Animal pathogen-derived polypeptides and uses thereof for genetic engineering

Abstract

To provide polypeptides, compositions, and methods for use thereof for genetic and epigenomic engineering.SOLUTION: Provided are polypeptides, compositions, and methods for use thereof for genetic and epigenomic engineering, including, genome editing and gene regulation. These polypeptides and compositions include nucleic acid binding domains that bind to a target nucleic acid of interest. The nucleic acid binding domains include repeat units derived from repeat units identified in proteins from animal pathogens such as bacterium of an order Legionellales and species Legionella and Francisella.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Application No. 62, filed April 18, 2018, pursuant to 35 U.S.C. § 119(e). No. 62 / 690,905, filed on August 8, 2018; No. 62 / 716,223, filed Sep. 28, 2018; and U.S. Provisional Application No. 62 / 738,822, filed Sep. 28, 2018. No. 5, and claims priority to U.S. Provisional Application No. 62 / 819,237, filed March 15, 2019. No. 6,399,433, the disclosures of which are incorporated herein by reference in their entireties. Incorporation by reference of sequence listing provided as a text file A text file titled "ALTI-720WO Se" was created on April 18, 2019 and has a size of 240 KB. A sequence listing is provided herein as "qList_ST25.txt". The entire contents are incorporated herein by reference.

[0002] Genome editing and gene regulation technologies involve the use of nucleic acid binding domains that bind to target nucleic acids. nothing. [Background technology]

[0003] Nucleic acid binding domains are useful for binding RNA guides such as those used in CRISPR-Cas 9-mediated gene editing. d domains, zinc finger proteins, TALE proteins, and meganuclei It contains domains that are unique to proteins such as ribozymes. Due to the importance of genome engineering, there are many desirable features such as ease of production, target specificity, and versatility. There is a need for nucleic acid binding domains with specific characteristics. Summary of the Invention

[0004] Polypeptides, their compositions, and methods for genetic and epigenomic engineering - Patents.com Methods are provided herein for genome editing using the polypeptides and compositions. and gene regulation, the polypeptides including Legionella or Legionella or It is derived from nucleic acid-binding proteins identified in animal pathogens, such as bacteria from the genus Francisella. The nucleic acid binding domain includes

[0005] In various aspects, the present disclosure provides non-natural modular polypeptides derived from animal pathogen proteins. The present invention provides a composition comprising a nucleic acid binding domain (MAP-NBD), wherein the MAP-NBD comprises a plurality of repeating units. The repeating units (RUs) of the nucleic acid recognize bases in the target nucleic acid. In some aspects, the animal pathogen protein is derived from a bacterium that infects an animal. In some aspects, the animal pathogen protein is derived from a bacterium that infects humans. In this aspect, the bacterium is from the order Legionella or the genus Legionella or Francisella. In one aspect, the bacterium is Legionella quateriensis (L. quateriensis) or is Legionella maceachernii (L. maceachernii). In some aspects, The unit is the consensus sequence 1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 19). Contains columns.

[0006] In some aspects, the target nucleic acid is a single nucleotide or a single base pair. In some embodiments, the target nucleic acid is DNA or RNA. D comprises at least three RUs, each RU binding to a base in a target nucleic acid, where the target nucleic acid is In further embodiments, the target nucleic acid sequence is at least Both are DNA or RNA that are three nucleotides long.

[0007] In certain aspects, the present disclosure provides a method for the preparation of a nucleic acid binding domain (NBD) and a heterologous functional domain. The present invention provides a recombinant polypeptide comprising an NBD, the NBD being attached to the N-terminus of the NBD such that the NBD specifically binds to a target nucleic acid. The NBD contains at least three repeating units (RUs) arranged from the carboxy terminus to the C-terminus, and each RU of the NBD is Census sequence: 1 xxxx 1xxxx11x12xx33xxx1xxxxxxxxxx14xxx (SEQ ID NO: 153), , where 1=A, F, I, L, M, T, V, or Y; 2=x or xx; 3=A, G, N, or S; 4=x, xx, or or xxx; and x=any amino acid, and each of the RUs is independently selected from SEQ ID NOs: 2-9, 23-3 5, 85-89, and 131-37. wherein SEQ ID NOs: 2-9, 33, and 89 are sequences of L. qua The amino acid sequence of RU identified in the teirensis bacterial protein (SEQ ID NO: 1) is provided; SEQ ID NO: 23-32, 34-35, and 133 were identified in the L. maceachernii bacterial protein (SEQ ID NO: 143). SEQ ID NOs: 25, 131-132, and 138 provide the amino acid sequences of RU from the order Legionellales. The amino acid sequence of RU identified in the protein from Bacteria (SEQ ID NO: 139) is provided. Rows 85 to 88, 134 to 137, and 151 are proteins derived from Francisella bacteria (SEQ ID NO:1 47) provides the amino acid sequence of the RU identified in. In one embodiment, the NBD is a half-repeat unit Further includes:

[0008] In certain aspects, the present disclosure provides a method for the preparation of a nucleic acid binding domain (NBD) and a heterologous functional domain. The present invention provides a recombinant polypeptide comprising an NBD, the NBD being attached to the N-terminus of the NBD such that the NBD specifically binds to a target nucleic acid. The NBD contains at least three repeating units (RUs) arranged from the α-terminus to the C-terminus, and each RU of the NBD has the following structure: Consensus sequences include: (F / L / Y)(D / G / N / S)(A / H / R / S / T / V)(D / E / K / Q)(E / H / Q)(I / L / V)(I / L / V)(C / H / K / R / S)(I / M / V)(A / V)(A / G / S)(H / N / R)(A / D / G / I / K / N / S / V)(G)(G)(A / G / S)(H / K / L / N / R )(N)(I / L)(A / D / E / I / K / V)(A / L / V)(I / M / V)(K / L / Q / T)(A / D / E / K / L / Q / S)(A / C / F / N / V / Y)(F / H / L / Q / Y)(A / D / H / P / Q)(A / D / I / K / R / T / V)(F / L)(K / M / Q / R / S)(D / E / N / S)(F / L / M)(D / E / G / H / K / N) column no. 154), where the consensus sequence is from the Legionellales bacterium, L. quateirensis RUs identified in proteins from L. bacterium and L. maceachernii It is based on the amino acid sequence.

[0009] In certain aspects, the present disclosure provides a method for the preparation of a nucleic acid binding domain (NBD) and a heterologous functional domain. The present invention provides a recombinant polypeptide comprising an NBD, the NBD being oriented such that it specifically binds to a target nucleic acid. It contains at least three repeating units (RUs) arranged from the N-terminus to the C-terminus, and each RU of the NBD is a component. Census sequence: YK(P / S)EDIIRLASH(D / G)GGSVNLEAVLRL(H / N)(P / S)QL(I / T)(G / R)LG (sequence number No. 156), and this consensus sequence is found in proteins from Francisella species. Based on the amino acid sequence of the identified RU.

[0010] In certain aspects, the present disclosure provides a method for the preparation of a nucleic acid binding domain (NBD) and a heterologous functional domain. The present invention provides a recombinant polypeptide comprising an NBD, the NBD being attached to the N-terminus of the NBD such that the NBD specifically binds to a target nucleic acid. The NBD contains at least three repeating units (RUs) arranged from the α-terminus to the C-terminus, and each RU in the NBD is a concept. Sequence: (F / L)(N / S / G)(S / V / A / T / H)(E / Q / K)(Q / E)(I / L)(I / V)(R / S / K)(M / I)(V / A)(S / A)X 12X13GG(G / A / S)(L / K / N)NL(K / I)AV(T / K / L)(A / D / K / S)(N / Y / C)(H / Y)(D / K)(D / A / V)L(Q / K / R)(N / D / E)(M / R)(G / K / E) (SEQ ID NO: 158), wherein X 12 X 13 =HK, HD, HA, HN, HG, NN, NG , RN, HI, HV, RT, HD, SN, HS, GS, or LN, and this consensus sequence is L. quatei This is based on the amino acid sequence of RU identified in a protein from the bacterium T. rensis.

[0011] In certain aspects, the last RU in the NBD is 15-20 amino acids in length and is a consensus The sequence may be a half repeat containing the sequence: 1xxxx11x12xx33x (SEQ ID NO: 157), where 1=A, F, I, L, M , T, V, or Y;2=x or xx;3=A, G, N, or S;4=x, xx, or xxx;x=any A It is an amino acid.

[0012] In some embodiments, the target nucleic acid is at least 3 nucleotides in length. In embodiments, the target nucleic acid sequence is DNA or RNA that is at least 3 nucleotides in length. .

[0013] In certain aspects, the 12th and 13th amino acid residues in the repeat unit are base contact residues. The repeating unit is called the base complement (BCR), which determines which base (A, G, T, or C) the repeating unit will bind. In certain aspects, the BCR in the repeat unit provided herein is The repeat units may be substituted with the BCR or RVD that are involved, thereby changing the bases to which they bind. In certain aspects, the BCR in the repeat unit provided herein binds to the Legionella protein The BCR may be substituted with a BCR identified in the repeat from the gene (e.g., SEQ ID NO: 1 or 143). In certain aspects, the BCR in the repeat units provided herein is a Legionellales protein. The repeats from the sequence (e.g., SEQ ID NO: 139) can be replaced with the BCR identified in the repeats from the sequence (e.g., SEQ ID NO: 139). In certain aspects, the BCR in the repeat units provided herein is a Francisella protein. The BCR may be substituted with the BCR identified in the repeat from the mutant (e.g., SEQ ID NO: 147). In the present specification, the BCR in the repeat unit is replaced with a BCR listed in Table 1 of the present specification. It may be replaced.

[0014] In some aspects, a naturally occurring or non-natural linker connects the NBD to the functional domain. In some aspects, the functional domain is an enzyme, a transcriptional activator, domains, transcriptional repression domains, biotinylation reagents, DNA nucleotide modifiers, or fluorophore In further embodiments, the enzyme includes a nuclease, a DNA modifying protein, or a chromatin modifying protein.

[0015] In further embodiments, the nuclease is a cleavage domain or a half-cleavage domain. In some embodiments, the cleavage domain or half-cleavage domain is a type IIS restriction enzyme. In further embodiments, the type IIS restriction enzyme comprises FokI or Bfil. In one aspect, FokI has a sequence similar to that of SEQ ID NO:11, at least 70%, at least 75%, at least 80%, or at least 90%. %, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or or at least 99% sequence identity. In some aspects, FokI has SEQ ID NO:11. The sequence is:

[0016] In some aspects, the chromatin modifying protein is a lysine-specific histone demethylase. In some aspects, the transcription activation domain is VP16, VP64, , p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldb1 self-association domain, SAM activation In some aspects, transcription factors (VP64, p65, HSF1), VPR (VP64, p65, Rta) The transcription repression domains are KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, and DNMT3A-DNMT3L. , DNMT3B, KOX, TGF-β-induced early gene (TIEG), v-erbA, SID, MBD 2, MBD3, Rb, and In some aspects, the DNA nucleotide modifier is adenosine deaminase. It is a nase.

[0017] In some aspects, the functional domain is a polypeptide that includes a RU, as described herein. A genomic locus containing a target nucleic acid bound by a denatured nucleic acid-binding domain. ) for genome editing, gene regulation, or imaging. In this case, each of the repeating units corresponds to any one of SEQ ID NOs: 2 to 10, 23 to 35, 85 to 89, and 131 to 137. At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least at least 92%, at least 94%, at least 95%, at least 96%, at least 98%, or In a further embodiment, the repeating unit has at least 99% sequence identity. The amino acid sequence of the present invention is any one of 10, 23 to 35, 85 to 89, 131 to 138, and 151 to 152.

[0018] In some aspects, the RU is derived from a wild-type protein from an animal pathogen. In some aspects, the RU comprises a modification of a wild-type protein. The modifications enhance the specific recognition of the target nucleotide, base pair, or both. In one embodiment, the modification comprises 1 to 29 modifications. The protein has at least 70%, at least 75%, at least 80%, or at least 85% identical to SEQ ID NO:1. , at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% In some aspects, the animal pathogen protein has a sequence identity of SEQ ID NO:1. It is.

[0019] In a further embodiment, the NBD comprises 3 to 40 RU, e.g., 3 to 35, 3 to 30, 4 to 35, 4 to 30, 5-35, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 10-28, or 10-25 RU In certain aspects, the NBD is at least 3 nucleotides in length, e.g., 3-35, 3-30, 4-35, 4-30, 5-35, 5-30, 6-30, 7-30, 8-30, 9-30, 10-30, 10-28, or 10-25 Nucle It binds to a target nucleic acid that is an octave long.

[0020] In further embodiments, the target nucleic acid is selected from the group consisting of the PDCD1 gene, the CTLA4 gene, the LAG3 gene, the TET2 gene, gene, BTLA gene, HAVCR2 gene, CCR5 gene, CXCR4 gene, TRA gene, TRB gene, B 2M gene, albumin gene, HBB gene, HBA1 gene, TTR gene, NR3C1 gene, CD52 gene, erythroid-specific enhancer of BCL11A gene, CBLB gene, TGFBR1 gene, SERPIN A1 gene, HBV genomic DNA in infected cells, CEP290 gene, DMD gene, CFTR gene, IL2 In another aspect, the chimeric antigen receptor is present in the RG gene, or a combination thereof. (CAR), α-L iduronidase (IDUA), iduronate-2-sulfatase (IDS), and Factor 9 (F9) is inserted upon cleavage of a region of the target nucleic acid sequence.

[0021] In various aspects, the present disclosure provides a method of genome editing in a subject, the method comprising: administering a non-naturally occurring modular nucleic acid binding domain comprising a functional domain, The domain comprises a cleavage domain or a cleavage half-domain; and and wherein the modular nucleic acid binding domain is derived from an animal pathogen protein (MAP-NBD). and a modular nucleic acid binding domain that binds to the MAP-NBD, the MAP-NBD comprising multiple repeat units, The repeating units recognize the target nucleic acid.

[0022] In certain embodiments, the method further comprises a second MAP-NBD, wherein the second MAP-NBD is In some embodiments, the MAP-NB comprises a second plurality of repeat units that recognizes a second target nucleic acid. D, the second MAP-NBD, or both, may contain a functional domain, e.g., a cleavage domain or a cleavage domain. In a further embodiment, the cleavage domain or cleavage half-domain further comprises In some aspects, the cleavage domain comprises a meganuclease Includes.

[0023] In a further embodiment, FokI has a sequence similar to that of SEQ ID NO: 11, at least 70%, at least 75%, At least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least In a further embodiment, FokI has at least 97%, or at least 99% sequence identity with In a further embodiment, the target nucleic acid sequence is the PDCD1 gene, CTL A4 gene, LAG3 gene, TET2 gene, BTLA gene, HAVCR2 gene, CCR5 gene, CXCR4 gene gene, TRA gene, TRB gene, B2M gene, albumin gene, HBB gene, HBA1 gene, Erythroid-specific enhancers of the TTR gene, NR3C1 gene, CD52 gene, and BCL11A gene, CBLB gene, TGFBR 1 gene, SERPINA1 gene, HBV genomic DNA in infected cells, CEP290 gene The gene is present in the DMD gene, the CFTR gene, the IL2RG gene, or a combination of these. In one aspect, chimeric antigen receptor (CAR), α-L iduronidase (IDUA), iduronate- 2-sulfatase (IDS), or factor 9 (F9), upon cleavage of a region of the target nucleic acid sequence. It is inserted.

[0024] In various aspects, the present disclosure provides a method of gene regulation in a subject, the method comprising: Administering a non-natural modular nucleic acid binding domain; and regulating gene expression. , where the modular nucleic acid binding domain is derived from an animal pathogen protein (MAP-NBD) The MAP-NBD contains multiple repeat units, and the multiple repeats The repeating units of the subunits recognize the target nucleic acid.

[0025] In a further embodiment, the MAP-NBD further comprises a functional domain. In some cases, the functional domain includes a transcription activation domain or a transcription repression domain. In some aspects, the activation domain is VP16, VP64, p65, the p300 catalytic domain, the TET1 catalytic domain, or the like. In, TDG, Ldb1 self-association domain, SAM activator (VP64, p65, HSF1), VPR (VP64, p6 5, Rta). In some aspects, the repression domain includes KRAB, Sin3a, LSD1, SUV39 H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-β-induced early genes (TIEG) , v-erbA, SID, MBD2, MBD3, Rb, or MeCP2.

[0026] In some aspects, the target nucleic acid sequence is a nucleic acid sequence encoding the PDCD 1 gene, the CTLA 4 gene, the LAG 3 gene, or a nucleic acid sequence encoding the PDCD 1 gene. , TET 2 gene, BTLA gene, HAVCR 2 gene, CCR 5 gene, CXCR 4 gene, TRA gene , TRB gene, B 2 M gene, albumin gene, HBB gene, HBA 1 gene, TTR gene, N R3C1 gene, CD52 gene, erythroid-specific enhancer of BCL11A gene, CBLB gene , TGFBR 1 gene, SERPINA 1 gene, HBV genomic DNA in infected cells, CEP 290 gene, D The MD gene, the CFTR gene, or a combination thereof.

[0027] In various aspects, the present disclosure provides a method for imaging genomic loci in vivo in a subject. The present invention provides a method for detecting a non-naturally occurring module conjugated to an imaging agent, the method comprising: administering a Lar nucleic acid binding domain to a subject; and imaging the subject. Here, the modular nucleic acid binding domain is derived from an animal pathogen protein (MAP-NBD). The MAP-NBD contains a modular DNA-binding domain that recognizes target nucleic acids. In some embodiments, the imaging agent is a fluorescent moiety. In some aspects, the fluorescent moiety is GFP or mCHERRY. In some aspects, the target nucleic acid is a nucleotide, a single base pair, or both. is DNA or RNA. In some aspects, the MAP-NBD recognizes a target nucleic acid sequence. In some embodiments, the MAP-NBD binds to a target nucleic acid sequence. In some aspects, the target nucleic acid sequence is DNA or RNA. In some aspects, the protein is an animal pathogen protein. The protein is derived from a bacterium. In a further embodiment, the bacterium is selected from the genus Legionella. In some aspects, the bacterium is L. quateirensis. The repeating unit contains the consensus sequence 1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 19).

[0028] In some aspects, the repeating units are selected from the group consisting of SEQ ID NOs: 2-10, 23-35, 85-89, and 131- At least 70%, at least 75%, at least 80%, at least 85% for any of 137 , at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% In some aspects, the repeating unit has a sequence identity of SEQ ID NOs: 2-9, 23-35. , 85 to 89, 131 to 138 or 151 amino acid sequence.

[0029] In some aspects, the repeat units are derived from a wild-type protein. In some embodiments, the repeat unit comprises a modification of a wild-type protein. In some embodiments, the modification is 1 to 29. In some aspects, the animal pathogen protein comprises a modification of SEQ ID NO:1. At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least at least 92%, at least 95%, at least 97%, or at least 99% sequence identity In a further embodiment, the animal pathogen protein is SEQ ID NO: 1. In some aspects, the cell is ex vivo The nucleic acid sequence may be in a plurality of cells of a mammal, in a human, or in a non-human animal.

[0030] A method for producing a polypeptide that specifically binds to a target DNA sequence is disclosed. The method comprises synthesizing a polypeptide comprising a DNA binding domain (DBD) that specifically binds to a target sequence. wherein the DBD is selected based on the DNA bases bound by the repeat unit. It contains repeat units assembled in the proper order to match the target DNA sequence, where the target If the target sequence contains an adenine (A), the repeat unit is FSSQQIIRMVSHAGGANNLKAVTANHDDLQNMG ( SEQ ID NO: 2) or LGHKELIKIAARNGGGNNLIAVLSCYAKLKEMG (SEQ ID NO: 89) It contains a 33-35 amino acid sequence with at least 70% identity or contains conservative amino acid substitutions. If the target sequence contains a thymine (T), the repeat The units are FNAEQIVRMVSHGGGSKNLKAVTDNHDDLKNMG (SEQ ID NO: 4); FNAEQIVSMVSNGGGSLNLKAVKKY HDALKDRG (SEQ ID NO: 6); or FNVEQIVSIVSHGGGSLNLKAVKKYHDVLKDRE (SEQ ID NO: 8) A sequence of 33 to 35 amino acids with at least 70% identity to the 4, 6, or 8, which contains an amino acid substitution; if the target sequence contains a cytosine (C), If the repeating unit is FNTEQIVRMVSHDGGSLNLKAVKKYHDALRERK (SEQ ID NO: 7) or FNAEQIV 33 having at least 70% identity to RMVSHDGGSLNLKAVTDNHDDLKNMG (SEQ ID NO: 9) or a sequence of SEQ ID NO: 7 or 9 containing conservative amino acid substitutions; If the target sequence contains a guanine (G), the repeat unit is FNVEQIVRMVSHNGGSKNLKAVT DNHDDLKNMG (SEQ ID NO:3);FNAEQIVSMVSNNGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:5);FNAEQIVRM VSHKGGSKNLALVKEYFPVFSSFH (SEQ ID NO:33); or LGHKELIKIAARNGGGNNLIAVLSCYAKLKEMG (SEQ ID NO: 89) containing a sequence of 33 to 35 amino acids having at least 70% identity to or the sequences of SEQ ID NO: 3, 5, 33, or 89 containing conservative amino acid substitutions.

[0031] Further methods for producing a polypeptide that specifically binds to a target DNA sequence are disclosed. The method comprises the steps of: generating a polypeptide comprising a DNA binding domain (DBD) that specifically binds to a target sequence; wherein the DBD is based on the DNA bases bound by the repeating units. It contains repeat units arranged in the proper order to match a selected target DNA sequence, Wherein, if the target sequence contains an adenine (A), the repeat unit is FSAKHIVRIAAHIGGSLNIKAVQQA QQALKELG (SEQ ID NO: 32), FSAEQIVSIAAHVGGSHNIEAVQKAHQALKELD (SEQ ID NO: 35), FSAEQIVRI AAHIGGSHNLKAVLQAQQALKELD (SEQ ID NO: 31), or FSAEQIVRIAAHIGGSRNIEATIKHYAMLTQPP (SEQ ID NO: 133) containing a sequence of 33 to 35 amino acids having at least 70% identity to or a sequence of SEQ ID NO: 32, 36, 31, or 133 containing conservative amino acid substitutions, If the target sequence contains a thymine (T), the repeat unit is Row number 28), FSAEQIVHIAAHGGGSLNIKAILQAHQTLKELN (sequence number 29), FSTEQIVCIAGHGGGSLNIKA VLLAQQALKDLG (SEQ ID NO:27), FSAEQIVSIAAHVGGSHNIEAVQKAHQALKELD (SEQ ID NO:35), if or at least 70% identical to FSAEQIVRIAAHIGGSRNIEATIKHYAMLTQPP (SEQ ID NO: 133) SEQ ID NO: 1, which contains a sequence of 33 to 35 amino acids having the same or a conservative amino acid substitution 28, 29, 27, 35, or 133 sequences; if the target sequence contains a cytosine (C), The units are FSAEQIVSIVAHDGGSRNIEAVQQAQHILKELG (SEQ ID NO: 24), FSAEQIVRIAAHDGGSLNIDAVQQ AQQALKELG (SEQ ID NO: 26), or FSAEQIVRIAAHIGGSHNLKAVLQAQQALKELD (SEQ ID NO: 31) Contains or conserves a sequence of 33 to 35 amino acids that has at least 70% identity to 24, 26 or 31 containing a target amino acid substitution; The repeating unit is FSAEQIVRIAAHIGGSRNIEAIQQAHHALKELG (SEQ ID NO: 30), FSADQIV RIAAHKGGSHNIVAVQQAQQALKELD (SEQ ID NO: 34), FSAEQIVSIAAHVGGSHNIEAVQKAHQALKELD (SEQ ID NO: 35), No. 35), or FSAEQIVRIAAHIGGSRNIEATIKHYAMLTQPP (SEQ ID NO: 133) A sequence of 33 to 35 amino acids with at least 70% identity or with conservative amino acid substitutions. The sequence of SEQ ID NO: 30, 34, 35, or 133.

[0032] In certain embodiments of the method, the DBD may comprise any repeating unit disclosed herein. For example, the DBD may be composed of repeats derived from different Legionella proteins as provided herein. The target sequence may be a promoter region or an open linker region of a gene of interest. The DBD may be in a loading frame. can be combined with [Brief description of the drawings]

[0033] [Figure 1] 1 is a schematic diagram of an embodiment of a polypeptide disclosed herein that includes a nucleic acid binding domain (NBD) composed of multiple repeat units. Each repeat unit has the consensus sequence 1xxxx11x12xx33xxx1xxxxxxxxxx14xxx (SEQ ID NO: 153). The last repeat unit is a half-repeat with a length of 15-20 amino acids. The NBD is flanked by an N-terminal domain and a C-terminal domain.

