Single base substitution protein and composition containing the same

Abstract

The present application relates to a single base substitution protein; a composition comprising the protein; and uses thereof.

Description

Technical Field

[0001] The present application relates to a technology for replacing cytosine (C) or adenine (A) with any base using a protein for single base substitution using a CRISPR enzyme, a deaminase, and a DNA glycosylase. Background Art

[0002] CRISPR enzyme-linked deaminases have been used to treat genetic disorders by editing gene loci where point mutations occur, or to induce targeted single nucleotide polymorphisms (SNPs) in genes in humans or eukaryotic cells.

[0003] Currently reported CRISPR enzyme-linked deaminases include:

[0004] 1) base editors (BEs), including (i) catalytically defective Cas9 (dCas9) or D10A Cas9 nickase (nCas9) derived from Streptococcus pyogenes (S. pyogenes), and (ii) rAPOBEC1, a rat cytidine deaminase;

[0005] 2) Target-AID, including (i) dCas9 or nCas9, and (ii) PmCDA1, which is an activation-induced cytidine deaminase (AID) ortholog of sea lamprey or human AID;

[0006] 3) CRISPR-X, including sgRNA and dCas9 linked to an MS2 RNA hairpin, to recruit a hyperactive AID variant fused to an MS2 binding protein; and

[0007] 4) Zinc finger proteins or transcription activator-like effectors (TALEs) fused to cytidine deaminase.

[0008] CRISPR enzyme-linked deaminases used with traditional DNA glycosylases can replace cytosine (C) with only thymine (T) in nucleotides, or replace adenine (A) with only guanine (G). In one example, a material in which Cas9, cytidine deaminase, and uracil DNA glycosylase inhibitor (UGI) are fused is used to replace cytosine (C) with thymine (T). The material is used to replace uracil (U) with thymine (T) using a mechanism that induces uracil (U) not to be removed by DNA glycosylase. Similarly, it has recently been reported that the use of adenosine deaminase instead of cytidine deaminase can replace adenine (A) with only guanine (G).

[0009] Therefore, the inventors of the present application intend to develop proteins for single base substitution by using CRISPR enzymes, deaminases, and DNA glycosylases, thereby replacing cytosine (C) or adenine (A) with any base. The development of this technology can be used for the identification of genetic diseases caused by mutations, and for the development of drugs and therapeutic agents by analyzing nucleic acid sequences that affect disease susceptibility or drug resistance due to SNPs, and will more effectively develop drugs and improve treatment effects in the future. Summary of the invention

[0010] [Technical issues]

[0011] Conventional CRISPR enzyme-linked deaminases are limited to converting cytosine (C) or adenine (A) into specific bases (A or G). Due to these limitations, the scope of research such as the identification of genetic diseases caused by mutations, disease susceptibility from SNPs, and the development of related therapeutic agents is limited.

[0012] Therefore, there is an urgent need to develop a means to replace cytosine (C) or adenine (A) with any base (A, T, C, G, or U) rather than a specific base.

[0013] The present application aims to provide a protein for single base substitution or a complex for single base substitution, or a composition for single base substitution comprising the same, and uses thereof.

[0014] The present application aims to provide a nucleic acid sequence encoding a protein for single base substitution or a vector comprising the nucleic acid sequence.

[0015] The present application aims to provide a method for single base substitution.

[0016] The present application aims to provide various uses of a protein for single base substitution or a complex for single base substitution or a composition for single base substitution comprising the same.

[0017] [Technical solution]

[0018] The present application provides a fusion protein for single base substitution or a nucleic acid encoding the fusion protein.

[0019] The present application provides a vector comprising a nucleic acid encoding a fusion protein for single base substitution.

[0020] The present application provides a complex for single base substitution.

[0021] The present application provides compositions for single base substitution.

[0022] The present application provides a method for single base substitution.

[0023] The present application provides the use of the fusion protein for single base substitution, the complex for single base substitution, and the composition for single base substitution of the present application for epitope screening, drug resistance gene or protein screening, drug sensitization screening, or virus resistance gene or protein screening.

[0024] The present application provides a fusion protein for single base substitution or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises (a) a CRISPR enzyme or a variant thereof, (b) a deaminase, and (c) a DNA glycosylase or a variant thereof. Wherein, the fusion protein for single base substitution induces the substitution of cytosine or adenine contained in one or more nucleotides in any base pair target nucleic acid sequence.

[0025] The present application provides a fusion protein for single base substitution or a nucleic acid encoding the fusion protein, wherein the fusion protein comprises any of the following components: (i) N-terminus-[CRISPR enzyme]-[deaminase]-[DNA glycosylase]-C-terminus; (ii) N-terminus-[CRISPR enzyme]-[DNA glycosylase]-[deaminase]-C-terminus; (iii) N-terminus-[deaminase]-[CRISPR enzyme]-[DNA glycosylase]-C-terminus; (iv) N-terminus-[deaminase]-[DNA glycosylase]-[CRISPR enzyme]-C-terminus; (v) N-terminus-[DNA glycosylase]-[CRISPR enzyme]-[deaminase]-C-terminus; and (vi) N-terminus-[DNA glycosylase]-[deaminase]-[CRISPR enzyme]-C-terminus.

[0026] The present application provides a complex for single base substitution, the complex comprising (a) a CRISPR enzyme or a variant thereof; (b) a deaminase; (c) a DNA glycosylase; and (d) two or more binding domains. Wherein, the fusion protein for single base substitution induces the substitution of cytosine or adenine contained in one or more nucleotides in any base pair target nucleic acid sequence.

[0027] According to the present application, in a complex for single base substitution, each of the CRISPR enzyme, the deaminase and the DNA glycosylase is connected to one or more binding domains. Wherein, the CRISPR enzyme, the deaminase and the DNA glycosylase form the complex through the interaction between the binding domains.

[0028] According to the present application, in the complex for single base substitution, any one selected from CRISPR enzyme, deaminase and DNA glycosylase is connected to the first binding domain and the second binding domain. Wherein, the first binding domain and the binding domain of another component are an interaction pair, and the second binding domain and the binding domain of another binding domain are an interaction pair. Wherein, the complex is formed by the pair.

[0029] According to the present application, the complex for single base substitution comprises (i) a first fusion protein, the first fusion protein comprising two components selected from CRISPR enzyme, deaminase and DNA glycosylase and a first binding domain, and (ii) a second fusion protein, the second fusion protein comprising another component not selected above and a second binding domain. Wherein, the first binding domain and the second binding domain are an interaction pair, and the complex is formed by the pair.

[0030] According to the present application, the complex for single base substitution comprises (i) a first fusion protein comprising a deaminase, a DNA glycosylase and a first binding domain, and (ii) a second fusion protein comprising a CRISPR enzyme and a second binding domain.

[0031] Wherein, the first binding domain is a single-chain variable fragment (scFv), and the second fusion protein further comprises at least one or more binding domains, wherein the further included binding domain is a GCN4 peptide. Wherein, two or more of the first fusion proteins can form a complex by interacting with any one of the GCN4 peptides.

[0032] The present application may provide a composition for single base substitution, the composition comprising (a) a guide RNA or a nucleic acid encoding the guide RNA, and (b) i) a fusion protein for single base substitution as claimed in claim 1 or a nucleic acid encoding the fusion protein, or ii) a complex for single base substitution as claimed in claim 13. Wherein, the guide RNA is complementary to the target nucleic acid sequence, wherein the target nucleic acid sequence bound to the guide RNA is 15 bp to 25 bp. Wherein, the fusion protein for single base substitution or the complex for single base substitution induces the substitution of one or more cytosines or adenines present in the target region containing the target nucleic acid sequence at any base pair.

[0033] According to the present application, the composition for single base substitution may include one or more vectors.

[0034] The present application may provide a method for single base substitution, the method comprising: contacting (i) and (ii) with a target region containing a target nucleic acid sequence in vitro (invitro) or ex vivo (ex vivo), wherein (i) is a guide RNA, and (ii) is a fusion protein for single base substitution as claimed in claim 1 or a complex for single base substitution as claimed in claim 12. The guide RNA binds complementarily to the target nucleic acid sequence, the target nucleic acid sequence bound to the guide RNA is 15 bp to 25 bp, and the fusion protein for single base substitution or the complex for single base substitution induces substitution of any base pair for one or more cytosines or adenines present in the target region containing the target nucleic acid sequence.

[0035] Wherein, the deaminase is cytidine deaminase, and the DNA glycosylase is uracil-DNA glycosylase or a variant thereof. Wherein, the fusion protein for single base substitution induces substitution of any base pair of C (cytosine) contained in one or more nucleotides in the target nucleic acid sequence.

[0036] Wherein, the cytidine deaminase may be APOBEC, activation-induced cytidine deaminase (AID) or a variant thereof.

[0037] Wherein, the deaminase may be adenosine deaminase, and the DNA glycosylase may be alkyladenine DNA glycosylase or a variant thereof. Wherein, the fusion protein for single base substitution may induce substitution of any base pair of adenine contained in one or more nucleotides in the target nucleic acid sequence.

[0038] The adenosine deaminase may be TadA, Tad2p, ADA, ADA1, ADA2, ADAR2, ADAT2, ADAT3 or variants thereof.

[0039] Among them, the binding domain can be any one selected from the FRB domain, FKBP dimerization domain, intein, ERT domain, VPR domain, GCN4 peptide, single-chain variable fragment (scFv), or any one of the domains that form heterodimers.

[0040] Wherein, in the complex for single base substitution, the pair can be any one selected from the following (i) to (vi): (i) FRB domain and FKBP dimerization domain; (ii) first intein and second intein; (iii) ERT domain and VPR domain; (iv) GCN4 peptide and single-chain variable fragment (scFv); and (v) first domain and second domain for forming heterodimer.

[0041] [Beneficial Effects]

[0042] The present application provides a protein for single base substitution and / or a nucleic acid encoding the protein.

[0043] The present application provides a composition for single base substitution, which comprises a protein for single base substitution and / or a nucleic acid encoding the protein.

[0044] The present application provides various uses of a protein for single base substitution or a composition for single base substitution comprising the protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 The figure illustrates a method for replacing cytosine (C) in a target nucleic acid region with N (A, T or G) using a protein for single base substitution.

[0046] Figure 2 The figure illustrates a method for replacing adenine (A) in a target nucleic acid region with N (C, T or G) using a protein for single base substitution.

[0047] Figure 3 Figures illustrating various designs of fusion proteins for single base substitutions to induce substitution of cytosine by any base.

[0048] Figure 4 Figures illustrating various designs of fusion proteins for single base substitutions to induce arbitrary base substitutions for adenine.

[0049] Figure 5 (a) is nCas9 with 10 identical GCN4 peptides fused to the carboxyl terminus; and, Figure 5 (b) and Figure 5 (c) Various designs of complexes (scFv-Apobec-UNG and scfv-UNG-Apobec), in which the single-chain variable fragment (scFv) was fused to Apobec and UNG, respectively.

[0050] Figure 6 (a) is a diagram illustrating the design of the complex, in which five identical GCN4 peptides are fused to the N-terminus and C-terminus of nCas9, one scFv is fused to APOBEC, and another scFv is fused to UNG. Figure 6 (b) is a diagram illustrating the design of the complex, in which five identical GCN4 peptides are fused to the C-terminus of nCas9, one scFv is fused to APOBEC, and another scFv is fused to UNG.

[0051] Figure 7 (a) shows the design of BE3 WT and bpNLS BE3; and, Figure 7(b) is a graph showing the efficiency of single base substitution using BE3 WT and bpNLS BE3 in HEK cells.

[0052] Figure 8 Graph showing the rates of C to G, C to T, or C to A substitutions in Hela cells using BE3 WT, ncas-delta UGI, UNG-ncas, and ncas-UNG. ncas-delta UGI is a protein that removes the uracil DNA-glycosylase inhibitor (UGI) from BE3 WT.

[0053] Fig. 9 The nucleic acid sequence (SEQ ID No: 1) that induces base substitution in the target region is shown. Fig. 9 Also shown are the base substitution rates of cytosine at position 15 and cytosine at position 16 in the nucleic acid sequence (SEQ ID NO: 1) using BE3 WT, bpNLS BE3, ncas-delta UGI, UNG-ncas, and ncas-UNG in hela cells.

[0054] Fig.10 Diagram confirming cytosine substitutions in hEMX1 target nucleic acid sequences targeting GX20 sgRNA in HEK cells.

[0055] Fig.11 A set of graphs showing the efficiency of single base substitutions using UNG-ncas and ncas-UNG in HEK cells. The left graph shows the C to N substitution rate in the hEMX1 target nucleic acid sequence targeted by GX20 sgRNA. The right graph shows the C to G or C to A substitution rates at positions 13C, 15C, 16C, and 17C in the hEMX1 target nucleic acid sequence targeted by GX20 sgRNA.

[0056] Fig.12 A set of graphs to confirm whether Nureki nCas9 has a C to N base substitution at the NG PAM in HEK cells.

[0057] Fig.13 To confirm the use Figure 5 A graph showing whether a complex for a single base substitution undergoes a C to N base substitution.

[0058] Fig.14 For identification use Figure 5 Figure 3. Complex for single base substitution. Schematic diagram of C substitutions occurring in the nucleic acid sequence of GX19 sgRNA targeting hEMX1 in PC9 cells.

[0059] Fig.15 To illustrate the use of Figure 5 Figure 3 is a graph of the C to G, C to T, or C to A substitution rates of the complexes for single base substitution at the 16C position in the sequence targeting hEMX1 sgRNA.

[0060] Fig.16 The design of a plasmid encoding a protein for single base substitution using nCas9 is shown. Figure 3 This is explained in 1) of (a).

[0061] Fig.17 The design of a plasmid for a CRISPR protein for single base substitution using Nurekin Cas9 is shown. The encoded protein for single base substitution is Figure 3 This is explained in 2) of (c).

[0062] Fig.18 The design of a plasmid encoding a protein for single base substitution using nCas9 is shown. Figure 3 This is explained in 3) of (a).

[0063] Fig.19 Shows the encoding Figure 4 Design of plasmid for single base substituted protein shown in (a).

[0064] Fig. 20 Shows the encoding Figure 4 Design of plasmid for single base substituted protein shown in (b).

[0065] Fig.21 A diagram illustrating the structure of a fused base substitution domain comprising a single-chain variable fragment (scFv).

[0066] Figure 22 to Figure 24 is a graph showing the single base substitution efficiency using the complex for single base substitution in HEK cells, wherein, Fig. 22 The substitution rates of C to G, C to A or C to G at position 11C in the hEMX1 target nucleic acid sequence (SEQ ID NO: 1) targeted by GX20 sgRNA are shown. Fig.23 The C to G, C to A or C to G substitution rates at the 15C position in the hEMX1 target nucleic acid sequence (SEQ ID NO: 1) targeted by GX20 sgRNA are shown, and Fig.24 Shown are the C to G, C to A, or C to G substitution rates at the 16C position in the hEMX1 target nucleic acid sequence (SEQ ID NO: 1) targeted by the GX20 sgRNA.

[0067] Fig.25Three of the sgRNAs (SEQ ID NO: 2-SEQ ID NO: 20) (SEQ ID NO: 2, SEQ ID NO: 3, and SEQ ID NO: 19) are shown in the extended data of an article titled "Base Editing of A, T to G, C in Genomic DNA without DNA Cleavage" published in the scientific journal Nature Figure 2 middle.

[0068] Fig.26 To illustrate the use of Fig.25 A set of graphs showing the A to N base substitution rates of sgRNA1 (SEQ ID NO: 2) selected in HEK293T cells.

[0069] Fig. 27 To illustrate the use of Fig.25 A set of graphs showing the A to N base substitution rates of sgRNA2 (SEQ ID NO: 3) selected in HEK293T cells.

[0070] Fig.28 To illustrate the use of Fig.25 A set of graphs showing the A to N base substitution rates of sgRNA3 (SEQ ID NO: 19) selected in HEK293T cells.

[0071] Fig.29 The figure is a graph showing the C to N base substitution rate in PC9 cells using sgRNA1 (SEQ ID NO: 21) and sgRNA2 (SEQ ID NO: 22) each capable of complementary binding to a region of the EGFR gene.

[0072] Fig.30 A set of graphs showing the C to A, C to T or C to G base substitution rates in PC9 cells using sgRNA1 (SEQ ID NO: 21) and sgRNA2 (SEQ ID NO: 22) that can complementarily bind to a region of the EGFR gene.

[0073] Fig.31 To analyze the results of cells surviving culture in medium supplemented with osimertinib after random base substitution of cytosine. DETAILED DESCRIPTION

[0074] Unless otherwise defined, all technical terms and scientific terms used in this specification have the same meaning as those generally understood by those of ordinary skill in the art to which the present invention belongs. Although methods and materials similar or equivalent to the methods and materials described in this specification can be used in the practice or experiment of the present invention, suitable methods and materials are described below. All publications, patent applications, patents and other references mentioned in this specification are incorporated by reference in their entirety. In addition, the materials, methods and embodiments are illustrative and non-limiting only.

[0075] The present application provides a protein for single-base substitution (single-base substitution protein), which comprises (a) a CRISPR enzyme or a variant thereof, (b) a deaminase, and (c) a DNA glycosylase or a variant thereof.

[0076] The present application provides a composition for single base substitution, which comprises a protein for single base substitution and (d) a guide RNA.

[0077] Here, a protein for single base substitution can act simultaneously with a guide RNA to induce any Nitrogenous bases (nitrogenous base) Target nucleic acid sequence The substitution of cytosine (C) or adenine (A) contained in one or more nucleotides.

[0078] The combination of (a) a CRISPR enzyme and (d) a guide RNA according to the protein for single base substitution provided by the present application can specifically guide the protein for single base substitution to a target region containing a target nucleic acid sequence.

[0079] Here, the combination of (b) a deaminase and (c) a DNA glycosylase, which are proteins for single base substitution, can induce substitution of another base pair with one or more nucleotides in the target region.

[0080] Nitrogenous bases

[0081] As used herein, "nitrogenous base" refers to a purine base or a pyrimidine base, which is a constituent of a nucleotide or a nucleobase.

[0082] The nitrogenous base used herein may be simply referred to as a base, and the base may refer to adenine (A), thymine (T), uracil (U), hydroxypurine (H), guanine (G), or cytosine (C).

[0083] The abbreviations of bases in this application, such as A, T, C, G, U or H, refer to nitrogenous bases when used in the context of base substitution. In addition, when used in the context of SEQ ID NO, universal nucleic acid, or nucleotide sequence set in the specification, they refer to nucleic acids or nucleotides commonly used in the art.

[0084] In one example, "replacing adenine (A) with guanine (G)" may mean replacing the nitrogenous base from A to G in the same position or the same type of nucleotide on the nucleic acid sequence.

[0085] In one example, "replacing adenine (A) with thymine (T)" may mean replacing the nitrogenous base from A to T in the same position or the same type of nucleotide on a nucleic acid sequence.

[0086] In one example, "replacing adenine (A) with cytosine (C)" may mean replacing the nitrogenous base from A to C in the same position or the same type of nucleotide on the nucleic acid sequence.

[0087] In one example, "replacing cytosine (C) with guanine (G)" may mean that the nitrogenous base in the same position or the same type of nucleotide on the nucleic acid sequence is replaced from C to G.

[0088] In one example, “replacing cytosine (C) with thymine (T)” may mean that the nitrogenous base in the same position or the same type of nucleotide on the nucleic acid sequence is replaced from C to T.

[0089] In one example, "replacing cytosine (C) with adenine (A)" may mean that the nitrogenous base in the same position or the same type of nucleotide on the nucleic acid sequence is replaced from C to A.

[0090] In one example, "3'-ATGCAAA-5'" does not refer to a nitrogenous base, but represents a nucleic acid sequence or a nucleotide sequence commonly used in the art.

[0091] Base substitution or base modification

[0092] "Base substitution" as used herein means replacing the base of a nucleotide in a target gene with another base. More specifically, the base of a nucleotide in a target region is replaced with another base.

[0093] In one example, base substitution may mean changing adenine (A), guanine (G), cytosine (C), thymine (T), hydroxypurine, or uracil (U) to another base.

[0094] In an exemplary embodiment, the base substitution may mean replacing adenine with cytosine, thymine, uracil, hydroxypurine, or guanine.

[0095] In an exemplary embodiment, the base substitution may mean replacing cytosine with adenine, thymine, uracil, hydroxypurine, or guanine.

[0096] In an exemplary embodiment, the base substitution may mean replacing guanine with cytosine, thymine, uracil, hydroxypurine, or adenine.

[0097] In an exemplary embodiment, the base substitution may mean replacing thymine with adenine, cytosine, uracil, hydroxypurine, or guanine.

[0098] In an exemplary embodiment, the base substitution may mean replacing uracil with cytosine, thymine, adenine, hydroxypurine, or guanine.

[0099] In an exemplary embodiment, the base substitution may mean replacing hydroxypurine with adenine, thymine, uracil, or guanine.

[0100] However, the present invention is not limited thereto.

[0101] The “base substitution” used herein may be a concept including “base modification.” Here, modification may mean changing to another base by modification of the base structure, and base substitution may mean a change in the base type.

[0102] In one example, the base modification is to change the chemical structure of adenine (A), guanine (G), cytosine (C), thymine (T), hydroxypurine or uracil (U).

[0103] In an exemplary embodiment, the base modification may be to convert adenine to hypoxanthine by deamination of adenine.

[0104] In an exemplary embodiment, the base modification may be hypoxanthine to guanine.

[0105] In an exemplary embodiment, the base modification may be to convert cytosine to uracil by deamination of cytosine.

[0106] In an exemplary embodiment, the base modification may be uracil to thymine.

[0107] However, the present invention is not limited thereto.

[0108] Target nucleic acid sequence - the nucleic acid sequence that binds complementary to the guide RNA

[0109] The target nucleic acid sequence refers to a nucleotide sequence that can or is capable of complementary binding to a guide RNA that is a component of the composition for single base substitution.

[0110] In one example, when the intracellular double-stranded DNA is subjected to a single base substitution, the intracellular double-stranded DNA is composed of a first DNA strand and a second DNA strand. Here, any one of the first DNA strand of the double-stranded DNA and the second DNA strand complementary to the first DNA strand may contain a target nucleic acid sequence. The first DNA strand or the second DNA strand containing the target nucleic acid sequence may be bound to a guide RNA. Here, the nucleic acid sequence in the first DNA strand or the second DNA strand bound to the guide RNA corresponds to the target nucleic acid sequence.

[0111] In one example, when the intracellular double-stranded RNA is subjected to a single base substitution, the intracellular double-stranded RNA is composed of a first RNA strand and a second RNA strand. Any of the first RNA strand of the double-stranded RNA and the second RNA strand complementary to the first RNA strand may contain a target nucleic acid sequence. The first RNA strand or the second RNA strand containing the target nucleic acid sequence may be bound to the guide RNA. Here, the nucleic acid sequence of the first RNA strand or the second RNA strand bound to the guide RNA corresponds to the target nucleic acid sequence.

[0112] In one example, when double-stranded DNA or RNA in a cell undergoes single base substitution, the single-stranded DNA or RNA may include a target nucleic acid sequence. That is, the single-stranded DNA or RNA may bind to a guide RNA, and here, the nucleic acid sequence bound to the guide RNA corresponds to the target nucleic acid sequence.

[0113] In one example, the target nucleic acid sequence may be a nucleotide sequence of 10bp, 11bp, 12bp, 13bp, 14bp, 15bp, 16bp, 17bp, 18bp, 19bp, 20bp, 21bp, 22bp, 23bp, 24bp, 25bp, 26bp, 27bp, 28bp, 29bp or 30bp or more.

[0114] Target region – the region containing the nucleotide to be substituted

[0115] The target region is a region comprising nucleotides in which base substitution is induced by the protein for single base substitution.

[0116] The target region is a region containing a target nucleic acid sequence to which the guide RNA binds. Here, the target nucleic acid sequence may contain nucleotides that induce base substitution by a protein for single base substitution.

[0117] The target region includes a nucleic acid sequence in the second DNA strand that is complementary to the target nucleic acid sequence in the first DNA strand, and the first DNA strand is complementary to the guide RNA. Here, the nucleic acid sequence in the second DNA strand may include nucleotides that induce base substitution by a protein for single base substitution.

[0118] In one example, the strand containing the target nucleic acid sequence in the double-stranded DNA or RNA may be referred to as the first strand, and the strand not containing the target nucleic acid sequence may be referred to as the second strand. Here, the target region may include the target nucleic acid sequence in the first strand that is complementary to the guide RNA and the nucleic acid sequence in the second strand that is complementary to the target nucleic acid sequence.

[0119] In one example, the strand containing the target nucleic acid sequence in the double-stranded DNA or RNA may be referred to as the second strand, and the strand not containing the target nucleic acid sequence may be referred to as the first strand. Here, the target region may include the target nucleic acid sequence in the second strand that is complementary to the guide RNA and the nucleic acid sequence in the first strand that is complementary to the target nucleic acid sequence.

[0120] The protein for single base substitution can induce base substitution of one or more nucleotides in the target region.

