WO2015020218A1

WO2015020218A1 - Highly-active tale protein by altering dna-bound protein domain

Info

Publication number: WO2015020218A1
Application number: PCT/JP2014/071116
Authority: WO
Inventors: 康志岡田; 一穂池田
Original assignee: 独立行政法人理化学研究所
Priority date: 2013-08-09
Filing date: 2014-08-08
Publication date: 2015-02-12

Abstract

The present invention provides: a protein containing an altered-type TALE section which exerts excellent binding activity with respect to a given target DNA base sequence with which sufficient DNA binding activity could not have been conventionally achieved; a nucleic acid coding for the aforementioned protein; an expression vector containing said nucleic acid; uses for the aforementioned protein, nucleic acid, and expression vector; and a method for screening the aforementioned protein. Specifically, the present invention provides an isolated altered-type protein exerting binding activity with respect to a target DNA, the altered-type protein containing a Transcription Activator-Like (TAL) Effector section derived from a Xanthomonas bacterium and having a repeating structure of multiple modules, wherein each of the modules has a target base recognition region for recognizing one base constituting the base sequence of the target DNA, and at least one of the modules has an amino acid sequence which was altered so that at least one amino acid residue, which is selected from a group comprising the 2^nd to 6^th and the 30^th to 34^th amino acids in an amino acid sequence represented by sequence number 1, has an artificial mutation.

Description

Highly active TALE protein by modification of DNA binding protein domain

The present invention relates to a protein comprising a modified TALE moiety with altered binding activity to the target DNA, a nucleic acid encoding the protein, an expression vector comprising the nucleic acid, and their use, and binding to the target DNA in the protein comprising the TALE moiety The present invention relates to a method for changing activity.

As a method for examining the functions of individual genes, genome editing techniques using knockout (destruction) of target genes and knockin (addition) of foreign genes using homologous recombination are widely used. In genome editing technology, there is a demand for technology that can selectively and freely modify arbitrary gene sequences in various biological species. In recent years, new tools using artificial nucleases (ZFN and TALEN) have attracted attention. ing.

Artificial nuclease is a chimeric protein in which a domain that specifically binds to DNA and a DNA cleavage domain are linked. The former specifically recognizes and binds to the target DNA sequence, and the latter cleaves the DNA in the vicinity. Utilizing the process by which cleaved DNA is repaired by an endogenous mechanism, it is easy to introduce mutations such as deletion, insertion, and base substitution into the target gene, and to introduce specific foreign DNA into the vicinity of the target region. Can be performed. As a result, in many plants, microorganisms, and animals, cultured cells or individual organisms in which the genomic sequence has been modified as intended can be obtained. In the future, it is expected to be applied to the creation of disease models, gene therapy by modifying disease-causing genes, and the creation of plants with the desired traits.

TALEN is an artificial nuclease that uses the transcription factor Transcription Activator-Like TAL (TAL) Effector (TALE) possessed by the genus Xanthomonas as a DNA binding domain, and is fused with a DNA cleavage domain エンド (endonuclease). TALEN has a higher degree of freedom in target genes compared to competing Zinc Finger Nuclease (ZFN), and is advantageous in terms of ease of production, cost, etc. There are databases, kits, protocols, etc. to support lab-based design. Provided (for example, Non-Patent Documents 1 to 3).

TALE was discovered as a transcription factor involved in gene expression regulation to suppress the host cell's immune mechanism from acting when pathogenic bacteria are infected (Non-patent Document 5). In TALEN, the DNA binding domain derived from TALE is linked to a target base sequence (approximately 17 bp) on the target genome by linking structural units (modules) consisting of about 34 amino acids with a repeat number of 10 to 30. Designed to. Each module includes a variable region having 2 amino acid residues that is modified to recognize one base constituting the target base sequence. The variable region is called Repeat-Variable-Diresidues (RVD), and the 12th and 13th amino acids usually correspond to this. That is, in TALEN, TALE makes the target base sequence specific by linking each module in which the variable region is modified for each base type (A, G, T, C) according to the target base sequence. Recognizes and binds to the DNA, and double-strand breaks of DNA are caused by the above-mentioned DNA cleavage domain (for example, Non-Patent

Documents

4, 6 to 8).

In addition, TALE can be applied to transcription regulation, epigenome control, and the like as an artificial transcription factor that binds to a specific transcription factor binding region in the promoter region, for example, by utilizing the binding ability to such a target base sequence. .

Recently, as disclosed in Non-Patent Documents 9 to 11, the three-dimensional structure of TALE has been clarified, and a repeating structure of a module having a Helix-turn-helix structure is wound around the main groove of the target DNA, There have been reports suggesting that the RVD region located in the turn part contributes to the stability and specificity of binding to DNA.

In the improvement of TALEN, various approaches have been attempted so far focusing on the RVD region in order to improve the binding stability with DNA (for example, Non-Patent Documents 12 to 14 and Patent Document 1). . However, depending on the target genome, sufficient cleavage efficiency may still not be obtained, and it is desired to modify the TALE part that has both binding stability and specificity for the target base sequence.

Special Table 2013-513389

The present invention has been made in view of the above-described present situation in the technical field, and has excellent DNA binding stability with respect to an arbitrary target DNA base sequence for which sufficient binding activity has not been obtained with conventional TALE. It is an object to provide a protein containing a modified TALE moiety having sex and specificity, a nucleic acid encoding the protein, an expression vector containing the nucleic acid, their use, and a screening method for the protein. To do.

The present inventors have intensively studied to solve the above-mentioned problems. In the process, the present inventors analyzed the sequence of the repetitive portion of the DNA binding domain of about 1000 types of TALE possessed by various bacteria of the genus Xanthomonas. As a result, in addition to the 12th to 13th amino acids (RVD) in each module of the repetitive part, which was previously known to contribute to the recognition of specific bases in DNA, the 4th and 32nd positions in each module These amino acids also have low sequence conservation. Therefore, the present inventors further examined the molecular structure of TALE, and the 4th and 32nd amino acids were involved in the binding between adjacent modules and played an important role in stabilizing the tertiary structure of the TALE molecule. It was suggested that Based on such findings, the present inventors have conceived the possibility that the activity of TALE can be artificially changed by introducing mutations into these amino acids, and the Voytas et al. Golden Gate TALEN construction method and its Based on the modification method (Non-patent Documents 1 and 2), mutations were introduced into the 4th and 32nd amino acid residues, and experiments were conducted to measure the activity. As a result, a highly active TALE protein with improved specific binding activity to the target DNA sequence was successfully constructed by introducing mutations. Based on the above findings, the present inventors have made further studies and completed the present invention.

That is, the present invention is as follows.
[1] An isolated modified protein having binding activity to a target DNA,
The modified protein includes a Transcription Activator-Like (TAL) Effector portion derived from a genus Xanthomonas having a repeating structure of a plurality of modules,
Each of the plurality of modules has a target base recognition region that recognizes one base constituting the base sequence of the target DNA, and
In at least one of the plurality of modules, in the amino acid sequence represented by SEQ ID NO: 1, at least one amino acid residue selected from the group consisting of 2 to 6 and 30 to 34 is artificially mutated Having an amino acid sequence modified to have
The modified protein.
[2] At least one of the modules having an amino acid sequence modified to have an artificial mutation is at least one selected from the group consisting of 2 to 6 in the amino acid sequence represented by SEQ ID NO: 1. The modified protein according to [1] above, wherein the amino acid residue and at least one amino acid residue selected from the group consisting of 30 to 34 are modified so as to have an artificial mutation.
[3] At least one of the plurality of modules is such that at least one amino acid residue selected from the group consisting of 20th to 26th in the amino acid sequence represented by SEQ ID NO: 1 has an artificial mutation The modified protein according to the above [1] or [2], which has been modified to.
[4] The above [1] to [1], wherein at least one of the plurality of modules is artificially modified so that the 11th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 is asparagine. 3]. The modified protein according to any one of [3].
[5] At least one of the modules having an amino acid sequence modified to have an artificial mutation has 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1, and the homology When the amino acid sequence represented by SEQ ID NO: 1 is aligned with the amino acid sequence represented by SEQ ID NO: 1, the 4th and / or 32nd amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 has an artificial mutation The modified protein according to any one of [1] to [4] above, which is modified.
[6] At least one of the modules having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 comprises the amino acid sequence having the homology and the amino acid sequence represented by SEQ ID NO: 1. (I) the amino acid sequence represented by SEQ ID NO: 1 has been modified so that the fourth amino acid residue is glutamic acid and the 32nd amino acid residue is alanine, or (Ii) The amino acid sequence represented by SEQ ID NO: 1 according to the above [5], which is modified so that the fourth amino acid residue is glutamine and the 32nd amino acid residue is alanine. Modified protein.
[7] The modified protein according to any one of [1] to [6] above, wherein the mutation has the same pattern in all of the plurality of modules.
[8] The modified protein according to any one of [1] to [7] above, wherein the number of modules in the repeating structure is 6 to 36.
[9] The modified protein according to any one of [1] to [8] above, further comprising a functional domain not derived from a genus Xanthomonas from which the TAL Effector moiety is derived.
[10] The modified protein according to [9] above, wherein the functional domain is all or a part of a nucleolytic enzyme or a nucleotransferase.
[11] The modified protein according to [10] above, wherein the nucleolytic enzyme contains a DNA cleavage domain of the restriction enzyme FokI.
[12] The modified protein according to [9] above, wherein the functional domain includes a transcriptional activator domain, a transcriptional repressor domain, or an epigenome regulator domain.
[13] The above [9] to [12], wherein the functional domain includes all or part of a fluorescent protein, photoprotein, or chromoprotein, or all or part of an enzyme protein that catalyzes fluorescence, luminescence, or color reaction. The modified protein according to any one of the above.
[14] An isolated nucleic acid encoding the amino acid sequence of the modified protein according to any one of [1] to [13] above.
[15] An expression vector comprising the nucleic acid according to [14] above.
[16] A transformant into which the expression vector according to [15] is introduced.
[17] A method for producing the modified protein according to any one of [1] to [13] above, wherein the method comprises:
A step of introducing a mutation in the nucleic acid encoding the original protein, wherein the mutation is such that the artificial mutation of the modified protein occurs in the protein encoded by the mutated nucleic acid; The original protein is such that the amino acid sequence of the protein into which the artificial mutation is introduced becomes the amino acid sequence of the modified protein,
Preparing an expression vector containing the mutated nucleic acid and obtaining the transformant by introducing the expression vector into a host cell; and
The method comprising culturing the transformant and isolating the modified protein from the culture.
[18] The modified protein according to any one of [1] to [13] is introduced into a cell, or RNA encoding the amino acid sequence of the modified protein or the amino acid sequence of the modified protein is encoded. An expression vector containing a nucleic acid to be expressed is introduced into a cell, the modified protein is expressed in the cell, and the modified protein is bound to a target gene and / or a region around it, (a) Whether to cleave, (b) modify the region by cleaving the region and applying homologous recombination repair or non-homologous end-joining repair to the cleaved region, or (c) modifying the region Or (d) a method of cleaving, altering or modifying the region in the cell, comprising the step of changing gene expression around the region.
[19] The cells are animal sperm, unfertilized eggs, fertilized eggs, cells constituting fetuses or adults, cells collected from plants or animal tissues, embryonic stem cells (ES cells), or artificial pluripotency The method according to [18] above, which is a stem cell (iPS cell).
[20] A method for producing an individual or strain of a mutant organism in which a target gene and / or a region around it is cleaved, altered or modified,
(1) preparing a cell of the organism including the region;
(2) The modified protein according to any one of the above [1] to [13] is introduced into a cell, or the RNA encoding the amino acid sequence of the modified protein or the amino acid sequence of the modified protein is encoded. An expression vector containing a nucleic acid to be introduced is introduced into a cell, the modified protein is expressed in the cell, and the modified protein is bound to the region, so that (a) the region is cleaved, or (b) Modifying the region by cleaving the region and applying homologous recombination repair or non-homologous end-joining repair to the cleaved region, (c) modifying the region, or (d) Changing the gene expression around the region;
(3) The method including the step of producing an individual or strain of the mutant organism from the cells obtained in step (2).
[21] An individual or strain of a mutant organism obtained by the method according to [20] above.
[22] A method for screening a modified protein whose binding activity to a target DNA is changed compared to an unmodified protein,
(1) a step of measuring the binding activity of the modified protein according to any one of [1] to [13] to the target DNA;
(2) measuring the binding activity of the unmodified protein having no artificial mutation in the modified protein to the target DNA;
(3) When the binding activity of the modified protein is changed as compared with the binding activity of the unmodified protein, the modified protein has a binding activity to the target DNA that is not The said method which has the process of selecting as a modified protein changed compared with the modified protein.
[23] The method according to [22] above, further comprising the step of preparing the modified protein using the method according to [17] before the step (1).
[24] A method for screening a modified protein whose cleavage activity against a target DNA is changed as compared with an unmodified protein,
(1) a step of measuring the cleavage activity of the modified protein according to [10] or [11] with respect to the target DNA;
(2) a step of measuring the cleavage activity of the unmodified protein having no artificial mutation in the modified protein with respect to the target DNA;
(3) When the cleavage activity of the modified protein is changed as compared to the cleavage activity of the unmodified protein, the modified protein has a cleavage activity against the target DNA. The said method which has the process of selecting as a modified protein changed compared with the modified protein.
[25] The method according to [24], further comprising the step of preparing the modified protein using the method according to [17] before the step (1).

The modified TALE portion according to the present invention has excellent binding activity (high binding stability and binding specificity) for any target DNA base sequence for which sufficient DNA binding activity could not be obtained with conventional TALE. Is obtained. Therefore, the protein of the present invention containing such a modified TALE moiety can be used for genome editing using a combination with a sequence-independent DNA cleavage domain, or artificial transcription for specifically activating or suppressing a target gene. High efficiency can be provided in use as a factor. The present invention also makes it possible to control (increase or decrease) the binding activity to the target DNA base sequence by modifying the TALE moiety. The present invention also provides a nucleic acid encoding the protein, an expression vector containing the nucleic acid, a method for screening the protein, a method for introducing mutation or modification into the genome using the protein, nucleic acid, expression vector, or the like. .

