands to form a double-strand break; And

(c) performing whole genome sequencing on the double-stranded DNA

It provides a DNA sequence analysis method comprising a.

Another example is,

(a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid comprising the nucleic acid molecule, and (2) a target-specific nuclease, a target-specific nuclease Introducing a nucleic acid molecule or a plasmid containing the nucleic acid molecule into an isolated cell or treating the isolated DNA to cut one of the DNA double strands;

(b) a single-strand specific endonuclease (e.g., endonuclease V or Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII) to the DNA from which one of the DNA double strands is cut ) To form a double-strand break by cutting the remaining DNA strands;

(c) performing whole genome sequencing on the double-stranded DNA; And

(d) confirming the cut position in the sequence read obtained by the sequencing

Containing,

A method of detecting a cleavage site or an off-target site of a target-specific nuclease is provided.

The method for detecting the off-target site is, after the step (d), when the identified cut location is not an on-target site, the off-target site It may further include a step (step (e)) determining as ).

Another example is,

(a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid containing the nucleic acid molecule, and (2) a target-specific nuclease, a target-specific nuclease Introducing a nucleic acid molecule or a plasmid containing the nucleic acid molecule into an isolated cell or treating the isolated DNA to cut one of the DNA double strands;

(b) a single-strand specific endonuclease (e.g., endonuclease V or Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII) to the DNA from which one of the DNA double strands is cut ) To form a double-strand break by cutting the remaining DNA strands;

(c) performing whole genome sequencing on the double-stranded DNA; And

(d) confirming the cut position in the sequence read obtained by the sequencing

Containing,

It provides a method of confirming the calibration efficiency and/or accuracy of a target-specific nuclease.

The method for confirming the calibration efficiency of the target-specific nuclease is, after the step (d), when the identified cleaved position is not an on-target site, an off-target site Determining (step (e)) and measuring the degree of cutting at the non-target location (the number of non-target locations and/or the frequency of cutting at the non-target location) and comparing it with the degree of cutting of the comparison object (e-1 ) May be additionally included, and in this case, it may be determined that the lower the degree of cutting at the non-target position, the higher the calibration efficiency and/or accuracy. The comparison object may be a target-specific nuclease for the target sequence of any target DNA, and in one example, a commonly used or known target-specific nuclease (e.g., a combination of RGEN and guide RNA) is selected. It can be either.

In the above step (a), (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid containing the nucleic acid molecule, and (2) a target-specific nuclease, a target-specific nuclease. The nucleic acid molecule encoding the clease, or the plasmid containing the nucleic acid molecule may be simultaneously (together) treated with the isolated cells or the isolated DNA, and in one example, adenosine deaminase and a target-specific nuclease are fused. Fusion protein (e.g., adenosine deaminase may be located at the N-terminus and target specific nuclease may be located at the C-terminus), or a nucleic acid sequence encoding the fusion protein or a plasmid containing the nucleic acid sequence I can. In this case, (3) according to the target-specific nuclease, an appropriate guide RNA, or a nucleic acid molecule encoding the same, or a plasmid containing the same may be further included.

In the step (a), the conversion of adenosine to inosine in the target site (17 to 23 nt toward the 5'end of PAM) of the strand in which the PAM sequence is present in the double strand of DNA by adenosine deaminase Occurs, and single-stranded cleavage (nick) occurs in the opposite strand (complementary strand) of the strand in which the adenosine-inosine conversion has occurred (ie, the strand in which the PAM sequence is present) by the target-specific nuclease.

In the step (b), in the case of using endonuclease V, the adenosine of the strand opposite to the strand in which the single strand was cleaved by the step (a) (that is, the strand in which adenohin-inosine conversion occurred) The 3'end of the nucleotide located within 5 nt, within 4 nt, within 3 nt, within 2 nt, or within 1 nt in the 5'direction and/or 3'direction from the mutant position converted to inosine is cut. In the above step (b), when a combination of Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII is used, inosine converted in step (a) is removed by AAG and the AP site (apurinic site) is generated, and endonuclease VIII recognizes the AP site and cleaves the 3'and 5'ends of the AP site. By performing this step (b), single-stranded cleavage occurs in the strand opposite to the strand where the single-stranded cleavage occurred in step (a), resulting in double-stranded cleavage.

Another example is

(1) adenosine deaminase, a nucleic acid molecule encoding an adenosine deaminase, or a plasmid containing the nucleic acid molecule,

(2) a target-specific nuclease, a nucleic acid molecule encoding a target-specific nuclease, or a plasmid containing the nucleic acid molecule, and

(3) Single-strand specific endonuclease (e.g., endonuclease V) or a combination of Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII (endonuclease VIII))

It provides a composition for DNA double strand breaks (double strand breaks; DSBs) comprising a.

In the methods and compositions provided herein, the DNA to be genomic correction (base correction) and/or cleavage may be genomic DNA or DNA fragment (PCT product), and present in or isolated from cells. Can be. The cells may be eukaryotic cells such as humans and mammals such as mice. In addition, in the methods and compositions provided herein, adenosine deaminase and target-specific nuclease are a fusion protein directly or indirectly linked through a peptide linker or a nucleic acid molecule encoding the same or a plasmid comprising the nucleic acid molecule. Can be used in the form.

Further, in the methods and compositions provided herein, a target specific nuclease may be used or included with a guide RNA.

In this case, adenosine deaminase, target specific nuclease, and guide RNA are

(i) a mixture of adenosine deaminase, target specific nuclease, and guide RNA,

(ii) a mixture of an adenosine deaminase-encoding nucleic acid sequence or a plasmid containing the same, a target-specific nuclease-encoding nucleic acid sequence or a plasmid containing the same, and a plasmid containing a guide RNA or a nucleic acid molecule encoding the same,

(iii) a mixture of a fusion protein and guide RNA to which adenosine deaminase and target specific nuclease are linked,

(iv) a mixture of a fusion protein-encoding nucleic acid molecule to which adenosine deaminase and target-specific nuclease are linked or a plasmid containing the nucleic acid molecule, and a plasmid containing a guide RNA or a nucleic acid molecule encoding the same, and

(v) Complex of adenosine deaminase and target-specific nuclease-linked fusion protein and guide RNA (ribonucleic acid protein, RNP) or mixture

It may be used in one or more forms selected from the group consisting of (eg, introduced into isolated cells or treated with isolated DNA or included in a composition).

The plasmid may be any plasmid including an expression system capable of inserting the adenosine deaminase-encoding gene and/or an inactivated target-specific nuclease-encoding gene and expressing it in a host cell. The plasmid contains elements for expression of a gene of interest, and may include a replication origin, a promoter, an operator, a transcription termination sequence, etc., and into the genome of a host cell. An appropriate enzyme site (e.g., a restriction enzyme site) for the introduction of and/or optionally a selectable marker to confirm successful introduction into a host cell and/or a ribosome binding site (RBS) for translation into a protein and / Or may further include an electronic control factor. The plasmid is a plasmid used in the art, such as pcDNA series, pSC101, pGV1106, pACYC177, ColE1, pKT230, pME290, pBR322, pUC8/9, pUC6, pBD9, pHC79, pIJ61, pLAFR1, pHV14, pGEX series, pET series. , pUC19, etc. may be one or more selected from the group consisting of, but is not limited thereto. The host cell is a cell to which base correction or double-strand break is introduced by the adenosine deaminase (e.g., a eukaryotic cell such as a mammal such as a human cell) or the adenosine deaminase encoding gene and/or an inactivated target It can be selected from all cells (eg, E. coli, etc.) capable of expressing a specific nuclease-encoding gene to express adenosine deaminase and an inactivated target-specific nuclease.

In the present specification, the term digested genome sequencing (Digenome-seq) technology refers to sequence analysis of a genome cut by a nuclease, and the nuclease non-target effect in the entire genome in a cell (off -target effect) can be applied to in vitro nuclease-digested whole-genome sequencing. Sequence reads having the same 5'end at the cleavage site of the nuclease are generated, and these can be identified by a computer by an appropriate program (eg, Digenome program). In one example, the cleavage genome sequencing is performed in steps (a), (b), and in the method for sequencing genomic DNA described above or for detecting off-target sites of nucleases. (c), or may be defined as including steps (a), (b), (c), and (d). In other words, steps (a), (b), and (c), or steps (a), (b), (c), and (d) may be performed by cutting dielectric sequencing.

As used herein, the term "base change (or base substitution)" means that a change (eg, substitution) has occurred in a nucleotide containing a corresponding base, and is the same as "nucleotide change (or nucleotide substitution)" It can be used in the sense, and this base variation can occur in one or both of the alleles.

The genome editing and/or gene editing technology is a technology capable of introducing a target-oriented mutation into the genome sequence of an animal or plant cell including human cells, and knock-out or knock-in a specific gene , It can be carried out in various forms, such as introducing a mutation into a non-coding DNA sequence that does not produce a protein. The method proposed herein is to detect the non-target position of the target-specific nuclease used in the genome editing and/or gene editing technology, which is a target-specific nuclease system that works specifically only at the target position. It can be usefully used to develop.

The step (a) is a step of cutting one of the DNA double strands by treating adenosine deaminase and a target-specific nuclease on the isolated cell or the living body or genomic DNA isolated from the cell. This is the step of cleaving genomic DNA into nucleases that act specifically on a specific target in vitro . The nuclease has the potential to cleave other sites, that is, non-target sites, depending on the specificity, even if it is produced specifically for the target. Therefore, as a result, by the step (a), a genomic DNA fragment in which a specific position is cut by cutting a target position or a plurality of non-target positions, which is a position where the target-specific nuclease used can have activity with respect to genomic DNA ( lead).

The isolated genomic DNA may be isolated from a non-transformed cell (wild type cell) and/or a cell transformed so that the target-specific nuclease is expressed or has nuclease activity, and the ratio of the target-specific nuclease Depending on the purpose of detecting the target position, it may be used without limitation in its origin. The cell from which the genome is isolated may be selected from all prokaryotic cells and eukaryotic cells (eg, mammalian cells such as humans, eukaryotic plant cells, etc.).

The adenosine deaminase is an enzyme involved in purine metabolism (EC 3.5.4.4), and refers to all enzymes that function to convert adenosine into inosine by deamination (deamination of an amino group with a keto group). . In one example, the adenosine deaminase may be from a prokaryotic cell or a eukaryotic cell, such as a eukaryotic animal (e.g., fish, amphibians, reptiles, birds, mammals, etc.), or from a eukaryotic plant, such as human adenosine deaminase. (E.g., GenBank Accession No. NP_000013.2 (coding gene: NM_000022.3), NP_001308979.1 (coding gene: NM_001322050.1), NP_001308980.1 (coding gene: NM_001322051.1), etc.), mouse adenosine deaminase (E.g., GenBank Accession No. NP_001258981.1 (coding gene: NM_001272052.1), NP_031424.1 (coding gene: NM_007398.4), etc.), one selected from the group consisting of E. coli TadA (NP_417054.2), etc. It can be more than that. The adenosine deaminase may be used in the form of a protein or a DNA encoding it (which may be optionally included in an appropriate recombinant vector), or in the form of an mRNA encoding the same. In addition, adenosine deaminase is a conventional adenosine deaminase, such as E. In prokaryotic cells such as coli , it may be directed evolution (DE). Directed evolution (DE) is one of the protein engineering methods, which is a mutagenesis process (generating a variant library), a selection process (expressing the variant and isolating a variant having a desired function (mutation) from among them), and amplification or It can involve a reproductive process (creating a template for the next round) and can be performed in vivo (in vivo) or in vitro. In one example, the artificially evolved adenosine deaminase may be an artificially evolved E. coli TadA (NP_417054.2) from E. coli . In one example, the artificially evolved adenosine deaminase is the 22nd amino acid residue (W), the 35th amino acid residue (H), and the 47th amino acid residue in the amino acid sequence excluding methionine (M) at the N terminal of NP_417054.2. (P), 50th amino acid residue (R), 83rd amino acid residue (L), 105th amino acid residue (A), 107th amino acid residue (D), 122nd amino acid residue (H), 145th amino acid residue (S ), 146th amino acid residue (D), 151st amino acid residue (R), 154th amino acid residue (E), 155th amino acid residue (I), 156th amino acid residue (K) (above, the amino acid position is NP_417054.2 One or more amino acids selected from the group consisting of (starting from the second amino acid of) and the like may be substituted or deleted with an amino acid different from the original (wild type) (see Reference Example 2), but are not limited thereto.

The target-specific nuclease is also called a programmable nuclease, and collectively refers to all types of nucleases capable of cutting by recognizing a specific location on a genomic DNA of interest.

The target-specific nuclease as used herein may be any nuclease having an activity of cleaving one strand of a DNA double strand.

In one embodiment, the target-specific nuclease is a Cas protein (eg, Cas9 protein (CRISPR (Clustered regularly interspaced short palindromic repeats) associated protein 9)), Cpf1 protein (CRISPR from Prevotella and Francisella 1), etc. And/or it may be one or more selected from the group consisting of nucleases (eg, endonucleases) involved in the type V CRISPR system. In this case, the target-specific nuclease may further include a target DNA-specific guide RNA for guiding to a target site of genomic DNA. The guide RNA may be transcribed in vitro, for example, may be transcribed from an oligonucleotide double strand or a plasmid template, but is not limited thereto. The target-specific nuclease may act in the form of a ribonucleic acid protein (RNP) by forming a ribonucleic acid-protein complex bound to a guide RNA (RNA-Guided Engineered Nuclease).

Cas protein is a major protein component of the CRISPR/Cas system, and is a protein capable of forming an activated endonuclease or nickase.

Cas protein or gene information can be obtained from a known database such as GenBank of NCBI (National Center for Biotechnology Information). For example, the Cas protein,

Streptococcus sp. ( Streptococcus sp.), such as a Cas protein derived from Streptococcus pyogenes , such as a Cas9 protein (eg, SwissProt Accession number Q99ZW2 (NP_269215.1));

Campylobacter genus, such as Campylobacter jejuni ( Campylobacter jejuni ) derived Cas protein, such as Cas9 protein;

Streptococcus genus, for example, Streptococcus Thermo filler's (Streptococcus thermophiles) or Streptococcus aureus (Streptocuccus aureus ) derived Cas protein, such as the Cas9 protein;

Neisseria meningiditis meningitidis ) derived Cas proteins, such as Cas9 protein;

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

Cas protein, such as Cas9 protein from the genus Francisella , for example, Francisella novicida

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

Cpf1 protein is a novel CRISPR system endonuclease that is distinct from the CRISPR/Cas system, and is relatively small in size compared to Cas9 and does not require tracrRNA, and can act by a single guide RNA. In addition, it recognizes a thymine-rich PAM (protospacer-adjacent motif) sequence and cuts a double chain of DNA to create a cohesive end (cohesive double-strand break).

For example, the Cpf1 protein is Candidatus genus, Lachnospira genus, Butyrivibrio genus, Peregrinibacteria , Acidominococcus genus, Porphyromonas ( Porphyromonas ) genus, Prevotella genus, Francisella genus, Candidatus metanoplasma ( Candidatus) Methanoplasma ), or Eubacterium ( Eubacterium ) may be derived from the genus, for example, Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus , Peregrinibacteria bacterium (GW2011_GWA_33_10), Acid aminococus . (BV3L6), Porphyromonas macacae , Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis , Prevotella disiens , Moraxella bovoculi (237), Smiihella sp. (SC_KO8D17), Leptospira inadai , Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum , Candidatus Paceibacter , Eubacterium It may be derived from microorganisms such as eligens , but is not limited thereto.

The target-specific 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 target-specific nuclease (eg, Cas9, Cpf1, etc.) may be a recombinant protein made by recombinant DNA. Recombinant DNA (rDNA) refers to a DNA molecule that has been artificially made by a gene recombination method such as molecular cloning to contain heterogeneous or allogeneic genetic material obtained from various organisms. For example, when the recombinant DNA is expressed in an appropriate organism to produce a target-specific nuclease ( in vivo or in vitro ), the recombinant DNA is a codon optimized for expression in the organism among the codons encoding the protein to be produced. It may be to have a nucleotide sequence reconstructed by selecting.

The target-specific nuclease used herein may be a mutated (inactivated) mutant target-specific nuclease. The mutant (inactivated) target-specific nuclease may mean that it has been mutated to lose the endonuclease activity that cuts all of the DNA double strands, for example, the endonuclease activity that cuts all the DNA double strands. 1 selected from mutated target-specific nucleases mutated to have nickase activity and mutated target-specific nucleases mutated to lose both endonuclease activity and nickase activity that cleaves both DNA double strands It can be more than a species. When the mutant target-specific nuclease has a nickase activity, the strand in which the base conversion occurs simultaneously or sequentially regardless of the sequence or at the same time as the base conversion by the deaminase (eg, adenosine to inosine conversion) Alternatively, a single-stranded nick can be introduced in the opposite strand (e.g., the opposite strand of the strand where base transformation has occurred) (e.g., in the opposite strand of the strand where the PAM is located, 3 in the direction of the 5'end of the PAM sequence). A nick is introduced at a position between the fourth nucleotide and the fourth nucleotide). Such target-specific nuclease mutation (eg, amino acid substitution) may occur at least in the catalytically active domain of the nuclease (eg, RuvC catalytic domain in the case of Cas9). In one example, when the target-specific nuclease is a Cas9 protein derived from Streptococcus pyogenes (SwissProt Accession number Q99ZW2 (NP_269215.1)), the mutation is a catalytic aspartate residue (10th position) Aspartic acid (D10), etc.), glutamic acid at position 762 (E762), histidine at position 840 (H840), asparagine at position 854 (N854), asparagine at position 863 (N863), aspartic acid at position 986 Acid (D986), phenylalanine at position 539 (F539), methionine at position 763 (M763), lysine at position 890 (K890), and the like. I can. In this case, any other amino acid to be substituted may be alanine, but is not limited thereto.

In another example, the mutated target-specific nuclease may have been mutated to improve specificity (accuracy). In one example, the mutated target-specific nuclease is in the group consisting of F539, M763, and K890 of Cas9 protein (eg, SwissProt Accession number Q99ZW2 (NP_269215.1)) derived from Streptococcus pyogenes . A variant in which at least one selected amino acid residue is substituted or deleted with an amino acid different from the original amino acid (more specifically, a variant comprising at least one mutation selected from the group consisting of F539S, M763I, and K890N, e.g., F539S, M763I , And a variant having both the K890N mutation (Sniper-Cas9)).

In another example, the mutated (inactivated) target-specific nuclease has a different D10 from the original Cas9 protein (eg, SwissProt Accession number Q99ZW2 (NP_269215.1)) derived from Streptococcus pyogenes . It is substituted with an amino acid (e.g., alanine (A)) to have Nikase activity, and in addition, at least one amino acid residue selected from the group consisting of F539, M763, and K890 is substituted with an amino acid different from the original (e.g., F539S , M763I, K890N), and the specificity (accuracy) may be improved.

In another example, the mutant target-specific nuclease may be mutated to recognize a PAM sequence different from that of the wild-type Cas9 protein. For example, the mutant target-specific nuclease is at least one of aspartic acid at position 1135 (D1135), arginine at position 1335 (R1335), and threonine at position 1337 (T1337) of the Cas9 protein derived from Streptococcus pyogenes. , For example, all three are substituted with other amino acids, and may be mutated to recognize an NGA different from the PAM sequence (NGG) of wild-type Cas9 (N is any base selected from A, T, G, and C).

In one example, the mutant target-specific nuclease is from the amino acid sequence of the Cas9 protein derived from Streptococcus piyogens,

(1) D10, H840, or a combination thereof (D10 + H840);

(2) D10 and at least one combination selected from the group consisting of F539, M763, and K890 (eg, D10 + F539 + M763 + K890);

(3) D1135, R1335, T1337, or a combination thereof (eg, D1135 + R1335 + T1337); or

(5) Combination of two or more selected from (1) to (3)

The amino acid substitution may have occurred in

As used herein, the'other amino acids' are alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, Arginine, histidine, lysine, among all known variants of the above amino acids, refers to an amino acid selected from amino acids except for amino acids in which the wild-type protein has an original mutation position. In one example, the'other amino acid' may be alanine, valine, glutamine, or arginine.

In one example, the mutant target-specific nuclease is a modified Cas9 protein that has lost endonuclease activity (e.g., has nickase activity, loses both endonuclease activity and nickase activity), or wild-type It may be to recognize a PAM sequence different from Cas9. For example, the modified Cas9 protein, in the Cas9 protein derived from Streptococcus pyogenes ,

(1) A mutation (eg, substitution with another amino acid) is introduced at the D10 or H840 position, resulting in loss of double-stranded endonuclease activity and a modified Cas9 having nickase activity, or Streptococcus pyogenes Modified Cas9 protein in which mutations (eg, substitutions with other amino acids) are introduced at both the D10 and H840 positions of the Cas9 protein, thereby losing both the double-stranded endonuclease activity and the nickase activity;

(2) In addition to the D10 position, a mutation (e.g., substitution with another amino acid) is introduced at one or more positions selected from the group consisting of F539, M763, and K890, resulting in loss of double-stranded endonuclease activity and nickase activity. Modified Cas9 protein having improved specificity while having;

(3) a modified Cas9 protein that recognizes a PAM sequence different from the wild type by introducing a mutation (eg, substitution with another amino acid) in one or more of D1135, R1335 and T1337 or both; or

(3) Modified Cas9 protein into which two or more of (1) to (3) are introduced

Can be

For example, the mutation at the D10 position of the CAs9 protein is a D10A mutation (means a mutation in which D, which is the 10th amino acid of the Cas9 protein, is substituted with A; hereinafter, the mutation introduced into Cas9 is indicated in the same manner). And, the mutation at the H840 position may be a H840A mutation, and the mutations at the D1135, R1335, and T1337 positions may be D1135V, R1335Q, and T1337R, respectively, and the mutations at the F539S, M763I, and K890N positions are respectively It may be F539S, M763I, and K890N.

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 may be a recombinant protein made by recombinant DNA. Recombinant DNA (rDNA) refers to a DNA molecule that has been artificially made by a gene recombination method such as molecular cloning to contain heterogeneous or allogeneic genetic material obtained from various organisms. For example, when recombinant DNA is expressed in an appropriate organism to produce a protein ( in vivo or in vitro ), the recombinant DNA is reconstructed by selecting a codon optimized for expression in the organism from among the codons encoding the protein to be produced. It may have a nucleotide sequence.

The nuclease may be used in the form of a protein, a nucleic acid molecule encoding the same (DNA or mRNA), a ribonucleic acid protein bound to a guide RNA, a nucleic acid molecule encoding the ribonucleic acid protein, or a recombinant vector containing the nucleic acid molecule. I can.

The nuclease or the nucleic acid molecule encoding the nuclease may be in a form capable of being delivered, acting, and/or expressed into the nucleus.

The nuclease may be in a form that is easy to be introduced into a cell. For example, the nuclease may be linked to a cell penetrating peptide and/or a protein transduction domain. The protein transduction domain may be poly-arginine or a TAT protein derived from HIV, but is not limited thereto. Since various types of cell penetrating peptides or protein transduction domains are known in the art in addition to the examples described above, those skilled in the art are not limited to the above examples and can apply various examples.

In addition, the nuclease or the encoding nucleic acid molecule may further include a nuclear localization signal (NLS) sequence or a sequence encoding the same. Therefore, the expression cassette containing the nucleic acid molecule encoding the nuclease may include a regulatory sequence such as a promoter sequence for expressing the nuclease, or in addition to it, an NLS sequence (eg, SV40 NLS, etc.). have. Such NLS sequences are well known in the art.

The nuclease or a nucleic acid molecule encoding the nuclease may be linked to a tag for separation and/or purification or a nucleic acid sequence encoding the tag. As an example, the tag may be appropriately selected from the group consisting of small peptide tags such as His tag, Flag tag, S tag, GST (Glutathione S-transferase) tag, MBP (Maltose binding protein) tag, etc. It doesn't work.

In the present invention, the term "guide RNA" refers to a target DNA-specific RNA (eg, RNA capable of hybridizing with a target site of DNA), which binds to a nucleotide such as a Cas protein, Cpf1, etc. and leads to the target DNA. Plays a role.

The guide RNA may be appropriately selected according to the type of nuclease to form the complex and/or the microorganism derived therefrom. For example, the guide RNA is

CRISPR RNA (crRNA) comprising a site capable of hybridizing with a DNA target site;

Trans- activating crRNA (tracrRNA) including a site that interacts with endonucleotides such as Cas protein, Cpf1, and the like; And

A single guide RNA (sgRNA) in the form of a fusion of the main regions of the crRNA and tracrRNA (eg, a hybridization site of crRNA and an interaction site of tracrRNA)

It may be one or more selected from the group consisting of,

Specifically, it may be a dual RNA (dual RNA) including CRISPR RNA (crRNA) and a trans- activating crRNA (tracrRNA), or a single guide RNA (sgRNA) including a major site 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 binding a Cas protein. More specifically, a portion having a sequence complementary to a sequence in the target DNA, a hairpin structure for binding to a Cas protein, and a Terminator sequence may be included. The structure described above may be sequentially present in the order of 5'to 3', but is not limited thereto. Any type of guide RNA can be used in the present invention as long as the guide RNA includes a major portion of crRNA and tracrRNA and a complementary portion of the target DNA.

For example, the Cas9 protein is used as two guide RNAs for target gene correction, that is, CRISPR RNA (crRNA) having a nucleotide sequence hybridizable with the target sequence site of the target gene and trans- activating crRNA (tracrRNA; Cas9 protein) that interacts with the Cas9 protein. Interacts with), and these crRNA and tracrRNA can be used in the form of a double-stranded crRNA:tracrRNA complex bonded to each other, or a single guide RNA (sgRNA) form by being linked through a linker. In one example, when using the Cas9 protein derived from Streptococcus pyogenes , the sgRNA includes at least a part or all of the crRNA comprising at least a hybridizable nucleotide sequence of the crRNA of the Cas9 and a site that interacts with the Cas9 protein of the tracrRNA of Cas9. Some or all of the tracrRNA may form a hairpin structure (stem-loop structure) through a nucleotide linker (in this case, a nucleotide linker may correspond to a loop structure).

The guide RNA, specifically, crRNA or sgRNA, includes a sequence complementary to a sequence in the target DNA, and at least one at the 5'end of the crRNA or sgRNA upstream region, specifically the crRNA of sgRNA or dualRNA, such as 1-10 , 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 Cpf1, the guide RNA may include crRNA, and may be appropriately selected according to the type of Cpf1 protein to form a complex 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 Cpf1) (i.e., the derived microorganism), which can be easily known by those of ordinary skill in the art to which this invention belongs. It is a matter.

In one example, when using the Cas9 protein derived from Streptococcus pyogenes as a target-specific nuclease, crRNA can be expressed by the following general formula 1:

5'-(N _cas9 ) _l -(GUUUUAGAGCUA)-(X _cas9 ) _m -3' (general formula 1)

In the general formula 1,

N _cas9 is a targeting sequence, that is, a site determined according to the sequence of the target site of the target gene (that is, a sequence capable of hybridizing with the sequence of the target site), and l is included in the targeting sequence. It represents the number of nucleotides and may be an integer of 17 to 23, 17 to 22, 18 to 23, or 18 to 22, such as 17, 18, 19, 20, 21, or 22;

The region containing 12 consecutive nucleotides (GUUUUAGAGCUA) adjacent to each other in the 3'direction of the target sequence is an essential part of crRNA,

X _cas9 is a site containing m nucleotides located at the 3'end of the crRNA (that is, adjacent to the 3'direction of the essential part of the crRNA), where m is an integer of 8 to 12, such as 11 days. And, the m nucleotides may be the same or different from each other, and each may be independently selected from the group consisting of A, U, C and G.

In one example, the X _cas9 may include UGCUGUUUUG, but is not limited thereto.

In addition, the tracrRNA can be represented by the following general formula 2:

5'-(Y _cas9 ) _p -(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC)-3' (general formula 2)

In the above general formula 2,

The site marked with 60 nucleotides (UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC) is an essential part of tracrRNA,

Y _cas9 is a site containing p nucleotides located adjacent to the 5'end of the essential part of the tracrRNA, p may be an integer of 6 to 20, such as an integer of 8 to 19, and the p nucleotides are the same. Or may be different, and each may be independently selected from the group consisting of A, U, C and G.

In addition, the sgRNA is a crRNA portion including the targeting sequence and essential site of the crRNA and the tracrRNA portion including the essential portion (60 nucleotides) of the tracrRNA to form a hairpin structure (stem-loop structure) through an oligonucleotide linker. (In this case, the oligonucleotide linker corresponds to the loop structure). More specifically, the sgRNA is a double-stranded RNA molecule in which a crRNA portion including a targeting sequence of crRNA and an essential portion and a tracrRNA portion containing an essential portion of tracrRNA are bonded to each other, and the 3'end of the crRNA site and 5 of the tracrRNA site 'It may have a hairpin structure whose ends are linked through an oligonucleotide linker.

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

5'-(N _cas9 ) _l -(GUUUUAGAGCUA)-(oligonucleotide linker)-(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC)-3' (general formula 3)

In the general formula 3, (N _cas9 ) _l is the targeting sequence as described in the above general formula 1.

The oligonucleotide linker included in the sgRNA may include 3 to 5, such as 4 nucleotides, and the nucleotides may be the same or different from each other, and each independently selected from the group consisting of A, U, C and G Can be.

The crRNA or sgRNA may further include 1 to 3 guanine (G) at the 5'end (ie, the 5'end of the targeting sequence site of the crRNA).

The tracrRNA or sgRNA may further include a terminating site including 5 to 7 uracils (U) at the 3'end of the essential portion (60 nt) of the tracrRNA.

The target sequence of the guide RNA is adjacent to the 5'of the PAM (Protospacer Adjacent Motif sequence (5'-NGG-3' in the case of S. pyogenes Cas9 (N is A, T, G, or C)) on the target DNA. It may be from about 17 to about 23 or from about 18 to about 22, such as 20 contiguous nucleic acid sequences located therein.

The targeting sequence of the guide RNA hybridizable with the target sequence of the guide RNA is the DNA strand where the target sequence is located (i.e., the PAM sequence (5'-NGG-3' (N is A, T, G, or C)) A nucleotide sequence of 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, 99% or more, or 100% of the nucleotide sequence of the complementary strand of the located DNA strand) By means of a sequence, complementary binding to the nucleotide sequence of the complementary strand is possible.

In the present 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 two DNA strands of the corresponding gene site of the target gene. At this time, since the DNA strand to which the guide RNA is actually bound is the complementary strand of the strand where the PAM sequence is located, the targeting sequence included in the guide RNA is the sequence of the target site except for changing T to U due to the nature of RNA. It will have the same nucleic acid sequence as Accordingly, in the present specification, the targeting sequence of the guide RNA and the sequence of the target site (or the sequence of the cleavage site) are represented by the same nucleic acid sequence except that T and U are mutually altered.

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

In the present invention, the term "on-target site" refers to a location at which mutation (cleavage, insertion, and/or deletion) is to be introduced using the target-specific nuclease, and according to the purpose It may be arbitrarily selected and may exist within the coding sequence of a specific gene, and may exist in a non-coding DNA sequence that does not produce a protein.

Since the target-specific nuclease has sequence specificity, it acts on the target site, but side effects may occur that act on off-target sites depending on the target sequence.

In the present specification, the term "off-target site" refers to a site having a sequence that is not identical to the target sequence of the target-specific nuclease, but the target-specific nuclease has activity. That is, it refers to a position other than the target position, which is cleaved by a target-specific nuclease. In one example, the non-target location may be used as a concept including not only an actual non-target location for a specific target-specific nuclease, but also a location likely to become a non-target location. The non-target position is not limited thereto, but may be any position other than the target position cleaved by a target-specific nuclease in vitro .

It can be caused by a variety of causes that the shear is active at positions other than the target position. For example, in the case of a non-target sequence having high sequence homology with the target position, which has a nucleotide mismatch with the target sequence designed for the target position, there is a possibility that the genetic scissors will work. The non-target position is not limited thereto, but may be a position having one or more nucleotide mismatches with the target sequence.

This can lead to unwanted gene mutations in the genome, which can be a serious problem in using the target-specific nuclease. Accordingly, the process of accurately detecting and analyzing a non-target position as well as the activity of the target-specific nuclease at the target position can be very important, and this is a target-specific nuclease that works specifically only at the target position without a non-target effect. It could be usefully used to develop clease.

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

The step (b) is a step of cutting the remaining DNA strands by treating the DNA whose one strand has been cut through the step (a) with a single-strand specific endonuclease.

The single-stranded specific endonuclease may collectively refer to all endonucleases having an activity of specifically cleaving single-stranded DNA. In one example, the single-strand specific endonuclease is endonuclease V, or 1 selected from the group consisting of a combination of endonuclease VIII and alkyladenine DNA glycosylase (Alkyladenine DNA Glycosylase; hAAG), etc. It can be more than a species. The endonuclease V may be a prokaryotic cell or a eukaryotic cell, such as a fungus, a eukaryotic animal (eg, fish, amphibians, reptiles, birds, mammals, etc.), or derived from a eukaryotic plant, for example, endonuclease V derived from E. coli. (NP_418426.2; E. coli ENDOV) may be one or more selected from the group consisting of. The single-strand-specific endonuclease (eg, endonuclease V) may be to cleave a strand converted from adenosine to inosine, and the 5'direction and/or 3 at the mutant position where the adenosine is converted to inosine It is possible to cut the 3'end of the nucleotide located within 5nt, 4nt, 3nt, 2nt, or 1nt in the direction. The endonuclease VIII serves to remove nucleotides converted from adenine to inosine by adenosine deaminase, and N-glycosylase activity to remove inosine from double-stranded DNA and an apurinic site (AP site) can be selected from one or more of all enzymes having both AP-lyase activity that cleaves the 3'and 5'ends. For example, the endonuclease VIII is E, coli endonuclease VIII (eg, GenBank Accession No. NP_415242.1, etc.), human endonuclease VIII (eg, GenBank Accession Nos. BAC06476.1, NP_001339449.1, etc.) , NP_001243481.1, NP_078884.2, NP_001339448.1, etc.), mouse endonuclease VIII (eg, GenBank Accession Nos. BAC06477.1, NP_082623.1, etc.), Escherichia coli endonuclease VIII (eg, GenBank Accession Nos.OBZ49008.1, OBZ43214.1, OBZ42025.1, ANJ41661.1, KYL40995.1, KMV55034.1, KMV53379.1, KMV50038.1, KMV40847.1, AQW72152.1, etc.) It may be more than a species, but is not limited thereto. The alkyladenine DNA glycosylase is an enzyme that catalyzes the hydrolysis of the N-glycosidic bond to remove the alkylation-damaged purine base to repair DNA.In particular, 3-methyladenine, 7-methyladenine, 7-methylguanine, 1N-ethenoadenine and remove hypoxanthine. Alkyl adenine DNA glycosylase that can be used is human alkyl adenine DNA glycosylase (hAAG; GenBank Accession no. NP_001015052.1 (coding nucleic acid sequence NM_001015052.2), NP_001015054.1 (coding nucleic acid sequence NM_001015054.2), NP_002425 .2 (coding nucleic acid sequence NM_002434.3) may be one or more selected from the group consisting of, but is not limited thereto.

Step (c) is a step of performing whole genome sequencing (WGS) using the DNA cut through step (b), and is a non-target position by searching for a sequence having homology with the sequence of the target position. Unlike the indirect method of predicting that it is carried out to detect non-target locations that are substantially cleaved by target-specific nucleases at the whole genome level.

In the present invention, the term "whole genome sequencing (WGS)" refers to a method of reading genomes in multiple multiples in 10 X, 20 X, 40 X format by next generation sequencing. "Next-generation sequencing" refers to a technology that fragments the full-length genome in a chip-based and PCR-based paired end format, and performs sequencing of the fragment at high speed based on a chemical reaction (hybridization). .

The step (d) is a step of determining the position where the DNA is cut from the sequence read obtained by the whole genome sequencing, and the target position and the non-target position of the target-specific nuclease by analyzing the sequencing data. Can be easily detected. Determining a specific location where DNA is cut from the nucleotide sequence data can be performed by various approaches, and the present invention provides various rational methods for determining 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, as an example for determining the cut position, when the nucleotide 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, etc.), The position where the 5'end is vertically aligned may mean the position where the DNA is cut. As used herein, the term "vertical alignment" means that when analyzing the whole genome sequencing result with a program such as BWA/GATK or ISAAC, adjacent Watson strand and Crick strand are each On the other hand, it refers to an arrangement in which the 5'end of two or more nucleotide sequence data starts at the same nucleotide position on the genome. Due to this, DNA fragments that are cut by a target-specific nuclease and have the same 5'end are each sequenced to appear.

That is, when a target-specific nuclease exhibits nuclease activity at a target position and a non-target position and cleaves the position, when the nucleotide sequence data is aligned, each of the commonly cut sites starts at the 5'end. Therefore, it is vertically aligned, but since the 5'end does not exist in the uncut area, it can be arranged in a staggered manner when aligned. Therefore, the vertically aligned position can be viewed as a site cut by a target-specific nuclease, which may mean a target position or a non-target position of the target-specific nuclease.

The "alignment" refers to mapping base sequence data to a reference genome, and then arranging bases having the same position in the genome according to each position. Therefore, any computer program may be used if the nucleotide sequence data can be sorted in the same manner as described above, which may be a known program known in the art, or may be selected from programs prepared for the purpose. In one embodiment, alignment was performed using ISAAC, but is not limited thereto.

As a result of alignment, it is possible to determine the position where the DNA was cut by the target-specific nuclease through a method such as finding the position where the 5'end is vertically aligned as described above, and the cut position is the target position (on- target site), it can be determined as an off-target site. In other words, a sequence identical to a nucleotide sequence designed as a target position of a target-specific nuclease is a target position, and a sequence not identical to the nucleotide sequence may be considered a non-target position. This is obvious from the definition of the non-target location described above. In particular, the non-target position may be composed of a sequence having homology to the sequence of the target position, specifically a sequence having one or more nucleotide mismatches with the target sequence, more specifically, 1 to 6 target positions. , 1 to 5, 1 to 4, 1 to 3, 1 to 2, or may have a nucleotide mismatch, but is not particularly limited thereto, and the target-specific nuclease can be cleaved Any position that can be included within the scope of the present invention. At this time, the target position may be a 15 to 30 nucleotide sequence complementary to the guide RNA, and may additionally include a sequence recognized by a target-specific nuclease (eg, a PAM sequence recognized by Cas9 in the case of Cas9).

In another example, in addition to the method of finding the position where the 5'end is vertically aligned, when a double peak pattern is shown in the 5'end plot, the position may be determined as a non-target position if the position is not a target position. When drawing a graph by 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, the double peak being cleaved by a target-specific nuclease. This is because it is represented by each strand of a double strand.