[0121] In one example, when the guide RNA is complementary to a target nucleic acid sequence contained in a first DNA strand of a double-stranded DNA, the protein for single base substitution can replace (i) one or more nucleotide bases in the target nucleic acid sequence, or (ii) one or more nucleotide bases in a nucleic acid sequence complementary to the target nucleic acid sequence in the second strand of the double-stranded DNA.

[0122] In one example, when the guide RNA is complementary to a target nucleic acid sequence contained in a first RNA strand of a double-stranded RNA, the protein for single base substitution can replace (i) one or more nucleotide bases in the target nucleic acid sequence, or (ii) one or more nucleotide bases in a nucleic acid sequence complementary to the target nucleic acid sequence in the second strand of the double-stranded RNA.

[0123] In an exemplary embodiment, the cytosine of one or more nucleotides in the target nucleic acid region may be replaced with guanine, thymine, uracil, hypoxanthine, or adenine.

[0124] In an exemplary embodiment, adenine of one or more nucleotides in the target nucleic acid sequence may be replaced with guanine, thymine, uracil, hypoxanthine, or cytosine.

[0125] The target gene used herein refers to a gene comprising a target region and a target nucleic acid sequence. In addition, the target gene in this specification refers to a gene in which the cytosine of one or more nucleotides in the target region is replaced with an arbitrary base by a protein for single base replacement.

[0126] Technical features - substitution with any base

[0127] The protein for single base substitution provided in the present application comprises (i) deaminase and (ii) DNA glycosylase as essential components.

[0128] The combination of the first component of the protein for single base substitution, which is a deaminase, and the second component of the protein for single base substitution, which is a DNA glycosylase, can induce the substitution of a base of a nucleotide in an arbitrary base pair nucleic acid sequence.

[0129] Here, the base substitution by deaminase and DNA glycosylase may be caused by the following two steps: (i) base deamination and / or (ii) cleavage or repair by DNA glycosylase, which are performed sequentially or simultaneously.

[0130] The first step: deamination of bases

[0131] Deamination refers to a biochemical reaction involving the cleavage of an amino group. In one example, in the case of DNA, deamination may refer to the amino group of a base (which is a constituent of a nucleotide) being changed to a hydroxyl or keto group.

[0132] In an exemplary embodiment, the deaminase may be a cytidine deaminase. The cytidine deaminase may provide uracil by deamination of cytosine. The cytidine deaminase may provide uracil by modification of cytosine.

[0133]

[0134] In an exemplary embodiment, the deaminase of the protein for single base substitution may be adenosine deaminase. Adenosine deaminase can provide hypoxanthine by deamination of adenine. Adenosine deaminase can provide hydroxypurine (hypoxanthine) by modification of adenine.

[0135]

[0136] In an exemplary embodiment, the deaminase may be guanine deaminase. Guanine deaminase may provide xanthine by deamination of guanine. Guanine deaminase may provide xanthine by modification of guanine.

[0137]

[0138] Second process: DNA glycosylation

[0139] DNA glycosylase is an enzyme involved in base excision repair (BER), and BER is a mechanism for removing and replacing damaged bases of DNA. DNA glycosylase catalyzes the first step of this mechanism by hydrolyzing the N-glycosidic bond between the base and deoxyribose of DNA. DNA glycosylase removes damaged nitrogenous bases while keeping the sugar-phosphate backbone intact. As a result, AP sites are generated, particularly purine sites or pyrimidinic sites. Afterwards, replacement with any base can be performed by AP endonuclease, terminal processing enzyme, DNA polymerase, flap endonuclease and / or DNA ligase.

[0140] In an exemplary embodiment, the DNA glycosylase may be uracil DNA glycosylase. Uracil DNA glycosylase hydrolyzes the N-glycosidic bond between uracil and deoxyribose in DNA. Uracil DNA glycosylase hydrolyzes the N-glycosidic bond between uracil and deoxyribose in a nucleotide containing uracil. Here, a cytidine deaminase acting on a nucleotide containing cytosine can be used to provide a nucleotide containing uracil by deamination.

[0141] In an exemplary embodiment, the DNA glycosylase may be an alkyladenine DNA glycosylase. Alkyladenine DNA glycosylase hydrolyzes the N-glycosidic bond between hydroxypurine (hypoxanthine) and deoxyribose in DNA. Alkyladenine DNA glycosylase hydrolyzes the N-glycosidic bond between hydroxypurine and deoxyribose in nucleotides containing hydroxypurine. Here, adenosine deaminase acting on nucleotides containing adenine can be used to provide nucleotides containing hydroxypurine by deamination.

[0142] Results of the first and second processes

[0143] One or more adenines or cytosines in a target region can be replaced with any base using the protein for single base substitution provided in the present application.

[0144] In one example, the deaminase of the protein for single base substitution can be adenosine deaminase, and the DNA glycosylase of the protein for single base substitution can be alkyladenine-DNA glycosylase or its variant. Here, the fusion protein for single base substitution (single base substitution fusion protein) can induce the substitution of any base (guanine, thymine or cytosine) to adenine in one or more nucleotides in the target nucleic acid sequence.

[0145] In an exemplary embodiment, substitution of cytosine for adenine in one or more nucleotides in a target region can be induced by a protein for single base substitution comprising: (a) a CRISPR enzyme or a variant thereof; (b) an adenosine deaminase; and (c) an alkyladenine DNA glycosylase.

[0146]

[0147] In an exemplary embodiment, substitution of thymine for adenine in one or more nucleotides in a target region can be induced by a protein for single base substitution comprising: (a) a CRISPR enzyme or a variant thereof; (b) an adenosine deaminase; and (c) an alkyladenine DNA glycosylase.

[0148]

[0149] In an exemplary embodiment, substitution of guanine for adenine in one or more nucleotides in a target region can be induced by a protein for single base substitution comprising: (a) a CRISPR enzyme or a variant thereof; (b) an adenosine deaminase; and (c) an alkyladenine DNA glycosylase.

[0150]

[0151] In one example, the deaminase of the protein for single base substitution can be cytidine deaminase, and its DNA glycosylase can be uracil DNA glycosylase or its variant. Here, the fusion protein for single base substitution can induce the substitution of cytosine of one or more nucleotides in any base pair target nucleic acid sequence.

[0152] In an exemplary embodiment, substitution of adenine for cytosine in one or more nucleotides in a target region can be induced by a protein for single base substitution comprising: (a) a CRISPR enzyme or a variant thereof; (b) a cytidine deaminase; and (c) a uracil DNA glycosylase.

[0153]

[0154] In an exemplary embodiment, substitution of thymine for cytosine in one or more nucleotides in a target region can be induced by a protein for single base substitution comprising: (a) a CRISPR enzyme or a variant thereof; (b) a cytidine deaminase; and (c) a uracil DNA glycosylase.

[0155]

[0156] In an exemplary embodiment, substitution of guanine for cytosine in one or more nucleotides in a target region can be induced by a protein for single base substitution comprising: (a) a CRISPR enzyme or a variant thereof; (b) a cytidine deaminase; and (c) a uracil DNA glycosylase.

[0157]

[0158] Hereinafter, the present invention will be described in detail.

[0159] One aspect of the present invention disclosed in the specification is a protein for single base substitution.

[0160] The protein used for single base substitution is a protein, polypeptide or peptide that can induce or produce single base substitution.

[0161] Limitations of traditional base editors

[0162] Traditional base editors are used in the form of fusion, connection or connection of deaminase, CRISPR enzyme and DNA glycosylase inhibitor. As a representative example, a base editor connected to a cytidine deaminase (e.g., rAPOBEC), nCas9 and uracil DNA glycosylase from a rat is used to replace the cytosine base with thymine. In addition, adenine (A) is replaced with guanine (G) using adenosine deaminase instead of cytidine deaminase.

[0163] Importantly, conventional base editors can treat diseases caused by point mutations (e.g., genetic disorders) by correcting point mutation sites in genes. However, conventional base editors have limitations, namely, they can only change cytosine (C) to a specific base, thymine (T), or adenine (A) to a specific base, guanine (G), by removing the amino group (-NH2) or replacing the amino group with a keto group using a DNA glycosylase inhibitor.

[0164] Utility of proteins for single base substitution

[0165] The use of traditional base editors has the limitation that the probability of the amino acid expressed by the replaced base being of a different type is low. Most diseases or disorders are not caused by point mutations, but are likely to be caused by structural or functional abnormalities at the peptide, polypeptide or protein level (rather than the nucleotide level). After all, since traditional base editors can only change adenine and cytosine into specific bases, the probability of changing the structure of peptides, polypeptides or proteins is significantly reduced.

[0166] The use of the protein for single base replacement provided in this specification can overcome the limitations of the prior art. The protein for single base replacement provided in this application has a novel combination consisting of: (a) editor protein, (b) deaminase, and (c) DNA glycosylase. In other words, the protein for single base replacement provided in this application has the advantage of replacing adenine (A), guanine (G), thymine (T) or cytosine (C) with any base (A, T, C, G, U or H).

[0167] Furthermore, the protein for single base substitution having novel components and novel combinations thereof has the advantage of simultaneously substituting one or more bases present in the target nucleic acid sequence.

[0168] Therefore, the protein for single base substitution provided by the present application can provide a "mutation" that randomly substitutes various bases. Peptides, polypeptides or proteins with various structures can be expressed from the mutated gene.

[0169] Due to the above technical effects, the protein for single base substitution provided by the present application can be used for epitope screening, drug resistance gene or protein screening, drug sensitization screening and / or virus resistance gene or protein screening.

[0170] The protein for single base substitution provided in the present application can induce the substitution of bases in the target region of any base pair target gene by being used together with a guide RNA.

[0171] [The first component of the protein for single base substitution - deaminase]

[0172] Deaminase is an enzyme involved in removing an amino group, and includes enzymes that change an amino group of a compound into a hydroxyl group or a keto group. There is an enzyme that catalyzes the amino group bound to each of cytosine, adenine, guanine, adenosine, cytidine, AMP, and ADP, etc., and such enzymes are generally contained in animal tissues.

[0173] The deaminase used herein may be referred to as a base substitution domain. Here, a base substitution domain refers to a peptide, polypeptide, domain or protein that participates in substituting a base of one or more nucleotides in a target gene with any other base.

[0174] The deaminase of the present application may be cytidine deaminase.

[0175] Here, cytidine deaminase refers to any enzyme having the activity of removing the amino (-NH2) group of cytosine, cytidine or deoxycytidine. Cytidine deaminase in this specification is used as a concept including cytosine deaminase. Cytidine deaminase in this specification can be used interchangeably with cytosine deaminase.

[0176] Cytidine deaminase converts cytosine to uracil.

[0177] Cytidine deaminase converts cytidine to uridine.

[0178] Cytidine deaminase converts deoxycytidine to deoxyuridine.

[0179] Cytidine deaminase refers to an enzyme having an activity of converting the base cytosine present in a nucleotide (e.g., cytosine present in double-stranded DNA or RNA) into uracil (C to U conversion or C to U editing), and converts cytosine in a chain having a PAM sequence of a target site sequence (target nucleic acid sequence) into uracil.

[0180] In one example, the cytidine deaminase may be derived from: a prokaryotic organism, such as Escherichia coli; or a mammal, such as a primate (such as a human and a monkey), and a rodent (such as a rat and a mouse), but the present invention is not limited thereto. For example, the cytidine deaminase may be APOBEC ("apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like") or one or more selected from the family of activation-induced cytidine deaminases (AID).

[0181] The cytidine deaminase may be APOBEC1, APOBEC2, APOBEC3B, APOBEC3C, APOBEC3D, APOBEC3F, APOBEC3G, APOBEC3H, APOBEC4, AID, or CDA, but the present invention is not limited thereto.

[0182] For example, the cytidine deaminase may be human APOBEC1, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI Accession No. NM_005889, NM_001304566 or NM_001644. Alternatively, the cytidine deaminase may be human APOBEC1, such as a protein or polypeptide represented by NCBI Accession No. NP_001291495, NP_001635 or NP_005880.

[0183] For example, the cytidine deaminase may be mouse APOBEC1, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI Accession No. NM_001127863 or NM_112436. Alternatively, the cytidine deaminase may be mouse APOBEC1, such as a protein or polypeptide represented by NCBI Accession No. NP_001127863 or NP_112436.

[0184] For example, the cytidine deaminase may be a human AID, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI Accession No. NM_020661 or NM_001330343. Alternatively, the cytidine deaminase may be a human AID, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI Accession No. NP_001317272 or NP_065712.

[0185] Examples of cytidine deaminases are listed below:

[0186] APOBEC1: a gene encoding human APOBEC1 (e.g., NCBI Accession Nos. NP_001291495, NP_001635, NP_005880), such as the APOBEC1 gene represented by NCBI Accession Nos. NM_005889 or NM_001304566; or a gene encoding mouse APOBEC1 (e.g., NCBI Accession Nos. NP_001127863, NP_112436), such as the APOBEC1 gene represented by NCBI Accession Nos. NM_001127863 or NM_112436.

[0187] APOBEC2: a gene encoding human APOBEC2 (e.g., NCBI Accession No. NP_006780), such as the APOBEC2 gene represented by NCBI Accession No. NM_006789; or a gene encoding mouse APOBEC2 (e.g., NCBI Accession No. NP_033824), such as the APOBEC2 gene represented by NCBI Accession No. NM_009694.

[0188] APOBEC3B: a gene encoding human APOBEC3B (e.g., NCBI Accession No. NP_001257340 or NP_004891), such as the APOBEC3B gene represented by NCBI Accession No. NM_004900 or NM_001270411; or a gene encoding mouse APOBEC3B (e.g., NCBI Accession No. NP_001153887, NP_001333970 or NP_084531), such as the APOBEC3B gene represented by NCBI Accession No. NM_001160415, NM_030255 or NM_001347041.

[0189] APOBE3C: a gene encoding human APOBEC3C (eg, NCBI Accession No. NP_055323), such as the APOBEC3C gene represented by NCBI Accession No. NM_014508.

[0190] APOBEC3D: a gene encoding human APOBEC3D (eg, NCBI Accession No. NP_689639 or NP_0013570710), such as the APOBEC3D gene represented by NCBI Accession No. NM_152426 or NM_001363781.

[0191] APOBEC3F: A gene encoding human APOBEC3F (eg, NCBI Accession No. NP_001006667 or NP_660341), such as the APOBEC3F gene represented by NCBI Accession No. NM_001006666 or NM_145298.

[0192] APOBEC3G: A gene encoding human APOBEC3G (eg, NCBI Accession No. NP_068594, NP_001336365, NP_001336366, or NP_001336367), such as the APOBEC3G gene represented by NCBI Accession No. NM_021822.

[0193] APOBEC3H: A gene encoding human APOBEC3H (eg, NCBI Accession No. NP_001159474, NP_001159475, NP_001159476, or NP_861438), such as an APOBEC3H gene represented by NCBI Accession No. NM_001166002, NM_001166003, NM_001166004, or NM_181773.

[0194] APOBEC4: a gene encoding human APOBEC4 (eg, NCBI Accession No. NP_982279), such as the APOBEC4 gene represented by NCBI Accession No. NM_203454; or a gene encoding mouse APOBEC4, such as the APOBEC4 gene represented by NCBI Accession No. NM_001081197.

[0195] Cytidine deaminase can be expressed by activation-induced cytidine deaminase (AID) gene. For example, the AID gene can be selected from the group consisting of the following genes, but the present invention is not limited thereto: a gene encoding a human AID gene (e.g., NP_001317272, NP_065712), such as an AID gene represented by NCBI accession number NM_020661 or NM_001330343; or a gene encoding a mouse AID gene (e.g., NP_03377512), such as an AID gene represented by NCBI accession number NM_009645.

[0196] Cytidine deaminase can be encoded by a CDA gene. For example, the CDA gene can be selected from the group consisting of the following genes, but the present invention is not limited thereto: a gene encoding human CDA (e.g., NCBI Accession No. NP_001776), such as the CDA gene represented by NCBI Accession No. NM_001785; or a gene encoding mouse CDA (e.g., NCBI Accession No. NP_082452), such as the CDA gene represented by NCBI Accession No. NM_028176.

[0197] The cytidine deaminase may be a cytidine deaminase variant.

[0198] A cytidine deaminase variant may be an enzyme having a higher cytidine deaminase activity than a wild-type cytidine deaminase.Cytidine deaminase activity is understood to include the deamination of cytosine or one of its analogs.

[0199] For example, a cytidine deaminase variant may be an enzyme in which one or more amino acid sequences in the cytidine deaminase are modified.

[0200] The modification of the amino acid sequence may be any one selected from substitution, deletion and insertion.

[0201] The deaminase of the present application may be adenosine deaminase.

[0202] Adenosine deaminase is any enzyme having the activity of removing an amino (-NH2) group of adenine, adenosine or deoxyadenosine or replacing the amino group with a keto (=O) group. Adenosine deaminase in this specification is used as a concept including adenine deaminase. Adenosine deaminase in this specification is used as a concept including adenine deaminase.

[0203] Adenosine deaminase converts adenine to hydroxypurine (hypoxanthine).

[0204] Adenosine deaminase converts adenosine to inosine.

[0205] Adenosine deaminase converts deoxyadenosine to deoxyinosine.

[0206] Adenosine deaminase can be derived from: prokaryotes, such as Escherichia coli; or mammals, such as primates (such as humans and monkeys), and rodents (such as rats and mice), but the present invention is not limited thereto. For example, adenosine deaminase can be tRNA-specific adenosine deaminase (TadA) or one or more selected from the adenosine deaminase (ADA) family.

[0207] The adenosine deaminase may be TadA, Tad2p, ADA, ADA1, ADA2, ADAR2, ADAT2 or ADAT3, but the present invention is not limited thereto.

[0208] For example, the adenosine deaminase may be Escherichia coli TadA, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI Accession No. NC_000913.3, etc. Alternatively, the adenosine deaminase may be Escherichia coli TadA, such as a protein or polypeptide represented by NCBI Accession No. NP_417054.2, etc.

[0209] For example, the adenosine deaminase may be human ADA, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI accession number NM_000022, NM_001322050 or NM_001322051, etc. Alternatively, the adenosine deaminase may be human ADA, such as a protein or polypeptide represented by NCBI accession number NP_000013, NP_001308979 or NP_001308980, etc.

[0210] For example, the adenosine deaminase may be mouse ADA, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI accession number NM_001272052 or NM_007398, etc. Alternatively, the adenosine deaminase may be mouse ADA, such as a protein or polypeptide represented by NCBI accession number NP_001258981 or NP_031424, etc.

[0211] For example, the adenosine deaminase may be human ADAR2, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI accession number NM_001033049, NM_001112, NM_001160230, NM_015833 or NM_015834, etc. Alternatively, the adenosine deaminase may be human ADAR2, such as a protein or polypeptide represented by NCBI accession number NP_001103, NP_001153702, NP_001333616, NP_001333617 or NP_056648, etc.

[0212] For example, the adenosine deaminase may be mouse ADAR2, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI accession number NM_001024837, NM_001024838, NM_001024839, NM_001024840 or NM_130895, etc. Alternatively, the adenosine deaminase may be mouse ADAR2, such as a protein or polypeptide represented by NCBI accession number NP_001020008, NP_570965 or NP_001020009, etc.

[0213] For example, the adenosine deaminase may be human ADAT2, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI accession number NM_182503.3 or NM_001286259.1, etc. Alternatively, the adenosine deaminase may be human ADAT2, such as a protein or polypeptide represented by NCBI accession number NP_001273188.1 or NP_872309.2, etc.

[0214] The adenosine deaminase may be any one of an adA variant, an ADAR2 variant, and an ADAT2 variant, but the present invention is not limited thereto.

[0215] For example, the ADAR2 variant may be one or more selected from the group consisting of the following genes, but the present invention is not limited thereto. The gene may be a gene encoding human ADAR2, such as the CDA gene represented by NCBI accession number NM_001282225, NM_001282226, NM_001282227, NM_001282228, NM_001282229, NM_017424 or NM_177405.

[0216] The adenosine deaminase may be an adenosine deaminase variant.

[0217] An adenosine deaminase variant may be an enzyme having higher adenosine deaminase activity than wild-type adenosine deaminase.

[0218] For example, an adenosine deaminase variant may be an enzyme in which one or more amino acid sequences in the adenosine deaminase are altered.

[0219] The adenosine deaminase may be an adenosine deaminase variant.

[0220] The adenosine deaminase variant may be an enzyme having a higher adenosine deaminase activity than the wild-type adenosine deaminase, wherein the adenosine deaminase activity may include removing an amino (-NH2) group of adenine, adenosine, deoxyadenosine or an analog thereof or replacing the amino (-NH2) group with a keto (=O) group, but the present invention is not limited thereto.

[0221] The adenosine deaminase variant may be an enzyme in which one or more amino acid sequences selected from the amino acid sequence constituting the wild-type adenosine deaminase are modified.

[0222] The modification of the amino acid sequence may be any one selected from the group consisting of replacement, deletion and insertion of one or more amino acids.

[0223] The adenosine deaminase variant may be a TadA variant, a Tad2p variant, an ADA variant, an ADA1 variant, an ADA2 variant, an ADAR2 variant, an ADAT2 variant, or an ADAT3 variant, but the present invention is not limited thereto.

[0224] For example, adenosine deaminase can be a TadA variant. For example, TadA variants can be ABE0.1, ABE1.1, ABE1.2, ABE2.1, ABE2.9, ABE2.10, ABE3.1, ABE4.3, ABE5.1, ABE5.3, ABE6.3, ABE6.4, ABE7.4, ABE7.8, ABE7.9 or ABE7.10, and specific details about TadA variants are described in detail in an article entitled "Base editing of A, T to C, G in genomic DNA without DNA cleavage" (Nicole M. Gaudelli et al., (2017) Nature, 551, 464-471), so reference can be made to the corresponding files.

[0225] The adenosine deaminase may be a fused adenosine deaminase.

[0226] The deaminases provided in the present application can be provided in a fusion form, wherein, for example, one or more functional domains are linked to a cytidine deaminase or an adenosine deaminase.

[0227] Here, the deaminase and the functional domains may be linked or fused so that each function is expressed.

[0228] The functional domain may be a domain having methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for separating and purifying proteins (including peptides), but the present invention is not limited thereto.

[0229] The functional domain may be a tag or reporter gene for isolation and purification of proteins (including peptides).

[0230] Here, the label may include any of the following: histidine (His) label, V5 label, FLAG label, influenza virus hemagglutinin (HA) label, Myc label, VSV-G label and thioredoxin (Trx) label. Here, the reporter gene may include any of the following autofluorescent proteins: for example, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP). However, the present invention is not limited thereto.

[0231] The functional domain can be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

[0232] Here, one or more NLSs may be included at or near the amino terminus of the CRISPR enzyme, at or near the carboxyl terminus of the CRISPR enzyme, or a combination thereof. The NLS may be derived from the following NLS sequences, but the present invention is not limited thereto: NLS of SV40 virus large T antigen having an amino acid sequence of PKKKRKV (SEQ ID NO: 23); NLS from nucleoplasmin (e.g., nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 24)); c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 25) or RQRRNELKRSP (SEQ ID NO: 26); hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 27); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 28) from the IBB domain of importin-α; NO:28); the sequence of myoma T protein VSRKRPRP (SEQ ID NO:29) and PPKKARED (SEQ ID NO:30); the sequence of human p53 PQPKKKPL (SEQ ID NO:31); the sequence of mouse c-abl IV SALIKKKKKMAP (SEQ ID NO:32); the sequences of influenza virus NS1 DRLRR (SEQ ID NO:33) and PKQKKRK (SEQ ID NO:34); the sequence of infectious virus delta antigen RKLKKKIKKL (SEQ ID NO:35); the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO:36); the sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO:37); and the sequence of the receptor for human steroid hormone, glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO:38).

[0233] A functional domain may be a binding domain capable of forming a complex with another domain, peptide, polypeptide or protein.

[0234] The binding domain may be one of a FRB domain and a FKBP dimerization domain; an intein; one of an ERT domain and a VPR domain; one of a GCN4 peptide and a single-chain variable fragment (scFv); or a domain that forms a heterodimer.

[0235] The binding domain may be a scFv, wherein the scFv may be paired with a GCN4 peptide and may specifically bind or connect to GCN4.

[0236] In one example, a first fusion protein in which a scFv functional domain is linked to an adenosine deaminase can be combined with a peptide, polypeptide, protein, or a second fusion protein comprising a GCN4 peptide.

[0237] [The second component of the protein for single base substitution - DNA glycosylase]

[0238] DNA glycosylases are enzymes involved in base excision repair (BER), a mechanism that removes and replaces damaged bases of DNA. DNA glycosylases catalyze the first step of this mechanism by hydrolyzing the N-glycosidic bond between the base and the deoxyribose in DNA. DNA glycosylases remove the damaged nitrogenous base while leaving the sugar-phosphate backbone intact.

[0239] The glycosylase of the present application may be uracil DNA glycosylase.

[0240] Uracil DNA glycosylase is an enzyme that acts to prevent DNA mutation by removing uracil (U) present in DNA, and may be one or more selected from all enzymes that act to initiate a base excision repair (BER) pathway by destroying the N-glycosidic bond of uracil.