It is a figure which shows the result of the conservation property of the amino acid residue in the repeat sequence of natural TALE. It is a figure which shows the combination of the 4th and 32nd amino acid residue of TALE repeat of 10 types of X. oryzae. It is a figure which shows the combination of the EA type | mold of the 4th and 32nd amino acid residues of a TALE repeat seen in X. citri etc. It is a figure which shows the combination of DQ type | mold of the 4th and 32nd amino acid residue of TALE repeat seen in X. axonopodis etc. It is a figure for demonstrating that the 4th and 32nd residue of a TALE repeat participates in the coupling | bonding between TALE repeats. It is a figure for demonstrating the structure of TALE at the time of DNA non-bonding and a coupling | bonding. It is a figure which shows the comparison of the DNA binding activity of dTALE 85F (conventional type) and dTALE 85mF (modified type (EA type)). Lane 1: dTALE 85F and target 85F, 2: dTALE 85F and control target, 3: dTALE 85mF and target 85F, 4: dTALE 85mF and control target It is a figure which shows the comparison of the DNA binding activity of dTALE 85R (conventional type) and dTALE 85mR (modified type (EA type)). Lane 1: dTALE 85R and target 85R, 2: dTALE 85R and control target, 3: dTALE 85mR and target 85R, 4: dTALE 85mR and control target It is a figure which shows the comparison of DNA binding activity of dTALE-209F (conventional type), dTALE-209mF (modified type (EA type)), and dTALE-209QF (modified type (EQ type)). Lane 1: dTALE 209F and target 209F, 2: dTALE 209F and control target, 3: dTALE 209mF and target 209F, 4: dTALE 209mF and control target, 5: dTALE 209QF and target 209F, 6: dTALE 209QF and control target

dTALE 209R (conventional) and dTALE 209mR (modified (EA)), dTALE 209QR (modified (EQ)), dTALE 209m-QR (combined modified (EA-EQ)), and dTALE 209Q-mR It is a figure which shows the comparison of the DNA binding activity of (complex modified type (EA-EQ type)). Lane 1: dTALE 209R and target 209R, 2: dTALE 209R and control target, 3: dTALE 209mR and target 209R, 4: dTALE 209mR and control target, 5: dTALE 209QR and target 209R, 6: dTALE 209QR and control target, 7 : DTALE 209m-QR and target 209R, 8: dTALE 209m-QR and control target, 9: dTALE 209Q-mR and target 209R, 10: TdTALE 209Q-mR and control target It is a figure which shows the comparison of the activity of the conventional TALEN and modified TALEN by HMA. It is a figure which shows the comparison of the in-vitro DNA cutting activity of conventional TALEN-209F and modified TALEN-209mF. It is a figure which shows the comparison of in-vitro DNA cutting activity of conventional TALEN-209R and modified TALEN-209mR. It is a figure which shows the comparison of DNA binding activity of dTALE 85F (conventional type), dTALE 85mF (modified type (EA type)), and dTALE 85m2F (modified type (QA type)). Lane 1: dTALE 85F and target 85F, 2: dTALE 85F and nonspecific target 209R, 3: dTALE 85mF and target 85F, 4: dTALE 85mF and nonspecific target 209R, 5: dTALE 85m2F and target 85F, 6: dTALE 85m2F and non-specific target 209R It is a figure which shows the comparison of DNA binding activity of dTALE 85R (conventional type), dTALE 85mR (modified type (EA type)), and dTALE 85m2R (modified type (QA type)). Lane 1: dTALE 85R and target 85R, 2: dTALE 85R and nonspecific target 209R, 3: dTALE 85mR and target 85R, 4: dTALE 85mR and nonspecific target 209R, 5: dTALE 85m2R and target 85R, 6: dTALE 85m2R and non-specific target 209R It is a figure which shows the comparison of the DNA binding activity of dTALE-209F (conventional type), dTALE-209mF (modified type (EA type)), and dTALE-209m2F (modified type (QA type)). Lane 1: dTALE 209F and target 209F, 2: dTALE 209F and non-specific target 209R, 3: dTALE 209mF and target 209F, 4: dTALE 209mF and non-specific target 209R, 5: dTALE 209m2F and target 209F, 6: dTALE 209m2F and non-specific target 209R It is a figure which shows the comparison of the DNA binding activity of dTALE S1PR3F (conventional type), dTALE S1PR3mF (modified type (EA type)), and dTALE S1PR3m2F (modified type (QA type)). Lane 1: dTALE S1PR3F and target S1PR3F, 2: dTALE S1PR3mF and target S1PR3F, 3: dTALE S1PR3m2F and target S1PR3F It is a figure which shows the comparison of DNA-binding activity of dTALE S1PR3R (conventional type), dTALE S1PR3mR (modified type (EA type)), and dTALE S1PR3m2R (modified type (QA type)). Lane 1: dTALE S1PR3R and target S1PR3R, 2: dTALE S1PR3mR and target S1PR3R, 3: dTALE S1PR3m2R and target S1PR3R Diagram showing comparison of DNA binding activity of dTALE SphkF (conventional type), dTALE SphkmF (modified type (EA type)), dTALE SphkpmF (modified type (incomplete EA type)) and dTALE Sphkm2F (modified type (QA type)) It is. Lane 1: dTALE SphkF and target SphkF, 2: dTALE SphkpmF and target SphkF, 3: dTALE SphkmF and target SphkF, 4: dTALE Sphkm2F and target SphkF It is a figure which shows the comparison of DNA binding activity of dTALE SphkR (conventional type), dTALE SphkmR (modified type (EA type)), and dTALE Sphkm2R (modified type (QA type)). Lane 1: dTALE SphkR and target SphkR, 2: dTALE SphkmR and target SphkR, 3: dTALE Sphkm2R and target SphkR It is a figure which shows the comparison of the DNA binding activity of dTALE CG16798F (conventional type), dTALE CG16798mF (modified type (EA type)), and dTALE CG16798m2F (modified type (QA type)). Lane 1: dTALE CG16798F and target CG16798F, 2: dTALE CG16798mF and target CG16798F, 3: dTALE CG16798m2F and target CG16798F It is a figure which shows the comparison of DNA-binding activity of dTALE CG16798R (conventional type), dTALE CG16798mR (modified type (EA type)), and dTALE CG16798m2R (modified type (QA type)). Lane 1: dTALE CG16798R and target CG16789R, 2: dTALE CG16798mR and target CG16798R, 3: dTALE CG16798m2R and target CG16798R Comparison of DNA binding activity of dTALE 209R (conventional), dTALE 209mR (modified (EA)), dTALE 209m2R (modified (QA)), and dTALE 209m3R (combined modified (EA-QA)) FIG. Lane 1: dTALE 209R and target 209R, 2: dTALE 209mR and target 209R, 3: dTALE 209m3R and target 209R, 4: dTALE 209m2R and target 209R It is a figure which shows the comparison of the DNA binding activity of dTALE 85F (conventional type), dTALE 85pmF (modified type (incomplete EA type)) and dTALE 85mF (modified type (EA type)). Lane 1: dTALE 85F and target 85F, 2: dTALE 85F and nonspecific target TRE, 3: dTALE 85pmF and target 85F, 4: dTALE 85pmF and nonspecific target TRE, 5: dTALE 85mF and target 85F, 6: dTALE 85mF and non-specific target TRE It is a figure which shows the comparison of DNA binding activity of dTALE 85R (conventional type), dTALE 85pmR (modified type (incomplete EA type)) and dTALE 85mR (modified type (EA type)). Lane 1: dTALE 85R and target 85R, 2: dTALE 85R and nonspecific target TRE, 3: dTALE 85pmR and target 85R, 4: dTALE 85pmR and nonspecific target TRE, 5: dTALE 85mR and target 85R, 6: dTALE 85mR and non-specific target TRE

dTALE 209R (conventional type), dTALE 209mR (modified type (EA type)), dTALE 209h1R (combined modified type (DD-DA-EA type)), dTALE 209h3R (combined modified type (mixed type 1)), and dTALE 209h2R It is a figure which shows the comparison of the specific and non-specific DNA binding activity of (complex modified type (DA-EA type)). Lane 1: dTALE 209R and target 209R, 2: dTALE 209R and target 209R-1c, 3: dTALE 209R and target 209R-1n, 4: dTALE 209R and target 209R-1m, 5: dTALE 209R and target 209R-3m, 6 : DTALE 209mR and target 209R, 7: dTALE 209mR and target 209R-1c, 8: dTALE 209mR and target 209R-1n, 9: RdTALE 209mR and target 209R-1m, 10: dTALE 209mR and target 209R-3m, 11: dTALE 209h1R and target 209R, 12: dTALE 209h1R and target 209R-1c, 13: dTALE 209h1R and target 209R-1n, 14: dTALE 209h1R and target 209R-1m, 15: dTALE 209h1R and target 209R-3m, 16: dTALE 209h3 Target 209R, 17: dTALE 209h3R and target 209R-1c, 18: dTALE 209h3R and target 209R-1n, 19: dTALE 209h3R and target 209R-1m, 20: dTALE 209h3R and target 209R-3m, 21: dTALE 209h2R and target 209R , 22: dTALE 209h2R and target 209R-1c, 23: dTALE 209h2R And target 209R-1n, 24: dTALE 209h2R and target 209R-1m, 25: dTALE 209h2R and target 209R-3m It is a figure which shows the comparison of the specific and non-specific DNA binding activity of dTALE-209mR (modified type (EA type)) and dTALE-209h4R (combined modified type (mixed type 2)). Lane 1: dTALE 209mR and target 209R, 2: dTALE 209mR and target 209R-1c, 3: dTALE 209mR and target 209R-1n, 4: dTALE 209mR and target 209R-1m, 5: dTALE 209mR and target 209R-3m, 6 : DTALE 209h4R and target 209R, 7: dTALE 209h4R and target 209R-1c, 8: dTALE 209h4R and target 209R-1n, 9: dTALE 209h4R and target 209R-1m, 10: dTALE 209h4R and target 209R-3m It is a figure which shows the comparison of the specific and non-specific DNA binding activity of dTALE-209R (conventional type), dTALE-209-4AR (modified type (4A type)), and dTALE-209-4ER type (modified type (type 4E)). Lane 1: dTALE 209 4ER and target 209R, 2: dTALE 209 4ER and target 209R-1c, 3: dTALET209 4ER and target 209R-1n, 4: dTALE 209 4ER and target 209R-1m, 5: dTALE 209 4ER and target 209R-2w, 6: dTALE 209 4ER and target 209R-3m, 7: dTALE 209 4AR and target 209R, 8: dTALE 209 4AR and target 209R-1c, 9: dTALE 209 4AR and target 209R-1n, 10: dTALE 209 4AR and target 209R-1m, 11: dTALE 209 4AR and target 209R-2w, 12: dTALE 209 4AR and target 209R-3m, 13: dTALE 209R and target 209R It is a figure which shows the result of the systematic sequence analysis about the arrangement | sequence (N-TAL) of the N terminal side out of the repeat motif of natural TALE protein. It is a figure which shows the result of the systematic sequence analysis about the sequence | arrangement (C-TAL) of the C terminal side out of the repeat motif of natural TALE protein. It is a figure which shows the comparison of the DNA binding activity of dTALE CG16798F, dTALE CG16798mF, dTALE CG16798m2F, dTALE EQ-CG16798F, dTALE EQ-CG16798mF, dTALE EQ-CG16798m2F. Lane 1: dTALE CG16798F and target CG16798F, 2: dTALE CG16798mF and target CG16798F, 3: dTALE CG16798m2F and target CG16798F, 4: dTALE EQ-CG16798F and target CG16798F, 5: dTALE EQ-CG16798F and target CG16798F CG16798m2F and target CG16798F It is a figure which shows the comparison of the DNA binding activity of dTALE CG16798R, dTALE CG16798mR, dTALE CG16798m2R, dTALE EQ-CG16798R, dTALE EQ-CG16798mR, and dTALE EQ-CG16798m2R. Lane 1: dTALE CG16798R and target CG16798R, 2: dTALE CG16798mR and target CG16798R, 3: dTALE CG16798m2R and target CG16798R, 4: dTALE EQ-CG16798R and target CG16798R, 5: dTALE EQ-CG16798R and EQ-CG16798R CG16798m2R and target CG16798R It is a figure which shows the comparison of the DNA binding activity of dTALE SphkF, dTALE SphkmF, dTALE Sphkm2F, dTALE EQ-SphkF, dTALE EQ-SphkmF, and dTALE EQ-Sphkm2F. Lane 1: dTALE SphkF and target SphkF, 2: dTALE SphkmF and target SphkF, 3: dTALE Sphkm2F and target SphkF, 4: dTALE EQ-SphkF and target SphkF, 5: dTALE EQ-SphkF, -6: SphkF Sphkm2F and target SphkF It is a figure which shows the comparison of the DNA binding activity of dTALE SphkR, dTALE SphkmR, dTALE Sphkm2R, dTALE EQ-SphkR, dTALE EQ-SphkmR, and dTALE EQ-Sphkm2R. Lane 1: dTALE SphkR and target SphkR, 2: dTALE SphkmR and target SphkR, 3: dTALE Sphkm2R and target SphkR, 4: dTALE EQ-SphkR and target SphkR, 5: dTALE EQ-SphkR-EQ-SphkR-E6 Sphkm2R and target SphkR It is a figure which shows the comparison of the DNA binding activity of dTALE 85mR, dTALE EQ-85mR, dTALE citri-85mR, dTALE 85m2R, dTALE EQ-85m2R, and dTALE citri-85m2R. Lane 1: dTALE 85mR and target 85R, 2: dTALE EQ-85mR and target 85R, 3: dTALE citri-85mR and target 85R, 4: dTALE 85m2R and target 85R, 5: dTALE EQ-85m2R and target 85R, 6: dALE citri-85m2R and target 85R Comparison of specific and non-specific DNA binding activities of dTALE 209m2R, dTALE Eq-oryzaeLR-209m2R, dTALE EQ-209m2R, dTALE oryzae-EQLR-209m2R, dTALE citri-EQLR-209m2R, dTALE citri-209m2R . Lane 1: dTALE 209m2R and target 209R, 2: dTALE EQ-oryzaeLR-209m2R and target 209R, 3: dTALE EQ-209m2R and target 209R, 4: dTALE oryzae-EQLR-209m2R and target 209R, 5: dTALE-citri-citri-citri-citri-citri 209m2R and target 209R, 6: dTALE citri-209m2R and target 209R, 7: dTALE 209m2R and target 209R-1c, 8: dTALE EQ-oryzaeLR-209m2R and target 209R-1c, 9: dTALE EQ-209m2R and target 209R-1c , 10: dTALE eoryzae-EQLR-209m2R and target 209R-1c, 11: dTALE citri-EQLR-209m2R and target 209R-1c, 12: dTALE citri-209m2R and target 209R-1c, 13: dTALE 209m2R and target 209R-1n , 14: dTALE EQ-oryzaeLR-209m2R and target 209R-1n, 15: dTALE EQ-209m2R and target 209R-1n, 16: dTALE oryzae-EQLR-209m2R and target 209R-1n, 17: dTALE citri-EQLR-209m2R Target 209R-1n, 18: dTALE citri-209m2R and target 209R-1n Comparison of specific and non-specific DNA binding activities of dTALE 209m2R, dTALE Eq-oryzaeLR-209m2R, dTALE EQ-209m2R, dTALE oryzae-EQLR-209m2R, dTALE citri-EQLR-209m2R, dTALE citri-209m2R . Lane 1: dTALE 209m2R and target 209R-1m, 2: dTALE EQ-oryzaeLR-209m2R and target 209R-1m, 3: dTALE EQ-209m2R and target 209R-1m, 4: dTALE oryzae-EQLR-209m2R and target 209R-1m , 5: dTALE citri-EQLR-209m2R and target 209R-1m, 6: dTALE citri-209m2R and target 209R-1m, 7: dTALE 2209m2R and target 209R-2w, 8: dTALE EQ-oryzaeLR-209m2R and target 209R-2w , 9: dTALE EQ-209m2R and target 209R-2w, 10: dTALE oryzae-EQLR-209m2R and target 209R-2w, 11: dTALE citri-EQLR-209m2R and target 209R-2w, 12: -2dTALE citri-209m2R and target 209R -2w, 13: dTALE 209m2R and target 209R-2s, 14: dTALE EQ-oryzaeLR-209m2R and target 209R-2s, 15: dTALE EQ-209m2R and target 209R-2s, 16: dTALE oryzae-EQLR-209m2R and target 209R -2s, 17: dTALE citri-EQLR-209m2R and target 209R-2s, 18: dTALE citri-209m2R and target 209R-2s, 19: dTALE 209m2R and target 209R It is a figure which shows the comparison of the DNA binding activity of conventional NN and N type NN. Lane 1: dTALE TRE and target TRE, 2: dTALE TRRE and target TRE-G5, 4: dTALE TREn1 and target TRE-A5, 5: dTALE TREn1 and control target, 6: dTALE TREn2 Target TRE-G5, 7: dTALE TREn2 and target TRE-A5, 8: dTALE TREn2 and control target, 9: dTALE TREn3 and target TRE-GA3, 10: dTALE TREn3 and control target, 11: dTALE TREn4 and target TRE-GA3 , 12: dTALE TREn4 and control target It is a figure which shows the comparison of the DNA binding activity of conventional NN and N type NN. Lane 1: dTALE TRE and target TRE, 2: dTALE TRE and control target, 3: dTALE TREn5 and target TRE-G3A3-TRE, 4: dTALE TREn5 and control target, 5: dTALE TREn6 and target TRE-G3A3-TRE, 6 : DTALE TREn6 and control target

Hereinafter, the present invention will be described in detail.

(protein)
The present invention provides an isolated modified protein (hereinafter also referred to as the protein of the present invention) having binding activity to a target DNA.