In one embodiment, genomic DNA is cut with a target-specific nuclease (e.g., RGEN), and then the whole genome is analyzed and then aligned with ISAAC, vertically aligned at the cut position, and staggered at the uncut position. It was confirmed that the pattern to be aligned was confirmed, and when this was represented by a 5'end plot, a unique pattern of double peaks appeared at the cut position.

Further, although not limited thereto, as a specific example, a position in which two or more sequence reads corresponding to the Watson strand and the Crick strand are vertically aligned is regarded as a non-target position. It can be determined, and more than 20% of the nucleotide sequence data is vertically aligned, and the position where the number of nucleotide sequence data having the same 5'end in each Watson strand and the creek strand is 10 or more is a non-target position, that is, a position where it is cut. It can be judged as.

The non-target location identification (detection) can be performed by treating genomic DNA with a target-specific nuclease in vitro . This can be checked practically appear a non-target effects in vivo (in vivo) with respect to the non-target location (detected) by the method. However, since this is only an additional verification process, it is not necessarily a step accompanying the scope of the present invention, and is only a step that can be additionally performed if necessary.

As used herein, the term "off-target effect" may be a concept distinguished from an off-target site. That is, as described above, the concept of a non-target position in the present invention refers to a position other than a target position among positions at which a target-specific nuclease can operate, and the position to be cleaved by the target-specific nuclease That being said, non-target effect refers to the effect of indel (Insertion and/or deletion) by a target-specific nuclease at a non-target location in a cell.

The term "indel" refers to mutations in which some bases are inserted and/or deleted in the nucleotide sequence of DNA. In addition, a non-target position in which the indel is generated by a target-specific nuclease is referred to as a non-target indel position. In conclusion, the non-target position in the present specification can be viewed as a concept including a non-target indel position, and it is sufficient as a position with the potential for the target-specific nuclease to have activity, and must be confirmed by indel by genetic scissors. It doesn't have to be. Meanwhile, in the present invention, a non-target location may be referred to as a candidate off-target site, and a non-target indel location may be referred to as a validated off-target site.

Specifically, the verification process is not limited thereto, but the genomic DNA is isolated from the cell expressing the target-specific nuclease for the non-target location, and the indel is identified at the non-target location of the DNA. It may be to confirm the non-targeting effect of. This may be to confirm a non-target effect by performing an indel identification method known in the art such as T7E1 analysis, mutation detection analysis using Cel-I enzyme, or targeted deep sequencing. The step of confirming the non-target effect may be to directly confirm whether indel has occurred at a non-target location. However, even if indels did not occur during the in-vivo verification process, it is not confirmed until indels occur at a frequency less than a detectable level, so it should be viewed as a supplementary means.

Checking the vertically aligned positions as described above, or simply by checking the double peaks in the 5'end plot, can sufficiently detect non-target positions, which is highly reproducible, but is a non-uniform cut pattern or low sequencing. There is a problem that some positions with depth may be omitted. Accordingly, in this specification, based on the alignment pattern of the nucleotide sequence data, a formula for calculating the DNA cleavage score at the position i of each nucleotide (ie, the nucleotide position on the genomic DNA) is provided as follows:

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 location.

In addition, through the above equation, a number of additional positions that have not been detected in the existing Digenome-seq can be detected, and through this, false-positive positions can be easily filtered out. The C value in the above formula is not limited by the embodiments of the present invention as those skilled in the art can apply an arbitrary 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 thereto, for example, for the nucleotide sequence of an arbitrary position (cut position), the score calculated by setting the C value as 1 is 2.5 or more or 0.1 or more, and homology with the on-target sequence In the case of having (e.g., in the case of including PAM (5'-NGN-3' or 5'-NNG-3') with 10 or less mismatches with the On-target sequence), the arbitrary position (cleaved Location) can be determined as a non-target location. However, the standard of the score may be appropriately adjusted or changed by a person skilled in the art according to the purpose.

In one example, the Digenome-seq method provided herein may be performed using a plurality of target-specific nucleases (eg, target-specific nucleases including a plurality of guide RNAs having different target sites), In this specification, this is referred to as "composite Digenome-seq". In this case, the target-specific nuclease may be a mixture of two or more, specifically, target-specific nucleases for 2 to 100 targets, but is not limited thereto.

In the case of the complex Digenome-seq, genomic DNA is cleaved by each target-specific nuclease, so it is important to determine which gene scissors the cleavage site was cut by. This can be achieved by classifying the non-target location according to the edit distance to the target location, and it is premised that the base sequence of the non-target location has homology with the target location. Through this, the target and non-target positions for each of the genetic scissors can be clearly distinguished.

In a specific embodiment of the present invention, for RNA-guided engineered nuclease (RGEN) targeting a specific position, among non-target positions detected through Digenome-seq in the entire genome, the nucleotide mismatch with the target position is 6 or less. When homologous positions are 13,000 or less and nucleotide mismatches do not have homologous positions of 2 or less, it was confirmed that non-targeting effects can be minimized by selecting the specific position as a target position of RGEN. This is an example showing the process of establishing a preferred criterion for selecting a target location using the Digenome-seq of the present invention, and it is expected that the non-targeting effect of the gene scissors can be minimized through the Digenome-seq.

On the other hand, it was confirmed that the number of positions having homology with the sequence of the target position was detected at a small rate through Digenome-seq as the level of nucleotide mismatch increased. This is because, in selecting the target position of RGEN, the more nucleotide sequences having homology with the target sequence in the genome, especially the more nucleotide sequences having high homology, the more specific they are. The target position of the RGEN selected through this may be a non-target effect is minimized.

In another example, the off-target identification method is CIRCLE-seq (circularization for in vitro reporting of cleavage effects by sequencing; see Nature Methods 14, 607-614 (2017)) and/or SITE-seq (Cameron, P.ett. al. Mapping the genomic landscape of CRISPR-Cas9 cleavage.Nature methods 14, 600-606 (2017) reference) can also be performed (see FIGS. 2A and 2B).

By the genome sequencing technology presented in the present invention, it is possible to detect the non-target position of the gene scissors at the genome level with high reproducibility, and thus, it can be used for the production of gene scissors with high target specificity and research therefor.

UGI/USER (red; 중심원으로부터 3번째 영역), 또는 Cas9 (Green; 중심원으로부터 4번째 영역)으로 처리된 유전체 DNA를 사용하여 HEK2 (12a) 및 RNF2 (12b) 부위에 대하여 얻어진 Genome-wide Circos plots을 보여주며, 화살표는 on-target 부위를 나타낸다.
도 12c 및 도 12d는 Digenome-captured sites에서의 DNA 서열을 비교하고 WebLogo를 사용하여 얻어진 Sequence logos이다.
도 12e는 targeted deep sequencing을 사용하여 측정된 ABE7.10 off-target sites에서의 염기 교정 효율을 나타낸 그래프로서, 3' 말단 끝의 3개 뉴클레오타이드는 PAM 서열을 나타내고, 소문자는 Mismatched base를 나타낸다(Means ± s.e.m. were from three independent experiments).
도 12f는 ABE7.10-매개 치환 빈도와 Cas9-매개 indel 빈도 간의 상관성 (위쪽) 및 ABE7.10-매개 치환 빈도와 BE3-매개 치환 빈도 간의 상관성 (이래쪽)을 보여주는 Scatterplots이다.
도 13a는 일반적으로 사용되는 형태 (GX₁₉), 절단된 형태 (gX₁₈ or gX₁₇), 및 연장된 형태 (gX₂₀ 또는 ggX₂₀)의 sgRNAs의 표적 DNA-상보적 RNA 서열을 예시적으로 보여준다.
도 13b는 HEK2, RNF2, TK_EphB4, TYRO3, WEE1, HPRT-exon6, 및 HPRT-exon8를 표적으로 하는 변형 sgRNA 사용시 targeted deep sequencing에 의하여 측정된 각 변형 sgRNA 별 ABE7.10의 염기 교정 빈도 평균을 나타낸 그래프이다.
도 13c 및 13d는 HEK293T 세포에서 targeted deep sequencing으로 측정된 HPRT-Exon 6 (13c) 및 TYRO3와 HPRT-exon8 (13d)의 on- 또는 off-target sites에서의 ABE7.10-매개 염기 교정 빈도(%)를 결과를 보여주는 그래프 및 히트맵으로서, 히트맵은 GX₁₉ sgRNA를 사용한 경우와 비교한 변형 sgRNA의 상대적 특이도 (relative specificities)를 보여주며, 상대적 특이도 (Specificity ratio)는 측정 대상 sgRNA의 특이도(on-target frequency/off-target frequency)를 GX₁₉ sgRNA의 특이도 (on-target frequency/off-target frequency)로 나누어 계산된 값이고, 3' 말단 끝의 3개 뉴클레오타이드는 PAM 서열을 나타내고, 소문자는 Mismatched base를 나타낸다 (Means ± s.e.m. were from three independent experiments).
도 13e 및 13f는 ABE7.10 및 Sniper ABE7.10을 사용한 경우의 HPRT-Exon 6 (13e) 및 HPRT-exon8, HEK2, EphB4 및 TYRO3 (이상, 13f)에 부위에서의 on- 및 off-target 활성 및 특이도 비율 (specificity ratio)을 보여주는 그래프 및 히트맵으로, 3' 말단 끝의 3개 뉴클레오타이드는 PAM 서열을 나타내고, 소문자는 Mismatched base를 나타내고, specificity ratio는 다음의 수식에 의하여 계산된다: Sniper ABE7.10의 specificity (on-target frequency/off-target frequency)/ABE7.10의 specificity (on-target frequency/off-target frequency) (Means ± s.e.m. were from three independent experiments).
도 13g 및 13h는 플라스미드를 통하여 세포내로 전달되는 경우와 RNP 형태로 세포내로 전달되는 경우의 HPRT-Exon 8 (13g) 및 HPRT-exon6, HEK2, EphB4 및 TYRO3 (이상, 13h)에 부위에서의 on- 및 off-target 활성 및 특이도 비율 (specificity ratio)을 보여주는 그래프 및 히트맵으로, 3' 말단 끝의 3개 뉴클레오타이드는 PAM 서열을 나타내고, 소문자는 Mismatched base를 나타내고, specificity ratio는 다음의 수식에 의하여 계산된다: RNP 전달시의 specificity (on-target frequency/off-target frequency)/플라스미드 전달시의 specificity (on-target frequency/off-target frequency) (Means ± s.e.m. were from three independent experiments).
도 13i 및 13j는 ABE7.10 또는 Sniper ABE7.10을 변형 sgRNA와 조합하여 사용한 경우의 HPRT-exon6 부위 (13i) 및 HPRT-exon8 부위 (13j)에서의 on- 및 off-target 활성 및 특이도 비율 (specificity ratio)을 보여주는 그래프 및 히트맵으로, 3' 말단 끝의 3개 뉴클레오타이드는 PAM 서열을 나타내고, 소문자는 Mismatched base를 나타내고, specificity ratio는 다음의 수식에 의하여 계산된다: Sniper ABE7.10와 변형 sgRNA의 조합의 specificity (on-target frequency/off-target frequency)/ABE7.10의 specificity (on-target frequency/off-target frequency) (Means ± s.e.m. were from three independent experiments).1A schematically shows a genomic DNA sequence analysis process using adenosine deaminase, Cas9 nikase (D10A) (ABE) and endonuclease V.
FIG. 1B schematically shows a genomic DNA sequence analysis process using adenosine deaminase, Cas9 nickase (D10A) (ABE), Alkyladenine DNA Glycosylase (AAG), and endonuclease VIII.
Figure 2a shows the result of confirming the generation of DSB (Double Strand Break) through real-time PCR after treatment with ABE and endonuclease V.
Figure 2b shows the result of confirming the generation of DSB through real-time PCR after treatment with ABE, Alkyladenine DNA Glycosylase (AAG), and endonuclease VIII.
2C is a result of confirming the base correction after treatment with ABE and endonuclease V or treatment with ABE, Alkyladenine DNA Glycosylase (AAG), and endonuclease VIII.
3A and 3B show the results of whole genome sequencing (WGS) after treatment with ABE and endonuclease V or treatment with ABE, Alkyladenine DNA Glycosylase (AAG), and endonuclease VIII.
4 is a result of confirming the cleavage position after treatment with ABE and endonuclease V or treatment with ABE, Alkyladenine DNA Glycosylase (AAG), and endonuclease VIII.
5 schematically shows the process of sequencing genomic DNA by CIRCLE-seq and SITE-seq.
6A to 6C are graphs showing the nucleotide correction frequency (%) and insertion/deletion frequency (%) obtained using sgRNA having 0 to 4 mismatched bases with respect to the on target site, and an asterisk is relative to one enzyme. The activity ([mismatched sgRNA result]/[matched sgRNA result]) represents a mismatched sgRNA that is three times higher than the other two enzymes, and the last three nucleotides at the 3'end represent the PAM sequence, and the lowercase letters represent the mismatched nucleotides, respectively. Shown (Means ± sem were from three independent experiments).
7A is an overview of the Digenome-seq analysis method using ABE7.10 and Endo V, wherein ABE7.10 catalyzes the conversion of guanine to deoxyinosine, and Endo V recognizes deoxyinosine, and It shows that the second phosphodiester bond 3'is cleaved for oxyinosine, and the position of the triangular vertex shows the position of the phosphodiester bond cleaved by ABE7.10 nickase and Endo V.
7B is a schematic diagram of an ABE-mediated Digenome-seq workflow,
Figure 7c is a schematic diagram of a Digenome-seq assay using ABE7.10 and hAAG/Endo VIII, where ABE7.10 mediates adenine-to-inosine transformation in one strand and creates a nick in the other strand, and hAAG is inosine. Is removed to create an AP site (apurinic/apyrimidinic site), and Endo VIII (DNA glycosylase and AP-lyase) shows that the AP site is cleaved, and the triangular vertex position is ABE7.10 nickase and hAAG/Endo VIII. Shows the site cut by.
7D shows the results of treatment with ABE7.10 and Endo V on the PCR product containing the ABE7.10 target sequence, showing that a double strand break is formed in DNA only when both ABE7.10 and Endo V are treated.
Figure 7e shows the results of treatment of ABE7.10, hAAG, and Endo VIII on the PCR product containing the ABE7.10 target sequence. Double strand in DNA only when all of ABE7.10, hAAG, and Endo VIII were treated. Figure 7f shows the Sanger sequencing result, showing A-to-G conversion by ABE7.10 and DNA cleavage by Endo V.
Figure 7g is a graph showing the qRT-PCR result, showing the DNA cleavage by ABE7.10 nickase and Endo V.
7H shows the results of Sanger sequencing, A-to-G conversion when genomic DNA was treated with ABE7.10, and G-to- when genomic DNA was treated with ABE7.10, hAAG, and Endo VIII. Shows A conversion.
7i is a graph showing the qRT-PCR results, showing the results of digestion of genomic DNA with ABE7.10, hAAG, and Endo VIII.
7J is an IGV image showing a result of straight alignment of sequence reads at a HEK2 target site obtained using WGS data.
7K is an IGV image showing the results of straight alignment of sequencing reads observed after ABE7.10, hAAG, and Endo VIII treatment.
Figure 8 shows the results of SDS-PAGE analysis of ABE7.10 purified using nickel affinity chromatography and heparin bead chromatography (M: marker, I(-): cell lysate before IPTG (isoproply-1-thio-β-D) -galactopyranoside) induction, I(+): cell lysate after IPTG induction, S: soluble lysate fraction, IS: insoluble lysate fraction, FT: flow-through, W1, W2: waste after washing, Ni: Ni-NTA agarose beads after elution of bound protein, NE: protein fraction eluted from nickel beads, Hp: Heparin Sepharose 6 Fast Flow affinity resins after elution of bound protein, HE: protein fraction after purification using heparin beads.Boxed area shows ABE7.10 protein band. Indicates).
9 schematically shows an in vitro DNA cleavage scoring system for Digenome-seq analysis of ABE.
10A and 10B show the ABE7.10 off-target site across the genome identified by Digenome-seq, where 10a is intact genomic DNA (grey; first region from the center circle) and ABE7.10 + Endo V treated. Genome showing DNA cleavage scores obtained via WGS using genomic DNA (blue; second region from central circle), and ABE7.10 + hAAG + Endo VIII treated genomic DNA (red; third region from central circle). -wide Circos plots (arrows indicate on-target sites), 10b shows the number of in vitro cleavage sites in the human genome identified using ABE7.10 + Endo V or ABE7.10 + hAAG + Endo VIII. It's a Venn diagram.
10C is a sequence logos obtained by comparing DNA sequences in Digenome-captured sites and using WebLogo.
11 is an IGV image showing vertical alignment results of sequencing reads in RNF2, TYRO3, WEE1, EphB4, HPRT-exon6, and HPRT-exon8 sites.
12A and 12B show intact genomic DNA (gray; first region from center) and genomic DNA treated with ABE7.10/Endo V (blue; second region from center), BE3

UGI / USER (red; 3-th region from the center circle), or Cas9; using a treatment with (Green 4 th region from the center circle) genome DNA HEK2 Genome-wide Circos plots obtained for the (12a) and RNF2 (12b) sites are shown, and arrows indicate on-target sites.
12C and 12D are sequence logos obtained by comparing DNA sequences in Digenome-captured sites and using WebLogo.
Figure 12e is a graph showing the base correction efficiency in ABE7.10 off-target sites measured using targeted deep sequencing, 3 nucleotides at the 3'end represent the PAM sequence, and lowercase letters represent the Mismatched base (Means ± sem were from three independent experiments).
12F is Scatterplots showing the correlation between ABE7.10-mediated substitution frequency and Cas9-mediated indel frequency (top) and correlation between ABE7.10-mediated substitution frequency and BE3-mediated substitution frequency (bottom).
Figure 13a exemplarily shows the target DNA-complementary RNA sequence of sgRNAs in commonly used forms (GX ₁₉ ), truncated forms (gX ₁₈ or gX ₁₇ ), and extended forms (gX ₂₀ or ggX ₂₀ ). .
13B is a graph showing the average of the base correction frequency of ABE7.10 for each modified sgRNA measured by targeted deep sequencing when using modified sgRNA targeting HEK2, RNF2, TK_EphB4, TYRO3, WEE1, HPRT-exon6, and HPRT-exon8 to be.
13C and 13D show ABE7.10-mediated nucleotide correction frequency (%) in on- or off-target sites of HPRT-Exon 6 (13c) and TYRO3 and HPRT-exon8 (13d) measured by targeted deep sequencing in HEK293T cells. ) As a graph and heat map showing the results, the heat map shows the relative specificities of the modified sgRNA compared to the case using GX ₁₉ sgRNA, and the relative specificity is the specificity of the sgRNA to be measured. It is a value calculated by dividing the degree (on-target frequency/off-target frequency) by the specificity (on-target frequency/off-target frequency) of GX ₁₉ sgRNA, and the 3 nucleotides at the 3'end represent the PAM sequence. , Lowercase letters indicate mismatched bases (Means ± sem were from three independent experiments).
13E and 13F show on- and off-target activities at sites in HPRT-Exon 6 (13e) and HPRT-exon8, HEK2, EphB4 and TYRO3 (above, 13f) when ABE7.10 and Sniper ABE7.10 were used. And a graph and a heat map showing the specificity ratio, wherein the 3 nucleotides at the 3'end represent the PAM sequence, the lowercase letters represent the mismatched base, and the specificity ratio is calculated by the following formula: Sniper ABE7 .10 specificity (on-target frequency/off-target frequency)/ABE 7.10 specificity (on-target frequency/off-target frequency) (Means ± sem were from three independent experiments).
Figures 13g and 13h show HPRT-Exon 8 (13g) and HPRT-exon6, HEK2, EphB4 and TYRO3 (above, 13h) when delivered intracellularly through plasmids and intracellularly in RNP form. -And a graph and heat map showing off-target activity and specificity ratio, 3 nucleotides at the 3'end represent the PAM sequence, lowercase letters represent mismatched bases, and the specificity ratio is in the following formula. It is calculated by: specificity at the time of RNP delivery (on-target frequency/off-target frequency)/specificity at the time of plasmid delivery (on-target frequency/off-target frequency) (Means ± sem were from three independent experiments).
13i and 13j show on- and off-target activity and specificity ratios at the HPRT-exon6 site (13i) and HPRT-exon8 site (13j) when ABE7.10 or Sniper ABE7.10 is used in combination with a modified sgRNA. As a graph and heat map showing the (specificity ratio), the 3 nucleotides at the 3'end represent the PAM sequence, the lowercase letters represent the mismatched base, and the specificity ratio is calculated by the following formula: Sniper ABE7.10 and modification The specificity of the combination of sgRNA (on-target frequency/off-target frequency)/specificity of ABE7.10 (on-target frequency/off-target frequency) (Means ± sem were from three independent experiments).

Hereinafter, the present invention will be described in more detail by way of examples, but these are only illustrative and are not intended to limit the scope of the present invention. It is obvious to those skilled in the art that the embodiments described below may be modified within the scope of the essential gist of the invention.

Example 1: adenosine With deaminase Cas9 Nikkei ( D10A ) After processing, Endonuclease Genomic DNA sequence analysis when treated with V

When the DNA extracted from the cell is mixed with adenosine deaminase-Cas9 nickase (D10A, the 10th amino acid of Cas9 has been mutated from D to A) and guide RNA complex, one strand of DNA is cut and A of the other strand is Is changed to I. When the reacted DNA is treated with endonuclease V (endonuclease 5), I is recognized and cleavage is induced. Eventually, double strand break (DSB) of DNA is induced by deaminase-Cas9 nickase (D10A) and endonuclease V (see Fig. 1A).

After the DNA is purified, the DNA in which the double-strand break (DSB) of DNA has occurred is subjected to end-repair process, followed by whole genome sequencing. In the case of DNA with double-strand break (DSB) of DNA, it can be seen that the sequence by sequencing at the cut part is straight alignment at the on-target position, and the part with straight alignment in the entire genome It is possible to detect the off-target of adenosine deaminase-Cas9 nickase by finding (Fig. 1a).

Example 2: adenosine With deaminase Cas9 Nikkei After processing, Alkyladenine DNA Glycosylase ( AAG ), genomic DNA sequence analysis

In addition, when Alkyladenine DNA Glycosylase (AAG) is treated on DNA treated with adenosine deaminase-Cas9 nickase, I is removed and AP-site (apurinic/apyrimidinic site) is created. Thereafter, treatment with endonuclease VIII (endonuclease 8) capable of cleaving the AP-site causes I to be removed, resulting in a double strand break (DSB) of DNA (Fig. 1b).

After the DNA is purified, the DNA in which the double-strand break (DSB) of DNA has occurred is subjected to end-repair process, followed by whole genome sequencing. In the case of DNA with double-strand break (DSB) of DNA, it can be seen that the sequence by sequencing at the cut part is straight alignment at the on-target position, and the part with straight alignment in the entire genome It is possible to detect the off-target of adenosine deaminase-Cas9 nickase by finding (Fig. 1b).

Example 3: adenosine With deaminase Cas9 Nikkei After processing, Endonuclease V or AAG Genomic DNA sequence analysis when processed

After treatment with ABE and endonuclease V or ABE, Alkyladenine DNA Glycosylase (AAG), and endonuclease VIII, it was confirmed whether DSB occurs through real-time PCR, and the results are shown in FIGS. 2A and 2B. As shown in Figures 2a and 2b, when only ABE was treated, it was found that about 50% was cut, but ABE and endonuclease V or ABE, Alkyladenine DNA Glycosylase (AAG), and endonuclease VIII were treated. Later, it can be seen that more than 90% of the DNA target sites were cut. To verify this phenomenon once again, sanger sequencing was performed. When ABE was treated, A was changed to G. After treatment with ABE and endonuclease V or ABE, Alkyladenine DNA Glycosylase (AAG), and endonuclease VIII, almost all of the parts changed to G It can be seen that (ABE and endonuclease V) or part (ABE, Alkyladenine DNA Glycosylase (AAG), endonuclease VIII) is changed to A.

Example 4: Whole Genome Sequencing ( WGS ) Perform

The ABE of Example 3, endonuclease V or ABE, Alkyladenine DNA Glycosylase (AAG), and DNA treated with endonuclease VIII were subjected to WGS, and the results are shown in FIGS. 3A, 3B, and 4 . As shown in Figure 3a, straight alignment can be confirmed from the sequence analysis result. 3B and 4, genome-wide off-target candidates can be found using a DNA cleavage scoring system.

A method of profiling off-targets in the whole genome of adenosine nucleotide-correcting gene scissors can be applied using CIRCLE-seq17 and SITE-seq18 (FIG. 5).

CIRCLE-seq (Tsai, SQ et al. CIRCLE-seq: a highly sensitive in vitro screen for genome-wide CRISPR-Cas9 nuclease off-targets) is a method of profiling off-targets in the whole genome of adenosine scissor. Nature methods 14, 607-614 (2017)) and SITE-seq (Cameron, P. et al. Mapping the genomic landscape of CRISPR-Cas9 cleavage. Nature methods 14, 600-606 (2017)). It is possible (see Fig. 5).

Reference example

1. Cell culture and transfection

HEK293T cells (ATCC CRL-11268) were cultured in DMEM medium supplemented with 10% (v/v) FBS and 1% (v/v) penicillin/streptomycin (Welgene), and HEK293T cells ( ^1.5 ×10 ⁵ ) were cultured in 24- Inoculated into a well plate, using Lipofectamine 2000 (Invitrogen; 3 ml for ABE and BE3, 2 μl for SpCas9), ABE (Addgene plasmid #113128; 1.5µg; DNA weight), BE3 (Addgene plasmid #73021) ; Plasmid encoding rAPOBEC1-XTEN-Cas9n-UGI-NLS; 1.5 μg) or SpCas9 (Addgene plasmid #43945 NP_269215.1; 0.5 μg) was transfected with sgRNA encoding plasmid (0.5 μg) with 70-80% confluency Made it.

In the following examples, the ABE or ABE7.10 is used to refer to the same protein, specifically adenosine deaminase (directed evolution of E. coli TadA (NP_417054.2)) and SpCas9 nikase ( D10A) (Modified Cas9 in which the 10th amino acid residue D of SpCas9 (NP_269215.1) is substituted with A) refers to a fusion protein to which it is linked (see Reference Example 2).

For ABE RNP-mediated genome editing, ABE7.10 protein (10 µg) and in vitro transcribed sgRNA (6 µg), or ABE7.10 protein (0.6 µg) and sgRNA (0.2 µg) coding plasmid were used in Neon ^TM Transfection System HEK293T cells (1×10 ⁵ ) were transfected by electroporation through (Thermo Fisher Scientific). At 72 h post-transfection, genomic DNA was extracted using DNeasy Blood & Tissue Kit (Qiagen).

The sgRNA used in the following examples is a PAM sequence (5'-NGG-3' at the 3'end of the target site (indicated as'sgRNA target site'or'DNA seq at a cleavage sites' in Tables 8 to 18). (N is A, T, G, or C)) in the sequence except for the sequence in which T was replaced with U, the targeting sequence in the following general formula 3'(N _cas9 ) _l' was used:

5'-(N _cas9 ) _l -(GUUUUAGAGCUA)-(GAAA)-(UAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC)-3' (general formula 3; oligonucleotide linker: GAAA).

2. ABE7 .10 protein expression and purification

ABE7.10 was designed as follows (N-terminal to C-terminal direction):

MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGS SEVEFSHEYWMRHALTLAKRA R DEREVPVGAVLV L NNRVIGEGWNR A IG F EPCVMCAGAMIHSRIGRVVFG HDPTAHAEIMALRQGGLVMQNYRLIDATLYVT L V R Y N AKTGAAGSLMDVLH PGMNHRVEITEGILADECAALL CY FFRM P RQ VFN AQKKAQSSTD SGGSSGGSSGSETPGTSESATPESSGGSSGGS DKKYSIGLAIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD PKKKRKV (SEQ ID NO: 535)

(In the amino acid sequence,

(1) Part of the polypeptide indicated in bold: wild type E. coli TadA (NP_417054.2) (SEQ ID NO: 536);

(2) Italicized polypeptide moiety: directed evodution E. coli TadA (NP_417054.2) (amino acid residues mutated in wild type (NP_417054.2) are indicated by italics + underline) (SEQ ID NO: 537);

(3) Underlined polypeptide moiety (SEQ ID NO: 538): SpCas9 nickase (D10A);

(4) Oligopeptides (SGGSSGGSSGSETPGTSESATPESSGGSSGGS) between polypeptides (1) and (2) and between polypeptides (2) and (3): peptide linker (SEQ ID NO: 539); And

(5) Oligopeptides (PKKKRKV) linked to the C-terminus of polypeptide (3): SV40 NLS) (Sungrye No. 540).

A plasmid for expression of ABE7.10 in which the human codon-optimized gene encoding ABE7.10 (including N-terminal His purification tag) designed as described above was cloned into pET28 (Addgene) was prepared, and the plasmid was BL21 Star (DE3) -competent E. coli cells (NEB) were transformed. After transformation, fresh single colonies were cultured overnight in Luria-Bertani (LB) medium (Thermo Fisher Scientific) containing 50 µg/mL kanamycin while shaking at 37°C. 8 mL of 1:50 diluted pre-cultures were inoculated into each flask containing 400 mL of LB medium supplemented with 50 ㎍/mL of kanamycin, and cultured with shaking at 37°C until OD ₆₀₀ =0.65 to 0.70. Then, the obtained culture was stored on ice. After inducing protein expression using IPTG (isopropyl-bD-1-thiogalactopyranoside; GoldBio) 1 mM, the culture was incubated with shaking at 18° C. for 16 hours.

The subsequent protein purification process was performed at 4°C. The obtained cell culture was centrifuged at 6,000 g for 10 minutes to harvest cells, and lysis buffer (50 mM sodium phosphate (Sigma-Aldrich), 500 mM NaCl (Sigma-Aldrich), 10 mM imidazole (Sigma-Aldrich)) , 1% Triton x-100 (Sigma-Aldrich), 20% glycerol, 1 mM 1,4-dithiothreitol (DTT; GoldBio), 1 mM phenylmethylsulfonyl fluoride (PMSF; Sigma-Aldrich), concentration of 1 mg/mL lysozyme ( Sigma-Aldrich), 10 μM ZnCl ₂ (Sigma-Aldrich), pH 8.0). Thereafter, the suspension was incubated three times alternately in a liquid nitrogen and a water bath (37° C.). Cells were lysed by sonication for 9 minutes (5 seconds (on), 10 seconds (off)), and the lysates were cleared by centrifugation at 15,000 g for 20 minutes. The supernatant was incubated with nickel agarose beads (Ni-NTA, QIAGEN) for 60 minutes at 4°C with shaking. The lysate-resin mixture was loaded onto a polypropylene column and 3 column volumes of washing buffer (50 mM sodium phosphate (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich), 35 mM imidazole (Sigma-Aldrich), 20% glycerol) , 1 mM DTT (GoldBio), 10 μM ZnCl ₂ (Sigma-Aldrich), pH 8.0), and the bound semi-white matter was washed with elution buffer (50 mM sodium phosphate (Sigma-Aldrich), 150 mM NaCl (Sigma-Aldrich)). ), 250 mM imidazole (Sigma-Aldrich), 20% glycerol, 1 mM DTT (GoldBio), 10 μM ZnCl ₂ (Sigma-Aldrich), pH 8.0). The eluted protein fraction was placed in a polypropylene column containing heparin agarose beads (Heparin Sepharose 6 Fast Flow, GE Healthcare) and 3 column volumes of washing buffer (50 mM sodium phosphate (Sigma-Aldrich), 150 mM NaCl (Sigma)). -Aldrich), 20% glycerol, 1 mM DTT (GoldBio), 10 μM ZnCl ₂ (Sigma-Aldrich), pH 8.0). The bound protein was dissolved in elution buffer (50 mM sodium phosphate (Sigma-Aldrich), 750 mM NaCl (Sigma-Aldrich), 20% glycerol, 1 mM DTT (GoldBio), 10 μM ZnCl ₂ (Sigma-Aldrich), pH 8.0). Eluted with. The resulting protein fraction was concentrated at 6,000 g using a centrifugal column (Amicon Ultra-4 Contrifugal Filter Devices, Millipore) to purify ABE7.10 protein.

3. PCR amplicon ABE7 .10-media In vitro Decomposition ( in vitro digestion)

PCR amplicon containing the target site of the target gene (see Tables 1 to 11) was mixed with ABE7.10 protein and in vitro transcribed HEK2-target sgRNA in a reaction volume of 100 ml for 1 hour at 37°C. Cultured. The obtained deamination product was purified using a PCR purification kit (MGmed). 2 μg of the deaminated product was added to endonuclease V (Endo V; NP_418426.2) (40 units) (New England BioLabs) or human Alkyladenine DNA Glycosylase (hAAG; NP_001015052.1, New England BioLabs) (10 units) (New England BioLabs) and endonuclease VIII (Endo VIII; NP_415242.1) (20 units) (New England BioLabs) in a reaction volume of 200 ml after incubation for 30 minutes at 37 ℃, DNA PCR purification kit Purified again using (MGmed) and analyzed with a Bioanalyzer (Agilent), and the in vitro digestion result was measured.

4. Genomic DNA ( genomic DNA) of ABE7 .10-media In vitro decomposition

Genomic DNA was isolated from HEK293T cells using DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer's instructions. 8 μg of genomic DNA was mixed with purified ABE7.10 protein (300 nM) and sgRNA (900 nM) in a reaction volume of 400 ml for 8 hours at 37° C. in a reaction buffer (50 mM Tris-HCl (pH 8.0), 25 mM KCl). , 2.5 mM MgSO ₄ , 0.1 mM EDTA, 10% glycerol, 2 mM DTT, 10 μM ZnCl ₂ ). After sgRNA was isolated using RNase A (50 μg/mL), DNA was purified using DNeasy Blood & Tissue Kit (Qiagen). After incubating 3㎍ of purified DNA with Endo V (40 units) or hAAG (10 units) and Endo VIII (20 units) at a reaction volume of 200 ml for 2 hours at 37 ℃, DNeasy Blood & Tissue Kit (Qiagen ) And purified again. Purified DNA was incubated with KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems) and analyzed by real-time quantitative PCR (qPCR) to analyze ABE7.10- and Endo V- or ABE7.10-, hAAG-, and Endo. The VIII-mediated DNA double strand break (DSB) was confirmed. The intact genomic DNA fraction was determined by comparative C _T analysis.

5. Whole genome sequencing; WGS ) And digenome sequencing

1 μg of the obtained in vitro digested DNA was fragmented in the range of 400 to 500 bp using a Covaris system (Thermo Fisher Scientific), and incubated with End Repair Mix (Illumina) to create a blunt end. The fragmented DNA was ligated with an adapter (illumina) to generate a library, and the entire genome sequencing (WGS) was performed using HiSeq X Ten Sequencer (Illumina) by requesting Macrogen. WGS was performed with a sequencing depth of 30-40x. Isaac aligner was used for genomic sequence mapping. DNA cleavage sites were identified using Digenome 1.0 and Digenome 2.0 programs (Kim, D., Kim, S., Kim, S., Park, J. & Kim, JS Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq.Genome research 26, 406-415 (2016)). The source code of the Digenome 2.0 version used in this specification can be found at'https://github.com/chizksh/digenome-toolkit2'.

6. Targeted deep sequencing

The on-target sites and potential off-target sites were amplified using KAPA HiFi HotStart PCR polymerase (#KK2502; KAPA BIOSYSTEMS). The amplified amplicon was amplified again using TruSeq HT Dual index-containing primer to create a deep sequencing library.