[0034] [Diagram 2]FIG. 2 shows a protein from L. quateirensis from which a nucleic acid binding repeat domain can be derived. SEQ ID NO: 1 provides the amino acid sequence of a complete protein comprising a repeat unit (RU) having the sequence set forth in SEQ ID NO: 89 and SEQ ID NO: 2 to SEQ ID NO: 10. The amino acid sequence set forth in SEQ ID NO: 89 extends from the residue at position 144 to the residue at position 176 of SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 2 extends from the residue at position 177 to the residue at position 209 of SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 3 extends from the residue at position 210 to the residue at position 242 of SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 4 extends from the residue at position 243 to the residue at position 275 of SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 5 extends from the residue at position 276 to the residue at position 308 of SEQ ID NO: 1. The amino acid sequence set forth in SEQ ID NO: 6 extends from the residue at position 309 to the residue at position 341 of SEQ ID NO: 1. The amino acid sequence shown in SEQ ID NO:7 extends from residue position 342 to residue position 374 of SEQ ID NO:1. The amino acid sequence shown in SEQ ID NO:8 extends from residue position 375 to residue position 407 of SEQ ID NO:1. The amino acid sequence shown in SEQ ID NO:9 extends from residue position 408 to residue position 440 of SEQ ID NO:1. The amino acid sequence shown in SEQ ID NO:10 extends from residue position 441 to residue position 459 of SEQ ID NO:1. The RU having the amino acid sequence of SEQ ID NO:10 is a half-repeated unit.

[0035] [Figure 3A] 3A shows consensus sequences of SEQ ID NO: 14 to SEQ ID NO: 18 that may be present in proteins derived from animal pathogens. Each consensus sequence of SEQ ID NO: 14 to SEQ ID NO: 18 contains a repeat motif, followed by a spacer of variable length, and a second repeat motif.

[0036] [Figure 3B] FIG. 3B shows the consensus sequence of SEQ ID NO: 19, which may be present in a repeat domain (e.g., MAP-NBD) of the present disclosure.

[0037] [Figure 4A] 4A shows bases to which the repeat sequences (SEQ ID NOs: 89 and 2-10, in order from N-terminus to C-terminus) in a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 may bind. The polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 was fused to a His tag. The larger the size of the base at a particular position, the higher the relative frequency with which that base is present at that position in the nucleic acid bound by the tested polypeptide.

[0038] [Figure 4B] FIG. 4B shows bases to which the repeat sequence (N-terminus to C-terminus: SEQ ID NO: 89 and 2-10) in a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 can bind. The polypeptide having the amino acid sequence set forth in SEQ ID NO: 1 was fused to an HA tag. The larger the size of the base at a particular position, the higher the relative frequency with which that base is present at that position in the nucleic acid bound by the tested polypeptide. The His-tagged and HA-tagged polypeptides showed the same binding specificity.

[0039] [Diagram 5] FIG. 5 summarizes the BCRs present in each of the repeat units having the amino acid sequences set forth in SEQ ID NOs: 89 and 2-10, and the corresponding bases to which the repeat units bind.

[0040] [Figure 6] Figure 6 shows that while the majority of the BCRs in the repeat units from L. quateirensis show specificity for the same bases as the RVD in the repeat from Xanthomonas reported by Miller et al. Nature Methods, 12(5), 465-471, 2015, the BCR "HA" in the repeat from L. quateirensis having the sequence of SEQ ID NO:2 specifies binding to "A" rather than "T" as observed for the repeat containing the RVD HA from Xanthomonas.

[0041] [Figure 7] Figure 7 provides the amino acid sequence of a protein from a Legionellales bacterium (SEQ ID NO: 139). The protein contains four repeating units.

[0042] [Figure 8A] FIG. 8A shows bases to which BCR can bind in the repeated sequence (having the amino acid sequences set forth in SEQ ID NO: 89 and 2-10) in the order from the N-terminus to the C-terminus in a polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. The larger the size of the base at a particular position, the higher the relative frequency with which that base occurs at that position in the nucleic acid bound by the tested polypeptide. The N-terminus (SEQ ID NO: 13) and C-terminus (SEQ ID NO: 159) of the protein did not bind to any bases.

[0043] [Figure 8B] Figure 8B shows the bases to which BCR may bind in the repeated sequence (having amino acid sequences set forth in SEQ ID NO: 28, 26, 32, 34, 30, 31, 23, 29, 24, 27, 35 and 133) in order from N-terminus to C-terminus in a polypeptide having the amino acid sequence set forth in SEQ ID NO: 143. The larger the size of the base at a particular position, the higher the relative frequency with which that base occurs at that position in the nucleic acid bound by the tested polypeptide. The N-terminus of the protein (SEQ ID NO: 144) mediated binding to G or the sequence GG, and the C-terminus of the protein (SEQ ID NO: 145) potentially mediated binding to the sequences T or TT.

[0044] [Figure 9] FIG. 9 shows that BCR proteins in the N-terminal to C-terminal order of repeat sequences (having amino acid sequences set forth in SEQ ID NOs: 28, 26, 32, 34, 30, 31, 23, 29, 24, 27, 35 and 133) in a polypeptide having the amino acid sequence set forth in SEQ ID NO: 143 bind in vivo to the same DNA sequence discovered by SELEX-seq. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0045] The present invention provides methods for genetic and epigenomic engineering, including genome editing and gene regulation. The present invention provides polypeptides, compositions and methods for their use. and the composition includes a nucleic acid binding domain that binds to a target nucleic acid of interest. , Legionellales bacteria and animal pathogens such as Legionella and Francisella species. The repeat units are derived from repeat units identified in previous proteins.

[0046] [Definition] As used herein in the context of polypeptides, the term "derived from" refers to a polypeptide that is derived from a particular source. Sequences based on protein sequences from a source (e.g., animal pathogens such as Legionella) A polypeptide derived from a protein from a particular source is a polypeptide derived from that Variants of proteins from specific sources (e.g., animal pathogens such as Legionella) For example, a polypeptide derived from a protein from a particular source may be The protein may have a sequence that is modified with respect to the sequence of the protein from which it is derived. A polypeptide derived from a protein has at least 30% sequence identity with the protein from which it is derived. identity, at least 40% sequence identity, at least 50% sequence identity, at least 60% share sequence identity, at least 80% sequence identity, or at least 90% sequence identity .

[0047] The term "modular" as used herein in the context of nucleic acid binding domains (e.g., Modular animal pathogen derived nucleic acid binding domain The multiple repeat units present in the NBD can be rearranged, and and / or other repeat units and in an order such that the NBD binds to the target nucleic acid. For example, any repeat unit in a modular nucleic acid binding domain may be organized in any order. can be replaced with a different repeating unit. The modularity of the nucleic acid binding domains disclosed herein allows for the binding of specific repeat units to the target nucleic acid. This allows the bases to be switched by simply replacing them with different repeat units. In some embodiments, the modularity of the nucleic acid binding domains disclosed herein is , by exchanging a particular repeat unit for another repeat unit to determine the affinity of the repeat unit for a particular target nucleic acid. In general, the nucleic acid binding domains disclosed herein allow for an increase in The modularity of the gene allows for genome editing that can precisely target any nucleic acid sequence of interest. This allows for the development of a concentration complex.

[0048] The terms "polypeptide," "peptide," and " "Protein" refers to polymeric forms of amino acids of any length, including those that are genetically coded. Genetically encoded and non-genetically encoded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having modified polypeptide backbones. The term includes fusion proteins, including, but not limited to, N-terminal Fusion proteins with heterologous amino acid sequences, with or without terminal methionine residues, heterologous and and homologous leader sequences; immunologically labeled proteins; etc. In certain embodiments, the term refers to any amino acid that includes a genetically encoded amino acid. In certain embodiments, the term refers to a polymeric form of amino acids of a given length. Polypeptides of amino acids of any length containing genetically encoded amino acids fused to a nucleic acid sequence. Refers to the mer form.

[0049] The term "heterologous" refers to structures that are defined by their origin from different sources. For example, with respect to a polypeptide, a "heterologous" polypeptide refers to a polypeptide that is a different polypeptide. The polypeptide may include an operably linked amino acid sequence derived from the polypeptide (e.g., NBD and and functional domains derived from different sources). With respect to polynucleotides, a "heterologous" polynucleotide may be derived from a different gene. Other exemplary "heterologous" nucleic acids may include a coding sequence. The nucleic acid may contain regulatory elements (e.g., promoters) from a genetic source other than its coding sequence. The expression construct includes an expression construct operably linked to a promoter , the coding sequence, or both, may be of different genetic origin in the host cell of interest. In the context of recombinant cells, "heterologous" refers to a heterologous host cell in which it resides. Refers to the presence of nucleic acids (or gene products, such as polypeptides) of a genetic origin different from that of the cell. obtain.

[0050] The term "operably linked" refers to a linkage between molecules to provide a desired function. For example, "operably linked" in the context of nucleic acids refers to a functional relationship between nucleic acid sequences. For example, a nucleic acid expression control sequence (promoter, signal sequence, or sequence) can be linked to (e.g., a transcription factor binding site of the transcription factor gene of the present invention) can be operably linked to a second polynucleotide, In some embodiments, the expression control sequence affects the transcription and / or translation of the second polynucleotide. In the context of a polypeptide, "operably linked" refers to a description of the polypeptide. Functional linkages between amino acid sequences (e.g., different domains) to provide a desired activity. It means.

[0051] As used herein, the term "cleavage" refers to the covalent division of a nucleic acid, e.g., a DNA molecule. Cleavage refers to the cleavage of the backbone, including but not limited to, the enzymatic cleavage of the phosphodiester bond. This can be initiated by a variety of methods, including elementary or chemical hydrolysis. Both types of cleavage are possible, and a double-stranded break can occur as a result of two different single-stranded breaks. When DNA is cut, it can have blunt or staggered ends. In one embodiment, the polypeptides provided herein are capable of targeting double-stranded DNA cleavage. Used for diagnostic purposes.

[0052] The "cleavage half-domain" can be combined with a second polypeptide (either the same or different) to form a It is a polypeptide sequence that forms a complex having cleavage activity (preferably double-strand cleavage activity). .

[0053] A "target nucleic acid," "target sequence," or "target site" refers to a nucleic acid that binds to a target nucleic acid, such as an NBD, as disclosed herein. The target nucleic acid is a nucleic acid sequence that defines the portion of the nucleic acid to which the binding molecule binds. The target nucleic acid may be present in a region of interest. A "region of interest" may be any region of cellular chromatin, e.g., a region that is bound to an exogenous molecule. A gene that is desired to be combined with a non-coding region within or adjacent to the gene. The binding may be a sequence. The region of interest may be, for example, a chromosome , episomes, organelle genomes (e.g., mitochondria, chloroplasts), or infectious viruses. The region of interest may be within the coding region of a gene, e.g., a promoter sequence, Within transcribed non-coding regions such as leader sequences, trailer sequences or introns, The region of interest can be in the non-transcribed region, either upstream or downstream of the coding region. They can be as small as one nucleotide pair or as long as 2,000 nucleotide pairs. or any integer value of nucleotide pairs.

[0054] An "exogenous" molecule is one that is not normally present in a cell but which has one or more genetic, biochemical or is a molecule that can be introduced into a cell by other means. Exogenous molecules can be, for example, A functional version of an endogenous molecule, e.g., lacking a mutation present in the endogenous gene. Exogenous nucleic acid may include a gene or gene segment. Exogenous molecules can be present in the genome, a plasmid or an episome. Methods are known to those of skill in the art and include, but are not limited to, lipid-mediated transfer (i.e., neutral and cationic lipid-containing liposomes), electroporation, direct injection, cell lysis , particle bombardment, calcium phosphate coprecipitation, DEAE-dextran mediated transfer and viruses Includes vector-mediated transfer.

[0055] In contrast, “endogenous” molecules are expressed in specific cells at specific developmental stages under specific environmental conditions. A molecule that is normally present. For example, endogenous nucleic acids are found in chromosomes, mitochondria, chloroplasts, or or other organelle genomes, or naturally occurring episomal nucleic acids. Endogenous molecules can include proteins, such as transcription factors and enzymes.

[0056] For purposes of this disclosure, a "gene" refers to a region of DNA that encodes a gene product, as well as a It includes all DNA regions that regulate the production of a product, such as coding sequences and / or Thus, a gene may be a promoter sequence, whether adjacent to a transcriptional sequence or not. , translation terminators, ribosome binding sites and internal ribosome entry sites. Regulatory sequence, enhancer, silencer, insulator, boundary element, replication origin, matrix Examples of target sequences include, but are not limited to, ribosome attachment sites and locus control regions.

[0057] "Gene expression" refers to the conversion of the information contained in a gene into a gene product. The product may be the direct transcription product of the gene or a transcription factor produced by translation of the mRNA. Gene products may include capping (e.g., mRNA, tRNA, rRNA, Antisense RNA, ribozyme, structural RNA, shRNA, RNAi, miRNA, or any other type RNA that is modified by processes such as polyadenylation, methylation, and editing, and methylation, acetylation, phosphorylation, ubiquitination, ADP-ribosylation, and myristoylation. and proteins that are modified, such as by glycosylation.

[0058] "Modulation" of gene expression means a change in the activity of a gene. Modulation of expression is limited Genome editing (e.g., cleavage, Expression can be regulated using a variety of techniques (e.g., modification, inactivation, donor integration, random mutation). Gene inactivation refers to the production of cells that do not contain the polypeptide or polypeptides described herein. Any changes in gene expression compared to cells not modified by such polypeptides. It refers to a reduction in the amount of DNA that is produced by the gene. Thus, gene inactivation can be partial or complete.

[0059] The term "patient" or "subject" refers to a human or non-human animal (e.g., a mammal). are used interchangeably to refer to

[0060] The terms "treat," "treating," "treatment," and the like refer to a disease, disorder, or condition or The cause of the disease, disorder, or condition suffered by the subject after the symptoms have been diagnosed, observed, etc. or a disease, disorder, or condition suffered by the subject. Eliminate, reduce, suppress, or alleviate, temporarily or permanently, at least one of the symptoms involved Actions initiated to reduce or ameliorate the administering a polypeptide comprising the NBD or a nucleic acid encoding the polypeptide to the subject. Point.

[0061] The terms "prevent," "preventing," "prevention," and the like generally refer to a particular disease, disorder, or condition. is associated with a subject prone to having a condition, the risk of the subject developing a disease, disorder, condition, etc. Temporarily or permanently prevent or suppress risk (e.g., as determined by the absence of clinical symptoms) or inhibiting, reducing, or delaying the onset of (e.g., a disease, disorder, condition, or or before the onset of those symptoms) (e.g., merging into heterogeneous functional domains) administering a polypeptide comprising the NBD or a nucleic acid encoding the polypeptide to a subject In some cases, the term also refers to slowing the progression of a disease, disorder or condition, or also refers to inhibiting their progression to a harmful or other undesirable condition.

[0062] The phrase "therapeutically effective amount" refers to a single amount, either alone or as part of a pharmaceutical composition, and in a single dose. When administered to a patient as a single dose or as part of a series of doses, it is believed to reduce the symptoms of a disease, disorder or condition. to a subject in an amount capable of having a detectable positive effect on the shape, aspect, or characteristics of the The therapeutically effective amount is determined by measuring the relevant physiological effect. It can be acknowledged.

[0063] [Animal pathogen-derived nucleic acid binding domains] The present disclosure relates to modular nucleic acid binding domains (NBDs) and methods for using modular NBDs. The modular NBDs can be engineered to target and bind to specific nucleic acid sequences. The nucleic acid sequence may be DNA or RNA. In some embodiments, A modular NBD can contain multiple repeat domains, where each repeat domain is a single repeat domain. Each repeat domain in the multiple repeat domains recognizes and binds to a single nucleotide or base pair. The antibodies can be specifically selected to target and bind to a particular nucleic acid sequence, and such antibodies can be Thus, it contributes to the modular nature of NBDs. A naturally occurring modular nucleic acid binding domain can include multiple repeating units, and the multiple repeats The repeating units of the repeating units recognize a single target nucleotide, base pair, or both. do.

[0064] In some embodiments, the repeat domain can be derived from an animal pathogen, Modular non-natural nucleic acid binding domains (MAP-NBDs), or “modular” nucleic acid binding domains derived from proteins, For example, in some cases, the MAP-NBD may be referred to as a MAP-NBD. In other cases, animal pathogens may be from the gram-negative bacterial genus Legionella. Holderia genus. In some cases, the animal pathogen is from the Paraburkholderia genus. In other cases, the animal pathogen may be a bacterium from the genus Francisella.

[0065] In certain aspects, NBDs such as MAP-NBDs are derived from the animal pathogen L. quateirensis. In certain aspects, RU is identified in a protein from L. quateirensis. The protein is derived from RU, which has the amino acid sequence set forth in SEQ ID NO:1. do.

[0066] With respect to repeat units, the terms "repeat," "repeat unit," "repeat motif," "Repeat domain" and "repeat sequence" are used interchangeably.

[0067] In certain embodiments, the repeat domain is selected from Legionellales bacteria, species of the genus Legionella, e.g. For example, L. quateirensis or L. maceachernii, Burkholderia spp., Paraburkholderia spp., or can be derived from Legionellales bacteria, which are species of the Legionella genus, such as Francisella. In some embodiments, the repeat domain is from 19 amino acid residues to 36 amino acid residues, For example, 19-35 amino acids, 20-36 amino acids, 30-36 amino acids, 31-36 amino acids amino acids, 32-36 amino acids, 33-35 amino acids, or 33-36 amino acids. In a particular embodiment, the repeat domain may comprise 33 amino acid residues. In other embodiments, the repeating unit comprises 35 amino acid residues. In the present invention, the MAP-NBD is not naturally occurring and is a nucleic acid that is encoded by a C-terminal fragment of the MAP-NBD in order to recognize a target nucleic acid. It contains multiple repeat units arranged at the termini.

[0068] In some embodiments, the repeat domain is a L. quateirensis protein having the sequence: It may be of quality. MPDLELNFAIPLHLFDDETVFTHDATNDNSQASSSYSSKSSPASANARKRTSRKEMSGPPSKEPANTKSRRANSQNNKLS LADRLTKYNIDEEFYQTRSDSLLSLNYTKKQIERLILYKGRTSAVQQLLCKHEELLNLISPDGLGHKELIKIAARNGGGN NLIAVLSCYAKLKEMGFSSQQIIRMVSHAGGANNLKAVTANHDDLQNMGFNVEQIVRMVSHNGGSKNLKAVTDNHDDLKN MGFNAEQIVRMVSHGGGSKNLKAVTDNHDDLKNMGFNAEQIVSMVSNNGGSKNLKAVTDNHDDLKNMGFNAEQIVSMVSN GGGSLNLKAVKKYHDALKDRGFNTEQIVRMVSHDGGSLNLKAVKKYHDALRERKFNVEQIVSIVSHGGGSLNLKAVKKYH DVLKDREFNAEQIVRMVSHDGGSLNLKAVTDNHDDLKNMGFNAEQIVRMVSHKGGSKNLALVKEYFPVFSSFHFTADQIV ALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKPNFNNTSSSTPSPSAPSFFQGPSTPIPTPVLDNSPAPIF SNPVCFFSSRSENNTEQYLQDSTLDLDSQLGDPTKNFNVNNFWSLFPFDDVGYHPHSNDVGYHLHSDEESPFFDF Column number 1).

[0069] As shown in Example 8 herein, the repeat sequence in this L. quateirensis protein The repeats found in the TALE protein from Xanthomonas are at positions 12 and 1. The amino acid sequence is highly conserved at all positions except the third position. The amino acids at the 12th and 13th positions of the repeat in the protein are the repeat variable di-residues ( The repeats in the Xanthomonas TALE protein are called RVDs, which are mostly distributed among the repeats in the Xanthomonas TALE protein. In each case, only the amino acids at these two positions are changed. The repeat units identified in this L. quateirensis protein are positions 12 and 13. The present invention also has additional sequence variations beyond the amino acid positions listed in Example 8. As shown in Figure 1, the aryl groups at positions 12 and 13 in the RUs disclosed herein are The amino acids mediate binding to specific bases. These residues are referred to herein as base-contacting residues. This is called the Building Code Recognition and Control (BCR).

[0070] In some embodiments, the repeats from the L. quateirensis protein are The repeat from the TALE protein from Such BCRs are called canonical BCRs. In some embodiments, the canonical BCR may contain residues NN, NG, or HD. In morphology, the repeats from the L. quateirensis protein are similar to the TALE protein from Xanthomonas. It is possible to include a BCR having a sequence of residues that differs from the RVD in the repeats from the protein. Such BCRs are referred to as non-canonical BCRs. In some embodiments, a non-canonical BCR comprises residues R It may include N, HA, HN, HG, or HK.

[0071] In some embodiments, the repeat of SEQ ID NO:89 comprises the BCR RN and the base guanine (G) In one embodiment, the repeat of SEQ ID NO:2 comprises the BCR HA and recognizes primarily the base adenine ( A) recognizes the repeat of SEQ ID NO:3. In some embodiments, the repeat of SEQ ID NO:3 includes the BCR HN and recognizes the base In some embodiments, the repeat of SEQ ID NO:4 comprises BCR HG and recognizes the base T In some embodiments, the repeat of SEQ ID NO:5 includes BCR NN. , which recognizes the base G. In some embodiments, the repeat of SEQ ID NO:6 comprises BCR NG, In some embodiments, the repeat of SEQ ID NO:7 comprises a BCR HD and recognizes the base T. In some embodiments, the repeat of SEQ ID NO:8 recognizes the base cytosine (C). and recognizes the base T. In some embodiments, the repeat of SEQ ID NO: 9 recognizes the BCR HD. and recognizes base C. In some embodiments, the half repeat of SEQ ID NO: 10 comprises BCR HK and recognizes the base G.

[0072] FIG. 2 shows the protein from L. quateirensis from which the nucleic acid binding repeat domain is derived. Lane number 1 shows the complete protein, which contains repeats of SEQ ID NO:2 to SEQ ID NO:10 and SEQ ID NO:89. include. Table 1 shows Legion alleles that may constitute the MAP-NBD of the present disclosure, including L. quateirensis, L. maceacher nii shows exemplary repeats from Burkholderia, Paraburkholderia or Francisella. The BCR of the repeat (i.e., the amino acids present at positions 12 and 13) are also shown. The MAP-NBD may comprise at least one of the repeats disclosed in Table 1. The AP-NBD can include any combination of the repeats disclosed in Table 1.

[0073] Table 1 shows Legionalles, L. quateirensis, L. maceachei, and other strains that may constitute the MAP-NBDs of the present disclosure. Representative repeats from rnii, Burkholderia, Paraburkholderia, or Francisella are shown. The BCRs of the particular repeats (i.e., the amino acids present at positions 12 and 13) are also shown. The MAP-NBD of the present disclosure may comprise at least one of the repeats disclosed in Table 1. The AP-NBD may comprise any combination of the repeats disclosed in Table 1. [Table 1] JPEG2025037883000003.jpg221170JPEG2025037883000004.jpg219170

[0074] In any of the animal pathogen-derived repeat domains of SEQ ID NOs: 2 to 10, 23 to 89, and 114 to 137, However, in addition to sequence variation in the BCR, there is considerable sequence divergence between the MAP-NBD repeats. Sequences other than the 12th and 13th amino acid positions in the RUs described herein may be used. The lack of sequence conservation is due to the fact that most of the sequence is conserved except for the 12th and 13th amino acid positions. This is in contrast to TALE proteins which contain repeats.

[0075] In some embodiments, the MAP-NBD of the disclosure comprises between 1 and 50 animal pathogen-derived repeat domains. In, for example, between 9 and 36, between 12 and 30, between 5 and 10, between 10 and 15, between 15 and 20, Between 20 and 25, between 25 and 30, between 30 and 35 repeat domains from animal pathogens, or between 35 and In certain embodiments, the repeat domains may include any of the repeat domains from animal pathogens described herein. The MAP-NBDs included are 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, and 21. , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or It may contain 40 animal pathogen derived repeat domains.

[0076] The repeat domain derived from an animal pathogen is a wild-type repeat domain, for example, SEQ ID NOs: 2-10, 23-89 The repeat domains derived from animal pathogens may be derived from any of the sequences ... In addition, modified animal pathogen-derived repeats enhanced for specific recognition of nucleotides or base pairs. The MAP-NBDs described herein can comprise one or more wild-type animal disease domains. a repeat domain derived from a pathogen, one or more modified repeat domains derived from an animal pathogen, or a combination thereof In some embodiments, the modified animal pathogen derived repeat domains may be included. These ligands can enhance the recognition of specific nucleic acid bases or base pairs. , 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, or In some embodiments, the modified animal pathogen derived repeat domain may comprise 29 mutations. A gene may have more than one modification, for example, 1 to 5 modifications, 5 to 10 modifications, 10 to 15 modifications, 15 to 20 modifications, In some embodiments, the amino acid sequence may include 20 to 25 modifications, or 25 to 29 modifications. The MAP-NBD can include multiple modified animal pathogen-derived repeat domains, Each of the derived repeat domains can have a different number of modifications.