Since the protein of the present invention can achieve sufficient improvement in binding activity for any target DNA sequence that has not been able to obtain sufficient binding activity in the past, the above-mentioned "target DNA" can be any single-stranded DNA sequence. It may be. The target DNA sequence is typically 6-36 bases in length, preferably 14-24 bases in length, more preferably 16-22 bases in length. If the length of the target DNA sequence is shorter than 6 bases, it may lead to a decrease in binding force or a decrease in binding specificity. On the other hand, when the length of the target DNA sequence is longer than 36 bases, the number of modules that the protein of the present invention should have increases, and as a result, the production of the protein can be inefficient or difficult.

When the protein of the present invention further contains an endonuclease moiety that functions by forming a dimer, as described later, the target DNA of the first protein of the present invention and the target of the second protein of the present invention Both DNAs meet the criteria for target DNA sequences as described above, and both target DNAs may be typically separated by 15-18 bp in the DNA desired to be cleaved. The endonuclease that formed the dimer can produce a double-strand break at the location between the two target DNAs.
As described later, when the protein of the present invention further contains a transcriptional activator or transcriptional repressor, the target DNA may be in the transcriptional regulatory region of the target gene.
On the other hand, as will be described later, when the protein of the present invention further contains an epigenome regulatory factor, the target DNA in the genome of the target cell, DNA methylation, post-translational modification of proteins such as histones, chromatin It may be located in or near the epigenomic region such as formation, formation of a nuclear structure.

Whether or not the protein of the present invention has the binding activity to the target DNA can be determined using the method described in the screening method described later. The protein of the present invention has a specific binding activity to a target DNA (that is, it binds to a target DNA with a significant binding force, but binds to a DNA containing a different sequence. Is significantly reduced or does not substantially bind). For example, the protein of the present invention has a binding affinity that is reduced by one or more orders of magnitude in the in vitro activity measurement method for a DNA sequence different from the target DNA, or significant in an intracellular activity measurement method such as a dTALE assay. That do not exhibit significant activity.

The protein of the present invention includes a Transcription Activator-Like (TAL) Effector part (hereinafter also referred to as a TALE part) derived from a genus Xanthomonas having a repetitive structure of a plurality of modules. The TALE portion usually has 2 or more, preferably 6 or more, more preferably 14 or more, and still more preferably 16 or more modules, and usually 36 or less, preferably 24 or less, more Preferably no more than 22, more preferably no more than 20, the above modules. Depending on the purpose of use of the protein of the present invention, if the number of modules is less than 12 (and therefore the number of bases in the target DNA sequence is 12), in general, a large number of the target DNA may exist in the genome. It is not preferable because of its properties. On the other hand, when the number of modules is more than 24, it is not preferable because not only production becomes difficult technically, but also the specific binding activity to DNA tends to decrease.

Each module has the following amino acid sequence:
LTPDQVVAIASNGGGKQALETVQRLLPVLCQDHG (SEQ ID NO: 1)
Or an amino acid sequence in which an amino acid mutation (deletion, substitution, addition) occurs in the amino acid sequence. The number of mutated amino acids is usually within 10 amino acids, preferably within 8 amino acids, more preferably within 6 amino acids, and even more preferably the number (4, 3, 2, or 1) amino acids. Each module preferably has an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NO: 1. The homology of the amino acid sequence was determined using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gap; matrix = BLOSUM62; filtering = OFF) Can be calculated.

In the amino acid sequence of SEQ ID NO: 1, the 12th and 13th bases constitute a target base recognition region that recognizes one base each constituting the base sequence of the target DNA, that is, repeat variable diresidue (RVD). It has been known. Specifically, in RVD, HD recognizes C, NG recognizes T, NI recognizes A, NN recognizes G or A, and NS recognizes A or C or G or T. , N * (where * represents a gap in the second position of RVD) recognizes C or T, HG recognizes T, and H * (where * is in the second position of RVD) (Represents a gap) recognizes T, IG recognizes T, NK recognizes G, HA recognizes C, ND recognizes C, HI recognizes C, HN recognizes G It is known that NA recognizes G, SN recognizes G or A, and YG recognizes T (for example, JP 2013-513389 A). Therefore, the RVD in each of the modules in the protein of the present invention is sequentially constructed so as to recognize each base in the base sequence of the target DNA, so that the protein of the present invention has the binding activity to the target DNA. be able to.

In the protein of the present invention, at least one of the plurality of modules (for example, 1, 2, 3, 4, 5 or more, 10 or more, 15 or more, 20 or more, 25 or more, 30 The above or all) are modified so that at least one amino acid residue selected from the group consisting of 2 to 6 and 30 to 34 has an artificial mutation in the amino acid sequence represented by SEQ ID NO: 1. Has been. As used herein, `` artificial mutation '' refers to a mutation introduced using a general point mutation introduction technique (e.g., Sawano et. Al. 2000, Nucleic Acids Research 28 (16), etc.) Does not include naturally occurring mutations (mutants and variants) based on species differences. As shown in the examples below, the 4th and 32nd amino acid residues are involved in binding between adjacent repeats (ie, modules) and are important for stabilizing the conformation of the TALE molecule. Plays an important role. Therefore, at least one (eg, one, two, three, four, or five) amino acid residues in the fourth and its vicinity (and hence 2-6) and / or the 32nd and its The module is designed to have an artificial mutation in at least one (eg, 1, 2, 3, 4 or 5) amino acid residues in the vicinity (thus 30-34). By artificially modifying the amino acid sequence, the binding strength of the protein of the present invention to the target DNA can be controlled. Among the binding activities with the target DNA, the binding strength and specificity are inherently contradictory properties, and it is desirable to adjust according to the application. This is made possible by the above-described artificial modification in the present invention and the module configuration of the protein (eg, hybrid of a high activity type module and a lower activity module). At least one (for example, one, two, three, four, or five) amino acids selected from the group consisting of 2 to 6 in order to more effectively increase the binding activity to the target DNA At least one amino acid residue selected from the group consisting of the residue and the 30th to 34th amino acid residues (for example, 1, 2, 3, 4 or 5) has an artificial mutation Preferably it is modified. The target to be mutated of the 2nd to 6th amino acid residues is preferably the 3rd to 6th amino acid residues, and more preferably the 4th amino acid residue. On the other hand, the target to be mutated among the 30th to 34th amino acid residues is preferably the 31st to 34th amino acid residues, and more preferably the 32nd amino acid residue. Therefore, as a particularly preferred embodiment, the 4th and 32nd amino acid residues are subjected to mutation in the module.

On the other hand, as shown in the examples described later, at the time of DNA binding, the 23rd glutamine in SEQ ID NO: 1 forms a hydrogen bond with the 33rd histidine of the adjacent repeat, and stabilization of the hydrogen bond The 32nd amino acid residue contributes. Therefore, the amino acid residue at the 23rd position and its vicinity (and hence the 20th to 26th positions) can also be a suitable target for controlling the binding activity to the target DNA. Of the 20th to 26th amino acid residues, the particularly preferred mutation target is the 24th amino acid residue. The number of mutations in the 20th to 26th amino acid residues is not particularly limited.

A suitable amino acid residue after mutation at each position described above can be appropriately determined by a person skilled in the art by a screening method described below based on a desired target of specific binding activity to a target DNA. As an example of a mutation that increases the binding activity, the fourth amino acid residue is modified to be glutamic acid and / or the 32nd amino acid residue is modified to be alanine. . More preferably, the fourth amino acid residue is modified to be glutamic acid and the 32nd amino acid residue is modified to be alanine. As another example of the mutation that increases the binding activity, the fourth amino acid residue is modified to be glutamine and / or the 32nd amino acid residue is modified to be alanine. It is. More preferably, the fourth amino acid residue is modified to be glutamic acid and the 32nd amino acid residue is modified to be alanine. Conversely, as an example of a mutation that reduces the binding activity, the fourth amino acid residue is aspartic acid, the 31st amino acid residue is glutamic acid, and the 32nd amino acid residue is glutamine. It has been done. In these embodiments, amino acid residues other than the 4th and / or 32nd positions may be further artificially modified or may not be modified. Here, the position of the amino acid residue means the position when the amino acid sequence of SEQ ID NO: 1 and the modified amino acid sequence are aligned. The alignment of amino acid sequences can be obtained using a general program such as BLAST2 Sequence or Align.

In all of the above modules, the mutations have the same pattern, that is, all the modules are subjected to the same mutation except for the RVD region (therefore, all the modules are other than the RVD region). Having the same amino acid sequence) is also preferable from the viewpoint of the production efficiency of the protein of the present invention.

Alternatively, since the protein of the present invention can exhibit high activity if a high activity module is contained in a certain ratio or more, the protein is a mixture of a high activity module and a lower activity module. There may be. Such a hybrid protein can significantly reduce nonspecific DNA binding activity while maintaining high specific DNA binding activity of the constructed TALE. As a lower activity module, the original module before introduction of an artificial mutation imparting higher activity according to the present invention, for example, one having the same sequence as the amino acid of SEQ ID NO: 1 except for the RVD region, etc. Is mentioned. As a highly active module, a module into which an artificial mutation imparting higher activity is introduced according to the present invention, for example, the amino acid residue at the 4th and / or 32nd has been modified as described above, etc. Is mentioned. The number of lower activity modules may be, for example, 1 to 10, preferably a number (for example, 1, 2, 3, 4 or 5), and the position is not particularly limited. It is a module.

The positions other than the above in the module, that is, positions other than the 2nd to 6th, 12th to 13th, 20th to 26th, and 30th to 34th are as long as the specific binding activity to the target DNA is not inhibited. It may have an artificial variation from one amino acid sequence. For example, by mutating serine (S), the 11th base in SEQ ID NO: 1, to asparagine (N), when RVD is NN, the binding activity to adenine is reduced without reducing the binding activity to guanine. Can be improved.

It is known that the TALE protein is composed of a repeat motif responsible for specific DNA binding and a DNA binding domain outside the repeat responsible for non-specific DNA binding. The TALE moiety in the proteins of the present invention may include such repeat outer DNA binding domains. In addition, as shown in Examples described later, the domain may have an artificial mutation. Alternatively or additionally, the TALE moiety has a structure such as a tag sequence for detection and purification, a nuclear localization sequence, a reporter sequence such as a fluorescent protein, or an intervening sequence that is cleaved by an endogenous enzyme in a cell. May be.

The protein of the present invention is the same as the TALE moiety in the protein of the present invention except that the protein of the present invention does not have the above-mentioned artificial mutation (eg, the above-described artificial modification is not performed). It is preferable that the binding activity to the target DNA is increased as compared with a protein containing a TALE moiety having an amino acid sequence. The increase in binding activity was measured by the method described in Example 1 described later (Mussolino et al., Nucleic Acids Res. 2011; 39 (21): 9283, Cong et. Al., Nat Commun. 2012 ; 3: 968), preferably at least 1.5 times, more preferably at least 2 times, even more preferably at least 5 times, and particularly preferably at least 10 times. Alternatively, the protein of the present invention may have a reduced binding activity to the target DNA as compared to the unmodified protein having no artificial mutation in the protein of the present invention. . The reduction in binding activity can be examined in the same manner as described above, for example, 2/3 or less, 1/2 or less, or 1/4 or less.

The protein of the present invention may further contain a functional domain not derived from a genus Xanthomonas from which the TAL Effector part is derived. The functional domain may be all or part of a nucleolytic enzyme or nucleotransferase in order to use the protein of the present invention in genome editing. The nucleolytic enzyme is preferably independent of the base sequence, and is exemplified by a cleavage domain derived from FokI. Also, use of base sequence-dependent nucleolytic enzyme domain, use of nucleolytic enzyme domain that causes DNA cleavage without forming a dimer, and also cuts only one strand without double strand break Functional domains such as nickase types have recently been reported and their use is also preferred. The functional domain also includes a transcriptional activator domain such as VP16, a transcriptional repressor domain such as KRAB, or a DNA methyltransferase or histone acetyltransferase in order to use the protein of the present invention in transcriptional activity regulation or epigenome control. Or an epigenome regulator domain such as histone deacetylase. Alternatively, the functional domain may also include all or part of a fluorescent protein, photoprotein, chromoprotein, or all or part of an enzyme protein that catalyzes a fluorescent, luminescent or chromogenic reaction. Here, the “part” means, for example, a part capable of exhibiting a target function by itself. These additional portions or domains are often bound to the C-terminal side of the above TALE portion, but may be on either the C-terminal side or the N-terminal side.

The protein of the present invention can be produced according to the production method of the present invention described later.

(Nucleic acid)
The present invention also provides an isolated nucleic acid (hereinafter also referred to as the nucleic acid of the present invention) encoding the amino acid sequence of the protein of the present invention.

The nucleic acid of the present invention is not particularly limited as long as it encodes the protein of the present invention. The nucleic acid may be DNA or RNA, or may be a DNA / RNA chimera. Preferably, DNA is used. The nucleic acid may be double-stranded or single-stranded. In the case of a double strand, it may be a double-stranded DNA, a double-stranded RNA or a DNA: RNA hybrid.

The nucleic acid of the present invention can typically be prepared using a commercially available kit. The kit is generally for producing TALEN, an artificial nuclease using TALE as a DNA binding domain, and the Golden Gate method (Cermak T, et al., Nucleic Acids Res. (2011) 39: e82) A combination of the Golden Gate method based on the Golden Gate TALEN and TAL Effector Kit and Yamamoto Lab TALEN Accessory Pack based on its modified method (Sakuma et al., Genes Cells 18 (4): 315-26, 2013) Various kits such as REALgeneAssembly TALEN Kit based on TALE Toolbox Kit, REAL method (Reyon D, et al., Curr. Protoc. Mol. Biol. (2012) Chapter 12: Unit 12.15) can be obtained from Addgene (For example, cell engineering, Vol. 32, No. 5 (2013): 510-514, etc.). These kits are convenient because conventional TALENs can be prepared simply by connecting modules corresponding to the target DNA sequence according to the manufacturer's manual. In order to produce a TALE-containing protein that does not contain a nuclease, the FokI functional domain may be simply removed from the vectors contained in these kits. Furthermore, general point mutation introduction techniques, for example, commercially available kits such as KOD-Plus- Mutagenesis Kit (Toyobo), PrimeSTAR Mutagenesis Basal Kit (Takara), QuikChange Multi Site-Directed Mutagenesis Kit (Agilent), or conventional methods such as PCR The above-mentioned artificial mutation in the protein of the present invention can be caused by introducing a mutation in the above-described kit vector as appropriate using a technique, or by total synthesis using an artificial gene synthesis technique.

(Expression vector, transformant and protein production method)
The present invention also provides an expression vector comprising the nucleic acid of the present invention (hereinafter also referred to as the expression vector of the present invention) and a transformant comprising the expression vector (hereinafter also referred to as the transformant of the present invention). The present invention further provides a method for producing the protein of the present invention using them (hereinafter also referred to as the production method of the present invention).

The expression vector of the present invention can be produced by operably linking the nucleic acid of the present invention downstream of a promoter in an appropriate expression vector. Examples of the vector include plasmid vectors and virus vectors, and can be appropriately selected depending on the host cell to be used.

Host cells include prokaryotic cells and eukaryotic cells. Examples of prokaryotic cells include Escherichia (Escherichia coli), Bacillus (Bacillus cilsubtilis), Agrobacterium (eg Rhizobium リ tumefacience, Rhizobium, etc.). rhizogenes) and the like. Examples of eukaryotic cells include yeasts (such as Saccharomyces cerevisiae), insect cells (such as larvae derived from night stealing larvae (Spodoptera frugiperda cells; Sf cells)), mammalian cells (human cells (293, etc.)). In addition to established cells such as monkey cells (COS-7, etc.), Chinese hamster cells (CHO cells, etc.), primary cultured cells (mouse fetal fibroblasts MEF, Primary cultured neurons), ES cells prepared from fertilized eggs, iPS cells established from the aforementioned primary cultured cells, plant cells, and the like are used. It is not limited to these.