The primers used for the target deep sequencing are summarized in Tables 1 to 7 below:

Primer for HEK2 Site No. 1st PCR_F 1st PCR_R 2nd PCR_F 2nd PCR_R HEK2_001 CCTTTCTCCACAGGCAGAGG CCATCACACCCAGCCTCATT TCAGGCGCTGTTGCTAGAAA CAGAGGAGGAAAGCAAGCTCA HEK2_002 GTTCCCACTGGACAGTCGAG GCAGGCAAATGAGGCTTCTG GTTCCCACTGGACAGTCGAG GGCTATTTGCTGGGGGAACT HEK2_003 ATGAACAGGGGGGCTTGATG CTGAGCCACTCCTTTCCCTG CCTCCATCTTCTCCGCAGAC TTCTTCGCCCTCTCAGCTTC HEK2_004 AGTCGTACTATGCAAGCCACA GGGCACATGTGTTTGCGTAA TGGGTTCAAAAACAAACAGAGAAGA TCTTGTGAAACAGAAATGTCAGTT HEK2_005 TGCTGTGGTCCTAGGTCTCA CTCAAGCAATCCTCCTGCCA AGTGGAAATCAGTGGGCCTG TGGAAAGAACCACCTTGTGAGT HEK2_006 AGGCTGGGTGCAATTTGTTT CCACAATTACATTTGCAGCAACC TCCCATCACCATTTACTGAGCA ACACTGCCTGATTTCAAGCT HEK2_007 GAGGCCCATTAACGTTTGGC AGACTCAAAACCTGGCCCAC ACCTGGCTGAGCTAACTGTG TCCAGCCCCATCTGTCAAAC HEK2_008 TCCACCTGTTCATCCTCCCT ATGCCACCTCTGAAGCTGAG TCTTTCATTGCATGGCTGTACC TGAATTTGTGTTGGGCCACA HEK2_009 TGGAACTGGGGAATTGGTGT AGGGGCAGGAACACAAGAAA CCTGATTTGATAGACATGACATTGT CAAACAAACAAGCAAAGCCTGT HEK2_010 GAATATCAGGGGATGGCGCA AGAGCTAGGAGGGCCTGAAA TGTCTTGAGTGGGCTGGTTC GGCCTGAAATTTGCCCTGAC HEK2_011 TCCTGGTGTCATCTTGTGCC TCCACAGTTCCCTAGGGCAT AATTCTCAAGAACAGTGAAAGTTGT TCCACAGTTCCCTAGGGCAT HEK2_012 TCACCCTCCTCCTCTCACTT ACAAGGACAGGCCAAGACTG ACCCAACAGAACAAAGAAATAACCC AGTGGGACTATGCCAGAGAT HEK2_013 CACTGGGGATGGCAATCTGT AAAGGCGGAAAGAGACAGGG ACCAGCAAAGAAAAGGGGGA GGAAAGAGACAGGGTCCTGAG HEK2_014 ATGGATGGTAGTGTCGTGGC TGGAATAGACCTGGGCTCCA CACACACACACACACACACA TGGAATAGACCTGGGCTCCA HEK2_015 CACGCAGCCAAGTCAAACTC GCAGAAGAATGGCGTGAACC GCCACTGGCCATCCTAACAA TTTCTGTGCCCTATGCTCCT HEK2_016 CTGACAGGCATTAGGTGGGG ATTATCTGCCTGCCTCAGCC TCCCAGTCCAGCCTATGC GCACCTGGCCCACTATATGT HEK2_017 CATGTCCCCCTTTCCTCCAC GTGGAAGAGCCTCTGGGAAC CCTGCCCTTTCCTTCCTTGT GGGCAGTTGTGAATCATGGC HEK2_018 GGTTGGGGTTGGGATTAGGG GGTGGAAGGACTCGCAGAAA TGACAACCACGTTCATGGGA CCCATCTTGTCTCTCTGGCC HEK2_019 GATCAGCCCAGGTGCTCTTT AGTTCAAGATCAGCCTGGCC CTGCCTAGCCCAAAGACCTC TGGTCCCAGCTACTCAGGAA HEK2_020 AAAGCAAGAGGGTGGGAGTG TGACGACCCCCTACTTCACT CGTTGGCTTAGACCAGGGTA TCCAGGATTTTGCCCCATCC HEK2_021 CAGCCTGGCCAACATAGTGA AAACTGTGCTCCCTTAGGCC ACCTCACTCTGTGTCCCCTT AAACTGTGCTCCCTTAGGCC HEK2_022 TATCAGCAGTTCCCCATCGC GGCTCTTACCAGTTTCCCCC ACATCTGCAGCCCAGTTTCT CACAGTGCTTTGCCCCATTG HEK2_023 CTGCCACTAAGCCACCTTCA AGGCTGAGGCAGGAGAATTG TCCCATGGATGAAAAGCCCA GTGACAGAGTGAGACTCCGC HEK2_024 GTGGAGGGTGCATGTGTACA CTAGGACACGGCTAAGGCAG TTTTCTGTGGGGCCAGTGTT CCAAGAGGAAACCTACCCCA HEK2_025 TTCGGCGTGTGAAGTTAGCT CGATCTTCCCACCTTGGCTT CTGCCGTGGGAGACAATTCA ACTGGTTCTGTTTGTGGCCA HEK2_026 AGCTTGCCAGATTGGTGGAA GAACACCATGCACGTTCACC AGAGAACAGAGAACCTGCAAGG CTGGCTTTGCTTCTCCCGTA HEK2_027 ATGGGCTCAGCTACGTCATG ACACAGAGTACACAGGCACG GGGCCCAGGGTAAGACTCTA CTTGCAGTGAGCCGAGATCA HEK2_028 CAGCCTATGCCCAGGAATCC CTAACCAAGAGCCCTGGTGT CTGCTTAGGTCCAGCCAACA TCTTGAAACTCCATGGCTGAA HEK2_029 CACTCCAGCTCAGGTGACAG TTACAGACGTGAGCCACCAC CATGTGAGGATGCAATGGGC GAGGCAGTTTGAGCTGAGCT HEK2_030 GACCCTGAGCTGCTACCATC AGAAATGCTGCTCTGGCCAT ACGAGTAAATGTTAAGCTTCTCCCT ATGCTGCTCTGGCCATAGAC HEK2_031 ACAATGGTCAGGTACGGCAG TCCAGTGCAACACAGTCTGG ACTGGTGTCCTGAAGTCAAGTC GAGCAGAGCCTCACACTTGT HEK2_032 GAACTCACAGAGCGGTGGAA CACCCAGGAAGACATGGTCC GTGTCTGGCGGAGAGAGAAC ACAAATCCTGGCACCAGACC HEK2_033 ATGGGAGAGCAATCCAGTGC AAAACTCTATGCCCCCCTGC TGGCCCCAAGATCAAAAGCT TGAGGGTAAGGACTGAGGCA HEK2_034 GGCTTGTAGGATGAGCTGCA TTCTTCTGCTTGGTCCAGCC CTACCTCTCCAGCAATGGTCC TGCTTGGTCCAGCCTATTGA HEK2_042 GAGACCTTATGCCCTTCACCA CGGTGAATTGGGTTATGGCC CCAAAGGCATGGTCCATGAA TTGGTGAGGCACTGTTTGGA HEK2_044 GCACTTTGGATGTGGAAGGG ACAAGGTCATTGCCTTCACA AGACCTACACTGACCTGAGGT ACAAGGTCATTGCCTTCACA HEK2_050 CCCAACACAATCCCCTCCAG TCTGGATGTCTACCTCTCTAGCA CCCTACCCAACACAATCCCC TCTCTGGATTGCTTTACAGGTT HEK2_052 AGCGGTGTAGGGATGAAAGC GGCCAACTCCTCCTCACTTC GCTTCACCTCTCTGAGCCTC TGGGCTGAAAACAAGAGGCA HEK2_065 AGCATGTTGCCTAGGCTTGT GCCCTGTATTCAGATCCGCT TCCTCTGCCCTGACCATGTA GAGAGCCTCGGGTGATTCTG HEK2_079 CATGTGTCAGTTCCAGCCCT GGCAAGAAACTGGGTAGCCT CGGTGCTTCAATGCTGTAAGG AGAGGATCCCCAACGACAGA HEK2_082 TTCTCCAGCTGTGCAGAGTG GAGCAGCTGTCCTCAACCAT TTCTGTACCTGCTGGGCATC TCAAGGAAATCACCCTGCCC HEK2_092 TCGACCTTGGAAAAACCTCCT TGGAAGCTCTCCCTTCAGGA TGGAGCTGCTCTTTTTTTCCTC CACCTTGAAGGCACCTTTTCA HEK2_093 CCACCATTTCAGTGAAGGCC TGCTCTTACCTGGCTCATGC CCACCATTTCAGTGAAGGCC AAGGTCAGGGGCCATGTTTT HEK2_099 GGAAGCTTTGCAAGTCAGGC ACATACACTGGGGCCTGTTG TGGGCACTGCTCAGTAAACA GCACATCCTGTACACATACCCA HEK2_101 AAGTGCTGGGATTACAGGCC GCCAGCAGGAAAGAGGACAT CTGGCTCTCAACTTCCTCCG CTAGCTGCAAGGAAGCCTGG HEK2_105 TGTGTGTCCTAAGCCGTGTC CTTCTGGGAAGATGCTGGGG CCAGAAGTCTCCGTGGCTG TTAATGCCGTATCCCCAGCC HEK2_106 TTCCATAGAGGGGAGGCGAA CAAGGGAGTTGCTAAGGGGG TGGGACTGACCGAGAGACAA CAAGGGAGTTGCTAAGGGGG HEK2_110 GGGAGACACATGGGCTTTCA CGCCCGGCCAATTTTTTGTA TCCCAAAACTGGCTCTTGCA ACCTGGCCCCCTTTAGATCT HEK2_119 GCTGGATGGCAGCTCAAAAC TTGGTCACATCACTCAGCCC GCTGGATGGCAGCTCAAAAC ACCTAGCACGCAATCAGTCA HEK2_121 GCCACAACATCTGGTGGGTA GAGGTGGAAGGTTGGCTTGA AGTTCTCCAGCAATGGTGTT AGAGCCAGACCCTGTCTCAA HEK2_139 AACCAGCATTATGGACCGGG TGGTGGCAATCTAGTGGAAGG AACCAGCATTATGGACCGGG ACGATGAACCATCTGCTGAGA HEK2_145 GGCATTTCTTTGAGCAGGGC AATCCCTTTTCCGTGGTGGG GGCATTTCTTTGAGCAGGGC CATTCCTGGCTCACAGTGGA

Primer for RNF2 Site No. 1st PCR_F 1st PCR_R 2nd PCR_F 2nd PCR_R RNF2-1 CTTTGCAGGTCACAAGGCAC TTTGGGTGGGTCTAAGTGGC CACAGCTCAGGGTGGCTAAA TGTGGCTTATGCTGCACTCA RNF2-2 AGTGTCTGGTGAGGGCCTAT ACCACTGTTCACCCAGTACC TCCAGCAATGTCTCAGGCTG AGCCAACATACAGAAGTCAGGA RNF2-3 AGAAGCAGAGGGTTGTTGCT GAGAGCTTGAGTGGGTCACC CTCTGGCCATTGATGCCAAA CAGCTCATCTGAAAGCTCTGT RNF2-4 TCTGGACCAAAAGAGCTTGCT TGGAGTTGTCCCGAAGAGAAC ACACCAGGTATCTGTCTTGACA TGCAGACCCTATCTTTACAGCA RNF2-5 GAGGGTCCCTCTCTGGAACT CAGTGGCTGTGAGGATGTGT CTTTGTAGCTGCAGGCCACA GGGAGTCAGGAGCTTGCAG RNF2-6 GGGTAAACCGAGGCATGTGA CCCTGCTCATGCTCAAAGGA GGCTTGAGGTAGTTGGCCAT CTTGACACAGGAGAGGCAGG RNF2-7 AGGGCCAGGCTACCAGATAA CCCCCAGCTGCTAAATGTCA TGGAGAGGGGGTAGAAGTGG ATGTCACCTGTTTGCCTGCT RNF2-8 AGGTCTTCTGACAACCTGCA AACTTAGTCAGGGGCCATGC AGCTGGTTCCAAACTTAGCCA GGAACAAGAACAGTGAGGCC RNF2-15 ACAAGCCTGAGTCACTGAGC ACCTTATGCACCTTGCCCTC ACCCTCATTTCCTGGAGTTCC TTTCATTGTCATTGTTACCATGACT RNF2-19 AGAAAGAGCTCCCAATGGCC CCCAGTGTTTTTCCCAGGGT TGAAGGGTGTGGGGTAGGAA CCCAGTGTTTTTCCCAGGGT RNF2-20 GGACTGAGTCCCAGCCTCTA GAGAGGTCAGGCAAACGACA CACCTAGAACAATGCCAGGC CCACGCCCATTTGCTTTCAT RNF2-24 AGGTAACAGTCCAGGCTGGA ACAACGCACCTAGGGAACTG TGACACCACCACAATTCTTGG AAAGCCACCGACCTTCAAGG RNF2-27 GCCACTGAGAGCAGAGACAG CTCTTTGAAGGTCTGGGGCA GCCACTGAGAGCAGAGACAG CTCTTTGAAGGTCTGGGGCA RNF2-41 TACCCATCACAGTGGTCCCA TGTTCCATAGTTCCTTTTTCCCT ACCACAATAGTACACAAGCCA ACTTTTTGTCCTTCTTTGGTTCA

Primer for TYRO3 Site No. 1st PCR_F 1st PCR_R 2nd PCR_F 2nd PCR_R TYRO3-1 CCACTCCAAGATGAGGCCTG TCCCCCTGAACCTGTTACCA ACCACAACAGCCAGGACTTC CCTGGCCTCTTCTCGCATAG TYRO3-2 AGTTACACAGGGCCTTCGTG CTGTCAACAAAGTGCTGGCC CTTGACTCCCATGCCTCCTG CTGTCAACAAAGTGCTGGCC TYRO3-3 CAGACAAGCCTGCTTCTCCA TCAGCAGTGTGAGAACGGAC CAACCTTGTAACAGGGCCCT AGCTTACTTTCCTGGGGAACA TYRO3-4 GCCCTGTTAGACTGTTGCCT TAGCTTAGTCTGGGCCACCT GCTGCTATAGGCTGGGACTG GCCCTTACCAGGAGCCAAAT TYRO3-5 CAGCTGCCTTACACACTGGA TGCCCCAATCATGTCTCCAC TTCGGAGTCATAGCCACTGC GACCAGGTGTCACGACTGTT TYRO3-6 GCACCCATGATCTCCAACCA TAGACAGGCCAGGAGTTCGA GATGACTTCTATCCGGGGCC AGCATGAAGAGCTGGTCAGG TYRO3-7 GTGAGTCAGTGGTCCAGGTG GAACAGGACTGTGGAGTGGG CGAGGTGTTCTGTCCAGGAT GGATGCAGTGAACTTGTGGC TYRO3-8 TTTGGGGCTTTGAGTGTGGT TACCATGTCCCCATGCCAAC GGACCACCGATCACAACACT TAGAAGCCAACACCCCACAC TYRO3-9 GCCTCACTTCACCTGGGATC TCATGTTGGTCAGGGTGGTC AGACTCCTTTCTCACGCACT TCCGTGGGGTCTATTAGGCC TYRO3-10 TTGACCCTCTCTGAGCCTCA TACACTCTGAGCCCTACCCC TGGGAAGCACCTGGGATAGA TACACTCTGAGCCCTACCCC TYRO3-11 AGGTGCTGGTGATGAGTGTG TGACTAGAGAAGCGGGTCCA GCTGGTGATGAGTGTGTTGC AGAGTTATGGTTACTGCTCAGTT TYRO3-12 CTTCCCAGAAGCCTCTGCAA GCTCATTTCCACTCCTGCCT GGGCATCAGAAGTGGGACAA TACCCACCTGTGTCCGTTTG

Primer for WEE1 Site No. 1st PCR_F 1st PCR_R 2nd PCR_F 2nd PCR_R WEE1-1 GAGCCATGTCTGCACCTCTT GGGAACAATGCCACCCAAAC TTCCCTGCCCCATTGCTATC ACACTCACAGGGCATGTCTG WEE1-2 TGGCATGCAAATATCTCCCA ATAAGCAGCACACCACCACA AGCACAGATGGCAAAAGCTG GTCACAGTGTTCAGGGGGAG WEE1-3 ATGCTGGCCAAGATACCCAC GCTACTTCCTACTGTCCCCC CACTGGACATCTGAGGAGGC CAGGCTGGAGTTCACAGAGG WEE1-4 TCATCAGAGGACCCCTGTGT ATCTGTTTTGGAGGCAAGGT ACTCCAAGAAGATTACTTACTTCCA ATCTGTTTTGGAGGCAAGGT WEE1-5 CTGGCCCAGACTTCTTGAGG ATGGAGTGCATGGAGTGGTG AGGTCCTGCTCCTCCTATGT CTGTCCTTTGACCCAGTCCC WEE1-6 ATGCTGTGTTCCAACCCAGT TGGCTCCACCATGTTGGAAA AGGGTGACTTTGACAGAACCA CCTCTGGGCACTATGCTAGG WEE1-7 ATGTCTAGACCGTTGCTGGC CGTTTGCTTTCAGTGACCCAG ATGTCTAGACCGTTGCTGGC CGTTTGCTTTCAGTGACCCAG WEE1-8 GTGATTGGCTACCAACCCCA CTTGAGGCCCAGGACACAAT TGTGCCACTTAACATGGACTCT GACCTAAAGACGTGCCACCT WEE1-9 ATGTCCACACGAACACCTGC CCACCACATACAGCCTCTCC TGGAAGTTACCAAGATGCCCC AGTTAGTTTTCCAGAATGCCGT WEE1-10 TGGGTGTTTTGAAGTGGCCT CAGTGAGAGCCCTGAGGTTG CCTCTGCTGAAATCCGTGGT TGGACCAGTACCAGTCCATG WEE1-11 CGTGAGACATGGGGAGCTTT GGGTTCCTTTCCCTGAGTGG GTGGGTCTTTCTCTGGCCTC CCTGGTTAGGCCCCATGATC WEE1-12 TGGCCAACATGGTGAAACCT TAGTGGTGTGACGAGGGCTA GCAGTGAACCAAGACCATGC AAACTTGCTGTCCTTGCCCT WEE1-13 AGCTGAAGTCTGGAAACGCA GGTGACGAGTGAAACCGTCT TCCCACGACCCTAGTAGTCC TTGCAGTGAGCCAAGATCGT WEE1-14 TGGTGTTGCTTACCTGATTTGC TGAAGAAAGGAGGTGTAGCTCT TGCACTCATACAATGTGGTCA CCATCTGACCTGGGGGAAAC WEE1-15 GACTCCTGGGCTCAAGCAAT TGTGCCCTGCTGACATCATT GACTCCTGGGCTCAAGCAAT TGCAGGCTATTGACTCCTTCA WEE1-16 TGAGCCCAGATTGTGCAACT GCCTGCTCCTGAGATCATGG CAACAAAGCGAGGCTCTGC CACACACACACACACACACA WEE1-17 TTTGGAAGCTCAGCCCTGTC AATGGGAGATTGGTGGGCAG AGTGCCAACAATTGTGAGTGT GCTGTTGCTGGTCTTTTGGT WEE1-18 ACTCCTCACTCAAGCAAGCC GTCCCTCAGGCCTCTCAAAC GCCTCATCTACCTGCAAGGG GTCCCTCAGGCCTCTCAAAC WEE1-19 CTCAGCCTCCCAAAGTGCTG TAGGAGAAGGGGGAGAGCAC AGCCTTGTCTTTGAGTTCTGA ACTCCTTCCCTGTTTCACCT WEE1-20 GGGCCAGTCAAATGAAGGGA TTTTGGGGGGCTGTCTCTTC GGTCACTGACTGCGTCTCAA TCAATCCTCCCTCATTGTGGT WEE1-21 TGCAGGGCCATTTGCATAGT AGGATGGCAGATTTCTCATTGGA CGGGTGGTATTTTTGCTAACCC AGTTTGTAGAAAAGGTGCTGCC

Primer for EphB4 Site No. 1st PCR_F 1st PCR_R 2nd PCR_F 2nd PCR_R EphB4-01 GCTGGTCTCGAACTCCTGAC GTTCATTGCTCAAAGCCGGG ACCTCAAGTGATCTGCCCAC TTCCTTTGGTGTGTGTGCCT EphB4-02 GTGCCAGTGGAAATGGAGGA CCACAACTCCACCTCTGCAA TCCCAAAAGCCCATAAGAGGAC ACCGCTACCCATTTGGATCTC EphB4-03 CCTTTCTTCCCCCTCCCCTA GCTGCCCCTAAGGTCACAAA TGCCCCATAGTTTTGCCCAT TCCTTCCCAAAAAGATGTTGGT EphB4-04 GTGGAGGTTCTTTTGGGGCT ACCAGCCTAGACAACATGGC CCCCAACTCTGAGTCTGAGC CCCCAATCTGCCCTAACTCC EphB4-05 AGGTTAGAAGCCAAGCCCTG AGCCCAGGAACAGTAGGCTA CTGCTACCTGCTCTTATGGCA TCACACAACGATGAAATCCCCT EphB4-06 GAGTAGCTGGGATTACGGGC TGGAGTGCAGTGGTGTGATC GAGTAGCTGGGATTACGGGC AGCTCAGGAGTTCCAGACCA EphB4-07 TGGTCAGGCTGGTCTTGAAC AGGACAAGTCACTGCTGCTG CTGACCTCAGCCTCCCAAAG TCATGTGCCATTATTGCCCAC EphB4-08 CTCAGCCTCCCAAGTAGCTG AAATGGAGGCTGAGGGAACG ACCCAGCCTCCATCAGAGAT AAATGGAGGCTGAGGGAACG EphB4-09 TCACAGGATTCTGGGCCTTG CAAGTCCGGCTCTTGCTTCT GCCTTGCTAACATGAGCCAG TGCGACCTCAAGTAAGTTGCT EphB4-10 AACTTGTGGCACTCTAGGCA TTTCACATGTGTCTCCCCGG ACACAATGCATTGATCGAGTTCT ACACTGTGCCCTGGTCAAAT EphB4-11 AGTGGATCCTCTGGGTTGGT TTAGCTGGATGTGGTGGCAG GGTTGGTTTGTCAGCAGCAG GAGCTGGGTACTGGTTACACA EphB4-12 CACTTACACAGACGGGCAGT GGGTCACTTTCCCCTCTTCG AACTGTACTGAAACTGAACTCTTTC CAGCCCCTTCGGAGTCTTTT EphB4-13 TACCAGGCTTTGCTTGAGGG ACCATGTGGAAATGTCAACCT TACCAGGCTTTGCTTGAGGG ACCATGTGGAAATGTCAACCT EphB4-14 GCAGTGTTGGTGGCAGATTC TTTGGCGGACAGGAAGGTAC ATTTGTTTGCCCTTACAGAGTTT TGTCCAAAACTGAAAAACCTGGT EphB4-15 CTCCTGACCTCGTGATCTGC CTGGGAGGGAGGCAGAAATG GGTCTCAAACTCTGGGCCTC TGGGGATTGTTTTGGGGATGA EphB4-16 TAGGCCCCTCCAACACTGTA AGGAAAATGGGCATGGGCTT TGCATCTGTTCATTTATTTTTGGCT AGGAAAATGGGCATGGGCTT EphB4-17 CAGAATGCAGTGACCCCAGT ACCATGCAAACACTAGCCGA CACCCTTCTGTCCCCATCAC AGAAAGAACTAGGTGGATCCCT EphB4-18 AAAGCATCACTCTCCAGCCC TCACCATGCCCGGCTAATTT GTCCCTCAAACTGTCCCCTG TCACCATGCCCGGCTAATTT EphB4-19 GGAGGGAGGGGAAGACAGAT CCTGAGACAAGAGCCACTGG CTGAGGGGCATCAGGGAAAG TCCCAGCATAGGACTCACCA EphB4-20 TACATGTGCCATGCTGGTGT TGGAGTGCAGTGGTGTGATC CCCAGAGTGTGATGTTCCCC GGTCTTGAACTCCTGGCCTC EphB4-21 AAGCATCTGTAACCCCAGCC ATTTTGGGAGGCTGAGGTGG TGCGCAACAGAGTGAGACTC TGGGTGACAGAGCAAGACTC EphB4-22 GGAGGGTTGCTCCACATTCT TGAAGTGGTCGTGATCTTAAATGT TGGCTGATAAGGCTGAGTGT ATGAAGAGGGAAGCTACAGC EphB4-23 GCAGCTAAAGGGGTCCAGAG TAACCATGCTCCTGCCACTG AGGGGTGAGACTAAGAGCCC CCACAGTGACTTCCTCAAAACC EphB4-24 TGCCATTCAAATGCAAGGCA CTCTCTACCCACCCAAGAAGG TTGTTGCAGTATAAGGTGAGGT TGTGACAGGCTTCAGTACACT EphB4-25 CCTCCTGGGTTCAAGCGATT TTTCCTTGTTGTGCCCCCTT TGCAACTGTTCTCACCTGGT CAGTGACCTGCTTTCCGTCA EphB4-26 GCAAAGGCAAACTGAGGGAC GGATCCACTGATGATTTTTGCCT ATGAATGGGGTCTGTGGGTT GCTTCCCCACTCTCTGAACC EphB4-27 GGGTTGAGGAAGGTTGAGCA CAGCTCACTGGAAAAAAGCCA AGGTTGAGCAAGGTCAGTGA TGATGTTTGTGGCCATGAGC EphB4-28 TTCCTTGGCTTCTGGTTGCA ATGCTGTGCTCACTCACGAA CACCCCTTCAAGACCTGGAC ATGCTGTGCTCACTCACGAA EphB4-29 TTGGCCTCCTAAAGTGCTGG AGATGTGGCATCAAGGTGCA TGCCCCCTATATGACAAATGCA TGTAAGCAGAGACCCCCAGT EphB4-30 GTGGTAGACAGTAGCTACCAGC ATATGAGCACCCACCAGCAC AGGTTTAGCTTCCTTTTAAGGCA TGAGTTTCTTTGCTGTCCTCCA EphB4-31 AGGGATATAGGGGGCACTTGA TGAGGAGTGCTCATCAGGTA AGACAAATCTCCCATGCACA TGGCCTTGATCAACTGGATGA EphB4-32 TGGTGCCACTAGCCTGAATG CTTTCCACTGCCATCTGGGT GCCCTTTCTGCATATGGGGA CCATTTCAGGGCCCTTCCTT EphB4-33 CCAAGAGGATTGAGTTGAGGCA AGCCCACCAGATTTTAGGAGA TGGGTAACTGCTTGTCCCTG AACCGATGGTGATAGGTTTGT EphB4-34 TGCCTCACGATTTGATCACGA CCCAGTGGCAGAAAATCAGT TCAGCCCCGTAAAACACACA TCTGCCAAATTTTAATGTTTCCCCA EphB4-35 GCAAGCTCTGACTGGTGAGT CAGGGACTCCAGGAAAGCAG AGTGCTTTTTCCCCTGGTCC GAGGAATTTGCAGGACCCCA EphB4-36 GGGCAGACTGACACTTCACA CGTGCCATGGTTTTCAGCTC GGCTGGGTACTCCTCTGAGA GGGTTTTGGTGTGGATGTCC EphB4-37 GCTCTTTGGGTAGCAGTGGT GGGAAAAGTGTAGGGGCACA TTCAGGCTCATGTGCTTGGT ACGCTTTTGGGAATGTTGGA EphB4-38 CAAGGCCCCAACTAGGTGTT AGCTGGGCTGGAAGAAAGTC CATTGCTCTGTGTTGCAAGCT AGCTGGGCTGGAAGAAAGTC EphB4-39 ACCACTTACTACGCCAAGCC CCTGATTTGAGGGGATGGGT ACCACTTACTACGCCAAGCC AGGCTGACATGTTCTTAGACTCT

Primer for HPRT-Exon6 Site No. 1st PCR_F 1st PCR_R 2nd PCR_F 2nd PCR_R HPRT_E6-01 TCTTACTGCTTGCTGAGGGC CTCACCCGCTTTGGAATCCT GCAGTTATACATGGGGGTTTTGG AGCAATCACTTAATCCCCCTTCA HPRT_E6-02 TGAGGCCTAGGCTGAGAAGT GTGCTTCAGGGGAGACTGTC GGATGGAAGGAATCATGGAGCT CCCCTGCCCATCCTGTTTTA HPRT_E6-03 GAACGTTGTTTTCCCTGGGC TGAGCCAAGATCGTGCCATT GCCCTCTTCAGTGAGCTTCA ATTGCACTCTAGCCTGGACG HPRT_E6-04 TGTGTGCCTCCAACTAGCTG CCACTTTACCCTCCAGCCAG AGCTGTCTCCTTTGCCTTCC TTGGCAAAGTGAGCAACTGC HPRT_E6-05 GATTCTCCTGCCTCAGCCTC TCTTATGTTGCCCAGGCTGG AGGTGCCGATTATCATGCCA GTGTTGTGCAACCATCACCA HPRT_E6-06 CCTGGACTTGAGGCAGTGAG CGCCACCACACCCTCTAATT GAAGGTGACCTGGTAGGACA TGGATGGATGACTTTGTCTACTT HPRT_E6-07 TGGATTAGGGGCCCACCATA ACGCCCGACCTGTTTAACAT AGCCAGCAGGTCATGAAGTC TTGAGCCCAGGAGTCTGAGA HPRT_E6-08 TGGTCCCACACGAAAGAAGG TCCATCCACCTAGCCATCCA TGTCAGGAACACAGGCACTT ATGTGAAGCAAGGGACAGGC HPRT_E6-09 CCCCACCCCCAAAAAATAGC ACACCCATGATGGAAGCCAA TGTTGCATGCCAAGTATTGTGA GCCTGCTCAGAGTCAAGGAA HPRT_E6-10 GAATGCATGAAACACCCCGG TCAAGGATGATTGCCATTGCT TCCCACAAATTAGGCCAGGAC TGATTGCCATTGCTTGTTGGA HPRT_E6-11 CCAAGAGACCATGGGCAGTT TGAGCCGAGATTGTGCCATT AATAGCAGGCCCTCTTCAGC ATTGCACTCTAGCCTGGACG HPRT_E6-12 CACACCCAGCCAAGATGGAT CCAACCTCCCAATCCCTGAC AGCAGGCTATATTTTGTGGTTGC ACCTGCAGAACTTAGTGAAACA HPRT_E6-13 ACACACACAGACGAAGGCAT CGACCTTCCTTTCCCGTTGA GCGGCTCCGTTTTGTTTGTT TGGACATGTCACCCAAGCTG HPRT_E6-14 TCCAGAGTGTAACCGGAAAGT TCAACTGAAGGTGGCAGGAA ATAGCCTATTGCCCCTCCCA GCCTCTAACTCTTTGTCTGGCT HPRT_E6-15 ATGTTGGCCAGGATGGTCTG ATGATGTTGTGCTGCGTTGG TCAGAAGCCTGCACTCTTCA TCAAAGGTCACAACACTTGCT HPRT_E6-16 TTCAACCTCTGTCCTGCTGC AGGCCTGGTGCTATGAGAGA TTCACTGCTCCAATGTGTGT AGGTGAGTTAAAGGGGGGAGA HPRT_E6-17 TGAACGAGGGTCAGTTTGGC CTGTGTCAGTGGCCATGTCT GCCTTTGCCTTATATTCGGC GTCAAACAGGTTGTACATGATGCA HPRT_E6-18 TCTTACCAACTGTGCCTTCCC AACTGGGAGGGAAAAGTGCC TGTCACGTGACCCTCTTCAT TGAGTGACTACAGTGAACGTTGT HPRT_E6-19 GGCAGGTGACTTAAGTCCCC GCACATGATCCTGTGTGTGC GGCAGGTGACTTAAGTCCCC GCACATGATCCTGTGTGTGC HPRT_E6-20 TGGCAGAATGGATTGGTTGAGA TGGAGACAGAGTCATTGCAAA TGGTCCCTACAGAGCTCTGA GAAGAGACAGAGACACAGAGCA HPRT_E6-21 CAAAGTTCCAACAAGGGCCC CTTCATGCTGGAGGCTGTCA ACCAACTCCTTGCTGTTGGG AGTGAAAGAAGGGAGCCAGC HPRT_E6-22 GAGACCAGGACACACAACGT TCGGCTCTCTTCATCACTGT CAAAATCCATTGCCCAGGCG TCGGCTCTCTTCATCACTGT HPRT_E6-23 AATCGCTTGAACCCAGGAGG CCAACCCTCCTCCTCTCAGA TGCTAACACATGCCTCCCTG CCAACCCTCCTCCTCTCAGA HPRT_E6-24 CTTCCCGGGTTGATCCTCAC ACCAAACGACTTGCCTGAGA CCTGCAACAAGTTTGTCTGGA TGGTGGCGATGATCCTTCAA HPRT_E6-25 TTTCATTGTCTGCTGCCAGA CGCTACTCTAACGGCCCAAA AGGGAACACAGTCCTTATTTGGA TGGAAATCACATCAAACCAACTAA HPRT_E6-26 TCTCTCCAATGGCAGCAGTG AGGCTGACAGTAGTTGCTTCC TCTCTCCAATGGCAGCAGTG CAGACAACAAAGTGTGGCCA HPRT_E6-27 ATGGTGGTCTTGGTCACAGC AAGATTCCCACACAGGAGCG GGTCTCAAACTCCTGGCCTC ATACTGACTGGCTGGCTCTG HPRT_E6-28 TCTGGCTTAATGTGGGGTGG ATGCTCCCATGAGGCCAAAT ATTGAGGGGGCTGGCTAAAC ACCCTTCATCATCACAGCTCT HPRT_E6-29 AACCCCACCAGTAGCAGGTA ACAGCCGCAGTCCTATGTTC AGCTTGTAGTGACCAGGTGA ACACTATGGGCCAATGTGAACT HPRT_E6-30 TGGTTCCTGCTGGGTTCTTC ACCCCACACTATACCCCCTC CCAGCGCCTGTTTCCAAAAG GCACACAAGATTCCAGCAGC HPRT_E6-31 CCCCACTGGTGACCTTACAC ATGAGGGAAGGGGAAGACCT CCACTGCAGGCTCAGGAATA ACAAACTGGGGGTGCTTCTG HPRT_E6-32 TGGTCGCCTCAGTCAGTTTC GGGAACAGCAAGTGCAAAGG TTACAGGCGTGAGTCACCAC AAGCCCAGGAATTTCGACGT HPRT_E6-33 AGGGGTTTCTGCCTCTTTGAG ACACCACCCAGTTCCACTTG AGTCTTATGGCGGGAATGTTT GGCTATGTACACATATTGTCCCA HPRT_E6-34 AGCCATAACCACAATCCCGT GGAGAGGAGAGAAAGCACCG AGCCATAACCACAATCCCGT AATGTGACCCTTGTGAGGCC HPRT_E6-35 TCCCTTGGAGCCCTACAGAA GCTTGGAGAGGCCCTTTGAT TCCCTTGGAGCCCTACAGAA AGAACAGGCATGCCAGCTTT HPRT_E6-36 GTTTCTCCTGACTCGGCCTC CACCAAAAGCACAGGCAACA TGGCCTTCCAAAGTGCTGG GGGGTGTGTAAAGGCAACCT HPRT_E6-37 GAGGCTGAGGCAGGAGAATC AGGCCTGGAGATAGAGCAGG GCAACAGAGTGAGACCCCAT AAAGCCGCTGGACTATGCTT HPRT_E6-38 CGCTAAATGAGGGCAAGGGA TGTCACGTCCTCCTAGGGTT CGCTAAATGAGGGCAAGGGA GACACCTTCCATGTCTCTGCA HPRT_E6-39 GAAGATGATGGAGACCGGCC TGGGCAGTGGTCACAAATGT CATTGGCATGACAGGGGAGA ACCTTTCCCAAACACACTCCT HPRT_E6-40 TGGCTACCCTGTCTCTGTGT ATTCCATGAGTCCCCGAGGA TGGTCCCTCAAGAGCTAGCT ATTCCATGAGTCCCCGAGGA HPRT_E6-41 GCGGATCATGAGGTCAGGAG TCTGAGCCAGGGAGCTTTTG GACAGAGTGAGACCCCGTCT TGTTTTTAAACTGCAGCAAATGCT HPRT_E6-52 ATCAGGCCTGGCATAGCATG CCTCCTGAGTAGCTGGGACT ATCAGGCCTGGCATAGCATG CGGCTTTGACTCCACCTCTT HPRT_E6-81 GACACCATCATGCAGGGTCA GGTCTTGAACTCCTGGCCTC TGCATTAGGAGAAGCTGGTTT TGTTATGTGCATTTGGCCACA

Primer for HPRT-Exon8 Site No. 1st PCR_F 1st PCR_R 2nd PCR_F 2nd PCR_R HPRT_E8-1 GGTGAAAAGGACCCCACGAA GAAGTGTCACCCTAGCCTGG AGAGAATATTTGTAGAGAGGCACA TGCTGGAAGGAGAAAACAATTC HPRT_E8-2 CCCTTGGTGTCAGTAATCCCA TGGCTGACAATGTCCTGCAT TGAGCTCTTATATGTGCAAGGCA CCCTGACCATTCTCACCACC HPRT_E8-3 TCTCCACGACCATCCAGTCT ATGTACCAAGCACTGTCCCG TAGCCTTTCTGAAGCAGGGC GCCAGTCTGACTCTTGAGCT HPRT_E8-4 TTAGACCTCCACTGGGTCGT GCAGAGAGCAACAGTGGACT GTCGTGTTGCACCCCATTTA TCCATCTGAAGCAGACTAGCC HPRT_E8-5 TCTGGGTGCCTCCATTGAAC CTGCCTGATGTCCACTCTCC GGAGACAGACACATGCATGGA TGCTTTCCCACATCCTGCAT HPRT_E8-6 TGAGTTTAGGATGGCAGCAACT CAATGCAAACCTGGCCTTCC CCAGATGTTCCCAAACTCTGC TGCCAGATGGATATGCACCA HPRT_E8-7 GAACCAGACCTCACCCCATG CCTGTGGCTCTGTCATGTGT GTCATCCAGTTGGTGCCAGA CCTGTGGCTCTGTCATGTGT HPRT_E8-8 CAGAGTGGATGGTGTGCTGT TAAGGCAGTGTGAAGCAGCA CAGAGTGGATGGTGTGCTGT TTTGCCAGAGAGGGTCTCCT HPRT_E8-9 TGCACGCCAGTCAAAGAGAT TGACCTGGCAATTCCACTCC AGCATGTGTCTGTACTTCATTGT TGACCTGGCAATTCCACTCC HPRT_E8-10 CCAGTTTGCAGTTCTGTGCC CCTGAAGCATCCTGACTGCT TGTTGACAGTCAAAAGTGAGGA CCTGAAGCATCCTGACTGCT HPRT_E8-11 ACTCTGGAGGCTCAGGTAGG ATTACAGGCGTGACCCACTG CGTTATCACATCTTCAGCAAAAGC ATTACAGGCGTGACCCACTG HPRT_E8-12 CCCGACCTCCAGGAAAAAGA GGTGGTGGGTGTTCAGATGT TGAGGGCCAAACCAGAAGAG CTCATTTTGAACTAACCCCTCCC HPRT_E8-13 GTCTCAAACTCGTGGGCTCA ACATGTCTGGCCTGCACATT TCCATTCCTGCTAAGTGCCC ACGACACTTGTGCACCTTTT HPRT_E8-14 TGTACCTCCTACCTGCTCCC GTGTGTGTGTGTGTGTGTGT CAAAGAAACAGAACCAATTGCATAT GCAGGTAGTTGTAACACAATGCT HPRT_E8-15 TCTTCGGACTGAACAAGCATCT TTTCAGATGAAGGCCGACCC CAGCCATGCCACTAAAGAGC GAGGTTTCTTTCAGCAGCCC HPRT_E8-16 GCCCATGCAATTGCTTAGGG CACTCCAAGGACAGTCCACC TCCCTTGGGATTTCAGGCTC GCATGAGCCAACTGCATTGT HPRT_E8-17 CCCACAAGCTTCATTCATGGC GCCTCTCAGGTTCATGCCAT CCCACAAGCTTCATTCATGGC ACGGACTGAGTTGGTACAGC HPRT_E8-18 TTGGGGGTCTCATTTGCCTC CCCTTACCCCTTCTCTCCCA TCCTTCTGCCTGAAAACAGACA CCAAGGGCAATTTCCAGTGG HPRT_E8-19 CACCACTGCACTCTACCCTG CAAGACCAGCCTAGGCAACA CACCACTGCACTCTACCCTG ACTTGTGGATTTTGCATATAGCCT HPRT_E8-20 AGTAGAGCTCCAGGGAAGGG GCCAAGAGCTCTGGAACCTT AGTAGAGCTCCAGGGAAGGG GCCCCTTCACTTGTTCCTCA

The resulting library was sequenced using Illumina MiniSeq equipped with a paired-end sequencing system. Base editing frequencies represent the frequency of modified target sites corrected by one or more edits within the editing window (positions 4-7). Computer program source code for calculating substitution and indel frequencies can be found at'https://github.com/ibs-cge/maund'.

Example 5. ABE7 .10, BE3, and Cas9's mismatched sgRNAs Tolerance

Using a series of mismatched sgRNAs targeting endogenous genomic loci, we investigated whether the off-target activities of ABE7.10, BE3 (cytosine base editor), and Cas9 are different from each other. More specifically, ABE7.10, BE3 (Base Editor plasmid; Addgene plasmid #73021; rAPOBEC1-) optimized for human codons and plasmids encoding sgRNAs containing 1 to 4 mismatched bases for the on target site XTEN-Cas9n-UGI-NLS) or SpCas9 (NP_269215.1)-encoding plasmid was transfected into HEK293T cells (refer to Reference Example 1), and the target in-depth sequencing of Reference Example 6 was performed, and base correction frequency and insertion /Deletion (Indel) frequency was measured.