[0241] The glycosylase may be uracil DNA glycosylase (UDG or UNG). Uracil DNA glycosylase (UNG) may be selected from the group consisting of the following genes, but the present invention is not limited thereto: genes encoding human UNG (e.g., NCBI accession numbers NP_003353 and NP_550433), such as the UNG genes represented by NCBI accession numbers NM_080911 and NM_003362; or genes encoding mouse UNG (e.g., NCBI accession numbers NP_001035781 and NP_035807), such as the UNG genes represented by NCBI accession numbers NM_001040691 and NM_011677; or genes encoding Escherichia coli UNG (e.g., NCBI accession number A DX49788.1, ACT28166.1, EFN36865.1, BAA10923.1, ACA76764.1, ACX38762.1, EFU59768.A, EFU53885.A, EFJ57281.1, EFU47398.1, EFK71412.1, EFJ92376.1, EFJ79936.1, EFO59084.1, EFK47562.1, KXH01728.1, ESE25979.1, ESD99489.1, ESD73882.1, and ESD69341.1).

[0242] The DNA glycosylase may be a uracil DNA glycosylase variant. The uracil DNA glycosylase variant may be an enzyme having higher DNA glycosylase activity than wild-type uracil DNA glycosylase.

[0243] For example, a uracil DNA glycosylase variant may be an enzyme in which one or more amino acid sequences of a wild-type uracil DNA glycosylase are modified. Here, the modification of the amino acid sequence may be substitution, deletion, insertion, or a combination thereof of at least one or more amino acids.

[0244] The glycosylase may be a fused uracil DNA glycosylase.

[0245] The glycosylase of the present application may be alkyladenine DNA glycosylase (AAG).

[0246] Alkyladenine DNA glycosylase is an enzyme that acts to prevent DNA mutation by removing alkylated or deaminated bases present in DNA, and may be one or more selected from all enzymes that act to initiate the base excision repair (BER) pathway by catalyzing the hydrolysis of the N-glycosidic bond of the alkylated or deaminated base.

[0247] The DNA glycosylase may be alkyladenine DNA glycosylase (AAG) or a variant thereof.

[0248] For example, alkyladenine DNA glycosylase (AAG) can be human AAG, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI accession number NM_002434, NM_001015052 or NM_001015054, etc. Alternatively, alkyladenine DNA glycosylase (AAG) can be human AAG, such as a protein or polypeptide represented by NCBI accession number NP_001015052, NP_001015054 or NP_002425, etc.

[0249] For example, alkyladenine DNA glycosylase (AAG) can be mouse AAG, such as a protein or polypeptide expressed by a gene or mRNA represented by NCBI accession number NM_010822, etc. Alternatively, alkyladenine DNA glycosylase (AAG) can be human AAG, such as a protein or polypeptide represented by NCBI accession number NP_034952, etc.

[0250] The DNA glycosylase may be an alkyladenine DNA glycosylase variant. The alkyladenine DNA glycosylase variant may be an enzyme having higher DNA glycosylase activity than wild-type alkyladenine DNA glycosylase.

[0251] For example, the alkyladenine DNA glycosylase variant may be an enzyme in which one or more amino acid sequences of the wild-type alkyladenine DNA glycosylase are modified, wherein the modification of the amino acid sequence may be substitution, deletion, insertion or a combination thereof of at least one amino acid.

[0252] The glycosylase may be a fused alkyladenine DNA glycosylase.

[0253] The present application may provide a fused uracil DNA glycosylase or a fused alkyladenine DNA glycosylase, wherein one or more functional domains are connected to the uracil DNA glycosylase or the alkyladenine DNA glycosylase. The uracil DNA glycosylase or the alkyladenine DNA glycosylase may be connected or fused to each functional domain, so that each function is expressed.

[0254] The functional domain may be a domain having methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for separating and purifying proteins (including peptides), but the present invention is not limited thereto.

[0255] Here, the functional domain may be a tag or reporter gene for isolating and purifying proteins (including peptides).

[0256] Here, the label may include any of the following: histidine (His) label, V5 label, FLAG label, influenza virus hemagglutinin (HA) label, Myc label, VSV-G label and thioredoxin (Trx) label. Here, the reporter gene may include any of the following autofluorescent proteins: for example, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP). However, the present invention is not limited thereto.

[0257] The functional domain can be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

[0258] Here, one or more NLSs may be included at or near the amino terminus of the CRISPR enzyme, at or near the carboxyl terminus of the CRISPR enzyme, or a combination thereof. The NLS may be derived from the following NLS sequences, but the present invention is not limited thereto: the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 23); the NLS from the nucleoplasmic protein (e.g., the nucleoplasmic protein bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 24)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 25) or RQRRNELKRSP (SEQ ID NO: 26); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 27); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 28) from the IBB domain of importin-α; the sequence VSRKRPRP (SEQ ID NO: 29) of the myoma T protein. NO: 29) and PPKKARED (SEQ ID NO: 30); the sequence of human p53 PQPKKKPL (SEQ ID NO: 31); the sequence of mouse c-abl IV SALIKKKKKMAP (SEQ ID NO: 32); the sequences DRLRR (SEQ ID NO: 33) and PKQKKRK (SEQ ID NO: 34) of influenza virus NS1; the sequence of infectious virus delta antigen RKLKKKIKKL (SEQ ID NO: 35); the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO: 36); the sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 37); and the sequence of the receptor for human steroid hormone, glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO: 38) One or more.

[0259] A functional domain may be a binding domain capable of forming a complex with another domain, peptide, polypeptide or protein.

[0260] The binding domain may be one of a FRB domain and a FKBP dimerization domain; an intein; one of an ERT domain and a VPR domain; one of a GCN4 peptide and a single-chain variable fragment (scFv); or a domain that forms a heterodimer.

[0261] The binding domain may be a scFv, wherein the scFv may be paired with a GCN4 peptide and may specifically bind or connect to GCN4.

[0262] In one example, a first fusion protein in which a scFv functional domain is linked to uracil DNA glycosylase or alkyladenine DNA glycosylase can be combined with a peptide, polypeptide, protein, or a second fusion protein comprising a GCN4 peptide.

[0263] [The third component of the protein for single base substitution - CRISPR enzyme]

[0264] The protein for single base substitution provided in the present application comprises a CRISPR enzyme or a CRISPR system comprising the CRISPR enzyme. The CRISPR enzyme in this specification may be referred to as a CRISPR protein.

[0265] The CRISPR system is a system that can introduce artificial mutations by targeting a target nucleic acid sequence near a protospacer adjacent motif (PAM) sequence on genomic DNA. Specifically, the guide RNA and the Cas protein bind to (or interact with) each other to form a guide RNA-Cas protein complex, and can induce mutations, insertions and deletions (indels) on genomic DNA by shearing of the target DNA sequence.

[0266] For a more detailed description of guide RNA, Cas protein, and guide RNA-Cas protein complex, reference may be made to Korean Patent Publication No. 10-2017-0126636.

[0267] Cas protein is used in this specification as a concept including all variants that can cooperate with guide RNA as active nuclease or nickase in addition to wild-type protein. Active nuclease or nickase can cleave target nucleic acid sequence and can be used to manipulate or modify nucleic acid sequence. In addition, inactive variants can be used to regulate transcription or separate targeted DNA.

[0268] The CRISPR protein in the present application can be Cas9 or Cpf1 derived from a variety of microorganisms, such as Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Campylobacter jejuni, Nocardiopsis dassonvillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacteriumsibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaerawatsonii, Cyanothece sp., Microcystis aeruginosa, Synechococcus sp.), Acetohalobium arabaticum, Ammonifex degensii, Caldicelulosiruptor bescii, Candidatus Desulforudis, Clostridium botulinum, Clostridium difficile, Finegoldia magna, Natranaerobius thermophilus, Pelotomaculum thermopropionicum, Acidithiobacillus caldus, Acidithiobacillus ferrooxidans, Allochromatium vinosum, Marinobacter sp., Nitrosococcus halophilus, Nitrosococcus watsonii, Pseudoalteromonas shaloplanktis, Ktedonobacter racemifer, Methanohalobium evestigatum, Anabaena variabilis, Nodularia spumigena, Nostoc sp., Arthrospira maxima, Arthrospira platensis, Arthrospira sp., Lyngbya sp., Microcoleus chthonoplastes, Oscillatoria sp., Petrotoga mobilis, Thermosipho africanus, or Acaryochloris marina.

[0269] The CRISPR enzyme may be a fully active CRISPR enzyme.

[0270] In one embodiment, a fully active CRISPR enzyme variant may be a Cas9 protein variant derived from SpCas9 Streptococcus pyogenes. Examples of variants are listed below:

[0271] A variant may be an enzyme in which one or more of the following amino acids are substituted:

[0272] E108G, E217A, A262T, R324L, S409I, E480K, E543D, M694I, E1219V, E480K, E543D, E12 19V, A262T, S409I, E480K, E543D, E1219V, A262T, S409I, E480K, E543D, M694I, E1219V , E108G, E217A, A262T, S409I, E480K, E543D, M694I, E1219V, A262T, R324L, S409I, E480K, E543D, M694I, E1219V, L111R, D1135V, G1218R, E1219F, A1322R, R1335V and T1337R. Among them, the CRISPR enzyme variants can recognize different PAM sequences, expand the target nucleic acid sequence in the genome by shortening the length of the PAM sequence that can be recognized by the CRISPR enzyme, and improve the nucleic acid access ability.

[0273] As a specific example, in the case of SpCas9, when SpCas9 is mutated (e.g., L111R, D1135V, G1218R, E1219F, A1322R, R1335V, and T1337R), the SpCas9 variant can operate by recognizing a PAM sequence of only "NG" (the initially recognized PAM sequence is "NGG") (N is one of A, T, C, and G).

[0274] Among them, SpCas9 variants (L111R, D1135V, G1218R, E1219F, A1322R, R1335V and T1337R) can be used interchangeably with "Nureki Cas9" ("CRISPR-Cas9 nuclease with expanded targeting space", Masu et al., (2018) Science 361, 1259-1262).

[0275] The CRISPR enzyme can be a nickase.

[0276] For example, when the type II CRISPR enzyme is a wild-type SpCas9, the nickase may be a SpCas9 variant in which the nuclease activity of the HNH domain is inactivated due to a mutation of histidine 840 in the amino acid sequence of the wild-type SpCas9 to alanine. Here, since the generated nickase (i.e., the SpCas9 variant) has the nuclease activity generated by the RuvC domain, the non-complementary strand of the target gene or nucleic acid (i.e., the strand that does not complementarily bind to the gRNA) can be cleaved.

[0277] In another example, when the type II CRISPR enzyme is wild-type CjCas9, the nickase may be a CjCas9 variant in which the nuclease activity of the HNH domain is inactivated due to a mutation of histidine 559 in the amino acid sequence of the wild-type CjCas9 to alanine. Here, since the generated nickase (i.e., CjCas9 variant) has nuclease activity from the RuvC domain, the non-complementary strand of the target gene or nucleic acid (i.e., the strand that does not complementarily bind to the gRNA) can be cleaved.

[0278] Furthermore, the nickase may have the nuclease activity of the HNH domain of the CRISPR enzyme. That is, the nickase may not comprise the nuclease activity of the RuvC domain of the CRISPR enzyme, and thus, the RuvC domain may be manipulated or modified.

[0279] In one example, when the CRISPR enzyme is a type II CRISPR enzyme, the nickase may be a type II CRISPR enzyme comprising a modified RuvC domain.

[0280] For example, when the type II CRISPR enzyme is a wild-type SpCas9, the nickase may be a SpCas9 variant in which the nuclease activity of the RuvC domain is inactivated due to the mutation of aspartic acid 10 in the amino acid sequence of the wild-type SpCas9 to alanine. Here, since the generated nickase (i.e., the SpCas9 variant) has nuclease activity from the HNH domain, the complementary chain of the target gene or nucleic acid (i.e., the chain complementary to the gRNA) can be cleaved.

[0281] In another example, when the type II CRISPR enzyme is wild-type CjCas9, the nickase may be a CjCas9 variant in which the nuclease activity of the RuvC domain is inactivated due to the mutation of aspartic acid 8 in the amino acid sequence of the wild-type CjCas9 to alanine. Here, since the generated nickase (i.e., the CjCas9 variant) has nuclease activity from the HNH domain, the complementary strand of the target gene or nucleic acid (i.e., the strand complementary to the gRNA) can be cleaved.

[0282] In one embodiment, the nickase may be a Nureki Cas9 variant in which the nuclease activity of the RuvC domain is inactivated due to the mutation of aspartic acid 10 in the amino acid sequence of Nureki Cas9 to alanine, and the enzyme is NurekiCas9 nickase (Nureki nCas9). Here, since the generated Nureki nCas9 has nuclease activity from the HNH domain, the complementary strand of the target gene or nucleic acid (i.e., the strand complementary to the gRNA) can be sheared.

[0283] In another embodiment, the nickase may be a Nureki Cas9 variant in which the nuclease activity of the HNH domain is inactivated due to a mutation of histidine 840 in the amino acid sequence of Nureki Cas9 to alanine, and the enzyme is NurekiCas9 nickase (Nureki nCas9). Here, since the generated Nureki nCas9 has nuclease activity from the RuvC domain, the non-complementary strand of the target gene or nucleic acid (i.e., the strand that does not complementarily bind to the gRNA) can be cut.

[0284] The CRISPR enzyme may be an inactive CRISPR enzyme.

[0285] "Inactive" refers to the state in which the function of the wild-type CRISPR enzyme is lost, that is, both the first function of cleaving the first strand of double-stranded DNA and the second function of cleaving the second strand of double-stranded DNA are lost. The CRISPR enzyme in this state is called an inactive CRISPR enzyme.

[0286] An inactive CRISPR enzyme may have nuclease inactivation due to a mutation in a domain having nuclease activity of a wild-type CRISPR enzyme.

[0287] The inactive CRISPR enzyme may have nuclease inactivation caused by mutations in the RuvC domain and the HNH domain. That is, the inactive CRISPR enzyme may not comprise the nuclease activity of the RuvC domain and the HNH domain of the CRISPR enzyme, and to this end, the RuvC domain and the HNH domain may be manipulated or modified.

[0288] In one example, when the CRISPR enzyme is a type II CRISPR enzyme, the inactive CRISPR enzyme may be a type II CRISPR enzyme comprising a modified RuvC domain and a HNH domain.

[0289] For example, when the type II CRISPR enzyme is a wild-type SpCas9, the inactive CRISPR enzyme may be a SpCas9 variant in which the nuclease activity of the RuvC domain and the HNH domain is inactivated due to mutation of both aspartic acid 10 and histidine 840 in the amino acid sequence of the wild-type SpCas9 to alanine. Here, since the generated inactive CRISPR enzyme (i.e., the SpCas9 variant) has inactivated nuclease activity of the RuvC domain and the HNH domain, it cannot cleave both double strands of the target gene or nucleic acid.

[0290] In another example, when the type II CRISPR enzyme is a wild-type CjCas9, the inactive CRISPR enzyme may be a CjCas9 variant in which the nuclease activities of the RuvC domain and the HNH domain are inactivated due to mutation of both aspartic acid 8 and histidine 559 in the amino acid sequence of the wild-type CjCas9 to alanine. Here, since the generated inactive CRISPR enzyme (i.e., the CjCas9 variant) has inactivated nuclease activities of the RuvC domain and the HNH domain, it cannot cut both double strands of the target gene or nucleic acid.

[0291] In addition, the present application can provide a CRISPR enzyme connected to a functional domain. Here, in addition to the original function of the wild-type CRISPR enzyme, the CRISPR enzyme variant can have additional functions.

[0292] The functional domain may be a domain having methylase activity, demethylase activity, transcription activation activity, transcription inhibition activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for separating and purifying proteins (including peptides), but the present invention is not limited thereto.

[0293] The functional domain may be a tag or reporter gene for isolation and purification of proteins (including peptides).

[0294] Here, the label may include any of the following: histidine (His) label, V5 label, FLAG label, influenza virus hemagglutinin (HA) label, Myc label, VSV-G label and thioredoxin (Trx) label. Here, the reporter gene may include any of the following autofluorescent proteins: for example, glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), β-galactosidase, β-glucuronidase, luciferase, green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP). However, the present invention is not limited thereto.

[0295] The functional domain can be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).

[0296] Here, one or more NLSs may be included at or near the amino terminus of the CRISPR enzyme, at or near the carboxyl terminus of the CRISPR enzyme, or a combination thereof. The NLS may be derived from the following NLS sequences, but the present invention is not limited thereto: the NLS of the SV40 virus large T antigen having the amino acid sequence PKKKRKV (SEQ ID NO: 23); the NLS from the nucleoplasmic protein (e.g., the nucleoplasmic protein bipartite NLS having the sequence KRPAATKKAGQAKKKK (SEQ ID NO: 24)); the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 25) or RQRRNELKRSP (SEQ ID NO: 26); the hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 27); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 28) from the IBB domain of importin-α; the sequence VSRKRPRP (SEQ ID NO: 29) of the myoma T protein. NO: 29) and PPKKARED (SEQ ID NO: 30); the sequence of human p53 PQPKKKPL (SEQ ID NO: 31); the sequence of mouse c-abl IV SALIKKKKKMAP (SEQ ID NO: 32); the sequences DRLRR (SEQ ID NO: 33) and PKQKKRK (SEQ ID NO: 34) of influenza virus NS1; the sequence of infectious virus delta antigen RKLKKKIKKL (SEQ ID NO: 35); the sequence of mouse Mx1 protein REKKKFLKRR (SEQ ID NO: 36); the sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 37); and the sequence of the receptor for human steroid hormone, glucocorticoid RKCLQAGMNLEARKTKK (SEQ ID NO: 38) One or more.

[0297] A functional domain may be a binding domain capable of forming a complex with another domain, peptide, polypeptide or protein.

[0298] The binding domain may be one of a FRB domain and a FKBP dimerization domain; an intein; one of an ERT domain and a VPR domain; one of a GCN4 peptide and a single-chain variable fragment (scFv); or a domain that forms a heterodimer.

[0299] The binding domain may be a GCN4 peptide. Here, the GCN4 peptide may be paired with the scFv and may specifically bind or connect to the scFv.

[0300] In one example, a first fusion protein in which a GCN4 peptide functional domain is linked to a CRISPR enzyme can be combined with a second fusion protein comprising a peptide, polypeptide, protein, or scFv.

[0301] [The first aspect of the protein for single base substitution—a fusion protein for single base substitution or a nucleic acid encoding the fusion protein]

[0302] One aspect of the protein for single base substitution disclosed in the present specification is a fusion protein for single base substitution.

[0303] In one example, the fusion protein for single base substitution or the nucleic acid encoding the fusion protein may comprise:

[0304] (a) a CRISPR enzyme or a variant thereof;

[0305] (b) a deaminase; and

[0306] (c) DNA glycosylase or a variant thereof.

[0307] Here, the fusion protein for adenine substitution can induce substitution of cytosine or adenine contained in one or more nucleotides in any base pair of the target nucleic acid sequence.

[0308] In an exemplary embodiment, the fusion protein for single base substitution may include a linker portion inserted between one selected from (a), (b) and (c) and another selected from (a), (b) and (c).

[0309] In an exemplary embodiment, the fusion protein for single base substitution may have any of the following components:

[0310] (i) N-terminus - [CRISPR enzyme] - [deaminase] - [DNA glycosylase] - C-terminus;

[0311] (ii) N terminus-[CRISPR enzyme]-[DNA glycosylase]-[deaminase]-C terminus;

[0312] (iii) N terminus - [deaminase] - [CRISPR enzyme] - [DNA glycosylase] - C terminus;

[0313] (iv) N terminus - [deaminase] - [DNA glycosylase] - [CRISPR enzyme] - C terminus;

[0314] (v) N terminus-[DNA glycosylase]-[CRISPR enzyme]-[deaminase]-C terminus; and (vi) N terminus-[DNA glycosylase]-[deaminase]-[CRISPR enzyme]-C terminus.

[0315] In an exemplary embodiment, the CRISPR enzyme or its variant may comprise any one or more selected from the group consisting of: a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Cas9 protein derived from Streptococcus thermophilus, a Cas9 protein derived from Staphylococcus aureus, a Cas9 protein derived from Neisseria meningitidis, and a Cpf1 protein.

[0316] In an exemplary embodiment, the CRISPR enzyme variant may be characterized by inactivation of any one or more of the RuvC domain and the HNH domain.

[0317] In an exemplary embodiment, the CRISPR enzyme variant may be a nickase.

[0318] In one embodiment, a fusion protein can be provided for adenine replacement.

[0319] The fusion protein for adenine replacement or the nucleic acid encoding the fusion protein may comprise:

[0320] (a) a CRISPR enzyme or a variant thereof;

[0321] (b) adenosine deaminase; and

[0322] (c) Alkyladenine DNA glycosylase or a variant thereof.

[0323] Among them, the fusion protein for adenine substitution can induce substitution of adenine contained in one or more nucleotides in any base pair target nucleic acid sequence.

[0324] The protein for adenine base replacement can be composed in the order of N-terminus-[CRISPR enzyme]-[adenosine deaminase]-[alkyladenine DNA glycosylase]-C-terminus.

[0325] The protein for adenine base replacement can be composed in the order of N-terminus-[alkyladenine DNA glycosylase]-[CRISPR enzyme]-[adenosine deaminase]-C-terminus.

[0326] The protein for adenine base replacement can be composed in the order of N-terminus-[alkyladenine DNA glycosylase]-[adenosine deaminase]-[CRISPR enzyme]-C-terminus.

[0327] The protein for adenine base replacement can be composed in the order of N-terminus-[adenosine deaminase]-[CRISPR enzyme]-[alkyladenine DNA glycosylase]-C-terminus.

[0328] The protein for adenine base replacement can be composed in the order of N-terminus-[CRISPR enzyme]-[alkyladenine DNA glycosylase]-[adenosine deaminase]-C-terminus.

[0329] The protein for adenine base replacement can be composed in the order of N-terminus-[adenosine deaminase]-[alkyladenine DNA glycosylase]-[CRISPR enzyme]-C-terminus.

[0330] The protein for adenine base substitution may further comprise a linker domain.

[0331] In one example, the linker domain can be a domain that operably links the CRISPR enzyme to an adenosine deaminase, an adenosine deaminase to an alkyladenine DNA glycosylase, and / or a CRISPR enzyme to an alkyladenine DNA glycosylase, and can be a domain that links the CRISPR enzyme, adenosine deaminase, and an alkyladenine DNA glycosylase to activate each function.

[0332] In one example, the linker domain may be an amino acid, peptide or polypeptide that does not affect the functional activity and / or structure of the CRISPR enzyme, adenosine deaminase and alkyladenine DNA glycosylase.

[0333] In one example, the domains for adenine base replacement can be composed in the following order: N-terminus-[CRISPR enzyme]-[linking domain]-[adenosine deaminase]-[alkyladenine DNA glycosylase]-C-terminus; N-terminus-[CRISPR enzyme]-[adenosine deaminase]-[linking domain]-[alkyladenine DNA glycosylase]-C-terminus; or N-terminus-[CRISPR enzyme]-[linking domain]-[adenosine deaminase]-[linking domain]-[alkyladenine DNA glycosylase]-C-terminus.

[0334] In one example, the protein for adenine base replacement can be composed in the following order: N terminus-[alkyladenine DNA glycosylase]-[linking domain]-[CRISPR enzyme]-[adenosine deaminase]-C terminus; N terminus-[alkyladenine DNA glycosylase]-[CRISPR enzyme]-[linking domain]-[adenosine deaminase]-C terminus; or N terminus-[alkyladenine DNA glycosylase]-[linking domain]-[CRISPR enzyme]-[linking domain]-[adenosine deaminase]-C terminus.

[0335] In one example, the protein for adenine base replacement can be composed in the following order: N terminus-[alkyladenine DNA glycosylase]-[linking domain]-[adenosine deaminase]-[CRISPR enzyme]-C terminus; N terminus-[alkyladenine DNA glycosylase]-[adenosine deaminase]-[linking domain]-[CRISPR enzyme]-C terminus; or N terminus-[alkyladenine DNA glycosylase]-[linking domain]-[adenosine deaminase]-[linking domain]-[CRISPR enzyme]-C terminus.

[0336] In one example, the protein for adenine base replacement can be composed in the following order: N terminus-[adenosine deaminase]-[linking domain]-[CRISPR enzyme]-[alkyladenine DNA glycosylase]-C terminus; N terminus-[adenosine deaminase]-[CRISPR enzyme]-[linking domain]-[alkyladenine DNA glycosylase]-C terminus; or N terminus-[adenosine deaminase]-[linking domain]-[CRISPR enzyme]-[linking domain]-[alkyladenine DNA glycosylase]-C terminus.

[0337] In one example, the protein for adenine base replacement can be composed in the following order: N terminus-[CRISPR enzyme]-[linking domain]-[alkyladenine DNA glycosylase]-[adenosine deaminase]-C terminus; N terminus-[CRISPR enzyme]-[alkyladenine DNA glycosylase]-[linking domain]-[adenosine deaminase]-C terminus; or N terminus-[CRISPR enzyme]-[linking domain]-[alkyladenine DNA glycosylase]-[linking domain]-[adenosine deaminase]-C terminus.

