Nucleic acid binding protein

Abstract

The present invention provides a new type of modified protein, which is used as a new detection tool related to gene expression and can detect chromatin open structure more sensitively and easily than the prior art. The present invention also provides a nucleic acid binding protein, characterized in that it contains a DNA binding domain formed by more than three TAL-repeat repeats, and binds to nucleic acids independently of the base sequence; and provides a method for fluorescent labeling of open chromatin in living cells, characterized in that it includes the step of introducing a gene encoding a nucleic acid binding protein into living cells, wherein the nucleic acid binding protein is a protein directly or indirectly bound to a DNA binding domain formed by more than three TAL-repeat repeats and a fluorescent protein, and the DNA binding domain binds to nucleic acids independently of the base sequence.

Description

Technical Field

[0001] The present invention relates to a probe for specifically and highly sensitively detecting and visualizing an open chromatin structure in a chromosome, and a method for labeling open chromatin in living cells using the probe.

[0002] This application claims priority based on Japanese Patent No. 2019-074004 filed in Japan on April 9, 2019, the entire contents of which are incorporated herein by reference. Background Art

[0003] Although the genomic information that constitutes an individual organism is basically the same, the research results from the epigenome have attracted much attention in recent years on the research topic of how each cell exerts its huge diversity. Through the progress of epigenome research, it has been clarified that it is important to correctly control the expression of essential genes and suppress the expression of non-essential genes in each cell. The cell-specific characteristics manifested by this regulation are stably maintained across cell divisions, and this phenomenon is called cell memory (or epigenetics).

[0004] TAL (Transcription Activator-Like) Effector (TALE) has been identified as a transcription factor associated with the regulation of gene expression used to suppress the immune mechanism of host cells during pathogenic bacterial infection (Non-Patent Document 1). TALE contains a structural unit (structural unit) (module) composed of about 34 amino acids called TAL-repeat at its center, and a DNA binding domain composed of parts connected by a number of repeats (also called "number of repeats", "number of repeats" or "number of repeats") of 10 to 30 (Non-Patent Document 2). TAL-repeat is composed of two α-helix structures. In the loop region connecting the two α-helix structures, there are two amino acid residues called Repeat Variable Diresidue (RVD) that are involved in nucleic acid base recognition. Each TAL-repeat recognizes a nucleic acid base based on the amino acid sequence of its respective RVD.

[0005] By connecting TAL-repeat with RVD that specifically recognizes specific nucleic acid bases in an appropriate order, TALE can be modified so that it selectively binds to nucleic acids containing specific base sequences. That is, by using a module corresponding to a DNA sequence that targets the DNA binding domain of TALE, sequence-dependent binding can be achieved. For example, in genome editing, TALEN (TALE-Nuclease) obtained by fusing the TALE that recognizes the nucleic acid sequence-dependently with the FokI nuclease is used (Patent Document 1).

[0006] On the other hand, it is known that organisms partially dissolve the aggregates composed of nucleic acids and histones that form a super three-dimensional structure in the nucleus to obtain an open chromatin structure during the expression control of various genes such as their development or differentiation. The analysis of whether an open chromatin structure is obtained is important in the analysis of unexplained gene functions, especially in the expression and identification of unknown differentiation-inducing genes related to specific diseases, the expression and identification of unidentified genes related to carcinogenesis, and the expression and identification of genes related to metabolic abnormalities or neurodegeneration based on unexplained regulatory mechanisms.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent Application No. 2013-529083

[0010] Non-patent literature:

[0011] Non-patent document 1: Christian, et al., Genetics, 2010, vol. 186, p. 757-761.

[0012] Non-patent document 2: Shinichi Tate, Biophysics, 2017, vol.57(3), p.127-130.

[0013] Non-patent document 3: Cermak, et al., Nucleic Acids Research, 2011, vol. 39, e82.

[0014] Non-patent document 4: Sakuma et al., Genes to Cells, 2013, vol. 18(4), p. 315-326.

[0015] Non-patent document 5: Reyon, et al., “Current Protocols in Molecular Biology”, Wiley Online Library, 2012, Chapter 12, 12.15.1-12.15.14

[0016] Non-patent document 6: Chen, et al., Nature Methods, 2016, vol. 13, p. 1013-1020.

[0017] Non-patent document 7: Abe et al., Genesis, 2011, vol. 49, p. 579-590. Summary of the invention

[0018] Problems to be solved by the present invention

[0019] The main purpose of the present invention is to provide a new type of modified protein as a new detection tool related to gene expression, which can detect the open structure of chromatin more sensitively and more easily than the existing technology.

[0020] Means used to solve problems

[0021] The inventors conducted in-depth research and found that: in the DNA binding domain of TALE, TAL-repeat, which is an RVD composed of a specific amino acid sequence, strongly binds to the four nucleic acid bases of adenine, guanine, thymine and cytosine in roughly the same manner; the DNA binding domain formed by the repeated connection of TAL-repeat, which is an RVD that recognizes all four nucleic acid bases, can become a module that binds firmly to the nucleic acid base sequence independently. In addition, it was found that the DNA binding domain can be used as a probe to recognize the open chromatin structure, thereby completing the present invention.

[0022] That is, the invention involved in the present invention relates to the technical solutions of the following items [1] to

[13] .