The obtained base correction frequency (%) and insertion/deletion frequency (%) are shown in FIGS. 6A to 6C and Tables 8 to 10:

Target gene: HEK2 (on target site: GAACACAAAGCATAGACTGCGGG (SEQ ID NO: 31)) sgRNA target site Sequence number (+) ABE7.10 (+) Cas9 (+) BE3 Base editing frequency (%) Relative activity Indel frequency (%) Relative activity Base editing frequency (%) Relative activity GggtgCAAAGCATAGACTGCGGG One 1.33 0.02 0.03 0.00 4.53 0.05 GAACgtggAGCATAGACTGCGGG 2 12.76 0.17 30.83 0.48 15.00 0.18 GAACACAAgatgTAGACTGCGGG 3 6.48 0.09 0.30 0.00 1.18 0.01 GAACACAAAGCAcgagCTGCGGG 4 0.70 0.01 0.00 0.00 0.18 0.00 GAACACAAAGCATAGAtcatGGG 5 0.86 0.01 0.03 0.00 0.19 0.00 GAgtgCAAAGCATAGACTGCGGG 6 2.40 0.03 2.60 0.04 13.68 0.16 GAACAtggAGCATAGACTGCGGG 7 75.71 1.02 76.37 1.18 74.30 0.88 GAACACAAgatATAGACTGCGGG 8 22.45 0.30 0.64 0.01 7.76 0.09 GAACACAAAGCgcgGACTGCGGG 9 1.33 0.02 0.00 0.00 0.75 0.01 GAACACAAAGCATAagtTGCGGG 10 1.16 0.02 0.05 0.00 0.19 0.00 GAACACAAAGCATAGACcatGGG 11 5.12 0.07 0.02 0.00 0.52 0.01 GAgtACAAAGCATAGACTGCGGG 12 61.71 0.83 63.07 0.97 69.18 0.82 GAACgtAAAGCATAGACTGCGGG 13 57.23 0.77 73.27 1.13 75.35 0.89 GAACACggAGCATAGACTGCGGG 14 76.76 1.04 82.69 1.28 80.95 0.96 GAACACAAgaCATAGACTGCGGG 15 68.19 0.92 69.70 1.08 71.33 0.85 GAACACAAAGtgTAGACTGCGGG 16 77.93 1.05 45.78 0.71 76.05 0.90 GAACACAAAGCAcgGACTGCGGG 17 3.01 0.04 0.03 0.00 1.10 0.01 GAACACAAAGCATAagCTGCGGG 18 49.41 0.67 1.68 0.03 23.86 0.28 GAACACAAAGCATAGAtcGCGGG 19 53.12 0.72 0.96 0.01 14.18 0.17 GAACACAAAGCATAGACTatGGG 20 33.85 0.46 0.54 0.01 4.27 0.05 GgACACAAAGCATAGACTGCGGG 21 77.99 1.05 56.26 0.87 80.99 0.96 GAAtACAAAGCATAGACTGCGGG 22 77.47 1.04 56.28 0.87 81.51 0.97 GAACAtAAAGCATAGACTGCGGG 23 75.90 1.02 60.44 0.93 77.98 0.93 GAACACAgAGCATAGACTGCGGG 24 78.61 1.06 52.17 0.81 80.94 0.96 GAACACAAAaCATAGACTGCGGG 25 77.42 1.04 43.30 0.67 77.51 0.92 GAACACAAAGCgTAGACTGCGGG 26 75.62 1.02 53.51 0.83 81.23 0.96 GAACACAAAGCATgGACTGCGGG 27 67.88 0.92 11.14 0.17 66.25 0.79 GAACACAAAGCATAGgCTGCGGG 28 78.22 1.05 28.65 0.44 74.33 0.88 GAACACAAAGCATAGACcGCGGG 29 79.11 1.07 47.36 0.73 81.18 0.96 GAACACAAAGCATAGACTGtGGG 30 79.86 1.08 25.81 0.40 73.01 0.87 GAACACAAAGCATAGACTGCGGG 31 74.14 1.00 64.70 1.00 84.20 1.00 no sgRNA 0.63 0.01 0.00 0.00 0.07 0.00

(In the above table, the'NGG' portion at the end is a PAM sequence;

The sgRNA sequence is a sequence obtained by converting T to U in a sequence excluding a PAM sequence among target site sequences;

mismatched bases for on-target sequences are shown in lowercase;

Relative activity = (mismatched sgRNA result/matched sgRNA result);

The same applies to all tables below)

Target gene: HEK3 (on target site: GGCCCAGACTGAGCACGTGATGG (SEQ ID NO: 62)) sgRNA target site Sequence number (+) ABE7.10 (+) Cas9 (+) BE3 Base editing frequency (%) Relative activity Indel frequency (%) Relative activity Base editing frequency (%) Relative activity GatttAGACTGAGCACGTGATGG 32 0.32 0.01 0.00 0.00 0.17 0.00 GGCCtgagCTGAGCACGTGATGG 33 0.41 0.01 0.03 0.00 0.13 0.00 GGCCCAGAtcagGCACGTGATGG 34 0.48 0.01 0.05 0.00 0.20 0.00 GGCCCAGACTGAatgtGTGATGG 35 0.31 0.01 0.09 0.00 0.12 0.00 GGCCCAGACTGAGCACacagTGG 36 0.39 0.01 0.02 0.00 0.25 0.00 GGtttAGACTGAGCACGTGATGG 37 0.33 0.01 0.02 0.00 0.15 0.00 GGCCCgagCTGAGCACGTGATGG 38 5.23 0.13 1.15 0.02 16.86 0.22 GGCCCAGAtcaAGCACGTGATGG 39 2.18 0.05 0.00 0.00 0.21 0.00 GGCCCAGACTGgatACGTGATGG 40 0.56 0.01 0.01 0.00 0.27 0.00 GGCCCAGACTGAGCgtaTGATGG 41 0.79 0.02 0.01 0.00 0.18 0.00 GGCCCAGACTGAGCACGcagTGG 42 1.54 0.04 0.18 0.00 0.22 0.00 GGttCAGACTGAGCACGTGATGG 43 6.02 0.15 49.87 0.66 2.11 0.03 GGCCtgGACTGAGCACGTGATGG 44 40.39 1.01 61.63 0.82 49.37 0.65 GGCCCAagCTGAGCACGTGATGG 45 12.81 0.32 58.23 0.77 6.68 0.09 GGCCCAGAtcGAGCACGTGATGG 46 15.88 0.40 46.57 0.62 12.28 0.16 GGCCCAGACTagGCACGTGATGG 47 17.80 0.45 19.65 0.26 3.98 0.05 GGCCCAGACTGAatACGTGATGG 48 0.46 0.01 0.04 0.00 0.19 0.00 GGCCCAGACTGAGCgtGTGATGG 49 3.63 0.09 1.66 0.02 0.64 0.01 GGCCCAGACTGAGCACacGATGG 50 9.28 0.23 0.51 0.01 1.53 0.02 GGCCCAGACTGAGCACGTagTGG 51 14.31 0.36 21.05 0.28 10.61 0.14 GaCCCAGACTGAGCACGTGATGG 52 50.32 1.26 62.84 0.83 67.85 0.89 GGCtCAGACTGAGCACGTGATGG 53 37.32 0.94 66.54 0.88 50.80 0.67 GGCCCgGACTGAGCACGTGATGG 54 39.21 0.98 76.56 1.02 74.92 0.98 GGCCCAGgCTGAGCACGTGATGG 55 41.09 1.03 63.21 0.84 71.77 0.94 GGCCCAGACcGAGCACGTGATGG 56 38.99 0.98 53.99 0.72 60.41 0.79 GGCCCAGACTGgGCACGTGATGG 57 64.05 1.61 57.72 0.77 72.00 0.94 GGCCCAGACTGAGtACGTGATGG 58 25.02 0.63 41.86 0.56 23.39 0.31 GGCCCAGACTGAGCAtGTGATGG 59 30.04 0.75 47.29 0.63 43.94 0.58 GGCCCAGACTGAGCACGcGATGG 60 48.08 1.21 61.45 0.81 66.11 0.87 GGCCCAGACTGAGCACGTGgTGG 61 43.88 1.10 60.87 0.81 72.75 0.95 GGCCCAGACTGAGCACGTGATGG 62 39.87 1.00 75.41 1.00 76.33 1.00 no sgRNA 0.21 0.01 0.00 0.00 0.16 0.00

Target gene: RNF2 (on target site: GTCATCTTAGTCATTACCTGAGG (SEQ ID NO: 93)) sgRNA target site Sequence number (+) ABE7.10 (+) Cas9 (+) BE3 Base editing frequency (%) Relative activity Indel frequency (%) Relative activity Base editing frequency (%) Relative activity GctgcCTTAGTCATTACCTGAGG 63 0.13 0.00 0.01 0.00 0.27 0.00 GTCActccAGTCATTACCTGAGG 64 0.12 0.00 0.04 0.00 0.60 0.01 GTCATCTTgactATTACCTGAGG 65 0.12 0.00 0.01 0.00 0.11 0.00 GTCATCTTAGTCgccgCCTGAGG 66 0.10 0.00 0.02 0.00 0.20 0.00 GTCATCTTAGTCATTAttcaAGG 67 0.14 0.00 0.01 0.00 0.16 0.00 GTtgcCTTAGTCATTACCTGAGG 68 0.15 0.00 0.01 0.00 1.13 0.02 GTCATtccAGTCATTACCTGAGG 69 0.10 0.00 0.03 0.00 0.65 0.01 GTCATCTTgacCATTACCTGAGG 70 0.12 0.00 0.00 0.00 0.23 0.00 GTCATCTTAGTtgcTACCTGAGG 71 0.12 0.00 0.06 0.00 0.27 0.00 GTCATCTTAGTCATcgtCTGAGG 72 0.14 0.00 0.01 0.00 0.16 0.00 GTCATCTTAGTCATTACtcaAGG 73 0.10 0.00 0.01 0.00 0.20 0.00 GTtgTCTTAGTCATTACCTGAGG 74 1.22 0.03 12.77 0.23 47.48 0.66 GTCActTTAGTCATTACCTGAGG 75 0.22 0.01 0.23 0.00 15.33 0.21 GTCATCccAGTCATTACCTGAGG 76 1.44 0.03 7.76 0.14 34.05 0.47 GTCATCTTgaTCATTACCTGAGG 77 0.20 0.00 0.10 0.00 5.97 0.08 GTCATCTTAGctATTACCTGAGG 78 0.38 0.01 5.16 0.09 2.19 0.03 GTCATCTTAGTCgcTACCTGAGG 79 0.21 0.00 2.74 0.05 1.67 0.02 GTCATCTTAGTCATcgCCTGAGG 80 0.17 0.00 0.02 0.00 0.33 0.00 GTCATCTTAGTCATTAttTGAGG 81 0.14 0.00 0.02 0.00 0.13 0.00 GTCATCTTAGTCATTACCcaAGG 82 0.12 0.00 0.08 0.00 0.37 0.01 GcCATCTTAGTCATTACCTGAGG 83 18.06 0.41 46.51 0.85 65.55 0.91 GTCgTCTTAGTCATTACCTGAGG 84 14.01 0.32 49.31 0.90 70.67 0.98 GTCATtTTAGTCATTACCTGAGG 85 3.53 0.08 51.54 0.94 56.95 0.79 GTCATCTcAGTCATTACCTGAGG 86 9.98 0.23 41.89 0.76 62.12 0.86 GTCATCTTAaTCATTACCTGAGG 87 1.37 0.03 33.09 0.60 16.41 0.23 GTCATCTTAGTtATTACCTGAGG 88 23.31 0.53 43.94 0.80 60.08 0.83 GTCATCTTAGTCAcTACCTGAGG 89 4.99 0.11 29.11 0.53 25.20 0.35 GTCATCTTAGTCATTgCCTGAGG 90 11.45 0.26 35.75 0.65 36.77 0.51 GTCATCTTAGTCATTACtTGAGG 91 13.85 0.31 23.51 0.43 19.42 0.27 GTCATCTTAGTCATTACCTaAGG 92 8.98 0.20 27.32 0.50 31.08 0.43 GTCATCTTAGTCATTACCTGAGG 93 44.00 1.00 54.83 1.00 72.17 1.00 no sgRNA 0.13 0.00 0.03 0.00 0.12 0.00

As shown in FIGS. 6A to 6C and Tables 8 to 10, when the Base editor and Cas9 nuclease are used with sgRNA having a mismatch of 1 or 2 bases with respect to the on target site, most of them retain their activity (1 Tolerance to dog or 2 base mismatched sgRNA), when combined with 3 or 4 mismatched sgRNA, the activity was significantly decreased. In particular, the tolerance of Cas9, ABE7.10 and BE3 to mismatched sgRNAs was different from each other. 6A and 8, in the case of the target site of the HEK2 gene, when using sgRNA having a mismatch of 2 to 4 bases, ABE7.10 has relatively high tolerance (maintaining activity), but Cas9 and BE3 are active. Low or inert. As shown in Figure 6b and Table 9, in the case of the target site of the HEK3 gene, Cas9 together with sgRNA having tandem mismatch at the 3-4 position (according to the numbering from 1 to 23 in the 5'to 3'direction). (Complex formation) showed high activity when used (66% of activity compared to the case of using fully-matched sgRNA). On the other hand, in the case of ABE7.10, when forming a complex with the same sgRNA as described above, compared with the case of using a fully-matched sgRNA, a relatively low relative activity of 15% was shown. These results suggest that ABE7.10, BE3, and Cas9 recognize individual sets of non-target sites in the human genome, and suggest that a method that can determine the overall ABE specificity of the genome in an unbiased manner is required.

Example 6. Genome-wide off-target site identification ( HEK2 gene)

In order to identify target sites across the genome of ABE7.10 through Digenome-seq that depends on DNA double-strand break (DSB) generated in vitro , adenine-deamination produced DSB was generated at the site containing inosine. Uracil-Specific Excision Reagent (USER), a mixture of E. coli Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII (Endo VIII) (New England Biolabs; https://www.neb.com/products/m5505- user-enzyme; Uracil DNA glycosylase (UDG) and DNA glycosylase-lyase Endonuclease VIII mixture with 50 mM KCl, 5 mM NaCl, 10 mM Tris-HCl (pH 7.4), 0.1 mM EDTA, 1 mM DTT, 175 mg/ml BSA And 50% (w/v) glycerol) and recombinant BE3 delta UGI protein (His6-rAPOBEC1-nCas9 protein; BE3 variant lacking UGI domain; pET28b-BE1 (Addgene plasmid #73018) using site directed mutagenesis Using dCas9 A840 substituted with H840), DSB was generated at a site containing uracil, which is a result of cytosine deamination ( in vitro ), and a genome-wide BE3 off-target site was identified (in an unbiased manner). Similarly, the combination of E. coli endonuclease V (Endo V) or human Alkyladenine DNA Glycosylase (hAAG) and Endo VIII cleaves the phosphodiester bond at the inosine-containing site. Can be used to infer (see Figs. 7A-7C). Endo V recognizes inosine in DNA and cleaves the second phosphodiester bond 3'to inosine, while hAAG scavenging inosine to create an AP site (apurinic/apyrimidinic site), which is recognized and processed by Endo VIII. This leads to a single-strand break.

To test such inference, the target DNA sequence (HEK2 PCR amplicon containing (SEQ ID NO: 31)E. coliRecombinant ABE7.10 protein expressed and purified in (see Fig. 8 for purification results) and their sgRNA to catalyze adenine deamination in one DNA strand and generate nick in the other DNA strand (Fig. 7a), Then, it was treated with Endo V (see Fig. 7a) or hAAG/Endo VIII (see Fig. 7c) to generate a composite DSB. The results are shown in FIGS. 7D and 7E. As expected, it can be seen that the PCR amplification product was cleaved by ABE7.10 + Endo V (Fig. 7D) or ABE7.10 + hAAG/Endo VIII (Fig. 7e).

After confirming that DSB can be generated at the ABE-target site containing inosine using the inosine-specific DNA repair enzyme, the human genomic DNA isolated from HEK293T cells was ABE7.10 + Endo V or ABE7.10 + hAAG. In vitro treatment with /Endo VIII and Digenome-seq analysis was performed to investigate whether ABE off-target sites across the genome could be identified. Human genomic DNA isolated from EK293T cells was converted to ABE7.10 ribonucleoprotein (RNP) (300 nM ABE7.10 and 900 nM sgRNA; targeted to an endogenous chromosomal site (known as HEK2). (SEQ ID NO: 31))) and then incubated for 8 hours, and then incubated for 2 hours with repair enzyme (Endo V or hAAG/Endo VIII). ABE-mediated adenine-to-inosine conversion (inosine is changed to guanine during PCR amplification) and inosine-containing sites (nicked DNA strands are amplified) at the target site (opposite strands with nicks cannot be sequenced) using Sanger sequencing. Impossible) was confirmed in Endo V-catalyzed DNA cleavage (Fig. 7f). When genomic DNA is treated with ABE7.10 and Endo V, DNA strands containing inosine are cleaved by Endo V. The DNA strand cut by Endo V cannot be amplified by PCR, but the DNA strand that does not contain inosine is amplified. As a result, only adenine was observed and no guanine was observed in the correction site observed by Sanger sequencing (FIG. 7F).

In addition, quantitative real-time PCR was performed to verify target DNA cleavage using ABE7.10 + Endo V, and the results are shown in FIG. 7G. Similar results were obtained when hAAG and Endo VIII were used instead of Endo V (FIGS. 7H and 7I ). Whole genome sequencing (WGS) was performed on the obtained genomic DNA digestion samples. After mapping sequencing reads to the human reference genome (hg19), use the Integrative Genomics Viewer (IGV) to monitor the alignment of sequence reads at the on-target site. It was confirmed that it exhibits a characteristic uniform alignment (FIGS. 7J and 7K ). This alignment was not observed with ABE7.10 alone genomic DNA or untreated genomic DNA (FIGS. 7J and 7K ). These results show that Digenome-seq can be used to comprehensively map ABE target (on-target) and off-target sites across the genome.

In order to determine the ABE7.10 off-target sites in the human genome, a DNA cleavage score for each base pair location was assigned across the genome using WGS data (see FIG. 9), and in vitro cleavage The sites with high scores, corresponding to the sites, were listed (the initial cutoff score was set to 2.5 (according to Digenome 1.0; Kim, D., Kim, S., Kim, S., Park, J. & Kim, JS Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq.Genome research 26, 406-415 (2016); Digenome 1.0 program does not consider sequence homology)). Use Endo V to 17 in vitro cleavage sites to use the hAAG / Endo VIII to give a 18 in vitro cleavage sites. 16 sites including the target site were identified in common in two different tests (using Endo V vs. using hAAG/Endo VIII) (FIGS. 10A and 10B), which means that Digenome-seq analysis is highly reproducible and reliable. Show that you have Sequence logos obtained by comparing the DNA sequences of these sites show that almost all nucleotide positions contribute to the specificity of ABE7.10 deaminase (see Fig. 10c).

Example 7. Various genes Target time Genome-wide off-target site identification

In addition to the HEK2 gene target ABE7.10 RNP used in Example 6, each of the six additional ABE7.10 RNPs was used, for genomic DNA, Digenome-seq (Digenome-seq 1.0: DNA cleavage score of 2.5 or higher) Determined as a non-target location candidate; (A site with a DNA cleavage score of 0.1 or more and less than 10 mismatches and a PAM (5'-NGN-3' or 5'-NNG-3') is a non-target location Determined), and the results are shown in Tables 11 to 18 and FIG.

The number of ABE on-target and off-target sites identified by Digenome-seq Target gene On-target DNA sequence Sequence number Number of in vitro cleavage sites Digenome 1.0 Digenome 2.0 HEK2 GAACACAAAGCATAGACTGCGGG 31 17 152 RNF2 GTCATCTTAGTCATTACCTGAGG 93 5 50 TYRO3 GGCCACACTAGCGTTGCTGCTGG 94 2 12 WEE1 GTCGAGATGTTCTATTACTCTGG 95 One 21 EphB4 GCAGAATATTCGGACAAACACGG 96 3 39 HPRT - Exon6 GTATAATCCAAAGATGGTCAAGG 97 28 148 HPRT - Exon8 GAAGTATTCATTATAGTCAAGGG 98 One 20 Average±S.E.M 8 ± 4 60 ± 20

Digenome-captured sites (HEK2) Site No. Chr Position DNA Cleavage Score DNA seq at a cleavage sites Sequence number HEK2_001 chr19 35505485 20.1 GAACAC-AAGCAcAGACTGaAGG 99 HEK2_002 chr15 93557679 15.1 GAACACA-tGCATAGACTGCTAG 100 HEK2_003 chr4 53536209 14.7 GAAtACtAAGCATAGACTcCAGG 101 HEK2_004 chr4 90522183 14.3 GAACACAAtGCATAGAtTGCCGG 102 HEK2_005 chr1 167742859 13.4 aAACACAgAGCAcAGACTGCTGA 103 HEK2_006 chr10 43159340 13.4 aAACACAAAGaCATAGACcaCTGG 104 HEK2_007 chr5 87240613 8.9 GAACACAAAGCATAGACTGCGGG 105 HEK2_008 chr9 290167 6.6 aAACAtAAAGaATAGACTGCAAG 106 HEK2_009 chr3 28560566 5.8 GAACACAAAGtATAGAaTGCTAG 107 HEK2_010 chr1 175310138 5.1 GgACACAAAGCtTAGACTcCAGG 108 HEK2_011 chr13 87616351 4.9 GAAgAtAAAGCATAGACTctAGG 109 HEK2_012 chr10 87125365 4.6 aAACAtAAAGCATAGACTGCaAAG 110 HEK2_013 chr18 32279782 3.9 aAAtACAAAGCATAGACTaatATG 111 HEK2_014 chr2 192248363 3.5 GAACACAtA-CATAGACaGCTGG 112 HEK2_015 chr5 160202385 3.0 GAAaACAAAGCAaAGAaaGCAGG 113 HEK2_016 chr15 63236612 2.9 aAcacaAtAGCATAGACTGgaCTG 114 HEK2_017 chr20 23101377 2.7 atACACAgAGCAaAGACTGCAGG 115 HEK2_018 chrX 48317375 2.5 aAACAtAAAGCcTAGACTGaCGG 116 HEK2_019 chr19 49972515 2.2 aAAaACAAAGCgTAGACTGtGGG 117 HEK2_020 chr12 81785997 2.1 GgACAaAAAGCATAGACTcaAGG 118 HEK2_021 chr1 40461424 2.1 atACACACAAGCAcAGACTGCAGG 119 HEK2_022 chr15 92968604 2.1 GAACACAgcaCATAaACTGCAGG 120 HEK2_023 chr5 142142325 2.0 aAACACtAA-CATAGACTGCAGG 121 HEK2_024 chr5 131174461 1.7 aAAtACAAtGCATAGACTGCTAG 122 HEK2_025 chr1 36097072 1.4 GtAaACAAAGCATAGACTGaGGG 123 HEK2_026 chrX 132724931 1.3 GAACA-tAAtCAcAGACTGCTGG 124 HEK2_027 chr6 139353017 1.3 ccAaACAAAaCATAGACTGCTGG 125 HEK2_028 chrX 36949816 1.3 GAAaACAAAaCATAGAgTGCTGG 126 HEK2_029 chr17 2670110 1.2 aAtCAaAtAGCATAGACTGCATG 127 HEK2_030 chr13 91254877 1.2 GAACACAAAcatagtACTGaAGG 128 HEK2_031 chrX 138877515 1.2 aAACAgAAAGCATgGACTGCGGA 129 HEK2_032 chr13 113428466 1.1 cAAtACAAAGgATAGACTGCAGG 130 HEK2_033 chr15 41796089 1.1 agACACAcAGCAcAGACTGCAGG 131 HEK2_034 chr11 128508576 1.0 GAAttCAAAGCATAGAtTGCAGG 132 HEK2_035 chr11 131870604 0.9 acACACAAAGCATAGACTatGTG 133 HEK2_036 chr4 91844238 0.9 GgAaACAAAGCATAGACattTGG 134 HEK2_037 chr3 105801924 0.8 tAAtACAAAGCATAGAtaGtTGG 135 HEK2_038 chr8 66280643 0.8 GAAtttAAAGCATAGACTGCAAG 136 HEK2_039 chr3 168165117 0.8 ctgaACAcAGCAaAGACTGCTGG 137 HEK2_040 chr15 65377019 0.8 GAgCgatAAGCAcAGACTGCTGG 138 HEK2_041 chr9 97332608 0.7 GtAattAAAGCAcAGACTGCTGG 139 HEK2_042 chr3 33744669 0.7 GAAaACAAAcCAcAGACTGgGGG 140 HEK2_043 chr2 19474362 0.7 ctAttgcAAGCAcAGACTGCTGG 141 HEK2_044 chr16 64198277 0.7 GAAtACAtAaCATAGACTGgGGG 142 HEK2_045 chr1 199217096 0.7 atACcatAAGCATAGACTGtTGG 143 HEK2_046 chr22 28895717 0.7 attaAgAtAGCATAGACTGCAGG 144 HEK2_047 chr13 94827431 0.6 aAtgACtAAtaATAGACTGCTGG 145 HEK2_048 chr20 895768 0.6 atttcaAcAGCATAGACTGtAGG 146 HEK2_049 chr1 108869934 0.5 agtatagcAGCATAGACTGCAGG 147 HEK2_050 chr20 97640 0.5 GAAttCAAAGCATAGAtTGCAGG 148 HEK2_051 chrX 58256414 0.5 GcAttCtAAtaATAGACTGCTGG 149 HEK2_052 chr3 10835885 0.5 aAACACAtAGCAcAGcCTGCAGG 150 HEK2_053 chr8 97317606 0.5 GAACACAgtaCATAGACTGgCAG 151 HEK2_054 chr7 1973215 0.5 GgctgCctAGCAcAGACTGCCGG 152 HEK2_055 chr5 145405225 0.5 tctCctAccGCATAGACTGCTGG 153 HEK2_056 chr14 106066714 0.4 GtcgcaAAcGCATAGACTtCCGG 154 HEK2_057 chr2 34403831 0.4 cttattAAAGCAcAGACTGCTGG 155 HEK2_058 chr7 83764326 0.4 actatatAAGCATAGACTGtTGG 156 HEK2_059 chr14 106188442 0.4 GtcgcaAAcGCATAGACTtCCGG 157 HEK2_060 chr9 111564240 0.4 cgAtttcAAGCATAGACTGCTGG 158 HEK2_061 chr4 91393910 0.4 tAAtcatAAGCATAGAaTGCTGG 159 HEK2_062 chr9 117268271 0.4 GAAtAC-AAGCATAGACTGCTGT 160 HEK2_063 chr1 208174511 0.4 tcACAaAtAaGCATAGACTGgCGG 161 HEK2_064 chr6 164073518 0.4 cttgctAAAGCAcAGACTGCTGG 162 HEK2_065 chr18 56307002 0.4 aAgaACAAAaCATAGACTGCAGG 163 HEK2_066 chrX 119188148 0.4 aAttAaAcAGaATAGACTGCTGG 164 HEK2_067 chr10 11472312 0.4 GAACACAgAaCcATAGACTGgGGG 165 HEK2_068 chrX 93938357 0.4 aAACAaAAAGCATAGACcaCAAG 166 HEK2_069 chr3 33958845 0.4 GAACACA-gGCATAGACTatGGA 167 HEK2_070 chr9 90257919 0.4 ctcagagAAGCAcAGACTGCAGG 168 HEK2_071 chr2 144687377 0.4 atcatatAtGCATAaACTGCAGG 169 HEK2_072 chr1 191029339 0.4 cAACAaAAtatATAGACTGCTGG 170 HEK2_073 chr3 124241469 0.3 attgttAAAGCAcAGACTGCAGG 171 HEK2_074 chr11 80540770 0.3 agAtcttAAGCATAGACTGtGGG 172 HEK2_075 chr4 146455378 0.3 tAACACAAtacCATAGACTGgCGG 173 HEK2_076 chr6 70806985 0.3 GAAtACAAAtaATAGACTatTGG 174 HEK2_077 chr3 10539384 0.3 GAAtttAAAGCATAGACTctGGG 175 HEK2_078 chr13 80619390 0.3 aAACAaAtAaaATAGACTGCAGG 176 HEK2_079 chr5 126385455 0.3 ccACACcAAGCATAGACTtCTGG 177 HEK2_080 chr9 92683115 0.3 aAcaAaAcAGCATAGACTGCATG 178 HEK2_081 chr17 2810242 0.3 tAAttgcAAGCATAGACgGCAGG 179 HEK2_082 chr12 131431445 0.3 GgAgAgAgAGCATAGACTGCTGG 180 HEK2_083 chr11 105148271 0.3 agAaAtAAtatATAGACTGCAGG 181 HEK2_084 chr1 75043747 0.3 tggttactAGCATAGACTtCAGG 182 HEK2_085 chr20 31946581 0.3 aAACACttAaCAcAGACTGCAGG 183 HEK2_086 chr8 58172690 0.3 ccACcagcAGCATAGACTcCAGG 184 HEK2_087 chr7 39877158 0.2 aAgacattttCATAGACTGtCGG 185 HEK2_088 chr3 65508026 0.2 tAAgcacAAGCATAGgCTGCTGG 186 HEK2_089 chr6 40925009 0.2 aAcaAgtAtGCATAGACTGCTGG 187 HEK2_090 chr15 81265977 0.2 aAAtACcAAcCAcAGACTGCAGG 188 HEK2_091 chr8 102768749 0.2 aAAtgCAtAGCATAGACTGCTGA 189 HEK2_092 chr1 237916998 0.2 tAACACAAAGacTAaACTGCAGG 190 HEK2_093 chr1 215323272 0.2 tAcCACgAAGCATAGACTGtAGG 191 HEK2_094 chr14 60249634 0.2 GAtCACtcAaCATAGACTGCATG 192 HEK2_095 chr3 29416510 0.2 GAACAg-AAaCATAGACcaaTGG 193 HEK2_096 chr11 79574188 0.2 tAcaAaAtAagATAGACTGCTGG 194 HEK2_097 chr10 96064388 0.2 acAgttAAAGaATAGACTGCAGG 195 HEK2_098 chr7 78443524 0.2 aAACACAAAgGCATAaACTcCAGG 196 HEK2_099 chr13 51093353 0.2 GAACACAcA-CAcAGACTGaGGG 197 HEK2_100 chr13 45266353 0.2 GgACAtAAAtCATAaACTGCTGG 198 HEK2_101 chr1 20011125 0.2 aAAgctAcAGCATAGACTGCAAG 199 HEK2_102 chr1 77190607 0.2 tcACACAAAcCATAGACTGaGGG 200 HEK2_103 chr11 68639975 0.2 GAtCtacctatATAGACTGCAGG 201 HEK2_104 chr9 31652144 0.2 tAtagaggAGCATAGACTGCTGG 202 HEK2_105 chr19 5068807 0.2 tAtCAgAAAGCAcAGACTGCGGG 203 HEK2_106 chr12 54405073 0.2 GAACAacAAGgATAaACTGCCGG 204 HEK2_107 chr12 33389548 0.2 tAcaAacAcGCATAGAaTGCCGG 205 HEK2_108 chr1 15519301 0.2 tAAgAaAtgGaATAGACTGCAGG 206 HEK2_109 chr20 21064111 0.2 aAAtAttAAGCATAGACTaCGGG 207 HEK2_110 chr20 17801623 0.2 GAAtgCcAAGCATAaACTGCAGG 208 HEK2_111 chr2 19844956 0.2 aActcCAAAGCATAtACTGCTGG 209 HEK2_112 chr2 116312126 0.2 GtgaACAcAGCATAGACTGgGGC 210 HEK2_113 chr15 35048979 0.2 atcCtaAgAaCATAGACTGCAGG 211 HEK2_114 chr11 19349648 0.2 tttgttAAAaCATAGACTGCTGG 212 HEK2_115 chr4 170242082 0.2 tAACAaAAtGCATAGACTGCTAG 213 HEK2_116 chr1 66454108 0.2 tgtataAcAGCATAcACTGCTGG 214 HEK2_117 chr9 20221246 0.2 aAACACtAAtaATAGACTGtGGG 215 HEK2_118 chr12 2617156 0.2 aAgCAatcAGCAcAGACTGCGGG 216 HEK2_119 chr19 32225924 0.2 GgACACAgAGtATAGACTGaGGG 217 HEK2_120 chr6 123554091 0.1 cAcaAatcAaaATAGACTGCTGG 218 HEK2_121 chr13 58440671 0.1 GAAttCAAAGCATgGACTGCAGG 219 HEK2_122 chr19 18370525 0.1 ttcCAaAAAaCATAGACTGCTGG 220 HEK2_123 chr8 74980814 0.1 GtAgAgtAAGCATAcACTGCTGG 221 HEK2_124 chrX 101465228 0.1 GgcCtttAgGCAcAGACTGCAGG 222 HEK2_125 chr21 16057037 0.1 aAgactttAGCATAGACTGCAAG 223 HEK2_126 chr5 76655640 0.1 GAAaAgtAAG-ATAGACTGCCGG 224 HEK2_127 chrX 37275566 0.1 aActcatcAGCATAGACTGtAGG 225 HEK2_128 chrX 39813705 0.1 aAAtACAgAGCtTAGACTaCTGG 226 HEK2_129 chr3 99649762 0.1 aAACAtgAAGCATAGACaGCAAG 227 HEK2_130 chr3 86016290 0.1 acAtAgAtAttATAGACTGCTGG 228 HEK2_131 chr2 42080736 0.1 cAcattAAAGCAcAGACTGCTGG 229 HEK2_132 chr7 154994106 0.1 GAgCctAgAGCAcAGACTGCAGG 230 HEK2_133 chr10 85566693 0.1 aAgacttAAGCAaAGACTGCTGG 231 HEK2_134 chr1 176037520 0.1 ttACACAtAGCAcAGACTaCAGG 232 HEK2_135 chr7 56616286 0.1 agAagttAAGCATAGACTGgTGG 233 HEK2_136 chrX 69481649 0.1 ctAtAttgAGaATAGACTGCAGG 234 HEK2_137 chr17 79546106 0.1 GtgCACAcAGCATAGACTGCATG 235 HEK2_138 chr9 74099753 0.1 ctAgcaAAAaCATAGACTGCTGG 236 HEK2_139 chr7 113235709 0.1 tAAaACAAAGaAcAGACTGCTGG 237 HEK2_140 chr5 165353994 0.1 tAcagatAgtaATAGACTGCAGG 238 HEK2_141 chr5 134255399 0.1 aAttAatAAcaATAGACTGCTGG 239 HEK2_142 chr6 39115051 0.1 aAgattgcAGCATAaACTGCTGG 240 HEK2_143 chr10 732884 0.1 aAgCACAtAaCATAGACTGaAGG 241 HEK2_144 chr14 88566211 0.1 GtAgAaAAAGtATAGACTGCAGG 242 HEK2_145 chr14 89446986 0.1 tActAtggtGCATAGACTGCTGG 243 HEK2_146 chr11 133057022 0.1 ctctACtgAGaATAGACTGCAGG 244 HEK2_147 chr13 47277564 0.1 tgAaAtAAtaCATAaACTGCCGG 245 HEK2_148 chr6 42655828 0.1 aAtaAacttGCATAGACTGtAGG 246 HEK2_149 chr10 72922827 0.1 agACcCtAAGCATAGACTGCAGA 247 HEK2_150 chr16 55456878 0.1 tAcCcaAAtGCAcAGACTGCTGG 248 HEK2_151 chr2 194416373 0.1 aAgatagAtttATAGACTGCTGG 249 HEK2_152 chr4 177589824 0.1 aAACACAAAaCATAaAtTaCTGG 250

Digenome-captured sites (RNF2) Site No. Chr Position DNA Cleavage Score DNA seq at a cleavage sites Sequence number RNF2-01 chr1 210858376 7.3 tcaccaTTAGTCATTACCTGCTG 251 RNF2-02 chr1 185056773 4.4 GTCATCTTAGTCATTACCTGAGG 93 RNF2-03 chr17 53928598 4.3 GTCATCTTAGTCATTAC-TGAGG 252 RNF2-04 chr6 143212078 4.1 GTaATaTTAGTCATTACCgGTGG 253 RNF2-05 chr17 17072075 3.6 GTCATCcTAGTCATTtaCTGGGG 254 RNF2-06 chr12 131619804 2.2 aTCAcCTTAGcCATTACCaGGGG 255 RNF2-07 chr9 102127746 1.4 GTCA-CTTAGTCATTgCCTGTGG 256 RNF2-08 chr15 92315580 1.0 GTCAcaTTAGcCATTACCTGTGA 257 RNF2-09 chr2 57559709 1.0 tgtATCTTAGaCATTACaTGTGG 258 RNF2-10 chr5 29673802 0.8 aTttTCTTAGTgATTAtCTGGGG 259 RNF2-11 chr15 25135467 0.8 catcTaaTAGTaATTACCTGGGG 260 RNF2-12 chr3 60339996 0.6 aTagTaTTAGTCATTACCTGTGA 261 RNF2-13 chr12 12595622 0.4 acCATCTTAGTCATTAtCTaATG 262 RNF2-14 chrX 131090978 0.4 aTttTCTTAGTCATTACCTaGAG 263 RNF2-15 chr2 51894204 0.4 aTCATCTTcaTCATTACaTGAGG 264 RNF2-16 chr2 134638146 0.4 cctATaTataTCATTACCTtTGG 265 RNF2-17 chr16 62531206 0.4 GTCATtTTAGTCATTAtCTtGAG 266 RNF2-18 chr22 44137982 0.4 aTCAcCTgAGTCATTACCcaTGG 267 RNF2-19 chr4 125521739 0.4 tTaATCTTAGTCATTACtTtTGG 268 RNF2-20 chr2 102668931 0.3 aTCATCaTcGTCATTAtCTGGGG 269 RNF2-21 chr14 88336132 0.3 aTCA--TTAGTCATTgCCTGAGG 270 RNF2-22 chr7 43967266 0.3 cTtAT-TTAGTCATTACCTGTAG 271 RNF2-23 chr2 41520178 0.3 GTCATaTTAaTCATTACaTaGAG 272 RNF2-24 chr1 111785389 0.3 tcCATCTcAcTCATTACCTGAGG 273 RNF2-25 chr8 51846780 0.3 GTtATCTTAGTCtTTACCTGAGA 274 RNF2-26 chr2 24749046 0.3 caCATCTTAccCATTACaTGAGG 275 RNF2-27 chr6 5869239 0.3 GcCAcCTcAGTCATTAgCTGGGG 276 RNF2-28 chrX 133138307 0.3 acatTagTAaaCATTACCTaGGG 277 RNF2-29 chr1 59227209 0.3 taCcTacctGTCATTACCTaTGG 278 RNF2-30 chr2 177556597 0.3 GatATCTTAGcCATTACCTaGGA 279 RNF2-31 chr3 113984053 0.3 tatcTacTAcTCATTACCTGAGG 280 RNF2-32 chr2 66353253 0.2 agCATCTctGTCATTACCcaGGG 281 RNF2-33 chr8 99451745 0.2 cgtgcaTTAGTCATTACCTGAGG 282 RNF2-34 chr5 109304296 0.2 GTCtgtaTAGTCATTACCTtTGG 283 RNF2-35 chr6 155177402 0.2 taCATCTatGTtATTACCTaTGG 284 RNF2-36 chr17 58390142 0.2 tTCATCTTtGTCATTACCTaAAG 285 RNF2-37 chr11 8498488 0.2 tatActaTAtaCATTACCTGGGG 286 RNF2-38 chr6 3726562 0.2 GctcatTcAcTCATTACCTaTGG 287 RNF2-39 chr16 5977420 0.2 GTgATCTaaAGTCATTACCTtAGG 288 RNF2-40 chrX 76946717 0.2 tTCAataTAaTCATTACCTGTGG 289 RNF2-41 chr18 62061226 0.2 aTtATtTTAGTCATTACCTtTGG 290 RNF2-42 chr19 14867049 0.2 ccCATCTcAGcCATTACCTGGGT 291 RNF2-43 chr13 70399620 0.1 agaATaTTAGTCcTTACCTGGGG 292 RNF2-44 chr12 86219647 0.1 GatcctTTAaTCATTACCTtTGG 293 RNF2-45 chr13 103624473 0.1 acCATCTTAGTCAcTACCTGGGC 294 RNF2-46 chr1 174478567 0.1 GTatTaTTgtaCATTACCTGAGG 295 RNF2-47 chr2 209871803 0.1 agCATCTTAGcCATTACCTcTAG 296 RNF2-48 chr5 91144278 0.1 agCATCTTAaTCcTTACCTcAGG 297 RNF2-49 chr4 160106364 0.1 GaatcCTcAtaCATTACCTGTGG 298 RNF2-50 chr4 37552932 0.1 aTCATCTTttTaATTACCTaTGG 299