[0338] In one example, the protein for adenine base replacement can be composed in the following order: N terminus-[adenosine deaminase]-[linking domain]-[alkyladenine DNA glycosylase]-[CRISPR enzyme]-C terminus; N terminus-[adenosine deaminase]-[alkyladenine DNA glycosylase]-[linking domain]-[CRISPR enzyme]-C terminus; or N terminus-[adenosine deaminase]-[linking domain]-[alkyladenine DNA glycosylase]-[linking domain]-[CRISPR enzyme]-C terminus.

[0339] In one embodiment, a fusion protein for cytosine replacement can be provided.

[0340] The fusion protein for cytosine replacement or the nucleic acid encoding the fusion protein may comprise:

[0341] (a) a CRISPR enzyme or a variant thereof;

[0342] (b) cytidine deaminase; and

[0343] (c) uracil DNA glycosylase or a variant thereof.

[0344] Among them, the fusion protein for single base substitution can induce the substitution of cytosine contained in one or more nucleotides in the target nucleic acid sequence for any base pair.

[0345] The protein used for cytosine base replacement can be composed in the order of N-terminus-[CRISPR enzyme]-[cytidine deaminase]-[uracil DNA glycosylase]-C-terminus.

[0346] The protein for cytosine base replacement can be composed in the order of N-terminus-[uracil DNA glycosylase]-[CRISPR enzyme]-[cytidine deaminase]-C-terminus.

[0347] The protein for cytosine base replacement can be composed in the order of N-terminus-[uracil DNA glycosylase]-[cytidine deaminase]-[CRISPR enzyme]-C-terminus.

[0348] The protein for cytosine base replacement can be composed in the order of N-terminus-[cytidine deaminase]-[CRISPR enzyme]-[uracil DNA glycosylase]-C-terminus.

[0349] The protein used for cytosine base replacement can be composed in the order of N-terminus-[CRISPR enzyme]-[uracil DNA glycosylase]-[cytidine deaminase]-C-terminus.

[0350] The protein used for cytosine base replacement can be composed in the order of N-terminus-[cytidine deaminase]-[uracil DNA glycosylase]-[CRISPR enzyme]-C-terminus.

[0351] The protein for cytosine base substitution may further comprise a linker domain.

[0352] In one example, the linker domain can be a domain that operably links the CRISPR enzyme to a cytidine deaminase, a cytidine deaminase to a uracil DNA glycosylase, and / or a CRISPR enzyme to a uracil DNA glycosylase, and can be a domain that links the CRISPR enzyme, cytidine deaminase, and uracil DNA glycosylase to activate each function.

[0353] In one example, the linker domain may be an amino acid, peptide or polypeptide that does not affect the functional activity and / or structure of the CRISPR enzyme, cytidine deaminase and uracil DNA glycosylase.

[0354] In one example, the domains for cytosine base replacement can be composed in the following order: N-terminus-[CRISPR enzyme]-[linking domain]-[cytidine deaminase]-[uracil DNA glycosylase]-C-terminus; N-terminus-[CRISPR enzyme]-[cytidine deaminase]-[linking domain]-[uracil DNA glycosylase]-C-terminus; or N-terminus-[CRISPR enzyme]-[linking domain]-[cytidine deaminase]-[linking domain]-[uracil DNA glycosylase]-C-terminus.

[0355] In one example, the protein for cytosine base replacement can be composed in the following order: N terminus-[uracil DNA glycosylase]-[linking domain]-[CRISPR enzyme]-[cytidine deaminase]-C terminus; N terminus-[uracil DNA glycosylase]-[CRISPR enzyme]-[linking domain]-[cytidine deaminase]-C terminus; or N terminus-[uracil DNA glycosylase]-[linking domain]-[CRISPR enzyme]-[linking domain]-[cytidine deaminase]-C terminus.

[0356] The protein for cytosine base replacement can be composed in the following order: N terminus-[uracil DNA glycosylase]-[linker domain]-[cytidine deaminase]-[CRISPR enzyme]-C terminus; N terminus-[uracil DNA glycosylase]-[cytidine deaminase]-[linker domain]-[CRISPR enzyme]-C terminus; or N terminus-[uracil DNA glycosylase]-[linker domain]-[cytidine deaminase]-[linker domain]-[CRISPR enzyme]-C terminus.

[0357] The protein for cytosine base replacement can be composed in the following order: N terminus-[cytidine deaminase]-[linker domain]-[CRISPR enzyme]-[uracil DNA glycosylase]-C terminus; N terminus-[cytidine deaminase]-[CRISPR enzyme]-[linker domain]-[uracil DNA glycosylase]-C terminus; or N terminus-[cytidine deaminase]-[linker domain]-[CRISPR enzyme]-[linker domain]-[uracil DNA glycosylase]-C terminus.

[0358] The protein for cytosine base replacement can be composed in the following order: N terminus-[CRISPR enzyme]-[linker domain]-[uracil DNA glycosylase]-[cytidine deaminase]-C terminus; N terminus-[CRISPR enzyme]-[uracil DNA glycosylase]-[linker domain]-[cytidine deaminase]-C terminus; or N terminus-[CRISPR enzyme]-[linker domain]-[uracil DNA glycosylase]-[linker domain]-[cytidine deaminase]-C terminus.

[0359] The cytosine base modification protein can be composed in the following order: N-terminus-[cytidine deaminase]-[linker domain]-[uracil DNA glycosylase]-[CRISPR enzyme]-C-terminus; N-terminus-[cytidine deaminase]-[uracil DNA glycosylase]-[linker domain]-[CRISPR enzyme]-C-terminus; or N-terminus-[cytidine deaminase]-[linker domain]-[uracil DNA glycosylase]-[linker domain]-[CRISPR enzyme]-C-terminus.

[0360] [Second Aspect of Protein for Single Base Substitution - Complex for Single Base Substitution]

[0361] One aspect of the protein for single base substitution disclosed in the present specification is a complex for single base substitution (single base substitution complex).

[0362] In one example, a complex for single base substitution may include:

[0363] (a) a CRISPR enzyme or a variant thereof;

[0364] (b) deaminases;

[0365] (c) DNA glycosylase; and

[0366] (d) Two or more binding domains.

[0367] Among them, the fusion protein for single base substitution can induce the substitution of cytosine or adenine contained in one or more nucleotides in the target nucleic acid sequence for any base pair.

[0368] In one example, a CRISPR enzyme may be linked to two or more binding domains in a complex for single base substitution.

[0369] Here, any one of the two or more binding domains linked to the CRISPR enzyme can be paired with the binding domain linked to the (b) deaminase, and another of them can be paired with the binding domain linked to the (c) DNA glycosylase. Here, due to the binding between the pairs, the components (a) CRISPR enzyme, (b) deaminase, and (c) DNA glycosylase form a complex to provide a complex for single base substitution.

[0370] In an exemplary embodiment, a CRISPR enzyme linked to two or more binding domains may have [binding domain (functional domain)] n -The configuration of the CRISPR enzyme (n can be an integer greater than 2).

[0371] For example, CRISPR enzymes can be found in Fig.32 As shown in (a).

[0372] Here, GCN4 may be an example of a binding domain connected to a CRISPR enzyme, and different types of binding domains may be connected thereto. However, the present invention is not limited thereto.

[0373] Here, the CRISPR enzyme can be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more binding domains.

[0374] In another example, the CRISPR enzyme may be Fig.32 As shown in (b).

[0375] Here, GCN4 may be an example of a binding domain connected to a CRISPR enzyme, and different types of binding domains may be connected thereto. However, the present invention is not limited thereto.

[0376] Here, the CRISPR enzyme may be linked to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more binding domains at the C-terminus and the N-terminus.

[0377] In an exemplary embodiment, the complex for single base substitution provided by the present application can be provided in the following manner:

[0378] Fig.33 Specific binding of the binding domains in components (a), (b) and (c).

[0379] Here, the binding domain GCN4 of (a), the binding domain scFv of (b), and the binding domain scFv of (c) are only examples and the present invention is not limited thereto. APOBEC may be replaced by adenosine deaminase, and UNG may be replaced by alkyladenine DNA glycosylase.

[0380] Herein, a plurality of (b) and / or a plurality of (c) may be combined to one (a).

[0381] Herein, "plurality" means 2, 3, 4, 5, 6, 7, 8, 9, 10 or more integers.

[0382] In an exemplary embodiment, the complex for single base substitution provided by the present application can be provided in the following manner:

[0383] Fig.34 Specific binding of the binding domains in components (a), (b) and (c).

[0384] Here, the binding domain GCN4 of (a), the binding domain scFv of (b), and the binding domain scFv of (c) are only examples and the present invention is not limited thereto. APOBEC may be replaced by adenosine deaminase, and UNG may be replaced by alkyladenine DNA glycosylase.

[0385] Here, a plurality of (b) and / or a plurality of (c) may be combined to one (a).

[0386] Here, "plurality" means 2, 3, 4, 5, 6, 7, 8, 9, 10 or more integers.

[0387] In one example, in a complex for single base substitution, the deaminase can be connected to two or more binding domains. Here, each of the two or more binding domains connected to the deaminase is paired with a binding domain connected to (a) the CRISPR enzyme and a binding domain connected to (c) the DNA glycosylase. Here, due to the binding between the pairs, the components (a) CRISPR enzyme, (b) deaminase and (c) DNA glycosylase form a complex, and a complex for single base substitution can be provided.

[0388] In one example, in a complex for single base substitution, the DNA glycosylase may be connected to two or more binding domains. Here, each of the two or more binding domains connected to the DNA glycosylase is paired with a binding domain connected to (a) the CRISPR enzyme and a binding domain connected to (b) the deaminase. Here, due to the binding between the pairs, the components (a) CRISPR enzyme, (b) deaminase, and (c) DNA glycosylase form a complex to provide a complex for single base substitution.

[0389] In one example, in a complex for single base substitution, the CRISPR enzyme may be linked to two or more binding domains and may be present in a fusion protein in which a deaminase and a DNA glycosylase are linked. Here, the fusion protein comprises one or more binding domains. In an exemplary embodiment, a binding domain linked to the CRISPR enzyme is paired with a binding domain of the fusion protein. Here, due to the binding between the pairs, components (a) CRISPR enzyme, (b) deaminase, and (c) DNA glycosylase form a complex to provide a complex for single base substitution.

[0390] In an exemplary embodiment, the complex for single base substitution provided by the present application can be Fig.35 It is formed by the specific binding of the binding domain of (a) and the binding domain of (b).

[0391] Here, the binding domain GCN4 of (a) and the binding domain scFv of (b) are only examples and the present invention is not limited thereto. APOBEC may be replaced by adenosine deaminase or different types of cytidine deaminase, and UNG may be replaced by alkyladenine DNA glycosylase.

[0392] Here, a plurality of (b) may be combined to one (a).

[0393] Here, "plurality" means 2, 3, 4, 5, 6, 7, 8, 9, 10 or more integers.

[0394] In an exemplary embodiment, the complex for single base substitution provided by the present application can be Fig.36 It is formed by the specific binding of the binding domain of (a) and the binding domain of (c).

[0395] Here, the binding domain GCN4 of (a) and the binding domain scFv of (b) are only examples and the present invention is not limited thereto. APOBEC may be replaced by adenosine deaminase or different types of cytidine deaminase, and UNG may be replaced by alkyladenine DNA glycosylase.

[0396] Here, a plurality of (b) may be combined to one (a).

[0397] Here, "plurality" means 2, 3, 4, 5, 6, 7, 8, 9, 10 or more integers.

[0398] In an exemplary embodiment, the complex for single base substitution provided by the present application can be Fig.37 It is formed by the specific binding of the binding domain of (a) and the binding domain of (b).

[0399] Here, the binding domain GCN4 of (a) and the binding domain scFv of (b) are only examples and the present invention is not limited thereto. APOBEC may be replaced by adenosine deaminase or different types of cytidine deaminase, and UNG may be replaced by alkyladenine DNA glycosylase.

[0400] Here, a plurality of (b) may be combined to one (a).

[0401] Here, "plurality" means 2, 3, 4, 5, 6, 7, 8, 9, 10 or more integers.

[0402] In an exemplary embodiment, the complex for single base substitution provided by the present application can be Fig.38 It is formed by the specific binding of the binding domain of (a) and the binding domain of (c).

[0403] Here, the binding domain GCN4 of (a) and the binding domain scFv of (b) are only examples and the present invention is not limited thereto. APOBEC may be replaced by adenosine deaminase or different types of cytidine deaminase, and UNG may be replaced by alkyladenine DNA glycosylase.

[0404] Here, a plurality of (b) may be combined to one (a).

[0405] Here, "plurality" means 2, 3, 4, 5, 6, 7, 8, 9, 10 or more integers.

[0406] In one example, the complex for single base substitution can be present in the form of a fusion protein, wherein the deaminase is connected to two or more binding domains and is connected to the CRISPR enzyme and the DNA glycosylase. Here, the fusion protein comprises one or more binding domains. In an exemplary embodiment, a binding domain connected to the deaminase is paired with a binding domain of the fusion protein. Here, due to the binding between the pairs, components (a) CRISPR enzyme, (b) deaminase and (c) DNA glycosylase form a complex to provide a complex for single base substitution.

[0407] In one example, the complex for single base replacement can exist in the form of a fusion protein, wherein the DNA glycosylase is connected to two or more binding domains, and the deaminase and CRISPR enzyme are connected. Here, the fusion protein comprises one or more binding domains. In an exemplary embodiment, a binding domain connected to the DNA glycosylase is paired with the binding domain of the fusion protein. Here, due to the binding between the pair, components (a) CRISPR enzyme, (b) deaminase and (c) DNA glycosylase form a complex to provide a complex for single base replacement.

[0408] In one example, a complex for single base substitution may include (i) a first fusion protein comprising two components selected from a CRISPR enzyme, a deaminase, and a DNA glycosylase and a first binding domain, and (ii) a second fusion protein comprising an unselected remaining component and a second binding domain. Wherein, the first binding domain and the second binding domain are an interacting pair, and here, the complex is formed by the pair. Wherein, the second fusion protein may further include a plurality of binding domains in addition to the second binding domain.

[0409] In an exemplary embodiment, a complex for single base substitution may include (i) a first fusion protein comprising a deaminase, a DNA glycosylase, and a first binding domain, and (ii) a second fusion protein comprising a CRISPR enzyme and a second binding domain. Here, the second fusion protein may further include a plurality of binding domains in addition to the second binding domain. Here, the first binding domain may be a single-chain variable fragment (scFv), and the second fusion protein may be a GCN4 peptide. Here, the scFv can provide a complex for single base substitution by interacting with the GCN4 peptide.

[0410] In an exemplary embodiment, a complex for single base substitution may include (i) a first fusion protein comprising a deaminase, a CRISPR enzyme, and a first binding domain, and (ii) a second fusion protein comprising a DNA glycosylase and a second binding domain. Here, the second fusion protein may further include a plurality of binding domains in addition to the second binding domain. Here, the first binding domain may be a single-chain variable fragment (scFv), and the second fusion protein may be a GCN4 peptide. Here, the scFv may provide a complex for single base substitution by interacting with the GCN4 peptide.

[0411] In an exemplary embodiment, a complex for single base substitution may include (i) a first fusion protein comprising a CRISPR enzyme, a DNA glycosylase, and a first binding domain, and (ii) a second fusion protein comprising a deaminase and a second binding domain. Here, the second fusion protein may further include a plurality of binding domains in addition to the second binding domain. Here, the first binding domain may be a single-chain variable fragment (scFv), and the second fusion protein may be a GCN4 peptide. Here, the scFv may provide a complex for single base substitution by interacting with the GCN4 peptide.

[0412] In one example, any one of a CRISPR enzyme, a deaminase, and a DNA glycosylase is linked to a first binding domain and a second binding domain, and here, the first binding domain is a pair that interacts with another binding domain. Here, the second binding domain is a pair that interacts with yet another binding domain, and a complex for single base substitution can be provided by the pair.

[0413] In one embodiment, the CRISPR enzyme can be connected to a first binding domain and a second binding domain. Here, the first binding domain is a pair that interacts with the binding domain of the deaminase, and the second binding domain is a pair that interacts with the binding domain of the DNA glycosylase. Here, a complex for single base substitution can be provided by the pair.

[0414] In one embodiment, the deaminase may be linked to a first binding domain and a second binding domain. Here, the first binding domain is a pair that interacts with the binding domain of the CRISPR enzyme, and the second binding domain is a pair that interacts with the binding domain of the DNA glycosylase. Here, a complex for single base substitution may be provided by the pair.

[0415] In one embodiment, the DNA glycosylase may be linked to a first binding domain and a second binding domain. Here, the first binding domain is a pair that interacts with the binding domain of the deaminase, and the second binding domain is a pair that interacts with the binding domain of the CRISPR enzyme. Here, a complex for single base substitution may be provided by the pair.

[0416] Here, the binding domain may be one of a FRB domain and a FKBP dimerization domain; an intein; one of an ERT domain and a VPR domain; one of a GCN4 peptide and a single-chain variable fragment (scFv); or a domain that forms a heterodimer.

[0417] Here, the pair may be any one of the following:

[0418] FRB domain and FKBP dimerization domain;

[0419] The first intein and the second intein;

[0420] ERT domain and VPR domain;

[0421] GCN4 peptide and single-chain variable fragment (scFv); and

[0422] The first domain and the second domain form a heterodimer.

[0423] The present application can provide a cytosine replacement complex.

[0424] For example, the deaminase may be a cytidine deaminase, and the DNA glycosylase may be a uracil DNA glycosylase or a variant thereof. Here, the fusion protein for single-base substitution may be a complex for single-base substitution, which induces substitution of any base pair of cytosine contained in one or more nucleotides in the target nucleic acid sequence.

[0425] In one example, the cytidine deaminase is APOBEC, activation-induced cytidine deaminase (AID), or a variant thereof.

[0426] In one example, any one of a CRISPR enzyme, a cytidine deaminase, and a uracil DNA glycosylase can be linked to a first binding domain and a second binding domain. Here, the first binding domain is a pair that interacts with another binding domain, and here, the second binding domain is a pair that interacts with yet another binding domain. Here, a complex for single base substitution can be provided by the pair.

[0427] In one embodiment, the CRISPR enzyme can be connected to a first binding domain and a second binding domain. Here, the first binding domain is an interaction pair that interacts with the binding domain of the deaminase, and the second binding domain is an interaction pair that interacts with the binding domain of the DNA glycosylase. Here, a complex for single base substitution can be provided by the pair.

[0428] In one example, a complex for single base substitution may include (i) a first fusion protein comprising a first binding domain and two components selected from a CRISPR enzyme, a cytidine deaminase, and a uracil DNA glycosylase, and (ii) a second fusion protein comprising an unselected remaining component and a second binding domain. Here, the first binding domain and the second binding domain are an interaction pair that interact with each other, and here, the complex can be formed by the pair. Here, the second fusion protein may further include a plurality of binding domains in addition to the second binding domain.

[0429] Here, the pair may be any one of the following:

[0430] FRB domain and FKBP dimerization domain;

[0431] The first intein and the second intein;

[0432] ERT domain and VPR domain;

[0433] GCN4 peptide and single-chain variable fragment (scFv); and

[0434] The first domain and the second domain form a heterodimer.

[0435] The present application can provide an adenine replacement complex.

[0436] In one example, the deaminase may be adenosine deaminase, and the DNA glycosylase may be alkyladenine DNA glycosylase or a variant thereof. Here, the fusion protein for single base substitution may be a complex for single base substitution, which induces substitution of any base pair of adenine contained in one or more nucleotides in the target nucleic acid sequence.

[0437] In one example, the adenine deaminase can be TadA, Tad2p, ADA, ADA1, ADA2, ADAR2, ADAT2, ADAT3, or a variant thereof.

[0438] In one example, any one of a CRISPR enzyme, adenosine deaminase, and alkyladenine DNA glycosylase can be linked to a first binding domain and a second binding domain. Here, the first binding domain is an interaction pair that interacts with a binding domain of another component, and the second binding domain is an interaction pair that interacts with a binding domain of yet another component. Here, a complex for single base substitution can be provided by the pair.

[0439] In one embodiment, the CRISPR enzyme may be linked to a first binding domain and a second binding domain. Here, the first binding domain is an interaction pair that interacts with the binding domain of the deaminase, and the second binding domain is an interaction pair that interacts with the binding domain of the DNA glycosylase. Here, a complex for single base substitution may be provided by the pair.

[0440] In one example, a complex for single base substitution may include (i) a first fusion protein comprising a first binding domain and two components selected from CRISPR enzymes, adenosine deaminase and alkyladenine DNA glycosylase, and (ii) a second fusion protein comprising a second binding domain and unselected remaining components. Here, the first binding domain and the second binding domain are an interaction pair that interact with each other, and the complex is formed by the pair. Here, the second fusion protein may further include a plurality of binding domains in addition to the second binding domain.

[0441] Here, the pair may be any one of the following:

[0442] FRB domain and FKBP dimerization domain;

[0443] The first intein and the second intein;

[0444] ERT domain and VPR domain;

[0445] GCN4 peptide and single-chain variable fragment (scFv); and

[0446] The first domain and the second domain form a heterodimer.

[0447] One aspect of the present invention disclosed in this specification is a composition for base substitution and a method of using the composition Method of things.

[0448] The composition for single base substitution can be used to artificially modify the bases of one or more nucleotides in a gene.

[0449] The term "artificially modified or artificially engineered" refers to a state that has been artificially modified, rather than a state that occurs in nature. For example, the artificially modified state may be a modification that artificially causes a mutation in a wild-type gene. Hereinafter, non-natural, artificially modified polymorphism-dependent genes may be used interchangeably with the term artificial polymorphism-dependent genes.

[0450] The composition for base modification may further comprise a guide RNA or a nucleic acid encoding the guide RNA.

[0451] In one example, the present invention provides a composition for single base substitution, the composition comprising:

[0452] (a) a guide RNA or a nucleic acid encoding the guide RNA, and (b) a fusion protein for single base substitution or a nucleic acid encoding the fusion protein, or a complex for single base substitution. Wherein, the guide RNA can complementarily bind to a target nucleic acid sequence, wherein the length of the target nucleic acid sequence bound to the guide RNA is 15 bp-25 bp, wherein the fusion protein for single base substitution or the complex for single base substitution induces the substitution of one or more cytosines or adenines present in a target region containing the target nucleic acid sequence with any base pair.

[0453] [The first component of the composition for base substitution: guide RNA]

[0454] The composition for base substitution may include a guide RNA or a nucleic acid encoding the guide RNA.

[0455] Guide RNA (gRNA) refers to RNA that can specifically guide the gRNA-CRISPR enzyme complex (i.e., CRISPR complex) to a target gene or nucleic acid. In addition, gRNA refers to a target gene or nucleic acid-specific RNA and can bind to a CRISPR enzyme to guide the CRISPR enzyme to a target gene or nucleic acid.

[0456] The guide RNA can be complementary to a partial sequence of either strand of the double strand of the target gene or nucleic acid. The partial sequence can refer to the target nucleic acid sequence.

[0457] Guide RNA can be used to direct the guide RNA-CRISPR enzyme complex to a location with a specific nucleotide sequence of a target gene or nucleic acid.

[0458] Guide RNA refers to an RNA that can specifically guide the gRNA-CRISPR enzyme complex (i.e., CRISPR complex) to a target gene, target region, or target nucleic acid sequence. In addition, gRNA refers to a target gene or nucleic acid-specific RNA, and can bind to the CRISPR enzyme to guide the CRISPR enzyme to a target gene, target region, or target nucleic acid sequence.

[0459] The guide RNA may be referred to as single-stranded guide RNA (single RNA molecule; single gRNA; sgRNA); or double-stranded guide RNA (comprising more than one, typically two, separate RNA molecules).

[0460] The guide RNA contains a site that binds complementary to the target sequence (hereinafter referred to as the guide site) and a site that participates in forming a complex with the Cas protein (hereinafter referred to as the complex formation site).

[0461] In one example, the guide RNA can interact with the SpCas9 protein and can be any one selected from SEQ ID NO.48-SEQ ID NO.81.

[0462] In another example, the guide RNA can interact with the CjCas9 protein and can comprise any one selected from SEQ ID NO.82-SEQ ID NO.92.

[0463] [Table 1]

[0464]

[0465]

[0466]

[0467] Here, the complex formation site can be determined according to the type of microorganism from which the Cas9 protein is derived. For example, in the case where the guide RNA interacts with the SpCas9 protein, the complex formation site may include 5′-GUUUUAGUCCCUGAAAAGGGACUAAAAUAAAGAGUUUGCGGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′ (SEQ. ID NO: 45); in the case where the guide RNA interacts with the CjCas9 protein, the complex formation site may include 5′-GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′ (SEQ ID NO: 46).

[0468] As the protospacer adjacent motif (PAM) sequence, NGG (N is A, T, C or G) was considered when the spCas9 protein was used, and NNNNRYAC (SEQ ID NO: 47) (N is independently A, T, C or G, R is A or G, and Y is C or T) was considered when the CjCas9 protein was used.

[0469] The composition may comprise one or more guide RNAs.

[0470] [Second component of the composition for base substitution - protein for single base substitution]

[0471] The composition for base substitution may include a protein for single base substitution or a nucleic acid encoding the protein.

[0472] The proteins used for single base substitution are the same as described above.

[0473] [The third component of the composition for base substitution: carrier]

[0474] The composition for base modification may be in the form of a carrier.