[0023] Item [1]. A nucleic acid binding protein, characterized in that it contains a DNA binding domain composed of three or more TAL-repeat repeats and binds to nucleic acid independently of the base sequence.

[0024] Item [2]. The nucleic acid binding protein of Item [1], wherein the RVD sequence of the TAL-repeat is HT, RS or HS.

[0025] Item [3]. The nucleic acid binding protein of Item [1] or Item [2], wherein the number of TAL-repeat repeats in the DNA binding domain is greater than 3 and less than 35.

[0026] Item [4]. The nucleic acid binding protein according to any one of Items [1] to [3], wherein the TAL-repeat consisting of the same amino acid sequence is repeatedly connected in the DNA binding domain.

[0027] Item [5]. A nucleic acid binding protein as described in any one of Items [1] to [4], wherein the DNA binding domain is the DNA binding domain of TALE, and the DNA binding domain of TALE is modified in such a way that the RVD sequence of the TAL-repeat is HT, RS or HS.

[0028] Item [6]. A nucleic acid binding protein as described in any one of Items [1] to [5], wherein the TAL-repeat contained in the DNA binding domain is a TAL-repeat of a TALE from a bacterium of the genus Xanthomonas, or a TAL-repeat in which an amino acid mutation is introduced into a region other than the RVD of the TAL-repeat.

[0029] Item [7]. A nucleic acid binding protein as described in any one of Items [1] to [6], wherein the nucleic acid binding protein is a protein obtained by directly or indirectly binding a protein containing the DNA binding domain to a fluorescent protein.

[0030] Item [8]. A protein probe which is composed of the nucleic acid-binding protein of any one of Items [1] to [7] and selectively binds to an open chromatin structure.

[0031] Item [9]. A nucleic acid molecule comprising a base sequence encoding the nucleic acid-binding protein of any one of Items [1] to [7].

[0032] Item

[10] . An expression vector incorporating the nucleic acid molecule of Item [9] and capable of expressing the nucleic acid binding protein of any one of Items [1] to [7] in a host cell.

[0033] Item

[11] . A method for fluorescently labeling open chromatin in living cells, characterized in that:

[0034] The method comprises the steps of introducing a gene encoding a nucleic acid binding protein into a living cell,

[0035] The nucleic acid binding protein is a protein comprising a DNA binding domain formed by three or more TAL-repeat repeats and a fluorescent protein.

[0036] The DNA binding domain binds to nucleic acid independently of the base sequence.

[0037] Item

[12] . A transformed cell into which a gene encoding the nucleic acid-binding protein of any one of Items [1] to [7] is introduced; wherein the cell is a cell other than a cell constituting a human.

[0038] Item

[13] . A transformed non-human animal into which a gene encoding the nucleic acid-binding protein of any one of Items [1] to [7] has been introduced.

[0039] Effects of the Invention

[0040] The nucleic acid binding protein of the present invention strongly binds to nucleic acid independently of the base sequence and can therefore be used as a probe for identifying the open chromatin structure of chromosomes. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a schematic diagram showing the structure of the GFP fusion protein containing the DNA binding domain of the TAL-repeat sequence used in Example 1.

[0042] Figure 2A 3A and 3B are fluorescent images showing cells expressing each GFP fusion protein in Example 1.

[0043] Figure 2B This is a graph showing the measurement results of FRAP of each GFP fusion protein in Example 1.

[0044] Figure 3A The figure shows the fluorescence images obtained by double staining using ChrocodiLE (a GFP fusion protein containing a DNA binding domain obtained by repeating 15 TAL-repeat sequences prepared in Example 1) and staining based on insertion of a fluorescent marker into a genomic region accessible to a transposase (ATAC) or fluorescent immunostaining of four modified histones (H3K4me 3, H3K27ac, H3K9me3, or H3K27me 3) in Example 2.

[0045] Figure 3B This is a diagram showing the results of the study of the correlation coefficient of colocalization between ChrocodiLE and four modified histones or the genome accessible to transposase in Example 2.

[0046] Figure 4 This is a diagram showing the results of identifying the base sequence of the genomic region to which ChrocodiLE binds in Example 2.

[0047] Figure 5 3 and 4 show fluorescent images of thin slices of the cerebral cortex, hippocampus, cerebellar cortex, and skin tissue of ChrocodiLE-Tg mice prepared in Example 3.

[0048] Figure 6 These are fluorescent images showing thin sections of the gonads and developing embryos of the ChrocodiLE-Tg nematode prepared in Example 4. DETAILED DESCRIPTION

[0049] [Nucleic acid binding protein]

[0050] The nucleic acid binding protein of the present invention is characterized in that it contains a DNA binding domain composed of three or more TAL-repeats repeatedly connected, and binds independently of the base sequence. The TAL-repeat in the nucleic acid binding protein of the present invention binds to the four nucleic acid bases of adenine, guanine, thymine and cytosine with approximately the same degree of sufficient strength. Therefore, the nucleic acid binding protein of the present invention, which has a DNA binding domain repeatedly connected to the TAL-repeat, strongly binds to nucleic acids independently of the base sequence. Below, the TAL-repeat that binds to the four nucleic acid bases with approximately the same degree of sufficient strength is referred to as a "non-selective binding TAL-repeat."