Digenome-captured sites (TYRO3) Site No. Chr Position DNA Cleavage Score DNA seq at a cleavage sites Sequence number TYRO3-01 chr15 76553332 4.3 GGCCACACTAGtGTTGCcGCTGG 300 TYRO3-02 chr15 41857272 3.4 GGCCACACTAGCGTTGCTGCTGG 94 TYRO3-03 chr20 45312757 2.0 GGCCACACcAGCcTTGCTGtCGG 301 TYRO3-04 chr5 173133468 0.8 GGCCAC--TAGCGTTGCTcCAGG 302 TYRO3-05 chr22 50686658 0.6 GGCCACACTgaCcTTGCTGCTGG 303 TYRO3-06 chr9 140109027 0.4 ttCCACAC-AGCtTTGCTGCTGG 304 TYRO3-07 chr17 17207899 0.3 GGCCAC-CTAGgGTTGCTGCTGG 305 TYRO3-08 chr11 99666699 0.2 ccaCcCtCTAGCaTTGCTGCTGG 306 TYRO3-09 chr11 27495183 0.2 cctaACACcAaCGTTGCTGCTGG 307 TYRO3-10 chr1 38684664 0.2 ctgagCtCTAGCaTTGCTGCTGG 308 TYRO3-11 chr3 22936710 0.1 GGCatataTAGCaTTGCTGCTGG 309 TYRO3-12 chr6 150629124 0.1 GaaCACACTAaCaTTGCTGtGGG 310

Digenome-captured sites (WEE1) Site No. Chr Position DNA Cleavage Score DNA seq at a cleavage sites Sequence number WEE1-01 chr1 234241713 11.1 tTaGAGATGTTCTATTAtTCCGG 311 WEE1-02 chr11 9610086 2.0 GTCGAGATGTTCTATTACTCTGG 95 WEE1-03 chr4 18855558 1.9 GagGAGATGcTCTATTACTCCGG 312 WEE1-04 chrX 42756785 0.7 aTCaAGATGaaCTATTACTCTGG 313 WEE1-05 chr1 215805743 0.4 GTgGAGATGTTaTgTTACTCTGG 314 WEE1-06 chr5 57863350 0.4 GaCataATGTTCTATTACTCAAG 315 WEE1-07 chr4 119010382 0.3 taatAGATGTTCTATTACTaAGG 316 WEE1-08 chr15 80845387 0.3 aTtaAGATGTTCTATTAaTaGGG 317 WEE1-09 chr13 87867165 0.2 cagctaATGTaaTATTACTCAGG 318 WEE1-10 chr4 107037600 0.2 aTCaAGATaTTCTATTACTgGGG 319 WEE1-11 chrX 136297269 0.2 tcttAtATtTTCTATTACTCAGG 320 WEE1-12 chr1 8500986 0.2 aTaGAGcaGTTaTATTACTCTGG 321 WEE1-13 chr11 71806684 0.2 GTaGAGATGTTCTcTaACTCAGG 322 WEE1-14 chr12 83599167 0.2 cTCttttatTTCTATTACTCTGG 323 WEE1-15 chr8 71394697 0.1 aatataATaTTCTATTACTCAGG 324 WEE1-16 chr8 86942319 0.1 caactataaTTCTATTACTCTGG 325 WEE1-17 chr1 61165563 0.1 GgCtActgtcTCTATTACTCAGG 326 WEE1-18 chr20 45730192 0.1 aTttAGATaTTCTATTACTCTGA 327 WEE1-19 chr8 134795474 0.1 cTttAagataTCTATTACTCTGG 328 WEE1-20 chr2 128238175 0.1 caaGAGAcGcTCTATcACTCAGG 329 WEE1-21 chr7 31677975 0.1 aaacAGcTGTTCTATTACTCAGG 330

Digenome-captured sites (EphB4) Site No. Chr Position DNA Cleavage Score DNA seq at a cleavage sites Sequence number EphB4-01 chr7 100411300 10.3 GCAGAATATTCGGACAAACACGG 96 EphB4-02 chr5 81021421 8.0 GCAGAATATTaCaGACAAACtAGG 331 EphB4-03 chr15 47712791 4.1 GCAGAATATcaGGACAAACAATG 332 EphB4-04 chr11 123329727 2.4 aCAGAATATTCaGACAAtCACAG 333 EphB4-05 chr1 93047514 2.2 GCAGtA-AaTaaGACAAACAAGG 334 EphB4-06 chr15 51198151 1.9 aCAaAATATTCGGACAggCACGG 335 EphB4-07 chr18 29712895 1.8 GCAGAATtTaaGGACAAgCAAGG 336 EphB4-08 chr3 55670295 1.8 GCAGAATA--aGGACAAACATGG 337 EphB4-09 chr13 25919264 1.4 cagtAATATTCaGACtAAACATGG 338 EphB4-10 chr3 122057177 0.9 ttAatATATaaGGACAAACATGG 339 EphB4-11 chr1 26891812 0.8 aCAtAATATTCaaACAAACAGGG 340 EphB4-12 chr10 131074805 0.6 aatGAATAaagGGACAAACAGGG 341 EphB4-13 chr4 123425007 0.5 tCAG-ATATaaGGACAAACATGG 342 EphB4-14 chr5 60692862 0.4 GCtGAATATTgGGACAAtCATGG 343 EphB4-15 chr9 930844 0.4 GCAGttATAaaaaGACAAACAGGG 344 EphB4-16 chr9 29344386 0.4 aCAGAATATaaGaACAAAtATGG 345 EphB4-17 chr4 160752620 0.4 atAGAATAaTCaGACAAAaAAGG 346 EphB4-18 chr16 23706770 0.4 GCAG-ATAaagGGACAAACATGG 347 EphB4-19 chr11 117354211 0.3 GacagtgAaTaGGACAAACATGG 348 EphB4-20 chr14 90497703 0.3 tgAGAATATgCGGACAAACACAG 349 EphB4-21 chr2 68459706 0.3 atAGAATATTaGGACAAAtAGAG 350 EphB4-22 chr13 51951355 0.2 caAtAATAaaaGGACAAACATGG 351 EphB4-23 chr17 7777056 0.2 aCAGgATAaaaGGACAAAtATGG 352 EphB4-24 chr3 188431448 0.2 aCAGAtATAcgtaGACAAACAGAG 353 EphB4-25 chr12 11705824 0.2 atAGAATAagaaGACAAACATGG 354 EphB4-26 chr10 95697506 0.2 ttAGAATATTCctACAAACAAGG 355 EphB4-27 chrX 123665071 0.2 cCAtgtTATTCaGACAAACATGG 356 EphB4-28 chr16 75869847 0.2 GCAGAA-AaTaGGACAAtCACGG 357 EphB4-29 chr6 128784409 0.2 agAGAATATa-GGACAAACtTGG 358 EphB4-30 chr5 144297821 0.2 GCAGAAataTttGACAAACAAGG 359 EphB4-31 chr14 29760524 0.1 aaAGAATATa-GaACAAACAGGG 360 EphB4-32 chr1 36914336 0.1 aCAGAA-ATaCGaACAAACATGG 361 EphB4-33 chr21 26227853 0.1 aacaAATgcaaGGACAAACATGG 362 EphB4-34 chr13 63528393 0.1 aCActAT-TTaaGACAAACAGGG 363 EphB4-35 chr10 72516747 0.1 aCAGAActacgaGACAAACACGG 364 EphB4-36 chr7 20108292 0.1 GCAGA-TAccCaGACAAACAGGG 365 EphB4-37 chr1 89896856 0.1 taAGAATAT-aGGACAAACAGGA 366 EphB4-38 chr4 145533104 0.1 cCAGAATATTa-GACAAACATGG 367 EphB4-39 chr20 9784096 0.1 agAGAATATagGGACAAAtAAGG 368

Digenome-captured sites (HPRT-Exon6) Site No. Chr Position DNA Cleavage Score DNA seq at a cleavage sites Sequence number HPRT_E6-001 chrX 133627607 29.0 GTATAATCCAAAGATGGTCAAGG 97 HPRT_E6-002 chr12 7468915 21.3 GTATAcatCcAAGATGGcCAGGG 369 HPRT_E6-003 chr3 10057621 17.0 GTATtA-CCAAAGATGGTCtGGG 370 HPRT_E6-004 chr22 39586062 12.7 GTATAATC-AAAGATGGcCcTGG 371 HPRT_E6-005 chr7 13928792 10.5 GTATtAT-CAAAaATGGTCAAGG 372 HPRT_E6-006 chr1 225831201 10.2 aTATAA-CCAAAGATGtTCACAG 373 HPRT_E6-007 chr17 53575925 9.3 aTATAATCttAAGATGGTCAAGG 374 HPRT_E6-008 chr12 13406973 9.0 aTATAATCCtAAGATGtTCATGG 375 HPRT_E6-009 chr14 27255077 6.1 GTATtATCCAAAGATGacCAGAG 376 HPRT_E6-010 chr7 85914581 5.6 aTAcAATCCAAAGtTGGTCtGGG 377 HPRT_E6-011 chr3 9906633 4.7 aTATAttaCCAAAGATGGTCtGGG 378 HPRT_E6-012 chr8 125496169 4.5 GTAgAATCCAtAGATGGaCAGGG 379 HPRT_E6-013 chr17 10064482 4.3 GTATtA-CCAAAGATGGTCATGG 380 HPRT_E6-014 chr2 53374352 4.3 GTAcAATCaAAAGATGaaCAAGG 381 HPRT_E6-015 chr4 102026801 4.1 aTATtATCCAAAGATGGagtTGG 382 HPRT_E6-016 chr6 19070067 3.8 aTgTAtTCCAAAGATGGTCAGGG 383 HPRT_E6-017 chr6 7761387 3.7 aagTAATCCAAAGATaGTCtTGG 384 HPRT_E6-018 chr12 59613203 3.6 GTATAAcCCAAAGAaaGTaATGG 385 HPRT_E6-019 chr16 20336811 3.6 aTATAtATtCcAAGATGGTtcTGG 386 HPRT_E6-020 chr4 136809212 3.5 tTATAATCCAAAGAaGGcCAAGG 387 HPRT_E6-021 chr12 117915656 3.5 GTATctAcCCAAAGATGtTCATGG 388 HPRT_E6-022 chr14 45005861 3.4 GTATAAT--AAAGATGGTCAAGG 389 HPRT_E6-023 chr14 104033102 3.3 aTAgAtTCCAAAGATGGaCATGG 390 HPRT_E6-024 chr11 93731924 3.1 cTATAATCtAAAaATGGTCAAGG 391 HPRT_E6-025 chr13 75257165 2.9 GTATAtTagAAAGATGGTCATGA 392 HPRT_E6-026 chr15 54911620 2.8 GgATAATCaAAAGATGGaCtTGG 393 HPRT_E6-027 chrX 6849511 2.6 aTATAATgCAAAGcTGGTCACGG 394 HPRT_E6-028 chr11 87526371 2.5 aTAcAATCCAAAGATatTCtAGG 395 HPRT_E6-029 chr3 19652583 2.4 GTATAATCCAAAGgTGGcCtAGG 396 HPRT_E6-030 chr17 45009128 2.4 aTATAATCCAtAGAaGGTCAAGA 397 HPRT_E6-031 chr8 9090663 2.3 GatatAatacAAGATGGaCAAGG 398 HPRT_E6-032 chr12 6810501 2.2 GgATAATCCAAAGATGGTgcAAG 399 HPRT_E6-033 chr6 118233456 1.5 aTATAAaCCAAAGATcGTtAAGG 400 HPRT_E6-034 chr14 85667729 1.4 tctcccTCCAAAGAaGGTCACGG 401 HPRT_E6-035 chrX 110551547 1.4 tTATAgTCCAAAGgaaGTCAGGG 402 HPRT_E6-036 chr8 18630339 1.2 tatTtATCCAAAGATGGTCtAGA 403 HPRT_E6-037 chr12 45338334 1.1 aTATAtaCCAAAGgTaGTCATGG 404 HPRT_E6-038 chr7 135056752 1.1 aggagATCCAAAGATGGTCAAGG 405 HPRT_E6-039 chr9 82090375 1.1 acATAgTCCAAAtATGGaCAGGG 406 HPRT_E6-040 chr12 103187212 1.0 GTATAtAattcAAGATGGTCAGGA 407 HPRT_E6-041 chr12 58996606 1.0 GaATAAgCCAAAGATGGTCAGTG 408 HPRT_E6-042 chr12 49657468 1.0 GTATctcCCAgAGATGGTCAAGG 409 HPRT_E6-043 chr7 120024149 0.9 aTgTAATCtAAAGATGaTtATGG 410 HPRT_E6-044 chr15 101375955 0.9 GTATAA-CCAAtGATGGTCAGGA 411 HPRT_E6-045 chr10 13873222 0.9 aTgTcATCCAAAGAaGGTCAGAG 412 HPRT_E6-046 chr21 41428166 0.9 agATAtTCCAAAGATGGTgAGGG 413 HPRT_E6-047 chr1 7314483 0.8 tTtccAcCCAAAGATGGTCcAGG 414 HPRT_E6-048 chr8 133182863 0.8 tTATAgctCcAAGATGGTCAAGG 415 HPRT_E6-049 chr6 1253997 0.8 tcAgAATCCtAAGATGGTCAGGA 416 HPRT_E6-050 chr12 15783424 0.8 GgATAATCCAAAGtTGGTCATAG 417 HPRT_E6-051 chr9 15160111 0.7 GTATAATCC-tAGATGGTgAGGG 418 HPRT_E6-052 chr12 29153372 0.7 GTATttTCCAAcGATGGTCATGG 419 HPRT_E6-053 chr3 1123204 0.7 GaATAATtCAAAGATaaTCAAGG 420 HPRT_E6-054 chr2 50999543 0.7 aaATAATa-AAAGATGGTCATGG 421 HPRT_E6-055 chr2 36768513 0.7 cTcatATCCAAAGATGGTaAAGG 422 HPRT_E6-056 chr6 4210565 0.7 tTATAA-CCAAAGATGGTaATGG 423 HPRT_E6-057 chr14 48260581 0.7 GccTAATCCAAAGATGagCAGGG 424 HPRT_E6-058 chr8 117575569 0.7 cTATAATgCCAAAGATGGTCATGG 425 HPRT_E6-059 chr5 160988739 0.7 GTATAgatCAAAGATGccCACGG 426 HPRT_E6-060 chr14 94364216 0.6 tctcctTCCAAAGATGGTCtGGG 427 HPRT_E6-061 chr9 125328946 0.6 aTAatcTCCAAAGATGtTCAGGG 428 HPRT_E6-062 chr9 134083092 0.6 aTATttTCCAAAGATGaTCACAG 429 HPRT_E6-063 chr16 6159271 0.6 tatTgATCCAAAGATGaTCAAGA 430 HPRT_E6-064 chr22 27822987 0.6 aTATAtTtCcAAGATGGgCAGGG 431 HPRT_E6-065 chr19 48640167 0.6 tTcTcATCCAAgGATGGTCAGGG 432 HPRT_E6-066 chr11 107913428 0.6 aTATAAaCaAAAGATGGTttAGG 433 HPRT_E6-067 chr22 25519897 0.6 aaATAgTCCAAgGATGGcCAGGG 434 HPRT_E6-068 chr2 222954701 0.5 GTATttTCtAAAGATGGTCATGA 435 HPRT_E6-069 chr1 21736973 0.5 GgATAAatCcAAGtTGGTCATGG 436 HPRT_E6-070 chr20 41218527 0.5 aTATttTCCAAAGATGGTCATGA 437 HPRT_E6-071 chr16 55855323 0.5 tgActcTCCAAAGATGGTCACAG 438 HPRT_E6-072 chr3 39725483 0.5 caATAtTCCAAAGATGGTtATGG 439 HPRT_E6-073 chrX 141390834 0.5 GcAgcATCCAAAGATGGgCAGGG 440 HPRT_E6-074 chr4 23070418 0.5 cTATAATtCAAAGATGtTCtTGG 441 HPRT_E6-075 chr8 9653862 0.5 cacatATatAAAGATGGTCATGG 442 HPRT_E6-076 chr22 32085052 0.5 GTATctTCCAAAGATGGcCAATG 443 HPRT_E6-077 chr16 8011143 0.5 aatattaCCtAAGATGGTCATGG 444 HPRT_E6-078 chr13 21574593 0.5 GTATAAaCCAAAaATatTCAGGG 445 HPRT_E6-079 chr12 58995581 0.4 GaATAAgCCAAAGATGGTCAGTG 446 HPRT_E6-080 chr10 107263858 0.4 aTATAATgCAAAGAaaGTCAAAG 447 HPRT_E6-081 chr11 44024764 0.4 GTATAAgCCAAAGcaGGTCACGG 448 HPRT_E6-082 chr6 138575959 0.4 aTATAAaCCAAAGAcGGcCcTGG 449 HPRT_E6-083 chr3 98960373 0.4 agATAATCCAAgGATGGTagAGG 450 HPRT_E6-084 chr8 43181791 0.4 aTATAATCCAgAaATGtTCACGG 451 HPRT_E6-085 chr5 118103678 0.4 actcttTtCcAAGATGGTCAGGG 452 HPRT_E6-086 chr7 129222414 0.4 cTgatgTCCAAAGATGGTCtCGG 453 HPRT_E6-087 chr3 160736673 0.4 GTgaAtTCCAAAGAaGGTCATGG 454 HPRT_E6-088 chrX 125678217 0.3 catTAATCCAAAGATGaTaAAGG 455 HPRT_E6-089 chr6 152915585 0.3 GctctATCCAAAGATGGaCAGGG 456 HPRT_E6-090 chr9 128839841 0.3 cTgTAATCCAgAaATaGTCAGGG 457 HPRT_E6-091 chr14 96980434 0.3 GTATAtgTCCAAAGATGaTCATGC 458 HPRT_E6-092 chr13 80168480 0.3 cTATAATCCAAgGAaGGcCAAGG 459 HPRT_E6-093 chr8 74179696 0.3 GTATccTCCAAAGATGagtAAGG 460 HPRT_E6-094 chr4 112559644 0.3 aaATAATaCAAAGATGGTtgGGG 461 HPRT_E6-095 chr13 74505576 0.3 caATAATCCAAAGAaGaTtAAGG 462 HPRT_E6-096 chr19 32267421 0.3 aTATAATCaCAAAGATGGcaATGG 463 HPRT_E6-097 chr14 95877075 0.3 GTtTAAgCCAAAGtTGGTCgGGG 464 HPRT_E6-098 chr8 36263307 0.3 GTAgcATCCAAAGATGGTggAGG 465 HPRT_E6-099 chr5 28287911 0.3 GTAgAATatcAAGATGGTCAAGG 466 HPRT_E6-100 chr5 98310114 0.3 agAatgcCCtAAGATGGTCtGGG 467 HPRT_E6-101 chr6 76199451 0.3 aTATAAcCagAAGATGtTCATGG 468 HPRT_E6-102 chr10 83966021 0.3 GTATtATCtAAAGgaGGTCATGG 469 HPRT_E6-103 chr1 55051337 0.2 agATAtaCagAAGATGGTCgGGG 470 HPRT_E6-104 chr8 91475188 0.2 aaATAATCCAAAaATaGTCATGG 471 HPRT_E6-105 chr9 20225753 0.2 cTtTAtTCCAAAGATaGTCATGG 472 HPRT_E6-106 chr6 81303564 0.2 GcATAttTCCAAAcATGGTCAAGG 473 HPRT_E6-107 chr1 241707903 0.2 GTATttTtCAAAGATGGTCATGA 474 HPRT_E6-108 chr9 28194167 0.2 GTATttTCCAAAGATGGcCACGA 475 HPRT_E6-109 chr3 17000484 0.2 tataAATCCAAgGATGGTCtGAG 476 HPRT_E6-110 chr16 73853834 0.2 tTATttTCCAAAGATGGTCACGA 477 HPRT_E6-111 chr15 29999660 0.2 tgcacATCCAcAGATGGTCAGGG 478 HPRT_E6-112 chr5 157715478 0.2 GTgTAATCCAgAGATGaTCACAG 479 HPRT_E6-113 chr10 2711644 0.2 tctTAATCCAtAGAaGGTCAAGG 480 HPRT_E6-114 chr17 61689903 0.2 tTcaAAT--AAAGATGGTCACGG 481 HPRT_E6-115 chrX 125134402 0.2 tcATAATCCAAAaGATGGaCAGGG 482 HPRT_E6-116 chr1 94251248 0.2 aTATAATaCAAAataGGTCAGGG 483 HPRT_E6-117 chr22 35100488 0.2 cTAgAtaCCAAAGATGGTCAGAG 484 HPRT_E6-118 chr2 38407889 0.2 aTtTAgTCCAAAGAgGGTCAGAG 485 HPRT_E6-119 chr7 112015061 0.2 aTATAcAaCCAAAGATGtTCtAGG 486 HPRT_E6-120 chr20 2163127 0.2 GTATAtAcatAAAGAaGGTCATGG 487 HPRT_E6-121 chr8 100230333 0.2 aTtatATCCAAAtATGGTCAGAG 488 HPRT_E6-122 chr2 206041685 0.2 tTATAATCCAtAAaATGGTCATAG 489 HPRT_E6-123 chr1 153531237 0.2 tTAgtcTCCAAAGATGGTCACTG 490 HPRT_E6-124 chrX 118562021 0.2 tatcttTCCAAAGATGGTCtATG 491 HPRT_E6-125 chr10 26718853 0.2 GTATttTCCAAAGcTaGTCATGG 492 HPRT_E6-126 chr17 61180425 0.2 GcATAATCCAAgGAaGGTCcTGG 493 HPRT_E6-127 chr2 125810813 0.2 aTATttTCCAAAaATGGTCACAG 494 HPRT_E6-128 chr7 28299854 0.2 aTATAATCCAAAagaaGgaAAAG 495 HPRT_E6-129 chr11 23995596 0.1 aTATAtcCaAAAGATGGTCtGGA 496 HPRT_E6-130 chr1 34307709 0.1 GatattaaCAAAGATGGTCtGGG 497 HPRT_E6-131 chr5 173751304 0.1 GaAgAATCCAAcGATGGcCACGG 498 HPRT_E6-132 chrX 14442705 0.1 acATAATCaCAAAtATGGTCACGG 499 HPRT_E6-133 chr20 15867186 0.1 GTATttTCCAAAGATGGcCACAG 500 HPRT_E6-134 chr5 157484648 0.1 tctaAtgaatAAGATGGTCgAGG 501 HPRT_E6-135 chr1 203269364 0.1 agATAATgCAAAGAcGGTtAGGG 502 HPRT_E6-136 chr12 28980303 0.1 GTATAtaCCAAAaATGGTgAAGG 503 HPRT_E6-137 chr18 52818481 0.1 aTATAc-CCAAAGATGGTCcAAG 504 HPRT_E6-138 chr10 131217430 0.1 tctgAcTCCcAAGATGGTCtTGG 505 HPRT_E6-139 chr3 109018997 0.1 GgAgAtaCCAAAGATGGTCAGGA 506 HPRT_E6-140 chr21 20933404 0.1 GTATAtTtCAAAtATGGTCATAG 507 HPRT_E6-141 chr2 220647283 0.1 aTATAA-CCAAAGATtGTCAGAG 508 HPRT_E6-142 chr22 23199165 0.1 cTggAgTCCAgAGATGGTCATGG 509 HPRT_E6-143 chr6 17237684 0.1 GTAgAAaCCAgAGATGGaCAAGG 510 HPRT_E6-144 chr10 82243028 0.1 tTATAATaCAAAGgTGGTaATGG 511 HPRT_E6-145 chr15 43004167 0.1 cTATAATCaAAAGtaGGTCATAG 512 HPRT_E6-146 chr7 25485727 0.1 aTATttTCCAAAaATGGTCATAG 513 HPRT_E6-147 chr2 71429668 0.1 GTAaAATCCAAAGAgGacCAGGG 514 HPRT_E6-148 chr3 141311087 0.1 cctaAATCtAAAGATGGTCgGGG 515

Digenome-captured sites (HPRT-Exon8) Site No. Chr Position DNA Cleavage Score DNA seq at a cleavage sites Sequence number HPRT_E8-01 chrX 133632686 2.6 GAAGTATTCATTATAGTCAAGGG 98 HPRT_E8-02 chr11 93382674 1.3 GAAGcATTCATTATAGTCAAAGG 516 HPRT_E8-03 chr21 34111536 0.6 GAAGaATTCATTATAGaCAATGG 517 HPRT_E8-04 chr11 100880874 0.6 aAgcTATTCATTATAGcaAATGG 518 HPRT_E8-05 chr22 29285654 0.6 GAAGTtaATTCATTATAGaCAATGG 519 HPRT_E8-06 chr4 15866630 0.5 aAAGTATTaATTATAGTCAAGGA 520 HPRT_E8-07 chr2 53543615 0.5 GAAGTAT-CcTTATAGTCAgAAG 521 HPRT_E8-08 chr3 149876617 0.4 GAAGTAT-CATTATAGTCttGGG 522 HPRT_E8-09 chr8 71429233 0.4 GcAGTATTCATaATAGTCAAAAG 523 HPRT_E8-10 chrX 146032194 0.4 aAAGTATTtcaTATAGcCAAAGG 524 HPRT_E8-11 chr5 132311070 0.4 GAAtTATTCATcATAGcCAAAGG 525 HPRT_E8-12 chr3 36349740 0.3 tAAGTAaTCATTATAGTCAgATG 526 HPRT_E8-13 chr3 181655989 0.3 atAGTATTCATTATAGTaAcAGG 527 HPRT_E8-14 chr11 116029576 0.3 aAAaTATcCATTATAGTCttACG 528 HPRT_E8-15 chr10 131905650 0.3 GAAcTATTCATTATtaTCAACGG 529 HPRT_E8-16 chr9 89312308 0.2 GttGTATTCATTATAGTCAAGAG 530 HPRT_E8-17 chr4 178543184 0.2 atAGTATTCATTATAGTaAcAGG 531 HPRT_E8-18 chr12 72733535 0.2 agAGTATcCAccATAGTCAAGGG 532 HPRT_E8-19 chr1 45916291 0.1 GAAGTAcatcTTATAaTCAATGG 533 HPRT_E8-20 chr4 153599265 0.1 aAtGTATTCATTATAGgCAAGAG 534

11 and Tables 11 to 18, when analyzed by Digenome 1.0 (DNA Cleavage Score cutoff: 2.5), a total of 7 sgRNAs forming a complex with ABE7.10 + Endo V were 1 to 28 including the target site ( Human genomic DNA was cut at 8±4 per sgRNA) site. These results show that Cas9 nuclease and BE3ΔUGI deaminase (+ USER) are 70±30 sites in the human genome, respectively (Kim, D., Kim, S., Kim, S., Park, J. & Kim, JS) Genome-wide target specificities of CRISPR-Cas9 nucleases revealed by multiplex Digenome-seq.Genome research 26, 406-415 (2016)) and the results of cutting genomic DNA at 8±3 sites.

Two of the seven sgRNAs analyzed in this example (HEK2 and RNF2) were used together with BE3ΔUGI or Cas9, respectively, to perform Digenome analysis, and the results were compared with the results obtained using ABE7.10. , Shown in Figures 12a and 12b. When HEK2 sgRNA was used in combination with ABE7.10, BE3ΔUGI, and Cas9, respectively, 17, 2, and 24 in vitro cleavage sites were generated, respectively (see FIG. 12C). When RNF2 sgRNA was used in combination with ABE7.10, BE3ΔUGI, and Cas9, respectively, 5, 1, and 13 cleavage sites were generated, respectively (see FIG. 12D). All three in vitro cleavage sites identified using two BE3 deaminases were also identified by their respective ABE7.10 deaminases. In contrast, the in vitro cleavage sites obtained using ABE7.10 and Cas9 were significantly different. Ten of the 17 sites (59%) obtained with HEK2 ABE7.10 were not identified when Cas9 was used. Two of the 5 sites (40%) obtained with RNF2 ABE7.10 were not identified when Cas9 and Cas9 were used. These results suggest that ABE is generally more specific than Cas9, yet able to recognize a set of off-target sites different from Cas9.

Using the above seven sgRNAs, ABE off-target base editing was verified in 57 in vitro cleavage sites identified through targeted amplicon sequencing. In addition, using the Digenome 2.0 program, it contains a sequence similar to PAM (5'-NGN-3'or 5'-NNG-3') and has a cutoff score of >0.1, compared to each of the on-target sequences. Candidate off-target sites including less than four mismatches were further identified.

Using 7 sgRNAs complexed with ABE7.10, 12 to 152 sites (60±20, on average) were obtained, which were 7 on-target sites obtained with Digenome 1.0 and 57 in vitro cleavage sites ( Tables 12 to 18) were all included. The frequency of base correction or gene correction of 7 ABE7.10 deaminases was measured at a total of 193 sites, including sites with a cleavage score of 1 or more obtained through deep sequencing or with high sequence homology, and the results are shown in Table 19 to It is shown in Table 25 and Figure 12e.

Mutation frequencies of ABE7.10, BE3, and Cas9 at on-target and off-target sites captured by Digenome-seq (HEK2; see Table 12 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Indel frequency (%) Untreated (+) ABE7.10 Untreated (+) BE3 Untreated (+) Cas9 HEK2_001 20.1 0.12 0.11 0.06 0.04 0.01 0.01 HEK2_002 15.1 0.00 0.00 0.04 0.04 0.02 0.01 HEK2_003 14.7 0.06 0.09 0.03 0.04 0.00 0.01 HEK2_004 14.3 0.10 0.31 0.07 0.18 0.02 3.09 HEK2_005 13.4 0.15 0.13 0.00 0.00 0.00 0.01 HEK2_006 13.4 0.00 0.00 0.00 0.00 0.06 0.11 HEK2_007 8.9 0.06 63.19 0.06 74.03 0.00 87.57 HEK2_008 6.6 0.06 0.12 0.03 0.03 0.02 0.01 HEK2_009 5.8 0.10 0.09 0.00 0.00 0.00 0.00 HEK2_010 5.1 0.00 0.00 0.03 0.03 0.00 0.01 HEK2_011 4.9 0.09 0.08 0.00 0.00 0.00 0.00 HEK2_012 4.6 0.05 0.06 0.00 0.00 0.02 0.00 HEK2_013 3.9 0.00 0.00 0.00 0.00 0.01 0.00 HEK2_014 3.5 0.08 0.08 0.18 0.11 0.00 0.00 HEK2_015 3.0 0.11 0.13 0.00 0.00 0.00 0.00 HEK2_016 2.9 No PCR HEK2_017 2.7 0.13 0.07 0.00 0.00 0.01 0.00 HEK2_018 2.5 0.18 0.20 0.08 0.06 0.00 0.00 HEK2_019 2.2 0.12 0.12 0.00 0.00 0.00 0.00 HEK2_020 2.1 0.12 0.20 0.02 0.03 0.09 0.08 HEK2_021 2.1 0.17 0.19 0.23 0.23 0.01 0.08 HEK2_022 2.1 0.13 0.11 0.08 0.08 0.00 0.01 HEK2_023 2.0 0.09 0.09 0.00 0.00 0.09 0.06 HEK2_024 1.7 0.00 0.00 0.08 0.06 0.00 0.00 HEK2_025 1.4 0.13 0.14 0.03 0.05 0.01 0.01 HEK2_026 1.3 0.13 0.16 0.08 0.08 0.00 0.00 HEK2_027 1.3 0.15 0.16 0.06 0.03 0.00 0.00 HEK2_028 1.3 0.00 0.00 0.03 0.02 0.01 0.02 HEK2_029 1.2 0.23 0.15 0.00 0.00 0.00 0.00 HEK2_030 1.2 0.09 0.11 0.00 0.00 0.01 0.02 HEK2_031 1.2 0.09 0.12 0.02 0.03 0.01 0.00 HEK2_032 1.1 0.05 0.09 0.04 0.06 0.00 0.00 HEK2_033 1.1 0.11 0.22 0.07 0.04 0.00 0.00 HEK2_034 1.0 0.05 0.05 0.07 0.13 2.42 4.10 HEK2_042 0.7 0.03 0.04 0.07 0.11 0.03 0.07 HEK2_044 0.7 0.00 0.00 0.08 0.03 0.00 0.00 HEK2_050 0.5 0.22 0.19 0.00 0.00 0.22 0.23 HEK2_052 0.5 0.13 0.11 0.03 0.02 0.13 0.11 HEK2_065 0.4 0.11 0.15 0.15 0.08 0.11 0.07 HEK2_079 0.3 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_082 0.3 0.00 0.00 0.34 0.24 0.00 0.00 HEK2_092 0.2 0.11 0.08 0.12 0.11 0.11 0.09 HEK2_093 0.2 0.02 0.03 0.14 0.13 0.02 0.05 HEK2_099 0.2 0.06 0.08 0.00 0.00 0.13 0.08 HEK2_101 0.2 0.00 0.00 0.07 0.06 0.00 0.00 HEK2_105 0.2 0.00 0.00 0.08 0.07 0.00 0.00 HEK2_106 0.2 0.21 0.21 0.06 0.03 0.21 0.15 HEK2_110 0.2 0.00 0.00 0.04 0.02 0.00 0.00 HEK2_119 0.2 0.00 0.00 0.05 0.05 0.00 0.00 HEK2_121 0.1 0.00 0.02 0.05 0.10 0.06 0.07 HEK2_139 0.1 0.11 0.09 0.02 0.03 0.11 0.11 HEK2_145 0.1 0.00 0.00 0.00 0.00 0.00 0.00

Mutation frequencies of ABE7.10, BE3, and Cas9 at on-target and off-target sites captured by Digenome-seq (RNF2; see Table 13 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Indel frequency (%) Untreated (+) ABE7.10 Untreated (+) BE3 Untreated (+) Cas9 RNF2-1 7.3 0.03 0.02 0.14 0.09 0.00 0.01 RNF2-2 4.4 0.02 26.26 0.05 78.82 0.01 88.29 RNF2-3 4.3 0.05 0.07 0.29 0.25 0.00 0.01 RNF2-4 4.1 0.05 0.05 0.00 0.00 0.01 0.01 RNF2-5 3.6 0.06 0.07 0.10 0.12 0.00 0.01 RNF2-6 2.2 0.06 0.09 0.09 0.10 0.02 0.02 RNF2-7 1.4 0.13 0.08 0.05 0.07 0.01 0.01 RNF2-8 1.0 0.08 0.10 0.04 0.05 0.01 0.00 RNF2-15 0.4 0.02 0.01 0.11 0.09 0.01 0.02 RNF2-19 0.4 0.00 0.02 0.06 0.06 0.07 0.09 RNF2-20 0.3 0.08 0.12 0.06 0.05 0.04 0.04 RNF2-24 0.3 0.03 0.03 0.02 0.05 0.29 0.35 RNF2-27 0.3 0.02 0.03 0.06 0.06 0.01 0.00 RNF2-41 0.2 0.03 0.01 0.00 0.00 0.03 0.03

Mutation frequencies of ABE7.10, BE3, and Cas9 at on-target and off-target sites captured by Digenome-seq (TYRO3; see Table 14 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Indel frequency (%) Untreated (+) ABE7.10 Untreated (+) BE3 Untreated (+) Cas9 TYRO3-1 4.3 0.30 7.77 0.08 3.84 0.06 12.94 TYRO3-2 3.4 0.48 71.60 0.07 39.78 0.05 49.28 TYRO3-3 2.0 3.38 3.51 0.18 0.27 0.00 0.14 TYRO3-4 0.8 0.26 0.21 0.15 0.16 0.00 0.00 TYRO3-5 0.6 0.50 0.38 0.09 0.10 0.00 0.02 TYRO3-6 0.4 0.25 0.24 0.08 0.06 0.01 0.00 TYRO3-7 0.3 0.27 0.27 0.51 0.38 0.01 0.01 TYRO3-8 0.2 0.00 0.00 0.26 0.19 0.00 0.00 TYRO3-9 0.2 0.26 0.23 0.06 0.10 0.00 0.00 TYRO3-10 0.2 0.08 0.10 0.05 0.02 0.00 0.01 TYRO3-11 0.1 0.07 0.11 0.00 0.00 0.00 0.00 TYRO3-12 0.1 0.33 0.35 0.19 0.16 0.00 0.00