[0077] In some embodiments, the MAP-NBD of the present disclosure is a naturally occurring L. quateirensis derived protein. The N-terminus can have the full length of the natural N-terminus of the protein, e.g., the N-terminus of SEQ ID NO: 1. It may be the full length N-terminal sequence and may have the following sequence: MPDLELNFAIPLHLFDDETVFTHDATNDN SQASSSYSSKSSPASANARKRTSRKEMSGPPSKEPANTKSRRANSQNNKLSLADRLTKYNIDEEFYQTRSDSLLSLNYTK KQIERLILYKGRTSAVQQLLCKHEELLNLISPDG (SEQ ID NO: 13). Any truncation of column 13 can be used as the N-terminus in the MAP-NBD of the present disclosure. For example, in some embodiments, MAP-NBD can be prepared by synthesis of naturally occurring L. quatei as follows: rensis, a truncated form containing amino acid residues at positions 15 (D) to 143 (G) at the N-terminus Contains the N-terminus: DATNDNSQASSSYSSKSSPASANARKRTSRKEMSGPPSKEPANTKSRRANSQNNKLSLADRLTKY NIDEEFYQTRSDSLLSLNYTKKQIERLILYKGRTSAVQQLLCKHEELLNLISPDG (SEQ ID NO: 20). In one embodiment, the MAP-NBD is synthesized from the native L. quateirensis N-terminus at position 29 as follows: (N) ~ containing a truncated N-terminus containing the amino acid residue at position 143 (G): NSQASSSYSSKSSPASANARKRTSRKEMSGPPSKEPANTKSRRANSQNNKLSLADRLTKYNIDEEFYQTRSDSLLSLNYT KKQIERLILYKGRTSAVQQLLCKHEELLNLISPDG (SEQ ID NO: 21). Any truncations of the naturally occurring L. quateirensis N-terminus are not intended to be construed as limiting the N-terminus of the NBD disclosed herein. The naturally occurring N-terminus of L. quateirensis can be used at positions 1-50, 1-70, 1-1 truncated to 00, 1-120, 1-130, 10-40, 60-100, or 100-120 amino acid residues and may be used at the N-terminus of the MAP-NBD.

[0078] In some embodiments, the present disclosure provides methods for identifying repeat domains from animal pathogens. For example, a method for producing a nucleic acid sequence comprising a first repeat motif, a spacer, and a second repeat motif is provided. A consensus sequence can be defined that includes the sequence 1xxx211x1xxx33x2x1. xxxxxxxxx1xxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 14), 1xxx211x1xxx33x2x1xxx xxxxxx1xxxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 15), 1xxx211x1xxx33x2x1xxxxx xxxx1xxxxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 16), 1xxx211x1xxx33x2x1xxxxxx xxx1xxxxxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 17), 1xxx211x1xxx33x2x1xxxxxx xxx1xxxxxxxx1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 18). SEQ ID NO: 14 to For any of SEQ ID NO:18, x can be any amino acid residue and 1, 2, and 3 can be any of the following: 1 is a flexible residue defined as: 1 is selected from A, F, I, L, M, T, or V. 2 may be selected from any of D, E, K, N, M, S, R, or Q, and 3 may be selected from A, G, , N, or S. Thus, in some embodiments, MAP-NBD is a polypeptide of SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, or SEQ ID NO:18. The consensus sequence of any of SEQ ID NOs: 14 to 18 may be derived from an animal pathogen. Compare one of them with all sequences in databases such as NCBI, MGRAST, JGI, and EBI. , identifying matches corresponding to animal pathogen proteins that contain repeat units of the DNA-binding repeat domain. It is possible.

[0079] In some embodiments, the MAP-NBD repeat domain is itself 1xxx211x1xxx33x2x1x xxxxxx1 (SEQ ID NO: 19), where x is any amino acid. and 1, 2, and 3 are flexible residues defined as follows: 1 is A , F, I, L, M, T, or V, and 2 is D, E, K, N, M, S, R, or Q may be selected from any of the groups listed in FIG. 3A and Q may be selected from any of the groups listed in FIG. 3A and Q may be selected from any of the groups listed in FIG. and 3B are the consensus sequences of SEQ ID NO: 14 to SEQ ID NO: 18 present in the animal pathogenic proteins of the present disclosure. FIG. 3A shows the consensus sequence of SEQ ID NO: 19 in the MNBDs of the present disclosure. SEQ ID NO: 14 to SEQ ID NO: 15 that may be present in proteins derived from animal pathogens for which the MAP-NBD of the present disclosure is provided. The consensus sequence of sequence number 18 is shown. Each consensus sequence of SEQ ID NO: 14 to SEQ ID NO: 18 is It contains a repeat motif, followed by a spacer of variable length, and a second repeat motif. shows the consensus sequence of SEQ ID NO: 19, which is a repeat domain (e.g., MAP-N BD).

[0080] The terms "MAP-NBD" and "NBD" are used herein to refer to Legionella bacteria or Identified in proteins from animal pathogens such as Dionysella spp. or Francisella spp. The terms "NBD" and "RU" are used interchangeably to refer to an NBD that contains a RU having a sequence derived from a RU.

[0081] In certain embodiments, the present disclosure provides a method for the preparation of a nucleic acid binding domain (NBD) and a heterologous functional domain. The present invention provides a recombinant polypeptide comprising an NBD, the NBD being attached to the N-terminus of the NBD such that the NBD specifically binds to a target nucleic acid. The NBD contains at least three repeating units (RUs) arranged from the α-terminus to the C-terminus, and each RU in the NBD is a Consensus array: 1xxxx11x12xx33xxx1xxxxxxxxxx14xxx, where 1= A, F, I, L, M, T, V or Y; 2= x or xx; 3= A, G, N or S; 4= x, xx or xxx, and x= any ami each RU is independently selected from SEQ ID NOs: 2 to 9, 23 to 35, 85 to 89, and 131 to 137; and a sequence identical to the amino acid sequence set forth in or 99%, or 100%) identical to SEQ ID NOs: 2 to 9, 33 and 89 are repeats identified in a protein from L. quateirensis bacteria (SEQ ID NO: 1). SEQ ID NOs: 23-32, 34-35 and 133 provide the amino acid sequences of the L. maceachernii strain. The amino acid sequence of the repeating unit identified in the protein from Bacillus subtilis (SEQ ID NO: 143) is provided. , SEQ ID NOs: 25, 131-132 and 138 are proteins from Legionella bacteria (SEQ ID NO: 139) SEQ ID NOs: 85-88, 134-137 provide the amino acid sequences of the repeat units identified in Fr Amino acids of the repeating units identified in the protein from the genus Ancisella (SEQ ID NO: 147) In one embodiment, a half RU is present at the C-terminus of the NBD (i.e., after the last RU). ), and a half RU is at least 70% (e.g., at least 15 to 20 amino acids that have at least 75%, 80%, 85%, 90%, 95%, or 99% identity with each other (or 100%) The BCR (i.e., amino acids at positions 12 and 13) contains a sequence of 10 amino acids long and is present in half the RU. , HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, HS, GS or LN.

[0082] In certain aspects, the present disclosure provides a method for the preparation of a nucleic acid binding domain (NBD) and a heterologous functional domain. The present invention provides a recombinant polypeptide comprising an NBD, the NBD being attached to the N-terminus of the NBD such that the NBD specifically binds to a target nucleic acid. It contains at least three repeating units (RUs) arranged from the α-terminus to the C-terminus, and each RU in the NBD is a constitutive unit. Suspension arrangement: (F / L / Y)(D / G / N / S)(A / H / R / S / T / V)(D / E / K / Q)(E / H / Q)(I / L / V)(I / L / V)(C / H / K / R / S) (I / M / V)(A / V)(A / G / S)(H / N / R)(A / D / G / I / K / N / S / V)(G)(G)(A / G / S)(H / K / L / N / R)(N)(I / L)(A / D / E / I / K / V)(A / L / V)(I / M / V)(K / L / Q / T)(A / D / E / K / L / Q / S)(A / C / F / N / V / Y)(F / H / L / Q / Y)(A / D / H / P / Q ) (A / D / I / K / R / T / V) (F / L) (K / M / Q / R / S) (D / E / N / S) (F / L / M) (D / E / G / H / K / N) (SEQ ID NO: 154) Here, the consensus sequence is based on the order Legionelles, the bacterium L. quateirensis, and the bacterium L. mac SEQ ID NOs: 2-10, 23-35, 85-89 and 13 were identified in the proteins from eachernii bacterium. It is based on the amino acid sequence of the repeating unit having the sequence shown in Figure 1-137.

[0083] In certain embodiments, the NBDs provided herein may further comprise a half-RU at the C-terminus, RU is FNAEQIVRMVSX 12 X 13 At least 70% (e.g., at least 15 to 20 amino acids that are at least 75%, 80%, 85%, 90%, 95%, or 99%, or 100%) identical or a sequence of SEQ ID NO: 155 with one or more conservative amino acid substitutions. where X 12 X 13 = HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD , SN, HS, GS or LN.

[0084] In certain aspects, at least three R present in the NBD provided herein Each U is independently FSSQQIIRMVSHAGGANNLKAVTANHDDLQNMG (SEQ ID NO:2); FNVEQIVRMVSHNGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:3); FNAEQIVRMVSHGGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:4); FNAEQIVSMVSNNGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:5); FNAEQIVSMVSNGGGSLNLKAVKKYHDALKDRG (SEQ ID NO:6); FNTEQIVRMVSHDGGSLNLKAVKKYHDALRERK (SEQ ID NO:7); FNVEQIVSIVSHGGGSLNLKAVKKYHDVLKDRE (SEQ ID NO:8); FNAEQIVRMVSHDGGSLNLKAVTDNHDDLKNMG (SEQ ID NO:9); FNAEQIVRMVSHKGGSKNLALVKEYFPVFSSFH (SEQ ID NO:33); or LGHKELIKIAARNGGGNNLIAVLSCYAKLKEMG (SEQ ID NO: 89) At least 70% identical (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99% , or 100% identical)

[0085] HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT at amino acid positions 12 and 13, respectively. , HD, SN, HS, GS or LN (any of SEQ ID NOs: 2 to 9, 33, and 89) In certain aspects, the RUs present in the NBD may be numbered as one or more of the following: Having any of the amino acid sequences of SEQ ID NOs: 2 to 9, 33, and 89 with conservative amino acid substitutions. In certain aspects, the RUs at amino acid positions 12 and 13 of the RU disclosed herein may be The BCR can be replaced with another BCR or RVD to change the base to which the RU binds. .

[0086] In certain embodiments, the NBD comprises at least three RUs and a half repeat as disclosed herein. In certain aspects, the half repeat unit may comprise the last repeat in the NBD. That is, it may be present as the last RU that binds to the last base of the target nucleic acid sequence. In the above, the half RU is present at the C-terminus of the NBD (i.e., after the last RU), where the half RU is At least 70% (e.g., at least 75%, e.g., at least 80% 0%, 85%, 90%, 95%, or 99%, or 100%) identical to a sequence of 15 to 20 amino acids in length. The BCRs present in the half RU (i.e., amino acids at positions 12 and 13) are HK, HD, HA, HN, and HG. , NN, NG, RN, HI, HV, RT, HD, SN, HS, GS or LN.

[0087] In certain aspects, the NBD binds to adenine and has the structure FSSQQIIRMVSX12 X 13 GGANNLKAVTANHDDLQN At least 70% (e.g., at least 75%, 80%, 85%, 90%, 95% 33 to 36 amino acid sequences that are identical (or 99% or 100%) to 2 with conservative amino acid substitutions of at least X 12 X 13 =HA do.

[0088] In certain aspects, the first RU in the NBD (i.e., the RU that binds to the first base of the target nucleic acid sequence) The first RU has at least one nucleotide sequence corresponding to LGHKELIKIAARNGGGNNLIAVLSCYAKLKEMG (SEQ ID NO: 89). 70% identity (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%, or 100% In one embodiment, the first RU of the NBD may be a sequence of 33 to 36 amino acids having a sequence identity with the first RU of the NBD. (i.e., amino acids 12 and 13 numbered with respect to SEQ ID NO:89) acid) is HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, HS, GS, or LN It could be one of the following.

[0089] In one embodiment, the polypeptide comprises an N-terminal domain, wherein the C The terminus (i.e., the last amino acid) is connected, either directly or via a linker, to the N of the first RU of the NBD. In certain aspects, the N-terminus is covalently linked to the The terminal domain is the amino acid sequence of the L. quateirensis protein shown in SEQ ID NO:1. It may be N-terminal and have the amino acid sequence shown in SEQ ID NO: 13. The N-terminal domain may be a fragment of SEQ ID NO: 13, such as the amino acid sequence set forth in SEQ ID NO: 20 or 21. In some embodiments, the N-terminal domain comprises a fragment having one or more conserved amino acids. A polypeptide comprising the amino acid sequence of SEQ ID NO: 13, 20 or 21 with an acid substitution. In one aspect, the N-terminal domain has the amino acid sequence of any of SEQ ID NOs: 13, 20, or 21. At least 85% (e.g., at least 90%, 95%, or 99%, or 100%) identical In certain aspects, the N-terminal domain is a polypeptide comprising the amino acid sequence of TALE proteins such as those expressed in deria, Paraburkholderia, or Xanthomonas The N-cap domain from protein or a fragment thereof.

[0090] In certain aspects, the polypeptide comprises a C-terminal domain, wherein the C-terminal domain The N-terminus (i.e., the first amino acid) is the most important amino acid in the NBD, either directly or via a linker. Covalently attached to the C-terminus (i.e., the last amino acid) of the subsequent RU or half-repeating unit (if any) In certain aspects, the C-terminal domain is linked to the amino acid sequence shown in SEQ ID NO:1. The C-terminus of the L. quateirensis protein having the sequence SEQ ID NO: 12 or SEQ ID NO: 159. In certain aspects, the C-terminal domain may have the amino acid sequence shown in SEQ ID NO: 12, for example, a fragment having the amino acid sequence set forth in SEQ ID NO:22. In one aspect, the C-terminal domain comprises a sequence similar to SEQ ID NO: 12, 159, or 160 with one or more conservative amino acid substitutions. or a polypeptide comprising a sequence of 22 amino acids. The sequence has at least 85% (e.g., 1 / 2) identity to the amino acid sequence of any of SEQ ID NOs: 12, 159, or 22. For example, a polypeptide that contains an amino acid sequence that is at least 90%, 95%, or 99%, or 100% identical to the polypeptide of the present invention. In certain aspects, the C-terminal domain is a peptide derived from Burkholderia, Paraburkholderia deria, or C-capped from TALE proteins such as those expressed in Xanthomonas It is the main or a fragment of it.

[0091] In certain aspects, three or more amino acids present in the NBD of the polypeptides disclosed herein Each of the RUs independently: FKADDAVRIACRTGGSHNLKAVHKNYERLRARG (SEQ ID NO:131); FNADQVIKIVGHDGGSNNIDVVQQFFPELKAFG (SEQ ID NO:132); FSADQVVKIAGHSGGSNNIAVMLAVFPRLRDFG (SEQ ID NO:138); or LDRQQILRIASHDGGSKNIAAVQKFLPKLMNFG (SEQ ID NO:25) At least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%, or 100%) identical to a sequence of 33 to 36 amino acids in length,

[0092] Amino acid positions numbered relative to one of SEQ ID NOs: 25, 131, 132 and 138, respectively. In positions 12 and 13, HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, HS, GS, In certain aspects, each of the three or more RUs may include a BCR selected from the group consisting of 1) 10 ... Each independently comprises one or more conservative amino acid substitutions of SEQ ID NOs: 25, 131, 132, and 138. As shown in Table 1, SEQ ID NOs: 25, 131, 132 and 138 can have one of the following sequences: This is a repeating unit identified in proteins from bacteria of the order Legionellales. The llales bacterial protein has the following amino acid sequence: MPKTKITTVSHGYDLDLMSSLPNGDPNQAK QGKIYLSGNGVYVVRDVAGIVHRGQLEFAINLEQLEQKINEPAFKAVILEKTSRAVGYTISNECFNVELNALAKAGFNNL DIDKLIFRRSSRGTVQTVLNSYNILLEKPYNLDRQQILRIASHDGGSKNIAAVQKFLPKLMNFGFNADQVIKIVGHDGGS NNIDVVQQFFPELKAFGFSADQVVKIAGHSGGSNNIAVMLAVFPRLRDFGFKADDAVRIACRTGGSHNLKAVHKNYERLR ARGYDNKKIISIAASNCGTETINTIMSTDEVEESDFLYFVTTVSTPVASQNLSSASNTNINYSNRFMTARKKTSDDNTDE VEEDQHRDKRRSNGR (SEQ ID NO: 139)

[0093] The RUs identified in this protein are SEQ ID NOs: 25, 131, 132 and 138 in FIG. As shown by:

[0094] In one embodiment, the polypeptide comprises an N-terminal domain, wherein the C The terminus (i.e., the last amino acid) is connected, either directly or via a linker, to the N of the first RU of the NBD. In certain aspects, the N-terminus is covalently linked to the The terminal domain has at least 85% (e.g., at least or a fragment thereof that is at least 90%, 95%, or 99% identical to the amino acid sequence of the SEQ ID NO:140 contains the amino acid sequence N-terminal to the repeat of SEQ ID NO:25 as shown in FIG. A fragment of SEQ ID NO:140 defines a sequence of about 10, 20 or 30 amino acids from the N-terminus of SEQ ID NO:140. In some embodiments, the N-terminal domain may include one or more conserved amino acids. A polypeptide comprising the amino acid sequence of SEQ ID NO: 13, 20, 21, or 140 with a substitution. In an aspect, the N-terminal domain has the amino acid sequence of any of SEQ ID NOs: 13, 20, 21, or 140. At least 85% (e.g., at least 90%, 95%, or 99%, or 100%) of the columns In certain aspects, the N-terminal domain is a polypeptide comprising an amino acid sequence identical to that of the are expressed in Burkholderia, Paraburkholderia, or Xanthomonas. The N-cap domain or a fragment thereof from such a TALE protein.

[0095] In certain aspects, the polypeptide comprises a C-terminal domain, wherein the C-terminal domain The N-terminus (i.e., the first amino acid) is the most important amino acid in the NBD, either directly or via a linker. fused to the C-terminus (i.e., the last amino acid) of the following RU or half-repeat unit (if any) In one embodiment, the C-terminal domain comprises a Legi domain having the amino acid sequence set forth in SEQ ID NO: 139. onellales bacterial protein and has the amino acid sequence set forth in SEQ ID NO: 141. SEQ ID NO:141 may be C-terminal to the last repeat of SEQ ID NO:131, as shown in FIG. The fragment of SEQ ID NO:141 defines an amino acid sequence located at the C-terminus of SEQ ID NO:141. In certain aspects, the C-terminal domain may exclude 1, 20, or 25 amino acids. and fragments of SEQ ID NO: 141, such as fragments having the amino acid sequence set forth in SEQ ID NO: 142. In certain aspects, the C-terminal domain comprises the sequence of SEQ ID NO: 141 with one or more conservative amino acid substitutions. or a polypeptide comprising a C-terminal domain of 142 amino acids. The amino acid sequence of SEQ ID NO: 141 or 142 is at least 85% (e.g., or a polypeptide that contains an amino acid sequence that is at least 90%, 95%, or 99%, or 100%) identical to the In certain aspects, the C-terminal domain is a peptide derived from Burkholderia, Paraburkholderia a, or C-capped domains from TALE proteins such as those expressed in Xanthomonas or a fragment thereof.

[0096] In one embodiment, the C-terminal domain comprises SEQ ID NO:1 with one or more conservative amino acid substitutions. 2, 159, 22, 141 or 142 amino acid sequence. and the C-terminal domain is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 12, 159, 22, 141 and 142. at least 85% (e.g., at least 90%, 95%, or 99%, or 100%) identical to In a particular aspect, the C-terminal domain is a polypeptide comprising an amino acid sequence of TALE proteins such as those expressed in Urkholderia, Paraburkholderia, or Xanthomonas The C cap domain from protein or a fragment thereof.

[0097] In certain aspects, three or more amino acids present in the NBD of the polypeptides disclosed herein Each of the RUs independently: FSAEQIVRIAAHDGGSRNIEAVQQAQHVLKELG (SEQ ID NO:23); FSAEQIVSIVAHDGGSRNIEAVQQAQHILKELG (SEQ ID NO:24); FSAEQIVRIAAHDGGSLNIDAVQQAQQALKELG (SEQ ID NO:26); FSTEQIVCIAGHGGGSLNIKAVLLAQQALKDLG (SEQ ID NO:27); YSSEQIVRVAAHGGGSLNIKAVLQAHQALKELD (SEQ ID NO:28); FSAEQIVHIAAHGGGSLNIKAILQAHQTLKELN (SEQ ID NO:29); FSAEQIVRIAAHIGGSRNIEAIQQAHHALKELG (SEQ ID NO:30); FSAEQIVRIAAHIGGSHNLKAVLQAQQALKELD (SEQ ID NO:31); FSAKHIVRIAAHIGGSLNIKAVQQAQQALKELG (SEQ ID NO:32); FSADQIVRIAAHKGGSHNIVAVQQAQQALKELD (SEQ ID NO:34); FSAEQIVSIAAHVGGSHNIEAVQKAHQALKELD (SEQ ID NO: 35); or FSAEQIVRIAAHIGGSRNIEATIKHYAMLTQPP (SEQ ID NO: 133) At least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or 99%, or 100%) identical to a sequence of 33 to 36 amino acids in length,

[0098] Numbered corresponding to any of SEQ ID NOs: 23 to 24, 26 to 32, 34 to 35, and 133, respectively. At amino acid positions 12 and 13, HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, In certain aspects, each of the three or more RUs comprises a BCR selected from HS, GS, or LN. SEQ ID NOs: 23-24, 26-32, 34-35, and ... and 133.

[0099] The RUs set forth in SEQ ID NOs: 23-24, 26-32, 34-35 and 133 are derived from L. maceachernii. The amino acid sequence of this protein is set forth in the following SEQ ID NO: As provided in Article 143:

[0100] JPEG2025037883000005.jpg91170

[0101] The 12 RUs identified in the protein from L. maceachernii are indicated by underlines. and SEQ ID NOs: 28, 26, 32, 34, 30, 31, 23, 29, 24, 27, 35, and 13, respectively. 3.

[0102] In certain aspects, the polypeptide comprises an N-terminal domain, wherein the N-terminal domain The C-terminus of is fused, directly or via a linker, to the N-terminus of the first RU of the NBD. In this aspect, the N-terminal domain has at least one amino acid sequence similar to that set forth in SEQ ID NO:144. and amino acid sequences with at least 85% (e.g., at least 90%, 95%, or 99%, or 100%) identity. SEQ ID NO:144 contains the repeat of SEQ ID NO:28 in SEQ ID NO:143. The amino acid sequence at the N-terminus is defined as follows:

[0103] MPKTNQPKNLEAKSTKNKISLPQDPQTLNELKIKGYPQDLAERLIKKGSSLAVKTVLKDHEQLVNFFTHLQIIRMAAQKG GAKNITTALNEYNSLTNLG (SEQ ID NO:144)

[0104] A fragment of SEQ ID NO:144 excludes about 10, 20 or 30 amino acids from the N-terminus of SEQ ID NO:144. In certain aspects, the N-terminal domain may have one or more conservative amino acid substitutions. A polypeptide comprising the amino acid sequence of SEQ ID NO: 13, 20, 21, 140 or 144. In one aspect, the N-terminal domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 20, 21, 140, or 144. At least 85% (e.g., at least 90%, 95%, or 99%, or 100%) of the sequence In a particular aspect, the N-terminal domain is a polypeptide comprising an amino acid sequence identical to that of The genes were expressed in Burkholderia, Paraburkholderia, or Xanthomonas. The N-cap domain or a fragment thereof is from a TALE protein such as

[0105] In certain aspects, the polypeptide comprises a C-terminal domain, wherein the C-terminal domain The N-terminus of ) half-repeat unit. In one embodiment, the C-terminal domain is fused to the C-terminus of SEQ ID NO: 143 145, which is the C-terminus of the L. maceachernii protein having the amino acid sequence set forth in SEQ ID NO: 145 may have the amino acid sequence set forth in SEQ ID NO: 13 in SEQ ID NO: 143. The amino acid sequence C-terminal to the last repeat of 3 is defined and has the following sequence:

[0106] YMLSQEQFLRLIDHHSGHLNLSILLDEQQWQAINDLCLQPHHFGRQNALEKFLQQGQRKYQNLQELEQFLFQDSADPMLL QETENQHEAEKINDCMDFILRLISATEPLDLQIEIEGIGLFSPSMHFDATQANFSTPAANEEKIDNSATEAGVNSRKRKI AAAHQKQPPRKKTATPLSATFISTLTTLAQSDNPRLEMASAEALMLKAPQKLAMGITVRKKTKCEGIAIITVTDKTKLNG WLSSASESTYSSVEAQGTRTVNNTHAFFSTPLTSDKKSPSFSSLDFYEDSGLGFDEEITNPPYMPELEPEFIL (Array Number 145)

[0107] A fragment of SEQ ID NO:145 is a fragment of about 10, 20, or 25 amino acids from the C-terminus of SEQ ID NO:145. In certain aspects, the C-terminal domain may be a fragment of SEQ ID NO: 145, such as the sequence In certain aspects, the C-terminal domain of the fragment includes a fragment having an amino acid sequence as set forth in SEQ ID NO:146. The polypeptide comprises the amino acid sequence of SEQ ID NO: 145 or 146 with one or more conservative amino acid substitutions. In certain aspects, the C-terminal domain is a polypeptide of SEQ ID NO: 145 or 146. A polypeptide containing an amino acid sequence that is at least 85% identical to any of the amino acid sequences In certain aspects, the C-terminal domain is a Burkholderia, Paraburkholderia, or the C-cap domain from a TALE protein, such as one expressed in Xanthomonas Or a fragment thereof.