[0051] There is no particular limitation on the non-selective binding TAL-repeat in the nucleic acid binding protein of the present invention as long as it is a substance that can bind to the four nucleic acid bases with sufficient strength to approximately the same degree. As the non-selective binding TAL-repeat, the RVD sequence is preferably HT, RS or HS (H: histidine, T: threonine, R: arginine, S: serine). The TAL-repeat composed of these amino acid sequences binds strongly to the four nucleic acid bases to approximately the same degree.

[0052] The non-selective binding TAL-repeat in the nucleic acid binding protein of the present invention can use a substance in which the RVD of the TAL-repeat possessed by a naturally occurring TALE or a variant thereof is mutated to HT, RS or HS. As a naturally occurring TALE, for example, a TALE from a bacterium of the genus Xanthomonas can be cited. As a TALE from a bacterium of the genus Xanthomonas, it has a DNA binding domain obtained by repeating the arrangement of TAL-repeat in the center, and a nuclear localization signal (NLS) sequence and a transcription activation domain on the C-terminal side of the DNA binding domain (for example, see non-patent document 2). The conservation of the 4th and 32nd amino acids of the TAL-repeat of the TALE from the genus Xanthomonas is low. TAL-repeat as a natural TALE from Xanthomonas bacteria includes, for example, DD type in which the 4th and 32nd amino acids are both aspartic acid; DA type in which the 4th is aspartic acid and the 32nd is alanine; AD type in which the 4th is alanine and the 32nd is aspartic acid; AA type in which the 4th is alanine and the 32nd is alanine; ED type in which the 4th is glutamic acid and the 32nd is aspartic acid; EA type in which the 4th is glutamic acid and the 32nd is alanine. Natural TALEs include TALEs composed only of TAL-repeat of the same type and TALEs containing two or more types of TAL-repeat. For example, among Xanthomonas bacteria, there are also bacteria that contain both DD-type TAL-repeat and EA-type TAL-repeat in one molecule of TALE.

[0053] A variant of a TAL-repeat possessed by a naturally occurring TALE (TAL-repeat derived from a natural TALE) may be a mutant (TAL-repeat mutant) in which a mutation is introduced into one or more, preferably 1 to 10, and more preferably 1 to 5 amino acids in a region other than the RVD region in the TAL-repeat while maintaining the function as a TAL-repeat. The mutation means replacement, deletion, or insertion of an amino acid. Examples of TAL-repeat mutants include: a mutant having an RVD region and composed of an amino acid sequence having a sequence identity of more than 80%, preferably more than 90%, and more preferably more than 95% with the amino acid sequence of a TAL-repeat derived from a natural TALE, and maintaining the function as a TAL-repeat. Examples of variants of naturally occurring TALEs include variants described in Patent Document 1. TAL-repeat or TAL-repeat mutants obtained by modifying RVD can be produced, for example, by general point mutation introduction technology using commercially available kits such as KOD-Plus-Mutagenesis Kit (manufactured by Toyobo Co., Ltd.), PrimeSTAR Mutagenesis Basal Kit (manufactured by Takara), and QuikChange Multi Site-Directed Mutagenesis Kit (manufactured by Agilent).

[0054] As a non-selective binding TAL-repeat in the nucleic acid binding protein of the present invention, for example, the RVD (12th and 13th amino acids) of the TAL-repeat composed of 34 amino acids from Xanthomonas bacteria can be modified into a dipeptide that binds to four nucleic acid bases with sufficient strength to approximately the same degree. The amino acid sequence of the TAL-repeat in which the RVD is modified to HT, RS or HS is shown in Table 1.

[0055] [Table 1]

[0056] RVD Amino acid sequence Serial Number HT LTPEQVVAIASHTGGKQALETVQRLLPVLCQAHG 1 RS LTPEQVVAIASRSGGKQALETVQRLLPVLCQAHG 2 HS LTPEQVVAIASHSGGKQALETVQRLLPVLCQAHG 3

[0057] In the amino acid sequences of sequence numbers 1 to 3, the 4th glutamic acid may be aspartic acid, glutamine or alanine. In the amino acid sequences of sequence numbers 1 to 3, the 32nd alanine may be aspartic acid.

[0058] The DNA binding domain of the nucleic acid binding protein of the present invention is composed of a structure in which three or more non-selective binding TAL-repeats are repeatedly connected. The more the number of repeats of the non-selective binding TAL-repeat in the DNA binding domain (also referred to as "number of repeats", "number of repeats" or "number of repeats") is, the stronger the binding to nucleic acid is. The number of repeats of the non-selective binding TAL-repeat in the nucleic acid binding protein of the present invention is preferably 3 or more and less than 35, more preferably 3 to 30, further preferably 3 to 25, further preferably 3 to 20, and particularly preferably 3 to 18. In addition, the number of repeats of the non-selective binding TAL-repeat in the nucleic acid binding protein of the present invention is preferably 8 or more, more preferably 8 or more and less than 35, further preferably 10 or more and less than 25. The DNA binding domain of the nucleic acid binding protein of the present invention may be composed only of non-selective binding TAL-repeats composed of the same amino acid sequence, or may include two or more non-selective binding TAL-repeats having different amino acid sequences. For example, the multiple non-selective binding TAL-repeat contained in the DNA-binding domain of the nucleic acid-binding protein of the present invention has the same amino acid sequence in the RVD region, but the amino acid sequence of the region outside the RVD region may be different from each other, preferably 1 to 5 amino acids different from each other. In addition, the multiple non-selective binding TAL-repeat contained in the DNA-binding domain of the nucleic acid-binding protein of the present invention has a common amino acid sequence outside the RVD region, but the RVD region may also be different from each other.