[0475] A "vector" can deliver a gene sequence to a cell. In general, "vector constructs," "expression vectors," and "gene transfer vectors" can direct the expression of a desired gene and refer to any nucleic acid structure capable of delivering a gene sequence to a target cell. Thus, the term "vector" includes vectors, as well as cloning and expression vehicles.

[0476] Here, the vector may be a viral or non-viral vector (eg, a plasmid).

[0477] Here, the vector may comprise one or more regulatory / control elements.

[0478] Here, the regulatory / control elements may include a promoter, an enhancer, an intron, a polyadenylation signal, a Kozak consensus sequence, an internal ribosome entry site (IRES), a splice acceptor and / or a 2A sequence.

[0479] The promoter may be a promoter recognized by RNA polymerase II.

[0480] The promoter may be a promoter recognized by RNA polymerase III.

[0481] The promoter may be an inducible promoter.

[0482] The promoter may be a target-specific promoter.

[0483] The promoter may be a viral or a non-viral promoter.

[0484] As the promoter, an appropriate promoter can be used depending on the control region (ie, the nucleic acid sequence encoding the guide RNA or the CRISPR enzyme).

[0485] For example, a useful promoter for guide RNA may be H1, EF-1a, tRNA, or U6 promoter. For example, a promoter for CRISPR enzyme may be CMV, EF-1a, EFS, MSCV, PGK, or CAG promoter.

[0486] The vector may be a viral vector or a recombinant viral vector.

[0487] The virus may be a DNA virus or an RNA virus.

[0488] Here, the DNA virus may be a double-stranded DNA (dsDNA) virus or a single-stranded DNA (ssDNA) virus.

[0489] Here, the RNA virus may be a single-stranded RNA (ssRNA) virus.

[0490] The virus may be a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus, or a herpes simplex virus, but the present invention is not limited thereto.

[0491] Typically, a virus can infect a host (e.g., a cell) to imprint a nucleic acid encoding viral genetic information into the host, or insert a nucleic acid encoding genetic information into the host's genome. A virus having the above characteristics can be used to introduce a guide RNA and / or a CRISPR enzyme into a target. The guide RNA and / or CRISPR enzyme introduced using such a virus can be temporarily expressed in a subject (e.g., a cell). Alternatively, the guide RNA and / or CRISPR enzyme introduced using such a virus can be continuously expressed in a subject (e.g., a cell) for a long period of time (e.g., 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months, 9 months, 1 year, 2 years, or permanently).

[0492] The viral packaging capacity can vary from at least 2 kb to 50 kb, depending on the virus type. Depending on this packaging capacity, a viral vector can be designed that contains either a guide RNA or a CRISPR enzyme alone or a viral vector that contains both a guide RNA and a CRISPR enzyme. Alternatively, a viral vector can be designed that contains a guide RNA, a CRISPR enzyme, and additional components.

[0493] For example, retroviral vectors have the packaging capacity for foreign sequences up to 6 kb to 10 kb and are composed of cis-acting long terminal repeats (LTRs). Retroviral vectors insert therapeutic genes into cells and provide permanent expression of the inserted genes.

[0494] In another example, regardless of cell division, adeno-associated virus vectors have very high efficiency of introduction into various cells (muscle, brain, liver, lung, retina, ear, heart and vascular cells) and are non-pathogenic, and since most viral genomes can be replaced by therapeutic genes and do not induce immune responses, repeated administration is possible. In addition, AAV is inserted into the chromosome of the target cell, thereby stably expressing the therapeutic protein for a long time. For example, AAV can be used to produce nucleic acids and peptides in vitro and transduce the nucleic acids or peptides to the target nucleic acids of cells in vivo and in vitro. However, AAV is small in size and has a packaging capacity of less than 4.5kb.

[0495] Among them, the composition for base modification may include a vector and an adenine base replacement protein, wherein the vector includes a nucleic acid encoding a guide RNA.

[0496] Among them, the composition for base modification may include a guide RNA and a vector, wherein the vector includes a nucleic acid encoding a protein for adenine base replacement.

[0497] Among them, the composition for base modification may include: a vector comprising a nucleic acid encoding a guide RNA; and a vector comprising a nucleic acid encoding a protein for adenine base substitution.

[0498] Among them, the composition for base modification may include a vector, wherein the vector includes a nucleic acid encoding a guide RNA and a nucleic acid encoding an adenine base replacement protein.

[0499] In another example, the composition for base modification may include

[0500] (a) a CRISPR enzyme or a nucleic acid encoding the CRISPR enzyme, the CRISPR enzyme comprising a first binding domain; and

[0501] (b) an adenosine deaminase or a nucleic acid encoding the adenosine deaminase, wherein the adenosine deaminase comprises a second binding domain.

[0502] Among them, the CRISPR enzyme can be a wild-type CRISPR enzyme or a CRISPR enzyme variant.

[0503] Among them, the CRISPR enzyme variant can be a nickase.

[0504] The adenosine deaminase may be TadA, Tad2p, ADA, ADA1, ADA2, ADAR2, ADAT2, ADAT3 or variants thereof.

[0505] The first binding domain can form a non-covalent bond with the second binding domain.

[0506] Among them, the first binding domain can be: one of the FRB domain and the FKBP dimerization domain; an intein; one of the ERT domain and the VPR domain; one of the GCN4 peptide and the single-chain variable fragment (scFv); or a domain that forms a heterodimer.

[0507] Among them, the second binding domain can be: one of the FRB domain and the FKBP dimerization domain; an intein; one of the ERT domain and the VPR domain; one of the GCN4 peptide and the single-chain variable fragment (scFv); or a domain that forms a heterodimer.

[0508] The composition for base modification may further comprise one or more guide RNAs or nucleic acids encoding the guide RNAs.

[0509] The composition for base modification may be in the form of a ribonucleoprotein (RNP) (i.e., a complex), comprising

[0510] Guide RNA;-

[0511] a CRISPR enzyme having a first binding domain; and -

[0512] Adenosine deaminase having a second binding domain.

[0513] Among them, the composition for base modification may include

[0514] a vector comprising a nucleic acid encoding a guide RNA;

[0515] a vector comprising a nucleic acid encoding a CRISPR enzyme having a first binding domain; and

[0516] A vector comprising a nucleic acid encoding an adenosine deaminase having a second binding domain.

[0517] Among them, the composition for base modification may include

[0518] A vector comprising a nucleic acid encoding a guide RNA; and

[0519] A complex of a CRISPR enzyme comprising a first binding domain and an adenosine deaminase comprising a second binding domain.

[0520] Among them, the composition for base modification may include a vector, which includes a nucleic acid encoding a guide RNA, a nucleic acid encoding a CRISPR enzyme having a first binding domain, and a nucleic acid encoding an adenosine deaminase having a second binding domain.

[0521] Among them, the composition for base modification may include: a vector containing a nucleic acid encoding a guide RNA and a nucleic acid encoding a CRISPR enzyme having a first binding domain; and a vector containing a nucleic acid encoding an adenosine deaminase having a second binding domain.

[0522] Among them, the composition for base modification may include: a vector containing a nucleic acid encoding a CRISPR enzyme having a first binding domain; and a vector containing a nucleic acid encoding a guide RNA and a nucleic acid encoding an adenosine deaminase having a second binding domain.

[0523] Among them, the composition for base modification may include: a vector containing a nucleic acid encoding a guide RNA; a CRISPR enzyme having a first binding domain; and a vector containing a nucleic acid encoding an adenosine deaminase having a second binding domain.

[0524] Among them, the composition for base modification may include: a vector containing a nucleic acid encoding a guide RNA; a vector containing a nucleic acid encoding a CRISPR enzyme having a first binding domain; and an adenosine deaminase having a second binding domain.

[0525] Among them, the composition for base modification may include: a vector containing a nucleic acid encoding a guide RNA and a nucleic acid encoding a CRISPR enzyme having a first binding domain; and an adenosine deaminase having a second binding domain.

[0526] Among them, the composition for base modification may include: a CRISPR enzyme having a first binding domain; and a vector containing a nucleic acid encoding a guide RNA and a nucleic acid encoding an adenosine deaminase having a second binding domain.

[0527] [The fourth component of the composition for base substitution: a complex of a guide RNA and a protein for single base substitution]

[0528] The composition for base modification may be a nucleic acid-protein complex. Wherein, the nucleic acid-protein complex may be a guide RNA-protein complex for adenine base substitution. Wherein, the nucleic acid-protein complex may be a guide RNA-protein complex for cytosine base substitution.

[0529] The guide RNA-protein for adenine base substitution complex may be formed by a non-covalent bond between the guide RNA and the protein for adenine base substitution.

[0530] The guide RNA-protein for cytosine base substitution complex may be formed by a non-covalent bond between the guide RNA and the protein for cytosine base substitution.

[0531] The composition for base modification may be of non-carrier type.

[0532] Here, the non-vector may be naked DNA, a DNA complex or mRNA.

[0533] The composition for base modification may be in the form of a carrier.

[0534] A description of the vector has been provided above.

[0535] In one example, a composition for base modification may include a protein for adenine base substitution having a CRISPR enzyme and adenosine deaminase, or a nucleic acid encoding the protein.

[0536] Wherein, the CRISPR enzyme may be a wild-type CRISPR enzyme or a CRISPR enzyme variant.

[0537] Wherein, the CRISPR enzyme variant may be a nickase.

[0538] The adenosine deaminase may be TadA, Tad2p, ADA, ADA1, ADA2, ADAR2, ADAT2, ADAT3 or a variant thereof.

[0539] The protein for adenine base replacement can be composed in the order of N-terminus-[CRISPR enzyme]-[adenosine deaminase]-C-terminus.

[0540] The protein for adenine base replacement can be composed in the order of N-terminus-[adenosine deaminase]-[CRISPR enzyme]-C-terminus.

[0541] Wherein, the protein for adenine base substitution may further comprise a linker domain.

[0542] The composition for base modification may further comprise one or more guide RNAs or nucleic acids encoding the guide RNAs.

[0543] Herein, the composition for base modification may be in the form of a complex of guide RNA-protein for adenine base substitution (ie, ribonucleoprotein (RNP)).

[0544] One aspect of the present invention disclosed in this specification is a protein for single base substitution or a protein comprising the protein Use of a composition for single base substitution.

[0545] The following uses of the protein for single base substitution provided in the present application can be provided.

[0546] The composition for base modification can be used to artificially modify the bases of one or more nucleotides in a target gene.

[0547] (i) The composition for base modification can be used to obtain information about a portion (i.e., an epitope with antibody resistance) that is mutated so as not to identify a substance expressed by the modified nucleic acid sequence by artificially modifying the bases of one or more nucleotides in a specific gene target region.

[0548] (ii) The composition for base modification can be used to obtain information about whether the sensitivity of a substance expressed by the modified nucleotide to a specific drug is reduced or lost by artificially modifying the base of one or more nucleotides in a specific gene target region. That is, the composition for base modification can be used to discover or confirm the region of the target gene that affects a specific drug or the region of the protein (target protein) encoded by the target gene.

[0549] (iii) The composition for base modification can be used to obtain information on whether the sensitivity of a substance expressed by the modified nucleotide to a specific drug is increased by artificially modifying the base of one or more nucleotides in the target region of a specific gene. That is, the composition for base modification can be used to discover or confirm the region of the target gene that affects the increase in sensitivity to a specific drug or the region of the protein (target protein) encoded by the target gene.

[0550] (iv) The composition for base modification can be used to obtain information on whether the substance expressed by the modified nucleic acid sequence has virus resistance by artificially modifying the base of one or more nucleotides in the target region of a specific gene. That is, the composition for base modification can be used to screen for virus resistance genes or virus resistance proteins.

[0551] [First use - epitope screening]

[0552] In one embodiment, the protein for single base substitution or the composition for base substitution comprising the protein can be used for epitope screening.

[0553] "Epitope" refers to a specific part of an antigen that allows the immune system (e.g., antibodies, B cells, or T cells) to recognize the antigen, and is also referred to as an antigenic determinant. Protein epitopes are roughly divided into conformational epitopes and linear epitopes based on the shape or mode of action of the antigen binding site (i.e., the specific part of the antibody that recognizes the epitope). Conformational epitopes consist of discontinuous amino acid sequences of the antigen (i.e., protein). Conformational epitopes react with the three-dimensional structure of the antigen binding site of the antibody. Most epitopes are conformational epitopes. In contrast, linear epitopes react with the one-dimensional structure of the antigen binding site of the antibody, and the amino acids that make up the linear epitope of the antigen are arranged in sequence.

[0554] "Epitope screening" means finding or detecting a specific portion of an antigen that allows the immune system (e.g., antibodies, B cells, or T cells) to recognize the antigen, or means a method, composition, or kit for finding or detecting a specific portion of an antigen that has mutated so that the immune system (e.g., antibodies, B cells, or T cells) cannot recognize the antigen. Among them, the specific portion of the antigen that has mutated so that the immune system (e.g., antibodies, B cells, or T cells) cannot recognize the antigen can be an epitope with antibody resistance.

[0555] The single-base substitution protein or a composition for base substitution comprising the protein can artificially generate single nucleotide polymorphisms (SNPs) to reveal the position of the SNP involved in changes in the body, such as the production, inhibition, increase or decrease of the expression of a specific factor, the generation or loss of a specific function, the presence or absence of a disease, or differences in responsiveness to external drugs or compounds (such as sequences that can be used as epitopes and the position of single nucleotide polymorphisms associated with drug resistance).

[0556] Descriptions of single base substitution proteins and compositions for base substitution are provided above.

[0557] For epitope screening, single base substitution proteins can be used to induce artificial SNPs in the genome.

[0558] Here, artificial SNPs can cause point mutations.

[0559] Point mutation refers to a mutation caused by the modification of a single nucleotide. Point mutations are divided into missense mutations, nonsense mutations, and silent mutations.

[0560] A missense mutation refers to a situation where a mutated codon encodes another amino acid due to one or more modified nucleotides. A nonsense mutation refers to a situation where a codon mutated by one or more modified nucleotides becomes a stop codon. A silent mutation refers to a situation where a codon mutated by one or more modified nucleotides encodes the same amino acid as the unmutated codon.

[0561] In one example, a codon may be changed to a codon encoding a different amino acid by replacing a base A with another base C, T or G. In other words, a missense mutation may occur. For example, when A is replaced by C, leucine may become glycine.

[0562] In another example, a codon may be changed to another codon encoding the same amino acid by replacing a base A with another base C, T or G. In other words, silent mutations may occur. For example, when A is replaced by C, a codon encoding the same proline may be generated.

[0563] In yet another example, when A is replaced by C, T or G to generate TAG, TGC or TAA, one of the stop codons (eg, UAA, UAG and UGA) may be generated. In other words, nonsense mutations may occur.

[0564] Single base substitution proteins can induce or produce artificial substitutions at the base of one or more nucleotides in a gene, causing point mutations.

[0565] The composition for base substitution can induce or generate artificial substitution at the base of one or more nucleotides in a gene, thereby causing a point mutation.

[0566] The induction of artificial substitutions of single bases has been described above.

[0567] The protein encoded by the point mutation caused by the single base substitution protein or the composition for base substitution comprising the protein may be a protein variant in which at least one or more amino acids are changed.

[0568] For example, when a point mutation is generated in a gene encoding EGFR by a single base substitution protein or a composition for base substitution comprising the protein, the protein encoded by the generated point mutation may be an EGFR variant in which at least one amino acid differs from that of the wild-type EGFR.

[0569] One or more modified amino acids may be changed to amino acids having similar properties.

[0570] The hydrophobic amino acid may be changed to a different hydrophobic amino acid. The hydrophobic amino acid may be one of glycine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, tyrosine and tryptophan.

[0571] A basic amino acid can be changed into another basic amino acid. A basic amino acid is one of arginine and histidine.

[0572] Acidic amino acids can be transformed into other acidic amino acids. Acidic amino acids are one of glutamic acid and aspartic acid.

[0573] Polar amino acids can be changed into other polar amino acids. Polar amino acids are one of serine, threonine, asparagine and glutamine.

[0574] One or more modified amino acids may be altered to amino acids with different properties.

[0575] In one example, a hydrophobic amino acid can be changed to a polar amino acid.

[0576] In another example, a hydrophobic amino acid can be changed to an acidic amino acid.

[0577] In one example, a hydrophobic amino acid can be changed to a basic amino acid.

[0578] In another example, a polar amino acid can be changed to a hydrophobic amino acid.

[0579] In one example, an acidic amino acid can be changed to a basic amino acid.

[0580] In another example, a basic amino acid can be changed to an acidic amino acid.

[0581] A protein variant in which at least one amino acid is changed may have a modified three-dimensional structure. When one or more amino acids in an amino acid sequence are changed to amino acids of different properties, the three-dimensional structure may be changed due to a change in the binding strength between the amino acid sequences. When the three-dimensional structure is changed, the conformational epitope may be modified. The modification may be induced using a single base substitution protein provided herein or a composition comprising the protein.

[0582] For example, when a point mutation in a gene encoding ATM is caused by a protein for single base substitution or a composition for base modification comprising the protein, the three-dimensional structure of the ATM variant encoded by the generated point mutation may be partially changed, and thus the conformational epitope may be modified. The single base substitution protein provided by the application or a composition comprising the protein may be used to induce the modification.

[0583] Genes with artificial SNPs can adjust the amount of protein synthesized.

[0584] In one example, the amount of mRNA transcription of a gene having an artificial SNP can be increased or decreased. Thus, the amount of protein synthesis can be increased or decreased.

[0585] In another example, when a regulatory region in a gene contains one or more artificial polymorphisms, the amount of protein synthesized by the gene containing the single nucleotide polymorphism can be increased or decreased.

[0586] Artificial SNPs present in genes can regulate protein activity.

[0587] In one example, one or more artificial SNPs can promote and / or reduce protein activity.

[0588] For example, when an artificial SNP is included in a gene encoding a nuclear membrane receptor, all factors or mechanisms (phosphorylation, acetylation, etc.) involved in a signal transduction process through ligand recognition and binding to the ligand can be activated or decreased.

[0589] For example, when an artificial SNP is included in a gene encoding a specific enzyme, the function of the enzyme (eg, acetylase, ie, the acetylation degree of the target gene) can be promoted or reduced.

[0590] Artificial SNPs present in genes can alter protein function.

[0591] In one example, the original function of a protein can be added and / or inhibited by one or more artificial SNPs.

[0592] For example, when an artificial SNP is included in a gene encoding a nuclear membrane receptor, the ability to recognize and / or bind to a ligand can be suppressed.

[0593] Alternatively, for example, when an artificial SNP is included in a gene encoding a nuclear membrane receptor, some functions of signal transduction to downstream factors through binding to a ligand may be inhibited.

[0594] In one embodiment, the epitope screening method may include:

[0595] a) preparing a cell capable of expressing one or more guide RNAs of one or more guide RNA libraries that complementarily bind to a target nucleic acid sequence present in a target gene, wherein the cell has a target nucleic acid sequence;

[0596] b) introducing a single base substitution protein or a nucleic acid encoding the protein into the cell;

[0597] c) treating the cells of b) with a drug or therapeutic agent;

[0598] d) isolating living cells; and

[0599] e) analyzing the nucleic acid sequence of the target gene in the isolated cells.

[0600] In one embodiment, the epitope screening method may include:

[0601] a) preparing a cell capable of expressing one or more guide RNAs of one or more guide RNA libraries that complementarily bind to a target nucleic acid sequence present in a target gene, wherein the cell has a target nucleic acid sequence;

[0602] b) introducing a protein for single base substitution or a nucleic acid encoding the protein into the cell;

[0603] c) treating the cells of b) with a drug or therapeutic agent;

[0604] d) isolating living cells; and

[0605] e) Obtaining information about the desired SNP from the isolated cells.

[0606] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0607] In one embodiment, the epitope screening method may include:

[0608] a) introducing a protein for single base substitution or a nucleic acid encoding the protein, and one or more guide RNAs of one or more guide RNA libraries or a nucleic acid encoding the guide RNA into a cell having a target nucleic acid sequence;

[0609] b) treating the cells of a) with a drug or therapeutic agent;

[0610] c) separating living cells; and

[0611] d) Obtaining information about the desired SNP from the isolated cells.

[0612] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0613] In another embodiment, the epitope screening method may include:

[0614] a) introducing a composition for base substitution into a cell having a target nucleic acid sequence;

[0615] b) treating the cells of a) with a drug or therapeutic agent;

[0616] c) separating living cells; and

[0617] d) analyzing the nucleic acid sequence of the target gene in the isolated cells.

[0618] In another embodiment, the epitope screening method may include:

[0619] a) introducing a composition for base substitution into a cell having a target nucleic acid sequence;

[0620] b) treating the cells of a) with a drug or therapeutic agent;

[0621] c) separating living cells; and

[0622] d) Obtaining information about the desired SNP from the isolated cells.

[0623] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0624] The guide RNA library may be a group of one or more guide RNAs that complementarily bind to a partial nucleic acid sequence of a target sequence. Although nucleic acids encoding the same guide RNA library are introduced into each cell, the cells may have different guide RNAs. As a result of introducing nucleic acids encoding the same guide RNA library into each cell, the cells may have the same guide RNA.

[0625] The description of guide RNA has been described above.

[0626] The protein for single base substitution may be a protein for adenine substitution or a protein for cytosine substitution.

[0627] The description of the protein for single base substitution, the protein for adenine substitution and the protein for cytosine substitution has been described above.

[0628] The introduction can be performed by one or more methods selected from electroporation, liposome, plasmid, viral vector, nanoparticle and protein translocation domain (PTD) fusion protein.

[0629] The antibody treated as described above may be an antibody that recognizes a protein encoding a target gene (hereinafter referred to as a target protein), and may be an antibody that is capable of reacting with an epitope of the target protein.

[0630] The living cells may be cells that do not react with the antibodies treated as above.

[0631] The isolated cell may be a cell having at least one nucleotide modification in the target gene.

[0632] Here, the modification of one or more nucleotides may be one or more artificial SNPs generated in the target gene.

[0633] Here, one or more artificial SNPs may induce a point mutation.

[0634] In the present application, modification of at least one nucleotide (ie, one or more artificial SNPs) present in the target gene can be confirmed. Thus, information on the target can be obtained.

[0635] Here, the nucleic acid sequence comprising a confirmed modification of at least one nucleotide (ie, one or more artificial SNPs) may be a nucleic acid sequence encoding an epitope.

[0636] [Second use - screening of drug-resistant genes or proteins]

[0637] In another embodiment, the protein for single base substitution or the composition for base substitution comprising the protein can be used for screening of drug-resistant genes or drug-resistant proteins.

[0638] Drug resistance screening can provide information about a region of a target gene or a protein encoding the target gene (hereinafter referred to as a target protein) that affects the reduction or loss of sensitivity to a specific drug. The region can be found or identified using a single base substitution protein provided by the application or a composition comprising the protein.

[0639] The present application provides a method for screening drug-resistant genes or drug-resistant proteins. Hereinafter, in an example of the screening method, specific steps will be described.

[0640] Preparation of sgRNA library

[0641] A guide RNA is prepared that can complementarily bind to a region of a target gene. In one embodiment, a guide RNA is prepared that can complementarily bind to a region of an exon in a target gene. Here, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, 2000 or 3000 or more guide RNAs can be prepared. Here, a plurality of prepared guide RNAs can complementarily bind to a region of an exon in a target gene.

[0642] In one example, the guide RNA comprises a site that is capable of complementary binding to a nucleotide sequence corresponding to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 or more regions of an exon in a target gene.

[0643] Preparation of transformed cells capable of expressing guide RNA

[0644] Prepare the following cells, which can prepare a guide RNA that can complementarily bind to a region of an exon in a target gene. The cells can be transfected with a vector encoding the prepared sgRNA library. Here, the cells can express one or more guide RNAs encoded in the sgRNA library.

[0645] Introduction of single base substitution proteins into transformed cells

[0646] The single-base substitution protein or a nucleic acid encoding the protein is introduced into a transformed cell capable of expressing one or more guide RNAs encoded in the sgRNA library. The single-base substitution protein can induce the substitution of any one or more bases in any base pair target region.

[0647] The single base substitution protein can induce the generation of at least one SNP in the target gene.

[0648] The single base substitution protein can induce the generation of at least one SNP in the target region.

[0649] In one example, when the introduced single base substitution protein is a cytidine substitution protein, at least one cytosine in the target region can be substituted with any base.

[0650] In one example, when the introduced single base substitution protein is an adenine substitution protein, at least one adenine in the target region can be substituted with any base.

[0651] Preparation of transformed cells

[0652] Instead of the steps of preparing transformed cells capable of expressing the guide RNA and introducing the protein for single base substitution into the transformed cells, the method of the present application may be performed by the following steps.

[0653] Prepare cells with target gene.

[0654] The single-base substitution protein and the guide RNA are introduced into the cell. Here, the single-base substitution protein and the guide RNA can be introduced in the form of an RNP complex (ribonucleoprotein complex) or a nucleic acid encoding them, respectively.