[0108] In a particular aspect, the L. maceachernii protein has the sequence shown in SEQ ID NO: 143. The N-terminal or C-terminal domains derived from The N-terminal domain can be constructed to mediate binding to the sequence G or GG. The N-terminal domain derived from the L. maceachernii protein having the sequence shown in column no. 143 In addition to containing one or both of the C-terminal and C-terminal domains, the polypeptide may also contain multiple antigen-binding domains. Each repeat can contain the bases T, C, A, G, G, A or C, C, T, C, T, G, or A, and SEQ ID NOs: 28, 26, 32, 34, 30, 31, 23, 29, 2 4, 27, 35, and 133, respectively.

[0109] In one embodiment, a recombinant polypeptide comprising an NBD and a heterologous functional domain, It contains at least three repeat units (RU) arranged from the N-terminus to the C-terminus and is specific for the target nucleic acid. Each RU binds to the consensus sequence: Contains YK(P / S)EDIIRLASH(D / G)GGSVNLEAVLRL(H / N)(P / S)QL(I / T)(G / R)LG (SEQ ID NO: 156) The consensus sequence is provided for the Francisella cell line having the amino acid sequence: Based on repeats identified in bacterial proteins:

[0110] JPEG2025037883000006.jpg149170 (SEQ ID NO:147)

[0111] The RU present in SEQ ID NO: 147 is the amino acid sequence shown in SEQ ID NO: 85 to 87, 134 to 137, and 151. The half RU present in SEQ ID NO: 147 has the amino acid sequence shown in SEQ ID NO: 152. These RUs are indicated by underlining in SEQ ID NO: 147 above. RU having the amino acid sequence shown in The amino acid sequence set forth in SEQ ID NO: 86) occurs once in SEQ ID NO: 147. RU( JPEG2025037883000008.jpg11161) occurs once in sequence number 147. JPEG2025037883000009.jpg9163) occurs twice in SEQ ID NO: 147. RU with sequence ( The amino acid sequence set forth in SEQ ID NO: 88 is present three times in SEQ ID NO: 147. RU( The amino acid sequence set forth in SEQ ID NO: 134 is present three times in SEQ ID NO: 147. RU( The amino acid sequence set forth in SEQ ID NO: 135 is represented by JPEG2025037883000012.jpg9163, which occurs once in SEQ ID NO: 147. RU( The amino acid sequence set forth in SEQ ID NO: 136 is represented by the amino acid sequence set forth in SEQ ID NO: 147. RU with JPEG2025037883000014.jpg10163) occurs twice in the sequence of SEQ ID NO:147.

[0112] In certain aspects, the NBD of the recombinant polypeptide disclosed herein comprises three RUs. Each RU may independently comprise at least 70% (e.g., at least 33-36 amplicons having at least 75%, 80%, 85%, 90%, 95%, or 99%, or 100% identity The amino acid sequence includes: YKSEDIIRLASHDGGSVNLEAVLRLHSQLTRLG (SEQ ID NO:85); YKPEDIIRLASHGGGSVNLEAVLRLNPQLIGLG (SEQ ID NO:86); YKSEDIIRLASHGGGSVNLEAVLRLHSQLTRLG (SEQ ID NO:87); YKSEDIIRLASHGGGSVNLEAVLRLNPQLIGLG (SEQ ID NO:88); YKSEDIIRLASHDGGSVNLEAVLRLNPQLIGLG (SEQ ID NO: 134); YKSEDIIRLASHDGGSINLEAVLRLNPQLIGLG (SEQ ID NO: 135); YKSEDIIRLASSNGGSVNLEAVLRLNPQLIGLG (SEQ ID NO: 136); YKSEDIIRLASSNGGSVNLEAVIAVHKALHSNG (SEQ ID NO: 137) or YKINHCVNLLKLNHDGFMLKNLIPYDSKLTGLG (SEQ ID NO: 151); and

[0113] Amino acid position 12 numbered with respect to one of SEQ ID NOs: 85-88, 134-137, and 151 and 13, respectively, HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, HS, GS, or LN In certain aspects, each of the RUs independently comprises one or more BCRs selected from the group consisting of: Any one of SEQ ID NOs: 85-88, 134-137, and 151 with conservative amino acid substitutions The amino acid sequence may be

[0114] In certain embodiments, the polypeptide is set forth in SEQ ID NO: 152 (YNKKQIVLIASGSSGG). At least 70% (e.g., at least 75%, 80%, 85%, 90%, 95%, or a half RU containing a sequence of 15 to 20 amino acids having identity of at least 99%, or 100%). In certain embodiments, the half RU is located at amino acid positions 12 and 13 numbered with respect to SEQ ID NO: 152. In each case, HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, HS, GS or LN.

[0115] In certain aspects, the polypeptide comprises an N-terminal domain and a C-terminal is fused directly or via a linker to the N-terminus of the first RU of the NBD. In the present invention, the N-terminal domain has a small amino acid sequence similar to that of SEQ ID NO: 148 or a fragment thereof. SEQ ID NO:148 contains an amino acid sequence that is at least 85% identical to the sequence present in SEQ ID NO:147. The amino acid sequence N-terminal to the first RU of sequence number 86. The terminal domain has at least one amino acid sequence selected from the group consisting of SEQ ID NO: 13, 20, 21, 140 and 144. 85% (e.g., at least 90%, 95%, or 99%, or 100%) amino acid identity Contains arrays.

[0116] In certain aspects, the polypeptide comprises a C-terminal domain and an N-terminus of the C-terminal domain. is fused directly or via a linker to the C-terminus of the last RU of the NBD. In the present invention, the C-terminal domain has a sequence similar to that of SEQ ID NO: 149, but is at least 85% similar to that of SEQ ID NO: 149. For example, an amino acid sequence having at least 90%, 95%, or 99%, or 100%) identity and fragments thereof, such as the amino acid sequence set forth in SEQ ID NO: 147. In certain aspects, the C-terminus The domain is selected from the group consisting of the amino acid sequences of SEQ ID NOs: 12, 159, 22, 141, 142, 145 and 146. At least 85% (e.g., at least 90%, 95%, or 99%, or 100%) In certain aspects, the C-terminal domain is a polypeptide comprising a unique amino acid sequence. TALEs such as those expressed in Burkholderia, Paraburkholderia, or Xanthomonas The C-cap domain from the protein or a fragment thereof.

[0117] In certain embodiments, the N-terminal domain is a DNA binding domain as disclosed, for example, in US 20180010152. It may be derived from the N-terminal region, such as the N-cap domain used in conjunction with the binding domain. In embodiments, the N-terminal domain may be an N-terminal region disclosed in US 20150225465, e.g. It may be derived from SEQ ID NO: 7, 8, or 9 disclosed therein.

[0118] In certain embodiments, the RUs present in the NBDs of the present disclosure are independent of the RUs provided herein. In certain aspects, the individual RUs listed in Table 1 may be selected from any of the The amino acid modifications (e.g., deletions, insertions, and / or substitutions) can be used to generate NBDs. In certain aspects, the individual RUs listed in Table 1 can be modified by deletions, insertions, and The amino acid sequence of the present invention may be modified via substitution and / or substitution and used to generate NBDs. As previously reported, the RUs identified here from different animal pathogen proteins can be mixed and matched. In another aspect, the NBD can be prepared by the synthesis of a glycerol derivative, as disclosed herein. In certain aspects, the NBDs provided herein may contain RUs from only one genus of bacteria. is a protein derived from the native protein (e.g., Legionella spp., Francisella spp., or proteins from Legionella order bacteria). In some embodiments, the NBDs disclosed herein comprise a half-repeat at the C-terminus of the NBD, and the half repeat is located between the NBD and the linker and / or C domain as provided herein, prior to the start of the C domain. This forms the last RU in

[0119] In certain aspects, one or more RUs in the NBDs provided herein are At least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95% of the RUs provided The identity between the pair of sequences may be 96%, 97%, 98%, or 99%, or 100%. The match identity is calculated by multiplying the number of matches in the pair by 100 and multiplying it by the length of the aligned region, including gaps. Identity scoring counts only exact matches and can be calculated by dividing the The similarity between amino acids is not taken into account. Only internal gaps are considered in length, not gaps at the ends of the sequence. Included.

[0120] Percent identity = (matches x 100) / length of aligned region (including gaps)

[0121] The phrase "conservative amino acid substitution" refers to substitutions of amino acid residues within the following groups: 1) L, I, M, V, F; 2) R, K; 3) F, Y, H, W, R; 4) G, A, T, S; 5) Q, N; and 6) D, E. Conservative amino acid substitutions replace amino acids in a protein with ones that have similar acidity, basicity, charge, or polarity. By substituting amino acids with side chains of different properties or sizes, the activity of the protein can be improved. It can be saved.

[0122] Guidance on substitutions, insertions or deletions is provided for amino acid sequence differences in proteins from different species. Based on sequence alignment or identification of multiple proteins with the same or similar functions It may be based on a quality-based consensus sequence.

[0123] In some embodiments, the N-terminal or C-terminal domains contained in the disclosed NBDs include A nuclear localization sequence (NLS) to facilitate import into the nucleus of a cell, e.g., an animal or plant cell. In certain aspects, the polypeptide is produced in a host cell and comprises a trangenesis amino acid sequence at the N-terminus. The translocation signal may be expressed in association with It may be cleaved during translocation.

[0124] In certain embodiments, a RU is a C-terminal RU with no additional amino acids separating it from the next adjacent RU. In certain aspects, the immediately adjacent RUs can be linked to the N-terminus by at least one amino acid In certain aspects, the spacer sequence may be at least At most 2, 3, 4, 5, 6, or 7 amino acids, or at most 5 amino acids, or at most 10 amino acids The spacer sequence may include amino acids with small side chains. Thus, the spacer sequence is a flexible linker.

[0125] [Linker] Any function, such as a nuclease domain, gene regulatory domain, or fluorophore The domain may be any of the NBDs provided herein, such as MAP-NBD (e.g., L. quateirensis, Burkholderia purpurea, and the like). kholderia, Paraburkholderia or Francisella) directly or via a linker. The linker may be natural or non-natural (e.g., synthetic). In some embodiments, MAP-NBD (e.g., L. quateirensis, Burkholderia, Parabu rkholderia or Francisella) appear to have "molecular Velcro" at their N- or C-termini. For example, a MAP-NBD having a first peptide sequence (e.g., L. quateirensis, Legionellales, Burkholderia, Paraburkholderia or Francisella Separately, a nuclease of interest (e.g., a half-cleavage domain such as FokI) can be produced by: The peptide is constructed with a second peptide sequence that is complementary to and binds to the first peptide sequence. It is possible to form a MAP-NBD-nuclease after administration in cells. to.

[0126] In another embodiment, MAP-NBD (e.g., L. quateirensis, Burkholderia, Paraburk holderia or Francisella) were engineered with linkers at their N- or C-termini. and subsequently, functional domains (e.g., gene editing domains, gene regulatory domains, or can be directly linked to the fluorescent domain).

[0127] In some embodiments, the linker is 3 to 540 nucleotides in length, e.g., 3 to 18, 18 to 33 , 33~48, 48~63, 63~78, 78~93, 93~108, 108~123, 123~138, 138~153, 153~16 8, 168-183, 183-198, 198-213, 213-228, 228-243, 243-258, 258-273, 273-28 8, 288~303, 303~318, 318~333, 333~348, 348~363, 363~378, 378~393, 393~40 8, 408~423, 423~438, 438~453, 453~468, 468~483, 483~513, 513~528, 528~54 3, 3, 18, 33, 48, 63, 78, 93, 108, 123, 138, 153, 168, 183, 198, 213, 228, 243, 258, 273, 288, 303, 318, 333, 348, 363, 378, 393, 408, 423, 438, 453, 468, 483, It can be encoded by a nucleic acid sequence of 498, 513, 528, or 543 nucleotides. In the present study, the linker length was 3, 18, 33, 48, 63, 78, 93, 108, 123, 138, 153, 168, and 18 3, 198, 213, 228, 243, 258, 273, 288, 303, 318, 333, 348, 363, 378, 393, 408, 42 3, 438, 453, 468, 483, 498, 513, 528, or 543 nucleotides. It can be coded as follows.

[0128] In certain aspects, the linker has a length of 1-180, 1-20, 20-40, 40-60, 60-80, 8 It may be 0-100, 100-120, 120-140, 140-160, or 160-180 amino acid residues.

[0129] Nuclease domains were then transferred to MAP-NBD (e.g., L. quateirensis, Burkholderia, Paraburkholderia The linkers for linking to the Streptococcus erythria or Francisella were 1, 10, 20, 30, 40, 5 0, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, or 180 amino acid residues The linker can be 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 1 It may be 20, 130, 140, 150, 160, 170, or 180 amino acid residues.

[0130] In certain aspects, the linker is a C-terminal fragment of a naturally occurring L. quateirensis protein. The C-terminus may be the full-length C-terminal sequence, and ALVKEYFPVFSSFHFTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKPNFNNTSSSTPSPSAPSF FQGPSTPIPTPVLDNSPAPIFSNPVCFFSSRSENNTEQYLQDSTLDLDSQLGDPTKNFNVNNFWSLFPFDDVGYHPHSND VGYHLHSDEESPFFDF (SEQ ID NO: 12). A padded SEQ ID NO: 12 can be used as a linker. The linker is located at position 1 (A) of the naturally occurring L. quateirensis protein as follows: and the C-terminus having amino acid residues from position 63 (P) to position 64 (P) of: ALVKEYFPVFSSFHFTADQIVA LICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKP (SEQ ID NO:22).

[0131] In certain aspects, the linker is a naturally occurring C-terminus of L. quateirensis, e.g. The C-terminus may be the full-length C-terminus sequence of sequence number 1. ALVKEYFPVFSSFHFTADQIVALICQSKQCFRNLKKNHQQWKNKGLSAEQIVDLILQETPPKPNFNNTSSSTPSPSAPSF FQGPSTPIPTPVLDNSPAPIFSNPVCFFSSRSENNTEQYLQDSTLDLDSQLGDPTKNFNVNNFWSLFPFDDVGYHPHSND It may have the sequence VGYHLHSDEESPFFDF (SEQ ID NO:12).

[0132] In certain embodiments, the linker is a C-terminal domain as disclosed herein. obtain.

[0133] In certain embodiments, the C-terminal domain may be present in the polypeptides disclosed herein. is used with a C-terminal region, e.g., a DNA binding domain as disclosed in US 20180010152 In some embodiments, the C-terminal domain can be derived from the C-cap domain. 25465, e.g., SEQ ID NOs: 64, 65, or It can be derived from 66.

[0134] [Functional domain] The NBD or MAP-NBD disclosed herein (e.g., L. quateirensis, Burkholderia, Parab The polypeptides (derived from Escherichia coli, Escherichia coli, or Escherichia coli) can be linked to a functional domain. , genome editing, gene regulation (e.g., activation or repression), or genome imaging In certain aspects, different types of activities can be provided, such as visualization of the gene locus. In this context, the functional domain is heterologous to the NBD. Heterogeneity in the context of domains and NBDs may be due to the fact that these domains originate from different sources. , indicating that they do not exist together in nature.

[0135] In one aspect, a method for regulating expression of an endogenous gene in a cell is disclosed. The method may further comprise introducing into a cell a polypeptide comprising the NBD and a heterologous functional domain, The endogenous gene contains a target nucleic acid sequence that binds to the NBD of the polypeptide, and the heterologous functional domain In some embodiments, the polypeptide is a polypeptide that regulates the expression of an endogenous gene. In certain aspects, the nucleic acid can be introduced as a nucleic acid encoding a gene. In certain aspects, the cell is a human cell and the sequence of the nucleic acid is a human cell. In certain aspects, the functional domain is codon-optimized for expression in a transcriptional regulator. The target nucleic acid sequence is present in the expression control region of a gene, where the polypeptide In certain aspects, functional domains act as transcriptional repressors. and the target nucleic acid sequence is present in an expression control region of a gene, wherein the polypeptide is In one aspect, the expression control region of a gene is a promoter region of the gene. Includes the area.

[0136] In some aspects, the functional domain comprises a cleavage domain or a half-cleavage domain. It is a nuclease and the endogenous gene is inactivated by cleavage. In some cases, inactivation occurs via non-homologous end joining (NHEJ). Inactivation can occur by base pair deletions or insertions, the introduction of single nucleotide polymorphisms (SNPs), or longer mutations. In some aspects, inactivation occurs by the introduction of a stretch of seed DNA. As taught in the specification, this occurs by truncation to generate a premature stop codon. In one embodiment, the polypeptide binds to a first target nucleic acid sequence of the gene and binds to a half-cleavage domain. and the method further comprises binding to a second target nucleic acid sequence of the gene. introducing a second polypeptide, which is a polypeptide comprising an NBD that comprises a half-cleavage domain; In some aspects, the first target nucleic acid sequence and the second target sequence are located within a gene. The two half-cleavage domains are spaced apart and positioned between the first and second target nucleic acid sequences. It mediates the cleavage of gene sequences in

[0137] Also provided herein are methods for introducing an exogenous nucleic acid into a region of interest in the genome of a cell. The method includes: (i) detecting a polypeptide comprising a disclosed NBD and a cleavage domain or half-cleavage domain; a target nucleic acid sequence in which the NBD of the polypeptide is adjacent to the region of interest (ii) a cleavage domain or half-cleavage domain that introduces cleavage in a region of interest; An exogenous nucleic acid, wherein the exogenous nucleic acid is integrated into the desired cleavage region by homologous recombination; The method includes introducing into a cell.

[0138] Further aspects of the polypeptides and methods are described below.

[0139] [A. Genome editing domain] The NBDs disclosed herein, or MAP-NBDs (e.g., L. quateirensis, Burkholderia, P Arabiburgholderia or Francisella) can be linked to a nuclease, where MAP-N The BD provides specificity and targeting, while the nuclease provides the genome editing function. In some embodiments, the nuclease is a cleavage half-domain and is capable of cleaving DNA. In another embodiment, the nuclease dimerizes to form an active complete domain. can be a cleavage domain that is capable of cleaving DNA without the need to dimerize. For example, The cleavage half-domain-containing nuclease is an endonuclease such as FokI or Bfil. In some embodiments, two cleavage half-domains (e.g., FokI and Bfil) can be fused together. The half-cleavage domains can be combined to form a single, fully functional cleavage domain. When used as a nuclease, two MAP-NBDs can be engineered, the first being The first MAP-NBD binds to the top strand of the target nucleic acid sequence and contains a FokI cleavage half-domain, and the second MAP-NBD binds to the top strand of the target nucleic acid sequence and contains a FokI cleavage half-domain. The nucleic acid sequence further comprises a second FokI half-cleavage domain that is linked to the lower strand of the nucleic acid sequence. The ase can be a type IIS restriction enzyme, such as FokI or Bfil.

[0140] In some embodiments, a cleavage domain capable of cleaving DNA without the need to dimerize. The meganuclease may be a homing endonuclease. In certain embodiments, the meganuclease can be I-AniI or I-OnuI.

[0141] MAP-NBD (e.g. L. quateirensis, Burkholderia, Paraburkholderia or Francisella The nuclease domain fused to the nuclease gene (derived from the nuclease) acts as an endonuclease or exonuclease. Endonucleases can be restriction endonucleases and homing endonucleases. Endonucleases may also include S1 nuclease, mung bean nuclease, Nuclease, pancreatic DNase I, micrococcal nuclease, or yeast HO endonuclease The exonuclease may be a 3'-5' exonuclease or a 5'-3' The exonuclease may also be a DNA exonuclease or or RNA exonuclease. Examples of exonucleases include exonuclease RNA polymerase I, II, III, IV, V, and VIII; DNA polymerase I, RNA exonuclease 2 etc.

[0142] The NBD or MAP-NBD disclosed herein (e.g., L. quateirensis, Burkholderia, Parab The nuclease domain fused to the restriction endonucleases (derived from urkholderia or Francisella) In some cases, the restriction enzyme may be a nuclease (or a restriction enzyme). It cleaves DNA at a specific site and has separate binding and cleavage domains. Such restriction enzymes are type IIS restriction enzymes.

[0143] The NBD or MAP-NBD disclosed herein (e.g., L. quateirensis, Burkholderia, Parab The nuclease domain fused to the nuclease of urkholderia or Francisella catalyzes type IIS nucleases. The type IIS nuclease may be FokI or Bfil. In some cases, the MAP -NBD (e.g. from L. quateirensis, Burkholderia, Paraburkholderia or Francisella) In other cases, the nuclease domain fused to the MAP-NBD (e.g., L. quatei rensis, Burkholderia, Paraburkholderia or Francisella) The zed domain is Bfil.

[0144] FokI may be wild-type FokI or may contain one or more mutations. okI can contain 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more mutations. Mutations can abolish cleavage activity. In some cases, Natural mutations can regulate homodimerization. For example, FokI is 446, 447, 479, 483, 484, 486, 487, 490, 491, 496, 498, 499, 500, 531, 534, 537, and 538 may have mutations in one or more amino acid residues.

[0145] In some cases, the FokI cleavage domain can be selected from the group consisting of the FokI cleavage domains described in, for example, Kim et al. zymes: Zinc finger fusions to Fok I cleavage domain,” PNAS 93: 1156-1160 (1996) In some cases, the FokI cleavage sites described herein are as described in In another example, the FokI fragment is that of SEQ ID NO: 11 (Table 2). The cleavage domain is, for example, FokI, as described in US Pat. No. 8,586,526.

[0146] Table 2 can be used herein in conjunction with any method or system described herein. An exemplary FokI sequence is shown. [Table 2]

[0147] MAP-NBD (e.g. L. quateirensis, Burkholderia, Paraburkholderia or Francisella (derived from ) is linked to a functional group that modifies DNA nucleotides, e.g., adenosine deaminase obtain.

[0148] [B. Regulatory domain] As another example, the NBD or MAP-NBD disclosed herein (e.g., L. quateirensis, Burkh olderia, Paraburkholderia or Francisella) are linked to gene regulatory domains Gene regulatory domains can act as activators or repressors. For example, the NBD or MAP-NBD disclosed herein (e.g., L. quateirensi s, Burkholderia, Paraburkholderia or Francisella) contain VP16, VP64, p65, p300 Catalytic domain, TET1 catalytic domain, TDG, Ldb 1 self-association domain, SAM activator (VP64, The term "activation domain" may be linked to an activation domain such as a phosphodiesterase (GPS) domain, ... The terms "activator," "activation domain," and "transcriptional activator" are used interchangeably and are used interchangeably herein. Alternatively, MAP-NBD (e.g., L. quateire) refers to a polypeptide that increases the expression of a gene. nsis, Burkholderia, Paraburkholderia or Francisella) are expressed by KRAB, Sin3a, LSD1 , SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-β-induced early genes (TIEG), v-erbA, SID, MBD2, MBD3, RB, or MeCP2. The terms "repressor," "repressor domain," and "transcriptional repressor" are used herein. The term is used interchangeably with and refers to a polypeptide that reduces the expression of a gene.