[0059] The DNA binding domain of the nucleic acid binding protein of the present invention, as a whole domain, may also contain a selective binding TAL-repeat that specifically binds to a specific base, as long as it does not hinder the function of binding to DNA independently of the base sequence. When containing multiple selective binding TAL-repeat, they may be composed of the same amino acid sequence, or may be composed of two or more different amino acid sequences. For example, the DNA binding domain of the nucleic acid binding protein of the present invention may include three or more non-selective binding TAL-repeat and one or more selective binding TAL-repeat. When containing both non-selective binding TAL-repeat and selective binding TAL-repeat, there is no particular limitation on the connection order of these TAL-repeats, and a block composed of non-selective binding TAL-repeat and a block composed of selective binding TAL-repeat can be connected, or the two can be connected alternately.

[0060] The nucleic acid binding protein of the present invention preferably contains an NLS peptide in addition to the above-mentioned DNA binding domain. By containing an NLS peptide, when the nucleic acid binding protein of the present invention is introduced into a cell or expressed in a cell, the nucleic acid binding protein can bind to the chromosome in the nucleus of the cell. As the NLS peptide, a known NLS peptide or a variant thereof possessed by a naturally occurring nuclear localized protein can be used.

[0061] The nucleic acid binding protein of the present invention preferably has a labeling site for directly or indirectly detecting the presence of the protein. As the labeling site, it can be appropriately selected from substances commonly used to label nucleic acids and proteins, such as fluorescent molecules, peptide tags, enzymes, biotin, avidin, streptavidin, gold sol, magnetic beads, and agarose beads. As fluorescent molecules, it can be organic compounds other than proteins such as fluorescein derivatives and rhodamine derivatives, or fluorescent proteins such as GFP (green fluorescent substance), mTQ2, Citrine, tagRFP, iRFP, and mMaple. As peptide tags, for example, His tags, GST tags, Flag tags, Myc tags, HA tags, tetrastyrene tags, etc. As enzymes, for example, HaloTag (haloalkane dehalogenase), SNAP-tag, CLIP-tag (methylguanine DNAmethyltransferase), HRP (Horseradish peroxidase), AP (Alkaline phosphatase), etc.

[0062] The nucleic acid binding protein of the present invention can be produced, for example, by replacing the DNA binding domain (repeat region of TAL-repeat) in a naturally occurring TALE or a variant thereof with a structure in which a non-selective binding TAL-repeat is repeatedly linked three or more times. In addition, the nucleic acid binding protein of the present invention also contains a protein that allows the labeling site to be directly or indirectly bound to the manufactured TALE variant via a peptide linker or the like.

[0063] In the case where the nucleic acid binding protein of the present invention is composed only of protein, the nucleic acid binding protein can be produced by introducing an expression vector into various protein expression systems that incorporates a nucleic acid molecule containing a base sequence encoding the full length of the nucleic acid binding protein. In the case where the nucleic acid binding protein of the present invention contains a portion other than the protein such as a non-protein fluorescent molecule, the nucleic acid binding protein of the present invention can be produced by introducing an expression vector into various protein expression systems that incorporates a nucleic acid molecule containing a base sequence encoding only a portion of the protein containing a DNA binding domain, and combining the produced protein with a non-protein molecule by conventional methods.

[0064] [Protein probe]

[0065] The nucleic acid-binding protein of the present invention strongly binds to nucleic acid independently of the base sequence. Therefore, in the nucleic acid-binding protein of the present invention, the protein having a labeling site can be used as a protein probe for widely identifying nucleic acids. In particular, the nucleic acid-binding protein of the present invention selectively identifies an open chromatin structure compared to condensed chromatin in chromosomes. Therefore, the nucleic acid-binding protein of the present invention is useful as a probe for identifying an open chromatin structure. In particular, the nucleic acid-binding protein of the present invention (hereinafter sometimes referred to as "the nucleic acid-binding fluorescent protein of the present invention") containing a target site as a fluorescent substance can be used as a tool for visualizing an open chromatin structure in real time in living cells. The nucleic acid-binding fluorescent protein of the present invention can also be used as a tool for visualizing the open chromatin structure in fixed cells.

[0066] For example, by expressing or introducing the nucleic acid-binding fluorescent protein of the present invention into living cells, and using it as a probe to visualize the dynamic changes of chromatin, the epigenomic analysis of the whole cell can be performed. In addition, by utilizing the characteristics of being able to monitor the open chromatin structure in real time, the nucleic acid-binding fluorescent protein of the present invention can also be used for the analysis of the expression mechanism and differentiation of iPS cells, etc. For example, it can be used for cancer treatment that restores tumor tissue to its original differentiated state, and it can also be used for the quality management (Quality Control) of artificial cells such as iPS cells. Furthermore, it is believed that drugs such as steroids are widely distributed in the body and their effects penetrate into the nucleus to regulate the expression state of various genes and play a role, but it is also believed that it can be used for the dynamic observation of such drugs.