[0655] Treating transformed cells with drugs or therapeutic agents

[0656] The transformed cells are treated with a material used as a drug or therapeutic agent (e.g., an antibiotic, an anticancer agent, or an antibody). Here, the treatment drug or therapeutic agent can specifically bind or react with the peptide, polypeptide, or protein expressed by the target gene. Alternatively, the treatment drug or therapeutic agent can reduce or lose the activity or function of the peptide, polypeptide, or protein expressed by the target gene. Alternatively, the treatment drug or therapeutic agent can improve or increase the activity or function of the peptide, polypeptide, or protein expressed by the target gene.

[0657] Transformed cells can be killed by the drug or therapeutic agent.

[0658] Despite treatment with the drug or therapeutic agent, the transformed cells may survive.

[0659] Cell selection

[0660] Despite the treatment with the drug or therapeutic agent, living cells can be isolated, selected or obtained.

[0661] In the living cells, at least one guide RNA and a protein for single base substitution can be used to replace at least one base in the target region of the target gene with an arbitrary base. The cells in which the base in the target gene is replaced with an arbitrary base using a protein for single base substitution can be tolerant to treatment drugs or therapeutic agents.

[0662] Here, the peptide, polypeptide or protein expressed by the target gene of the surviving cells may be resistant to the drug or therapeutic agent.

[0663] Access to information

[0664] The genome of the living cell or the nucleic acid sequence of the target gene can be analyzed to obtain information about sites that are resistant to treatment drugs or therapeutic agents.

[0665] The genome of the living cell or the nucleic acid sequence of the target gene can be analyzed to obtain information on whether the structure or function of the peptide, polypeptide or protein expressed by the target gene is changed. The changed structure or function can play a key role in determining whether drug resistance exists.

[0666] In one embodiment, the method for screening drug resistance genes or drug resistance proteins may include:

[0667] a) preparing cells having a target gene;

[0668] b) introducing into the cell one or more guide RNAs of one or more guide RNA libraries that can complementarily bind to a target nucleic acid sequence or nucleic acids encoding the guide RNAs, and a single base substitution protein or a nucleic acid encoding the protein;

[0669] c) treating the cells of b) with a drug or therapeutic agent;

[0670] d) isolating living cells; and

[0671] e) analyzing the nucleic acid sequence of the target gene in the isolated cells.

[0672] In one embodiment, the method for screening drug resistance genes or drug resistance proteins may include:

[0673] a) preparing cells capable of expressing one or more guide RNAs from one or more guide RNA libraries, wherein the one or more guide RNA libraries can complementarily bind to a target nucleic acid sequence present in a target gene;

[0674] b) introducing a single base substitution protein or a nucleic acid encoding the protein into the cell;

[0675] c) treating the cells of b) with a drug or therapeutic agent;

[0676] d) isolating living cells; and

[0677] e) analyzing the nucleic acid sequence of the target gene in the isolated cells.

[0678] In one embodiment, the method for screening drug resistance genes or drug resistance proteins may include:

[0679] a) preparing cells capable of expressing one or more guide RNAs from one or more guide RNA libraries, wherein the one or more guide RNA libraries can complementarily bind to a target nucleic acid sequence present in a target gene;

[0680] b) introducing a single base substitution protein or a nucleic acid encoding the protein into the cell;

[0681] c) treating the cells of b) with a drug or therapeutic agent;

[0682] d) isolating living cells; and

[0683] e) Obtaining information about the desired SNP from the isolated cells.

[0684] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0685] In one embodiment, the method for screening drug resistance genes or drug resistance proteins may include:

[0686] a) introducing into the cell a single base substitution protein or a nucleic acid encoding the protein, and any one or more guide RNAs of a guide RNA library or a nucleic acid encoding the guide RNA;

[0687] b) treating the cells of a) with a drug or therapeutic agent;

[0688] c) separating living cells; and

[0689] d) Obtaining information about the desired SNP from the isolated cells.

[0690] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0691] In another embodiment, the method for screening drug resistance genes or drug resistance proteins may include:

[0692] a) introducing a composition for base substitution into a cell having a target nucleic acid sequence;

[0693] b) treating the cells of a) with a drug or therapeutic agent;

[0694] c) separating living cells; and

[0695] d) analyzing the nucleic acid sequence of the target gene in the isolated cells.

[0696] In another embodiment, the method for screening drug resistance genes or drug resistance proteins may include:

[0697] a) introducing a composition for base substitution into a cell having a target nucleic acid sequence;

[0698] b) treating the cells of a) with a drug or therapeutic agent;

[0699] c) separating living cells; and

[0700] d) Obtaining information about the desired SNP from the isolated cells.

[0701] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0702] The guide RNA library can be a group of one or more guide RNAs that can complementarily bind to a partial nucleic acid sequence of a target sequence. Although nucleic acids encoding the same guide RNA library are introduced into cells separately, each cell may contain different guide RNAs. As a result of introducing nucleic acids encoding the same guide RNA library into each cell, each cell may have the same guide RNA.

[0703] A description of guide RNA is provided above.

[0704] The single base substitution protein may be a protein for adenine substitution or a protein for cytidine substitution.

[0705] Descriptions of single base substitution proteins, adenine substitution proteins, and cytidine substitution proteins are provided above.

[0706] The introduction can be performed by one or more methods selected from the group consisting of electroporation, liposomes, plasmids, viral vectors, nanoparticles, and protein translocation domain (PTD) fusion proteins.

[0707] The treatment drug as described above can be a material that curbs or inhibits the activity or function of a protein (hereinafter referred to as a target protein) encoded by a target gene. Here, the material can be a biological material (such as RNA, DNA, protein, peptide or antibody) or a non-biological material (such as a compound).

[0708] The treatment drug as described above can be a material that promotes or increases the activity or function of a protein encoded by a target gene (hereinafter referred to as a target protein). Here, the material can be a biological material (such as RNA, DNA, protein, peptide or antibody) or a non-biological material (such as a compound).

[0709] The living cells may be cells having target protein activity, for example, cells having drug resistance whose functions are not changed by the treatment drugs as described above.

[0710] The isolated cell may be a cell having a modification of at least one nucleotide in the target gene.

[0711] Here, the modification of one or more nucleotides may be one or more artificial SNPs generated in the target gene.

[0712] Here, one or more artificial SNPs may induce a point mutation.

[0713] Here, the modification of at least one nucleotide present in the target gene, i.e., one or more artificial SNPs, can be identified. Thus, the desired information can be obtained.

[0714] Here, the nucleic acid sequence comprising the identified modification of at least one nucleotide (ie, one or more artificial SNPs) may be a nucleic acid sequence encoding a region of a protein that affects drug resistance.

[0715] The treatment drug as described above may be an anticancer agent. However, it is not limited to anticancer agents, and includes materials or therapeutic agents for treating all known diseases or disorders.

[0716] In one example, the drug may use a mechanism that interrupts the growth of cancer cells by inhibiting epidermal growth factor receptor (EGFR), inhibiting angiogenesis to cancer cells by blocking vascular endothelial growth factor (VEGF), or inhibiting anaplastic lymphoma kinase.

[0717] In one embodiment, the method for screening drug resistance mutations may include: inducing artificial SNPs on the target gene by introducing a composition for single base substitution into cells containing the target gene, treating the cells with a specific drug, selecting living cells with the desired SNP, and obtaining information about the desired SNP by analyzing the selected cells. Wherein, the desired SNP may be related to the structure or function of the protein expressed by the target gene.

[0718] In one embodiment, the target gene may be an EGFR gene, a VEGF gene or an anaplastic lymphoma kinase gene. However, the present invention is not limited thereto.

[0719] In one embodiment, the treatment drug as described above may be cisplatin, carboplatin, vinorelbine, paclitaxel, docetaxel, gemcitabine, pemetrexed, Iressa, Tarceva, giotrif, Tagrisso, Xalkori, zykadia, alectinib, Alunbrig (brigatinib), Avastin (bevacizumab), Avastin (bevacizumab), keytruda (pembrolizumab), Opdivo (nivolumab), Tecentriq (atezolizumab), Imfinzi (durvalumab) or osimertinib. However, the present invention is not limited thereto.

[0720] In one embodiment, the method for screening EGFR mutant genes with osimertinib resistance can be performed as follows.

[0721] In one embodiment, the method for screening drug-resistant mutants may include: inducing artificial SNPs on the EGFR gene by introducing a composition for single base substitution into cells having the EGFR gene, treating the cells with a drug, selecting living cells having the desired SNP, and obtaining information about the desired SNP by analyzing the selected cells. The desired SNP may be related to the structure or function of EGFR.

[0722] Here, the treated drug may be osimertinib. However, the present invention is not limited thereto, and may be any material for inhibiting or losing the function of EGFR.

[0723] In one embodiment, the method for screening drug-resistant mutants may include: inducing an artificial SNP on the EGFR gene by introducing a composition for single base substitution comprising C797S sgRNA1 and / or C797S sgRNA2 into a cell having the EGFR gene, treating the cell with a drug, selecting living cells having a desired SNP, and obtaining information about the desired SNP by analyzing the selected cells. The desired SNP may be related to the structure or function of EGFR.

[0724] The treatment drug may be osimertinib. However, the present invention is not limited thereto, and the treatment drug may be any material for inhibiting EGFR function or causing EGFR function to be lost.

[0725] According to one embodiment, the EGFR region with osimertinib resistance is identified. It is confirmed that the SNP is induced in the EGFR region with osimertinib resistance by the introduced composition for single base substitution or single base substitution protein.

[0726] That is, by using the single base substitution protein provided by the present application, information on various positions that show resistance to osimertinib can be obtained by substituting cytosine present in the EGFR gene in cells with any base.

[0727] In one embodiment, the present application may provide a method for obtaining EGFR resistance SNP information, the method may include:

[0728] a) introducing into the cell a single base substitution protein or a nucleic acid encoding the protein, and any one or more guide RNAs of a guide RNA library or a nucleic acid encoding the guide RNA;

[0729] b) treating the cells of a) with a drug or therapeutic agent;

[0730] c) separating living cells; and

[0731] d) Obtaining information about the desired SNP from the isolated cells.

[0732] Here, the desired SNP may be associated with the structure and function of the protein expressed by the target gene.

[0733] [Third use - drug sensitivity screening]

[0734] In one embodiment, the single base substitution protein or a composition for base modification comprising the protein can be used for drug sensitization screening.

[0735] "Drug sensitization" refers to a state of hypersensitivity to a specific drug and increased sensitivity to a specific drug. On the other hand, "desensitization" refers to a state of loss of sensitivity to a specific drug and a state of tolerance to a specific drug.

[0736] Drug sensitization screening refers to a method, composition or kit for discovering or confirming a target gene or a region of a protein encoding the target gene (hereinafter referred to as a target protein) that affects increased sensitivity to a specific drug.

[0737] In one embodiment, the drug sensitivity screening method may include:

[0738] a) preparing a cell capable of expressing any one or more guide RNAs of one or more guide RNA libraries, wherein the one or more guide RNA libraries are capable of complementary binding to a target nucleic acid present in a target gene;

[0739] b) introducing a single base substitution protein or a nucleic acid encoding the protein into the cell;

[0740] c) treating the cells of b) with a drug or therapeutic agent;

[0741] d) isolating living cells; and

[0742] e) analyzing the nucleic acid sequence of the target gene in the isolated cells.

[0743] In one embodiment, the drug sensitivity screening method may include:

[0744] a) preparing a cell capable of expressing any one or more guide RNAs of one or more guide RNA libraries, wherein the one or more guide RNA libraries are capable of complementary binding to a target nucleic acid present in a target gene, wherein the cell comprises a target nucleic acid sequence;

[0745] b) introducing a single base substitution protein or a nucleic acid encoding the protein into the cell;

[0746] c) treating the cells of b) with a drug or therapeutic agent;

[0747] d) isolating living cells; and

[0748] e) Obtaining information about the desired SNP from the isolated cells.

[0749] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0750] In one embodiment, the drug sensitivity screening method may include:

[0751] a) introducing a protein for single base substitution or a nucleic acid encoding the protein, and any one or more guide RNAs of a guide RNA library or a nucleic acid encoding the guide RNA into a cell having a target nucleic acid sequence;

[0752] b) treating the cells of a) with a drug or therapeutic agent;

[0753] c) separating living cells; and

[0754] d) Obtaining information about the desired SNP from the isolated cells.

[0755] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0756] In another embodiment, the drug sensitivity screening method may include:

[0757] a) introducing a composition for base substitution into a cell having a target nucleic acid sequence;

[0758] b) treating the cells of a) with a drug or therapeutic agent;

[0759] c) separating living cells; and

[0760] d) analyzing the nucleic acid sequence of the target gene in the isolated cells.

[0761] In another embodiment, the drug sensitivity screening method may include:

[0762] a) introducing a composition for base substitution into a cell having a target nucleic acid sequence;

[0763] b) treating the cells of a) with a drug or therapeutic agent;

[0764] c) separating living cells; and

[0765] d) Obtaining information about the desired SNP from the isolated cells.

[0766] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0767] The guide RNA library may be a group of one or more guide RNAs that can complementarily bind to a portion of the nucleic acid of the target sequence. Although nucleic acids encoding the same guide RNA library are introduced into each cell, the cells may contain different guide RNAs. As a result of introducing nucleic acids encoding the same guide RNA library into each cell, each cell may have the same guide RNA.

[0768] The description of guide RNA has been described above.

[0769] The single base substitution protein may be an adenine substitution protein or a cytosine substitution protein.

[0770] Descriptions of single base substitution proteins, adenine substitution proteins, and cytosine substitution proteins have been described above.

[0771] The introduction can be performed by one or more methods selected from electroporation, liposome, plasmid, viral vector, nanoparticle and protein translocation domain (PTD) fusion protein.

[0772] The treatment drug as described above can be a material that suppresses or inhibits the activity or function of a protein encoded by a target gene (hereinafter referred to as a target protein). Here, the material can be a biological material (such as RNA, DNA, protein, peptide or antibody) or a non-biological material (such as a compound).

[0773] The treatment drug as described above may be a material that promotes or increases the activity or function of the target gene. Here, the material may be a biological material (such as RNA, DNA, protein, peptide or antibody) or a non-biological material (such as a compound).

[0774] The isolated cell may be a cell having a significantly altered activity or function of the target protein (ie, increased drug sensitivity) due to the treatment drug in c).

[0775] Here, the cells having increased drug sensitivity may be living cells treated with the drug.

[0776] The isolated cell may be a cell having a modification of at least one nucleotide in the target gene.

[0777] The modification of one or more nucleotides may be one or more artificial SNPs generated in the target gene.

[0778] Among them, one or more artificial SNPs can induce point mutations.

[0779] The modification of at least one nucleotide present in the target gene (ie, one or more artificial SNPs) can be confirmed. Thus, the desired information can be obtained.

[0780] Here, the nucleic acid sequence comprising the confirmed modification of at least one nucleotide (ie, one or more artificial SNPs) may be a nucleic acid sequence encoding a region of a protein that affects increased drug sensitivity.

[0781] [Fourth use - screening of virus resistance genes or proteins]

[0782] In another embodiment, the single base substitution protein or the composition for base modification comprising the protein can be used to screen for virus resistance genes or proteins.

[0783] In one embodiment, the method of screening for a virus resistance gene or protein may include:

[0784] a) preparing a cell capable of expressing any one or more guide RNAs of one or more guide RNA libraries, wherein the one or more guide RNA libraries are capable of complementary binding to a target nucleic acid present in a target gene;

[0785] b) introducing a protein for single base substitution or a nucleic acid encoding the protein into the cell;

[0786] c) treating the cells of b) with a drug or therapeutic agent;

[0787] d) isolating living cells; and

[0788] e) analyzing the nucleic acid sequence of the target gene in the isolated cells.

[0789] In one embodiment, the method of screening for a virus resistance gene or protein may include:

[0790] a) preparing a cell capable of expressing any one or more guide RNAs of one or more guide RNA libraries, wherein the one or more guide RNA libraries are capable of complementary binding to a target nucleic acid present in a target gene, wherein the cell comprises the target nucleic acid sequence;

[0791] b) introducing a protein for single base substitution or a nucleic acid encoding the protein into the cell;

[0792] c) treating the cells of b) with a drug or therapeutic agent;

[0793] d) isolating living cells; and

[0794] e) Obtaining information about the desired SNP from the isolated cells.

[0795] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0796] In one embodiment, the method of screening for a virus resistance gene or protein may include:

[0797] a) introducing a protein for single base substitution or a nucleic acid encoding the protein, and any one or more guide RNAs of a guide RNA library or a nucleic acid encoding the guide RNA into a cell having a target nucleic acid sequence;

[0798] b) treating the cells of a) with a drug or therapeutic agent;

[0799] c) separating living cells; and

[0800] d) Obtaining information about the desired SNP from the isolated cells.

[0801] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0802] In another embodiment, the method of screening for a viral resistance gene or protein may comprise:

[0803] a) introducing a composition for base substitution into a cell having a target nucleic acid sequence;

[0804] b) treating the cells of a) with a drug or therapeutic agent;

[0805] c) separating living cells; and

[0806] d) analyzing the nucleic acid sequence of the target gene in the isolated cells.

[0807] In another embodiment, the method of screening for a viral resistance gene or protein may comprise:

[0808] a) introducing a composition for base substitution into a cell having a target nucleic acid sequence;

[0809] b) treating the cells of a) with a drug or therapeutic agent;

[0810] c) separating living cells; and

[0811] d) Obtaining information about the desired SNP from the isolated cells.

[0812] Here, the desired SNP may be associated with the structure or function of the protein expressed by the target gene.

[0813] The guide RNA library may be a group of one or more guide RNAs that complementarily bind to a partial nucleic acid sequence of a target sequence. Although nucleic acids encoding the same guide RNA library are introduced into each cell, the cells may have different guide RNAs. As a result of introducing nucleic acids encoding the same guide RNA library into each cell, the cells may have the same guide RNA.

[0814] The description of guide RNA has been described above.

[0815] The protein for single base substitution may be a protein for adenine substitution or a protein for cytosine substitution.

[0816] Descriptions of proteins for single base substitution, proteins for adenine substitution, and proteins for cytosine substitution have been described above.

[0817] The introduction can be performed by one or more methods selected from electroporation, liposome, plasmid, viral vector, nanoparticle and protein translocation domain (PTD) fusion protein.

[0818] The virus treated as described above can be introduced into the cell through interaction with a protein encoding a target gene (hereinafter referred to as a target protein).

[0819] The living cells may be cells that do not interact with the virus treated in c), that is, cells that have virus resistance.

[0820] The isolated cell may be a cell having a modification of at least one nucleotide in the target gene.

[0821] The isolated cell may be a cell having a modification of at least one nucleotide in the target gene.

[0822] The modification of one or more nucleotides may be one or more artificial SNPs generated in the target gene.

[0823] Among them, one or more artificial SNPs can induce point mutations.

[0824] The modification of at least one nucleotide present in the target gene, that is, one or more artificial SNPs, can be confirmed. Thus, the desired information can be obtained.

[0825] The nucleic acid sequence comprising a confirmed modification of at least one nucleotide (ie, one or more artificial SNPs) may be a nucleic acid sequence encoding a region of a protein that is critical for interacting with a virus.

[0826] One aspect of the present invention disclosed in this specification is a method for single base substitution.

[0827] The composition for base substitution can induce or generate an artificial modification in the base of one or more nucleotides in a gene.

[0828] Artificial modifications or substitutions can be induced or generated by a guide RNA-single base substitution protein complex.

[0829] Here, the guide RNA-single base replacement protein complex can be applied to one or more of the following steps: i) targeting a target nucleic acid sequence, ii) shearing the target nucleic acid sequence, iii) deamination of one or more nucleotides in the target nucleic acid sequence, iv) removal of the deaminated base, and v) repair or recovery of the target nucleic acid sequence after base removal. Here, the steps can be performed sequentially or simultaneously, and the order of the steps can be changed.

[0830] i) Targeting of target nucleic acid sequences

[0831] "Target nucleic acid sequence" is a nucleotide sequence present in a target gene or nucleic acid, and specifically, is a partial nucleotide sequence of a target region in a target gene or nucleic acid. Here, "target region" is a site in a target gene or nucleic acid that can be modified by a guide RNA-protein complex for base substitution.

[0832] Hereinafter, "target sequence" may be used as a term to represent two types of nucleotide sequence information. For example, in the case of a target gene, the target nucleic acid sequence may refer to the sequence information of the DNA transcription strand in the target gene, or the nucleotide sequence information of the non-transcription strand.

[0833] For example, the target nucleic acid sequence may refer to 5′-ATCATTGGCAGACTAGTTCG-3′ (SEQ ID NO: 17), which is a partial nucleotide sequence of the target region of target gene A (transcribed chain); and 5′-CGAACTAGTCTGCCAATGAT-3′ (SEQ ID NO: 18), which is a nucleotide sequence complementary to the aforementioned chain (non-transcribed chain).

[0834] The target nucleic acid sequence may be a sequence of 5 to 50 nucleotides.

[0835] In one embodiment, the target nucleic acid sequence can be a 16nt sequence, a 17nt sequence, a 18nt sequence, a 19nt sequence, a 20nt sequence, a 21nt sequence, a 22nt sequence, a 23nt sequence, a 24nt sequence, or a 25nt sequence.

[0836] The target nucleic acid sequence comprises a guide RNA binding sequence or a guide RNA non-binding sequence.

[0837] A "guide RNA binding sequence (guide nucleic acid binding sequence)" is a nucleotide sequence that is partially or completely complementary to a guide sequence contained in the guide domain of the guide RNA, and is capable of complementary binding to a guide sequence contained in the guide domain of the guide RNA. The target nucleic acid sequence and the guide RNA binding sequence are nucleotide sequences that can be changed according to the target gene or nucleic acid (i.e., a target subjected to genetic manipulation or modification), and can be designed in various ways according to the target gene or nucleic acid.

[0838] The "guide RNA non-binding sequence (guide nucleic acid non-binding sequence)" is partially or completely complementary to the guide sequence contained in the guide domain of the guide RNA, and may not be complementary to the guide sequence contained in the guide domain of the guide RNA. In addition, the guide RNA non-binding sequence is a nucleotide sequence that is complementary to the guide RNA binding sequence, and may have complementary binding to the guide RNA binding sequence.

[0839] The guide RNA binding sequence may be a partial nucleotide sequence of the target nucleic acid sequence, and may be one of two nucleotide sequences having two different sequences of the target nucleic acid sequence, i.e., two nucleotide sequences that can complementarily bind to each other. Wherein, the guide RNA non-binding sequence may be a nucleotide sequence in the target nucleic acid sequence other than the guide RNA binding sequence.

[0840] For example, when a partial nucleotide sequence 5′-ATCATTGGCAGACTAGTTCG-3′ (SEQ ID NO: 17) of the target region of target gene A and a nucleotide sequence 5′-CGAACTAGTCTGCCAATGAT-3′ (SEQ ID NO: 18) complementary thereto are used as target nucleic acid sequences, the guide RNA binding sequence may be one of the two target nucleic acid sequences, for example, 5′-ATCATTGGCAGACTAGTTCG-3′ (SEQ ID NO: 17) or 5′-CGAACTAGTCTGCCAATGAT-3′ (SEQ ID NO: 18). Here, when the guide RNA binding sequence is 5′-ATCATTGGCAGACTAGTTCG-3′ (SEQ ID NO: 17), the guide RNA non-binding sequence may be 5′-CGAACTAGTCTGCCAATGAT-3′ (SEQ ID NO: 18), or when the guide RNA binding sequence is 5′-CGAACTAGTCTGCCAATGAT-3′ (SEQ ID NO: 18), the guide RNA non-binding sequence may be 5′-ATCATTGGCAGACTAGTTCG-3′ (SEQ ID NO: 17).

[0841] The guide RNA binding sequence may be a nucleotide sequence selected from the target nucleic acid sequence, i.e., a nucleotide sequence identical to the transcribed strand and a nucleotide sequence identical to the non-transcribed strand. Here, the guide RNA non-binding sequence may be a nucleotide sequence that does not include a nucleotide sequence selected from the guide RNA binding sequence of the target nucleic acid sequence, i.e., a nucleotide sequence identical to the transcribed strand and a nucleotide sequence identical to the non-transcribed strand.

[0842] The guide RNA binding sequence can have the same length as the target nucleic acid sequence.

[0843] The guide RNA non-binding sequence can have the same length as the target nucleic acid sequence or the guide RNA binding sequence.

[0844] The guide RNA binding sequence can be a sequence of 5 to 50 nucleotides.

[0845] In one embodiment, the guide RNA binding sequence can be a 16 nucleotide sequence, a 17nt sequence, a 18nt sequence, a 19nt sequence, a 20nt sequence, a 21nt sequence, a 22nt sequence, a 23nt sequence, a 24nt sequence, or a 25nt sequence.

[0846] The guide RNA non-binding sequence can be a sequence of 5 to 50 nucleotides.

[0847] In one embodiment, the guide RNA non-binding sequence can be a 16 nucleotide sequence, a 17nt sequence, a 18nt sequence, a 19nt sequence, a 20nt sequence, a 21nt sequence, a 22nt sequence, a 23nt sequence, a 24nt sequence, or a 25nt sequence.

[0848] The guide RNA binding sequence may have partial or complete complementary binding with a guide sequence contained in the guide domain of the guide RNA, and the length of the guide RNA binding sequence may be the same as the length of the guide sequence.