Examples of mammals include laboratory animals such as rodents and rabbits such as mice, rats, hamsters and guinea pigs; domestic animals such as pigs, cows, goats, horses, sheep and minks; pets such as dogs and cats; Non-human primates (eg, monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, etc.). Examples of animals other than mammals include, but are not limited to, laboratory animals such as C. elegans, fish (zebrafish), and amphibians (Xenopus laevis, Nettle Xenopus). is not.
The plant cell is not particularly limited as long as it is a cell other than an animal cell, in other words, a cell having a cell wall, and angiosperm and gymnosperm (seed plant) including monocotyledonous and dicotyledonous plants, Any of moss plants, fern plants, herbaceous plants and woody plants may be used. Specific examples of plants include, for example, eggplant family [eggplant, tomato, pepper, pepper, tobacco, etc.], rice family [rice, wheat, barley, perennial ryegrass, italian ryegrass, meadow phesc, tall fescue, orchardgrass, timothy, etc.] Brassicaceae [Arabidopsis, Brassica, Chinese cabbage, cabbage, Japanese radish, rapeseed, etc.], Leguminosae [soybean, azuki bean, green beans, broad bean, etc.], Cucurbitaceae [cucumber, melon, watermelon, pumpkin, etc.], Convolvulaceae [sweet potato, etc.], Liliaceae [Leek, Onion, Leek, Garlic, Asparagus, etc.], Lamiaceae [Perilla, etc.], Asteraceae [Asteraceae, Chrysanthemum, Lettuce, etc.], Rosaceae [Roses, Strawberries, etc.], Citrus Family [Tangerines, Citrus Etc.], Myrtaceae [eucalyptus etc.], Willowaceae [poplar etc.], Akaza department [E Lenovo, sugar beet, etc.], gentianaceae [gentian, etc.], and a plant of the Caryophyllaceae [carnations, etc.]. In particular, solanaceous plants and cruciferous plants are preferable, and among them, tomato, tobacco, and Arabidopsis are preferably used.
Such plant cells include cells in plant bodies in addition to plant-derived cultured cells. Furthermore, plant cells derived from various forms of plants, such as suspension culture cells, protoplasts, leaf sections, callus, immature embryos, pollen and the like are included.

As plasmid vectors, plasmid vectors derived from E. coli (eg, pBR322, pBR325, pUC12, pUC13), plasmid vectors derived from Bacillus subtilis (eg, pUB110, pTP5, pC194), yeast-derived plasmid vectors (eg, pSH19, pSH15), Examples include plasmid vectors for eukaryotic cells (pCS2, pCMV, pcDNA, pCAGGS), binary vectors for plant cells, and the like, which can be appropriately selected according to the type of host used and the purpose of use.

The type of virus vector can be appropriately selected according to the type of host cell used and the purpose of use. For example, when insect cells are used as the host, baculovirus vectors can be used. When mammalian cells are used as hosts, Moloney murine leukemia virus vectors, lentivirus vectors, Sindbis virus vectors and other retrovirus vectors, adenovirus vectors, herpes virus vectors, adeno-associated virus vectors, parvovirus vectors, Vaccinia virus vectors, Sendai virus vectors, and the like can be used.

Also, a promoter that can initiate transcription in the host cell can be selected according to the type of host cell to be used. For example, when the host is Escherichia, trp promoter, lac promoter, T7 promoter and the like are preferable. When the host is Bacillus, SPO1 promoter, SPO2 promoter, penP promoter and the like are preferable. When the host is yeast, PHO5 promoter, PGK promoter, etc. are preferable. When the host is an insect cell, a polyhedrin promoter, a P10 promoter and the like are preferable. When the host is a mammalian cell, a subgenomic (26S) promoter, CMV promoter, SRα promoter, CAG promoter, EF1 promoter and the like are preferable.

The expression vector of the present invention may contain an enhancer, a splicing signal, a poly A addition signal, a selection marker, an SV40 replication origin and the like in a functional manner, if desired. Examples of the selection marker include dihydrofolate reductase gene [methotrexate (MTX) resistance], ampicillin resistance gene, neomycin resistance gene, kanamycin resistance gene, hygromycin resistance gene and the like.

The expression vector of the present invention is preferably isolated or purified.

Since the expression vector of the present invention can express the protein of the present invention in an appropriate host cell as described later, it is useful for producing the protein of the present invention, for example.

The above-described expression vector of the present invention is prepared according to a gene transfer method known per se (for example, lipofection method, calcium phosphate method, microinjection method, protoplast fusion method, electroporation method, DEAE dextran method, gene transfer method using Gene Gun). By introducing it into a host cell, a transformant into which the expression vector has been introduced (the transformant of the present invention) can be produced. The transformant can express the protein of the present invention. The transformant of the present invention is useful for producing the protein of the present invention.

The protein of the present invention can also be expressed in cells by synthesizing RNA from the above-described vector of the present invention by in vitro transcription reaction and introducing it into a fertilized egg or cell.

The protein of the present invention can be produced by culturing the transformant of the present invention by a method known per se according to the type of host and isolating the protein of the present invention from the culture. The transformant whose host is an Escherichia bacterium is cultured in an appropriate medium such as LB medium or M9 medium, usually at about 15 to 43 ° C. for about 3 to 24 hours. The transformant whose host is Bacillus is cultured in an appropriate medium, usually at about 30 to 40 ° C. for about 6 to 24 hours. Culturing of the transformant whose host is yeast is carried out in an appropriate medium such as a Birkholder medium, usually at about 20 ° C. to 35 ° C. for about 24 to 72 hours. Cultivation of transformants whose hosts are insect cells or insects is usually carried out in a suitable medium such as Grace's Insect medium supplemented with about 10% bovine serum, usually at about 27 ° C for about 3 to 5 days. The transformant whose host is an animal cell is cultured in an appropriate medium such as MEM medium supplemented with about 10% bovine serum, usually at about 30 ° C. to 40 ° C. for about 15 to 60 hours. In any culture, aeration and agitation may be performed as necessary. Isolation or purification of the protein of the present invention from the culture can be achieved, for example, by subjecting the cell lysate or culture supernatant to multiple chromatography such as reverse phase chromatography, ion exchange chromatography, affinity chromatography and the like. Can be achieved.

In addition, as for the protein (unmodified protein) used as the origin for manufacturing the protein of this invention according to this invention, the protein into which the above-mentioned artificial variation | mutation was introduce | transduced in the amino acid sequence is included by the protein of this invention There is no particular limitation as long as it can be a thing. Therefore, the unmodified protein includes a TALE portion derived from a genus Xanthomonas having a repetitive structure of a plurality of modules, and each of the plurality of modules recognizes one base constituting the base sequence of the target DNA. It may have the aforementioned target base recognition region. For example, each module of the TALE part in the unmodified protein has SEQ ID NO: 1 or an amino acid sequence in which an amino acid mutation (deletion, substitution, addition) has occurred in the amino acid sequence. The number of mutated amino acids is usually within 10 amino acids, preferably within 8 amino acids, more preferably within 6 amino acids, and even more preferably the number (4, 3, 2, or 1) amino acids. Each module preferably has an amino acid sequence having 80% or more homology with the amino acid sequence of SEQ ID NO: 1. Amino acid sequence homology is calculated using the homology calculation algorithm NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) under the following conditions (expected value = 10; allow gaps; matrix = BLOSUM62; filtering = OFF) Can be calculated. The TALE portion in the unmodified protein can have, for example, the same sequence as a given natural TALE except that the target base recognition region in each module is configured to recognize the target DNA. .

(Genome editing method)
The present invention also provides a method for cleaving, modifying or modifying a genome in a target gene or a region around the target gene in a target cell (hereinafter collectively referred to as the genome editing method of the present invention). As will be apparent from the description below, the method can be performed in vitro. The method includes binding the protein of the present invention to a target gene and / or a region around it, and for that purpose, introduces the protein of the present invention into a cell or encodes the amino acid sequence of the protein of the present invention. An expression vector containing RNA or a nucleic acid encoding the amino acid sequence of the protein of the present invention is introduced into the cell, and the protein of the present invention is expressed in the cell.

The target cell is not particularly limited, and examples thereof include the host cells described above for the transformant.

Introduction of the protein of the present invention into cells can be carried out using a method for introducing protein into cells known per se. Examples of such methods include a method using a protein introduction reagent, a method using a protein introduction domain (PTD) or a cell-penetrating peptide (CPP) fusion protein, an electroporation method, a microporation method, a microinjection method, Delivery by a bacterial type III secretion system. Protein introduction reagents include cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systmes), Pro-Ject ^™ Protein Transfection Reagent (PIERCE) and ProVectin (IMGENEX), and lipid-based Profect-1 (Targeting Systems) ), Penetrain Peptide (Q biogene) and Chariot Kit (Active Motif) based on membrane-permeable peptides, GenomONE (Ishihara Sangyo) using HVJ envelope (inactivated Sendai virus), and the like are commercially available. The introduction can be carried out according to the protocol attached to these reagents, but the general procedure is as follows. The protein of the present invention is diluted in an appropriate solvent (for example, a buffer solution such as PBS, HEPES, etc.), an introduction reagent is added, and the mixture is incubated at room temperature for about 5-15 minutes to form a complex. Add to the exchanged cells and incubate at 37 ° C for 1 to several hours. Thereafter, the medium is removed and replaced with a serum-containing medium. In order to introduce a protein into a plant cell, a protoplast can be produced from the cell according to a method known per se.

As PTD, Drosophila-derived AntP, HIV-derived TAT (Frankel, A. et al, Cell 55, 1189-93 (1988); Green, M. & Loewenstein, PM Cell 55, 1179-88 (1988)), Penetratin (Derossi, D. et al, J. Biol. Chem. 269, 10444-50 (1994)), Buforin II (Park, C. B. et al. Proc. Natl Acad. Sci. USA 97, 8245-50 (2000)), Transportan (Pooga, M. et al. FASEB J. 12, 67-77 (1998)), MAP (model amphipathic peptide) (Oehlke, J. et al. Biochim. Biophys. Acta. 1414, 127 -39 (1998)), K-FGF (Lin, Y. Z. et al. J. Biol. Chem. 270, 14255-14258 (1995)), Ku70 (Sawada, M. et al. Nature Cell Biol. 5 , 352-7 (2003)), Prion (Lundberg, P. et al. Biochem. Biophys Res. Commun. 299, 85-90 2002 (2002)), pVEC (Elmquist, A. et al. Exp. Cell Res. 269, 237-44 (2001)), Pep-1 (Morris, M. C. et al. Nature Biotechnol. 19, 1173-6 (2001)), Pep-7 (Gao, C. et al. Bioorg. Med . Chem. 10, 4057-65 ( 2002)), SynBl (Rousselle, C. et al. MoI. Pharmacol. 57, 679-86 (2000)), HN-I (Hong, F. D. & Clayman, G L. Cancer Res. 60, 6551- 6 (2000)), those using cell-passing domains of proteins such as VP22 derived from HSV have been developed. Examples of CPP derived from PTD include polyarginine such as 11R (Cell Stem Cell, 4: 381-384 (2009)) and 9R (Cell Stem Cell, 4: 472-476 (2009)).

A fusion protein expression vector incorporating the cDNA of the protein of the present invention and a PTD sequence or CPP sequence is prepared and recombinantly expressed, and the fusion protein is recovered and used for introduction. Introduction can be performed in the same manner as described above except that no protein introduction reagent is added.

Microinjection is a method in which a protein solution is put into a glass needle having a tip diameter of about 1 μm and puncture is introduced into a cell, and the protein can be reliably introduced into the cell.

In addition, electroporation method, semi-intact cell method (Kano, F. et al. Methods in Molecular Biology, Vol. 322, 357-365 (2006)), introduction method using Wr-T peptide (Kondo, E. et al. Protein introduction methods such as Mol. Cancer Ther. 3 (12), 1623-1630 (2004)) can also be used.

The protein introduction operation can be performed any number of one or more times (for example, 1 to 10 times or 1 to 5 times), and preferably the introduction operation is performed 2 or more times (for example, 3 or 4 times). ) Can be done repeatedly. Examples of the interval when the introduction operation is repeated include 6 to 48 hours, preferably 12 to 24 hours.

The introduction of RNA or expression vector into cells is as described above.

The introduction of the above-mentioned artificial nuclease or the introduction / expression of RNA encoding it or an expression vector into a target cell generates double strand breaks (DSBs) in the target DNA (target gene) in the cell. DSBs introduced into cells by artificial nucleases are repaired mainly by two DNA repair pathways: non-homologous end joining (NHEJ) and homology-directed repair (HDR) . In repairs with NHEJ, base deletions and insertions often occur and the gene may be disrupted by frameshifting. Therefore, it is preferably used particularly for destroying genes. On the other hand, since DSB is accurately repaired in HDR, base modification and gene insertion are possible by introducing a donor construct (a gene having a homologous sequence of the target sequence at both ends) together with an artificial nuclease (Joung JK, et al., Nat. Rev. Mol. Cell Biol. (2013) 14: 49-55). Introduction of the donor construct into the cell can be performed in the same manner as the introduction of the expression vector into the cell. Furthermore, it is possible to induce translocations by cutting large chromosomes or inversions by cutting the same chromosome with two pairs of artificial nucleases, or by cutting different chromosomes.

Whether the target gene has been deleted or modified can be confirmed, for example, using the HMA method, Cel-I assay, or the like, or by determining the base sequence of the target sequence.

Alternatively, for example, the region can be modified by binding the protein of the present invention containing the above-described epigenome regulatory factor to the region.

The cells obtained by editing the genome obtained by using the above method can be used for the production of mutant individuals or mutant strains of organisms from which the cells are derived.

(Screening method)
The present invention also provides a method (hereinafter also referred to as screening method I of the present invention) for screening a modified protein whose binding activity to a target DNA is changed (improved or reduced) as compared with an unmodified protein. . Therefore, the method also includes a method for producing a modified protein having altered (improved or reduced) compared to the protein (unmodified protein) whose binding activity to the target DNA is the original, or a TALE moiety. It is also a method for controlling the binding activity of a protein to a target DNA.

The screening method I of the present invention includes (1) a step of measuring the binding activity of the protein of the present invention (modified protein) to a target DNA, and (2) no artificial mutation in the modified protein. Measuring the binding activity of the unmodified protein to the target DNA. These steps are performed in vitro. Moreover, the order which performs these processes is not ask | required. It is preferable to examine not only the binding ability to target DNA (binding stability) but also the specificity of binding using non-target DNA sequences.
The above steps (1) and (2) can be performed, for example, by the following method. A TALE-derived artificial DNA binding domain containing a mutagenized or unmodified module is expressed as a protein in a test tube translation reaction or in E. coli, etc., and the binding affinity for target DNA sequences and non-target DNA sequences is determined by gel shift assay or Measure by biosensor method. Alternatively, create an expression vector or RNA by fusing a TALE-derived artificial DNA-binding domain containing a mutagenized or unmodified module with an enhancer activity domain such as VP16, and downstream of the target DNA sequence and non-target DNA sequence. It is introduced into a cell at the same time as a plasmid linked to a reporter expression unit such as luciferase, and measured by a dTALE assay to measure expression-inducing activity. See also Example 1 below.
The binding activities obtained in steps (1) and (2) above are compared with each other. When the binding activity obtained in the step (1) changes (improves or decreases) compared to the binding activity obtained in the step (2), the modified protein binds to the target DNA. Is selected as a modified protein that is altered compared to the unmodified protein. The comparison is preferably performed based on the presence or absence of a significant difference. The improvement of the binding activity is preferably at least 1.5 times, for example, at least 2 times when measured by the in vitro activity measurement method as described above or the method described in Example 1 (the above dTALE assay). More preferably, it is more preferably at least 5 times, and particularly preferably at least 10 times. The reduction of the binding activity is, for example, 2/3 or less, 1/2 or less, or 1/4 or less in the same measurement as described above.

The protein of the present invention to be subjected to screening can be produced according to the production method of the present invention described above based on the original protein (the above-mentioned unmodified protein). That is, the method includes a step of producing the protein of the present invention (modified protein) in which the above-described artificial mutation is introduced into the original unmodified protein before the step (1). The step can be performed, for example, by mutating a nucleic acid encoding an unmodified protein (that is, a mutation that causes the above-described artificial mutation that the protein of the present invention should have in the protein encoded by the mutated nucleic acid). To produce a nucleic acid encoding the protein of the present invention, obtain an expression vector containing the produced nucleic acid, introduce it into the host cell, obtain a transformant, and It can be performed by culturing the transformant and isolating the protein of the present invention from the culture. These procedures are described in detail herein.