[0067] [Nucleic acid molecules]

[0068] The nucleic acid molecule containing the base sequence encoding the nucleic acid binding protein of the present invention (hereinafter sometimes referred to as the "nucleic acid molecule of the present invention") is not particularly limited as long as it is a molecule containing the base sequence of the amino acid sequence of the target nucleic acid binding protein obtained by translation. In the nucleic acid molecule of the present invention, the base sequence encoding the protein portion of the nucleic acid binding protein of the present invention is preferably appropriately designed in consideration of the codon usage frequency of the host expressing the protein. In order to reduce the risk of homologous recombination, the base sequence encoding the TAL-repeat portion in the DNA binding domain is preferably designed to differ from each other by utilizing codon fluctuations.

[0069] The nucleic acid molecules of the present invention can be chemically synthesized according to the base sequence information, or conventional techniques such as PCR can be used to modify naturally occurring TALE genes. For example, the nucleic acid molecules of the present invention can be prepared using a commercially available kit, which can be used to prepare DNA binding proteins using TALE as a DNA binding domain. As the kit, for example, various kits such as the Golden Gate TALEN and TALEffector Kit based on the Golden Gate method (non-patent document 3), the Yamamoto Lab TALEN Accessory Pack based on its modification method (non-patent document 4) that combines the Golden Gate method and the PCR method, and the REALAssembly TALEN Kit based on the REAL method (non-patent document 5) can be obtained from Addgene. These kits are very simple, and the previous TALE fusion proteins can be prepared by simply connecting the modules (DNA binding domains) corresponding to the target DNA sequence according to the manufacturer's manual. In the case where the nucleic acid binding protein of the present invention does not contain a nuclease, it is only necessary to simply remove the Fokl functional domain in the vectors contained in these kits. Furthermore, by using the conventional point mutation introduction technology listed above or conventional techniques such as PCR, the mutation is appropriately introduced into the vector of the kit to obtain the nucleic acid molecule of the present invention. In addition, the nucleic acid molecule of the present invention can also be manufactured by total synthesis using artificial gene synthesis technology.

[0070] [Expression vector]

[0071] The expression vector of the present invention is an expression vector that is integrated with the nucleic acid molecule of the present invention and can express the nucleic acid binding protein of the present invention in a host cell. Specifically, starting from the upstream, an expression cassette comprising a DNA having a promoter sequence, a nucleic acid molecule of the present invention, and a DNA having a terminator sequence is integrated into the expression vector. The production of the expression cassette and the integration of the expression cassette into the expression vector can be implemented by using known gene recombination techniques. Commercially available expression vector preparation kits can be used to integrate polynucleotides into the expression vector.

[0072] Host cells may be prokaryotic cells or eukaryotic cells. Prokaryotic cells may include Escherichia coli and other Escherichia bacteria, Bacillus subtilis and other Bacillus bacteria, Agrobacterium of Rhizobium bacteria (e.g., Rhizobium tumefacience, Rhizobium rhizogenes), etc. Eukaryotic cells may include yeasts such as Saccharomyces cerevisiae and Schizosaccharomyces pombe, insect cells such as established cell lines derived from larvae of cabbage armyworms (Spodoptera frugiperda cells; Sf cells), animal cells, plant cells, etc. As animal cells, there can be mentioned: cultured cells of established cell lines such as HEK293 cells (human-derived culture strains), COS-7 cells (monkey-derived culture strains), and CHO cells (Chinese hamster-derived culture strains); primary cultured cells collected from fetal or adult tissues such as mouse fetal fibroblasts MEF and primary cultured neural cells; ES cells made from fertilized eggs; iPS cells established from primary cultured cells, etc. Plant cells include cells in plants in addition to cultured cells from plants. Furthermore, plant cells from various forms of plants are also included, such as suspension culture cells, protoplasts, leaf slices, callus tissue, immature embryos, pollen, etc.

[0073] Animal cells used as host cells may be cells from mammals or cells from animals other than mammals. Examples of mammals include experimental animals such as rodents such as mice, rats, hamsters, and guinea pigs, or rabbits; livestock such as pigs, cattle, goats, horses, sheep, and minks; pets such as dogs and cats; humans or non-human primates (e.g., monkeys, cynomolgus monkeys, rhesus monkeys, marmosets, orangutans, chimpanzees, etc.). In addition, animals other than mammals include experimental animals such as nematodes (C.elegans), insects (fruit flies), fish (zebra fish, medaka), and amphibians (African clawed frogs, tropical clawed frogs), but are not limited thereto.