[0849] The guide RNA binding sequence can be a nucleotide sequence that is complementary to the guide sequence contained in the guide domain of the guide RNA, such as a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90% or 95% complementarity or complete complementarity.

[0850] In one example, the guide RNA binding sequence may have or include a sequence of 1-8 nucleotides that is not complementary to a guide sequence included in the guide domain of the guide RNA.

[0851] The guide RNA non-binding sequence may have partial or complete sequence homology with a guide sequence contained in the guide domain of the guide RNA, and the length of the guide RNA non-binding sequence may be the same as the length of the guide sequence.

[0852] The guide RNA non-binding sequence can be a nucleotide sequence having homology to the guide sequence contained in the guide domain of the guide RNA, for example, a nucleotide sequence having at least 70%, 75%, 80%, 85%, 90% or 95% sequence homology or complete identity.

[0853] In one example, the guide RNA non-binding sequence can have or include a sequence of 1-8 nucleotides that is not homologous to the guide sequence included in the guide domain of the guide RNA.

[0854] The guide RNA non-binding sequence can be complementary to the guide RNA binding sequence and has the same length as the guide RNA binding sequence.

[0855] The guide RNA non-binding sequence can be a nucleotide sequence that is complementary to the guide RNA binding sequence, such as a nucleotide sequence that has at least 90% or 95% complementarity or complete complementarity.

[0856] In one example, the guide RNA non-binding sequence may have or include a sequence of 1-2 nucleotides that is not complementary to the guide RNA binding sequence.

[0857] In addition, the guide RNA binding sequence can be a nucleotide sequence located near a nucleotide sequence that can be recognized by the CRISPR enzyme.

[0858] In one example, the guide RNA binding sequence may be a sequence of 5-50 consecutive nucleotides located adjacent to the 5' end and / or 3' end of the nucleotide sequence capable of being recognized by the CRISPR enzyme.

[0859] In addition, the guide RNA non-binding sequence may be a nucleotide sequence located near a nucleotide sequence that can be recognized by the CRISPR enzyme.

[0860] In one example, the guide RNA non-binding sequence may be a sequence of 5-50 consecutive nucleotides located adjacent to the 5' end and / or 3' end of the nucleotide sequence capable of being recognized by the CRISPR enzyme.

[0861] "Targeting" refers to the complementary binding of a target nucleic acid sequence present in a target gene or nucleic acid to a guide RNA binding sequence. Here, the complementary binding can be 100% complete complementary binding, or incomplete complementary binding of more than 70% and less than 100%. Therefore, "targeting gRNA" refers to the complementary binding of gRNA to a guide RNA binding sequence in a target nucleic acid sequence present in a target gene or nucleic acid.

[0862] The guide RNA-protein complex for single base substitution can be targeted to a target nucleic acid sequence.

[0863] ii) Cleavage of target nucleic acid sequence

[0864] The guide RNA-single base replacement protein complex can cleave the target nucleic acid sequence.

[0865] Here, when the target nucleic acid sequence is a double-stranded nucleic acid, the shearing may be shearing of both double strands. Alternatively, the shearing may be shearing of one of the double strands.

[0866] Here, when the target nucleic acid sequence is a single-stranded nucleic acid, the cleavage may be single-stranded cleavage.

[0867] Alternatively, the cleavage pattern of the target nucleic acid sequence cleavage may vary depending on the type of CRISPR enzyme that constitutes the guide RNA-single-base substitution protein complex.

[0868] For example, when the CRISPR enzyme constituting the guide RNA-single-base substitution protein complex is a wild-type CRISPR enzyme (such as SpCas9), the cleavage of the target nucleic acid sequence can be the cleavage of both strands of the target nucleic acid sequence.

[0869] In another example, when the CRISPR enzyme constituting the guide RNA-single-base substitution protein complex is a nickase (e.g., NurekinCas9), the cleavage of the target nucleic acid sequence may be the cleavage of one of the double strands of the target nucleic acid sequence.

[0870] iii) Deamination of one or more nucleotides in the target nucleic acid sequence

[0871] The guide RNA-single base replacement protein complex can deaminate the amino (-NH2) group of the base of one or more nucleotides in the target nucleic acid sequence.

[0872] Here, deamination can occur at either cytosine or adenine bases.

[0873] For example, when there are 5 nucleotides containing adenine in the target nucleic acid sequence (here, the 5 nucleotides may be consecutive or discontinuous), the guide RNA-single base substitution protein complex can deaminate the amino (-NH2) groups of all adenines in the 5 nucleotides containing adenine.

[0874] In another example, when there are 8 nucleotides containing cytosine in the target nucleic acid sequence (here, 5 nucleotides may be consecutive or discontinuous), the guide RNA-single-base replacement protein complex can deaminate the amino (-NH2) group of cytosine in 3 of the 8 nucleotides containing cytosine.

[0875] The base to be deaminated may vary depending on the type of deaminase constituting the guide RNA-single-base substitution protein complex.

[0876] For example, when the deaminase constituting the guide RNA-single base substitution protein complex is an adenosine deaminase (e.g., TadA or a TadA variant), deamination can occur at adenine. Here, when the amino (-NH2) group of adenine is deaminated, a keto (=O) group can be formed. Hypoxanthine can be generated by deamination of adenine.

[0877] In another example, when the deaminase constituting the guide RNA-single base replacement protein complex is a cytidine deaminase (e.g., APOBEC1 or an APOBEC1 variant), deamination can occur at cytosine. Here, when the amino (-NH2) group of cytosine is deaminated, a keto (=O) group can be formed. Uracil can be generated by deamination of cytosine.

[0878] iv) Removal of bases by deamination

[0879] The guide RNA-single base substitution protein complex can remove the deaminated base generated in step iii). Here, the removal of the deaminated base can remove all or part of the deaminated base generated in step iii).

[0880] Here, the deaminated base may be deaminated cytosine or adenine.

[0881] Here, the deaminated base may be uracil or hypoxanthine.

[0882] The removal of the deaminated base may vary depending on the type of DNA glycosylase constituting the guide RNA-single-base substitution protein complex.

[0883] For example, when the DNA glycosylase constituting the guide RNA-single base substitution protein complex is alkyladenine DNA glycosylase (AAG) or an AAG variant, the N-glycosidic bond connecting deoxyribose or ribose to the base (deaminated adenine or hypoxanthine) constituting the nucleotide can be hydrolyzed. In addition, an AP site (apurinic / apyrimidinic site) can be formed. The AP site can be located in the DNA (or RNA) in the form of an apurinic or pyrimidinic base either spontaneously or due to DNA (or RNA) damage.

[0884] In another example, when the DNA glycosylase constituting the guide RNA-single base substitution protein complex is uracil DNA glycosylase (UDG or UNG) or a UDG variant, the N-glycosidic bond connecting deoxyribose or ribose to the base (deaminated cytosine or uracil) constituting the nucleotide can be hydrolyzed. In addition, an AP site (apurinic / apyrimidinic site) can be formed.

[0885] v) Repair or restoration of target nucleic acid sequences after base removal

[0886] Repair or restoration of the target nucleic acid sequence after base removal includes repair or restoration of the target nucleic acid sequence after shearing.

[0887] The base-removed target nucleic acid sequence may be a sheared target nucleic acid sequence.

[0888] The cleaved target nucleic acid sequence may be a target nucleic acid sequence in which both the double strands are cleaved.

[0889] Wherein, the sheared target nucleic acid sequence may be a target nucleic acid sequence in which one of the double strands is sheared. Wherein, the sheared strand may be a strand in which a base is removed. Alternatively, the sheared strand may be a strand in which a base is not removed.

[0890] Repair or restoration of the base-removed target nucleic acid sequence can be repair or restoration of any base (ie, adenine, cytosine, guanine, thymine or uracil) at the AP site of one or more base-removed nucleotides in the target nucleic acid sequence.

[0891] For example, the AP site of one or more deaminized adenine removed nucleotides in the target nucleic acid sequence can be repaired to guanine. Alternatively, the AP site of one or more deaminized adenine removed nucleotides in the target nucleic acid sequence can be repaired to cytosine. The AP site of one or more deaminized adenine removed nucleotides in the target nucleic acid sequence can be repaired to thymine. The AP site of one or more deaminized adenine removed nucleotides in the target nucleic acid sequence can be repaired to uracil. The AP site of one or more deaminized adenine removed nucleotides in the target nucleic acid sequence can be repaired to adenine.

[0892] In another example, the AP site of one or more nucleotides from which deaminated cytosines are removed in the target nucleic acid sequence may be repaired to adenine. Alternatively, the AP site of one or more nucleotides from which deaminated cytosines are removed in the target nucleic acid sequence may be repaired to guanine. Alternatively, the AP site of one or more nucleotides from which deaminated cytosines are removed in the target nucleic acid sequence may be repaired to thymine. Alternatively, the AP site of one or more nucleotides from which deaminated cytosines are removed in the target nucleic acid sequence may be repaired to uracil. Alternatively, the AP site of one or more nucleotides from which deaminated cytosines are removed in the target nucleic acid sequence may be repaired to cytosine.

[0893] Artificial modification may occur in the exons or introns, splice sites, regulatory regions (enhancers or repressor regions), 5' end or its adjacent region, or 3' end or its adjacent region of the gene.

[0894] For example, artificial modification can be the replacement of one or more bases in the exon region.For example, in the exon region of a gene, one or more A and / or C can be replaced with different bases (A, C, T, G or U).

[0895] In another example, artificial modification can be the replacement of one or more bases in the intron region.For example, in the intron region of a gene, one or more A and / or C can be replaced with different bases (A, C, T, G or U).

[0896] For example, artificial modification can be the replacement of one or more bases at the splice site.For example, at the splice site of a gene, one or more A and / or C can be replaced with different bases (A, C, T, G or U).

[0897] In another example, artificial modification can be the replacement of one or more bases in a regulatory region (enhancer or repression region). For example, in a regulatory region (enhancer or repression region), one or more A and / or C can be replaced with different bases (A, C, T, G or U).

[0898] The artificial modification may be a modification of the codon sequence of a gene encoding a protein.

[0899] "Codon" refers to one of the genetic codes from a gene that encodes an amino acid. When DNA is transcribed into messenger RNA (mRNA), three nucleotides of this mRNA form each codon. A codon can encode one type of amino acid or a stop codon that terminates amino acid synthesis.

[0900] The artificial modification may be a modification of a protein encoding codon sequence by modification of one or more single bases, and the modified codon sequence may encode the same amino acid or a different amino acid.

[0901] For example, when one or more nucleic acid sequences are changed from C to T, a CCC codon encoding proline may be changed to CUU or CUC encoding leucine, UCC or UCU encoding serine, or UUC or UUU encoding phenylalanine.

[0902] For example, when one or more bases are changed from A to C, ACC or ACA encoding threonine can be changed to CCC or CCA encoding proline.

[0903] For example, when one or more bases are changed from A to G, an AAA codon encoding lysine may become GAA or GAG encoding glutamate, GGA or GGG encoding glycine, or AGA or AGG encoding arginine.

[0904] Example

[0905] The present invention will be further described in detail below in conjunction with embodiments.

[0906] The present invention will be further described in detail below in conjunction with examples. The examples are provided only to more specifically illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not limited to the examples according to the gist of the present invention.

[0907] Experimental methods

[0908] [Example 1]

[0909] Example 1-1: Plasmid construction

[0910] Plasmids were constructed using Gibson Assembly (NEBuilder HiFi DNA Assembly Kit, NEB). Figure 3 (a) Figure 7 (a) and Fig.21After each fragment was amplified by PCR, the DNA fragments amplified by PCR were added to the Gibson Assembly Master Mix and incubated at 50°C for 60 minutes. All plasmids contained a CMV promoter, a p15A replication origin, and a selection marker for the ampicillin resistance gene. Some plasmids contained human codon-optimized WT-Cas9 (P3s-Cas9HC; Addgene plasmid #43945) or its variants.

[0911] Example 1-2: Cell culture and transfection

[0912] (1) HEK293T cells: single base substitution CRISPR protein transfection

[0913] HEK293T cells were incubated in Dulbecco's modified Eagle's medium (DMEM, Welgene) supplemented with 10% FBS and 1% antibiotics at 37°C under 5% CO2. Before transfection, HEK293T cells were plated at 2×10 5 The cells were distributed into 6-well plates at a density of 100 cells / well. Subsequently, 4 μL of Lipofectamine TM 2000 (Thermo Fisher Scientific, 11668019), 1 μg BE3 (WT, bpNLS, xCas-UNG, UNG-xCas, scFv-APO-UNG, or scFv-UNG-APO) and 1 μg sgRNA expression plasmid (hEMX1 GX19 or GX20) were transfected in 200 μL of Opti-MEM medium.

[0914] (2) Hela cells: single base substitution CRISPR protein transfection

[0915] Hela cells were incubated in Dulbecco's modified Eagle's medium (DMEM, Welgene) supplemented with 10% FBS and 1% antibiotics at 37°C under 5% CO2. Before transfection, Hela cells were plated at 2×10 5 The cells were distributed into 6-well plates at a density of 100 cells / well. Then, 4 μL Lipofectamine TM 2000 (Thermo Fisher Scientific, 11668019), 1 μg of base replacement plasmid (BE3 WT, bpNLS BE3, ung-ncas, ncas-ung, or ncas-delta UNG) and 1 μg of sgRNA expression plasmid were transfected in 200 μL of Opti-MEM medium.

[0916] (3) HEK293T cells: single base substitution CRISPR protein transfection

[0917] HEK293T cells were incubated in Dulbecco's modified Eagle's medium (DMEM, Welgene) supplemented with 10% FBS and 1% antibiotics at 37°C under 5% CO2. Before transfection, HEK293T cells were plated at 2×10 5 The cells were distributed into 6-well plates at a density of 100 cells / well. Subsequently, 2 μL of Lipofectamine TM 2000 (Thermo Fisher Scientific, 11668019), 500 ng of base replacement plasmid (bpNLS-UNG-APOBEC-Nureki nCas9-bpNLS) and 500 ng of sgRNA expression plasmid (hEMX1 GX19 or GX20) were transfected in 200 μL of Opti-MEM medium.

[0918] Example 1-3: Design and synthesis of hEMX1 GX19 sgRNA and hEMX1 GX20 sgRNA

[0919] (1) Design and synthesis of sgRNA

[0920] Guide RNAs were designed considering the "NGG" PAM or "NG" PAM of the hEMX gene using the CRISPR RGEN tool (http: / / www.rgenome.net; Park et al., Bioinformatics 31:4014-4016, 2015). The designed guide RNAs were considered to have no 1-base or 2-base mismatches except for the on-target site.

[0921] After annealing and extension of the oligonucleotides used to generate the sgRNA expression plasmids (see Table 1), they were cloned into the Bsa1 site of the pRG2 plasmid.

[0922] [Table 2]

[0923]

[0924] (2) Deep sequencing

[0925] The on-target and off-target sites were amplified to 200 bp to 300 bp by PCR using HiPi Plus DNA polymerase (Elpis-Bio). The PCR products obtained by the above method were sequenced using a MiSeq (Illumina) instrument and amplified using the CRISPR RGEN tool ( www.rgenome.net Substitutions within 5 bp of the CRISPR / Cas9 cleavage site were considered mutations induced by the single-base substitution CRISPR protein.

[0926] Example 1-4: Experimental results

[0927] Using the single base substitution CRISPR protein according to this example, the effect of replacing cytosine (C) with adenine (A), thymine (T), or guanine (G) was confirmed.

[0928] (1) bpNLS verification

[0929] Using BE3 WT and bpNLS BE3 WT in HEK cells, it was confirmed that bpNLS BE3 WT increased the C to T substitution rate compared to BE3 WT (see Figure 7 B).

[0930] (2) Confirmation of base substitution efficiency of single-base substitution CRISPR protein

[0931] 1) Confirmation of C to N (A, T, G) efficiency in Hela cells

[0932] The rate of C to N substitutions in the hEMX1 GX19 sgRNA target was confirmed using a single-base substitution CRISPR protein in HeLa cells.

[0933] As a result of the experiment, it was confirmed that there was almost no difference in the C to G or C to A substitution rate between the UGI-removed ncas-delta UGI and BE3 WT. However, it was confirmed that the C to G or C to A substitution rate of UNG-ncas and ncas-UNG fused with UNG increased compared with BE3 WT (see Figure 8 ). This result confirmed that when UGI was substituted with UNG in BE3 WT, the probability of C to G or C to A substitution increased.

[0934] In addition, in the hEMX1 GX19 sgRNA sequence, the substitution rate of 15C or 16C was confirmed. As an experimental result, UNG-ncas or ncas-UNG was confirmed to have an increased probability of C to G or C to A substitution at 15C or 16C compared with BE3 WT or bpNLS BE3 (see Fig. 9 ).

[0935] It was confirmed that in the hEMX1 GX19 sgRNA sequence, C to G or C to A substitutions were more likely to occur at 15C than at 16C, and that the probability of C to G or C to A substitutions was the highest in single-base substitution CRISPR proteins with UNG-ncas structures (see Fig. 9 ).

[0936] 2) Confirmation of C to N (A, T, G) efficiency in HEK cells

[0937] The C to N substitution rate of single-base substitution CRISPR proteins was confirmed using the hEMX1 GX20 sgRNA target in HEK cells.

[0938] As a result of the experiment, it was confirmed that base substitutions occurred at 13C, 15C, 16C, and 17C in the hEMX1 GX19 sgRNA sequence (see Fig.10 ).

[0939] In addition, it was confirmed that in HEK cells, the C to N substitution rate of ncas-UNG was increased compared with UNG-ncas (see Fig.11 In particular, it was confirmed that C to G or C to A base substitutions occurred more easily in UNG-ncas than in ncas-UNG at 15C, 16C, and 17C (see Fig.11 ).

[0940] In addition, as a result of confirming the efficiency of single base substitution in the hEMX1 target nucleic acid sequence using a single base substitution CRISPR protein complex (i.e., a fused base substitution domain (scFv-APO-UNG or scFv-UNG-APO) with a single-chain variable fragment (scFv)), it was confirmed that base substitution from C to A was more likely to occur at 11C, and base substitution from C to G was more likely to occur at 15C and 16C (see Figure 22 to Figure 24 ).

[0941] (3) Nureki nCas9 verification

[0942] To expand the target sites capable of generating random errors using single-base substitution CRISPR proteins, experiments were performed using Nureki nCas9 with the NGPAM sequence.

[0943] As a result of experiments using hEMX1 GX17 sgRNA and hEMX1 GX20 sgRNA, it was confirmed that they work well in HEK cells. In particular, it was confirmed that C to N substitution occurs in NG PAM (see Fig.12 ).

[0944] [Example 2]

[0945] Example 2-1: Plasmid construction

[0946] Plasmids were constructed using Gibson Assembly (NEBuilder HiFi DNA Assembly Kit, NEB). Figure 4After each fragment was amplified by PCR, the DNA fragments amplified by PCR were added to the Gibson Assembly Master Mix and incubated at 50°C for 60 minutes. All plasmids contain human codon-optimized WT-Cas9 (P3s-Cas9HC; Addgene plasmid #43945), CMV promoter, p15A replication origin, and ampicillin resistance gene selection marker (see Fig.19 and Fig. 20 ).

[0947] Example 2-2: Design and synthesis of sgRNA

[0948] (1) Design of sgRNA

[0949] We selected the extended data from the article titled "Base editing of A,T to C, Gingenomic DNA without DNA cleavage" published in the scientific journal Nature Figure 2 The three sgRNAs shown (see Fig.25 ).

[0950] (2) Synthesis of sgRNA

[0951] Two complementary oligonucleotides are annealed to the PCR amplification template and extended for sgRNA synthesis.

[0952] In vitro transcription of template DNA (excluding the "NGG" at the 3' end of the target sequence) was performed using T7 RNA polymerase (New England Biolabs), RNA was synthesized according to the manufacturer's protocol, and then template DNA was removed using Turbo DNAse (Ambion). Transcribed RNA was purified using the Expin Combo kit (GeneAll) and isopropanol precipitation.

[0953] In this example, the chemically synthesized sgRNA used herein was modified with 2'OMe and phosphorothioate.

[0954] Example 2-3: Cell culture and transfection

[0955] (1) HEK293T cells: single base substitution CRISPR protein transfection

[0956] HEK293T cells were incubated in Dulbecco's modified Eagle's medium (DMEM, Welgene) supplemented with 10% FBS and 1% antibiotics at 37°C under 5% CO2. Before transfection, HEK293T cells were plated at 5×10 4The cells were then plated in 24-well plates at a density of 100 μL. HD transfection reagent (Catalog No. E231A, Promega) was used to transfect 1 μg of each of three different sgRNA expression plasmids with 3 μg of ABE (WT, N-AAG or C-AAG) in 200 μL of Opti-MEM medium.

[0957] (2) Deep sequencing

[0958] The on-target and off-target sites were PCR amplified to a size of 200 bp to 300 bp using HiPi Plus DNA polymerase (Elpis-Bio). The PCR products obtained by the above method were sequenced using a MiSeq (Illumina) instrument and analyzed using the Cas analyzer provided by the CRISPR RGEN tool (www.rgenome.net). Substitutions within 5 bp of the CRISPR / Cas9 cleavage site were considered to be mutations induced by single-base substitution CRISPR proteins.

[0959] Example 2-4: Experimental results

[0960] Adenine base editor (ABE) refers to adenine repair genetic scissors and is a technology that replaces adenine (A) with guanine (G). Alkyl adenine DNA glycosylase (AAG) is an enzyme that removes inosine bases from DNA ( Figure 2 ). The present inventors developed an adenine base replacement protein by inserting the AAG gene at the N-terminus and C-terminus of the ABE WT plasmid to induce random mutation of adenine (A). Cas9 nickase, adenosine deaminase and DNA glycosylase were used to produce fusion proteins in various orders ( Figure 4 ).

[0961] In order to confirm the random mutation of adenine (A), three sgRNAs (sgRNA1, sgRNA2, and sgRNA3) were transfected into HEK 293T cells together with a plasmid having a nucleic acid encoding a base substitution protein (i.e., a modified ABE plasmid). As a result of the experiment, it was confirmed that adenine (A) 14 in the base sequence of sgRNA 1 in HEK293T cells transfected with modified ABE plasmids (N-AAG and C-AAG) was randomly replaced with a different base (thymine, T; cytosine, C; or guanine, G) compared to ABE WT. It was confirmed that adenine (A) 19 and adenine (A) 13 in the base sequence of sgRNA1 were replaced with different bases ( Fig. 27 ), and only in the plasmid with AAG inserted at the N-terminus, adenine 16 and adenine 12 in sgRNA1 were replaced. ( Fig.28). Thus, it was confirmed that random substitution of different base pairs of adenine (A) was induced by inserting AAG into ABE. In addition, when adenine substitution protein was used without considering the order of Cas9 nickase, adenosine deaminase, and DNA glycosylase, random substitution of different base pairs of adenine (A) was confirmed to be induced (see Figure 26 to Figure 28 ).

[0962] [Example 3]

[0963] Single base substitution using the SunTag system

[0964] Example 3-1: Plasmid construction

[0965] Plasmids were constructed using Gibson Assembly (NEBuilder HiFi DNA Assembly Kit, NEB). Figure 5 (a) Figure 5 (b) and Figure 5 After each fragment of (c), the DNA fragments amplified by PCR were added to the Gibson Assembly Master Mix and incubated at 50°C for 15-60 minutes. All plasmids contain human codon-optimized WT-Cas9 (P3s-Cas9HC; Addgene plasmid #43945), CMV promoter, p15A replication origin, and ampicillin resistance gene selection marker.

[0966] Example 3-2: Cell culture and transfection

[0967] PC9 cells were incubated in Rosewell Park Memorial Institute 1640 (RPMI 1640, Welgene) supplemented with 10% FBS and 1% antibiotics at 37°C under 5% CO2. Before transfection, PC9 cells were plated at 2×10 5 The cells were distributed into 24-well plates at a density of 100 cells / mL. TM2000 (Thermo Fisher Scientific, 11668019), 1500 ng of each base replacement plasmid (Apobec-nCas9-UGI and Apobec-Nureki nCas9-UNG) and 500 ng of sgRNA expression plasmid (hEMX1 GX19); 1000 ng of SunTag plasmid (GCN4-nCas9) and 1000 ng of each ScFv plasmid (ScFv-Apobec-UNG and ScFv-UNG-Apobec); or 500 g of sgRNA expression plasmid (hEMX1 GX19) were transfected in 200 μL of Opti-MEM medium.

[0968] Example 3-3: Deep Sequencing

[0969] The on-target and off-target sites were amplified by PCR to a size of 200 bp to 300 bp using HiPi Plus DNA polymerase (Elpis-Bio). The PCR products obtained by the above method were sequenced using a MiSeq (Illumina) instrument and analyzed using the Cas analyzer provided by the CRISPR RGEN tool (www.rgenome.net). Substitutions within 10 bp of the sgRNA sequence region were considered to be mutations induced by single-base substitution CRISPR proteins.

[0970] Example 3-4: Experimental results

[0971] Confirm the C to N substitution rate in PC9 cells using the single base substitution protein.

[0972] Using the SunTag system, the induction of random mutations was increased by maximizing the UNG effect with only one nCas9. The results confirmed that ScFv-UNG-Apobec was able to have a single base substitution efficiency similar to WT and induce random base substitutions (C to T or A or G) (see Fig.13 ).