The present invention also provides a method (hereinafter also referred to as screening method II of the present invention) for screening a modified protein having altered (improved or reduced) cleavage activity against the target DNA compared to the unmodified protein. . The modified protein is a protein of the present invention containing a nuclease moiety. Therefore, the method also includes a method for producing a modified protein having altered (improved or reduced) as compared with a protein (unmodified protein) having a cleavage activity against a target DNA, or a TALE moiety and a nuclease. It is also a method for controlling the cleavage activity of a target-containing protein against a target DNA.

The screening method II of the present invention comprises (1) a step of measuring the cleavage activity of the protein of the present invention (modified protein) against the target DNA, and (2) no artificial mutation in the modified protein. Measuring the cleavage activity of the unmodified protein against the target DNA. These steps are performed in vitro. Moreover, the order which performs these processes is not ask | required. It is preferable to examine not only the cleavage activity against the target DNA but also the specificity of the cleavage using non-target DNA sequences.
The above steps (1) and (2) are carried out by measuring target DNA and non-target DNA cleavage activity in a test tube using TALEN, or measuring target DNA cleavage activity in a cell or an individual ( SSA method, HMA method, etc.) can be used. See also Examples 2 and 3 below.
The cleavage activities measured in the above steps (1) and (2) are compared with each other. When the cleavage activity obtained in the step (1) changes (improves or decreases) compared to the cleavage activity obtained in the step (2), the modified protein is cleaved against the target DNA. Is selected as a modified protein that is altered compared to the unmodified protein. The comparison is preferably performed based on the presence or absence of a significant difference. By measuring the target DNA and non-target DNA cleavage activity in a test tube using purified protein, non-target DNA cleavage is not observed, and the cleavage activity per unit time and amount of protein of the target DNA is unmodified protein. Significantly higher, preferably at least 1.5 times, more preferably at least 2 times, even more preferably at least 5 times, and particularly preferably at least 10 times.

The protein of the present invention to be used for screening can be produced according to the production method of the present invention described above based on the original protein having the nuclease moiety (the above-mentioned unmodified protein). That is, the method includes a step of producing the protein of the present invention (modified protein) in which the above-described artificial mutation is introduced into the original unmodified protein before the step (1). The step can be performed, for example, by mutating a nucleic acid encoding an unmodified protein (that is, a mutation that causes the above-described artificial mutation that the protein of the present invention should have in the protein encoded by the mutated nucleic acid). To produce a nucleic acid encoding the protein of the present invention, obtain an expression vector containing the produced nucleic acid, introduce it into the host cell, obtain a transformant, and It can be performed by culturing the transformant and isolating the protein of the present invention from the culture. These procedures are described in detail herein.

Hereinafter, although an example etc. are shown and the present invention is explained in detail, the present invention is not limited by the following examples etc.

1. Analyzes and structural studies of repeat sequences of natural TALE

The repeat sequences of about 1000 TALE DNA binding domains possessed by various bacteria of the genus Xanthomonas were analyzed. Sequence information was obtained from PubMed. FIG. 1 shows the results of an analysis of the conservation of amino acid residues in the repeat sequence of the examined natural TALE. In the figure, gray portions indicate conserved amino acid residues. As shown in FIG. 1, it was found that not only the 12th and 13th so-called RVDs (repeatesvariable diresidue) but also the 4th and 32nd amino acids had low conservation. The 12th and 13th RVD residues have already been shown to be involved in the specificity of DNA base recognition, and the results of X-ray structural analysis support this. However, the low conservation of amino acid residues at the 4th and 32nd amino acids was not noticed and was not considered to affect the activity of TALE (eg, Streubel et al., Nature Biotechnology 30: 593- 595, 2012).

When analyzing the TALE sequence of each Xanthomonas genus in more detail, in the orizae type, aspartic acid (D) has the highest frequency as the fourth amino acid residue, and aspartic acid (D) as the 32nd amino acid residue. Was the most frequent. Tale of Golden Gate method and its modification method by Voytas et al., Which has been widely used in the past, is a combination of aspartic acid (D) 4th and aspartic acid (D) 32nd (hereinafter referred to as DD type or conventional type) However, in natural TALE, DD repeats are rare in orizae type, and other types of Xanthomonas such as citri type have different combinations such as EA type and DQ type. It was found that the repeats were continuously observed (Figure 2-4).

Subsequently, the molecular structure of TALE was examined. As shown in FIG. 5, the 4th and 32nd residues are involved in binding between adjacent repeats, suggesting that they play an important role in stabilizing the conformation of the TALE molecule.

Furthermore, the structure of TALE when DNA was not bound and bound was reanalyzed in detail. As shown in FIG. 6, in the EA type, the 4th glutamic acid (E) forms a hydrogen bond with the 5th glutamine (Q) of the adjacent repeat, and the 32nd alanine (A) is the 33rd histidine ( It was suggested that H) stabilized the hydrogen bond formed with the 23rd glutamine (Q) of the adjacent repeat. These hydrogen bonds are structures that can be seen only during DNA binding, and are thought to contribute to the stabilization of the three-dimensional structure when bound along the major groove of DNA.
On the other hand, from this structure, the DA type and DD type are expected to have a more unstable TALE structure than the EA type. In the DA type, the 4th aspartic acid (D) has a shorter side chain than the glutamic acid (E), so hydrogen bonds do not reach.In the DD type, the 32nd aspartic acid (D) is hydrogen bonded to the 33rd histidine (H). This is because the hydrogen bond with the 23rd glutamine (Q) of the adjacent repeat is destabilized.

The above analysis strongly suggests that natural TALE regulates the stability of the three-dimensional structure of TALE according to its physiological function by providing diversity at the 4th and 32nd amino acid residues. It was. On the contrary, it is expected that the activity of TALE can be artificially changed by adding mutation to the 4th and 32nd amino acid residues. Therefore, experiments were carried out to introduce mutations at the 4th and 32nd amino acid residues based on the construction method of Golden Gate TALEN by Voytas et al. And the modification method by Sakuma et al. As a result, as shown below, a highly active TALE protein with improved specific binding ability to the target sequence was successfully constructed.

Example 1: Modification by changing the fourth amino acid of each repeat module of the TALE-DNA binding motif from aspartic acid (D) to glutamic acid (E) and the 32nd amino acid from aspartic acid (D) to alanine (A) Type TALE (EA type), modified TALE (EQ type) in which the 31st amino acid was changed from glutamine (Q) to glutamic acid (E) and the 32nd amino acid was changed from aspartic acid (D) to glutamine (Q) Comparison of binding activity to specific DNA sequences of conventional TALE and modified TALE by reporter assay using cultured cells

The FokI functional domain was removed from the C terminus of pCS2TAL3-DD (Dahlem et al (PLoS Genet. 2012 Aug; 8 (8)) and VP16 transcription activation domain x3 (VP48) was inserted (BamHI / XbaI) pCS2-TALE -VP48 vector was constructed, and the sequence of the vector is shown in SEQ ID NO: 29. In addition, each vector (pFUS_A2A, pFUS_A2B, pGUS_A2A, pFUS_A2B, etc.) used in the modified Golden gate method (Sakuma et. Al. 2013 Genes to Cells. 18 (4) p315) pFUS_B1, pFUS_B2, pFUS_B3, pFUS_B4, pFUS_B5, pFUS_B6, pHD1, pHD2, pHD3, pHD4, pNN3, NIp4 The following mutations were introduced into pNG2, pNG3, pNG4, pNG5, and pNG6) by the usual point mutation introduction technique using primers (reference: Sawano et. al. 2000, Nucleic Acids Research 28 (16), etc.). Specifically, PCR was carried out for 18 cycles using the following phosphorylated primers in the presence of Taq DNA ligase using KOD Plus Neo. After decomposing the mutation not transfected plasmid by management, to recover the transformed plasmids into E. coli.

(Mutation example 1) In each repeat module of the TALE-DNA binding motif, the codon corresponding to the fourth amino acid residue is changed from GAC (aspartic acid (D)) to GAG (glutamic acid (E)), and the 32nd amino acid. The codons corresponding to the residues were all changed from GAC (aspartic acid (D)) to GCC (alanine (A)).

Primer used for mutation introduction of the 4th amino acid (amino acid was changed from aspartic acid (D) to glutamic acid (E))
CCTGACCCCGGAGCAAGTGGTGGCTAT (4DE-1F) (SEQ ID NO: 2)
CCTGACTCCGGAGCAAGTGGTGGCTAT (4DE-2F) (SEQ ID NO: 3)

Plasmid with mutations introduced by these primers
(4DE-1F): pFUS_A2A, pFUS_A2B, pFUS_B1-6, pNN2-6, pNG2-6, pNI2-6
(4DE-2F): pHD2-6

Primer used for mutagenesis of the 32nd amino acid (modified from aspartic acid (D) to alanine (A))
GTGCTGTGCCAGGCCCATGGCCTGAC (32DA-1F) (SEQ ID NO: 4)
GGTCTCAGGCCCATGGCCTGAC (32DA-2F) (SEQ ID NO: 5)
GGTCTCGCCAGGCCCATGGCCTGAC (32DA-3F) (SEQ ID NO: 6)
GTGCTGTGCCAGGCCCATGGGAGACCC (32DA-4F) (SEQ ID NO: 7)
GTGCTGTGCCAGGCCCATGGCCGAGAC (32DA-5F) (SEQ ID NO: 8)
GTGCTGTGCCAGGCCCGAGACCCTCG (32DA-6F) (SEQ ID NO: 9)

Plasmid with mutations introduced by these primers
(32DA-1F): pHD5-6, pNN5-6, pNI5-6, pNG5-6, pFUS_B4-6, pFUS_A2A
(32DA-2F): pHD3, pNN3, pNI3, pNG3, pFUS_B2
(32DA-3F): pHD4, pNN4, pNI4, pNG4, pFUS_B3
(32DA-4F): pHD1, pNN1, pNI1, pNG1
(32DA-5F): pFUS_A2B
(32DA-6F): pHD2, pNN2, pNI2, pNG2

(Mutation example 2) In each repeat module of the TALE-DNA binding motif, the codon corresponding to the 31st amino acid residue is changed from CAG (glutamine (Q)) to GAG (glutamic acid (E)), and the 32nd amino acid residue All codons corresponding to the group were changed from GAC (aspartic acid (D)) to CAG (glutamine (Q)).

Primers used for mutagenesis
GTGCTGTGCGAGCAGCATGGCCTGAC (31E32Q-1F) (SEQ ID NO: 10)
GGTCTCAGCAGCATGGCCTGAC (31E32Q-2F) (SEQ ID NO: 11)
GGTCTCGCGAGCAGCATGGCCTGAC (31E32Q-3F) (SEQ ID NO: 12)
GTGCTGTGCGAGCAGCATGGGAGACCC (31E32Q-4F) (SEQ ID NO: 13)
GTGCTGTGCGAGCAGCATGGCCGAGAC (31E32Q-5F) (SEQ ID NO: 14)
GTGCTGTGCGAGCAGCGAGACCCTCG (31E32Q-6F) (SEQ ID NO: 15)
GGTGCTGTGCGAGGGAGACCC (31E32Q-7F) (SEQ ID NO: 16)

Plasmid with mutations introduced by these primers
(31E32Q-1F): pHD5-6, pNN5-6, pNI5-6, pNG5-6, pFUS_B4-6, pFUS_A2A
(31E32Q-2F): pHD3, pNN3, pNI3, pNG3, pFUS_B2
(31E32Q-3F): pHD4, pNN4, pNI4, pNG4, pFUS_B3
(31E32Q-4F): pHD1, pNN1, pNI1, pNG1
(31E32Q-5F): pFUS_A2B
(31E32Q-6F): pHD2, pNN2, pNI2, pNG2
(31E32Q-7F): pHD3, pNN3, pNI3, pNG3

Based on the modified Golden gate method (Sakuma et. Al. 2013 Genes to Cells. 18 (4) p315) using the conventional plasmid and the plasmid mutated by the above method, pCS2-TALE-VP48 The following dTALE vector was constructed using the vector.

The following sequence shows the RVDs that make up TALE.

85F: NN NG HD NN NG HD HD NI HD NG HD NI NI NG (conventional)
85mF: NN NG HD NN NG HD HD NI HD NG HD NI NI NG (modified (EA))
85R: NN HD NG NG NN NG NN NG HD HD HD NI NG NG (conventional)
85mR: NN HD NG NG NN NG NN NG HD HD HD NI NG NG (modified (EA))
209F: HD NI HD HD NG NG HD NN NI HD NI HD NI NN NG NN (conventional)
209mF: HD NI HD HD NG NG HD NN NI HD NI HD NI NN NG NN (modified (EA))
209QF: HD NI HD HD NG NG HD NN NI HD NI HD NI NN NG NN (modified (EQ))
209R: NI HD NI HD NI NG HD HD NI NN HD NG NN NG NG (conventional)
209mR: NI HD NI HD NI NG HD HD NI NN HD NG NN NG NG (modified (EA))
209QR: NI HD NI HD NI NG HD HD NI NN HD NG NN NG NG (modified (EQ))
209m-QR: NI HD NI HD NI NG HD HD NI NN HD NG NN NG NG (Composite modified type (EA-EQ type)); 1-6th repeat module is EA type, 7th and later are EQ type Composite modified type
209Q-mR: NI HD NI HD NI NG HD HD NI NN HD NG NN NG NG (Composite modified type (EA-EQ type)); 1-6th repeat module is configured as EQ type, 7th and later are configured as EA type Composite modified type

A reporter vector was constructed in which the target sequence x2 of each TALE was inserted into the pCS2 vector, and a minimal CMV promoter and FLuc were inserted downstream thereof. The sequence of the reporter vector pCS2-85Fx2-mCMV-FLuc when the target sequence is 85F is shown in SEQ ID NO: 28.
These and the phRL-TK vector were co-expressed in MDCK2 cultured cells, and the DNA binding activity of each TALE sequence was quantified as the amount of luminescence of FLuc. Each signal was normalized by the amount of luminescence of Renilla luciferase. (Reference: Mussolino et. Al. Nucleic Acids Res. 2011; 39 (21): 9283., Cong et. Al. Nat Commun. 2012; 3: 968)
Target sequence
85F: (T) GTCGTCCACTCAAT (SEQ ID NO: 17)
85R: (T) GCTTGTGTCCCATT (SEQ ID NO: 18)
209F: (T) CACCTTCGACACAGTG (SEQ ID NO: 19)
209R: (T) ACACATCCAGCTGTT (SEQ ID NO: 20)
Control: (T) CCCTATCAGTGATAGAGA (SEQ ID NO: 21)

The results are shown in FIGS. In the above experiment for measuring the binding activity between dTALE and DNA, modification of the EA type TALE sequence resulted in an increase in binding activity to a specific DNA sequence up to about 2.5 times (comparison between lanes 1 and 3). On the other hand, there was no significant difference in the binding activity to nonspecific DNA sequences (comparison between lanes 2 and 4). In addition, the binding activity to specific DNA sequences of EQ type TALE decreased compared to the conventional type (comparison between

lanes

1 and 5 in FIGS. 9 and 10). The combined TALE of the EA type and the EQ type showed a binding activity to a DNA sequence in the middle of the EA type and the EQ type.
From the above results, it was found that the binding activity to the target DNA sequence can be controlled by introducing a mutation into a specific site of the TALE sequence in this example.