[0074] Plant cells used as host cells are any cells other than animal cells, in other words, any cells having cell walls. There is no particular limitation on the type of plant cells, and the cells may be cells from any plant including angiosperms and gymnosperms (seed plants), including monocotyledons and dicotyledons, mosses, ferns, herbs, and woody plants. Specific examples of plants include: Solanaceae plants such as eggplant, tomato, green pepper, hot pepper, and tobacco; Gramineae plants such as rice, wheat, barley, perennial ryegrass, Italian ryegrass, meadow fescue, reed fescue, orchardgrass, and timothy grass; Cruciferae plants such as Arabidopsis, rapeseed, Chinese cabbage, cabbage, radish, and rapeseed; Leguminosae plants such as soybean, adzuki bean, green bean, and broad bean; Cucurbitaceae plants such as cucumber, melon, watermelon, and pumpkin; Convolvulaceae plants such as sweet potato; Liliaceae plants such as shallot, onion, leek, garlic, and asparagus; Lamiaceae plants such as perilla; Asteraceae plants such as chrysanthemum, chrysanthemum, and lettuce; Rosaceae plants such as rose and strawberry; Rutaceae plants such as citrus and prickly ash; Myrtaceae plants such as eucalyptus; Salicaceae plants such as poplar; Spinach, sugar beet, and sugar beet. beet) and other Chenopodiaceae plants; Gentianaceae plants such as gentian; Carnation and other Caryophyllaceae plants. Solanaceae plants and cruciferous plants are particularly preferred, and tomatoes, tobacco and Arabidopsis are preferably used.

[0075] As the kind of expression vector, plasmid vector, viral vector etc. can be enumerated, and can be suitably selected according to the host cell used.Expression vector of the present invention can be manufactured by operably connecting nucleic acid of the present invention to the downstream of the promoter in the appropriate expression vector.In addition, the expression vector of the present invention imported into the host cell is preferably separated or purified.

[0076] Plasmid vectors include: plasmid vectors from Escherichia coli such as pBR322, pBR325, pUC12, and pUC13; plasmid vectors from Bacillus subtilis such as pUB110, pTP5, and pC194; plasmid vectors from yeast such as pSH19 and pSH15; plasmid vectors for eukaryotic cells such as pCS2, pCMV, pcDNA, and pCAGGS; binary vectors for plant cells, etc., which can be appropriately selected according to the type of host used or the purpose.

[0077] When the expression vector of the present invention is a viral vector, the type of viral vector used can be appropriately selected according to the type of host cell used or the purpose. For example, when insect cells are used as the host, baculovirus vectors can be used. In addition, when mammalian cells are used as the host, retrovirus vectors such as Moloney mouse leukemia virus vectors, lentivirus vectors, Sindbis virus vectors, adenovirus vectors, herpes virus vectors, adeno-associated virus vectors, parvovirus vectors, vaccinia virus vectors, Sendai virus vectors, etc. can be used.

[0078] In addition, the promoter used in the expression vector of the present invention can be selected according to the type of host cell used. A promoter that can initiate transcription in the host cell is selected. For example, when the host is an Escherichia bacterium, trp promoter, lac promoter, T7 promoter, etc. are preferred. When the host is a Bacillus bacterium, SPO1 promoter, SPO2 promoter, penP promoter, etc. are preferred. When the host is yeast, PHO5 promoter, PGK promoter, etc. are preferred. When the host is an insect cell, polyhedrin promoter, P10 promoter, etc. are preferred. When the host is a mammalian cell, subgenomic (26S) promoter, CMV promoter, SRα promoter, CAG promoter, EF1 promoter, etc. are preferred.

[0079] The expression vector of the present invention may contain an enhancer, a splicing signal, a polyA addition signal, a selection marker, an SV40 replication origin, etc., as necessary, in a manner that enables the respective functions to be exerted. Examples of the selection marker include: a dihydrofolate reductase gene [methotrexate (MTX) resistance gene], an ampicillin resistance gene, a neomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, etc.

[0080] By utilizing known gene introduction methods to introduce the expression vector of the present invention into host cells, transformants (transformed cells) into which the expression vector has been introduced can be manufactured. As known gene introduction methods, for example, liposome transfection, calcium phosphate method, microinjection, protoplast fusion, electroporation, DEAE dextran method, Gene Gun gene transfer method, etc. can be cited. The transformants produced can express the nucleic acid-binding protein of the present invention. That is, the expression vector of the present invention and the transformants into which the expression vector has been introduced can be used for the manufacture of nucleic acid-binding protein of the present invention, etc.

[0081] The nucleic acid binding protein of the present invention can be expressed in cells by synthesizing RNA from the expression vector of the present invention through in vitro transcription reaction and introducing it into fertilized eggs or cells.

[0082] The nucleic acid-binding protein of the present invention can be produced by culturing transformed cells into which the expression vector of the present invention has been introduced by a known method depending on the type of host. The produced nucleic acid-binding protein can be used directly in the transformed cells as it is, or can be isolated or purified from the culture of the transformed cells.

[0083] The transformed cells whose host is Escherichia bacteria are 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 transformed cells whose host is Bacillus bacteria are cultured in an appropriate medium, usually at about 30 to 40°C for about 6 to 24 hours. The transformed cells whose host is yeast are cultured in an appropriate medium such as Burkholder medium, usually at about 20 to 35°C for about 24 to 72 hours. The transformed cells whose host is insect cells or insects are cultured in an appropriate 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 transformed cells whose host is animal cells are cultured in an appropriate medium such as MEM medium supplemented with about 10% bovine serum, usually at about 30 to 40°C for about 15 to 60 hours. In any culture, aeration or stirring can be performed as needed. Isolation or purification of the nucleic acid-binding protein of the present invention from the culture can be achieved, for example, by subjecting the bacterial cell lysate or culture supernatant to various types of chromatography, such as reverse phase chromatography, ion exchange chromatography, and affinity chromatography.