[0149] In some embodiments, the NBD or MAP-NBD disclosed herein (e.g., L. quate irensis, Burkholderia, Paraburkholderia or Francisella) are Can be linked to modified proteins. MAP-NBD (e.g., L. quateirensis, Burkholderia, Parabu rkholderia or Francisella) express lysine-specific histone demethylase 1 (LSD1) MAP-NBD (e.g., L. quateirensis , Burkholderia, Paraburkholderia or Francisella) are similar to other tandem strains such as KRAB. It can bind to proteins that can recruit proteins. Proteins that can recruit proteins (e.g., DNMT3a) and other proteins (e.g., KRA B) can act as a repressor of transcription. Thus, DNA-modifying proteins (e.g. :DNMT3a) or domains that can recruit other proteins (e.g., KRAB , a domain found in transcriptional repressors such as Kox1) linked to a MAP-NBD (e.g., L. qua teirensis, Burkholderia, Paraburkholderia or Francisella) are gene suppressors. can provide the ability to function as a transcription factor, where MAP-NBD (e.g. : L. quateirensis, Burkholderia, Paraburkholderia or Francisella) and targeting, recruiting DNA-modifying and other proteins. Proteins that can inhibit the transcription of genes provide gene suppression functionality, which is essential for engineered genome sequencing. Nodal complex or MAP-NBD-gene regulator (MAP-NBD-GR), more specifically MAP-NBD-transcription factor The child (MAP-NBD-TF) can be called

[0150] In some embodiments, the expression of a fusion domain of the present disclosure is reduced compared to untreated cells. By using a DNA-binding domain with a specific function (e.g., MAP-NBD-TF), the expression of the target gene can be reduced. at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, At least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or In some embodiments, the IL-10 expression level can be reduced by at least 99% compared to untreated cells. Therefore, by using the MAP-NBD-TF of the present disclosure, the expression of checkpoint genes can be suppressed by 90%. It can be reduced by more than %.

[0151] In some embodiments, a DNA binding domain (e.g., :MAP-NBD-TF) and subsequent target gene suppression and subsequent target gene expression reduction are at least At least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days , at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days , at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days , at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days , at least 27 days, or at least 28 days. Therefore, the suppression of target genes by the MAP-NBD-TF of the present disclosure and the subsequent reduction in expression of the target genes are , 1-3 days, 3-5 days, 5-7 days, 7-9 days, 9-11 days, 11-13 days , 13 days to 15 days, 15 days to 17 days, 17 days to 19 days, 19 days to 21 days, 21 days to 23 days , 23 to 25 days, or 25 to 28 days.

[0152] In various aspects, the present disclosure provides methods for identifying target binding sites in target genes of cells. The present invention provides a method for producing a nucleic acid sequence comprising: (a) a DNA binding domain, a repressor domain, and a linker domain; (b) contacting the cell with an engineered transcriptional repressor comprising a transcription factor receptor agonist (TGR) and a transcription factor receptor agonist (TGR); (c) measuring whether expression of the target gene is reduced by at least 50% over a period of at least three days. At least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 9 2%, at least 95%, at least 97%, or at least 99% suppression. wherein the target genes are selected from checkpoint genes and T cell surface receptors. Be selected.

[0153] In some aspects, expression of the target gene is in at least 75% of the plurality of cells, at least At least 80%, at least 85%, at least 90%, at least 95%, or at least 99% In some aspects, the engineered genomic regulatory complex is inhibited by at least three In some aspects, the engineered genome regulatory complex is undetectable after days. Determination of inability to identify a target gene was determined by qPCR, FLAG-tag imaging, or a combination of both. In some aspects, the expression of the target gene is measured by Flow cytometric quantification of expression.

[0154] In some embodiments, a DNA binding domain (e.g., Target gene suppression using MAP-NBD-TFs was observed after the DNA-binding domain-TFs became undetectable. DNA-binding domain fused to a repression domain (e.g., MAP-NBD-TF) The repression domain is no longer detectable after at least 3 days. The selected DNA-binding domain (e.g., MAP-NBD-TF) is expressed for at least 1 day, at least 2 days, or at least 3 days. days, at least 4 days, at least 5 days, at least 6 days, at least 1 week, at least 2 weeks may be undetectable after at least three weeks, or at least four weeks. Using qPCR or FLAG-tag imaging, we identified fusion domains in the morphology of Verify that the fused DNA-binding domain (e.g., MAP-NBD-TF) is no longer detectable. can be done.

[0155] In some embodiments, a MAP-NBD containing repeat units from L. quateirensis is used. The promoter region of the human PDCD1 gene the sequence of the target DNA present in Either the main or the C-terminal domain from SEQ ID NO:1 truncated at amino acid position 63 The amino acid sequences of NBDs containing the following are shown in Table 3: [Table 3] JPEG2025037883000017.jpg217170JPEG2025037883000018.jpg216170JPEG2025037883000019.jpg218170JPEG20250378830 00020.jpg215170JPEG2025037883000021.jpg218170JPEG2025037883000022.jpg216170JPEG2025037883000023.jpg158170

[0156] [C. Imaging section] MAP-NBD (e.g. L. quateirensis, Burkholderia, Paraburkholderia or Francisella The fluorophore (derived from hydroxycoumarin, Toxicoumarine, Alexafluor, Aminocoumarin, Cy2, FAM, Alexafluor 488 , Fluorescein FITC, Alexa Fluor 430, Alexa Fluor 532, HEX, Cy3, TRITC , Alexa Fluor 546, Alexa Fluor 555, R-Phycoerythrin (PE), Rhodamine Red-X, Tamara, Cy3.5, Rox, Alexa Fluor 568, Red 613, Texas Red ... Alexafluor 594, Alexafluor 633, Allophycocyanin, Alexafluor 633, C y5, Alexa Fluor 660, Cy5.5, TruRed, Alexa Fluor 680, Cy7, GFP, or mCH ERRY. MAP-NBD (e.g. L. quateirensis, Burkholderia, Paraburkholderia or Francise The IgG1-binding domain (derived from Ila) can be linked to a biotinylation reagent.

[0157] [target] In some embodiments, the methods described herein comprise administering to a subject a MAP-NBD as described herein. The present invention also includes a method of modifying the genetic material of a target cell using a gene encoding a target cell. The target cell may be a eukaryotic or prokaryotic cell. The target cell may be an animal cell or a plant cell. Animal cells may be marine invertebrate cells. The cells may be from vertebrates, fish, insects, amphibians, reptiles, or mammals. Mammalian cells may be obtained from primates, monkeys, horses, cows, pigs, dogs, cats, or rodents. Mammals include primates, apes, dogs, cats, rabbits, ferrets, etc. Rodents include mice, rats, hamsters, gerbils, hamsters, chinchillas, The avian cells may be cells from a canary, parakeet, or parrot. The reptile cells may be from a turtle, lizard, or snake. The cells can be from tropical fish. For example, fish cells can be from zebrafish (e.g., Danio rerio). The nematode cells can be from a nematode (e.g., C. elegans). The zoan cells could be from a frog. The arthropod cells could be from a tarantula or hermit crab. It may be one of the following.

[0158] Mammalian cells also include cells obtained from a primate (e.g., a human or non-human primate). Mammalian cells can include epithelial cells, connective tissue cells, hormone-secreting cells, neuronal cells, and the like. The cells may include cells, skeletal muscle cells, blood cells, immune system cells, or stem cells.

[0159] Exemplary mammalian cells include the 293A cell line, the 293FT cell line, 293F cells, 293H cells, HEK293 cells, and the like. cells, CHO DG 44 cells, CHO-S cells, CHO-K1 cells, Expi293F TM cells, Flp-In TM T-REx TM 293 thin cell line, Flp-In TM -293 cell line, Flp-In TM -3T3 cell line, Flp-In TM -BTK cell line, Flp-In TM -CHO Thin cell line, Flp-In TM -CV-1 cell line, Flp-In TM -Jurkat cell line, FreeStyle TM 293-F cells, FreeSty le TM CHO-S cell line, GripTite TM 293 MSR cell line, GS-CHO cell line, HepaRG TM Cell line, T-REx T M Jurkat cell line, Per.C6 cells, T-REx TM -293 cell line, T-REx TM -CHO cell line, T-REx TM -HeLa Thin cell lines, NC-HIMT cell lines, PC12 cell lines, and primary T cells, primary hematopoietic stem cells, and primary human embryonic stem cells Primary cells (e.g., from humans) including human ES cells (hESCs) and primary induced pluripotent stem cells (iPSCs) These may include, but are not limited to:

[0160] In some embodiments, the MAP-NBDs of the present disclosure can be used to modify target cells. The target cells themselves may be modified or unmodified. Editing unmodified cells with the indicated MAP-NBDs to introduce insertions, deletions, or mutations into their genome In some embodiments, modified cells that already have a mutation can be can be repaired with the MAP-NBD of the present disclosure.

[0161] In some cases, the target cell is a cell that contains one or more single nucleotide polymorphisms (SNPs). Thus, the MAP-NBD nucleases described herein can target and catalyze target cells containing SNPs. It is designed to read and edit

[0162] In some cases, the target cell is a cell that does not contain a modification. The genome may include a genome that is free of defects (e.g., free of genetic mutations) and may be any of the genomes described herein. MAP-NBD nucleases, which are capable of cleaving DNA from DNA, are used to introduce modifications (e.g., mutations) into genomes. obtain.

[0163] In some cases, the target cell is a cancer cell. The cancer may be a solid tumor or a hematological malignancy. Solid tumors can include sarcomas or carcinomas. Exemplary sarcoma target cells are defined as However, it is not known whether the tumor is caused by alveolar rhabdomyosarcoma, alveolar soft part sarcoma, ameloblastoma, angiosarcoma, or chondroma. Sarcoma, Chordoma, Clear cell sarcoma of soft tissue, Dedifferentiated liposarcoma, Desmoid, Desmoplastic angioplasty Epithelioid cell tumor, embryonal rhabdomyosarcoma, epithelioid fibrosarcoma, epithelioid hemangioendothelioma, epithelioid sarcoma, extraosseous Myxoid chondrosarcoma, Extraskeletal osteosarcoma, Fibrosarcoma, Giant cell tumor, Hemangiopericytoma, Infantile fibrosarcoma, Inflammatory Myofibroblastoma, Kaposi's sarcoma, leiomyosarcoma of bone, liposarcoma, malignant fibrosarcoma (MFH), malignant bone fibrous histiocytoma (MFH), malignant mesenchymoma, malignant peripheral nerve sheath tumor, mesenchymal chondrosarcoma, mucinous gland Fibrosarcoma, myxoid liposarcoma, myxoinflammatory fibroblastic sarcoma, and perivascular epiphyseal cell differentiation Bone sarcoma, parosteal osteosarcoma, neoplasm with differentiation into perivascular epiphyseal cells, periosteal osteosarcoma , pleomorphic rhabdomyosarcoma, PNET / extraskeletal Ewing tumor, rhabdomyosarcoma, liposarcoma round, small cell Contains cells from osteosarcoma, solitary fibrous tumor, synovial sarcoma, or osteosarcoma telangiectatica. It can be seen.

[0164] Exemplary carcinoma target cells include anal cancer, appendix cancer, bile duct cancer (i.e., cholangiocarcinoma), bladder cancer, and the like. , brain cancer, breast cancer, cervical cancer, colon cancer, cancer of unknown primary site (CUP), esophageal cancer, eye cancer, fallopian tube cancer, gastrointestinal Cancer of the kidney, liver, lung, medulloblastoma, melanoma, oral cancer, ovarian cancer, pancreatic cancer, parathyroid cancer, Penile cancer, pituitary tumor, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, throat cancer, thyroid cancer, These include, but are not limited to, cells obtained from uterine, vaginal, or vulvar cancer.

[0165] Alternatively, the cancerous cells may include cells obtained from a hematological malignancy. may include leukemia, lymphoma, myeloma, non-Hodgkin's lymphoma, or Hodgkin's lymphoma. In some cases, the hematological malignancy may be a T-cell hematological malignancy. In other cases, the hematological malignancy may be a B-cell based hematological malignancy. Exemplary B-cell hematological malignancies include, but are not limited to, chronic lymphocytic leukemia (CLL). LL), small lymphocytic lymphoma (SLL), high-risk CLL, non-CLL / SLL lymphoma, prolymphocytic Leukemia (PLL), follicular lymphoma (FL), diffuse large B-cell lymphoma (DLBCL), myeloma Mantle cell lymphoma (MCL), nodal marginal zone B-cell lymphoma, nodal marginal zone Zone B-cell lymphoma, Burkitt lymphoma, non-Burkitt high-grade B-cell lymphoma, primary longitudinal Partial B-cell lymphoma (PMBL), immunoblastic large cell lymphoma, B-lymphoblastic lymphoma, B-cell Prolymphocytic leukemia, plasma cell lymphoma, splenic marginal zone lymphoma, plasma cell myeloma, plasma Lymphoma, Mediastinal Lymphoma (Thymus) Large Cell Lymphoma, Intravascular Large B-Cell Lymphoma, Primary Large Cell Exemplary T cell-based hematologic malignancies may include lymphoma, or lymphomatoid granulomatosis. Tumors include, but are not limited to, peripheral T-cell lymphoma, anaplastic large cell lymphoma, hematologic Tumor immunoblastic lymphoma, cutaneous T-cell lymphoma, adult T-cell leukemia / lymphoma (ATLL), blast cell Enteropathic NK-cell lymphoma, enteropathic T-cell lymphoma, splenic gamma delta T-cell lymphoma, lymphoblastic lymphoma , nasal NK / T cell lymphoma, or therapy-related T cell lymphoma.

[0166] In some cases, the cells can be a tumor cell line. Exemplary tumor cell lines include, but are not limited to, Not included, but 600MPE, AU565, BT-20, BT-474, BT-483, BT-549, Evsa-T, Hs578T, MCF- 7, MDA-MB-231, SkBr3, T-47D, HeLa, DU145, PC3, LNCaP, A549, H1299, NCI-H460, A27 80, SKOV-3 / Luc, Neuro2a, RKO, RKO-AS45-1, HT-29, SW1417, SW948, DLD-1, SW480, Ca pan-1, MC / 9, B72.3, B25.2, B38.1, DMS153, SU86.86, SNU-182, SNU-423, SNU-449, SN U-387, Hs817.T, LMH, LMH / 2A, SNU-398, PLHC-1, HepG2 / SF, OCI-Ly1, OCI-Ly2, OCI-Ly 3, OCI-Ly4, OCI-Ly6, OCI-Ly7, OCI-Ly10, OCI-Ly18, OCI-Ly19, U2932, DB, HBL-1, RI VA, SUDHL2, TMD8, MEC1, MEC2, 8E5, CCRF-CEM, MOLT-3, TALL-104, AML-193, THP-1, B DCM, HL-60, Jurkat, RPMI8226, MOLT-4, RS4, K-562, KASUMI-1, DA, DA, DA-10, Raudi , Jeko-1, NK-92, and Mino.

[0167] In some embodiments, the methods described herein comprise administering to a subject a MAP-NBD as described herein. In some embodiments, the nucleic acid of the present disclosure can be used to modify a target gene. The enzyme is fused to a DNA binding domain for a specific genomic locus of interest. Genome editing can be achieved by using a combination of techniques. Genetic modification can be used to insert functional genes for therapeutic purposes. gene transfer, gene knockout for therapeutic gene, or administration to subjects in need This may include ex vivo engineering of cells that repair the tumor (e.g., HSC or CAR T cells). The genome editing complex includes PDCD1, CTLA4, LAG3, TET2, BTLA, HAVCR2, CCR5, CXCR4, TRA, and TRB. Erythroid-specific enhancement of the B2M, albumin, HBB, HBA1, TTR, NR3C1, CD52, and BCL11A genes Sensor, CBLB, TGFBR1, SERPINA1, HBV genomic DNA in infected cells, CEP290, DMD, CFTR, IL2 The target site can be within RG, CS-1, or any combination thereof. In this study, the genome editing complex contains a chimeric antigen receptor (CAR), α-L iduronidase (IDUA) , iduronate-2-sulfatase (IDS), or factor 9 (F9). The genome editing complex can be used to edit hematopoietic stem cells (HSCs) and T cells. Alternatively, the genome editing complexes can be used to engineer cells, such as human cells, ex vivo. It may be administered directly to a subject in need thereof.

[0168] [Method of producing polypeptide] In certain embodiments, the polypeptides disclosed herein, such as MAP-NBD, are recombinant and may be produced using any suitable method, including non-recombinant methods (eg, chemical synthesis).

[0169] [A. Chemical synthesis] When the polypeptide is chemically synthesized, the synthesis may proceed via liquid phase or solid phase. Solid-phase peptide synthesis (SPPS) is a method for the synthesis of unnatural amino acids and / or peptide / protein backbone modifications. Various forms of SPPS, such as Fmoc and Boc, allow for the incorporation of the polypeptides of the present disclosure. The details of chemical synthesis are known in the art. (e.g. Ganesan A. 2006 Mini Rev. Med. Chem. 6:3-10; and Camarero JA et al., 2005 Protein Pept Lett. 12:723-8).

[0170] [B. Recombinant Production] When the polypeptide is produced using recombinant techniques, the polypeptide may be produced in any suitable configuration. Using the construct and any suitable host cell, the polypeptide can be expressed as an intracellular or secreted protein. These can be produced in prokaryotic, such as bacterial (e.g., E. coli) or yeast host cells, respectively. In certain aspects, the host for the production of the polypeptide may be a yeast cell or a eukaryotic cell. The eukaryotic cells used as cells include insect cells, mammalian cells, and / or plant cells. In certain aspects, mammalian host cells are used, including human cells (e.g., HeLa, 293, H9, and Jurkat cells); mouse cells (e.g., NIH3T3, L cells, and and C127 cells; primate cells (e.g., Cos 1, Cos 7, and CV1) and hamster cells (e.g. In certain embodiments, the present invention may include a method for producing a cell line comprising the steps of: The polypeptides disclosed herein are produced in CHO cells.

[0171] A variety of host-vector systems suitable for expression of the polypeptides can be prepared according to standard procedures known in the art. For example, Sambrook et al., 1989 Current Protocols in Molecular Biology Cold Spring Harbor Press, New York; and Ausubel et al. 1995 C See Current Protocols in Molecular Biology, Eds. Wiley and Sons. Methods for introducing the gene into cells include, for example, transformation, electroporation, conjugation, phosphorylation, etc. Stable expression of the introduced polypeptide-encoding nucleic acid The method of transfer can be selected to provide a nucleic acid encoding a polypeptide. The vector may be provided as an inheritable episomal element (e.g., a plasmid) or may be genomically integrated. A variety of suitable vectors for use in producing the polypeptide of interest are commercially available. It is being done.

[0172] The vector may provide for extrachromosomal maintenance in the host cell or may be integrated into the host cell genome. The expression vector can provide transcriptional and translational regulatory sequences. and the coding region is operatively under the transcriptional control of a transcription initiation region and a transcription and translation termination region. When linked to a transcription factor, it can provide inducible or constitutive expression. and translational regulatory sequences, such as promoter sequences, ribosome binding sites, transcription start and stop sites. sequences, translation initiation and termination sequences, and enhancer or activator sequences. The promoter may be, but is not limited to, a constitutive promoter or an inducible promoter. The promoter may be a strong constitutive promoter (e.g., T7).

[0173] Also provided herein are nucleic acids encoding the polypeptides disclosed herein. In certain aspects, the nucleic acid encoding the polypeptide disclosed herein is In certain aspects, the nucleic acid sequence is operably linked to a promoter sequence that effects expression of the nucleic acid. Therefore, the sequence of the nucleic acid is codon optimized for expression of the polypeptide in human cells. In some embodiments, the nucleic acid is deoxyribonucleic acid (DNA). The nucleic acid is ribonucleic acid (RNA). Also disclosed herein are polypeptides for binding to target nucleic acids. Also provided herein is a vector comprising a nucleic acid encoding the peptide. The vector is a viral vector.

[0174] In certain aspects, a nucleic acid or vector encoding a polypeptide disclosed herein is In certain aspects, a host cell is provided that contains the polypeptide disclosed herein. In a particular aspect, a host cell expressing the polypeptide is provided. Also disclosed are cells.

[0175] [Delivery] The polypeptides disclosed herein, compositions comprising the disclosed polypeptides, and Nucleic acids encoding the indicated polypeptides can be introduced into the host by, for example, injection, infection, transfection, and delivered to target cells by any suitable means, including vesicle- or liposome-mediated delivery. It can be done.

[0176] In certain aspects, mRNA or vectors encoding the polypeptides disclosed herein are The vector can be introduced into target cells via injection, transfection, or viral infection. The method can include, but is not limited to, the step of: mide vector, retroviral vector, lentiviral vector, adenoviral vector vectors, poxvirus vectors, herpesvirus and adeno-associated virus vectors Any vector system can be used, including those described herein, including those described herein. When the nucleic acid encoding the polypeptide is introduced into a cell, the nucleic acid encoding the polypeptide may be introduced into the cell from the same vector or a different vector. Non-viral vector delivery systems include DNA plasmids, Naked nucleic acid and nucleic acid complexed with a delivery vehicle such as a liposome or poloxamer Viral vector delivery systems include DNA and RNA viruses, which After delivery to a cell, the genome is episomal or integrated. Suitable vectors for introducing polynucleotides include non-integrating lentiviral vectors. (IDLV) is one example.

[0177] Non-viral vector delivery systems include electroporation, lipofection, and microinjection. Injection, gene gun, virosome, liposome, immunoliposome, polycacti On or lipid: Nucleic acid conjugates, naked DNA, artificial virions, and drug-enhanced uptake of DNA This includes the insertion.

[0178] Primary cells can be isolated and used ex vivo for reintroduction into the subject to be treated. Suitable primary cells include peripheral blood mononuclear cells (PBMCs) and other blood cell subsets, e.g. Examples include, but are not limited to, CD4+ T cells or CD8+ T cells. For example, embryonic stem cells, induced pluripotent stem cells, hematopoietic stem cells, neural stem cells, mesenchymal stem cells, Stem cells include muscle stem cells and skin stem cells. In certain aspects, stem cells are The polypeptides disclosed herein may be isolated from or treated with a subject. The cells may be derived from the somatic cells of a given subject.

[0179] In certain aspects, a polypeptide of the present disclosure or a polypeptide encoding the present disclosure The cell into which the nucleic acid is introduced can be an animal cell, for example a cell from a human in need of treatment. In another aspect, the cell may be a plant cell. The DNA construct may be prepared according to various conventional techniques. For example, the DNA construct can be introduced into a desired plant host (e.g., into its genome) by The constructs were synthesized by electroporation and microinjection of plant cell protoplasts. They can be introduced directly into the genomic DNA of plant cells using techniques such as transcriptional cleavage or by DNA constructs. Entities can be introduced directly into plant tissue using biolistic methods such as DNA particle bombardment.

[0180] In certain aspects, the polypeptides of the present disclosure are only present transiently in the target cell. For example, the polypeptide may be expressed for a short period of time, such as for example, 1 day, 3 days, 1 week, 3 weeks, or up to 1 month. A polypeptide is expressed from the nucleic acid. Transient expression of the polypeptides of the present disclosure is desired. In practice, adenovirus-based systems can be used. AAV ("AAV") vectors also are used to transduce cells with nucleic acids encoding the polypeptides of the present disclosure. For example, in the in vitro production of nucleic acids and peptides, and in It can be used for in vivo and ex vivo gene therapy procedures. , recombinant adeno-associated virus vectors such as replication-deficient recombinant adenovirus vectors ( rAAV) is used to introduce nucleic acids encoding the polypeptides disclosed herein. It is possible.

[0181] In certain aspects, the nucleic acid encoding the polypeptide disclosed herein is Delivery using gene therapy vectors with high specificity for tissue or cell type Viral vectors typically contain a viral envelope protein on the outer surface of the virus. By including a sequence encoding a ligand that is expressed as a fusion protein with the protein The ligand is engineered to have specificity for a given cell type. The antibody is selected to have affinity for a receptor known to be present on the

[0182] In certain aspects, gene therapy vectors are administered to individual patients in vivo. In certain aspects, administration can be by systemic administration (e.g., intravenous administration), as described below. Intravenous, intraperitoneal, intramuscular, subcutaneous, or intracranial injection), direct injection (e.g., intrathecal), Alternatively, the vectors can be administered intracellularly via injection into cells explanted from an individual patient (e.g., , lymphocytes, bone marrow aspirates, tissue biopsies) or universal donor hematopoietic stem cells ex vivo The vector is then delivered to a cell, usually a cell that has incorporated the vector or a cell that contains a gene encoding the vector. After selection for cells modified by expression of the polypeptide of the present disclosure, the cells are It may be reimplanted into the patient.

[0183] In certain aspects, the nucleic acid encoding the polypeptide provided herein comprises: Codon optimization may be used to enhance expression of the polypeptide in a target cell. For example, In order to enhance the production of a polypeptide in a human cell, the polypeptide may be expressed in an animal cell, such as a human cell. The sequence of the nucleic acid is altered to provide codons known to be highly used in For example, the methods disclosed herein for codon optimization in mammalian cells can be used. By making silent mutations in the nucleotide sequence encoding the polypeptide to be Similar codon optimization can be performed in other host cell systems (e.g., plants, fungi, etc.). can be used for optimal expression.

[0184] [Composition] In certain aspects, the polypeptides and nucleic acids described herein are pharma- ceutically acceptable. In certain aspects, the polypeptide and The nucleic acid and the nucleic acid are present in the pharmaceutical composition in a therapeutically effective amount. A therapeutically effective amount can be determined based on the efficacy observed in cells (e.g., the present invention). In a reporter cell line in which expression of the reporter is regulated in response to the disclosed polypeptides. The desired effect of the compound can be determined using an assay that measures the desired effect of the compound. To carry out the methods and uses of treatment and prevention described herein, subjects are It may be administered in vivo.