Example 2: Modification of the sequence of each DNA-binding repeat (Modification of the 4th amino acid from aspartic acid (D) to glutamic acid (E) and the 32nd amino acid from aspartic acid (D) to alanine (A)) Comparison of activity of conventional TALEN and modified TALEN

Conventional TALEN vectors (pCS2TAL3-DD and pCS2TAL3-RR) that recognize and cleave the following specific genomic sequences of zebrafish and modified TALEN vectors were constructed. These vectors produced TALEN composed of conventional TALE repeats and TALEN composed of modified TALE repeats in accordance with the Golden-GATE TALEN construction method. For the production of modified TALEN, each pFUS vector and RVD vector into which mutations were introduced in Example 1 were used. The difference from Example 1 in constructing the vector was that unmodified pCS2TAL3-DD and pCS2TAL3-RR that contained the FokI functional domain were used as the backbone.
That is, TALEN vectors pCS2TAL3-DD and pCS2TAL3-RR are used to construct TALEN composed of conventional TALE repeats and TALEN composed of modified TALE repeats corresponding to the following specific genomic sequences of zebrafish: did.
Subsequently, each RNA was synthesized using these vectors and mMESSAGE mMACHINE (SP6). About 400pg each of reverse and forward TALEN pair RNA was injected into the early embryo of zebrafish. After 24 hours, the embryos were collected, and the activity of TALEN was evaluated by heteroduplex mobility assay (HMA), and the conventional type and the modified type were compared. (Ota et. Al. Genes to Cells. 2013; 18 (6): 450)
TALEN1: 209 (Kif3A)
For. TALEN: HD NI HD HD NG NG HD NN NI HD NI HD NI NN NG NN
Rev. TALEN: NI HD NI HD NI NG HD HD NI NN HD NG NN NG NG
Target genome sequence T CACCTTCGACACAGTG ttcggcccagacagta AACAGCTGGATGTGT A (SEQ ID NO: 22)
Primer pair used for HMA For .: TGATATAAGCAGTGAGCCTCCA (SEQ ID NO: 23)
Rev .: CAGAGTCTATGATGGGTCGTG (SEQ ID NO: 24)

TALEN2: S1PR3a-1
For. TALEN: NI HD NI NI HD HD NI HD NG HD NI NN NN HD NI
Rev. TALEN: NI HD NI HD NI NG NI HD HD NI NN NG NN HD NG NG HD
Target genome sequence T ACAACCACTCAGGCA agtggggaagaccaa GAAGCACTGGTATGTGT A (SEQ ID NO: 25)
Primer pair used for HMA For: GGAATGAATGATGTTATAGTTAGCC (SEQ ID NO: 26)
Rev: GTAATGTTCTCAATCACGATCAGAG (SEQ ID NO: 27)

The results are shown in FIG. In the above two cases, TALEN activity was significantly increased by modification of the TALE sequence.

Example 3: Measurement and comparison of specific DNA cleavage activity of expressed and purified conventional and modified TALEN proteins in vitro

Conventional and modified types having the following RVD sequences (in each DNA binding repeat, the fourth amino acid is from aspartic acid (D) to glutamic acid (E), and the 32nd amino acid is from aspartic acid (D) to alanine (A) TALEN (pCS2-TAF), which has been modified to), was subcloned into pET21b, expressed in the BL21 strain, and purified using cobalt resin. The purified TALEN protein was reacted with an approximately 1400 bp DNA fragment containing the target DNA sequence under the following conditions, and the cleavage activity was measured.
TALEN protein: 209 (Kif3A)
For .: HD NI HD HD NG NG HD NN NI HD NI HD NI NN NG NN
Rev.: NI HD NI HD NI NG HD HD NI NN HD NG NN NG NG
Reaction conditions: 20 mM Tris-acetate, 10 mM magnesium acetate, 50 mM potassium acetate, 1 mM DTT, 100 μg / ml BSA (bovine serum albumin), 37 ° C
TALEN concentration 27nM, DNA concentration 27nM (However, Lanes 1-3 are 13.5nM, 27nM, 54nM)

The results are shown in Figs. In the above two cases, it was confirmed that the cleavage activity of the modified TALEN was increased compared to the conventional TALEN.

Example 4: Production of further modified TALE and comparison of binding activity to specific DNA sequences of conventional TALE and modified TALE

Based on the above results, further studies were made, a new modified TALE was prepared, and the binding activity to the target DNA sequence was analyzed. Among the newly created modified TALE, in particular, the QA type in which the 4th amino acid residue of all repeat modules is modified to glutamine (Q) and the 32nd amino acid residue is alanine (A), and partly This includes the conventional type of repeats only, and a mixture of 2 to 4 different types. QA-type repeats are found in natural TALEs such as campestris and citri. This analysis demonstrates that QA type is also as highly active as EA type, and that it exhibits high activity if all modules contain a high activity type at a certain rate.
A new modified TALE was produced in the same manner as in Example 1. More specifically, it is as follows.

1. Mutation Introduction into Golden Gate Plasmid Vector Mutation introduction into the Golden gate plasmid vector was performed using the same method as in Example 1. The introduced mutations and the primers and plasmids used for the mutations are shown below.

(Mutation example 4-1) 4A mutagenesis
Primer used for mutation introduction of the 4th amino acid (amino acid is changed from aspartic acid (D) to alanine (A))
CCTGACCCCGGCCCAAGTGGTGGCTAT (4DA-1F) (SEQ ID NO: 30)
CCTGACTCCGGCCCAAGTGGTGGCTAT (4DA-2F) (SEQ ID NO: 31)

Plasmid with mutations introduced by these primers
(4DA-1F): pFUS_A2A, pFUS_A2B, pFUS_B1-6, pNN2-6, pNG2-6, pNI2-6
(4DA-2F): pHD2-6

(Mutation example 4-2) 4Q32A mutation introduction
Primer used for mutation introduction of the 4th amino acid (amino acid was changed from aspartic acid (D) to glutamine (Q))
CCTGACCCCGCAACAAGTGGTGGCTAT (4DQ-1F) (SEQ ID NO: 32)
CCTGACTCCGCAACAAGTGGTGGCTAT (4DQ-2F) (SEQ ID NO: 33)

Plasmid with mutations introduced by these primers
(4DQ-1F): pFUS_A2A, pFUS_A2B, pFUS_B1-6, pNN2-6, pNG2-6, pNI2-6
(4DQ-2F): pHD2-6

Primer used for mutagenesis of the 32nd amino acid (modified from aspartic acid (D) to alanine (A))
GTGCTGTGCCAGGCCCATGGCCTGAC (32DA-1F) (SEQ ID NO: 4)
GGTCTCAGGCCCATGGCCTGAC (32DA-2F) (SEQ ID NO: 5)
GGTCTCGCCAGGCCCATGGCCTGAC (32DA-3F) (SEQ ID NO: 6)
GTGCTGTGCCAGGCCCATGGGAGACCC (32DA-4F) (SEQ ID NO: 7)
GTGCTGTGCCAGGCCCATGGCCGAGAC (32DA-5F) (SEQ ID NO: 8)
GTGCTGTGCCAGGCCCGAGACCCTCG (32DA-6F) (SEQ ID NO: 9)

Plasmid with mutations introduced by these primers
(32DA-1F): pHD5-6, pNN5-6, pNI5-6, pNG5-6, pFUS_B4-6, pFUS_A2A
(32DA-2F): pHD3, pNN3, pNI3, pNG3, pFUS_B2
(32DA-3F): pHD4, pNN4, pNI4, pNG4, pFUS_B3
(32DA-4F): pHD1, pNN1, pNI1, pNG1
(32DA-5F): pFUS_A2B
(32DA-6F): pHD2, pNN2, pNI2, pNG2

2. Construction of dTALE The construction of the dTALE vector was also performed in the same manner as in Example 1. The configuration of the dTALE repeat module used for this analysis is shown below. Each symbol used in that case has the following sequence.
DD: Conventional type (4th D, 32nd D)
4E: 4th E, others are conventional
4A: 4th A, others are conventional
EA: 4th E, 32nd A, others are conventional
QA: 4th Q, 32nd A, others are conventional
EQ: 31st E, 32nd Q, etc.
DA: 32nd A, others are conventional
4D: Conventional (4th D (the last half repeat does not include the 32nd amino acid residue))
The parentheses indicate the RVDs that make up the TALE.

85F: DD (NN) DD (NG) DD (HD) DD (NN) DD (NG) DD (HD) DD (HD) DD (NI) DD (HD) DD (NG) DD (HD) DD (NI) DD (NI) 4D (NG) (Conventional)
85mF: EA (NN) EA (NG) EA (HD) EA (NN) EA (NG) EA (HD) EA (HD) EA (NI) EA (HD) EA (NG) EA (HD) EA (NI) EA (NI) 4E (NG) (modified (EA))
85pmF: EA (NN) EA (NG) EA (HD) EA (NN) EA (NG) EA (HD) EA (HD) EA (NI) EA (HD) EA (NG) EA (HD) EA (NI) DA (NI) 4D (NG) (modified (EA))
85m2F: QA (NN) QA (NG) QA (HD) QA (NN) QA (NG) QA (HD) QA (HD) QA (NI) QA (HD) QA (NG) QA (HD) QA (NI) QA (NI) 4Q (NG) (Modified type (QA type))
85R: DD (NN) DD (HD) DD (NG) DD (NG) DD (NN) DD (NG) DD (NN) DD (NG) DD (HD) DD (HD) DD (HD) DD (NI) DD (NG) 4D (NG) (Conventional)
85mR: EA (NN) EA (HD) EA (NG) EA (NG) EA (NN) EA (NG) EA (NN) EA (NG) EA (HD) EA (HD) EA (HD) EA (NI) EA (NG) 4E (NG) (Modified type (EA type))
85pmR: EA (NN) EA (HD) EA (NG) EA (NG) EA (NN) EA (NG) EA (NN) EA (NG) EA (HD) EA (HD) EA (HD) EA (NI) DA (NG) 4D (NG) (modified (EA))
85m2R: QA (NN) QA (HD) QA (NG) QA (NG) QA (NN) QA (NG) QA (NN) QA (NG) QA (HD) QA (HD) QA (HD) QA (NI) QA (NG) 4Q (NG) (Modified type (QA type))
209F: DD (HD) DD (NI) DD (HD) DD (HD) DD (NG) DD (NG) DD (HD) DD (NN) DD (NI) DD (HD) DD (NI) DD (HD) DD (NI) DD (NN) DD (NG) 4D (NN) (Conventional)
209mF: EA (HD) EA (NI) EA (HD) EA (HD) EA (NG) EA (NG) EA (HD) EA (NN) EA (NI) EA (HD) EA (NI) EA (HD) EA (NI) EA (NN) EA (NG) 4E (NN) (Modified type (EA type))
209m2F: QA (HD) QA (NI) QA (HD) QA (HD) QA (NG) QA (NG) QA (HD) QA (NN) QA (NI) QA (HD) QA (NI) QA (HD) QA (NI) QA (NN) QA (NG) 4Q (NN) (Modified type (QA type))
S1PR3F: DD (NI) DD (HD) DD (NI) DD (NI) DD (HD) DD (HD) DD (NI) DD (HD) DD (NG) DD (HD) DD (NI) DD (NN) DD (NN) DD (HD) 4D (NI) (conventional)
S1PR3mF: EA (NI) EA (HD) EA (NI) EA (NI) EA (HD) EA (HD) EA (NI) EA (HD) EA (NG) EA (HD) EA (NI) EA (NN) EA (NN) EA (HD) 4E (NI) (Modified type (EA type))
S1PR3m2F: QA (NI) QA (HD) QA (NI) QA (NI) QA (HD) QA (HD) QA (NI) QA (HD) QA (NG) QA (HD) QA (NI) QA (NN) QA (NN) QA (HD) 4Q (NI) (Modified type (QA type))
S1PR3R: DD (NI) DD (HD) DD (NI) DD (HD) DD (NI) DD (NG) DD (NI) DD (HD) DD (HD) DD (NI) DD (NN) DD (NG) DD (NN) DD (HD) DD (NG) DD (NG) 4D (HD) (Conventional)
S1PR3mR: EA (NI) EA (HD) EA (NI) EA (HD) EA (NI) EA (NG) EA (NI) EA (HD) EA (HD) EA (NI) EA (NN) EA (NG) EA (NN) EA (HD) EA (NG) EA (NG) 4E (HD) (Modified type (EA type))
S1PR3m2R: QA (NI) QA (HD) QA (NI) QA (HD) QA (NI) QA (NG) QA (NI) QA (HD) QA (HD) QA (NI) QA (NN) QA (NG) QA (NN) QA (HD) QA (NG) QA (NG) 4Q (HD) (modified (QA))
SphkF: DD (HD) DD (NI) DD (NN) DD (NI) DD (NI) DD (NG) DD (HD) DD (NI) DD (HD) DD (NN) DD (HD) DD (HD) DD (HD) DD (NN) DD (NI) 4D (NN) (conventional)
SphkmF: EA (HD) EA (NI) EA (NN) EA (NI) EA (NI) EA (NG) EA (HD) EA (NI) EA (HD) EA (NN) EA (HD) EA (HD) EA (HD) EA (NN) EA (NI) 4E (NN) (Modified type (EA type))
SphkpmF: EA (HD) EA (NI) EA (NN) EA (NI) EA (NI) EA (NG) EA (HD) EA (NI) EA (HD) EA (NN) EA (HD) EA (HD) DA (HD) EA (NN) EA (NI) 4D (NN) (modified (incomplete EA))
Sphkm2F: QA (HD) QA (NI) QA (NN) QA (NI) QA (NI) QA (NG) QA (HD) QA (NI) QA (HD) QA (NN) QA (HD) QA (HD) QA (HD) QA (NN) QA (NI) 4Q (NN) (Modified type (QA type))
SphkR: DD (HD) DD (HD) DD (HD) DD (NI) DD (HD) DD (NG) DD (NN) DD (HD) DD (NN) DD (NI) DD (HD) DD (NI) DD (NN) DD (NN) DD (NG) DD (HD) DD (NG) 4D (NN) (Conventional)
SphkmR: EA (HD) EA (HD) EA (HD) EA (NI) EA (HD) EA (NG) EA (NN) EA (HD) EA (NN) EA (NI) EA (HD) EA (NI) EA (NN) EA (NN) EA (NG) EA (HD) EA (NG) 4E (NN) (modified type (EA type))
Sphkm2R: QA (HD) QA (HD) QA (HD) QA (NI) QA (HD) QA (NG) QA (NN) QA (HD) QA (NN) QA (NI) QA (HD) QA (NI) QA (NN) QA (NN) QA (NG) QA (HD) QA (NG) 4Q (NN) (modified type (QA type))
CG16798F: DD (NN) DD (NN) DD (HD) DD (HD) DD (NG) DD (NN) DD (NG) DD (HD) DD (NI) DD (NN) DD (HD) DD (NN) DD (NI) DD (HD) DD (NI) 4D (NI) (conventional)
CG16798mF: EA (NN) EA (NN) EA (HD) EA (HD) EA (NG) EA (NN) EA (NG) EA (HD) EA (NI) EA (NN) EA (HD) EA (NN) EA (NI) EA (HD) EA (NI) 4E (NI) (Modified type (EA type))
CG16798m2F: QA (NN) QA (NN) QA (HD) QA (HD) QA (NG) QA (NN) QA (NG) QA (HD) QA (NI) QA (NN) QA (HD) QA (NN) QA (NI) QA (HD) QA (NI) 4Q (NI) (Modified type (QA type))
CG16798R: DD (NG) DD (NG) DD (HD) DD (NI) DD (HD) DD (NI) DD (NI) DD (NN) DD (NG) DD (NG) DD (NG) DD (NI) DD (NG) DD (NN) DD (NN) DD (NN) DD (HD) 4D (NI) (Conventional)
CG16798mR: EA (NG) EA (NG) EA (HD) EA (NI) EA (HD) EA (NI) EA (NI) EA (NN) EA (NG) EA (NG) EA (NG) EA (NI) EA (NG) EA (NN) EA (NN) EA (NN) EA (HD) 4E (NI) (modified type (EA type))
CG16798m2R: QA (NG) QA (NG) QA (HD) QA (NI) QA (HD) QA (NI) QA (NI) QA (NN) QA (NG) QA (NG) QA (NG) QA (NI) QA (NG) QA (NN) QA (NN) QA (NN) QA (HD) 4Q (NI) (modified (QA type))
209R: DD (NI) DD (HD) DD (NI) DD (HD) DD (NI) DD (NG) DD (HD) DD (HD) DD (NI) DD (NN) DD (HD) DD (NG) DD (NN) DD (NG) 4D (NG) (Conventional)
209mR: EA (NI) EA (HD) EA (NI) EA (HD) EA (NI) EA (NG) EA (HD) EA (HD) EA (NI) EA (NN) EA (HD) EA (NG) EA (NN) EA (NG) 4E (NG) (modified type (EA type))
209m2R: QA (NI) QA (HD) QA (NI) QA (HD) QA (NI) QA (NG) QA (HD) QA (HD) QA (NI) QA (NN) QA (HD) QA (NG) QA (NN) QA (NG) 4Q (NG) (Modified type (QA type))
209m3R: EA (NI) QA (HD) EA (NI) QA (HD) EA (NI) QA (NG) EA (HD) QA (HD) EA (NI) QA (NN) EA (HD) QA (NG) EA (NN) QA (NG) 4E (NG) (Composite modified type (EA-QA type))