Mutation frequencies of ABE7.10, BE3, and Cas9 at on-target and off-target sites captured by Digenome-seq (WEE1; see Table 15 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Indel frequency (%) Untreated (+) ABE7.10 Untreated (+) BE3 Untreated (+) Cas9 WEE1-1 11.1 0.26 0.26 0.00 0.00 0.00 0.06 WEE1-2 2.0 0.05 62.59 0.11 9.95 0.00 56.24 WEE1-3 1.9 0.38 0.42 0.00 0.00 0.00 4.13 WEE1-4 0.7 0.13 0.11 0.06 0.03 0.00 0.01 WEE1-5 0.4 0.08 0.11 0.00 0.00 0.00 0.01 WEE1-6 0.4 0.10 0.10 0.06 0.05 0.00 0.01 WEE1-7 0.3 0.04 0.06 0.00 0.00 0.00 0.02 WEE1-8 0.3 0.25 0.24 0.00 0.00 0.01 0.00 WEE1-9 0.2 0.05 0.04 0.12 0.17 0.01 0.01 WEE1-10 0.2 0.11 0.07 0.17 0.15 0.04 0.05 WEE1-11 0.2 0.24 0.29 0.00 0.00 0.46 0.43 WEE1-12 0.2 No PCR WEE1-13 0.2 0.67 0.69 0.00 0.00 0.40 0.29 WEE1-14 0.2 0.00 0.00 0.13 0.13 0.00 0.01 WEE1-15 0.1 0.14 0.11 0.00 0.00 0.01 0.01 WEE1-16 0.1 0.00 0.03 1.37 1.29 0.00 0.00 WEE1-17 0.1 0.14 0.08 0.13 0.20 0.02 0.00 WEE1-18 0.1 0.09 0.11 0.00 0.00 0.00 0.01 WEE1-19 0.1 0.11 0.12 0.00 0.00 0.01 0.00 WEE1-20 0.1 0.26 0.25 0.00 0.00 0.00 0.00 WEE1-21 0.1 0.09 0.12 0.27 0.19 0.02 0.00

Mutation frequencies of ABE7.10, BE3, and Cas9 at on-target and off-target sites captured by Digenome-seq (EphB4; see Table 16 for'DNA sequence at a cleavage sites') DNA Cleavage Score Base editing frequency (%) Indel frequency (%) Untreated (+) ABE7.10 Untreated (+) BE3 Untreated (+) Cas9 EphB4-01 10.3 0.12 57.62 0.14 0.17 0.01 74.73 EphB4-02 8.0 0.15 0.23 0.00 0.00 0.00 1.02 EphB4-03 4.1 0.09 0.11 0.26 0.30 0.01 0.14 EphB4-04 2.4 0.09 0.10 0.05 0.06 0.01 0.01 EphB4-05 2.2 0.14 0.13 0.08 0.09 0.01 0.00 EphB4-06 1.9 No PCR EphB4-07 1.8 0.33 0.30 0.09 0.10 0.00 0.01 EphB4-08 1.8 0.12 0.15 0.07 0.14 0.00 0.00 EphB4-09 1.4 0.50 0.42 0.00 0.00 0.01 0.01 EphB4-10 0.9 0.07 0.08 0.00 0.00 0.01 0.00 EphB4-11 0.8 0.07 0.08 0.16 0.16 0.01 0.02 EphB4-12 0.6 0.52 0.54 0.00 0.00 0.01 0.01 EphB4-13 0.5 0.15 0.14 0.11 0.09 0.01 0.01 EphB4-14 0.4 0.15 0.14 0.10 0.13 0.01 0.00 EphB4-15 0.4 0.12 0.10 0.00 0.00 0.01 0.01 EphB4-16 0.4 0.06 0.08 0.13 0.11 0.01 0.00 EphB4-17 0.4 0.20 0.20 0.00 0.00 0.32 0.33 EphB4-18 0.4 0.60 0.69 0.35 0.30 0.00 0.01 EphB4-19 0.3 0.11 0.09 0.10 0.10 0.00 0.00 EphB4-20 0.3 0.23 0.20 0.00 0.00 0.98 1.10 EphB4-21 0.3 No PCR EphB4-22 0.2 0.04 0.04 0.00 0.00 0.00 0.00 EphB4-23 0.2 0.05 0.06 0.05 0.05 0.12 0.11 EphB4-24 0.2 0.27 0.31 99.98 99.98 0.00 0.01 EphB4-25 0.2 0.06 0.06 0.00 0.00 0.00 0.00 EphB4-26 0.2 0.13 0.10 0.00 0.00 0.00 0.00 EphB4-27 0.2 No PCR EphB4-28 0.2 0.29 0.27 0.12 0.13 0.00 0.00 EphB4-29 0.2 0.55 0.48 0.00 0.00 0.00 0.01 EphB4-30 0.2 0.10 0.08 0.03 0.04 0.00 0.00 EphB4-31 0.1 0.15 0.18 0.00 0.00 0.24 0.21 EphB4-32 0.1 0.54 0.45 0.03 0.01 0.00 0.00 EphB4-33 0.1 0.23 0.15 0.15 0.11 0.03 0.01 EphB4-34 0.1 0.15 0.13 0.21 0.27 0.01 0.00 EphB4-35 0.1 6.14 6.02 0.20 0.31 0.00 0.00 EphB4-36 0.1 No PCR EphB4-37 0.1 0.22 0.19 0.00 0.00 0.00 0.00 EphB4-38 0.1 0.12 0.12 0.43 0.40 0.11 0.13 EphB4-39 0.1 0.11 0.11 0.00 0.00 0.01 0.00

Mutation frequencies of ABE7.10, BE3, and Cas9 at on-target and off-target sites captured by Digenome-seq (HPRT-Exon6; see Table 17 for'DNA sequence at a cleavage sites') Site No.
DNA Cleavage Score Base editing frequency (%) Indel frequency (%) Untreated (+) ABE7.10 Untreated (+) BE3 Untreated (+) Cas9 HPRT_E6-01 29.0 0.11 62.07 0.05 29.25 0.00 71.69 HPRT_E6-02 21.3 0.22 0.21 0.01 0.01 0.00 0.00 HPRT_E6-03 17.0 0.08 0.18 0.03 0.01 0.00 0.00 HPRT_E6-04 12.7 0.38 0.40 0.00 0.00 0.00 0.00 HPRT_E6-05 10.5 0.15 0.14 0.00 0.00 0.00 0.00 HPRT_E6-06 10.2 0.30 0.31 0.59 0.44 0.00 0.00 HPRT_E6-07 9.3 0.02 1.59 0.28 0.28 0.81 17.56 HPRT_E6-08 9.0 0.12 0.12 0.04 0.03 0.00 0.17 HPRT_E6-09 6.1 0.07 0.05 0.04 0.06 0.00 0.00 HPRT_E6-10 5.6 0.24 0.14 0.13 0.12 0.00 0.01 HPRT_E6-11 4.7 0.15 0.17 0.01 0.02 0.00 0.01 HPRT_E6-12 4.5 0.26 0.31 0.07 0.06 0.00 0.27 HPRT_E6-13 4.3 0.35 1.89 0.05 0.29 0.00 0.00 HPRT_E6-14 4.3 0.10 0.11 0.15 0.17 0.00 0.00 HPRT_E6-15 4.1 0.07 0.06 0.11 0.07 0.00 0.00 HPRT_E6-16 3.8 0.10 0.34 0.41 1.74 0.01 0.00 HPRT_E6-17 3.7 0.08 0.09 0.10 0.08 0.00 0.00 HPRT_E6-18 3.6 0.26 0.31 0.36 0.39 0.00 0.00 HPRT_E6-19 3.6 0.06 0.05 0.00 0.00 0.00 0.00 HPRT_E6-20 3.5 0.00 0.00 0.05 0.08 0.00 0.15 HPRT_E6-21 3.5 0.07 0.08 0.30 0.32 0.03 0.03 HPRT_E6-22 3.4 0.16 0.21 0.00 0.00 0.00 0.01 HPRT_E6-23 3.3 0.33 0.22 0.07 0.08 0.00 0.01 HPRT_E6-24 3.1 0.09 0.06 0.06 0.05 0.00 0.01 HPRT_E6-25 2.9 0.13 0.10 0.00 0.00 0.01 0.00 HPRT_E6-26 2.8 0.11 0.07 0.04 0.07 0.00 0.00 HPRT_E6-27 2.6 0.22 0.27 0.00 0.00 0.00 0.00 HPRT_E6-28 2.5 0.33 0.29 0.27 0.20 0.00 0.01 HPRT_E6-29 2.4 0.23 0.24 0.04 0.03 0.07 0.09 HPRT_E6-30 2.4 0.50 0.49 0.03 0.04 0.00 0.00 HPRT_E6-31 2.3 0.21 0.24 0.00 0.00 0.00 0.00 HPRT_E6-32 2.2 0.24 0.29 0.05 0.07 0.01 0.00 HPRT_E6-33 1.5 0.22 0.23 0.08 0.07 0.01 0.01 HPRT_E6-34 1.4 0.00 0.00 0.63 0.54 0.00 0.00 HPRT_E6-35 1.4 0.20 0.22 0.02 0.05 0.00 0.00 HPRT_E6-36 1.2 0.05 0.07 0.05 0.03 0.15 0.10 HPRT_E6-37 1.1 0.02 0.01 0.07 0.07 0.00 0.00 HPRT_E6-38 1.1 0.16 0.25 0.06 0.89 0.01 0.00 HPRT_E6-39 1.1 0.04 0.04 0.11 0.12 0.00 0.00 HPRT_E6-40 1.0 0.12 0.09 0.00 0.00 0.00 0.01 HPRT_E6-41 1.0 0.25 0.30 0.00 0.00 0.00 0.00 HPRT_E6-52 0.7 0.00 0.00 0.01 0.06 0.01 0.00 HPRT_E6-81 0.4 0.21 0.23 0.08 0.08 0.00 0.00

Mutation frequencies of ABE7.10, BE3, and Cas9 at on-target and off-target sites captured by Digenome-seq (HPRT-Exon8; see Table 18 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Indel frequency (%) Untreated (+) ABE7.10 Untreated (+) BE3 Untreated (+) Cas9 HPRT_E8-1 2.4 0.02 70.04 0.05 27.31 0.07 64.71 HPRT_E8-2 1.3 0.20 5.77 0.09 0.58 0.07 50.40 HPRT_E8-3 0.6 0.16 0.16 0.05 0.02 0.01 0.04 HPRT_E8-4 0.6 0.04 0.06 63.15 63.00 0.01 0.01 HPRT_E8-5 0.6 0.10 0.08 0.10 0.11 0.00 0.02 HPRT_E8-6 0.5 0.03 0.07 N/A N/A 0.01 0.19 HPRT_E8-7 0.5 0.16 0.15 0.07 0.11 0.00 0.00 HPRT_E8-8 0.4 0.07 0.07 0.08 0.05 0.03 0.04 HPRT_E8-9 0.4 0.02 0.03 0.06 0.09 0.02 0.04 HPRT_E8-10 0.4 0.01 0.01 N/A N/A 0.01 0.03 HPRT_E8-11 0.4 0.08 0.07 0.09 0.09 0.04 0.03 HPRT_E8-12 0.3 0.10 0.09 0.05 0.05 0.00 0.01 HPRT_E8-13 0.3 0.07 0.03 0.06 0.05 0.00 0.03 HPRT_E8-14 0.3 0.08 0.14 0.67 0.48 0.02 0.00 HPRT_E8-15 0.3 0.10 0.07 0.09 0.07 0.00 0.01 HPRT_E8-16 0.2 0.02 0.02 0.08 0.09 0.02 0.05 HPRT_E8-17 0.2 0.10 0.11 0.05 0.05 0.00 0.02 HPRT_E8-18 0.2 0.05 0.08 0.14 0.10 0.01 0.20 HPRT_E8-19 0.1 0.05 0.06 0.06 0.03 0.03 0.01 HPRT_E8-20 0.1 0.15 0.17 0.04 0.04 0.01 0.00

As shown in Tables 19 to 25, at 173 sites, a single nucleotide substitution was not detected at a frequency above the noise level (generally in the range of 0.1 to 1%) caused by sequencing errors. Among the other 20 validated sites, 7 on-target sites exhibited base correction frequencies between 29% and 72%. The other 13 validated off-target sites showed a base calibration frequency in the range of 0.1% to 7.8%, showing very low calibration efficiency. These results show that the Digenome-seq method is sensitive enough to detect even ABE off-target sites with very low calibration frequency near the detection limit.

In parallel with the above test, the Cas9-induced indel frequency and BE3-induced substitution frequency were measured at 193 site and compared with ABE7.10, and the results are shown in FIG. 12F. As shown in Fig. 12f, ABE7.10 activity was found to have a stronger association with Cas9 activity (R ² =0.77) than BE3 activity (R ² =0.39). In the upper graph of FIG. 12F, the base correction frequency is relatively high by ABE7.10, but the base correction frequency is very low (0.001% or less) by BE3. Among 6 sites (indicated by a hollow point), two sites Does not contain cytosine in the BE3 editing window. In the lower graph of FIG. 12F, the frequency of base correction by ABE7.10 was high in six sites (indicated by hollow dots), but the indel frequency by Cas9 was very low (indel frequency of 0.001% or less), of which Three sites (50%=3/6) have one inserted or deleted nucleotide compared to each of the on-target sites, and can form DNA or RNA bulges when hybridized with sgRNA. These bulge-forming sites are a common feature of BE3 off-target sites, but are rarely observed at Cas9 off-target sites. Three or two of the 193 sites identified using ABE7.10 through Digenome-seq were subjected to base or gene correction at high frequency in cells by Cas9 or BE3, respectively, but by ABE7.10, It exhibited a very low base correction frequency (substitution frequency 0.001% or less) (indicated by gray dots in Fig. 12f). These results again confirm that ABE7.10, BE3, and Cas9 are able to recognize different sets of off-target sites in the human genome.

Example 8. ABE off-target effect test

To minimize or eliminate the off-target effect of ABE, three methods were tested: sgRNA modification, RNP intracellular delivery instead of plasmid, and Sniper-Cas9 (engineered Cas9 variant obtained through directed evolution in E. coli ; It has E539S, M761I, and K890N mutations for the wild-type SpCas9 amino acid sequence (NP_269215.1), and further has a D10A mutation).

First (sgRNA modification), the targeting sequence of the sgRNA is a standard GX ₁₉ sgRNA consisting of 20 nucleotides (X ₁₉ : 19 arbitrary nucleotides (each independently selected from A, U(T), C, and G) means The number after X means any number of nucleotides, the same hereinafter), a truncated sgRNA (indicated by gX ₁₈ or gX ₁₇ ) from which 1 or 2 nucleotides have been removed from the 5'end of'X' (g is An extended sgRNA (gX ₂₀ or ggX) further comprising guanine inconsistent with the corresponding sequence of the target site, G denotes guanine that matches the corresponding sequence of the target site) or 1 or 2 extra guanines at the 5'end. ₂₀ ) was used to measure the frequency of base correction in HEK293T cells. The modified sgRNA used as a representative example of the HPRT exon 6 target sequence is shown in FIG. 13A. Modified sgRNAs for other genes were prepared with reference to FIG. 13A based on the On-target DNA sequence of each gene shown in Table 11.

The results of base correction frequency (measured by targeted deep sequencing) in HEK293T cells obtained using ABE7.10 and the modified sgRNA are shown in FIGS. 13B to 13D and Tables 26 to 32:

ABE7.10 off-target effect analysis when modified sgRNAs were used (HEK2; see Table 12 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Untreated (+) ABE7.10 gX17 gX18 GX19 gX20 ggX20 HEK2_001 20.1 0.12 0.22 0.15 0.11 0.12 0.13 HEK2_002 15.1 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_003 14.7 0.06 0.07 0.04 0.09 0.08 0.07 HEK2_004 14.3 0.10 0.10 0.11 0.31 0.21 0.11 HEK2_005 13.4 0.15 0.14 0.16 0.13 0.13 0.12 HEK2_006 13.4 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_007 8.9 0.06 9.80 47.71 63.19 61.77 55.40 HEK2_008 6.6 0.06 0.07 0.05 0.12 0.12 0.08 HEK2_009 5.8 0.10 0.08 0.08 0.09 0.11 0.12 HEK2_010 5.1 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_011 4.9 0.09 0.10 0.07 0.08 0.09 0.09 HEK2_012 4.6 0.05 0.05 0.03 0.06 0.04 0.04 HEK2_013 3.9 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_014 3.5 0.08 0.09 0.13 0.08 0.05 0.07 HEK2_015 3.0 0.11 0.09 0.14 0.13 0.13 0.14 HEK2_016 2.9 No PCR HEK2_017 2.7 0.13 0.07 0.15 0.07 0.12 0.10 HEK2_018 2.5 0.18 0.25 0.21 0.20 0.23 0.23 HEK2_019 2.2 0.12 0.13 0.13 0.12 0.11 0.12 HEK2_020 2.1 0.12 0.20 0.11 0.20 0.14 0.14 HEK2_021 2.1 0.17 0.18 0.18 0.19 0.21 0.20 HEK2_022 2.1 0.13 0.10 0.09 0.11 0.14 0.13 HEK2_023 2.0 0.09 0.13 0.05 0.09 0.12 0.08 HEK2_024 1.7 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_025 1.4 0.13 0.14 0.13 0.14 0.11 0.13 HEK2_026 1.3 0.13 0.12 0.15 0.16 0.11 0.16 HEK2_027 1.3 0.15 0.19 0.21 0.16 0.18 0.18 HEK2_028 1.3 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_029 1.2 0.23 0.20 0.16 0.15 0.18 0.14 HEK2_030 1.2 0.09 0.11 0.10 0.11 0.08 0.09 HEK2_031 1.2 0.09 0.12 0.10 0.12 0.08 0.09 HEK2_032 1.1 0.05 0.07 0.12 0.09 0.10 0.06 HEK2_033 1.1 0.11 0.11 0.14 0.22 0.14 0.16 HEK2_034 1.0 0.05 0.05 0.06 0.05 0.05 0.05 HEK2_042 0.7 0.03 0.07 0.04 0.04 0.05 0.04 HEK2_044 0.7 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_050 0.5 0.22 0.23 0.13 0.19 0.16 0.19 HEK2_052 0.5 0.13 0.10 0.09 0.11 0.07 0.11 HEK2_065 0.4 0.11 0.12 0.07 0.15 0.14 0.12 HEK2_079 0.3 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_082 0.3 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_092 0.2 0.11 0.08 0.11 0.08 0.06 0.10 HEK2_093 0.2 0.02 0.00 0.02 0.03 0.03 0.04 HEK2_099 0.2 0.06 0.15 0.13 0.08 0.10 0.13 HEK2_101 0.2 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_105 0.2 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_106 0.2 0.21 0.23 0.26 0.21 0.27 0.19 HEK2_110 0.2 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_119 0.2 0.00 0.00 0.00 0.00 0.00 0.00 HEK2_121 0.1 0.00 0.02 0.04 0.02 0.07 0.02 HEK2_139 0.1 0.11 0.12 0.09 0.09 0.17 0.14 HEK2_145 0.1 0.00 0.00 0.00 0.00 0.00 0.00

ABE7.10 off-target effect analysis when modified sgRNAs are used (RNF2; see Table 13 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Untreated (+) ABE7.10 gX17 gX18 GX19 gX20 ggX20 RNF2-1 7.3 0.03 0.01 0.02 0.02 0.01 0.03 RNF2-2 4.4 0.02 0.07 0.63 26.26 29.43 16.19 RNF2-3 4.3 0.05 0.07 0.07 0.07 0.05 0.06 RNF2-4 4.1 0.05 0.05 0.05 0.05 0.04 0.05 RNF2-5 3.6 0.06 0.06 0.08 0.07 0.07 0.08 RNF2-6 2.2 0.06 0.08 0.04 0.09 0.06 0.05 RNF2-7 1.4 0.13 0.15 0.09 0.08 0.17 0.16 RNF2-8 1.0 0.08 0.11 0.10 0.10 0.08 0.07 RNF2-15 0.4 0.02 0.02 0.03 0.01 0.05 0.02 RNF2-19 0.4 0.00 0.02 0.02 0.02 0.00 0.01 RNF2-20 0.3 0.08 0.10 0.10 0.12 0.10 0.10 RNF2-24 0.3 0.03 0.01 0.02 0.03 0.03 0.03 RNF2-27 0.3 0.02 0.03 0.05 0.03 0.03 0.01 RNF2-41 0.2 0.03 0.01 0.01 0.01 0.01 0.01

ABE7.10 off-target effect analysis when modified sgRNAs are used (TYRO3; see Table 14 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Untreated (+) ABE7.10 gX17 gX18 GX19 gX20 ggX20 TYRO3-1 4.3 0.30 0.26 5.32 7.77 9.26 8.32 TYRO3-2 3.4 0.48 0.58 36.53 71.60 67.78 54.99 TYRO3-3 2.0 3.38 3.44 3.40 3.51 3.34 3.53 TYRO3-4 0.8 0.26 0.20 0.21 0.21 0.24 0.24 TYRO3-5 0.6 0.50 0.41 0.50 0.38 0.51 0.42 TYRO3-6 0.4 0.25 0.25 0.24 0.24 0.26 0.25 TYRO3-7 0.3 0.27 0.31 0.25 0.27 0.31 0.27 TYRO3-8 0.2 0.00 0.00 0.00 0.00 0.00 0.00 TYRO3-9 0.2 0.26 0.21 0.24 0.23 0.21 0.21 TYRO3-10 0.2 0.08 0.06 0.07 0.10 0.05 0.08 TYRO3-11 0.1 0.07 0.10 0.10 0.11 0.09 0.08 TYRO3-12 0.1 0.33 0.30 0.37 0.35 0.32 0.30

Analysis of ABE7.10 off-target effects when modified sgRNAs are used (WEE1; see Table 15 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Untreated (+) ABE7.10 gX17 gX18 GX19 gX20 ggX20 WEE1-1 11.1 0.26 0.26 0.24 0.26 0.26 0.23 WEE1-2 2.0 0.05 0.25 0.95 62.59 37.80 26.30 WEE1-3 1.9 0.38 1.80 0.40 0.42 0.42 0.39 WEE1-4 0.7 0.13 0.08 0.09 0.11 0.11 0.11 WEE1-5 0.4 0.08 0.10 0.12 0.11 0.10 0.07 WEE1-6 0.4 0.10 0.12 0.12 0.10 0.07 0.10 WEE1-7 0.3 0.04 0.03 0.04 0.06 0.05 0.06 WEE1-8 0.3 0.25 0.23 0.22 0.24 0.20 0.29 WEE1-9 0.2 0.05 0.03 0.04 0.04 0.03 0.06 WEE1-10 0.2 0.11 0.10 0.14 0.07 0.11 0.10 WEE1-11 0.2 0.24 0.22 0.26 0.29 0.22 0.18 WEE1-12 0.2 No PCR WEE1-13 0.2 0.67 0.71 0.66 0.69 0.69 0.70 WEE1-14 0.2 0.00 0.00 0.00 0.00 0.00 0.00 WEE1-15 0.1 0.14 0.13 0.12 0.11 0.15 0.13 WEE1-16 0.1 0.00 0.01 0.03 0.03 0.01 0.02 WEE1-17 0.1 0.14 0.10 0.11 0.08 0.12 0.11 WEE1-18 0.1 0.09 0.09 0.06 0.11 0.11 0.08 WEE1-19 0.1 0.11 0.08 0.10 0.12 0.09 0.08 WEE1-20 0.1 0.26 0.26 0.24 0.25 0.27 0.24 WEE1-21 0.1 0.09 0.09 0.15 0.12 0.12 0.13

Analysis of ABE7.10 off-target effects when modified sgRNAs are used (EPHB4; see Table 16 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Untreated (+) ABE7.10 gX17 gX18 GX19 gX20 ggX20 EphB4-01 10.3 0.12 0.26 0.95 57.62 33.54 22.95 EphB4-02 8.0 0.15 0.16 0.13 0.23 0.14 0.13 EphB4-03 4.1 0.09 0.11 0.09 0.11 0.10 0.11 EphB4-04 2.4 0.09 0.06 0.11 0.10 0.10 0.10 EphB4-05 2.2 0.14 0.12 0.12 0.13 0.11 0.13 EphB4-06 1.9 No PCR EphB4-07 1.8 0.33 0.29 0.29 0.30 0.35 0.29 EphB4-08 1.8 0.12 0.12 0.09 0.15 0.19 5.82 EphB4-09 1.4 0.50 0.49 0.49 0.42 0.44 0.40 EphB4-10 0.9 0.07 0.07 0.09 0.08 0.09 0.07 EphB4-11 0.8 0.07 0.07 0.07 0.08 0.07 0.05 EphB4-12 0.6 0.52 0.57 0.57 0.54 0.53 0.58 EphB4-13 0.5 0.15 0.16 0.16 0.14 0.14 0.15 EphB4-14 0.4 0.15 0.14 0.12 0.14 0.13 0.14 EphB4-15 0.4 0.12 0.11 0.10 0.10 0.11 0.10 EphB4-16 0.4 0.06 0.06 0.06 0.08 0.05 0.06 EphB4-17 0.4 0.20 0.16 0.18 0.20 0.17 0.18 EphB4-18 0.4 0.60 0.64 0.60 0.69 0.74 0.64 EphB4-19 0.3 0.11 0.10 0.09 0.09 0.07 0.10 EphB4-20 0.3 0.23 0.11 0.19 0.20 0.19 0.18 EphB4-21 0.3 No PCR EphB4-22 0.2 0.04 0.04 0.06 0.04 0.05 0.03 EphB4-23 0.2 0.05 0.05 0.06 0.06 0.08 0.08 EphB4-24 0.2 0.27 0.22 0.30 0.31 0.35 0.32 EphB4-25 0.2 0.06 0.05 0.09 0.06 0.05 0.06 EphB4-26 0.2 0.13 0.10 0.10 0.10 0.10 0.11 EphB4-27 0.2 No PCR EphB4-28 0.2 0.29 0.26 0.33 0.27 0.31 0.31 EphB4-29 0.2 0.55 0.40 0.37 0.48 0.37 0.40 EphB4-30 0.2 0.10 0.05 0.05 0.08 0.07 0.07 EphB4-31 0.1 0.15 0.13 0.15 0.18 0.19 0.15 EphB4-32 0.1 0.54 0.44 0.36 0.45 0.46 0.48 EphB4-33 0.1 0.23 0.21 0.15 0.15 0.15 0.23 EphB4-34 0.1 0.15 0.14 0.14 0.13 0.14 0.14 EphB4-35 0.1 6.14 5.82 6.08 6.02 6.27 5.68 EphB4-36 0.1 No PCR EphB4-37 0.1 0.22 0.16 0.21 0.19 0.17 0.17 EphB4-38 0.1 0.12 0.16 0.14 0.12 0.14 0.13 EphB4-39 0.1 0.11 0.08 0.09 0.11 0.09 0.11

ABE7.10 off-target effect analysis when modified sgRNAs are used (HPRT-Exon6; see Table 17 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Untreated (+) ABE7.10 gX17 gX18 GX19 gX20 ggX20 HPRT_E6-01 29.0 0.11 16.94 29.23 62.07 60.39 58.46 HPRT_E6-02 21.3 0.22 0.15 0.17 0.21 0.18 0.17 HPRT_E6-03 17.0 0.08 0.11 0.10 0.18 0.10 0.07 HPRT_E6-04 12.7 0.38 0.45 0.42 0.40 0.43 0.39 HPRT_E6-05 10.5 0.15 0.22 0.14 0.14 0.15 0.15 HPRT_E6-06 10.2 0.30 0.23 0.35 0.31 0.36 0.28 HPRT_E6-07 9.3 0.02 0.03 0.05 1.59 0.06 0.06 HPRT_E6-08 9.0 0.12 0.10 0.08 0.12 0.11 0.10 HPRT_E6-09 6.1 0.07 0.03 0.06 0.05 0.04 0.06 HPRT_E6-10 5.6 0.24 0.19 0.15 0.14 0.18 0.12 HPRT_E6-11 4.7 0.15 0.22 0.25 0.17 0.15 0.16 HPRT_E6-12 4.5 0.26 0.22 0.26 0.31 0.26 0.26 HPRT_E6-13 4.3 0.35 0.55 0.45 1.89 1.26 0.59 HPRT_E6-14 4.3 0.10 0.11 0.13 0.11 0.08 0.09 HPRT_E6-15 4.1 0.07 0.05 0.09 0.06 0.04 0.08 HPRT_E6-16 3.8 0.10 6.37 0.26 0.34 0.08 0.08 HPRT_E6-17 3.7 0.08 0.17 0.10 0.09 0.10 0.12 HPRT_E6-18 3.6 0.26 0.31 0.37 0.31 0.37 0.39 HPRT_E6-19 3.6 0.06 0.06 0.09 0.05 0.04 0.05 HPRT_E6-20 3.5 0.00 0.09 0.08 0.00 0.09 0.00 HPRT_E6-21 3.5 0.07 0.06 0.11 0.08 0.09 0.08 HPRT_E6-22 3.4 0.16 0.15 0.19 0.21 0.17 0.14 HPRT_E6-23 3.3 0.33 0.26 0.27 0.22 0.25 0.28 HPRT_E6-24 3.1 0.09 0.07 0.07 0.06 0.09 0.11 HPRT_E6-25 2.9 0.13 0.11 0.11 0.10 0.16 0.12 HPRT_E6-26 2.8 0.11 0.08 0.05 0.07 0.12 0.09 HPRT_E6-27 2.6 0.22 0.28 0.27 0.27 0.27 0.22 HPRT_E6-28 2.5 0.33 0.39 0.32 0.29 0.35 0.29 HPRT_E6-29 2.4 0.23 0.21 0.16 0.24 0.20 0.19 HPRT_E6-30 2.4 0.50 0.46 0.50 0.49 0.51 0.45 HPRT_E6-31 2.3 0.21 0.25 0.23 0.24 0.24 0.17 HPRT_E6-32 2.2 0.24 0.27 2.20 0.29 0.25 0.28 HPRT_E6-33 1.5 0.22 0.24 0.17 0.23 0.22 0.17 HPRT_E6-34 1.4 0.00 0.00 0.00 0.00 0.00 0.00 HPRT_E6-35 1.4 0.20 0.16 0.24 0.22 0.23 0.23 HPRT_E6-36 1.2 0.05 0.04 0.03 0.07 0.05 0.05 HPRT_E6-37 1.1 0.02 0.08 0.03 0.01 0.03 0.08 HPRT_E6-38 1.1 0.16 0.33 0.22 0.25 0.22 0.19 HPRT_E6-39 1.1 0.04 0.06 0.04 0.04 0.04 0.04 HPRT_E6-40 1.0 0.12 0.13 0.12 0.09 0.10 0.12 HPRT_E6-41 1.0 0.25 0.19 0.11 0.30 0.40 0.10 HPRT_E6-52 0.7 0.00 0.00 0.00 0.00 0.00 0.00 HPRT_E6-81 0.4 0.21 0.20 0.19 0.23 0.22 0.23

ABE7.10 off-target effect analysis when modified sgRNAs are used (HPRT-Exon8; see Table 18 for'DNA sequence at a cleavage sites') Site No. DNA Cleavage Score Base editing frequency (%) Untreated (+) ABE7.10 gX17 gX18 GX19 gX20 ggX20 HPRT_E8-1 2.4 0.02 37.90 62.81 70.04 63.66 56.04 HPRT_E8-2 1.3 0.20 0.20 0.25 5.77 0.56 0.29 HPRT_E8-3 0.6 0.16 0.13 0.19 0.16 0.15 0.12 HPRT_E8-4 0.6 0.04 0.05 0.05 0.06 0.04 0.06 HPRT_E8-5 0.6 0.10 0.12 0.12 0.08 0.11 0.13 HPRT_E8-6 0.5 0.03 0.03 0.04 0.07 0.02 0.06 HPRT_E8-7 0.5 0.16 0.11 0.12 0.15 0.16 0.15 HPRT_E8-8 0.4 0.07 0.07 0.09 0.07 0.09 0.09 HPRT_E8-9 0.4 0.02 0.02 0.02 0.03 0.04 0.01 HPRT_E8-10 0.4 0.01 0.01 0.01 0.01 0.01 0.02 HPRT_E8-11 0.4 0.08 0.05 0.08 0.07 0.05 0.09 HPRT_E8-12 0.3 0.10 0.06 0.10 0.09 0.08 0.09 HPRT_E8-13 0.3 0.07 0.04 0.03 0.03 0.04 0.03 HPRT_E8-14 0.3 0.08 0.11 0.10 0.14 0.11 0.10 HPRT_E8-15 0.3 0.10 0.10 0.07 0.07 0.10 0.10 HPRT_E8-16 0.2 0.02 0.05 0.05 0.02 0.02 0.02 HPRT_E8-17 0.2 0.10 0.09 0.11 0.11 0.10 0.10 HPRT_E8-18 0.2 0.05 0.04 0.45 0.08 0.04 0.04 HPRT_E8-19 0.1 0.05 0.05 0.03 0.06 0.03 0.06 HPRT_E8-20 0.1 0.15 0.14 0.18 0.17 0.17 0.17

Figure 13b is a graph showing the average of the base correction frequency of ABE7.10 for each modified sgRNA measured by targeted deep sequencing when using modified sgRNA targeting HEK2, RNF2, TK_EphB4, TYRO3, WEE1, HPRT-exon6, and HPRT-exon8 to be.

13C and 13D, the modified sgRNA reduced the off-target activity of ABE7.10 in almost all sites while maintaining the targeting activity (in FIG. 13D, the y-axis graph is ggX20, gX20, GX19 in order from the top). , gX18, gX17, and untreated results are shown). For example, as shown in Figure 13c, gX ₂₀ or ggX ₂₀ sgRNA with respect to four verified off-target sites identified using GX ₁₉ sgRNA targeting HPRT Exon 6 has a specificity ratio compared to existing GX ₁₉ sgRNA. It improved 2 to 26 times. 13C and 13D, truncated sgRNAs reduce the on target and off-target activity of ABE7.10 at most sites, and worsen the off-target effect at the site of mismatch near or near the 5'end. Appeared to be letting.