[0185] Pharmaceutical compositions of the disclosure can be formulated to be compatible with the intended method or route of administration. Exemplary routes of administration are described herein. The therapeutically acceptable diluent, carrier or excipient may include nuclease inhibitors, proteases, Inhibitors, suitable vehicles such as saline or citrate buffered saline, , but not limited to these.

[0186] [array] The sequences of further polypeptides described herein are as follows: JPEG2025037883000024.jpg203170 EXAMPLES

[0187] These examples are provided for illustrative purposes only and are not intended to be limiting of the scope of the claims provided herein. It is not intended to limit the scope.

[0188] [Example 1] Production of modular animal pathogen-nucleic acid binding domains (MAP-NBDs) This example illustrates the production of modular animal pathogen-nucleic acid binding domains (MAP-NBDs). MAP-NBD is derived from L. quateirensis. MAP-NBD is represented by SEQ ID NO: 2 to SEQ ID NO: 10 or SEQ ID NO: The repeat domains are from L. quateirensis, such as one of the repeat domains numbered 89. The domains are selected to recognize and bind to specific nucleotides or base pairs. Thus, a MAP-NBD is provided that targets and binds to a nucleic acid sequence of interest. The nucleic acid may be DNA or RNA. The MAP-NBD may be recombinantly expressed or synthetically constructed.

[0189] [Example 2] Production of modular animal pathogen-nucleic acid binding domain (MAP-NBD)-nucleases This example shows the construction of a modular animal pathogen-nucleic acid binding domain (MAP-NBD)-nuclease. The production is exemplified. MAP-NBD is derived from L. quateirensis. MAP-NBD is represented by SEQ ID NO: 2 to SEQ ID NO: Multiple antibodies from L. quateirensis, such as either SEQ ID NO: 10, SEQ ID NO: 89, or SEQ ID NO: 33, were Each repeat domain recognizes a specific nucleotide or base pair. and a MAP-NBD that is selected to bind to and thereby target and bind to a nucleic acid sequence of interest. The MAP-NBD nuclease may be recombinantly expressed or synthetically constructed. The nuclease is linked via a linker. The linker is a synthetic linker, SEQ ID NO:1. The full length C-terminus of the native L. quateirensis protein as set forth in SEQ ID NO: 1, or the native L. quateirensis protein as set forth in SEQ ID NO: 2, ateirensis protein. The nuclease is a cleavage domain (e.g., I-An meganucleases such as I-I or I-OnuI) or cleavage half-domains (e.g., FokI nucleases The enzyme is, for example, SEQ ID NO: 11, or Bfil.

[0190] [Example 3] Modular animal pathogen-nucleic acid binding domain (MAP-NBD)-gene regulator (MAP-NBD-GR) manufacturing This example shows the modular animal pathogen-nucleic acid binding domain (MAP-NBD)-gene regulator The production of MAP-NBD-GR is exemplified. MAP-NBD is derived from L. quateirensis. MAP-NBD is a derivative of the L. quateirensis, such as any of sequence numbers 2 to 10, 89, or 33 wherein each repeat domain is a specific nucleotide or The nucleic acid sequence is selected to recognize and bind to a base pair, thereby targeting and binding to a nucleic acid sequence of interest. The MAP-NBD-GR is either recombinantly expressed or synthetically constructed. The MAP-NBD is linked to a gene regulatory domain via an optional linker. The linker is a synthetic linker, the full-length C-terminus of the native L. quateirensis protein set forth in SEQ ID NO:1. or a C-terminal fragment of the native L. quateirensis protein set forth in SEQ ID NO: 1. Child regulators include gene activators (e.g., VP16, VP64, p65, p300 catalytic domain, TE T1 catalytic domain, TDG, Ldb 1 self-association domain, SAM activators (VP64, p65, HSF1), if VPR (VP64, p65, Rta) or gene repressors (KRAB, Sin3a, LSD1, SUV39H1, G9 A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-β-induced early genes (TIEG), ve rbA, SID, MBD2, MBD3, RB, or MeCP2).

[0191] [Example 4] Fabrication of modular animal pathogen-nucleic acid binding domain (MAP-NBD)-fluorophores This example shows the modular animal pathogen-nucleic acid binding domain (MAP-NBD)-fluorophore The production of MAP-NBD is exemplified. MAP-NBD is derived from L. quateirensis. MAP-NBD is represented by SEQ ID NO: 2 to SEQ ID NO: Multiple clones from L. quateirensis, such as SEQ ID NO: 10, SEQ ID NO: 89, or SEQ ID NO: 33 The repeat domains each recognize a particular nucleotide or base pair. The MAP-NB is selected to recognize and bind to a nucleic acid sequence of interest, thereby targeting and binding the nucleic acid sequence of interest. D. The MAP-NBD-fluorophore may be recombinantly expressed or synthetically constructed. The MAP-NBD is linked to a fluorophore via an optional linker. The linker may be a synthetic linker, the full-length C-terminus of the native L. quateirensis protein set forth in SEQ ID NO:1, or or a C-terminal fragment of the native L. quateirensis protein set forth in SEQ ID NO: 1. The phore is a fluorescent moiety such as green fluorescent protein or mCHERRY.

[0192] [Example 5] Genome editing with modular animal pathogen-nucleic acid binding domain (MAP-NBD)-nucleases This example uses modular animal pathogen-nucleic acid binding domain (MAP-NBD)-nucleases. The genome editing used is shown. MAP-NBD nuclease was used as described in Examples 1 and 2. MAP-NBD is engineered to contain multiple repeats that target a nucleic acid sequence of interest. When a cleavage half-domain (e.g., FokI) is used as a nuclease, each Two MAP-NBDs with cleavage half-domains were prepared and administered. The first MAP-NBD bound to the first target nucleic acid sequence is designed to bind to another FokI cleavage site. A second MAP-NBD covalently attached to the hemidomain is engineered to bind to a second target nucleic acid sequence. wherein the first and second target nucleic acid sequences are prepared such that the first and second MAP-NBDs are the respective targets. The FokI cleavage half-domains are appropriately spaced to provide for dimerization of the FokI cleavage half-domains upon binding to a target nucleic acid sequence. When administered to a subject, the MAP-NBDs bind to their target nucleic acid sequences. Dimerization of two FokI cleavage half-domains results in enzymatic activity and induction of double-strand breaks (DSBs) A cleavage domain (e.g., a meganuclease such as I-AniI or I-OnuI or two FokIs) When a MAP-NBD-binding domain is used, a single MAP-NBD bound to the cleavage domain is administered to the subject. The subject may be a cell, a plurality of cells, or a human. The nuclease induces a double-strand break in the target sequence, Provides non-homologous end joining or homology-guided repair, thereby providing genome editing functionality do.

[0193] [Example 6] Modular animal pathogen-nucleic acid binding domain (MAP-NBD)-gene regulator (MAP-NBD-GR) Gene regulation by An example of this is the modular animal pathogen-nucleic acid binding domain (MAP-NBD)-gene regulator (MAP The results show gene regulation by MAP-NBD-GR. MAP-NBD-GR was synthesized as described in Examples 1 and 3. MAP-NBDs are engineered to contain multiple repeats that target a nucleic acid sequence of interest. MAP-NBD-GR is administered to a subject. The subject may be a cell, a plurality of cells, or a human or non-human The animal is a human primate or other animal. MAP-NBD-GR is then transfected with the gene Regulating expression (eg, activation or repression domains).

[0194] [Example 7] Modular Animal Pathogen-Nucleic Acid Binding Domain (MAP-NBD)-Fluorophore Imaging G This example shows the modular animal pathogen-nucleic acid binding domain (MAP-NBD)-fluorophore This example illustrates imaging of genomic loci in cells using MAP-NBD fluorophores. The MAP-NBD is prepared as described in Examples 1 and 4. The MAP-NBD is a nucleotide sequence that targets a nucleic acid sequence of interest. The MAP-NBD-fluorophore is administered to the cells. The cells may be in a plurality of cells ex vivo or in culture in an animal, such as a human or non-human primate. The MAP-NBD-fluorophore hybridizes to the target nucleic acid sequence, thereby Imaging allows visualization of the labeled target cells. and labeled target cells indicate the presence of the targeted genomic locus.

[0195] [Example 8] Base specificity of repeats from L. quateirensis This example shows the determination of the base specificity of a repetitive sequence from L. quateirensis. A polypeptide comprising the L. quateirensis protein of the sequence set forth in SEQ ID NO: 1 fused to a tag. The base specificity of each of the repeats of SEQ ID NO:89 and SEQ ID NO:2 to SEQ ID NO:10 was examined. Decided by ELEX.

[0196] In vitro transcription and translation (IVTT) of target polypeptides was performed using the Thermo Fisher 1-Step Human Co-translation Kit. The following reagents were added to a nuclease-free tube and mixed: The following was added: HeLa lysate (15 μl), accessory protein (3 μl), reaction mixture (6 μl), plus MidDNA (0.2-0.5 μg / μl) (2.4 μl), and nuclease-free HO (3.6 μl). Incubate at 30°C for 120 minutes to 6 hours.

[0197] Protein expression was monitored by Western blot analysis using appropriate primary and HRP-conjugated secondary antibodies. It was detected by

[0198] A mixture of all four phosphoramidites was used to synthesize oligonucleotide targeting The library was randomly chemically synthesized. Each oligonucleotide sequence was independently It is flanked by a 20 base pair sequence of: ACACGACGCTCTTCCGATCTNNNNNNNNNNNNNNNNNNNNNN NNNAGATCGGAAGAGCACACGTC, where N is one of the four phosphoramidites. The sequence was annealed to the 3' library primer (5'- GACGTGTGCTCTTCCGATCT) as follows: The oligonucleotide sequences in the library are isolated by incubating the library under the conditions Converted to double stranded: [Table 4]

[0199] The oligonucleotide library was digested with ExoI for 2.5 h at 37 °C to produce ssD NA was removed. [Table 5]

[0200] DNases were prepared using phenol:chloroform:isoamyl alcohol and EtOH precipitation (-70 °C, 1.5 h). Clean up A. The double stranded library was resuspended in QIAGEN EB buffer.

[0201] SELEX was carried out using the following SELEX buffer. [Table 6] [Table 7] [Table 8]

[0202] For each polypeptide tested, 25 μl of Dynabeads® His-Tag or Pierce e™ Anti-HA magnetic beads were washed once with 200 μl of SELEX binding buffer. The beads were washed for 25 min. The mixture was resuspended in 1 μl of SELEX binding buffer and placed on ice.

[0203] SELEX Cycle 1 The bead-protein complex was incubated with 200 pmol of oligonucleotide in a total volume of 100 μl of SELEX buffer. The products of the in vitro transcription and translation reactions were mixed with the double-stranded library. , preincubated with SELEX buffer for 10 min and then added to the library duplexes. . [Table 9] The tube was placed on a rotator at a low angle and incubated at room temperature for 50 minutes. Washed beads (in SELEX binding buffer) were added to the complexes and incubated on a rotator for 20 min. The tube was placed on a magnet for 2 minutes and the supernatant was discarded. The beads were then incubated for 2 hours. The bound DNA target (with beads) was washed six times with EX wash buffer. The library primers were used for PCR amplification: fwd: ACACGACGCTCTTCCGATCT Rev: GACGTGTGCTCTTCCGATCT [Table 10] The PCR-1 reaction was separated from the beads on a magnet. 5 μl of the PCR-1 reaction was used for PCR-2 amplification. and 1 μl of PCR-2 was used in SELEX cycle 2. [Table 11]

[0204] SELEX Cycle 2 The bead-protein complexes were mixed with the PCR products from the first cycle in a total volume of 100 μl for SELEX. The in vitro transcription-translation (IVTT) reaction was mixed with SELEX buffer for 10 min. Reincubate. [Table 12] The tube was placed on a rotator at a low angle and incubated at room temperature for 50 minutes. Washed beads (in SELEX binding buffer) were added to the complexes and incubated on a rotator for 20 min. The tube was placed on a magnet for 2 minutes and the supernatant was discarded. The beads were The beads were washed six times with ELEX buffer. The bound DNA target (with beads) was then purified by the following 5' / 3' ligation: The fragment was PCR amplified using the following primers: fwd: ACACGACGCTCTTCCGATCT and rev: GAC GTGTGCTCTTCCGATCT. [Table 13] The PCR-1 reaction was separated from the beads on a magnet. 5 μl of the PCR-1 reaction was used for PCR-2 amplification. and 25 μl of PCR-2 was used in the third SELEX cycle. [Table 14]

[0205] SELEX Cycle 3 The IVTT protein and the first cycle PCR product were mixed in a total of 100 μl of SELEX buffer. Preincubate the in vitro transcription-translation (IVTT) reaction with SELEX buffer for 10 min. did. [Table 15] The tube was placed on a rotator at a low angle and incubated at room temperature for 50 minutes. Add the washed beads (in SELEX binding buffer) to the complex and incubate on a rotator for 2 The tubes were placed on a magnet for 2 minutes and the supernatant was discarded. The beads were washed six times with SELEX wash buffer. The bound DNA target (with beads) was analyzed by the following 5' / 3 The library was amplified by PCR using primers: ACACGACGCTCTTCCGATCT, rev: GACGTGTGCT CTTCCGATCT. [Table 16] The PCR-1 reaction was separated from the beads on a magnet. 5 μl of the PCR-1 reaction was used for PCR-2 amplification. Ta. [Table 17] The PCR-1 reaction from the third SELEX cycle was separated from the beads on the magnet. , and PCR-1 reactions from the third SELEX cycle were analyzed by MiniSeq.

[0206] Paired-end reads were merged using BBmerge. We extracted the variable region sequences of the base pairs. 5000 sequences were randomly selected from the entire set (including duplicates). ), the GADEM motif discovery program with the parameters '-maskR 1 -fullScan 1 -gen 3' The sequence logos in Figures 4A and 4B were provided as input to GADEM MME format. The logo was drawn using the "ceqlogo" tool in the MEME suite from a logo file.

[0207] 4A and 4B show the binding motif determined for the polypeptide of SEQ ID NO:1, This is the sequence LGHKELIKIAARNGGGNNLIAVLSCYAKLKEMG (sequence No. 89) and each of the following repeats of SEQ ID NO: 2 to SEQ ID NO: 10. The larger the size of the base in the nucleic acid that binds to the tested polypeptide, the greater the The base occurs at a high relative frequency in

[0208] FIG. 4A shows a repeat sequence of a polypeptide having the amino acid sequence set forth in SEQ ID NO:1 (from the N-terminus to The sequence of SEQ ID NO: 89 and 2 to 10) arranged toward the end indicate the bases to be bound. The polypeptide having the amino acid sequence was fused to a His tag.

[0209] FIG. 4B shows a repeat sequence of a polypeptide having the amino acid sequence set forth in SEQ ID NO:1 (from the N-terminus to At the end: SEQ ID NO: 89 and 2 to 10) indicate the bases to which the amino acid sequence shown in SEQ ID NO: 1 is bound. The His-tagged polypeptide and the HA-tagged polypeptide were fused to an HA tag. Tides showed the same binding specificity.

[0210] The numerical representation of the position weighting matrix (PWM) of Figures 4A and 4B is shown in the table below. The background character frequencies from the background are: A - 0.25000; C - 0.25000; G - 0.25000; and T - 0.25000. [Table 18] [Table 19]

[0211] [Example 9] Creation of modular animal pathogen-nucleic acid binding domains (MAP-NBDs) This example illustrates the production of modular animal pathogen-nucleic acid binding domains (MAP-NBDs). MAP-NBD is a marker for L. quateirensis, Burkholderia, Paraburkholderia, Francisella, or MAP-NBD is derived from L. quateirensis, Burkholderia, Pa multiple repeat domains from Raburkholderia, Francisella, or any combination thereof; For example, any one of the repeats in Table 1, wherein each repeat domain is a specific nucleotide sequence. The nucleic acid sequence of interest is targeted to the target nucleic acid sequence. The nucleic acid may be DNA or RNA. The MAP-NBD may be recombinantly expressed or synthesized. It will be constructed.

[0212] [Example 10] Production of modular animal pathogen-nucleic acid binding domain (MAP-NBD)-nucleases This example shows the construction of a modular animal pathogen-nucleic acid binding domain (MAP-NBD)-nuclease. The production of MAP-NBD is exemplified by the following: L. quateirensis, Burkholderia, Paraburkholderia, Francisella cisella, or any combination thereof. MAP-NBD is derived from L. quateirensis, Burkholderia cerevisiae, L. ... Multiple antibodies from Kholderia, Paraburkholderia, Francisella, or any combination thereof Each repeat domain may include any one of SEQ ID NO: 2 to SEQ ID NO: 95, The nucleotides are selected to recognize and bind to specific nucleotides or base pairs, thereby The MAP-NBD nuclease is a recombinant MAP-NBD nuclease that is capable of targeting and binding to a nucleic acid sequence of interest. The MAP-NBD is linked to the nuclease via an optional linker. The linkers are synthetic linkers, natural linkers from L. quateirensis, Burkholderia, and Paraburkholderi. a or the full-length C-terminus of the Francisella protein, or a truncated form thereof. The enzyme may be a cleavage domain (e.g., a meganuclease such as I-AniI or I-OnuI) or a cleavage domain. A half-domain (e.g., a FokI nuclease, e.g., SEQ ID NO: 11, or Bfil).

[0213] [Example 11] Modular animal pathogen-nucleic acid binding domain (MAP-NBD)-gene regulator (MAP-NBD-GR) manufacturing This example shows the modular animal pathogen-nucleic acid binding domain (MAP-NBD)-gene regulator The production of MAP-NBD-GR is exemplified. MAP-NBD was synthesized from L. quateirensis, Burkholderia, Parabur MAP-NBD is derived from L. quholderia, Francisella, or any combination thereof. ateirensis, Burkholderia, Paraburkholderia, Francisella, or any combination thereof any one of the repeat domains listed in Table 1, Each repeat domain is selected to recognize and bind a specific nucleotide or base pair. , thereby providing a MAP-NBD that targets and binds to a nucleic acid sequence of interest. It can be recombinantly expressed or synthetically constructed. MAP-NBD can be linked to the MAP-NBD via any linker. The linker is a synthetic linker, a natural L. quateirensi linker, or a nucleotide sequence that is linked to the gene regulatory domain. s, the full length C-terminus of a Burkholderia, Paraburkholderia or Francisella protein, or is a truncated form of its C-terminus. Gene regulators are gene activators (e.g. VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG, Ldb1 self-association domain, SAM activators (VP64, p65, HSF1), or VPRs (VP64, p65, Rta) or gene replicators Sensor (KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DNMT3L, DNMT3B, K OX, TGF-β-inducible early gene (TIEG), v-erbA, SID, MBD2, MBD3, RB, or MeCP2) be.

[0214] [Example 12] Fabrication of modular animal pathogen-nucleic acid binding domain (MAP-NBD)-fluorophores This example shows the modular animal pathogen-nucleic acid binding domain (MAP-NBD)-fluorophore The production of MAP-NBD is exemplified by L. quateirensis, Burkholderia, Paraburkholderia, Fr ancisella, or any combination thereof. MAP-NBD is derived from L. quateirensis, B Multiple repeats from Urkholderia, Paraburkholderia, Francisella or any combination thereof domain, e.g., any one of the repeats listed in Table 1, wherein each repeat domain are selected to recognize and bind to specific nucleotides or base pairs, thereby The MAP-NBD provides a MAP-NBD that targets and binds to a nucleic acid sequence of interest. The MAP-NBD may be recombinantly expressed or synthetically constructed. The linkers are synthetic linkers, natural L. quateirensis linkers, Burkholderia spp. The full length C-terminus of a Kholderia, Paraburkholderia or Francisella protein, or its C The fluorophore is a truncated form of the GFP. is the fluorescent part.

[0215] [Example 13] Genome editing with modular animal pathogen-nucleic acid binding domain (MAP-NBD)-nucleases This example uses modular animal pathogen-nucleic acid binding domain (MAP-NBD)-nucleases. The genome editing used is shown. MAP-NBD nuclease was used as described in Examples 9 and 10. MAP-NBDs are engineered to contain multiple repeats that target a nucleic acid sequence of interest. When a cleavage half-domain is used as the nuclease (e.g., FokI), each of the FokI cleavage domains Two MAP-NBDs with truncated half-domains are prepared and administered. The combined first MAP-NBD is designed to bind to a first target nucleic acid sequence and cleaves another FokI fragment. A second MAP-NBD covalently attached to the domain is engineered to bind to a second target nucleic acid sequence. wherein the first and second target nucleic acid sequences are selected from the group consisting of the first and second MAP-NBDs and the second MAP-NBDs. The FokI cleavage half-domains are appropriately spaced to provide for dimerization of the FokI cleavage half-domains upon binding to the nucleic acid sequence. When administered to a subject, the MAP-NBDs bind to their target nucleic acid sequences. Dimerization of the FokI cleavage half-domains of the cleavage domains results in enzymatic activity and the induction of double-strand breaks (DSBs). A cleavage domain (e.g., a meganuclease such as I-AniI or I-OnuI or two FokI domains) When a MAP-NBD-binding domain is used, a single MAP-NBD bound to the cleavage domain is generated after administration to a subject. The subject may be a cell, a plurality of cells, or a human or The target sequence is a double-stranded break that is introduced by the nuclease, which then binds to the target sequence. Provides homogeneous end joining or homology-directed repair, thereby providing genome editing functionality do.

[0216] [Example 14] Modular animal pathogen-nucleic acid binding domain (MAP-NBD)-gene regulator (MAP-NBD-GR) Gene regulation by An example of this is the modular animal pathogen-nucleic acid binding domain (MAP-NBD)-gene regulator (MAP The results show gene regulation by MAP-NBD-GR. MAP-NBD-GR was prepared as described in Examples 9 and 11. MAP-NBDs are engineered to contain multiple repeat sequences that target a nucleic acid sequence of interest. MAP-NBD-GR is administered to a subject. The subject may be a cell, a plurality of cells, or a human or non-human In turn, MAP-NBD-GR regulates gene expression (i.e., its activity). via activation or inhibition domains).

[0217] [Example 15] Modular Animal Pathogen-Nucleic Acid Binding Domain (MAP-NBD)-Fluorophore Imaging G This example shows the modular animal pathogen-nucleic acid binding domain (MAP-NBD)-fluorophore This example illustrates imaging of genomic loci in cells using MAP-NBD fluorophores. The MAP-NBD is prepared as described in Examples 9 and 12. The MAP-NBD-fluorophore is administered to the cells. The cells may be in a plurality of cells ex vivo or in an animal, such as a human or non-human primate. The MAP-NBD-fluorophore hybridizes to the target nucleic acid sequence, thereby Imaging allows visualization of labeled target cells and the labeled A target cell that is positive indicates that the targeted genomic locus is present.

[0218] [Example 16] In vitro base specificity of repeats from L. maceachernii Repeats identified in hypothetical protein WP_058451450 “LEGm” from L. maceachernii To determine the base specificity of the ribosomal protein, we engineered and generated a DNA construct that encoded the full-length protein. The full-length protein (having the sequence set forth in SEQ ID NO: 143) was synthesized by in vitro transcription and translation. The sequence motifs identified were synthesized by the reaction and characterized using the SELEX-seq method. , compared to the modular structure of each protein, DNA bases are added to each repeat and to the N- and C-termini. A preference was assigned to the hypothetical protein WP_W from L. quateirensis, described in Example 8. P_058473422 (SEQ ID NO: 1) The base specificity of the repeats identified in "LEGq" is further confirmed. Ta.

[0219] Constructs Based on their domain structure, LEGq and LEGm proteins are divided into N-terminal, individual repeat The DNA modules encoding these modules were was engineered and synthesized with flanking restriction sites (Integrated DNA Technologies, Coralv ille, Iowa). Reyon et al. 2013, (Current Protocols in Molecular Biology, 12.16.1 These DNA modules were ligated using a modified protocol from In the case of the LEGq protein, a DNA construct was prepared that encodes the entire full-length protein. The LEGm construct was synthesized with an N-terminal FLAG tag that allows protein retention during SELEX. and a C-terminal HA tag, and the fully assembled construct was named pVax_NG_6 The LEGq construct was cloned into 3aa, propagated in E. coli, and purified using standard methods. The products were cloned into the pT7CFE1-CHA vector (Thermo Scientific) (this vector The proteins were grown in E. coli and subjected to standard procedures. The purified plasmid constructs were subjected to Sanger sequencing to identify the expected DNA fragments. The sequence of each module was confirmed as follows: JPEG2025037883000041.jpg129167

[0220] SELEX-seq An in vitro transcription / translation (IVTT) kit was used to synthesize LEGq (1 Step Human Coupled IVT, Promega) and LEGm (TnT Quick Coupled Transcription Full-length proteins were extracted from plasmid constructs for the ELISA kit (Gene / Translation kit, Thermo Scientific). The SELEX-seq protocol was adapted from Miller et al. 2011 (above). As described above, the nucleotide sequences are expressed as single-stranded DNA oligonucleotides flanked by a common 20 nt adapter sequence. A library of ordered random sequences of 25 nucleotides (nt) in length was generated. A double-stranded DNA library was generated using mer extension.