3. Construction of target luciferase vector The construction of the target luciferase vector was performed in the same manner as in Example 1.
In the same manner as in Example 1, these and the phRL-TK vector were coexpressed in MDCK2 cultured cells, and the DNA binding activity of each TALE sequence was quantified as the amount of luminescence of FLuc. Each signal was normalized by the amount of luminescence of Renilla luciferase. (Reference: Mussolino et. Al. Nucleic Acids Res. 2011; 39 (21): 9283., Cong et. Al. Nat Commun. 2012; 3: 968)
Target sequence
85F: (T) GTCGTCCACTCAAT (SEQ ID NO: 17)
85R: (T) GCTTGTGTCCCATT (SEQ ID NO: 18)
209F: (T) CACCTTCGACACAGTG (SEQ ID NO: 19)
209R: (T) ACACATCCAGCTGTT (SEQ ID NO: 20)
S1PR3F: (T) ACAACCACTCAGGCA (SEQ ID NO: 34)
S1PR3R: (T) ACACATACCAGTGCTTC (SEQ ID NO: 35)
SphkF: (T) CAGAATCACGCCCGAG (SEQ ID NO: 36)
SphkR: (T) CCCACTGCGACAGGTCTG (SEQ ID NO: 37)
CG16798F: (T) GGCCTGTCAGCGACAA (SEQ ID NO: 38)
CG16798R: (T) TTCACAAGTTTATGGGCA (SEQ ID NO: 39)
TRE: (T) CCCTATCAGTGATAGAGA (SEQ ID NO: 21)

The results are shown in Fig. 14-25. In the measurement experiment of the above-mentioned dTALE-DNA binding activity, up to about 7-fold increase in binding activity to a specific DNA sequence was observed by modification of the EA type TALE sequence. In addition, the modification of the QA type TALE sequence showed an increase in binding activity to a specific DNA sequence of up to 10 times or more. On the other hand, there was no significant difference in the binding activity to non-specific DNA sequences (FIGS. 14-16, 24, 25). In addition, the incomplete EA modified type, which partially includes the conventional type in the C-terminal repeat module, showed a specific DNA binding ability comparable to that of the EA type (FIGS. 19, 24, and 25).
From the above results, it was found that the binding activity to the target DNA sequence can be controlled by introducing a mutation into a specific site of the TALE sequence in this example.

Example 5: Measurement of non-specific DNA binding activity of dTALE The binding activity of dTALE from a target sequence to a slightly different non-target sequence was measured to examine the specificity of binding.
A part of the dTALE constructed in common with the plasmid introduced with the mutation in Example 4 was used in common, and some modified dTALEs were further constructed. The following measurements were carried out by the same method as in Example 1. Using a target DNA sequence designed so that a mismatch occurs at 1 to 3 bases with various modified dTALEs including the conventional type, the stepwise non-specific DNA binding activity of each dTALE was quantified and compared.

The configuration of the newly built dTALE repeat module is shown below.
209 4AR: 4A (NI) 4A (HD) 4A (NI) 4A (HD) 4A (NI) 4A (NG) 4A (HD) 4A (HD) 4A (NI) 4A (NN) 4A (HD) 4A (NG ) 4A (NN) 4A (NG) 4A (NG) (modified type (4A type))
209 4ER: 4E (NI) 4E (HD) 4E (NI) 4E (HD) 4E (NI) 4E (NG) 4E (HD) 4E (HD) 4E (NI) 4E (NN) 4E (HD) 4E (NG ) 4E (NN) 4E (NG) 4E (NG) (Modified type (4E type))
209h1R: DD (NI) DA (HD) EA (NI) DD (HD) DA (NI) EA (NG) DD (HD) DA (HD) EA (NI) DD (NN) DA (HD) EA (NG) DD (NN) DA (NG) 4D (NG) (Composite modified type (DD-DA-EA type))
209h2R: DA (NI) EA (HD) DA (NI) EA (HD) DA (NI) EA (NG) DA (HD) EA (HD) DA (NI) EA (NN) DA (HD) EA (NG) DA (NN) EA (NG) 4D (NG) (Composite modified type (DA-EA type))
209h3R: DD (NI) DA (HD) EA (NI) DD (HD) DA (NI) EA (NG) DA (HD) EA (HD) DA (NI) EA (NN) DA (HD) EA (NG) DD (NN) DA (NG) 4D (NG) (Composite modified type (mixed type 1))
209h4R: DD (NI) EA (HD) DA (NI) AD (HD) DD (NI) EA (NG) DA (HD) AD (HD) DA (NI) EA (NN) DA (HD) AD (NG) DD (NN) EA (NG) 4D (NG) (Composite modified type (mixed type 2))

The target sequence of the newly constructed target luciferase vector (underlined part is mismatch base) is shown below.
209R-1c: (T) ACACATCCAGCT C TT (SEQ ID NO: 40)
209R-1n: (T) A G ACATCCAGCTGTT (SEQ ID NO: 41)
209R-1m: (T) ACACAT G CAGCTGTT (SEQ ID NO: 42)
209R-2w: (T) ACACATCC T GCTG A T (SEQ ID NO: 43)
209R-2s: (T) ACACAT G CAGCT C TT (SEQ ID NO: 44)
209R-3m: (T) AC T CAT G CAGCT C TT (SEQ ID NO: 45)

The results are shown in Figures 26-28. In the above experiment for measuring the binding activity of dTALE and DNA, it was shown that the conventional type, EA type, and complex modified type gradually decrease the target binding ability according to the number and arrangement of mismatch bases in the target DNA. It was. The mismatched bases were arranged in the order of 3 ′ (close to the C end of TALE), 5 ′ (close to the N end), and the center, decreasing TALE-DNA binding activity. On the other hand, types 4E and 4A showed relatively high binding activity against non-specific target DNA.
Based on the above results, the binding activity to the target sequence and the specificity of the binding are often in a trade-off relationship, but by combining the conventional repeat module and the modified repeat module, the high specificity of the constructed TALE is high. It was shown that non-specific DNA binding activity can be significantly reduced while retaining DNA binding activity.

Example 6: Analysis of the effects of TALE C-terminal and N-terminal repeat sequences on DNA binding activity The TALE protein consists of a repeat motif responsible for specific DNA binding and a repeat outside responsible for non-specific DNA binding. It is known to be composed of DNA binding domains. As a result of systematic sequence analysis of the natural TALE protein, it was found that the N-terminal sequence (N-TAL) and C-terminal sequence (C-TAL) outside the repeat motif are classified into the following three systems: (Figure 30: N-TAL, Figure 31: C-TAL): 1.oryzae type, which is characteristically found in Xanthomonas oryzae, 2.citri type, which is found in species other than oryzae (citri, campestris, axonopodis, etc.) 3. EQ type accepted by TALE with EQ type repeat module.
The DNA binding activity of conventional dTALE, which employs oryzae type N-TAL and C-TAL, and citri type and EQ type dTALE was measured and compared in the same manner as in Example 1.

1. Construction of citri type and EQ type dTALE vectors

1-1. Construction of citri-type dTALE vector Totally synthesize double-stranded DNA of the following sequence encoding citri-type N-TAL and C-TAL and incorporate it into pCS2-TALE-VP48 vector (N-TAL; KpnI / BsmBI, C-TAL; BsmBI / BamHI), pCS2-TALE (citri) -VP48 vector was constructed (the sequence is shown in SEQ ID NO: 51). The pLR vector used in the modified Golden gate method encodes the last half repeat and a part of C-TAL, but the C-TAL sequence part encoded in the pLR vector is common to the oryzae and citri types. Therefore, the conventional pLR vector was used for the construction of citri type TALE.

Citri-type N-TAL (KpnI / BsmBI)
AAGGGTACCGTAGATTTGAGAACTTTGGGATATTCACAGCAGCAGCAGGAAAAGATCAAGCCCAAAGTGAGGTCGACAGTCGCGCAGCATCACGAAGCGCTGGTGGGTCATGGGTTTACACATGCCCACATCGTAGCCTTGTCGCAGCACCCTGCAGCCCTTGGCACGGTCGCCGTCAAGTACCAGGACATGATTGCGGCGTTGCCGGAAGCCACACATGAGGCGATCGTCGGTGTGGGGAAACAGTGGAGCGGAGCCCGAGCGCTTGAGGCCCTGTTGACGGTCGCGGGAGAGCTGAGAGGGCCTCCCCTTCAGCTGGACACGGGCCAGTTGCTGAAGATCGCGAAGCGGGGAGGAGTCACGGCGGTCGAGGCGGTGCACGCGTGGCGCAATGCGCTCACGGGAGCACCCCTGGAGACGTAC (SEQ ID NO: 46)

citri-Type_C-TAL (BsmBI / BamHI)
TATCGTCTCCAACGATCATCTTGTAGCGCTGGCCTGCCTCGGCGGACGACCCGCCTTGGATGCGGTGAAGAAGGGGCTCCCGCACGCGCCTGCATTGATTAAGCGGACCAACAGAAGAATCCCCGAGAGGACATCACATCGAGTGGCAGGATCCAAG (SEQ ID NO: 47)

1-2. Construction of EQ-type dTALE vector Based on the results of sequence analysis, double-stranded DNA of the following sequences encoding the typical EQ-type N-TAL and C-TAL was synthesized completely, and pCS2-TALE- It was incorporated into a VP48 vector (N-TAL; KpnI / BsmBI, C-TAL; BsmBI / BamHI), and a pCS2-TALE (EQ) -VP48 vector was constructed (the sequence is shown in SEQ ID NO: 52).

EQ-type_N-TAL (KpnI / BsmBI)
CAAGGGTACCGTGGATCTATGCACGCTCGGCTACAGTCAGCAGCAGCAAGACGAGATCAAACCGAAGGCCCGTGCTACAGTGGCGCAGCACCACCAGGCACTGATGGGCCATGGGTTTACACGCGCGCACATCGTTGCGCTCAGCCAACACCCGGCAGCGTTAGGGACCGTCGCTGTCAAGTATCAGGCCATGATCGCAGCGTTGCCAGAGGCGACACACGAAGACATCGTTGGCGTCGGCAAACAGTGGTCCGGCGCACGCGCCCTGGAGGCCTTGCTCACGGTTTCCGGGGAGTTGAGAGGTCCGCCGTTACAGTTGGACACAGGCCAACTTCTGAAGATTGCAAAACGTGGCGGCGTGACCGCAGTGGAGGCAGTGCATGCATGGCGCAATGCACTGACGGGTGCCCCCCTGGAGACGGGCGC (SEQ ID NO: 48)

EQ-Type_C-TAL (BsmBI / BamHI)
CCGGTCGTCTCCAACGACCACCTCGTCGCCTTGGCCTGCCTCGGCGGACGTCCTGCCCTGGAAGCAGTGAAAAAGGGATTGCCGCACGCGCCGACCTTGATCAAAAGAACCAATCGCCGTCTGCCAGAACGCACGTCCCATCGCGTTGCCGGATCCCAACT (SEQ ID NO: 49)

1-3. Construction of EQ-type pLR-xx vector In the TALE-DNA binding motif, a mutation was introduced into the C-TAL part of the pLR vector encoding the last half-repeat and part of C-TAL, and the EQ-type C- A pLR vector encoding TAL was constructed. Specifically, the codon corresponding to the third amino acid residue from the C-TAL N-terminal is changed from GTG (valine) to TTC (phenylalanine), and the codon corresponding to the 11th amino acid residue is changed from CCG (proline) to CAG. All changed to (Glutamine).

Primers used
GCGCTCGAAAGCATTTTCGCCCAGCTGAGCCGGCCTGATCAGGCGTTGGCCGCGTTG (EQ-Type pLRxx) (SEQ ID NO: 50)

Plasmids introduced with mutations
pLR-NN, pLR-NG, pLR-NI, pLR-HD

2. Construction of dTALE The following dTALE vector was constructed using the pCS2-TALE-VP48 vector, pCS2-TALE (citri) -VP48 vector, and pCS2-TALE (EQ) -VP48 vector based on the method of Example 1.
EQ-: EQ type dTALE constructed using pCS2-TALE (EQ) -VP48 vector
citri-: citri-type dTALE constructed using pCS2-TALE (citri) -VP48 vector

EQ-CG16798F: DD (NN) DD (NN) DD (HD) DD (HD) DD (NG) DD (NN) DD (NG) DD (HD) DD (NI) DD (NN) DD (HD) DD ( NN) DD (NI) DD (HD) DD (NI) DD (NI)
EQ-CG16798mF: EA (NN) EA (NN) EA (HD) EA (HD) EA (NG) EA (NN) EA (NG) EA (HD) EA (NI) EA (NN) EA (HD) EA ( NN) EA (NI) EA (HD) EA (NI) EA (NI)
EQ-CG16798m2F: QA (NN) QA (NN) QA (HD) QA (HD) QA (NG) QA (NN) QA (NG) QA (HD) QA (NI) QA (NN) QA (HD) QA ( NN) QA (NI) QA (HD) QA (NI) QA (NI)
EQ-CG16798R: DD (NG) DD (NG) DD (HD) DD (NI) DD (HD) DD (NI) DD (NI) DD (NN) DD (NG) DD (NG) DD (NG) DD ( NI) DD (NG) DD (NN) DD (NN) DD (NN) DD (HD) DD (NI)
EQ-CG16798mR: EA (NG) EA (NG) EA (HD) EA (NI) EA (HD) EA (NI) EA (NI) EA (NN) EA (NG) EA (NG) EA (NG) EA ( NI) EA (NG) EA (NN) EA (NN) EA (NN) EA (HD) EA (NI)
EQ-CG16798m2R: QA (NG) QA (NG) QA (HD) QA (NI) QA (HD) QA (NI) QA (NI) QA (NN) QA (NG) QA (NG) QA (NG) QA ( NI) QA (NG) QA (NN) QA (NN) QA (NN) QA (HD) QA (NI)
EQ-SphkF: DD (HD) DD (NI) DD (NN) DD (NI) DD (NI) DD (NG) DD (HD) DD (NI) DD (HD) DD (NN) DD (HD) DD ( HD) DD (HD) DD (NN) DD (NI) DD (NN) (EQ type N-TAL, C-TAL)
EQ-SphkmF: EA (HD) EA (NI) EA (NN) EA (NI) EA (NI) EA (NG) EA (HD) EA (NI) EA (HD) EA (NN) EA (HD) EA ( HD) EA (HD) EA (NN) EA (NI) EA (NN)
EQ-Sphkm2F: QA (HD) QA (NI) QA (NN) QA (NI) QA (NI) QA (NG) QA (HD) QA (NI) QA (HD) QA (NN) QA (HD) QA ( HD) QA (HD) QA (NN) QA (NI) QA (NN)
EQ-SphkR: DD (HD) DD (HD) DD (HD) DD (NI) DD (HD) DD (NG) DD (NN) DD (HD) DD (NN) DD (NI) DD (HD) DD ( NI) DD (NN) DD (NN) DD (NG) DD (HD) DD (NG) DD (NN)
EQ-SphkmR: EA (HD) EA (HD) EA (HD) EA (NI) EA (HD) EA (NG) EA (NN) EA (HD) EA (NN) EA (NI) EA (HD) EA ( NI) EA (NN) EA (NN) EA (NG) EA (HD) EA (NG) EA (NN)
EQ-Sphkm2R: QA (HD) QA (HD) QA (HD) QA (NI) QA (HD) QA (NG) QA (NN) QA (HD) QA (NN) QA (NI) QA (HD) QA ( NI) QA (NN) QA (NN) QA (NG) QA (HD) QA (NG) QA (NN)
EQ-85mR: EA (NN) EA (HD) EA (NG) EA (NG) EA (NN) EA (NG) EA (NN) EA (NG) EA (HD) EA (HD) EA (HD) EA ( NI) EA (NG) EA (NG)
citri-85mR: EA (NN) EA (HD) EA (NG) EA (NG) EA (NN) EA (NG) EA (NN) EA (NG) EA (HD) EA (HD) EA (HD) EA ( NI) EA (NG) EA (NG)
EQ-85m2R: QA (NN) QA (HD) QA (NG) QA (NG) QA (NN) QA (NG) QA (NN) QA (NG) QA (HD) QA (HD) QA (HD) QA ( NI) QA (NG) QA (NG)
citri-85m2R: QA (NN) QA (HD) QA (NG) QA (NG) QA (NN) QA (NG) QA (NN) QA (NG) QA (HD) QA (HD) QA (HD) QA ( NI) QA (NG) QA (NG)
EQ-209m2R: QA (NI) QA (HD) QA (NI) QA (HD) QA (NI) QA (NG) QA (HD) QA (HD) QA (NI) QA (NN) QA (HD) QA ( NG) QA (NN) QA (NG) QA (NG)
citri-209m2R: QA (NI) QA (HD) QA (NI) QA (HD) QA (NI) QA (NG) QA (HD) QA (HD) QA (NI) QA (NN) QA (HD) QA ( NG) QA (NN) QA (NG) QA (NG)