[0973] [Example 4]

[0974] Induction of EGFR C797S mutation using single-base substitution CRISPR protein and confirmation of osimertinib resistance

[0975] Example 4-1: PC9 cells: transduction of single-base substitution CRISPR protein and drug culture

[0976] PC9 cells were incubated in Rosewell Park Memorial Institute 1640 (RPMI 1640, Welgene) supplemented with 10% FBS and 1% antibiotics at 37°C under 5% CO2. Before transfection, PC9 cells were plated at 3×10 6 The density of cells is distributed into 15cm 2 Then, 4 μL Lipofectamine TM 2000 (Thermo Fisher Scientific, 11668019), 5 μg of each of two different sgRNA expression plasmids were transfected with 15 μg N-UNG in 3 mL of Opti-MEM medium. Three days after transfection, the plasmid was treated with 4 μg / mL blasticidin for 7 days. After obtaining a stable cell line by sufficient antibiotic culture, the cells were treated with 100nM osimertinib (Selleckchem, S5078) for 20 days, which is a targeted therapeutic agent for non-small cell lung cancer. A positive control experiment was performed using sgRNAs (C797S sgRNA 1 (SEQ ID NO: 21) and C797S sgRNA 2 (SEQ ID NO: 22) capable of producing C797S mutants known to be resistant to osimertinib. It was confirmed that the C797S mutant was enriched using the screening system.

[0977] Example 4-2: Deep sequencing

[0978] The on-target and off-target sites were PCR amplified to a size of 200 bp to 300 bp using HiPi Plus DNA polymerase (Elpis-Bio). The PCR products obtained by the above method were sequenced using a MiSeq (Illumina) instrument and analyzed using the BE analyzer provided by the CRISPR RGEN tool (www.rgenome.net). Substitutions within 10 bp of the sgRNA sequence site were considered to be mutations induced by the single-base substitution CRISPR protein.

[0979] Example 4-3: Experimental results

[0980] Osimertinib, a third-generation EGFR tyrosine kinase inhibitor (TKI), is used as a therapeutic agent for patients with EGFR T790M-positive non-small cell lung cancer who are resistant to second-generation drugs. By inducing random base substitutions of cytosines in the target sgRNA sequence through N-UNG, mutants that are resistant to specific drugs are screened.

[0981] By using the known mutant C797S that is resistant to osimertinib as a positive control, it was confirmed that the corresponding tool was effective. When the base substitution of C15 to G in C797S sgRNA 1 or the base substitution of C13 to G in C797S sgRNA 2 occurred, the amino acid 797 cysteine ​​of EGFR was changed to serine. As a result of the experiment, although only 10% of 15C and 13C were replaced by G by C797S sgRNA 1 and divalent N-UNG in the group treated with blasticidin alone, it was confirmed that the portion of C changed to G increased by 50% or 80% in the group treated with osimertinib (see Fig.30 ).

[0982] [Example 5]

[0983] Preparation of transformed cells and screening of drug-resistant mutants by introduction of EGFR sgRNA library

[0984] Example 5-1: Design and synthesis of EGFR sgRNA library

[0985] A total of 1803 sgRNAs from 27 exons of the epidermal growth factor receptor (EGFR) gene were designed using the CRISPR RGEN tool (www.rgenome.net). After adding CACCG to the 5' end of the forward oligonucleotide sequence, AAAC to the 5' end of the reverse oligonucleotide sequence, and C to the 3' end of the designed 1803 sgRNA oligonucleotide pool, Twist Bioscience was commissioned to synthesize them.

[0986] Example 5-2: Preparation of EGFR sgRNA library plasmid

[0987] The synthesized EGFR sgRNA oligonucleotide pool was reacted at 95°C for 5 minutes and annealed by gradually cooling down to 25°C. Subsequently, the PiggyBac transposon backbone vector and the EGFR sgRNA oligonucleotide pool cut with the Bsa1 restriction enzyme were ligated with T4 ligase. The ligation reaction solution was inserted into the Endura TM DUO electrocompetent cells (Lucigen, catalog number 60242-2). The transformed E. coli cells were evenly spread on LB medium supplemented with ampicillin and incubated overnight at 37°C. EGFR sgRNA library plasmids were obtained from E. coli colonies using NuceloBond Xtra Midi EF (Macherey-Nagel, catalog number 740420.50).

[0988] Example 5-3: Cell culture

[0989] PC9 cells were incubated in Rosewell Park Memorial Institute 1640 (RPMI 1640, Welgene) supplemented with 10% FBS and 1% antibiotics at 37°C under 5% CO2.

[0990] Example 5-4: Preparation of transformed cells using the PiggyBac transposon

[0991] Cells expressing EGFR sgRNA were prepared by applying a gene delivery system (i.e., PiggyBac transposon) to PC9 cells. Before transformation, PC9 cells were cultured at 4 × 10 6 The cells were distributed into T175 flasks at a density of 100 cells / mL. Subsequently, 40 μL of Lipofectamine TM 2000 (Thermo Fisher Scientific, 11668019), the piggyBac transposon vector and the transposase expression vector were transfected in 3 mL of Opti-MEM medium at a ratio of 1:5. The next day, the cells were treated with 2 μg / mL puromycin and incubated for 7 days. Stable cell lines were obtained by subculturing with sufficient antibiotics.

[0992] Example 5-5: Transfection of single-base substitution CRISPR protein and screening of drug-resistant mutants

[0993] When using Lipofectamine TM About 18 to 24 hours before transfection, 4 × 10 6 Transformed PC9 cells were distributed into T175 flasks. Subsequently, 20 μg of N-UNG was transfected. Three days after transfection, the cells were treated with 4 μg / mL blasticidin as an antibiotic and incubated for 7 days. When stable cells were obtained through sufficient antibiotic culture, 4×10 6 The cells were distributed into T175 flasks. Subsequently, the cells were incubated with 100 nM osimertinib (Selleckchem, S5078), a non-small cell lung cancer therapeutic agent, for 20 days to obtain resistant mutant cells.

[0994] Example 5-6: Deep Sequencing

[0995] HiPi Plus DNA polymerase (Elpis-Bio) was used to PCR amplify the on-target and off-target sites to a size of 200 bp to 300 bp. The PCR products obtained by the above method were sequenced using a MiSeq (Illumina) instrument, and the resulting 1803 EGFR sgRNA sequences were commissioned for analysis.

[0996] Example 5-7: Experimental results

[0997] In PC9 cells expressing EGFR sgRNA, cytosines in the sgRNA were randomly replaced with N-UNG, and the cells were then incubated in medium supplemented with osimertinib, followed by live cell analysis (see Fig.29 and Fig.30 ). Fig.31 Shown are the results of live cell analysis by randomly replacing cytosine in sgRNA with N-UNG in PC9 cells capable of expressing EGFR sgRNA and incubating the cells in medium supplemented with osimertinib. <110> Turkin Co., Ltd. <120> Single base substitution protein and composition containing the same <130> OPP19-003-PCT <150> US 62 / 851,372 <151> 2019-05-22 <150> US 62 / 898,094 <151> 2019-09-10 <160> 92 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Homo sapiens <400> 1 aagaagaaga cgagcctgag 20 <210> 2 <211> twenty three <212> DNA <213> Homo sapiens <400> 2 gaacacaaag catagactgc ggg 23 <210> 3 <211> twenty three <212> DNA <213> Homo sapiens <400> 3 gagtatgagg catagactgc agg 23 <210> 4 <211> twenty three <212> DNA <213> Homo sapiens <400> 4 gtcaagaaag cagagactgc cgg 23 <210> 5 <211> 23 <212> DNA <213> Homo sapiens <400> 5 gagcaaagac aatagactgt agg 23 <210> 6 <211> 23 <212> DNA <213> Homo sapiens <400> 6 gatgagataa tgatgagtca ggg 23 <210> 7 <211> 23 <212> DNA <213> Homo sapiens <400> 7 ggattgaccc aggccagggc tgg 23 <210> 8 <211> 23 <212> DNA <213> Homo sapiens <400> 8 gaatactaag catagactcc agg 23 <210> 9 <211> 23 <212> DNA <213> Homo sapiens <400> 9 gtaaacaaag catagactga ggg 23 <210> 10 <211> 23 <212> DNA <213> Homo sapiens <400> 10 gaagaccaag gatagactgc tgg 23 <210> 11 <211> 23 <212> DNA <213> Homo sapiens <400> 11 gaacataaag aatagaatga tgg 23 <210> 12 <211> 23 <212> DNA <213> Homo sapiens <400> 12 ggacaggcag catagactgt ggg 23 <210> 13 <211> 23 <212> DNA <213> Homo sapiens <400> 13 gtagaaaaag tatagactgc agg 23 <210> 14 <211> 23 <212> DNA <213> Homo sapiens <400> 14 gaagatagag aatagactgc tgg 23 <210> 15 <211> 23 <212> DNA <213> Homo sapiens <400> 15 ggctaaagac catagactgt ggg 23 <210> 16 <211> 23 <212> DNA <213> Homo sapiens <400> 16 gggaataaat catagaatcc tgg 23 <210> 17 <211> 14 <212> DNA <213> Homo sapiens <400> 17 kraatkkaga kkkk 14 <210> 18 <211> twenty three <212> DNA <213> Homo sapiens <400> 18 gacaaagagg aagagagacg ggg 23 <210> 19 <211> twenty three <212> DNA <213> Homo sapiens <400> 19 acacacacac ttagaatctg tgg 23 <210> 20 <211> twenty three <212> DNA <213> Homo sapiens <400> 20 cacacacact tagaatctgt ggg 23 <210> twenty one <211> 19 <212> DNA <213> Homo sapiens <400> twenty one gtacgggaag ccgacggag 19 <210> twenty two <211> 19 <212> DNA <213> Homo sapiens <400> twenty two acgggaagcc gacggagga 19 <210> twenty three <211> 7 <212> PRT <213> Artificial sequence <220> <223> NLS sequence from SV40 virus <400> twenty three Pro Lys Lys Lys Arg Lys Val 1 5 <210> twenty four <211> 16 <212> PRT <213> Artificial sequence <220> <223> NLS sequences derived from nucleoplasmic proteins <400> twenty four Lys Arg Pro Ala Ala Thr Lys Lys Ala Gly Gln Ala Lys Lys Lys Lys 1 5 10 15 <210> 25 <211> 9 <212> PRT <213> Artificial sequence <220> <223> Bipartite NLS sequence derived from c-myc <400> 25 Pro Ala Ala Lys Arg Val Lys Leu Asp 1 5 <210> 26 <211> 11 <212> PRT <213> Artificial sequence <220> <223> Bipartite NLS of nucleoplasmic proteins <400> 26 Arg Gln Arg Arg Asn Glu Leu Lys Arg Ser Pro 1 5 10 <210> 27 <211> 38 <212> PRT <213> Artificial sequence <220> <223> hRNPA1 M9 NLS <400> 27 Asn Gln Ser Ser Asn Phe Gly Pro Met Lys Gly Gly Asn Phe Gly Gly 1 5 10 15 Arg Ser Ser Gly Pro Tyr Gly Gly Gly Gly Gln Tyr Phe Ala Lys Pro 20 25 30 Arg Asn Gln Gly Gly Tyr 35 <210> 28 <211> 42 <212> PRT <213> Artificial sequence <220> <223> Importin alpha IBB domain sequence <400> 28 Arg Met Arg Ile Glx Phe Lys Asn Lys Gly Lys Asp Thr Ala Glu Leu 1 5 10 15 Arg Arg Arg Arg Val Glu Val Ser Val Glu Leu Arg Lys Ala Lys Lys 20 25 30 Asp Glu Gln Ile Leu Lys Arg Arg Asn Val 35 40 <210> 29 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Myoma T protein sequence <400> 29 Val Ser Arg Lys Arg Pro Arg Pro 1 5 <210> 30 <211> 8 <212> PRT <213> Artificial sequence <220> <223> Myoma T protein sequence <400> 30 Pro Pro Lys Lys Ala Arg Glu Asp 1 5 <210> 31 <211> 7 <212> PRT <213> Artificial sequence <220> <223> Human p53 sequence <400> 31 Pro Pro Lys Lys Lys Pro Leu 1 5 <210> 32 <211> 12 <212> PRT <213> Artificial sequence <220> <223> Mouse c-abl IV sequence <400> 32 Ser Ala Leu Ile Lys Lys Lys Lys Lys Lys Met Ala Pro 1 5 10 <210> 33 <211> 5 <212> PRT <213> Artificial sequence <220> <223> NS1 sequence of influenza virus <400> 33 Asp Arg Leu Arg Arg 1 5 <210> 34 <211> 7 <212> PRT <213> Artificial sequence <220> <223> NS1 sequence of influenza virus <400> 34 Pro Lys Gln Lys Lys Arg Lys 1 5 <210> 35 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Hepatitis viruses <400> 35 Arg Lys Leu Lys Lys Lys Ile Lys Lys Leu 1 5 10 <210> 36 <211> 10 <212> PRT <213> Artificial sequence <220> <223> Sequence of mouse Mx1 protein <400> 36 Arg Glu Lys Lys Lys Phe Leu Lys Arg Arg 1 5 10 <210> 37 <211> 20 <212> PRT <213> Artificial sequence <220> <223> Human ADP ribose <400> 37 Lys Arg Lys Gly Asp Glu Val Asp Gly Val Asp Glu Val Ala Lys Lys 1 5 10 15 Lys Ser Lys Lys 20 <210> 38 <211> 17 <212> PRT <213> Homo sapiens <400> 38 Arg Lys Cys Leu Gln Ala Gly Met Asn Leu Glu Ala Arg Lys Thr Lys 1 5 10 15 Lys <210> 39 <211> 20 <212> DNA <213> Artificial sequence <220> <223> GX19 <400> 39 gagtccgagc agaagaagaa 20 <210> 40 <211> 20 <212> DNA <213> Artificial sequence <220> <223> GX20 <400> 40 tgcccctccc tccctggccc 20 <210> 41 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Nureki sgRNA 1 <400> 41 gaggacaaag tacaaacggc 20 <210> 42 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Nureki sgRNA 2 <400> 42 gggctcccat cacatcaacc 20 <210> 43 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Nureki sgRNA 3 <400> 43 ggccccagtg gctgctctgg 20 <210> 44 <211> 20 <212> DNA <213> Artificial sequence <220> <223> Nureki sgRNA 4 <400> 44 gctttaccca gttctctggg 20 <210> 45 <211> 77 <212> RNA <213> Artificial sequence <220> <223> Sequences that form a complex with the spCas9 protein <400> 45 guuuuagucc cugaaaaggg acuaaaauaa agaguuugcg ggacucugcg ggguuacaau 60 ccccuaaaac cgcuuuu 77 <210> 46 <211> 76 <212> RNA <213> Artificial sequence <220> <223> Sequences that form a complex with cjCas9 protein <400> 46 guuuuagagc uagaaauagc aaguuaaaau aaggcuaguc cguuaucaac uugaaaaagu 60 ggcaccgagu cggugc 76 <210> 47 <211> 8 <212> PRT <213> Artificial sequence <220> <223> PAM of cjCas9 protein <400> 47 Asn Asn Asn Asn Arg Tyr Ala Cys 1 5 <210> 48 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 48 guaacacgag cagggguccu 20 <210> 49 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 49 ccccgccugu aacacgagca 20 <210> 50 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 50 accccgccug uaacacgagc 20 <210> 51 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 51 aggaccccug cucguguuac 20 <210> 52 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 52 accccugcuc guguuacagg 20 <210> 53 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 53 caccacgagu cuagacucug 20 <210> 54 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 54 ggacuucucu caauuuucua 20 <210> 55 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 55 ccuacgaacc acugaacaaa 20 <210> 56 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 56 ccauuuguuc agugguucgu 20 <210> 57 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 57 cauuuguuca gugguucgua 20 <210> 58 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 58 ggguugcguc agcaaacacu 20 <210> 59 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 59 uuugcugacg caacccccac 20 <210> 60 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 60 uccgcaguau ggaucggcag 20 <210> 61 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 61 aggaguuccg caguauggau 20 <210> 62 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 62 uccucugccg auccauacug 20 <210> 63 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 63 cgucccgcgc aggauccagu 20 <210> 64 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 64 ccgcgggauu cagcgccgac 20 <210> 65 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 65 uccgcgggau ucagcgccga 20 <210> 66 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 66 cccgucggcg cugaaucccg 20 <210> 67 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 67 guaaagagag gugcgccccg 20 <210> 68 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 68 ggggcgcacc ucucuuuacg 20 <210> 69 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 69 gaagcgaagu gcacacgguc 20 <210> 70 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 70 ggucuccaug cgacgugcag 20 <210> 71 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 71 aaugucaacg accgaccuug 20 <210> 72 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 72 aggaggcugu aggcauaaau 20 <210> 73 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 73 cggaaguguu gauaagauag 20 <210> 74 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 74 ccggaagugu ugauaagaua 20 <210> 75 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 75 gcgagggagu ucuucuucua 20 <210> 76 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 76 gaccuucguc ugcgaggcga 20 <210> 77 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 77 gauugagacc uucgucugcg 20 <210> 78 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 78 cucccucgcc ucgcagacga 20 <210> 79 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 79 gauugagauc uucugcgacg 20 <210> 80 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 80 gucgcagaag aucucaaucu 20 <210> 81 <211> 20 <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 81 ucgcagaaga ucucaaucuc 20 <210> 82 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 82 ugucaacaag aaaaaccccg cc 22 <210> 83 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 83 aagcccuacg aaccacugaa ca 22 <210> 84 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 84 uuaccaauuu ucuuuugucu uu 22 <210> 85 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 85 acgucccgcg caggauccag uu 22 <210> 86 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 86 gugcacacgg uccggcagau ga 22 <210> 87 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 87 gugccuucuc aucugccgga cc 22 <210> 88 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 88 cgacgugcag aggugaagcg aa 22 <210> 89 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 89 ugcgacgugc agaggugaag cg 22 <210> 90 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 90 gaccgugugc acuucgcuuc ac 22 <210> 91 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 91 auguccaugc cccaaagcca cc 22 <210> 92 <211> twenty two <212> RNA <213> Artificial sequence <220> <223> Sp20-viHBV-B <400> 92 gaccaccaaa ugccccuauc uu 22

Claims

1. A fusion protein for single base substitution or a nucleic acid encoding the fusion protein, wherein: The fusion protein comprises: (a) Cas9 nickase; (b) APOBEC; and (c) uracil DNA glycosylase, Arrange them in the order of N-terminus-[uracil DNA glycosylase]-[APOBEC]-[Cas9 nickase]-C-terminus, Wherein, the uracil DNA glycosylase is an enzyme that destroys the N-glycosidic bond of uracil, The fusion protein for single base substitution can induce substitution of any base other than cytosine for cytosine, wherein the cytosine is contained in one or more nucleotides in the target nucleic acid sequence.

2. The fusion protein for single base substitution according to claim 1 or a nucleic acid encoding the fusion protein, in, The fusion protein for single base substitution further comprises one or more nuclear localization sequences (NLS).

3. The fusion protein for single base substitution according to claim 1 or a nucleic acid encoding the fusion protein, in, The Cas9 nickase is derived from one selected from the group consisting of: a Cas9 protein derived from Streptococcus pyogenes, a Cas9 protein derived from Campylobacter jejuni, a Cas9 protein derived from Streptococcus thermophilus, a Cas9 protein derived from Staphylococcus aureus, and a Cas9 protein derived from Neisseria meningitidis.

4. The fusion protein for single base substitution according to claim 3 or a nucleic acid encoding the fusion protein, in, The Cas9 nickase is characterized by inactivation of either a RuvC domain or a HNH domain.

5. The fusion protein for single base substitution according to claim 1 or a nucleic acid encoding the fusion protein, in, The fusion protein for single base substitution comprises a linking portion, which is inserted between one selected from (a), (b) and (c) and another selected from (a), (b) and (c).

6. A vector comprising a nucleic acid encoding the fusion protein for single base substitution according to any one of claims 1 to 5.

7. A complex for single-base substitution, the complex for single-base substitution comprising: (a) Cas9 nickase; (b) APOBEC; and (c) uracil DNA glycosylase; Their order is N-terminus-[uracil DNA glycosylase]-[APOBEC]-[Cas9 nickase]-C-terminus, in, The uracil DNA glycosylase is an enzyme that destroys the N-glycosidic bond of uracil. Wherein, the complex for single base substitution further comprises: (d) two or more binding domains, The complex for single base substitution can induce substitution of any base other than cytosine for cytosine, wherein the cytosine is contained in one or more nucleotides in the target nucleic acid sequence.

8. The complex for single base substitution according to claim 7, in, The Cas9 nickase, the APOBEC, and the uracil DNA glycosylase are each connected to one or more binding domains, Wherein, the Cas9 nickase, the APOBEC, and the uracil DNA glycosylase form the complex through the interaction between the binding domains.

9. The complex for single base substitution according to claim 8, in, Any one of the Cas9 nickase, the APOBEC, and the uracil DNA glycosylase is connected to the first binding domain and the second binding domain, wherein the first binding domain and the binding domain of another component are capable of forming a pair, and the second binding domain and the binding domain of yet another component are capable of forming a pair, wherein the complex is formed by the formation of the pair.

10. The complex for single base substitution according to claim 7, in, The complex for single base substitution comprises: (i) a first fusion protein, the first fusion protein comprising two components selected from the group consisting of the Cas9 nickase, the APOBEC and the uracil DNA glycosylase and a first binding domain, and (ii) a second fusion protein, the second fusion protein comprising a component not selected in (i) selected from the group consisting of the Cas9 nickase, the APOBEC and the uracil DNA glycosylase, and a second binding domain, wherein the first binding domain and the second binding domain are capable of forming a pair, and wherein the complex is formed by the formation of the pair.

11. The complex for single base substitution according to claim 10, in, The complex for single base substitution comprises: (i) the first fusion protein, the first fusion protein comprising the APOBEC, the uracil DNA glycosylase and the first binding domain, and (ii) the second fusion protein, the second fusion protein comprising the Cas9 nickase and the second binding domain.

12. The complex for single base substitution according to claim 7, in, The binding domain is any one of a FRB domain, a FKBP dimerization domain, an intein, an ERT domain, a VPR domain, a GCN4 peptide and a single-chain variable fragment (scFv), or any one of a domain that forms a heterodimer.

13. The complex for single base substitution according to claim 9 or 10, in, The pair is selected from any one of the following: (i) FRB domain and FKBP dimerization domain; (ii) a first intein and a second intein; (iii) ERT domain and VPR domain; (iv) GCN4 peptide and single-chain variable fragment (scFv); and (v) The first domain and the second domain form a heterodimer.

14. The complex for single base substitution according to claim 13, in, The pair is the GCN4 peptide and the single chain variable fragment (scFv).

15. The complex for single base substitution according to claim 11, in, The first binding domain is a single chain variable fragment (scFv), Wherein, the second fusion protein further comprises one or more binding domains, wherein the binding domain further comprised in the second fusion protein is a GCN4 peptide, and Wherein, two or more of the first fusion proteins form the complex by interacting with any one of the GCN4 peptides.

16. A composition for single base substitution, comprising: (a) a guide RNA or a nucleic acid encoding the guide RNA, and (b) i) the fusion protein for single base substitution according to any one of claims 1 to 5 or the nucleic acid encoding the fusion protein, or ii) the complex for single base substitution according to any one of claims 7 to 15, in, The guide RNA binds complementary to the target nucleic acid sequence. Wherein, the target nucleic acid sequence bound to the guide RNA is 15bp-25bp, The fusion protein for single base substitution or the complex for single base substitution can induce substitution of any base other than cytosine for cytosine, wherein the cytosine is contained in one or more nucleotides in the target region.

17. The composition for single base substitution according to claim 16, in, The composition for single base substitution comprises one or more vectors.

18. A method for single base substitution, the method comprising: contacting (i) and (ii) with a target region comprising a target nucleic acid sequence in vitro or ex vivo, (i) guide RNA, (ii) the fusion protein for single base substitution according to any one of claims 1 to 5 or the complex for single base substitution according to any one of claims 7 to 15, Wherein, the guide RNA binds complementary to the target nucleic acid sequence. Wherein, the target nucleic acid sequence bound to the guide RNA is 15bp-25bp, The fusion protein for single base substitution or the complex for single base substitution can induce substitution of any base other than cytosine for cytosine, wherein the cytosine is contained in one or more nucleotides in the target region.

19. Use of the fusion protein for single base substitution according to any one of claims 1 to 5 or the complex for single base substitution according to any one of claims 7 to 15 in preparing a kit for single base substitution, the kit comprising: (i) guide RNA, (ii) the fusion protein for single base substitution according to any one of claims 1 to 5 or the complex for single base substitution according to any one of claims 7 to 15, in, contacting (i) and (ii) with a target region comprising a target nucleic acid sequence in vitro or ex vivo, Wherein, the guide RNA binds complementary to the target nucleic acid sequence. Wherein, the target nucleic acid sequence bound to the guide RNA is 15bp-25bp, The fusion protein for single base substitution or the complex for single base substitution can induce substitution of any base other than cytosine for cytosine, wherein the cytosine is contained in one or more nucleotides in the target region.

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