[0221] The IVTT protein was subjected to three SELEX cycles as follows: binding of the protein to double-stranded DNA; immobilization of protein:DNA complexes on anti-HA magnetic beads and washing away unbound DNA; PCR amplification of bound DNA. In this way, the DNA molecules bound to the protein were amplified by LEGq and L Used as input for subsequent binding to EGq protein in each cycle , and enriched the preferred binding sequences of these proteins in a pool of DNA molecules.

[0222] PCR samples from each SELEX cycle were diluted 1:50 and cloned into a common endonucleases flanked by 25 nt random sequences. The PCR products were amplified using indexing primers that recognize the dsDNA adapter. and sequenced on an Illumina MiniSeq instrument.

[0223] SELEX-seq data analysis Sequence reads from MiniSeq are multiplexed using the index sequences of the primers. The sequence reads were then assigned to individual SELEX samples. The method requires constant sequences matching the common adapter flanking a 25-nucleotide variable region. A random sample of 5,000 reads from each sample was filtered using the GADEM motif. -Finder (Li et al. 2009, Journal of Computational Biology, 16(2):317-29) The discovered motifs were used as input to the MEME suite (Bailey et al. 2 015, Nucleic Acids Research, 43(W1):W39-49) using the “ceqlogo” function. Ta. result The binding motifs for LEGq and LEGm proteins discovered by SELEX-seq were These are shown in Figures 8A and 8B, respectively, and the recognized DNA bases are listed below. JPEG2025037883000042.jpg205169JPEG2025037883000043.jpg62169

[0224] [Example 17] In vivo base specificity of repeats from L. maceachernii DNA binding preference of LEGm protein (SEQ ID NO: 143) for DNA in the context of chromatin The gene was expressed in vivo and its binding site in the human genome was identified using the FLAG-seq method. was evaluated by detecting

[0225] FLAG-seq

[0226] CUT&RU for detection of FLAG-tagged proteins, an approach called "FLAG-seq" The in vivo binding of LEGm was detected using the N-seq method (Skene and Henikoff 2017). In other words, the above LEGm construct was linearized by restriction digestion and synthesized according to the manufacturer's protocol. In vitro transcription using the T7 mScript™ Standard mRNA Production System (CellScript) One microgram of the mRNA encoding LEGM was then subjected to transcriptional Prepare nucleofection vectors using a BTX ECM-830 nucleofection device (Harvard Apparatus) according to the manufacturer's instructions. 0.5 million CD3+ T cells were transfected with 0.5% PBS. The cells were incubated at 37°C for 6 hours. The resulting samples were then subjected to the CUT&RUN-seq protocol using the FLAG tag for detection of LEGm protein. DNA fragments isolated from control and LEGm-transfected cells were end-repaired and prepared. Sequencing was performed using the SMARTer ThruPLEX DNA-seq kit (Takara) according to the manufacturer's protocol. The DNA was ligated to fixed adapters and sequenced on an Illumina NextSeq instrument using standard protocols. .

[0227] FLAG-seq data analysis

[0228] Sequencing reads from NextSeq were demultiplexed and sequenced into individual FLAG-seq samples. The sequences were assigned and aligned to the reference human genome using the BWA algorithm (Li and Durbin 2009). The MACS peak calling algorithm (Zhang et al. 2008) was used to identify the control and L By comparing FLAG-seq signals from EGm-transfected cells, the location of LEGm binding was identified. The MME tool suite (Bailey et al. 2015) was then used to identify the enriched sequence models. This analysis was performed to determine the LEGm sequence observed in multiple SELEX-seq experiments. We reobtained the same binding motif for (Figure 9).

[0229] Although preferred embodiments of the present invention have been shown and described herein, it will be apparent to those skilled in the art that It should be apparent that such an embodiment is provided by way of example only. Numerous variations, modifications, and substitutions may occur without departing from the invention. It will be appreciated that various alternatives to the embodiments of the invention described herein may be used in practicing the invention. It should be understood that the following claims define the scope of the invention and that these patents that methods and structures within the scope of the claims and their equivalents are thereby covered; is intended.

[0230] For the sake of completeness, certain aspects of the disclosed polypeptides, compositions and methods are set forth below in the following numbers: They are described in the sections marked with 1. Non-natural modular nucleic acid binding domains (MAP-NBDs) derived from animal pathogen proteins wherein the MAP-NBD comprises a plurality of repeating units, and A composition, wherein the repeat unit recognizes a target nucleic acid. 2. The composition of paragraph 1, wherein the animal pathogen protein is derived from a bacterium. 3. The composition of paragraph 2, wherein the bacterium is selected from the genus Legionella. 4. The composition described in any one of items 2 to 3, wherein the bacterium is L. quateirensis. 5. The repeating unit has the consensus sequence 1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 19). 5. The composition of any one of claims 1 to 4, comprising: 6. Any of items 1 to 5, wherein the target nucleic acid is a single nucleotide, a single base pair, or both. The composition of any one of claims 1 to 4. 7. The composition of any one of claims 1 to 6, wherein the target nucleic acid is DNA or RNA. 8. The composition of any one of 1 to 7, wherein the MAP-NBD binds to a target nucleic acid sequence. 9. The composition of paragraph 8, wherein the target nucleic acid sequence is DNA or RNA. 10. The composition of any one of 1 to 9, further comprising a functional domain. 11. A natural or non-natural linker is further provided between the MAP-NBD and the functional domain. 10. The composition of claim 10, comprising 12. The functional domain is an enzyme, an activation domain, an inhibition domain, a biotinylation reagent, A combination of any one of claims 10 to 11, comprising a DNA nucleotide modifier or a fluorophore. Composition. 13. The enzyme is a nuclease, a DNA-modifying protein, or a chromatin-modifying protein. 12. The composition of claim 12, 14. The composition of claim 13, wherein the nuclease is a cleavage domain or a half-cleavage domain. 15. The composition of claim 14, wherein the cleavage domain or half-cleavage domain comprises a type IIS restriction enzyme. . 16. The composition of claim 15, wherein the type IIS restriction enzyme comprises FokI or Bfil. 17. FokI is at least 70%, at least 75%, at least 80% relative to SEQ ID NO: 11; At least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or 17. The composition of claim 16, wherein said sequence identity is at least 99%. 18. The composition of any one of items 16 to 17, wherein FokI has the sequence of SEQ ID NO:11. 19. The chromatin modifying protein is lysine-specific histone demethylase 1 (LSD1). 14. The composition of claim 13, 20. The activation domain is VP16, VP64, p65, p300 catalytic domain, or TET1 catalytic domain. , TDG, Ldb1 self-association domain, SAM activator (VP64, p65, HSF1), VPR (VP64, p65, R ta). 21. The repressor domain is KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT 1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-β-induced early gene (TIEG), v-erbA, SID, MBD2, 13. The composition of claim 12, comprising MBD3, Rb, or MeCP2. 22. The composition of claim 12, wherein the DNA nucleotide modifier is adenosine deaminase. . 23. A genome sequence comprising a functional domain defined by a modular nucleic acid binding domain. enabling genome editing, gene regulation, or imaging at the chromosome locus,10-22 The composition of any one of claims 1 to 5. 24. The repeating unit is at least 7 times the number of repeating units for any one of SEQ ID NO: 2 to SEQ ID NO: 10. 0%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, 1 to 23, which have at least 95%, at least 97%, or at least 99% sequence identity with The composition of any one of claims. 25. Any one of 1 to 24, wherein the repeating unit is any one of SEQ ID NO: 2 to SEQ ID NO: 10. The composition of paragraph . 26. The composition of any one of clauses 1 to 25, wherein the repeat unit is derived from a wild-type protein. 27. The composition of any one of claims 1 to 25, wherein the repeat unit comprises a modification of a wild-type protein. thing. 28. The modification enhances specific recognition of a target nucleotide, base pair, or both. , The composition of claim 27. 29. The composition of any one of claims 27 to 28, wherein the modifications include 1 to 29 modifications. 30. The animal pathogen protein has a sequence identity of at least 70%, at least 75%, or both of SEQ ID NO:1. %, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least A combination of any one of claims 1 to 29 having at least 97%, or at least 99% sequence identity with Composition. 31. The composition of any one of claims 1 to 30, wherein the animal pathogen protein is SEQ ID NO: 1. . 32. The target nucleic acid sequence is selected from the group consisting of PDCD1 gene, CTLA4 gene, LAG3 gene, TET2 gene, and BT LA gene, HAVCR2 gene, CCR5 gene, CXCR4 gene, TRA gene, TRB gene, B2M gene , albumin gene, HBB gene, HBA1 gene, TTR gene, NR3C1 gene, CD52 gene, B Erythroid-specific enhancer of the CL11A gene, the CBLB gene, the TGFBR1 gene, and the SERPINA1 gene , HBV genomic DNA in infected cells, CEP290 gene, DMD gene, CFTR gene, IL2RG gene 32. The composition of any one of claims 1 to 31, or any combination thereof. 33. Upon cleavage of the region of the target nucleic acid sequence, chimeric antigen receptor (CAR), α-L iduronin IDUA, iduronate-2-sulfatase (IDS) or factor 9 (F9) are inserted. 33. The composition of any one of items 1 to 32. 34. A method for genome editing in a subject, comprising: administering a non-naturally occurring modular nucleic acid binding domain; and Inducing double-strand breaks wherein the modular nucleic acid binding domain is a modular nucleic acid binding domain derived from an animal pathogen protein. - a nucleic acid binding domain (MAP-NBD), said MAP-NBD comprising a plurality of repeat units, said plurality The repeat unit of the repeat unit recognizes a target nucleic acid. method. 35. The method according to claim 1, further comprising the step of: 35. The method of claim 34, wherein the repeat units of the second plurality of repeat units recognize a second target nucleic acid. 36. The MAP-NBD, the second MAP-NBD, or both, further comprise a functional domain. 34-35. The method according to any one of claims 34-35. 37. The method of claim 36, wherein the functional domain comprises a cleavage domain or a cleavage half-domain. . 38. The method of claim 35, wherein the cleavage domain or cleavage half-domain comprises FokI or Bfil. 39. The method of claim 38, wherein the cleavage domain comprises a meganuclease. 40. FokI is at least 70%, at least 75%, at least 80%, relative to SEQ ID NO: 11; At least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or 39. The method of claim 38, wherein said sequence identity is at least 99%. 41. The method according to any one of clauses 38 and 40, wherein FokI has the sequence of SEQ ID NO: 11. . 42. The target nucleic acid sequence is selected from the group consisting of PDCD1 gene, CTLA4 gene, LAG3 gene, TET2 gene, and BT LA gene, HAVCR2 gene, CCR5 gene, CXCR4 gene, TRA gene, TRB gene, B2M gene , albumin gene, HBB gene, HBA1 gene, TTR gene, NR3C1 gene, CD52 gene, B Erythroid-specific enhancer of the CL11A gene, the CBLB gene, the TGFBR1 gene, and the SERPINA1 gene , HBV genomic DNA in infected cells, CEP290 gene, DMD gene, CFTR gene, IL2RG gene 34 to 41, or a combination thereof. 43. Upon cleavage of the region of the target nucleic acid sequence, chimeric antigen receptor (CAR), α-L iduronin IDUA, iduronate-2-sulfatase (IDS) or factor 9 (F9) are inserted. 43. The method according to any one of items 34 to 42. 44. A method for regulating a gene in a subject, comprising: administering a non-naturally occurring modular nucleic acid binding domain; and Regulating gene expression wherein the modular nucleic acid binding domain is derived from an animal pathogen protein. a DNA binding domain (MAP-NBD), the MAP-NBD comprising a plurality of repeat units, the plurality of The repeat unit of the repeat unit recognizes a target nucleic acid; method. 45. The method of claim 44, wherein the MAP-NBD further comprises a functional domain. 46. ​​The method according to claim 45, wherein the functional domain comprises an activation domain or a repression domain. . 47. The activation domain is VP16, VP64, p65, p300 catalytic domain, or TET1 catalytic domain. , TDG, Ldb1 self-association domain, SAM activator (VP64, p65, HSF1), VPR (VP64, p65, R ta). 48. The repressor domain is KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT 1, DNMT3A-DNMT3L, DNMT3B, KOX, TGF-β-induced early gene (TIEG), v-erbA, SID, MBD2, 47. The method of claim 46, comprising MBD3, Rb, or MeCP2. 49. The target nucleic acid sequence is selected from the group consisting of PDCD1 gene, CTLA4 gene, LAG3 gene, TET2 gene, and BT LA gene, HAVCR2 gene, CCR5 gene, CXCR4 gene, TRA gene, TRB gene, B2M gene , albumin gene, HBB gene, HBA1 gene, TTR gene, NR3C1 gene, CD52 gene, B Erythroid-specific enhancer of the CL11A gene, the CBLB gene, the TGFBR1 gene, and the SERPINA1 gene , HBV genomic DNA in infected cells, CEP290 gene, DMD gene, CFTR gene, IL2RG gene 44 to 48, or a combination thereof. 50. Upon cleavage of the region of the target nucleic acid sequence, chimeric antigen receptor (CAR), α-L iduronin IDUA, iduronate-2-sulfatase (IDS) or factor 9 (F9) are inserted. 50. The method according to any one of items 44 to 49. 51. A method for imaging a genomic locus comprising: administering a non-naturally occurring modular nucleic acid binding domain; and Imaging the subject wherein the modular nucleic acid binding domain is derived from an animal pathogen protein. a DNA binding domain (MAP-NBD), the MAP-NBD comprising a plurality of repeat units, the plurality of The repeat unit of the repeat unit recognizes a target nucleic acid; method. 52. The method of claim 51, wherein the MAP-NBD further comprises a functional domain. 53. The method of paragraph 52, wherein the functional domain is an imaging agent. 54. The method of claim 53, wherein the imaging agent is a fluorescent moiety. 55. The method of claim 54, wherein the fluorescent moiety is GFP or mCHERRY. 56. The target nucleic acid is a single nucleotide, a single base pair, or both, The method according to any one of the items. 57. The method of any one of clauses 38 to 56, wherein the target nucleic acid is DNA or RNA. 58. The method of any one of items 38 to 57, wherein the MAP-NBD recognizes a target nucleic acid sequence. 59. The method according to item 58, wherein said MAP-NBD binds to said target nucleic acid sequence. 60. The method of any one of 58 to 59, wherein the target nucleic acid sequence is DNA or RNA. 61. The method according to any one of claims 34 to 43, further comprising a linker between the MAP-NBD and the functional domain. The method according to any one of paragraphs 50 to 51 to 60. 62. The method of any one of items 32 to 61, wherein the animal pathogen protein is derived from a bacterium. 63. The method of claim 62, wherein the bacterium is selected from the genus Legionella. 64. A method according to any one of items 62 to 63, wherein the bacterium is L. quateirensis. 65. The repeating unit has the consensus sequence 1xxx211x1xxx33x2x1xxxxxxxxx1 (SEQ ID NO: 19). Any one of the methods of 32 to 64, including columns. 66. The repeating unit is at least 7 times the number of repeating units for any one of SEQ ID NO: 2 to SEQ ID NO: 10. 0%, at least 75%, at least 80%, at least 85%, at least 90%, at least 92%, 32-65 having at least 95%, at least 97%, or at least 99% sequence identity 2. The method according to claim 1 . 67. Any of items 32 to 66, wherein the repeating unit is any of SEQ ID NO: 2 to SEQ ID NO: 10. Method of one term. 68. The method of any one of items 32 to 67, wherein the repeat unit is derived from a wild-type protein. 69. The method according to any one of claims 32 to 68, wherein the repeat unit comprises a modification of a wild-type protein. How to. 70. The method of claim 69, wherein the modification enhances specific recognition of the target nucleotide. 71. The method of any one of clauses 69 to 70, wherein the modifications comprise between 1 and 29 modifications. 72. The animal pathogen protein has a sequence identity of at least 70% and at least 75% with respect to SEQ ID NO:1. %, at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 32 to 71, having at least 97%, or at least 99% sequence identity with the Method of posting. 73. The method of any one of 32 to 66, wherein the animal pathogen protein is SEQ ID NO:1. 74. The method of any one of clauses 32 to 73, wherein the genomic locus is intracellular. 75. The cell is an ex vivo cell population, in a human, or in a non-human animal. Method 68 in the above. 76. The target nucleic acid sequence is selected from the group consisting of PDCD1 gene, CTLA4 gene, LAG3 gene, TET2 gene, and BT LA gene, HAVCR2 gene, CCR5 gene, CXCR4 gene, TRA gene, TRB gene, B2M gene , albumin gene, HBB gene, HBA1 gene, TTR gene, NR3C1 gene, CD52 gene, B CL11 Erythroid-specific enhancer of A gene, CBLB gene, TGFBR1 gene, SERPINA1 gene HBV genomic DNA in infected cells, CEP290 gene, DMD gene, CFTR gene, IL2RG gene, 51 to 75, or a combination thereof. 77. Upon cleavage of the region of the target nucleic acid sequence, chimeric antigen receptor (CAR), α-L iduronin IDUA, iduronate-2-sulfatase (IDS) or factor 9 (F9) are inserted. 77. The method according to any one of items 51 to 76.

Claims

1. A recombinant polypeptide comprising a nucleic acid binding domain (NBD) and a heterologous functional domain. 、 The NBD has a small number of amino acids that specifically bind to a target nucleic acid, which are arranged from the N-terminus to the C-terminus of the NBD. It contains at least three repeating units (RUs), each RU having the consensus sequence: 1xxxx11x12xx33xxx1xxxxxxxxxx14xxx Including, 1= A, F, I, L, M, T, V, or Y 2= ​​x or xx 3= AGNS 4=x, xx, or xxx x = any amino acid and each of the RUs is independently selected from the group consisting of SEQ ID NOs: 2-10, 23-35, 85-89, and 131-137. A sequence of 33 to 36 amino acids that has at least 70% identity to one of the amino acid sequences described in including the sequence of amino acid lengths, Recombinant polypeptides.

2. A recombinant polypeptide comprising a nucleic acid binding domain (NBD) and a heterologous functional domain. 、 The NBD has a small number of amino acids that specifically bind to a target nucleic acid, which are arranged from the N-terminus to the C-terminus of the NBD. At least three repeating units (RUs), each of which has the consensus sequence: (F / L / Y)(D / G / N / S)(A / H / R / S / T / V)(D / E / K / Q)(E / H / Q)(I / L / V)(I / L / V)(C / H / K / R / S)(I / M / V)(A / V)(A / G / S)(H / N / R)(A / D / G / I / K / N / S / V)(G)(G)(A / G / S)(H / K / L / N / R)(N)(I / L)(A / D / E / I / K / V)(A / L / V)(I / M / V)(K / L / Q / T)(A / D / E / K / L / Q / S)(A / C / F / N / V / Y)(F / H / L / Q / Y)(A / D / H / P / Q)(A / D / I / K / R / T / V)(F / L)(K / M / Q / R / S)(D / E / N / S)(F / L / M)(D / E / G / H / K / N) (SEQ ID NO: 154) Including, Recombinant polypeptides.

3. Further comprising a half RU at the C-terminus of the NBD, the half RU comprising FNAEQIVRMVSX 12 X 13 GGSKNL (SEQ ID NO: 155) or a sequence of 15-20 amino acids having at least 70% identity to The sequence of SEQ ID NO: 155 with the following conservative amino acid substitutions: where X 12 X 13 = HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, HS, GS, or or LN, The recombinant polypeptide of claim 1 or 2.

4. Each RU, independently, FSSQQIIRMVSHAGGANNLKAVTANHDDLQNMG (SEQ ID NO:2); FNVEQIVRMVSHNGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:3); FNAEQIVRMVSHGGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:4); FNAEQIVSMVSNNGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:5); FNAEQIVSMVSNGGGSLNLKAVKKYHDALKDRG (SEQ ID NO:6); FNTEQIVRMVSHDGGSLNLKAVKKYHDALRERK (SEQ ID NO:7); FNVEQIVSIVSHGGGSLNLKAVKKYHDVLKDRE (SEQ ID NO:8); FNAEQIVRMVSHDGGSLNLKAVTDNHDDLKNMG (SEQ ID NO:9); FNAEQIVRMVSHKGGSKNLALVKEYFPVFSSFH (SEQ ID NO:33); or LGHKELIKIAARNGGGNNLIAVLSCYAKLKEMG (SEQ ID NO:89) A sequence of 33 to 36 amino acids having at least 70% identity to Amino acid positions 12 and 13 numbered with respect to one of SEQ ID NOs: 2-9, 33, and 89 HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, HS, GS, or LN, respectively. comprising a base contacting residue (BCR) selected from 3. A recombinant polypeptide according to claim 1 or 2.

5. Further comprising a half RU at the C-terminus of the NBD, the half RU comprising FNAEQIVRMVSX 12 X 13 GGSKNL (SEQ ID NO: 155) or a sequence of 15-20 amino acids having at least 70% identity to The sequence of SEQ ID NO: 155 with the following conservative amino acid substitutions: Here, X 12 X 13 = HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, HS, GS, or is LN, The recombinant polypeptide of claim 4.

6. at least one of said RUs binds to an adenine; FSSQQIIRMVSX 12 X 13 GGANNLKAVTANHDDLQNMG (SEQ ID NO:2) or one or more sequences of 33 to 36 amino acids having at least 70% identity to 2 with the above conservative amino acid substitutions, wherein X 12 X 13 =HA be, A recombinant polypeptide according to claim 4 or 5.

7. the polypeptide comprises an N-terminal domain, the C-terminus of the N-terminal domain being the first The recombinant polypeptide according to any one of claims 1 to 6, wherein the recombinant polypeptide is fused to the N-terminus of RU of 。

8. A linker amino acid sequence between the C-terminus of the N-terminal domain and the N-terminus of the first RU of the NBD.

8. The recombinant polypeptide of claim 7, comprising:

9. The N-terminal domain is an amino acid sequence set forth in any one of SEQ ID NOs: 13, 20, and 21. The combination of claim 7, comprising an amino acid sequence having at least 85% identity thereto or a fragment thereof. Recombinant polypeptide.

10. the polypeptide comprises a C-terminal domain, the N-terminus of the C-terminal domain being the end of the NBD; The recombinant polypeptide according to any one of claims 1 to 9, wherein the RU is fused to the C-terminus of 。

11. A linker amino acid sequence between the N-terminus of the C-terminal domain and the C-terminus of the last RU of the NBD.

11. The recombinant polypeptide of claim 10, comprising:

12. The C-terminal domain is selected from the group consisting of amino acids set forth in SEQ ID NOs: 12, 159, and 22.

11. The method according to claim 10, comprising administering to said patient an amino acid sequence having at least 85% identity to said Recombinant polypeptide of.

13. Each RU is independent. FKADDAVRIACRTGGSHNLKAVHKNYERLRARG (SEQ ID NO: 131); FNADQVIKIVGHDGGSNNIDVVQQFFPELKAFG (SEQ ID NO: 132); FSADQVVKIAGHSGGSNNIAVMLAVFPRLRDFG (SEQ ID NO: 138); or LDRQQILRIASHDGGSKNIAAVQKFLPKLMNFG (SEQ ID NO:25) A sequence of 33 to 36 amino acids having at least 70% identity to Amino acid position 12 numbered with respect to one of SEQ ID NOs: 25, 131, 132 and 138 and 13, respectively, HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, HS, GS, or LN, 3. A recombinant polypeptide according to claim 1 or 2.

14. an N-terminal domain, the C-terminus of said N-terminal domain fused to the N-terminus of the first RU of said NBD The recombinant polypeptide of claim 13.

15. A linker amino acid sequence between the C-terminus of the N-terminal domain and the N-terminus of the first RU of the NBD.

15. The recombinant polypeptide of claim 14, comprising:

16. The N-terminal domain has at least 85% identity to the amino acid sequence set forth in SEQ ID NO:

140.

15. The recombinant polypeptide of claim 14, comprising an amino acid sequence having the same identity as or a fragment thereof.

17. a C-terminal domain, the N-terminus of the C-terminal domain fused to the C-terminus of the last RU of the NBD The recombinant polypeptide according to any one of claims 13 to 16,

18. A linker amino acid sequence between the N-terminus of the C-terminal domain and the C-terminus of the last RU of the NBD.

18. The recombinant polypeptide of claim 17, comprising:

19. The C-terminal domain has at least 85% identity to the amino acid sequence set forth in SEQ ID NO:

141.