In addition, using pCS2-TALE-VP48 vector, pCS2-TALE (citri) -VP48 vector, pCS2-TALE (EQ) -VP48 vector, conventional pLR vector, EQ-type pLR vector and having mixed C-TAL sequence A dTALE vector was constructed.

oryzae-EQLR-209m2R: QA (NI) QA (HD) QA (NI) QA (HD) QA (NI) QA (NG) QA (HD) QA (HD) QA (NI) QA (NN) QA (HD) QA (NG) QA (NN) QA (NG) QA (NG) (conventional type (oryzae type) N-TAL, C-TAL (part of N end is EQ type))
citri-EQLR-209m2R: QA (NI) QA (HD) QA (NI) QA (HD) QA (NI) QA (NG) QA (HD) QA (HD) QA (NI) QA (NN) QA (HD) QA (NG) QA (NN) QA (NG) QA (NG) (citri type N-TAL, C-TAL (N-terminal part is EQ type))
EQ-oryzaeLR-209m2R: QA (NI) QA (HD) QA (NI) QA (HD) QA (NI) QA (NG) QA (HD) QA (HD) QA (NI) QA (NN) QA (HD) QA (NG) QA (NN) QA (NG) QA (NG) (EQ type N-TAL, C-TAL (N-terminal part is conventional type (oryzae type))

The following measurements were performed by the same method as in Example 1 using a part of dTALE constructed in Example 4. The target sequence used is as described above.

3. Results The results are shown in Figure 31-37. In Fig. 31-34, there are two types of non-repeat DNA binding domains: conventional (oryzae type) and EQ type, and specific when the repeat module is conventional, modified (EA type) and modified (QA). DNA binding activity was measured and compared (four sequence combinations in FIGS. 31-34). Figure 35 shows specific DNA binding activity when the nonrepeat domain is the conventional type (oryzae type), EQ type, and citri type, and the repeat module is the modified type (EA type) or the modified type (QA type). Were compared (1 case). In Figure 36-37, there are six types of non-repeat domains: conventional (oryzae type), EQ type, citri type, conventional type + EQ type pLR, citri type + EQ type pLR, EQ type + conventional type pLR. dTALE was constructed, and stepwise non-specific DNA binding activity was compared by the same method as in Example 5.
In the above experiment for measuring the binding activity between dTALE and DNA, it was found that the specific DNA binding activity of conventional (oryzae type), citri type, and EQ type varies depending on the target sequence, but is almost the same. It was. Oryzae-EQLR type dTALE was found to have low target specificity.

Example 7: Analysis of the effect of modification of the 11th amino acid residue on binding ability and specificity when the sequence of RVD is NN Focusing on RVD NN that recognizes guanine (G) and adenine (A), The DNA binding activity of type N and type N was compared.
That is, in the conventional TALEN, the 11th amino acid residue (one before RVD) in each repeat module is serine (S), but the sequence analysis of natural TALE protein shows that RVD is NN. Showed that the 11th amino acid residue was asparagine (N) in a ratio of about 60%. A modified NN repeat module in which the residue is changed from serine to asparagine is created, and the conventional NN (NN) in which the 11th to 13th sequences are SNN and the 11th to 13th sequences are NNN The binding ability and specificity of each type of NN (3N) RVD for guanine (G) and adenine (A) were measured and compared.

1. Mutagenesis In the same manner as in Example 1, in each repeat module of the TALE-DNA binding motif, the codon corresponding to the 11th amino acid residue was changed from AGC (serine) to AAC (asparagine).

Primer used for mutagenesis of the 11th amino acid (amino acid changed from serine (S) to asparagine (N))
GCTATCGCCAACAACAATGGCGGCAAG (11SN-F) (SEQ ID NO: 53)

Plasmid with mutation introduced by this primer
pNN1, pNN2, pNN3, pNN4, pNN5, pNN6, pLR-NN

2. dTALE Construction The configuration of a dTALE repeat module constructed by the same method as in Example 1 is shown below. Conventional NN is written as NN, and N-type NN is written as 3N.

TRE: DD (HD) DD (HD) DD (HD) DD (NG) DD (NI) DD (NG) DD (HD) DD (NI) DD (NN) DD (NG) DD (NN) DD (NI) DD (NG) DD (NI) DD (NN) DD (NI) DD (NN) 4D (NI)
TREn1: DD (HD) DD (HD) DD (HD) DD (NG) DD (NI) DD (NG) DD (HD) DD (NI) DD (NN) DD (NG) DD (NN) DD (NI) DD (3N) DD (3N) DD (3N) DD (3N) 4D (3N)
TREn2: DD (HD) DD (HD) DD (HD) DD (NG) DD (NI) DD (NG) DD (HD) DD (NI) DD (NN) DD (NG) DD (NN) DD (NI) DD (NN) DD (NN) DD (NN) DD (NN) 4D (NN)
TREn3: DD (HD) DD (HD) DD (HD) DD (NG) DD (NI) DD (NG) DD (HD) DD (NI) DD (NN) DD (NG) DD (NN) DD (NI) DD (3N) DD (NN) DD (3N) DD (NN) DD (3N) 4D (NN)
TREn4: DD (HD) DD (HD) DD (HD) DD (NG) DD (NI) DD (NG) DD (HD) DD (NI) DD (NN) DD (NG) DD (NN) DD (NI) DD (NN) DD (3N) DD (NN) DD (3N) DD (NN) 4D (3N)
TREn5: DD (HD) DD (HD) DD (HD) DD (NG) DD (NI) DD (NG) DD (3N) DD (3N) DD (3N) DD (NN) DD (NN) DD (NN) DD (NG) DD (NI) DD (NN) DD (NI) DD (NN) 4D (NI)
TREn6: DD (HD) DD (HD) DD (HD) DD (NG) DD (NI) DD (NG) DD (NN) DD (NN) DD (NN) DD (3N) DD (3N) DD (3N) DD (NG) DD (NI) DD (NN) DD (NI) DD (NN) 4D (NI)

3. Target vector construction A target vector was constructed in the same manner as in Example 1. The target sequence is shown below.
TRE: (T) CCCTATCAGTGATAGAGA (SEQ ID NO: 54)
TRE-G5: (T) CCCTATCAGTGAGGGGG (SEQ ID NO: 55)
TRE-A5: (T) CCCTATCAGTGAAAAAA (SEQ ID NO: 56)
TRE-GA3: (T) CCCTATCAGTGAGAGAGA (SEQ ID NO: 57)
TRE-G3A3-TRE: (T) CCCTATGGGAAATAGAGA (SEQ ID NO: 58)
Control: (T) TTATTCCCTGACAG (SEQ ID NO: 59)
The binding activity was measured in the same manner as in Example 1.

4. Results The results are shown in Figure 38-39. In the experiment for measuring the binding activity of dTALE and DNA as described above, both conventional NN and N-type NN were found to bind to both guanine and adenine. However, N-type NN showed about 1.3 times the binding activity of conventional NN in the ability to bind 5 consecutive adenines. It was shown that the ability to bind to adenine can be improved by about 30% without reducing the binding activity to guanine by using N-type NN. Therefore, it was found that N-type NN can be used as an RVD that recognizes G or A as an alternative RVD superior to conventional NN.

This application is based on Japanese Patent Application No. 2013-167144 filed in Japan (filing date: August 9, 2013), the contents of which are incorporated in full herein.

Claims

An isolated modified protein having a binding activity to a target DNA,
The modified protein includes a Transcription Activator-Like (TAL) Effector portion derived from a genus Xanthomonas having a repeating structure of a plurality of modules,
Each of the plurality of modules has a target base recognition region that recognizes one base constituting the base sequence of the target DNA, and
In at least one of the plurality of modules, in the amino acid sequence represented by SEQ ID NO: 1, at least one amino acid residue selected from the group consisting of 2 to 6 and 30 to 34 is artificially mutated Having an amino acid sequence modified to have
The modified protein.
Wherein at least one of the modules having an amino acid sequence modified so as to have an artificial mutation is at least one amino acid residue selected from the group consisting of 2 to 6 in the amino acid sequence represented by SEQ ID NO: 1 The modified protein according to claim 1, wherein at least one amino acid residue selected from the group consisting of and 30 to 34 is modified so as to have an artificial mutation.
At least one of the plurality of modules is modified so that at least one amino acid residue selected from the group consisting of the 20th to 26th amino acids has an artificial mutation in the amino acid sequence represented by SEQ ID NO: 1. The modified protein according to claim 1 or 2, wherein
At least one of the plurality of modules is artificially modified so that the 11th amino acid residue in the amino acid sequence represented by SEQ ID NO: 1 is asparagine. The modified protein according to Item.
At least one of the modules having an amino acid sequence modified so as to have an artificial mutation has 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1, and has the homology When the sequence and the amino acid sequence represented by SEQ ID NO: 1 were aligned, the amino acid sequence represented by SEQ ID NO: 1 was modified so that the 4th and / or 32nd amino acid residue had an artificial mutation The modified protein according to any one of claims 1 to 4, which is a protein.
At least one of the modules having an amino acid sequence having 80% or more homology with the amino acid sequence represented by SEQ ID NO: 1 aligned the amino acid sequence having the homology with the amino acid sequence represented by SEQ ID NO: 1. (I) the amino acid sequence represented by SEQ ID NO: 1 has been modified so that the fourth amino acid residue is glutamic acid and the 32nd amino acid residue is alanine, or (ii) The modified protein according to claim 5, which is modified so that the fourth amino acid residue is glutamine and the 32nd amino acid residue is alanine in the amino acid sequence represented by SEQ ID NO: 1.
The modified protein according to any one of claims 1 to 6, wherein the mutation has the same pattern in all of the plurality of modules.
The modified protein according to any one of claims 1 to 7, wherein the number of modules in the repeating structure is 6 to 36.
The modified protein according to any one of claims 1 to 8, further comprising a functional domain not derived from a bacterium belonging to the genus Xanthomonas from which the TAL Effector portion is derived.
The modified protein according to claim 9, wherein the functional domain is all or a part of a nucleolytic enzyme or a nucleotransferase.
The modified protein according to claim 10, wherein the nucleolytic enzyme comprises a DNA cleavage domain of a restriction enzyme FokI.
The modified protein according to claim 9, wherein the functional domain includes a transcription activator domain, a transcription repressor domain, or an epigenome regulator domain.
The functional domain comprises all or part of a fluorescent protein, luminescent protein, chromoprotein, or all or part of an enzyme protein that catalyzes a fluorescence, luminescence or chromogenic reaction. Modified protein of
An isolated nucleic acid encoding the amino acid sequence of the modified protein according to any one of claims 1 to 13.
An expression vector comprising the nucleic acid according to claim 14.
A transformant into which the expression vector according to claim 15 has been introduced.
A method for producing the modified protein according to any one of claims 1 to 13, wherein the method comprises:
A step of introducing a mutation in the nucleic acid encoding the original protein, wherein the mutation is such that the artificial mutation of the modified protein occurs in the protein encoded by the mutated nucleic acid; The original protein is such that the amino acid sequence of the protein into which the artificial mutation is introduced becomes the amino acid sequence of the modified protein,
Preparing an expression vector containing the mutated nucleic acid and obtaining the transformant by introducing the expression vector into a host cell; and
The method comprising culturing the transformant and isolating the modified protein from the culture.
The modified protein according to any one of claims 1 to 13 is introduced into a cell, or RNA encoding the amino acid sequence of the modified protein or a nucleic acid encoding the amino acid sequence of the modified protein is included. (A) cutting the region by introducing an expression vector into the cell, expressing the modified protein in the cell, and binding the modified protein to the target gene and / or its surrounding region; (B) modifying the region by cleaving the region and applying homologous recombination repair or non-homologous end-joining repair to the cleaved region, (c) modifying the region, or ( d) A method for cleaving, altering or modifying the region in the cell, comprising the step of changing gene expression around the region.
The cells are animal sperm, unfertilized eggs, fertilized eggs, fetal or adult cells, or cells collected from plant or animal tissues, embryonic stem cells (ES cells) or induced pluripotent stem cells (iPS) The method according to claim 18, which is a cell).
A method for producing an individual or strain of a mutant organism in which a target gene and / or its surrounding region is cleaved, altered or modified,
(1) preparing a cell of the organism including the region;
(2) The modified protein according to any one of claims 1 to 13 is introduced into a cell, or the RNA encoding the amino acid sequence of the modified protein or the amino acid sequence of the modified protein is encoded. An expression vector containing a nucleic acid is introduced into a cell, the modified protein is expressed in the cell, and the modified protein is bound to the region, thereby (a) cutting the region, or (b) Altering the region by cleaving the region and applying homologous recombination repair or non-homologous end-joining repair to the cleaved region, (c) modifying the region, or (d) the region Changing the surrounding gene expression;
(3) The method including the step of producing an individual or strain of the mutant organism from the cells obtained in step (2).
An individual or strain of a mutant organism obtained by the method according to claim 20.
A method for screening a modified protein whose binding activity to a target DNA is changed compared to an unmodified protein,
(1) a step of measuring the binding activity of the modified protein according to any one of claims 1 to 13 to the target DNA;
(2) measuring the binding activity of the unmodified protein having no artificial mutation in the modified protein to the target DNA;
(3) When the binding activity of the modified protein is changed as compared with the binding activity of the unmodified protein, the modified protein has a binding activity to the target DNA that is not The said method which has the process of selecting as a modified protein changed compared with the modified protein.
The method according to claim 22, further comprising the step of preparing the modified protein using the method according to claim 17 before the step (1).
A method for screening a modified protein having altered cleavage activity against a target DNA compared to an unmodified protein,
(1) a step of measuring the cleavage activity of the modified protein according to claim 10 or 11 against the target DNA;
(2) a step of measuring the cleavage activity of the unmodified protein having no artificial mutation in the modified protein with respect to the target DNA;
(3) When the cleavage activity of the modified protein is changed as compared to the cleavage activity of the unmodified protein, the modified protein has a cleavage activity against the target DNA. The said method which has the process of selecting as a modified protein changed compared with the modified protein.
The method according to claim 24, further comprising the step of preparing the modified protein using the method according to claim 17 before the step (1).