[0084] By introducing the expression vector of the present invention into the fertilized egg of a non-human animal using a known gene introduction method, the nucleic acid molecule of the present invention is integrated into the cells constituting the body, and a transformed non-human animal expressing the nucleic acid-binding protein of the present invention can be obtained under constant or specific conditions. The nucleic acid molecule of the present invention can be integrated into the chromosome or outside the chromosome. In addition, when the nucleic acid molecule of the present invention contains the promoter of a gene expressed by tissue specificity, a transformed non-human animal expressing the nucleic acid-binding protein of the present invention only in the specific tissue can be obtained. The introduction of the expression vector into the fertilized egg can be carried out using a known method such as microinjection. As the fertilized egg into which the expression vector of the present invention is introduced, a fertilized egg of an animal other than humans from the animal cells listed as the host cell can be used.

[0085] By introducing the expression vector of the present invention into the callus of a plant according to a known gene introduction method, and redifferentiating the obtained transformed callus, it is possible to obtain redifferentiated individuals of the transformed plant expressing the nucleic acid binding protein of the present invention in the cell. The expression vector can be introduced into the callus by a known method such as the Agrobacterium method. As the callus into which the expression vector of the present invention is introduced, callus from the plant listed in the plant cell used as the host cell prepared by a conventional method can be used.

[0086] Transformed animals or plants expressing the nucleic acid-binding protein of the present invention, or tissues or cells collected therefrom, can be used to analyze open chromatin structures. For example, transformed animals or plants expressing the nucleic acid-binding fluorescent protein of the present invention can visualize open chromatin structures in real time, and thus by analyzing open chromatin structures at specific development and differentiation stages, or studying open chromatin structures before and after treatment with specific drugs, changes in open chromatin structures caused by the drug treatment can be observed.

[0087] For example, by expressing or introducing the nucleic acid-binding fluorescent protein of the present invention into living cells and using it as a probe to visualize the dynamic changes of chromatin, the epigenomic analysis of the entire cell can be performed. In addition, the nucleic acid-binding fluorescent protein of the present invention can also be used to analyze the expression mechanism and differentiation of iPS cells by utilizing the characteristic of being able to detect open chromatin structure in real time.

[0088] [Method for fluorescently labeling open chromatin in living cells]

[0089] By introducing the nucleic acid-binding fluorescent protein of the present invention into living cells, the open chromatin structure can be fluorescently labeled. When the nucleic acid-binding fluorescent protein of the present invention is a protein comprising a DNA binding domain and a fluorescent protein composed of a structure consisting of more than 3, more preferably more than 8, non-selective binding TAL-repeat repeats, the nucleic acid molecule (gene) encoding the protein can be introduced into living cells to express the protein in the living cells. As the introduced living cells, the same cells as those listed in the method for producing the above-mentioned transformant can be used. In addition, as a method for introducing genes (nucleic acid molecules) into living cells, it can be carried out in the same manner as the method for producing the above-mentioned transformant.

[0090] The purified nucleic acid-binding fluorescent protein of the present invention can be directly injected into living cells.

[0091] The introduction of the nucleic acid binding fluorescent protein of the present invention into living cells can be implemented using a known method for introducing proteins into cells. As this method, for example, it can be enumerated: a method using a protein introduction reagent, a method using a protein introduction domain (PTD) or a cell penetrating peptide (CPP) fusion protein, electroporation, microporation, microinjection, delivery of bacterial type III secretion system, etc. As a protein introduction reagent, there are commercially available cationic lipid-based BioPOTER Protein Delivery Reagent (Gene Therapy Systmes, Inc.), Pro-Ject (registered trademark) Protein Transfection Reagent (PIERCE, Inc.), and ProVectin (IMGENEX, Inc.), lipid-based Profect-1 (Targeting Systems), Penetrain Peptide (Q biogene, Inc.) and Chariot Kit (Active Motif, Inc.), using HVJ envelope (inactivation of Sendai virus) GenomONE (Ishihara Sangyo Co., Ltd.), etc. The introduction can be carried out according to the protocol attached to these reagents, but the general steps are as follows. The nucleic acid-binding fluorescent protein of the present invention is diluted in an appropriate solvent (e.g., PBS, HEPES or other buffer), an introduction reagent is added, and the mixture is incubated at room temperature for about 5 to 15 minutes to form a complex, which is then added to cells replaced with a serum-free medium and incubated at 37° C. for 1 to several hours. The medium is then removed and replaced with a serum-containing medium. In addition, in order to introduce proteins into plant cells, protoplasts can also be prepared from the cells according to known methods.