18. The recombinant polypeptide of claim 17, comprising an amino acid sequence having a sequence identity thereto or a fragment thereof. 。

20. Each RU, independently, FSAEQIVRIAAHDGGSRNIEAVQQAQHVLKELG (SEQ ID NO:23); FSAEQIVSIVAHDGGSRNIEAVQQAQHILKELG (SEQ ID NO:24); FSAEQIVRIAAHDGGSLNIDAVQQAQQALKELG (SEQ ID NO:26); FSTEQIVCIAGHGGGSLNIKAVLLAQQALKDLG (SEQ ID NO:27); YSSEQIVRVAAHGGGSLNIKAVLQAHQALKELD (SEQ ID NO:28); FSAEQIVHIAAHGGGSLNIKAILQAHQTLKELN (SEQ ID NO:29); FSAEQIVRIAAHIGGSRNIEAIQQAHHALKELG (SEQ ID NO:30); FSAEQIVRIAAHIGGSHNLKAVLQAQQALKELD (SEQ ID NO:31); FSAKHIVRIAAHIGGSLNIKAVQQAQQALKELG (SEQ ID NO:32); FSADQIVRIAAHKGGSHNIVAVQQAQQALKELD (SEQ ID NO:34); FSAEQIVSIAAHVGGSHNIEAVQKAHQALKELD (SEQ ID NO: 35); or FSAEQIVRIAAHIGGSRNIEATIKHYAMLTQPP (SEQ ID NO: 133) A sequence of 33 to 36 amino acids having at least 70% identity to Amino acids numbered according to one of SEQ ID NOs: 23-24, 26-32, 34-35, and 133 At acid positions 12 and 13, respectively, HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, comprising a base contacting residue (BCR) selected from SN, HS, GS, or LN; The recombinant polypeptide of claim 1 or 2:

21. an N-terminal domain, the C-terminus of said N-terminal domain fused to the N-terminus of the first RU of said NBD 21. The recombinant polypeptide of claim 20,

22. A linker amino acid sequence between the C-terminus of the N-terminal domain and the N-terminus of the first RU of the NBD.

22. The recombinant polypeptide of claim 21 , comprising:

23. The N-terminal domain has at least 85% identity to the amino acid sequence set forth in SEQ ID NO:

144.

22. The recombinant polypeptide of claim 21, comprising an amino acid sequence having a sequence identity thereto, or a fragment thereof. 。

24. a C-terminal domain, the N-terminus of the C-terminal domain fused to the C-terminus of the last RU of the NBD The recombinant polypeptide according to any one of claims 20 to 23,

25. A linker amino acid sequence between the N-terminus of the C-terminal domain and the C-terminus of the last RU of the NBD.

25. The recombinant polypeptide of claim 24, comprising:

26. The C-terminal domain has at least 85% identity to the amino acid sequence set forth in SEQ ID NO:

145.

25. The recombinant polypeptide of claim 24, comprising an amino acid sequence having a sequence identity thereto, or a fragment thereof. 。

27. The NBDs are in the order in which they naturally occur in Legionella species or in the order Legionellales. A recombinant polypeptide according to any one of claims 1 to 26, which does not contain RU.

28. Any of claims 1 to 27, comprising a linker amino acid sequence located between two or more adjacent RUs. A recombinant polypeptide described in any one of claims 1 to 4.

29. A recombinant polypeptide comprising a nucleic acid binding domain (NBD) and a heterologous functional domain. The NBD may include a small number of amino acids that specifically bind to a target nucleic acid, the small number being arranged from the N-terminus to the C-terminus of the NBD. It contains at least three repeating units (RUs), each RU having the consensus sequence: YK(P / S)EDIIRLASH(D / G)GGSVNLEAVLRL(H / N)(P / S)QL(I / T)(G / R)LG (SEQ ID NO: 156) A recombinant polypeptide comprising:

30. Each RU, independently, YKSEDIIRLASHDGGSVNLEAVLRLHSQLTRLG (SEQ ID NO:85); YKPEDIIRLASHGGGSVNLEAVLRLNPQLIGLG (SEQ ID NO:86); YKSEDIIRLASHGGGSVNLEAVLRLHSQLTRLG (SEQ ID NO:87); YKSEDIIRLASHGGGSVNLEAVLRLNPQLIGLG (SEQ ID NO:88); YKSEDIIRLASHDGGSVNLEAVLRLNPQLIGLG (SEQ ID NO: 134); YKSEDIIRLASHDGGSINLEAVLRLNPQLIGLG (SEQ ID NO: 135); YKSEDIIRLASSNGGSVNLEAVLRLNPQLIGLG (SEQ ID NO: 136); YKSEDIIRLASSNGGSVNLEAVIAVHKALHSNG (SEQ ID NO: 137); or YKINHCVNLLKLNHDGFMLKNLIPYDSKLTGLG (SEQ ID NO: 151) A sequence of 33 to 36 amino acids having at least 70% identity to Amino acid positions numbered with respect to one of SEQ ID NOs: 85-88, 134-137, and 151 In positions 12 and 13, HK, HD, HA, HN, HG, NN, NG, RN, HI, HV, RT, HD, SN, comprising a base contacting residue (BCR) selected from HS, GS, or LN; 30. The recombinant polypeptide of claim 29.

31. the polypeptide comprises an N-terminal domain, the C-terminus of the N-terminal domain being the first 30. The recombinant polypeptide of claim 29, wherein the RU is fused to the N-terminus of the RU.

32. A linker amino acid sequence between the C-terminus of the N-terminal domain and the N-terminus of the first RU of the NBD.

32. The recombinant polypeptide of claim 31 , comprising:

33. The N-terminal domain has at least 85% identity to the amino acid sequence set forth in SEQ ID NO:

148.

32. The recombinant polypeptide of claim 31, comprising an amino acid sequence having the same identity as the polypeptide of claim 31, or a fragment thereof.

34. the polypeptide comprises a C-terminal domain, the N-terminus of the C-terminal domain being the end of the NBD; The recombinant polypeptide according to any one of claims 29 to 33, wherein the RU is fused to the C-terminus of Do.

35. A linker amino acid sequence between the N-terminus of the C-terminal domain and the C-terminus of the last RU of the NBD.

35. The recombinant polypeptide of claim 34, comprising:

36. The C-terminal domain has at least 85% identity to the amino acid sequence set forth in SEQ ID NO:

149.

35. The recombinant polypeptide of claim 34, comprising an amino acid sequence having the same identity as the polypeptide of claim 34, or a fragment thereof.

37. 3. The method of claim 1, wherein the NBD does not contain RUs in the order in which they naturally occur in Francisella bacteria.

7. A recombinant polypeptide according to any one of 6.

38. Any of claims 1 to 37, comprising a linker amino acid sequence located between two or more adjacent RUs. A recombinant polypeptide described in any one of claims 1 to 4.

39. The composition according to any one of claims 1 to 38, wherein the heterologous functional domain is a polypeptide. Recombinant polypeptide.

40. 40. The method according to claim 1, wherein the heterologous functional domain is located at the N-terminus of the NBD. A recombinant polypeptide as described above.

41. The method according to any one of claims 1 to 40, wherein the heterologous functional domain is located at the C-terminus of the NBD. The recombinant polypeptide of claim 1.

42. The functional domain is an enzyme, a transcriptional activator, a transcriptional repressor, or a DNA nucleotide.

42. A recombinant polypeptide according to any one of claims 39 to 41, comprising a modifier.

43. The enzyme is a nuclease, a DNA-modifying protein, or a chromatin-modifying protein.

43. The recombinant polypeptide of claim 42.

44. The recombinant nucleic acid of claim 43, wherein the nuclease is a cleavage domain or a half-cleavage domain. Polypeptides.

45. The recombinant vector of claim 44, wherein the cleavage domain or half-cleavage domain comprises a type IIS restriction enzyme. E. Polypeptide.

46. 46. ​​The recombinant polypeptide of claim 45, wherein said type IIS restriction enzyme comprises FokI or Bfil.

47. The type IIS restriction enzyme includes FokI, and the FokI has a sequence corresponding to the amino acid sequence set forth in SEQ ID NO:

11. At least 70%, at least 75%, at least 80%, at least 85%, at least 90%, having at least 92%, at least 95%, at least 97%, or at least 99% identity 47. The recombinant polypeptide of claim 46, comprising the amino acid sequence.

48. 47. The recombinant polypeptide of claim 46, wherein FokI has the sequence of SEQ ID NO:

11.

49. The chromatin modifying protein is lysine-specific histone demethylase 1 (LSD1).

43. The recombinant polypeptide of claim 42.

50. The transcriptional activator is VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG , Ldb1 self-association domain, SAM activators (VP64, p65, HSF1), or VPR (VP64, p65, 43. The recombinant polypeptide of claim 42, comprising a polypeptide comprising the polypeptide of claim 42, the polypeptide of claim 43 comprising the polypeptide of claim 44.

51. The transcriptional repressor is KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DN MT3L, DNMT3B, KOX, TGF-β-induced early gene (TIEG), v-erbA, SID, MBD2, MBD3, RB, or MeCP2.

52. The recombinant polypeptide of claim 43, wherein the DNA nucleotide modifier is adenosine deaminase. Lipeptide.

53. The target nucleic acid is the PDCD1 gene, the CTLA4 gene, the LAG3 gene, the TET2 gene, the ETLA gene, the HA V gene, CR2 gene, CCR5 gene, CXCR4 gene, TRA gene, TRE gene, E2M gene, albumin gene, HEE gene, HEAl gene, TTR gene, NR3Cl gene, CD52 gene, ECLllA gene The erythroid-specific enhancer of the CELE gene, TGFERl gene, and SERPINAl gene in infected cells The HEV genome DNA, CEP290 gene, DMD gene, CFTR gene, or IL2 RG gene in A recombinant polypeptide according to any one of claims 1 to 52.

54. 54. Any of claims 1 to 53, wherein the heterologous functional domain comprises a fluorophore or a detectable tag. A recombinant polypeptide according to any one of claims 1 to 4.

55. A nucleic acid encoding a polypeptide according to any one of claims 1 to 53.

56. The nucleic acid is operably linked to a promoter sequence that confers expression of the polypeptide.

56. The nucleic acid of claim 55.

57. The sequence of the nucleic acid is codon-optimized for expression of the polypeptide in a human cell.

57. The nucleic acid of claim 55 or 56.

58. The nucleic acid according to any one of claims 55 to 57, wherein the nucleic acid is deoxyribonucleic acid (DNA). acid.

59. 58. The nucleic acid of any one of claims 55 to 57, wherein the nucleic acid is a ribonucleic acid (RNA).

60. A vector comprising the nucleic acid of any one of claims 55 to 58.

61. The vector of claim 60, wherein the vector is a viral vector.

62. A nucleic acid according to any one of claims 55 to 59 or a vector according to claim 60 or 61. A host cell comprising the

63. A host cell comprising a polypeptide according to any one of claims 1 to 54.

64. A host cell expressing a polypeptide according to any one of claims 1 to 53.

65. A polypeptide according to any one of claims 1 to 53 and a pharma- ceutically acceptable excipient. A pharmaceutical composition comprising:

66. A nucleic acid according to any one of claims 55 to 59 or a vector according to claims 60 or 61. and a pharma- ceutical acceptable excipient.

67. 1. A method for regulating expression of an endogenous gene in a cell, comprising: introducing the polypeptide of claim 39 into a cell, wherein the NBD of the polypeptide binds to a target nucleic acid sequence present in the endogenous gene. and wherein the heterologous functional domain regulates expression of the endogenous gene. method.

68. 67. The method of claim 67, wherein the polypeptide is introduced as a nucleic acid encoding the polypeptide. The method described above.

69. 69. The method of claim 68, wherein the nucleic acid is deoxyribonucleic acid (DNA).

70. 69. The method of claim 68, wherein the nucleic acid is ribonucleic acid (RNA).

71. 67. The sequence of the nucleic acid is codon-optimized for expression in a human cell. A method according to any one of claims 1 to 70.

72. The functional domain is a transcription activator, and the target nucleic acid sequence is an expression control region of a gene. and wherein the polypeptide increases expression of a gene. Method of posting.

73. The transcriptional activator is VP16, VP64, p65, p300 catalytic domain, TET1 catalytic domain, TDG , Ldb1 self-association domain, SAM activator (VP64, p65, HSF1), or VPR (VP64, p65, R ta).

74. The functional domain is a transcriptional repressor, and the target nucleic acid sequence is located in an expression control region of a gene. and wherein the polypeptide reduces expression of a gene. The method described.

75. The transcriptional repressor is KRAB, Sin3a, LSD1, SUV39H1, G9A (EHMT2), DNMT1, DNMT3A-DN MT3L, DNMT3B, KOX, TGF-β-induced early gene (TIEG), v-erbA, SID, MBD2, MBD3, RB, or MeCP2.

76. The genes include the PDCD1 gene, the CTLA4 gene, the LAG3 gene, the TET2 gene, the ETLA gene, and the HA VCR2 gene, CCR5 gene, CXCR4 gene, TRA gene, TRE gene, E2M gene, albumin NF-α gene, HEE gene, HEAl gene, TTR gene, NR3Cl gene, CD52 gene, ECLllA gene Erythroid-specific enhancer, CELE gene, TGFERl gene, SERPINAl gene, infected cells HEV genomic DNA, CEP290 gene, DMD gene, CFTR gene, or IL2RG gene in The method according to any one of claims 67 to 75,

77. The expression control region of the gene comprises a promoter region of the gene.

76. The method of any one of claims 76 to 76.

78. The functional domain is a nuclease comprising a cleavage domain or a half-cleavage domain, The method of any one of claims 67 to 71, wherein the endogenous gene is inactivated by truncation.

79. 79. The method of claim 78, wherein the inactivation occurs via non-homologous end joining (NHEJ).

80. The polypeptide binds to a first target nucleic acid sequence within the gene and includes a half-cleavage domain. a first polypeptide comprising a first polypeptide, the method comprising introducing a second polypeptide, The second polypeptide binds to a second target nucleic acid sequence in the gene and produces a half-cleavage domain.

80. The method of claim 78 or 79, which is a polypeptide according to claim 44 comprising

81. The first target nucleic acid sequence and the second target sequence are spaced apart in the gene. and the two half-cleavage domains are positioned between the first and second target nucleic acid sequences.

81. The method of claim 80, wherein the nucleic acid sequence mediates cleavage of the gene sequence at said position.

82. 82. Any of claims 78 to 81, wherein the cleavage domain or cleavage half-domain comprises FokI or Bfil. The method according to any one of claims 1 to 5.

83. FokI is at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or 83. The method of claim 82, wherein said sequence identity is at least 99%.

84. The method of claim 82 or 83, wherein FokI has the sequence of SEQ ID NO:

11.

85. The method of any one of claims 78 to 81, wherein the cleavage domain comprises a meganuclease. Law.

86. The genes include the PDCD1 gene, the CTLA4 gene, the LAG3 gene, the TET2 gene, the ETLA gene, and the HA VCR2 gene, CCR5 gene, CXCR4 gene, TRA gene, TRE gene, E2M gene, albumin NF-α gene, HEE gene, HEAl gene, TTR gene, NR3Cl gene, CD52 gene, ECLllA gene Erythroid-specific enhancer, CELE gene, TGFERl gene, SERPINAl gene, infected cells HEV genomic DNA, CEP290 gene, DMD gene, CFTR gene, or IL2RG gene in The method according to any one of claims 78 to 85,

87. 1. A method for introducing an exogenous nucleic acid into a region of interest in the genome of a cell, comprising: The NBD of the polypeptide binds to a target nucleic acid sequence adjacent to the region of interest. A polypeptide according to claim 44, The exogenous nucleic acid; into the cell, The cleavage domain or half-cleavage domain induces cleavage in the region of interest, and the exogenous nucleic acid The acid is integrated into the cleaved region of interest by homologous recombination. method.

88. 87. The method of claim 87, wherein the polypeptide is introduced as a nucleic acid encoding the polypeptide. The method described above.

89. 89. The method of claim 88, wherein the nucleic acid is deoxyribonucleic acid (DNA).

90. 89. The method of claim 88, wherein the nucleic acid is ribonucleic acid (RNA).

91. 88. The sequence of said nucleic acid is codon-optimized for expression in a human cell. A method according to any one of claims 1 to 90.

92. The exogenous nucleic acid may be a chimeric antigen receptor (CAR), an alpha-L iduronidase (IDUA), an iduronate (IDUA), or a combination thereof.

91. The method of claim 88-90, which encodes an isoform of ... Any method as described above.

93. The target nucleic acid sequence is selected from the group consisting of PDCD1 gene, CTLA4 gene, LAG3 gene, TET2 gene, and ETLA gene. gene, HA VCR2 gene, CCR5 gene, CXCR4 gene, TRA gene, TRE gene, E2M gene, Albumin gene, HEE gene, HEAl gene, TTR gene, NR3Cl gene, CD52 gene, ECLll A gene erythroid specific enhancer, CELE gene, TGFERl gene, SERPINAl gene, HEV genomic DNA, CEP290 gene, DMD gene, CFTR gene, or IL2RG gene in stained cells The method according to any one of claims 88 to 90, wherein the method is in a child.

94. The polypeptide binds to a first target nucleic acid sequence within the gene and includes a half-cleavage domain. a first polypeptide comprising a target nucleic acid sequence that binds to a second target nucleic acid sequence within the gene, Introducing a second polypeptide, which is the polypeptide of claim 44 containing a half-cleavage domain. The method of any of claims 87 to 93, comprising:

95. The first target nucleic acid sequence and the second target sequence flank the region of interest, 95. The method of claim 94, wherein one half-cleavage domain mediates cleavage in the region of interest.

96. 87-95, wherein the cleavage domain or the cleavage half-domain comprises FokI or Bfil.

2. The method according to claim 1 ,

97. FokI is at least 70%, at least 75%, at least 80%, At least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or 97. The method of claim 96, wherein said sequence identity is at least 99%.

98. The method of claim 96 or 97, wherein FokI has the sequence of SEQ ID NO:

11.

99. The method of any one of claims 87 to 95, wherein the cleavage domain comprises a meganuclease. Law.

100. The method according to any one of claims 67 to 99, wherein the cell is an animal cell or a plant cell. 。

101. The method of any one of claims 67 to 99, wherein the cell is a human cell.

102. 102. The method of any one of claims 67 to 101, wherein the cell is an ex vivo cell.

103. The introducing step comprises administering to a subject the polypeptide or a nucleic acid encoding same. The method according to any one of claims 67 to 101, comprising:

104. 104. The method of claim 103, wherein said administering comprises parenteral administration.

105. 104. The method of claim 103, wherein said administering comprises intravenous, intramuscular, intrathecal, or subcutaneous administration. The method described.

106. 104. The method of claim 103, wherein said administering comprises direct injection at a site in the subject. 。

107. 104. The method of claim 103, wherein said administering comprises direct injection into the tumor.

108. 1. A method for imaging a genomic locus in a cell, comprising:

54. The cell, wherein the target nucleic acid sequence to which the NBD binds is present at the genomic locus. contacting a polypeptide according to claim 1 with a polypeptide of formula (I); and The genomic locus is identified by detecting the fluorophore or detectable tag. Imaging The method includes:

109. 109. The method of claim 108, wherein the detectable tag is a member of a specific binding pair.

110. 109. The method of claim 108, wherein the tag is biotin or avidin.

111. The method of claim 108, wherein the tag is an HA tag or a FLAG tag.

112. 109. The method of claim 108, wherein the fluorophore is GFP or mCHERRY.

113. 1. A method for producing a polypeptide that specifically binds to a target DNA sequence, comprising: synthesizing a polypeptide comprising a DNA binding domain (DBD) that specifically binds to The DBD is selected based on the DNA bases bound by its repeating units and is used to bind the target DNA sequence. It contains repeating units combined in the appropriate order to match the columns, where: If the target sequence contains an adenine (A), the repeat unit is FSSQQIIRMVSHAGGANNLKAVTANHDDLQNMG (SEQ ID NO: 2), LGHKELIKIAARNGGGNNLIAVLSCYAKLKEMG (SEQ ID NO: 89) Containing or conserving a sequence of 33 to 35 amino acids having at least 70% identity to comprising the sequence of SEQ ID NO:2 or SEQ ID NO:89 containing targeted amino acid substitutions; If the target sequence contains a thymine (T), the repeat unit is FNAEQIVRMVSHGGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:4); FNAEQIVSMVSNGGGSLNLKAVKKYHDALKDRG (SEQ ID NO: 6); or FNVEQIVSIVSHGGGSLNLKAVKKYHDVLKDRE (SEQ ID NO: 8) Containing or conserving a sequence of 33 to 35 amino acids having at least 70% identity to comprising the sequence of SEQ ID NO: 4, 6 or 8 containing targeted amino acid substitutions; If the target sequence contains a cytosine (C), the repeat unit is FNTEQIVRMVSHDGGSLNLKAVKKYHDALRERK (SEQ ID NO: 7); or FNAEQIVRMVSHDGGSLNLKAVTDNHDDLKNMG (SEQ ID NO: 9) Containing or conserving a sequence of 33 to 35 amino acids having at least 70% identity to The sequence of SEQ ID NO: 7 or 9 includes a targeted amino acid substitution; If the target sequence contains a guanine (G), the repeat unit is FNVEQIVRMVSHNGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:3); FNAEQIVSMVSNNGGSKNLKAVTDNHDDLKNMG (SEQ ID NO:5); FNAEQIVRMVSHKGGSKNLALVKEYFPVFSSFH (SEQ ID NO:33); or LGHKELIKIAARNGGGNNLIAVLSCYAKLKEMG (SEQ ID NO: 89) Containing or conserving a sequence of 33 to 35 amino acids having at least 70% identity to 3, 5, 33, or 89, each of which contains a targeted amino acid substitution; method.

114. 1. A method for producing a polypeptide that specifically binds to a target DNA sequence, comprising: synthesizing a polypeptide comprising a DNA binding domain (DBD) that specifically binds to The DBD is selected based on the DNA bases bound by its repeating units and is used to bind the target DNA sequence. It contains repeating units combined in the appropriate order to match the columns, where: If the target sequence contains an adenine (A), the repeat unit is FSAKHIVRIAAHIGGSLNIKAVQQAQQALKELG (SEQ ID NO: 32), FSAEQIVSIAAHVGGSHNIEAVQKAHQALKELD (SEQ ID NO: 35), FSAEQIVRIAAHIGGSHNLKAVLQAQQALKELD (SEQ ID NO: 31), or FSAEQIVRIAAHIGGSRNIEATIKHYAMLTQPP (SEQ ID NO: 133) Containing or conserving a sequence of 33 to 35 amino acids having at least 70% identity to comprising the sequence of SEQ ID NO: 32, 36, 31 or 133 containing targeted amino acid substitutions; If the target sequence contains a thymine (T), the repeat unit is YSSEQIVRVAAHGGGSLNIKAVLQAHQALKELD (SEQ ID NO:28), FSAEQIVHIAAHGGGSLNIKAILQAHQTLKELN (SEQ ID NO:29), FSTEQIVCIAGHGGGSLNIKAVLLAQQALKDLG (SEQ ID NO:27), FSAEQIVSIAAHVGGSHNIEAVQKAHQALKELD (SEQ ID NO: 35), or FSAEQIVRIAAHIGGSRNIEATIKHYAMLTQPP (SEQ ID NO: 133) Containing or conserving a sequence of 33 to 35 amino acids having at least 70% identity to SEQ ID NO: 28, 29, 27, 35, or 133, each of which contains a targeted amino acid substitution; If the target sequence contains a cytosine (C), the repeat unit is FSAEQIVSIVAHDGGSRNIEAVQQAQHILKELG (SEQ ID NO:24), FSAEQIVRIAAHDGGSLNIDAVQQAQQALKELG (SEQ ID NO: 26), or FSAEQIVRIAAHIGGSHNLKAVLQAQQALKELD (SEQ ID NO:31) Containing or conserving a sequence of 33 to 35 amino acids having at least 70% identity to comprising the sequence of SEQ ID NO: 24, 26, or 31 containing targeted amino acid substitutions; If the target sequence contains a guanine (G), the repeat unit is FSAEQIVRIAAHIGGSRNIEAIQQAHHALKELG (SEQ ID NO: 30), FSADQIVRIAAHKGGSHNIVAVQQAQQALKELD (SEQ ID NO: 34), FSAEQIVSIAAHVGGSHNIEAVQKAHQALKELD (SEQ ID NO: 35), or FSAEQIVRIAAHIGGSRNIEATIKHYAMLTQPP (SEQ ID NO: 133) Containing or conserving a sequence of 33 to 35 amino acids having at least 70% identity to 30, 34, 35, or 133, each of which contains a targeted amino acid substitution; method.

115. 115. The method of claim 113 or 114, wherein said target sequence is in the promoter region of a gene of interest. 。

116. 113 or 114, wherein the target sequence is in an open reading frame of a gene of interest.

14. The method according to claim 14.

117. The method of any one of claims 113 to 116, wherein the DBD is linked to a functional domain. 。

Images