In addition, the same test as above was performed using Sniper ABE7.10 in which Sniper-Cas9 was fused with an adenosine deaminase moiety instead of wild-type SpCas9, and the results measured by Digenome-seq were shown in FIGS. 13E and 13F (FIG. , The y-axis graph shows the results of (+) SpCas9 ABE7.10, (+)ABE7.10, and untreated in order from above) and Tables 33 to 39:

Mutation frequencies of ABE7.10 and Sniper ABE7.10 at on-target and off-target sites captured by Digenome-seq (HEK2; see Table 12 for'DNA sequence at a cleavage sites') Base editing frequency (%) Site No. DNA Cleavage Score Untreated (+) ABE7.10 (+) Sniper ABE7.10 HEK2_001 20.1 0.17 0.17 0.16 HEK2_002 15.1 0.55 0.55 0.59 HEK2_003 14.7 0.02 0.05 0.07 HEK2_004 14.3 0.16 0.33 0.13 HEK2_005 13.4 0.16 0.22 0.22 HEK2_006 13.4 0.22 0.18 0.17 HEK2_007 8.9 0.31 72.76 75.92 HEK2_008 6.6 0.09 0.10 0.11 HEK2_009 5.8 0.11 0.10 0.09 HEK2_010 5.1 0.39 0.42 0.34 HEK2_011 4.9 0.12 0.13 0.11 HEK2_012 4.6 0.24 0.23 0.22 HEK2_013 3.9 0.20 0.24 0.20 HEK2_014 3.5 0.02 0.03 0.00 HEK2_015 3.0 0.17 0.21 0.22 HEK2_016 2.9 No PCR HEK2_017 2.7 0.20 0.22 0.18 HEK2_018 2.5 0.32 0.30 0.31 HEK2_019 2.2 0.37 0.36 0.43 HEK2_020 2.1 0.15 0.13 0.12 HEK2_021 2.1 0.50 0.42 0.47 HEK2_022 2.1 0.19 0.20 0.24 HEK2_023 2.0 6.76 6.74 7.27 HEK2_024 1.7 0.10 0.11 0.11 HEK2_025 1.4 0.22 0.24 0.23 HEK2_026 1.3 0.23 0.19 0.22 HEK2_027 1.3 0.40 0.39 0.41 HEK2_028 1.3 0.14 0.16 0.15 HEK2_029 1.2 0.22 0.20 0.23 HEK2_030 1.2 0.18 0.20 0.16 HEK2_031 1.2 0.10 0.16 0.13 HEK2_032 1.1 0.08 0.12 0.10 HEK2_033 1.1 0.18 0.21 0.20 HEK2_034 1.0 0.13 0.11 0.10 HEK2_042 0.7 0.20 0.21 0.24 HEK2_044 0.7 0.00 0.00 0.00 HEK2_050 0.5 0.12 0.13 0.13 HEK2_052 0.5 0.13 0.15 0.16 HEK2_065 0.4 0.23 0.26 0.22 HEK2_079 0.3 0.12 0.09 0.13 HEK2_082 0.3 0.21 0.22 0.21 HEK2_092 0.2 0.10 0.10 0.11 HEK2_093 0.2 0.12 0.10 0.09 HEK2_099 0.2 0.12 0.14 0.10 HEK2_101 0.2 0.19 0.18 0.17 HEK2_105 0.2 0.16 0.14 0.14 HEK2_106 0.2 0.27 0.29 0.30 HEK2_110 0.2 0.00 0.00 0.00 HEK2_119 0.2 0.10 0.12 0.13 HEK2_121 0.1 0.09 0.11 0.10 HEK2_139 0.1 0.25 0.31 0.28 HEK2_145 0.1 0.15 0.14 0.15

Mutation frequencies of ABE7.10 and Sniper ABE7.10 at on-target and off-target sites captured by Digenome-seq (RNF2; see Table 13 for'DNA sequence at a cleavage sites') Base editing frequency (%) Site No. DNA Cleavage Score Untreated (+) ABE7.10 (+) Sniper ABE7.10 RNF2-1 7.3 0.03 0.05 0.05 RNF2-2 4.4 0.22 32.79 16.24 RNF2-3 4.3 0.44 0.45 0.51 RNF2-4 4.1 0.18 0.17 0.19 RNF2-5 3.6 0.98 0.94 0.89 RNF2-6 2.2 0.35 0.33 0.42 RNF2-7 1.4 0.36 0.37 0.33 RNF2-8 1.0 0.32 0.35 0.27 RNF2-15 0.4 0.29 0.27 0.26 RNF2-19 0.4 0.22 0.23 0.24 RNF2-20 0.3 1.39 1.23 1.22 RNF2-24 0.3 0.11 0.18 0.18 RNF2-27 0.3 0.40 0.48 0.46 RNF2-41 0.2 0.02 0.02 0.04

Mutation frequencies of ABE7.10 and Sniper ABE7.10 at on-target and off-target sites captured by Digenome-seq (TYRO3; see Table 14 for'DNA sequence at a cleavage sites') Base editing frequency (%) Site No. DNA Cleavage Score Untreated (+) ABE7.10 (+) Sniper ABE7.10 TYRO3-1 4.3 0.64 3.04 3.12 TYRO3-2 3.4 1.17 72.66 68.95 TYRO3-3 2.0 11.24 11.54 11.48 TYRO3-4 0.8 0.36 0.36 0.32 TYRO3-5 0.6 1.02 1.07 1.02 TYRO3-6 0.4 0.68 0.63 0.61 TYRO3-7 0.3 0.54 0.44 0.76 TYRO3-8 0.2 0.00 0.00 0.00 TYRO3-9 0.2 0.98 0.85 0.95 TYRO3-10 0.2 0.16 0.18 0.07 TYRO3-11 0.1 0.32 0.22 0.28 TYRO3-12 0.1 0.70 0.88 0.82

Mutation frequencies of ABE7.10 and Sniper ABE7.10 at on-target and off-target sites captured by Digenome-seq (WEE1; see Table 15 for'DNA sequence at a cleavage sites') Base editing frequency (%) Site No. DNA Cleavage Score Untreated (+) ABE7.10 (+) Sniper ABE7.10 WEE1-1 11.1 0.80 0.73 0.76 WEE1-2 2.0 0.35 58.59 53.38 WEE1-3 1.9 2.38 2.32 2.08 WEE1-4 0.7 0.21 0.24 0.22 WEE1-5 0.4 0.18 0.17 0.17 WEE1-6 0.4 0.76 0.96 0.88 WEE1-7 0.3 0.12 0.12 0.11 WEE1-8 0.3 1.05 0.92 1.02 WEE1-9 0.2 0.59 0.61 0.58 WEE1-10 0.2 0.24 0.28 0.23 WEE1-11 0.2 0.23 0.21 0.13 WEE1-12 0.2 No PCR WEE1-13 0.2 1.45 1.50 1.65 WEE1-14 0.2 0.00 0.00 0.00 WEE1-15 0.1 0.46 0.41 0.56 WEE1-16 0.1 0.00 0.00 0.00 WEE1-17 0.1 0.18 0.17 0.13 WEE1-18 0.1 0.23 0.28 0.24 WEE1-19 0.1 0.32 0.45 0.32 WEE1-20 0.1 0.81 0.97 1.13 WEE1-21 0.1 0.21 0.24 0.25

Mutation frequencies of ABE7.10 and Sniper ABE7.10 at on-target and off-target sites captured by Digenome-seq (EphB4; see Table 16 for'DNA sequence at a cleavage sites') Base editing frequency (%) Site No. DNA Cleavage Score Untreated (+) ABE7.10 (+) Sniper ABE7.10 EphB4-01 10.3 0.13 52.42 54.66 EphB4-02 8.0 0.15 0.19 0.13 EphB4-03 4.1 0.16 0.16 0.16 EphB4-04 2.4 0.26 0.27 0.21 EphB4-05 2.2 0.12 0.14 0.12 EphB4-06 1.9 No PCR EphB4-07 1.8 0.13 0.14 0.14 EphB4-08 1.8 No PCR EphB4-09 1.4 0.27 0.24 0.26 EphB4-10 0.9 0.07 0.08 0.07 EphB4-11 0.8 0.14 0.13 0.14 EphB4-12 0.6 0.18 0.19 0.22 EphB4-13 0.5 0.18 0.19 0.17 EphB4-14 0.4 0.12 0.10 0.13 EphB4-15 0.4 0.02 0.02 0.03 EphB4-16 0.4 0.05 0.07 0.08 EphB4-17 0.4 0.09 0.08 0.08 EphB4-18 0.4 0.39 0.37 0.35 EphB4-19 0.3 0.06 0.07 0.10 EphB4-20 0.3 0.11 0.13 0.12 EphB4-21 0.3 0.00 0.16 0.15 EphB4-22 0.2 0.08 0.11 0.08 EphB4-23 0.2 0.06 0.10 0.07 EphB4-24 0.2 0.12 0.14 0.14 EphB4-25 0.2 0.30 0.38 0.35 EphB4-26 0.2 0.07 0.09 0.08 EphB4-27 0.2 No PCR EphB4-28 0.2 0.17 0.14 0.14 EphB4-29 0.2 0.22 0.22 0.21 EphB4-30 0.2 0.13 0.13 0.09 EphB4-31 0.1 0.24 0.20 0.22 EphB4-32 0.1 0.21 0.16 0.23 EphB4-33 0.1 0.17 0.18 0.12 EphB4-34 0.1 0.17 0.15 0.13 EphB4-35 0.1 2.32 2.36 2.33 EphB4-36 0.1 No PCR EphB4-37 0.1 0.13 0.12 0.12 EphB4-38 0.1 0.22 0.25 0.24 EphB4-39 0.1 0.19 0.22 0.22

Mutation frequencies of ABE7.10 and Sniper ABE7.10 at on-target and off-target sites captured by Digenome-seq (HPRT-Exon6; see Table 17 for'DNA sequence at a cleavage sites') Base editing frequency (%) Site No. DNA Cleavage Score Untreated (+) ABE7.10 (+) Sniper ABE7.10 HPRT_E6-01 29.0 0.73 58.63 63.33 HPRT_E6-02 21.3 0.14 0.17 0.15 HPRT_E6-03 17.0 0.22 0.34 0.28 HPRT_E6-04 12.7 0.34 0.36 0.35 HPRT_E6-05 10.5 0.12 0.12 0.12 HPRT_E6-06 10.2 0.26 0.29 0.23 HPRT_E6-07 9.3 0.12 1.45 0.19 HPRT_E6-08 9.0 0.12 0.11 0.10 HPRT_E6-09 6.1 0.12 0.12 0.15 HPRT_E6-10 5.6 0.09 0.11 0.11 HPRT_E6-11 4.7 0.26 0.32 0.24 HPRT_E6-12 4.5 0.18 0.18 0.16 HPRT_E6-13 4.3 0.12 1.19 0.23 HPRT_E6-14 4.3 0.14 0.12 0.16 HPRT_E6-15 4.1 0.04 0.03 0.04 HPRT_E6-16 3.8 0.07 0.19 0.07 HPRT_E6-17 3.7 0.12 0.12 0.12 HPRT_E6-18 3.6 0.25 0.26 0.20 HPRT_E6-19 3.6 0.07 0.07 0.08 HPRT_E6-20 3.5 0.14 0.10 0.13 HPRT_E6-21 3.5 0.07 0.08 0.07 HPRT_E6-22 3.4 0.12 0.15 0.12 HPRT_E6-23 3.3 0.08 0.08 0.07 HPRT_E6-24 3.1 0.11 0.12 0.12 HPRT_E6-25 2.9 0.09 0.08 0.09 HPRT_E6-26 2.8 0.09 0.07 0.07 HPRT_E6-27 2.6 0.46 0.45 0.45 HPRT_E6-28 2.5 0.13 0.13 0.14 HPRT_E6-29 2.4 0.28 0.27 0.29 HPRT_E6-30 2.4 0.27 0.30 0.30 HPRT_E6-31 2.3 0.15 0.18 0.12 HPRT_E6-32 2.2 0.23 0.24 0.20 HPRT_E6-33 1.5 0.22 0.24 0.27 HPRT_E6-34 1.4 0.00 0.00 0.00 HPRT_E6-35 1.4 0.10 0.09 0.09 HPRT_E6-36 1.2 0.09 0.11 0.08 HPRT_E6-37 1.1 0.02 0.06 0.09 HPRT_E6-38 1.1 0.21 0.22 0.21 HPRT_E6-39 1.1 0.04 0.04 0.02 HPRT_E6-40 1.0 0.23 0.23 0.28 HPRT_E6-41 1.0 0.06 0.13 0.16 HPRT_E6-52 0.7 0.00 0.00 0.00 HPRT_E6-81 0.4 0.13 0.18 0.15

Mutation frequencies of ABE7.10 and Sniper ABE7.10 at on-target and off-target sites captured by Digenome-seq (HPRT-Exon8; see Table 18 for'DNA sequence at a cleavage sites') Base editing frequency (%) Site No. DNA Cleavage Score Untreated (+) ABE7.10 (+) Sniper ABE7.10 HPRT_E8-1 2.4 0.03 70.09 71.15 HPRT_E8-2 1.3 0.11 10.33 3.53 HPRT_E8-3 0.6 0.17 0.17 0.19 HPRT_E8-4 0.6 0.05 0.03 0.06 HPRT_E8-5 0.6 0.08 0.08 0.10 HPRT_E8-6 0.5 0.04 0.10 0.09 HPRT_E8-7 0.5 0.16 0.16 0.14 HPRT_E8-8 0.4 No PCR HPRT_E8-9 0.4 0.07 0.06 0.04 HPRT_E8-10 0.4 0.11 0.11 0.12 HPRT_E8-11 0.4 0.05 0.07 0.05 HPRT_E8-12 0.3 0.14 0.15 0.13 HPRT_E8-13 0.3 0.05 0.05 0.03 HPRT_E8-14 0.3 0.26 0.25 0.25 HPRT_E8-15 0.3 0.04 0.05 0.06 HPRT_E8-16 0.2 0.11 0.11 0.10 HPRT_E8-17 0.2 0.09 0.09 0.08 HPRT_E8-18 0.2 0.04 0.15 0.05 HPRT_E8-19 0.1 0.05 0.04 0.07 HPRT_E8-20 0.1 0.08 0.05 0.05

In addition, the frequency of base correction was measured when ABE7.10 and sgRNA were introduced into the cell in the form of ABE7.10 RNP conjugated (see Reference Example 1), and ABE7.10 and sgRNA were introduced into the cell through a plasmid (reference example) 1) compared to the case of Fig. 13g and 13h (in Fig. 13h, the y-axis graph shows the results of (+)ABE7.10_RNP, (+)ABE7.10_plasmid, and untreated in order from the top) Indicated.

13E to 13H and Tables 26 to 32, when Sniper ABE7.10 is used and ABE7.10 RNP is used, when ABE7.10 is used, and ABE7.10 plasmid is used, and In comparison, the specificity of the base calibration can be improved by about 7 times.

In addition, it was confirmed that the off-target effect of ABE7.10 can be further reduced by using Sniper ABE7.10 in combination with modified sgRNAs (see Figs. 13i and 13j; in Figs. 13i and 13j, the y-axis graph is from above. From ggX20-Sniper ABE7.10, gX20-Sniper ABE7.10, GX19-Sniper ABE7.10, gX18-Sniper ABE7.10, gX17-Sniper ABE7.10, GX19-ABE7.10, and untreated results are shown. ).

Through this example, using mismatched sgRNAs, Digenome-seq, and targeted amplicon sequencing, ABE7.10, BE3, and Cas9 can recognize different sets of off-target sites in the human genome, and each independently It was confirmed that an evaluation means was required. It was confirmed that the ABE7.10 deaminase targeting the seven genes tested above catalyzes the in vitro A-to-G conversion very specifically in a limited number of sites in the human genome. In addition, it was confirmed that the off-target activity of ABE7.10 can be reduced or eliminated by using modified sgRNAs, ABE7.10 RNPs, and/or Sniper ABE7.10.

<110> TOOLGEN INCORPORATED Seoul National University R&DB Foundation <120> Method of identifying base editing using adenosine deaminase <130> DPP20190026KR <150> 10-2018-0009508 <151> 2018-01-25 <160> 540 <170> KopatentIn 3.0 <210> 1 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 1 gggtgcaaag catagactgc ggg 23 <210> 2 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 2 gaacgtggag catagactgc ggg 23 <210> 3 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 3 gaacacaaga tgtagactgc ggg 23 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 4 gaacacaaag cacgagctgc ggg 23 <210> 5 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 5 gaacacaaag catagatcat ggg 23 <210> 6 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 6 gagtgcaaag catagactgc ggg 23 <210> 7 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 7 gaacatggag catagactgc ggg 23 <210> 8 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 8 gaacacaaga tatagactgc ggg 23 <210> 9 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 9 gaacacaaag cgcggactgc ggg 23 <210> 10 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 10 gaacacaaag cataagttgc ggg 23 <210> 11 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 11 gaacacaaag catagaccat ggg 23 <210> 12 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 12 gagtacaaag catagactgc ggg 23 <210> 13 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 13 gaacgtaaag catagactgc ggg 23 <210> 14 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 14 gaacacggag catagactgc ggg 23 <210> 15 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 15 gaacacaaga catagactgc ggg 23 <210> 16 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 16 gaacacaaag tgtagactgc ggg 23 <210> 17 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 17 gaacacaaag cacggactgc ggg 23 <210> 18 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 18 gaacacaaag cataagctgc ggg 23 <210> 19 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 19 gaacacaaag catagatcgc ggg 23 <210> 20 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 20 gaacacaaag catagactat ggg 23 <210> 21 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 21 ggacacaaag catagactgc ggg 23 <210> 22 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 22 gaatacaaag catagactgc ggg 23 <210> 23 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 23 gaacataaag catagactgc ggg 23 <210> 24 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 24 gaacacagag catagactgc ggg 23 <210> 25 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 25 gaacacaaaa catagactgc ggg 23 <210> 26 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 26 gaacacaaag cgtagactgc ggg 23 <210> 27 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 27 gaacacaaag catggactgc ggg 23 <210> 28 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 28 gaacacaaag cataggctgc ggg 23 <210> 29 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 29 gaacacaaag catagaccgc ggg 23 <210> 30 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK2) <400> 30 gaacacaaag catagactgt ggg 23 <210> 31 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA on target site for HEK2) <400> 31 gaacacaaag catagactgc ggg 23 <210> 32 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 32 gatttagact gagcacgtga tgg 23 <210> 33 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 33 ggcctgagct gagcacgtga tgg 23 <210> 34 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 34 ggcccagatc aggcacgtga tgg 23 <210> 35 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 35 ggcccagact gaatgtgtga tgg 23 <210> 36 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 36 ggcccagact gagcacacag tgg 23 <210> 37 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 37 ggtttagact gagcacgtga tgg 23 <210> 38 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 38 ggcccgagct gagcacgtga tgg 23 <210> 39 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 39 ggcccagatc aagcacgtga tgg 23 <210> 40 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 40 ggcccagact ggatacgtga tgg 23 <210> 41 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 41 ggcccagact gagcgtatga tgg 23 <210> 42 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 42 ggcccagact gagcacgcag tgg 23 <210> 43 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 43 ggttcagact gagcacgtga tgg 23 <210> 44 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 44 ggcctggact gagcacgtga tgg 23 <210> 45 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 45 ggcccaagct gagcacgtga tgg 23 <210> 46 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 46 ggcccagatc gagcacgtga tgg 23 <210> 47 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 47 ggcccagact aggcacgtga tgg 23 <210> 48 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 48 ggcccagact gaatacgtga tgg 23 <210> 49 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 49 ggcccagact gagcgtgtga tgg 23 <210> 50 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 50 ggcccagact gagcacacga tgg 23 <210> 51 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 51 ggcccagact gagcacgtag tgg 23 <210> 52 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 52 gacccagact gagcacgtga tgg 23 <210> 53 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 53 ggctcagact gagcacgtga tgg 23 <210> 54 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 54 ggcccggact gagcacgtga tgg 23 <210> 55 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 55 ggcccaggct gagcacgtga tgg 23 <210> 56 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 56 ggcccagacc gagcacgtga tgg 23 <210> 57 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 57 ggcccagact gggcacgtga tgg 23 <210> 58 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 58 ggcccagact gagtacgtga tgg 23 <210> 59 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 59 ggcccagact gagcatgtga tgg 23 <210> 60 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 60 ggcccagact gagcacgcga tgg 23 <210> 61 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for HEK3) <400> 61 ggcccagact gagcacgtgg tgg 23 <210> 62 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA on target site for HEK3) <400> 62 ggcccagact gagcacgtga tgg 23 <210> 63 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 63 gctgccttag tcattacctg agg 23 <210> 64 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 64 gtcactccag tcattacctg agg 23 <210> 65 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 65 gtcatcttga ctattacctg agg 23 <210> 66 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 66 gtcatcttag tcgccgcctg agg 23 <210> 67 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 67 gtcatcttag tcattattca agg 23 <210> 68 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 68 gttgccttag tcattacctg agg 23 <210> 69 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 69 gtcattccag tcattacctg agg 23 <210> 70 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 70 gtcatcttga ccattacctg agg 23 <210> 71 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 71 gtcatcttag ttgctacctg agg 23 <210> 72 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 72 gtcatcttag tcatcgtctg agg 23 <210> 73 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 73 gtcatcttag tcattactca agg 23 <210> 74 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 74 gttgtcttag tcattacctg agg 23 <210> 75 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 75 gtcactttag tcattacctg agg 23 <210> 76 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 76 gtcatcccag tcattacctg agg 23 <210> 77 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 77 gtcatcttga tcattacctg agg 23 <210> 78 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 78 gtcatcttag ctattacctg agg 23 <210> 79 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 79 gtcatcttag tcgctacctg agg 23 <210> 80 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 80 gtcatcttag tcatcgcctg agg 23 <210> 81 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 81 gtcatcttag tcattatttg agg 23 <210> 82 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 82 gtcatcttag tcattaccca agg 23 <210> 83 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 83 gccatcttag tcattacctg agg 23 <210> 84 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 84 gtcgtcttag tcattacctg agg 23 <210> 85 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 85 gtcattttag tcattacctg agg 23 <210> 86 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 86 gtcatctcag tcattacctg agg 23 <210> 87 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 87 gtcatcttaa tcattacctg agg 23 <210> 88 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 88 gtcatcttag ttattacctg agg 23 <210> 89 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 89 gtcatcttag tcactacctg agg 23 <210> 90 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 90 gtcatcttag tcattgcctg agg 23 <210> 91 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 91 gtcatcttag tcattacttg agg 23 <210> 92 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA target site for RNF2) <400> 92 gtcatcttag tcattaccta agg 23 <210> 93 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA on target site for RNF2) <400> 93 gtcatcttag tcattacctg agg 23 <210> 94 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA on target site for TYRO3) <400> 94 ggccacacta gcgttgctgc tgg 23 <210> 95 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA on target site for WEE1) <400> 95 gtcgagatgt tctattactc tgg 23 <210> 96 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA on target site for EphB4) <400> 96 gcagaatatt cggacaaaca cgg 23 <210> 97 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA on target site for HPRTExon6) <400> 97 gtataatcca aagatggtca agg 23 <210> 98 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (sgRNA on target site for HPRTExon8) <400> 98 gaagtattca ttatagtcaa ggg 23 <210> 99 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 99 gaacacaagc acagactgaa gg 22 <210> 100 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 100 gaacacatgc atagactgct ag 22 <210> 101 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 101 gaatactaag catagactcc agg 23 <210> 102 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 102 gaacacaatg catagattgc cgg 23 <210> 103 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 103 aaacacagag cacagactgc tga 23 <210> 104 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 104 aaacacaaag acatagacca ctgg 24 <210> 105 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 105 gaacacaaag catagactgc ggg 23 <210> 106 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 106 aaacataaag aatagactgc aag 23 <210> 107 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 107 gaacacaaag tatagaatgc tag 23 <210> 108 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 108 ggacacaaag cttagactcc agg 23 <210> 109 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 109 gaagataaag catagactct agg 23 <210> 110 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 110 aaacataaag catagactgc aaag 24 <210> 111 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 111 aaatacaaag catagactaa tatg 24 <210> 112 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 112 gaacacatac atagacagct gg 22 <210> 113 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 113 gaaaacaaag caaagaaagc agg 23 <210> 114 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 114 aacacaatag catagactgg actg 24 <210> 115 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 115 atacacagag caaagactgc agg 23 <210> 116 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 116 aaacataaag cctagactga cgg 23 <210> 117 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 117 aaaaacaaag cgtagactgt ggg 23 <210> 118 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 118 ggacaaaaag catagactca agg 23 <210> 119 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 119 atacacacaa gcacagactg cagg 24 <210> 120 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 120 gaacacagca cataaactgc agg 23 <210> 121 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 121 aaacactaac atagactgca gg 22 <210> 122 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 122 aaatacaatg catagactgc tag 23 <210> 123 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 123 gtaaacaaag catagactga ggg 23 <210> 124 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 124 gaacataatc acagactgct gg 22 <210> 125 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 125 ccaaacaaaa catagactgc tgg 23 <210> 126 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 126 gaaaacaaaa catagagtgc tgg 23 <210> 127 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 127 aatcaaatag catagactgc atg 23 <210> 128 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 128 gaacacaaac atagtactga agg 23 <210> 129 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 129 aaacagaaag catggactgc gga 23 <210> 130 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 130 caatacaaag gatagactgc agg 23 <210> 131 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 131 agacacacag cacagactgc agg 23 <210> 132 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 132 gaattcaaag catagattgc agg 23 <210> 133 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 133 acacacaaag catagactat gtg 23 <210> 134 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 134 ggaaacaaag catagacatt tgg 23 <210> 135 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 135 taatacaaag catagatagt tgg 23 <210> 136 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 136 gaatttaaag catagactgc aag 23 <210> 137 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 137 ctgaacacag caaagactgc tgg 23 <210> 138 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 138 gagcgataag cacagactgc tgg 23 <210> 139 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 139 gtaattaaag cacagactgc tgg 23 <210> 140 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 140 gaaaacaaac cacagactgg ggg 23 <210> 141 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 141 ctattgcaag cacagactgc tgg 23 <210> 142 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 142 gaatacataa catagactgg ggg 23 <210> 143 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 143 ataccataag catagactgt tgg 23 <210> 144 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 144 attaagatag catagactgc agg 23 <210> 145 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 145 aatgactaat aatagactgc tgg 23 <210> 146 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 146 atttcaacag catagactgt agg 23 <210> 147 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 147 agtatagcag catagactgc agg 23 <210> 148 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 148 gaattcaaag catagattgc agg 23 <210> 149 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 149 gcattctaat aatagactgc tgg 23 <210> 150 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 150 aaacacatag cacagcctgc agg 23 <210> 151 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 151 gaacacagta catagactgg cag 23 <210> 152 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 152 ggctgcctag cacagactgc cgg 23 <210> 153 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 153 tctcctaccg catagactgc tgg 23 <210> 154 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 154 gtcgcaaacg catagacttc cgg 23 <210> 155 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 155 cttattaaag cacagactgc tgg 23 <210> 156 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 156 actatataag catagactgt tgg 23 <210> 157 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 157 gtcgcaaacg catagacttc cgg 23 <210> 158 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 158 cgatttcaag catagactgc tgg 23 <210> 159 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 159 taatcataag catagaatgc tgg 23 <210> 160 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 160 gaatacaagc atagactgct gt 22 <210> 161 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 161 tcacaaataa gcatagactg gcgg 24 <210> 162 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 162 cttgctaaag cacagactgc tgg 23 <210> 163 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 163 aagaacaaaa catagactgc agg 23 <210> 164 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 164 aattaaacag aatagactgc tgg 23 <210> 165 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 165 gaacacagaa ccatagactg gggg 24 <210> 166 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 166 aaacaaaaag catagaccac aag 23 <210> 167 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 167 gaacacaggc atagactatg ga 22 <210> 168 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 168 ctcagagaag cacagactgc agg 23 <210> 169 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 169 atcatatatg cataaactgc agg 23 <210> 170 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 170 caacaaaata tatagactgc tgg 23 <210> 171 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 171 attgttaaag cacagactgc agg 23 <210> 172 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 172 agatcttaag catagactgt ggg 23 <210> 173 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 173 taacacaata ccatagactg gcgg 24 <210> 174 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 174 gaatacaaat aatagactat tgg 23 <210> 175 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 175 gaatttaaag catagactct ggg 23 <210> 176 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 176 aaacaaataa aatagactgc agg 23 <210> 177 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 177 ccacaccaag catagacttc tgg 23 <210> 178 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 178 aacaaaacag catagactgc atg 23 <210> 179 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 179 taattgcaag catagacggc agg 23 <210> 180 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 180 ggagagagag catagactgc tgg 23 <210> 181 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 181 agaaataata tatagactgc agg 23 <210> 182 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 182 tggttactag catagacttc agg 23 <210> 183 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 183 aaacacttaa cacagactgc agg 23 <210> 184 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 184 ccaccagcag catagactcc agg 23 <210> 185 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 185 aagacatttt catagactgt cgg 23 <210> 186 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 186 taagcacaag cataggctgc tgg 23 <210> 187 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 187 aacaagtatg catagactgc tgg 23 <210> 188 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 188 aaataccaac cacagactgc agg 23 <210> 189 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 189 aaatgcatag catagactgc tga 23 <210> 190 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 190 taacacaaag actaaactgc agg 23 <210> 191 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 191 taccacgaag catagactgt agg 23 <210> 192 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 192 gatcactcaa catagactgc atg 23 <210> 193 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 193 gaacagaaac atagaccaat gg 22 <210> 194 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 194 tacaaaataa gatagactgc tgg 23 <210> 195 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 195 acagttaaag aatagactgc agg 23 <210> 196 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 196 aaacacaaag gcataaactc cagg 24 <210> 197 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 197 gaacacacac acagactgag gg 22 <210> 198 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 198 ggacataaat cataaactgc tgg 23 <210> 199 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 199 aaagctacag catagactgc aag 23 <210> 200 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 200 tcacacaaac catagactga ggg 23 <210> 201 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 201 gatctaccta tatagactgc agg 23 <210> 202 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 202 tatagaggag catagactgc tgg 23 <210> 203 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 203 tatcagaaag cacagactgc ggg 23 <210> 204 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 204 gaacaacaag gataaactgc cgg 23 <210> 205 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 205 tacaaacacg catagaatgc cgg 23 <210> 206 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 206 taagaaatgg aatagactgc agg 23 <210> 207 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 207 aaatattaag catagactac ggg 23 <210> 208 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 208 gaatgccaag cataaactgc agg 23 <210> 209 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 209 aactccaaag catatactgc tgg 23 <210> 210 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 210 gtgaacacag catagactgg ggc 23 <210> 211 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 211 atcctaagaa catagactgc agg 23 <210> 212 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 212 tttgttaaaa catagactgc tgg 23 <210> 213 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 213 taacaaaatg catagactgc tag 23 <210> 214 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 214 tgtataacag catacactgc tgg 23 <210> 215 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 215 aaacactaat aatagactgt ggg 23 <210> 216 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 216 aagcaatcag cacagactgc ggg 23 <210> 217 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 217 ggacacagag tatagactga ggg 23 <210> 218 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 218 cacaaatcaa aatagactgc tgg 23 <210> 219 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 219 gaattcaaag catggactgc agg 23 <210> 220 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 220 ttccaaaaaa catagactgc tgg 23 <210> 221 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 221 gtagagtaag catacactgc tgg 23 <210> 222 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 222 ggcctttagg cacagactgc agg 23 <210> 223 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 223 aagactttag catagactgc aag 23 <210> 224 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 224 gaaaagtaag atagactgcc gg 22 <210> 225 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 225 aactcatcag catagactgt agg 23 <210> 226 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 226 aaatacagag cttagactac tgg 23 <210> 227 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 227 aaacatgaag catagacagc aag 23 <210> 228 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 228 acatagatat tatagactgc tgg 23 <210> 229 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 229 cacattaaag cacagactgc tgg 23 <210> 230 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 230 gagcctagag cacagactgc agg 23 <210> 231 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 231 aagacttaag caaagactgc tgg 23 <210> 232 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 232 ttacacatag cacagactac agg 23 <210> 233 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 233 agaagttaag catagactgg tgg 23 <210> 234 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 234 ctatattgag aatagactgc agg 23 <210> 235 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 235 gtgcacacag catagactgc atg 23 <210> 236 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 236 ctagcaaaaa catagactgc tgg 23 <210> 237 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 237 taaaacaaag aacagactgc tgg 23 <210> 238 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 238 tacagatagt aatagactgc agg 23 <210> 239 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 239 aattaataac aatagactgc tgg 23 <210> 240 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 240 aagattgcag cataaactgc tgg 23 <210> 241 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 241 aagcacataa catagactga agg 23 <210> 242 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 242 gtagaaaaag tatagactgc agg 23 <210> 243 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 243 tactatggtg catagactgc tgg 23 <210> 244 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 244 ctctactgag aatagactgc agg 23 <210> 245 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 245 tgaaataata cataaactgc cgg 23 <210> 246 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 246 aataaacttg catagactgt agg 23 <210> 247 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 247 agaccctaag catagactgc aga 23 <210> 248 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 248 tacccaaatg cacagactgc tgg 23 <210> 249 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 249 aagatagatt tatagactgc tgg 23 <210> 250 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HEK2) <400> 250 aaacacaaaa cataaattac tgg 23 <210> 251 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 251 tcaccattag tcattacctg ctg 23 <210> 252 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 252 gtcatcttag tcattactga gg 22 <210> 253 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 253 gtaatattag tcattaccgg tgg 23 <210> 254 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 254 gtcatcctag tcatttactg ggg 23 <210> 255 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 255 atcaccttag ccattaccag ggg 23 <210> 256 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 256 gtcacttagt cattgcctgt gg 22 <210> 257 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 257 gtcacattag ccattacctg tga 23 <210> 258 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 258 tgtatcttag acattacatg tgg 23 <210> 259 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 259 attttcttag tgattatctg ggg 23 <210> 260 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 260 catctaatag taattacctg ggg 23 <210> 261 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 261 atagtattag tcattacctg tga 23 <210> 262 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 262 accatcttag tcattatcta atg 23 <210> 263 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 263 attttcttag tcattaccta gag 23 <210> 264 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 264 atcatcttca tcattacatg agg 23 <210> 265 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 265 cctatatata tcattacctt tgg 23 <210> 266 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 266 gtcattttag tcattatctt gag 23 <210> 267 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 267 atcacctgag tcattaccca tgg 23 <210> 268 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 268 ttaatcttag tcattacttt tgg 23 <210> 269 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 269 atcatcatcg tcattatctg ggg 23 <210> 270 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 270 atcattagtc attgcctgag g 21 <210> 271 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 271 cttatttagt cattacctgt ag 22 <210> 272 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 272 gtcatattaa tcattacata gag 23 <210> 273 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 273 tccatctcac tcattacctg agg 23 <210> 274 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 274 gttatcttag tctttacctg aga 23 <210> 275 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 275 cacatcttac ccattacatg agg 23 <210> 276 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 276 gccacctcag tcattagctg ggg 23 <210> 277 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 277 acattagtaa acattaccta ggg 23 <210> 278 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 278 tacctacctg tcattaccta tgg 23 <210> 279 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 279 gatatcttag ccattaccta gga 23 <210> 280 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 280 tatctactac tcattacctg agg 23 <210> 281 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 281 agcatctctg tcattaccca ggg 23 <210> 282 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 282 cgtgcattag tcattacctg agg 23 <210> 283 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 283 gtctgtatag tcattacctt tgg 23 <210> 284 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 284 tacatctatg ttattaccta tgg 23 <210> 285 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 285 ttcatctttg tcattaccta aag 23 <210> 286 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 286 tatactatat acattacctg ggg 23 <210> 287 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 287 gctcattcac tcattaccta tgg 23 <210> 288 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 288 gtgatctaaa gtcattacct tagg 24 <210> 289 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 289 ttcaatataa tcattacctg tgg 23 <210> 290 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 290 attattttag tcattacctt tgg 23 <210> 291 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 291 cccatctcag ccattacctg ggt 23 <210> 292 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 292 agaatattag tccttacctg ggg 23 <210> 293 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 293 gatcctttaa tcattacctt tgg 23 <210> 294 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 294 accatcttag tcactacctg ggc 23 <210> 295 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 295 gtattattgt acattacctg agg 23 <210> 296 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 296 agcatcttag ccattacctc tag 23 <210> 297 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 297 agcatcttaa tccttacctc agg 23 <210> 298 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 298 gaatcctcat acattacctg tgg 23 <210> 299 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for RNF2) <400> 299 atcatctttt taattaccta tgg 23 <210> 300 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 300 ggccacacta gtgttgccgc tgg 23 <210> 301 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 301 ggccacacca gccttgctgt cgg 23 <210> 302 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 302 ggccactagc gttgctccag g 21 <210> 303 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 303 ggccacactg accttgctgc tgg 23 <210> 304 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 304 ttccacacag ctttgctgct gg 22 <210> 305 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 305 ggccacctag ggttgctgct gg 22 <210> 306 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 306 ccaccctcta gcattgctgc tgg 23 <210> 307 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 307 cctaacacca acgttgctgc tgg 23 <210> 308 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 308 ctgagctcta gcattgctgc tgg 23 <210> 309 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 309 ggcatatata gcattgctgc tgg 23 <210> 310 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for TYRO3) <400> 310 gaacacacta acattgctgt ggg 23 <210> 311 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 311 ttagagatgt tctattattc cgg 23 <210> 312 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 312 gaggagatgc tctattactc cgg 23 <210> 313 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 313 atcaagatga actattactc tgg 23 <210> 314 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 314 gtggagatgt tatgttactc tgg 23 <210> 315 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 315 gacataatgt tctattactc aag 23 <210> 316 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 316 taatagatgt tctattacta agg 23 <210> 317 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 317 attaagatgt tctattaata ggg 23 <210> 318 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 318 cagctaatgt aatattactc agg 23 <210> 319 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 319 atcaagatat tctattactg ggg 23 <210> 320 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 320 tcttatattt tctattactc agg 23 <210> 321 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 321 atagagcagt tatattactc tgg 23 <210> 322 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 322 gtagagatgt tctctaactc agg 23 <210> 323 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 323 ctcttttatt tctattactc tgg 23 <210> 324 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 324 aatataatat tctattactc agg 23 <210> 325 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 325 caactataat tctattactc tgg 23 <210> 326 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 326 ggctactgtc tctattactc agg 23 <210> 327 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 327 atttagatat tctattactc tga 23 <210> 328 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 328 ctttaagata tctattactc tgg 23 <210> 329 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 329 caagagacgc tctatcactc agg 23 <210> 330 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for WEE1) <400> 330 aaacagctgt tctattactc agg 23 <210> 331 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 331 gcagaatatt acagacaaac tagg 24 <210> 332 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 332 gcagaatatc aggacaaaca atg 23 <210> 333 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 333 acagaatatt cagacaatca cag 23 <210> 334 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 334 gcagtaaata agacaaacaa gg 22 <210> 335 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 335 acaaaatatt cggacaggca cgg 23 <210> 336 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 336 gcagaattta aggacaagca agg 23 <210> 337 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 337 gcagaataag gacaaacatg g 21 <210> 338 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 338 cagtaatatt cagactaaac atgg 24 <210> 339 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 339 ttaatatata aggacaaaca tgg 23 <210> 340 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 340 acataatatt caaacaaaca ggg 23 <210> 341 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 341 aatgaataaa gggacaaaca ggg 23 <210> 342 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 342 tcagatataa ggacaaacat gg 22 <210> 343 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 343 gctgaatatt gggacaatca tgg 23 <210> 344 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 344 gcagttataa aaagacaaac aggg 24 <210> 345 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 345 acagaatata agaacaaata tgg 23 <210> 346 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 346 atagaataat cagacaaaaa agg 23 <210> 347 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 347 gcagataaag ggacaaacat gg 22 <210> 348 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 348 gacagtgaat aggacaaaca tgg 23 <210> 349 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 349 tgagaatatg cggacaaaca cag 23 <210> 350 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 350 atagaatatt aggacaaata gag 23 <210> 351 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 351 caataataaa aggacaaaca tgg 23 <210> 352 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 352 acaggataaa aggacaaata tgg 23 <210> 353 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 353 acagatatac gtagacaaac agag 24 <210> 354 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 354 atagaataag aagacaaaca tgg 23 <210> 355 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 355 ttagaatatt cctacaaaca agg 23 <210> 356 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 356 ccatgttatt cagacaaaca tgg 23 <210> 357 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 357 gcagaaaata ggacaatcac gg 22 <210> 358 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 358 agagaatata ggacaaactt gg 22 <210> 359 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 359 gcagaaatat ttgacaaaca agg 23 <210> 360 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 360 aaagaatata gaacaaacag gg 22 <210> 361 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 361 acagaaatac gaacaaacat gg 22 <210> 362 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 362 aacaaatgca aggacaaaca tgg 23 <210> 363 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 363 acactattta agacaaacag gg 22 <210> 364 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 364 acagaactac gagacaaaca cgg 23 <210> 365 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 365 gcagataccc agacaaacag gg 22 <210> 366 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 366 taagaatata ggacaaacag ga 22 <210> 367 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 367 ccagaatatt agacaaacat gg 22 <210> 368 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for EphB4) <400> 368 agagaatata gggacaaata agg 23 <210> 369 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 369 gtatacatcc aagatggcca ggg 23 <210> 370 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 370 gtattaccaa agatggtctg gg 22 <210> 371 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 371 gtataatcaa agatggccct gg 22 <210> 372 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 372 gtattatcaa aaatggtcaa gg 22 <210> 373 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 373 atataaccaa agatgttcac ag 22 <210> 374 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 374 atataatctt aagatggtca agg 23 <210> 375 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 375 atataatcct aagatgttca tgg 23 <210> 376 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 376 gtattatcca aagatgacca gag 23 <210> 377 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 377 atacaatcca aagttggtct ggg 23 <210> 378 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 378 atatattacc aaagatggtc tggg 24 <210> 379 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 379 gtagaatcca tagatggaca ggg 23 <210> 380 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 380 gtattaccaa agatggtcat gg 22 <210> 381 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 381 gtacaatcaa aagatgaaca agg 23 <210> 382 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 382 atattatcca aagatggagt tgg 23 <210> 383 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 383 atgtattcca aagatggtca ggg 23 <210> 384 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 384 aagtaatcca aagatagtct tgg 23 <210> 385 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 385 gtataaccca aagaaagtaa tgg 23 <210> 386 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 386 atatatattc caagatggtt ctgg 24 <210> 387 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 387 ttataatcca aagaaggcca agg 23 <210> 388 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 388 gtatctaccc aaagatgttc atgg 24 <210> 389 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 389 gtataataaa gatggtcaag g 21 <210> 390 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 390 atagattcca aagatggaca tgg 23 <210> 391 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 391 ctataatcta aaaatggtca agg 23 <210> 392 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 392 gtatattaga aagatggtca tga 23 <210> 393 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 393 ggataatcaa aagatggact tgg 23 <210> 394 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 394 atataatgca aagctggtca cgg 23 <210> 395 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 395 atacaatcca aagatattct agg 23 <210> 396 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 396 gtataatcca aaggtggcct agg 23 <210> 397 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 397 atataatcca tagaaggtca aga 23 <210> 398 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 398 gatataatac aagatggaca agg 23 <210> 399 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 399 ggataatcca aagatggtgc aag 23 <210> 400 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 400 atataaacca aagatcgtta agg 23 <210> 401 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 401 tctccctcca aagaaggtca cgg 23 <210> 402 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 402 ttatagtcca aaggaagtca ggg 23 <210> 403 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 403 tatttatcca aagatggtct aga 23 <210> 404 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 404 atatatacca aaggtagtca tgg 23 <210> 405 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 405 aggagatcca aagatggtca agg 23 <210> 406 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 406 acatagtcca aatatggaca ggg 23 <210> 407 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 407 gtatataatt caagatggtc agga 24 <210> 408 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 408 gaataagcca aagatggtca gtg 23 <210> 409 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 409 gtatctccca gagatggtca agg 23 <210> 410 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 410 atgtaatcta aagatgatta tgg 23 <210> 411 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 411 gtataaccaa tgatggtcag ga 22 <210> 412 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 412 atgtcatcca aagaaggtca gag 23 <210> 413 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 413 agatattcca aagatggtga ggg 23 <210> 414 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 414 tttccaccca aagatggtcc agg 23 <210> 415 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 415 ttatagctcc aagatggtca agg 23 <210> 416 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 416 tcagaatcct aagatggtca gga 23 <210> 417 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 417 ggataatcca aagttggtca tag 23 <210> 418 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 418 gtataatcct agatggtgag gg 22 <210> 419 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 419 gtattttcca acgatggtca tgg 23 <210> 420 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 420 gaataattca aagataatca agg 23 <210> 421 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 421 aaataataaa agatggtcat gg 22 <210> 422 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 422 ctcatatcca aagatggtaa agg 23 <210> 423 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 423 ttataaccaa agatggtaat gg 22 <210> 424 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 424 gcctaatcca aagatgagca ggg 23 <210> 425 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 425 ctataatgcc aaagatggtc atgg 24 <210> 426 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 426 gtatagatca aagatgccca cgg 23 <210> 427 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 427 tctccttcca aagatggtct ggg 23 <210> 428 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 428 ataatctcca aagatgttca ggg 23 <210> 429 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 429 atattttcca aagatgatca cag 23 <210> 430 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 430 tattgatcca aagatgatca aga 23 <210> 431 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 431 atatatttcc aagatgggca ggg 23 <210> 432 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 432 ttctcatcca aggatggtca ggg 23 <210> 433 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 433 atataaacaa aagatggttt agg 23 <210> 434 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 434 aaatagtcca aggatggcca ggg 23 <210> 435 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 435 gtattttcta aagatggtca tga 23 <210> 436 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 436 ggataaatcc aagttggtca tgg 23 <210> 437 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 437 atattttcca aagatggtca tga 23 <210> 438 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 438 tgactctcca aagatggtca cag 23 <210> 439 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 439 caatattcca aagatggtta tgg 23 <210> 440 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 440 gcagcatcca aagatgggca ggg 23 <210> 441 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 441 ctataattca aagatgttct tgg 23 <210> 442 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 442 cacatatata aagatggtca tgg 23 <210> 443 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 443 gtatcttcca aagatggcca atg 23 <210> 444 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 444 aatattacct aagatggtca tgg 23 <210> 445 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 445 gtataaacca aaaatattca ggg 23 <210> 446 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 446 gaataagcca aagatggtca gtg 23 <210> 447 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 447 atataatgca aagaaagtca aag 23 <210> 448 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 448 gtataagcca aagcaggtca cgg 23 <210> 449 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 449 atataaacca aagacggccc tgg 23 <210> 450 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 450 agataatcca aggatggtag agg 23 <210> 451 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 451 atataatcca gaaatgttca cgg 23 <210> 452 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 452 actcttttcc aagatggtca ggg 23 <210> 453 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 453 ctgatgtcca aagatggtct cgg 23 <210> 454 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 454 gtgaattcca aagaaggtca tgg 23 <210> 455 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 455 cattaatcca aagatgataa agg 23 <210> 456 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 456 gctctatcca aagatggaca ggg 23 <210> 457 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 457 ctgtaatcca gaaatagtca ggg 23 <210> 458 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 458 gtatatgtcc aaagatgatc atgc 24 <210> 459 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 459 ctataatcca aggaaggcca agg 23 <210> 460 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 460 gtatcctcca aagatgagta agg 23 <210> 461 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 461 aaataataca aagatggttg ggg 23 <210> 462 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 462 caataatcca aagaagatta agg 23 <210> 463 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 463 atataatcac aaagatggca atgg 24 <210> 464 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 464 gtttaagcca aagttggtcg ggg 23 <210> 465 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 465 gtagcatcca aagatggtgg agg 23 <210> 466 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 466 gtagaatatc aagatggtca agg 23 <210> 467 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 467 agaatgccct aagatggtct ggg 23 <210> 468 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 468 atataaccag aagatgttca tgg 23 <210> 469 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 469 gtattatcta aaggaggtca tgg 23 <210> 470 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 470 agatatacag aagatggtcg ggg 23 <210> 471 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 471 aaataatcca aaaatagtca tgg 23 <210> 472 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 472 ctttattcca aagatagtca tgg 23 <210> 473 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 473 gcatatttcc aaacatggtc aagg 24 <210> 474 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 474 gtatttttca aagatggtca tga 23 <210> 475 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 475 gtattttcca aagatggcca cga 23 <210> 476 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 476 tataaatcca aggatggtct gag 23 <210> 477 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 477 ttattttcca aagatggtca cga 23 <210> 478 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 478 tgcacatcca cagatggtca ggg 23 <210> 479 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 479 gtgtaatcca gagatgatca cag 23 <210> 480 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 480 tcttaatcca tagaaggtca agg 23 <210> 481 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 481 ttcaaataaa gatggtcacg g 21 <210> 482 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 482 tcataatcca aaagatggac aggg 24 <210> 483 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 483 atataataca aaataggtca ggg 23 <210> 484 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 484 ctagatacca aagatggtca gag 23 <210> 485 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 485 atttagtcca aagagggtca gag 23 <210> 486 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 486 atatacaacc aaagatgttc tagg 24 <210> 487 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 487 gtatatacat aaagaaggtc atgg 24 <210> 488 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 488 attatatcca aatatggtca gag 23 <210> 489 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 489 ttataatcca taaaatggtc atag 24 <210> 490 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 490 ttagtctcca aagatggtca ctg 23 <210> 491 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 491 tatctttcca aagatggtct atg 23 <210> 492 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 492 gtattttcca aagctagtca tgg 23 <210> 493 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 493 gcataatcca aggaaggtcc tgg 23 <210> 494 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 494 atattttcca aaaatggtca cag 23 <210> 495 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 495 atataatcca aaagaaggaa aag 23 <210> 496 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 496 atatatccaa aagatggtct gga 23 <210> 497 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 497 gatattaaca aagatggtct ggg 23 <210> 498 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 498 gaagaatcca acgatggcca cgg 23 <210> 499 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 499 acataatcac aaatatggtc acgg 24 <210> 500 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 500 gtattttcca aagatggcca cag 23 <210> 501 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 501 tctaatgaat aagatggtcg agg 23 <210> 502 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 502 agataatgca aagacggtta ggg 23 <210> 503 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 503 gtatatacca aaaatggtga agg 23 <210> 504 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 504 atatacccaa agatggtcca ag 22 <210> 505 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 505 tctgactccc aagatggtct tgg 23 <210> 506 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 506 ggagatacca aagatggtca gga 23 <210> 507 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 507 gtatatttca aatatggtca tag 23 <210> 508 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 508 atataaccaa agattgtcag ag 22 <210> 509 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 509 ctggagtcca gagatggtca tgg 23 <210> 510 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 510 gtagaaacca gagatggaca agg 23 <210> 511 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 511 ttataataca aaggtggtaa tgg 23 <210> 512 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 512 ctataatcaa aagtaggtca tag 23 <210> 513 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 513 atattttcca aaaatggtca tag 23 <210> 514 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 514 gtaaaatcca aagaggacca ggg 23 <210> 515 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon6) <400> 515 cctaaatcta aagatggtcg ggg 23 <210> 516 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 516 gaagcattca ttatagtcaa agg 23 <210> 517 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 517 gaagaattca ttatagacaa tgg 23 <210> 518 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 518 aagctattca ttatagcaaa tgg 23 <210> 519 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 519 gaagttaatt cattatagac aatgg 25 <210> 520 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 520 aaagtattaa ttatagtcaa gga 23 <210> 521 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 521 gaagtatcct tatagtcaga ag 22 <210> 522 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 522 gaagtatcat tatagtcttg gg 22 <210> 523 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 523 gcagtattca taatagtcaa aag 23 <210> 524 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 524 aaagtatttc atatagccaa agg 23 <210> 525 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 525 gaattattca tcatagccaa agg 23 <210> 526 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 526 taagtaatca ttatagtcag atg 23 <210> 527 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 527 atagtattca ttatagtaac agg 23 <210> 528 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 528 aaaatatcca ttatagtctt acg 23 <210> 529 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 529 gaactattca ttattatcaa cgg 23 <210> 530 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 530 gttgtattca ttatagtcaa gag 23 <210> 531 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 531 atagtattca ttatagtaac agg 23 <210> 532 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 532 agagtatcca ccatagtcaa ggg 23 <210> 533 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 533 gaagtacatc ttataatcaa tgg 23 <210> 534 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> Synthetic (DNA seq at a cleavage site for HPRTExon8) <400> 534 aatgtattca ttataggcaa gag 23 <210> 535 <211> 1771 <212> PRT <213> Artificial Sequence <220> <223> Synthetic (ABE7.10) <400> 535 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly Ser Ser Gly Gly Ser Ser 165 170 175 Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser Ala Thr Pro Glu Ser Ser 180 185 190 Gly Gly Ser Ser Gly Gly Ser Ser Glu Val Glu Phe Ser His Glu Tyr 195 200 205 Trp Met Arg His Ala Leu Thr Leu Ala Lys Arg Ala Arg Asp Glu Arg 210 215 220 Glu Val Pro Val Gly Ala Val Leu Val Leu Asn Asn Arg Val Ile Gly 225 230 235 240 Glu Gly Trp Asn Arg Ala Ile Gly Leu His Asp Pro Thr Ala His Ala 245 250 255 Glu Ile Met Ala Leu Arg Gln Gly Gly Leu Val Met Gln Asn Tyr Arg 260 265 270 Leu Ile Asp Ala Thr Leu Tyr Val Thr Phe Glu Pro Cys Val Met Cys 275 280 285 Ala Gly Ala Met Ile His Ser Arg Ile Gly Arg Val Val Phe Gly Val 290 295 300 Arg Asn Ala Lys Thr Gly Ala Ala Gly Ser Leu Met Asp Val Leu His 305 310 315 320 Tyr Pro Gly Met Asn His Arg Val Glu Ile Thr Glu Gly Ile Leu Ala 325 330 335 Asp Glu Cys Ala Ala Leu Leu Cys Tyr Phe Phe Arg Met Pro Arg Gln 340 345 350 Val Phe Asn Ala Gln Lys Lys Ala Gln Ser Ser Thr Asp Ser Gly Gly 355 360 365 Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr Ser Glu Ser 370 375 380 Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser Asp Lys Lys 385 390 395 400 Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly Trp Ala Val 405 410 415 Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys Val Leu Gly 420 425 430 Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly Ala Leu Leu 435 440 445 Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys Arg Thr Ala 450 455 460 Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr Leu Gln Glu 465 470 475 480 Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe Phe His Arg 485 490 495 Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His Glu Arg His 500 505 510 Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His Glu Lys Tyr 515 520 525 Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser Thr Asp Lys 530 535 540 Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met Ile Lys Phe 545 550 555 560 Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp Asn Ser Asp 565 570 575 Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn Gln Leu Phe 580 585 590 Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys Ala Ile Leu 595 600 605 Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu Ile Ala Gln 610 615 620 Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu Ile Ala Leu 625 630 635 640 Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp Leu Ala Glu 645 650 655 Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp Asp Leu Asp 660 665 670 Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu Phe Leu Ala 675 680 685 Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile Leu Arg Val 690 695 700 Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met Ile Lys Arg 705 710 715 720 Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala Leu Val Arg 725 730 735 Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp Gln Ser Lys 740 745 750 Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln Glu Glu Phe 755 760 765 Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly Thr Glu Glu 770 775 780 Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys Gln Arg Thr 785 790 795 800 Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly Glu Leu His 805 810 815 Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu Lys Asp Asn 820 825 830 Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro Tyr Tyr Val 835 840 845 Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met Thr Arg Lys 850 855 860 Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val Val Asp Lys 865 870 875 880 Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn Phe Asp Lys 885 890 895 Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu Leu Tyr Glu 900 905 910 Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr Val Thr Glu 915 920 925 Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys Lys Ala Ile 930 935 940 Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val Lys Gln Leu 945 950 955 960 Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser Val Glu Ile 965 970 975 Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr Tyr His Asp 980 985 990 Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn Glu Glu Asn 995 1000 1005 Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu Phe Glu Asp 1010 1015 1020 Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His Leu Phe Asp 1025 1030 1035 1040 Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr Gly Trp Gly 1045 1050 1055 Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys Gln Ser Gly 1060 1065 1070 Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala Asn Arg Asn 1075 1080 1085 Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys Glu Asp Ile 1090 1095 1100 Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His Glu His Ile 1105 1110 1115 1120 Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile Leu Gln Thr 1125 1130 1135 Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg His Lys Pro 1140 1145 1150 Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr Thr Gln Lys 1155 1160 1165 Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu Glu Glu Gly Ile 1170 1175 1180 Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val Glu Asn Thr 1185 1190 1195 1200 Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln Asn Gly Arg 1205 1210 1215 Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu Ser Asp Tyr 1220 1225 1230 Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp Asp Ser Ile 1235 1240 1245 Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly Lys Ser Asp 1250 1255 1260 Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn Tyr Trp Arg 1265 1270 1275 1280 Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe Asp Asn Leu 1285 1290 1295 Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys Ala Gly Phe 1300 1305 1310 Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys His Val Ala 1315 1320 1325 Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu Asn Asp Lys 1330 1335 1340 Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys Leu Val Ser 1345 1350 1355 1360 Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu Ile Asn Asn 1365 1370 1375 Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val Gly Thr Ala 1380 1385 1390 Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val Tyr Gly Asp 1395 1400 1405 Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser Glu Gln Glu 1410 1415 1420 Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn Ile Met Asn 1425 1430 1435 1440 Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile Arg Lys Arg 1445 1450 1455 Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val Trp Asp Lys 1460 1465 1470 Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met Pro Gln Val 1475 1480 1485 Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe Ser Lys Glu 1490 1495 1500 Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala Arg Lys Lys 1505 1510 1515 1520 Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro Thr Val Ala 1525 1530 1535 Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys Ser Lys Lys 1540 1545 1550 Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met Glu Arg Ser 1555 1560 1565 Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys Gly Tyr Lys 1570 1575 1580 Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr Ser Leu Phe 1585 1590 1595 1600 Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala Gly Glu Leu 1605 1610 1615 Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val Asn Phe Leu 1620 1625 1630 Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro Glu Asp Asn 1635 1640 1645 Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr Leu Asp Glu 1650 1655 1660 Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile Leu Ala Asp 1665 1670 1675 1680 Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His Arg Asp Lys 1685 1690 1695 Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe Thr Leu Thr 1700 1705 1710 Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr Thr Ile Asp 1715 1720 1725 Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala Thr Leu Ile 1730 1735 1740 His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp Leu Ser Gln 1745 1750 1755 1760 Leu Gly Gly Asp Pro Lys Lys Lys Arg Lys Val 1765 1770 <210> 536 <211> 167 <212> PRT <213> Artificial Sequence <220> <223> wild type E. coli TadA (NP_417054.2) <400> 536 Met Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu 1 5 10 15 Thr Leu Ala Lys Arg Ala Trp Asp Glu Arg Glu Val Pro Val Gly Ala 20 25 30 Val Leu Val His Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Pro 35 40 45 Ile Gly Arg His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg 50 55 60 Gln Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu 65 70 75 80 Tyr Val Thr Leu Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His 85 90 95 Ser Arg Ile Gly Arg Val Val Phe Gly Ala Arg Asp Ala Lys Thr Gly 100 105 110 Ala Ala Gly Ser Leu Met Asp Val Leu His His Pro Gly Met Asn His 115 120 125 Arg Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu 130 135 140 Leu Ser Asp Phe Phe Arg Met Arg Arg Gln Glu Ile Lys Ala Gln Lys 145 150 155 160 Lys Ala Gln Ser Ser Thr Asp 165 <210> 537 <211> 166 <212> PRT <213> Artificial Sequence <220> <223> modified E. coli TadA (NP_417054.2) <400> 537 Ser Glu Val Glu Phe Ser His Glu Tyr Trp Met Arg His Ala Leu Thr 1 5 10 15 Leu Ala Lys Arg Ala Arg Asp Glu Arg Glu Val Pro Val Gly Ala Val 20 25 30 Leu Val Leu Asn Asn Arg Val Ile Gly Glu Gly Trp Asn Arg Ala Ile 35 40 45 Gly Leu His Asp Pro Thr Ala His Ala Glu Ile Met Ala Leu Arg Gln 50 55 60 Gly Gly Leu Val Met Gln Asn Tyr Arg Leu Ile Asp Ala Thr Leu Tyr 65 70 75 80 Val Thr Phe Glu Pro Cys Val Met Cys Ala Gly Ala Met Ile His Ser 85 90 95 Arg Ile Gly Arg Val Val Phe Gly Val Arg Asn Ala Lys Thr Gly Ala 100 105 110 Ala Gly Ser Leu Met Asp Val Leu His Tyr Pro Gly Met Asn His Arg 115 120 125 Val Glu Ile Thr Glu Gly Ile Leu Ala Asp Glu Cys Ala Ala Leu Leu 130 135 140 Cys Tyr Phe Phe Arg Met Pro Arg Gln Val Phe Asn Ala Gln Lys Lys 145 150 155 160 Ala Gln Ser Ser Thr Asp 165 <210> 538 <211> 1367 <212> PRT <213> Artificial Sequence <220> <223> SpCas9 nickase (D10A) <400> 538 Asp Lys Lys Tyr Ser Ile Gly Leu Ala Ile Gly Thr Asn Ser Val Gly 1 5 10 15 Trp Ala Val Ile Thr Asp Glu Tyr Lys Val Pro Ser Lys Lys Phe Lys 20 25 30 Val Leu Gly Asn Thr Asp Arg His Ser Ile Lys Lys Asn Leu Ile Gly 35 40 45 Ala Leu Leu Phe Asp Ser Gly Glu Thr Ala Glu Ala Thr Arg Leu Lys 50 55 60 Arg Thr Ala Arg Arg Arg Tyr Thr Arg Arg Lys Asn Arg Ile Cys Tyr 65 70 75 80 Leu Gln Glu Ile Phe Ser Asn Glu Met Ala Lys Val Asp Asp Ser Phe 85 90 95 Phe His Arg Leu Glu Glu Ser Phe Leu Val Glu Glu Asp Lys Lys His 100 105 110 Glu Arg His Pro Ile Phe Gly Asn Ile Val Asp Glu Val Ala Tyr His 115 120 125 Glu Lys Tyr Pro Thr Ile Tyr His Leu Arg Lys Lys Leu Val Asp Ser 130 135 140 Thr Asp Lys Ala Asp Leu Arg Leu Ile Tyr Leu Ala Leu Ala His Met 145 150 155 160 Ile Lys Phe Arg Gly His Phe Leu Ile Glu Gly Asp Leu Asn Pro Asp 165 170 175 Asn Ser Asp Val Asp Lys Leu Phe Ile Gln Leu Val Gln Thr Tyr Asn 180 185 190 Gln Leu Phe Glu Glu Asn Pro Ile Asn Ala Ser Gly Val Asp Ala Lys 195 200 205 Ala Ile Leu Ser Ala Arg Leu Ser Lys Ser Arg Arg Leu Glu Asn Leu 210 215 220 Ile Ala Gln Leu Pro Gly Glu Lys Lys Asn Gly Leu Phe Gly Asn Leu 225 230 235 240 Ile Ala Leu Ser Leu Gly Leu Thr Pro Asn Phe Lys Ser Asn Phe Asp 245 250 255 Leu Ala Glu Asp Ala Lys Leu Gln Leu Ser Lys Asp Thr Tyr Asp Asp 260 265 270 Asp Leu Asp Asn Leu Leu Ala Gln Ile Gly Asp Gln Tyr Ala Asp Leu 275 280 285 Phe Leu Ala Ala Lys Asn Leu Ser Asp Ala Ile Leu Leu Ser Asp Ile 290 295 300 Leu Arg Val Asn Thr Glu Ile Thr Lys Ala Pro Leu Ser Ala Ser Met 305 310 315 320 Ile Lys Arg Tyr Asp Glu His His Gln Asp Leu Thr Leu Leu Lys Ala 325 330 335 Leu Val Arg Gln Gln Leu Pro Glu Lys Tyr Lys Glu Ile Phe Phe Asp 340 345 350 Gln Ser Lys Asn Gly Tyr Ala Gly Tyr Ile Asp Gly Gly Ala Ser Gln 355 360 365 Glu Glu Phe Tyr Lys Phe Ile Lys Pro Ile Leu Glu Lys Met Asp Gly 370 375 380 Thr Glu Glu Leu Leu Val Lys Leu Asn Arg Glu Asp Leu Leu Arg Lys 385 390 395 400 Gln Arg Thr Phe Asp Asn Gly Ser Ile Pro His Gln Ile His Leu Gly 405 410 415 Glu Leu His Ala Ile Leu Arg Arg Gln Glu Asp Phe Tyr Pro Phe Leu 420 425 430 Lys Asp Asn Arg Glu Lys Ile Glu Lys Ile Leu Thr Phe Arg Ile Pro 435 440 445 Tyr Tyr Val Gly Pro Leu Ala Arg Gly Asn Ser Arg Phe Ala Trp Met 450 455 460 Thr Arg Lys Ser Glu Glu Thr Ile Thr Pro Trp Asn Phe Glu Glu Val 465 470 475 480 Val Asp Lys Gly Ala Ser Ala Gln Ser Phe Ile Glu Arg Met Thr Asn 485 490 495 Phe Asp Lys Asn Leu Pro Asn Glu Lys Val Leu Pro Lys His Ser Leu 500 505 510 Leu Tyr Glu Tyr Phe Thr Val Tyr Asn Glu Leu Thr Lys Val Lys Tyr 515 520 525 Val Thr Glu Gly Met Arg Lys Pro Ala Phe Leu Ser Gly Glu Gln Lys 530 535 540 Lys Ala Ile Val Asp Leu Leu Phe Lys Thr Asn Arg Lys Val Thr Val 545 550 555 560 Lys Gln Leu Lys Glu Asp Tyr Phe Lys Lys Ile Glu Cys Phe Asp Ser 565 570 575 Val Glu Ile Ser Gly Val Glu Asp Arg Phe Asn Ala Ser Leu Gly Thr 580 585 590 Tyr His Asp Leu Leu Lys Ile Ile Lys Asp Lys Asp Phe Leu Asp Asn 595 600 605 Glu Glu Asn Glu Asp Ile Leu Glu Asp Ile Val Leu Thr Leu Thr Leu 610 615 620 Phe Glu Asp Arg Glu Met Ile Glu Glu Arg Leu Lys Thr Tyr Ala His 625 630 635 640 Leu Phe Asp Asp Lys Val Met Lys Gln Leu Lys Arg Arg Arg Tyr Thr 645 650 655 Gly Trp Gly Arg Leu Ser Arg Lys Leu Ile Asn Gly Ile Arg Asp Lys 660 665 670 Gln Ser Gly Lys Thr Ile Leu Asp Phe Leu Lys Ser Asp Gly Phe Ala 675 680 685 Asn Arg Asn Phe Met Gln Leu Ile His Asp Asp Ser Leu Thr Phe Lys 690 695 700 Glu Asp Ile Gln Lys Ala Gln Val Ser Gly Gln Gly Asp Ser Leu His 705 710 715 720 Glu His Ile Ala Asn Leu Ala Gly Ser Pro Ala Ile Lys Lys Gly Ile 725 730 735 Leu Gln Thr Val Lys Val Val Asp Glu Leu Val Lys Val Met Gly Arg 740 745 750 His Lys Pro Glu Asn Ile Val Ile Glu Met Ala Arg Glu Asn Gln Thr 755 760 765 Thr Gln Lys Gly Gln Lys Asn Ser Arg Glu Arg Met Lys Arg Ile Glu 770 775 780 Glu Gly Ile Lys Glu Leu Gly Ser Gln Ile Leu Lys Glu His Pro Val 785 790 795 800 Glu Asn Thr Gln Leu Gln Asn Glu Lys Leu Tyr Leu Tyr Tyr Leu Gln 805 810 815 Asn Gly Arg Asp Met Tyr Val Asp Gln Glu Leu Asp Ile Asn Arg Leu 820 825 830 Ser Asp Tyr Asp Val Asp His Ile Val Pro Gln Ser Phe Leu Lys Asp 835 840 845 Asp Ser Ile Asp Asn Lys Val Leu Thr Arg Ser Asp Lys Asn Arg Gly 850 855 860 Lys Ser Asp Asn Val Pro Ser Glu Glu Val Val Lys Lys Met Lys Asn 865 870 875 880 Tyr Trp Arg Gln Leu Leu Asn Ala Lys Leu Ile Thr Gln Arg Lys Phe 885 890 895 Asp Asn Leu Thr Lys Ala Glu Arg Gly Gly Leu Ser Glu Leu Asp Lys 900 905 910 Ala Gly Phe Ile Lys Arg Gln Leu Val Glu Thr Arg Gln Ile Thr Lys 915 920 925 His Val Ala Gln Ile Leu Asp Ser Arg Met Asn Thr Lys Tyr Asp Glu 930 935 940 Asn Asp Lys Leu Ile Arg Glu Val Lys Val Ile Thr Leu Lys Ser Lys 945 950 955 960 Leu Val Ser Asp Phe Arg Lys Asp Phe Gln Phe Tyr Lys Val Arg Glu 965 970 975 Ile Asn Asn Tyr His His Ala His Asp Ala Tyr Leu Asn Ala Val Val 980 985 990 Gly Thr Ala Leu Ile Lys Lys Tyr Pro Lys Leu Glu Ser Glu Phe Val 995 1000 1005 Tyr Gly Asp Tyr Lys Val Tyr Asp Val Arg Lys Met Ile Ala Lys Ser 1010 1015 1020 Glu Gln Glu Ile Gly Lys Ala Thr Ala Lys Tyr Phe Phe Tyr Ser Asn 1025 1030 1035 1040 Ile Met Asn Phe Phe Lys Thr Glu Ile Thr Leu Ala Asn Gly Glu Ile 1045 1050 1055 Arg Lys Arg Pro Leu Ile Glu Thr Asn Gly Glu Thr Gly Glu Ile Val 1060 1065 1070 Trp Asp Lys Gly Arg Asp Phe Ala Thr Val Arg Lys Val Leu Ser Met 1075 1080 1085 Pro Gln Val Asn Ile Val Lys Lys Thr Glu Val Gln Thr Gly Gly Phe 1090 1095 1100 Ser Lys Glu Ser Ile Leu Pro Lys Arg Asn Ser Asp Lys Leu Ile Ala 1105 1110 1115 1120 Arg Lys Lys Asp Trp Asp Pro Lys Lys Tyr Gly Gly Phe Asp Ser Pro 1125 1130 1135 Thr Val Ala Tyr Ser Val Leu Val Val Ala Lys Val Glu Lys Gly Lys 1140 1145 1150 Ser Lys Lys Leu Lys Ser Val Lys Glu Leu Leu Gly Ile Thr Ile Met 1155 1160 1165 Glu Arg Ser Ser Phe Glu Lys Asn Pro Ile Asp Phe Leu Glu Ala Lys 1170 1175 1180 Gly Tyr Lys Glu Val Lys Lys Asp Leu Ile Ile Lys Leu Pro Lys Tyr 1185 1190 1195 1200 Ser Leu Phe Glu Leu Glu Asn Gly Arg Lys Arg Met Leu Ala Ser Ala 1205 1210 1215 Gly Glu Leu Gln Lys Gly Asn Glu Leu Ala Leu Pro Ser Lys Tyr Val 1220 1225 1230 Asn Phe Leu Tyr Leu Ala Ser His Tyr Glu Lys Leu Lys Gly Ser Pro 1235 1240 1245 Glu Asp Asn Glu Gln Lys Gln Leu Phe Val Glu Gln His Lys His Tyr 1250 1255 1260 Leu Asp Glu Ile Ile Glu Gln Ile Ser Glu Phe Ser Lys Arg Val Ile 1265 1270 1275 1280 Leu Ala Asp Ala Asn Leu Asp Lys Val Leu Ser Ala Tyr Asn Lys His 1285 1290 1295 Arg Asp Lys Pro Ile Arg Glu Gln Ala Glu Asn Ile Ile His Leu Phe 1300 1305 1310 Thr Leu Thr Asn Leu Gly Ala Pro Ala Ala Phe Lys Tyr Phe Asp Thr 1315 1320 1325 Thr Ile Asp Arg Lys Arg Tyr Thr Ser Thr Lys Glu Val Leu Asp Ala 1330 1335 1340 Thr Leu Ile His Gln Ser Ile Thr Gly Leu Tyr Glu Thr Arg Ile Asp 1345 1350 1355 1360 Leu Ser Gln Leu Gly Gly Asp 1365 <210> 539 <211> 32 <212> PRT <213> Artificial Sequence <220> <223> peptide linker <400> 539 Ser Gly Gly Ser Ser Gly Gly Ser Ser Gly Ser Glu Thr Pro Gly Thr 1 5 10 15 Ser Glu Ser Ala Thr Pro Glu Ser Ser Gly Gly Ser Ser Gly Gly Ser 20 25 30 <210> 540 <211> 7 <212> PRT <213> Artificial Sequence <220> <223> SV40 NLS <400> 540 Pro Lys Lys Lys Arg Lys Val 1 5

Claims (22)

(1) adenosine deaminase, a nucleic acid molecule encoding the same, or a plasmid containing the nucleic acid molecule,
(2) an inactivated target-specific nuclease, a nucleic acid molecule encoding the same, or a plasmid containing the nucleic acid molecule,
(3) guide RNA, and
(4) Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII (endonuclease VIII) combination or endonuclease V (endonuclease V)
Including,
The inactivated target-specific nuclease has lost endonuclease activity that cuts all DNA double strands,
A composition for cutting DNA double strands using adenosine deaminase. The composition of claim 1, wherein the inactivated target-specific nuclease is a Cas9 protein or Cpf1 protein that has lost endonuclease activity that cleaves DNA double strands, adenosine deaminase. . The method of claim 2, wherein the inactivated target-specific nuclease is at least one selected from the group consisting of amino acid residues D10, H840, F539, M763, and K890 in Cas9 protein derived from Streptococcus pyogenes . A mutation substituted with an amino acid different from the original was introduced,
The other amino acids are selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. Which is an amino acid different from the original amino acid,
A composition for cutting DNA double strands using adenosine deaminase. The method of claim 1,
The adenosine deaminase, inactivated target-specific nuclease, and guide RNA are
(i) a mixture of adenosine deaminase, target-specific nuclease, and guide RNA, (ii) adenosine deaminase-encoding nucleic acid sequence or a plasmid comprising the same, a target-specific nuclease-encoding nucleic acid sequence, or comprising the same A mixture of a plasmid and a plasmid comprising a guide RNA or a nucleic acid molecule encoding it, (iii) a mixture of a fusion protein and guide RNA to which adenosine deaminase and target-specific nuclease are linked, (iv) adenosine deaminase and target specific A mixture of a fusion protein-encoding nucleic acid molecule to which an enemy nuclease is linked or a plasmid containing the nucleic acid molecule, and a plasmid containing a guide RNA or a nucleic acid molecule encoding the same, and (v) adenosine deaminase and a target-specific nuclease are linked Complex or mixture of fusion protein and guide RNA
A composition for cutting DNA double strands using one or more forms selected from the group consisting of, adenosine deaminase. The method of claim 1,
The inactivated target-specific nuclease is in the Cas9 protein derived from Streptococcus pyogenes ,
A mutation in which amino acid residue D10 is substituted with an amino acid different from the original amino acid is introduced, or
A mutation in which both amino acid residues D10 and H840 are substituted with amino acids different from the original amino acid is introduced, or
Amino acid residues F539, M763, and K890 are all substituted with amino acids different from the original amino acid mutation is introduced,
The other amino acids are selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. Which is an amino acid different from the original amino acid,
A composition for cutting DNA double strands using adenosine deaminase. The DNA using adenosine deaminase according to any one of claims 1 to 5, wherein the guide RNA is a double-stranded crRNA:tracrRNA complex in which crRNA and tracrRNA are bound to each other, or a single-stranded guide RNA (sgRNA). Composition for cutting double strands. (a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid comprising the nucleic acid molecule, and (2) i) an inactivated target-specific nuclease, an inactivated target A nucleic acid molecule encoding a specific nuclease, or a plasmid containing the nucleic acid molecule and ii) a guide RNA, a nucleic acid molecule encoding the guide RNA, or a plasmid containing the nucleic acid molecule are introduced into an isolated cell, or Processing the separated DNA to cut one of the DNA double strands; And
(b) a combination of Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII, or endonuclease V, to the DNA from which one of the DNA double strands is cut, and the remaining DNA strands It comprises the step of cutting the,
The inactivated target-specific nuclease has lost endonuclease activity that cuts all DNA double strands,
A method of generating a double strand break in DNA using adenosine deaminase. (a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid comprising the nucleic acid molecule, and (2) i) an inactivated target-specific nuclease, an inactivated target A nucleic acid molecule encoding a specific nuclease, or a plasmid containing the nucleic acid molecule and ii) a guide RNA, a nucleic acid molecule encoding the guide RNA, or a plasmid containing the nucleic acid molecule are introduced into an isolated cell, or Processing the separated DNA to cut one of the DNA double strands;
(b) a combination of Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII, or endonuclease V, to the DNA from which one of the DNA double strands is cut, and the remaining DNA strands Cutting it; And
(c) comprising the step of performing whole genome sequencing on the double-stranded DNA,
The inactivated target-specific nuclease has lost endonuclease activity that cuts all DNA double strands,
A method for analyzing nucleic acid sequence of DNA into which base editing has been introduced by adenosine deaminase. (a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid comprising the nucleic acid molecule, and (2) i) an inactivated target-specific nuclease, an inactivated target A nucleic acid molecule encoding a specific nuclease, or a plasmid containing the nucleic acid molecule and ii) a guide RNA, a nucleic acid molecule encoding the guide RNA, or a plasmid containing the nucleic acid molecule are introduced into an isolated cell, or Processing the separated DNA to cut one of the DNA double strands;
(b) a combination of Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII, or endonuclease V, to the DNA from which one of the DNA double strands is cut, and the remaining DNA strands Cutting it;
(c) performing whole genome sequencing on the double-stranded DNA; And
(d) confirming the cut position in the sequence read obtained by the sequencing,
The inactivated target-specific nuclease has lost endonuclease activity that cuts all DNA double strands,
Adenosine Deaminase Base Correction Location Identification Method. (a) (1) adenosine deaminase, a nucleic acid molecule encoding adenosine deaminase, or a plasmid comprising the nucleic acid molecule, and (2) i) an inactivated target-specific nuclease, an inactivated target A nucleic acid molecule encoding a specific nuclease, or a plasmid containing the nucleic acid molecule and ii) a guide RNA, a nucleic acid molecule encoding the guide RNA, or a plasmid containing the nucleic acid molecule are introduced into an isolated cell, or Processing the separated DNA to cut one of the DNA double strands;
(b) a combination of Alkyladenine DNA Glycosylase (AAG) and endonuclease VIII, or endonuclease V, to the DNA from which one of the DNA double strands is cut, and the remaining DNA strands Cutting it;
(c) performing whole genome sequencing on the double-stranded DNA; And
(d) confirming the cut position in the sequence read obtained by the sequencing,
The inactivated target-specific nuclease has lost endonuclease activity that cuts all DNA double strands,
Method for identifying the off-target site of adenosine deaminase. The method of claim 9, wherein the inactivated target-specific nuclease is a Cas9 protein or a Cpf1 protein that has lost its endonuclease activity for cleaving DNA double strands. The method of claim 9, wherein the inactivated target-specific nuclease is at least one selected from the group consisting of amino acid residues D10, H840, F539, M763, and K890 in Cas9 protein derived from Streptococcus pyogenes . A mutation substituted with an amino acid different from the original was introduced,
The other amino acids are selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. Which is an amino acid different from the original amino acid,
Way. The method of claim 9,
The adenosine deaminase, inactivated target-specific nuclease, and guide RNA are
(i) a mixture of adenosine deaminase, target-specific nuclease, and guide RNA, (ii) adenosine deaminase-encoding nucleic acid sequence or a plasmid comprising the same, a target-specific nuclease-encoding nucleic acid sequence, or comprising the same A mixture of a plasmid and a plasmid comprising a guide RNA or a nucleic acid molecule encoding it, (iii) a mixture of a fusion protein and guide RNA to which adenosine deaminase and target-specific nuclease are linked, (iv) adenosine deaminase and target specific A mixture of a fusion protein-encoding nucleic acid molecule to which an enemy nuclease is linked or a plasmid containing the nucleic acid molecule, and a plasmid containing a guide RNA or a nucleic acid molecule encoding the same, and (v) adenosine deaminase and a target-specific nuclease are linked Complex or mixture of fusion protein and guide RNA
The method is used in one or more forms selected from the group consisting of. The method of claim 9,
The inactivated target-specific nuclease is in the Cas9 protein derived from Streptococcus pyogenes ,
A mutation in which amino acid residue D10 is substituted with an amino acid different from the original amino acid is introduced, or
A mutation in which both amino acid residues D10 and H840 are substituted with amino acids different from the original amino acid is introduced, or
Amino acid residues F539, M763, and K890 are all substituted with amino acids different from the original amino acid mutation is introduced,
The other amino acids are selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, valine, aspartic acid, cysteine, glutamine, glycine, serine, threonine, tyrosine, aspartic acid, glutamic acid, arginine, histidine, and lysine. Which is an amino acid different from the original amino acid,
Way. The method of claim 9, wherein the guide RNA is a double-stranded crRNA:tracrRNA complex in which crRNA and tracrRNA are bound to each other, or a single-stranded guide RNA (sgRNA). The method according to claim 9, which is carried out in vitro. The method of claim 9, wherein the DNA isolated in step (a) is genomic DNA. The method of claim 10, wherein after step (d),
The method further comprising the step of determining an off-target site when the cut position is not an on-target site. The method of claim 10,
The cleavage position identified in step (d) is a position in which the 5'end is vertically aligned by aligning the obtained nucleotide sequence data, or a position showing a double peak pattern in the 5'end plot. The method of claim 19, wherein the alignment is performed using BWA/GATK or ISAAC after mapping the nucleotide sequence data to a reference genome. The method of claim 19, wherein the step of determining that a position in which two or more sequence reads corresponding to a Watson strand and a Crick strand are vertically aligned is a non-target position. The method further comprising. The method of claim 19, wherein the step of determining that a position in which at least 20% of the nucleotide sequence data are vertically aligned and the number of nucleotide sequence data having the same 5'end in each Watson strand and a Creek strand is 10 or more is a non-target position. The method further comprising.

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