[0092] As PTDs, proteins such as AntP from Drosophila, TAT from HIV (Frankel, et al., Cell, vol. 55, p. 1189-1193 (1988); Green & Loewenstein, Cell, vol. 55, p. 1179-1188 (1988)), Penetratin (Derossi, et al., J. Biol. Chem., vol. 269, p. 10444-10450 (1994); Park, et al., Proc. Natl Acad Sci, USA vol. 97, p. 8245-8250 (2000)), Transportan (Pooga, et al., FASEB J., vol. 12, p. 67-77 (1998)), MAP (model amphipathic peptide) (Oehlke, et al., Cell, vol. 13, p. 67-77 (1998)), and MAP (model amphipathic peptide) (Oehlke, et al., Cell, vol. 13, p. 67-77 (1998)) have been developed. al, Biochim Biophys Acta. Vol. 1414, p. 127-139 (1998)), K-FGF (Lin, et al., J. Biol. Chem., vol. 270, p. 14255-14258 (1995)), Ku70 (Sawada, et al., Nature Cell Biol. Vol. 5, p. 352-357 (2003)), Prion (Lundberg, et al., Biochem. Biophys. Res. Commun. Vol. 299, p. 85-90 (2002)), pVEC (Elmquist, et al., Exp. Cell Res., vol.269, p.237-244(2001)), Pep-1 (Morris, et al. al., Nature Biotechnol. Vol. 19, p. 1173-1176 (2001)), Pep-7 (Gao et al, Bioorg. Med. Chem. Vol. 10, p. 4057-4065 (2001)), SynBl (Rousselle, et al., Mol. Pharmacol. Vol. 57, p. 679-686 (2000)), HN-I (Hong & Clayman, Cancer Res., vol. 60, p. 6551-6556 (2000)), VP22 from HSV, etc.Examples of CPP derived from PTD include polyarginine such as 11R (Cell Stem Cell, vol. 4, p. 381-384 (2009)) and 9R (Cell Stem Cell, vol. 4, p. 472-476 (2009)).

[0093] A fusion protein expression vector incorporating the cDNA of the nucleic acid-binding fluorescent protein of the present invention and the PTD sequence or CPP sequence is prepared, recombinantly expressed, and the fusion protein is recovered and used for introduction. The introduction can be performed in the same manner as above except that no protein introduction reagent is added.

[0094] Microinjection is a method in which a protein solution is loaded into a glass needle with a tip diameter of about 1 μm and then punctured into cells. This method can reliably introduce proteins into cells.

[0095] In addition, protein introduction methods such as electroporation, semi-active cell method (Kano, et al., Methods in Molecular Biology, vol. 322, p. 357-365 (2006)), and Wr-T peptide-based introduction method (Kondo, et al., Mol. Cancer Ther. Vol. 3 (12), p. 1623-1630 (2004)) can also be used.

[0096] The protein introduction operation can be performed any number of times, for example, from 1 to 10 times, preferably from 2 to 5 times, more preferably from 2 to 5 times, and further preferably from 3 to 4 times. The interval between repeated introduction operations is, for example, 6 to 48 hours, preferably 12 to 24 hours.

[0097] Example

[0098] Next, the present invention will be described in further detail with reference to Examples and the like, but the present invention is not limited to these Examples.

[0099] [Example 1]

[0100] The RVD sequences of the DNA binding domains of approximately 1,000 TALEs possessed by various bacteria of the genus Xanthomonas were detected, and TALE variants containing TAL-repeats having these RVD sequences were prepared to study the binding properties to nucleic acid bases.

[0101] <Preparation of proteins with DNA binding domain>

[0102] As a TAL-repeat having various RVD sequences, a TALE sequence based on a known Xanthomonas bacterium is prepared by PCR, and the obtained TAL-repeat is repeatedly connected to produce a DNA binding domain having a TAL-repeat with a target RVD sequence arranged in 15 repeats, and a protein having a peptide tag attached to the N-terminus and a fluorescent protein (EGFP, etc.) or a luminescent protein (cyan Nano-Lantern, etc.) attached to the C-terminus (Takai et al., PNAS, 2015, vol. 112, p. 4352-4356.).

[0103] Figure 1 The schematic diagram shows the structure of the GFP fusion protein used as a visualization probe. The GFP fusion protein is a protein comprising a DNA binding domain in which TAL-repeat is repeatedly arranged, a peptide tag is linked to the N-terminus, and a GFP protein is linked to the C-terminus.

[0104] <Evaluation of nucleic acid binding specificity>

[0105] The binding of the prepared TALE variants to nucleic acid bases was measured by a reporter assay using the intracellular luminescent protein orange Nano-Lantern (Takai et al., ibid) and by an in vitro ELISA method using a luminescent protein fused to the C-terminus of each protein.

[0106] As a result, as a TALE variant that binds to the four base residues with approximately the same degree of binding force, a variant consisting of the amino acid sequence of sequence numbers 1 to 3, i.e., the RVD sequence is HT, RS or HS, and the 4th and 32nd amino acid sequences are glutamic acid and alanine, respectively, was selected.

[0107] <The effect of the number of TAL-repeat repeats on binding to open chromatin structure>

[0108] The DNA binding domain composed of TAL-repeat that non-selectively binds to four bases was introduced into living cells as an EGFP fusion protein to test whether the domain can be used as a probe to identify open chromatin structure. At this time, DNA binding domains with different numbers of TAL-repeat repeats were introduced to study the effect of different numbers of TAL-repeat repeats on binding to open chromatin structure.

[0109] As the DNA binding domain, a domain having a structure consisting of 1, 2, 3, 8, 12 or 15 non-selective binding TAL-repeat (RVD sequence is HT) consisting of the amino acid sequence of SEQ ID NO: 1 was used. In addition, the above-mentioned kit was used to prepare an expression vector incorporating a nucleic acid molecule encoding a GFP fusion protein having each DNA binding domain.

[0110] The fluorescence recovery after photofading (FRAP) method was performed on cells expressing GFP fusion proteins with different numbers of non-selective binding TAL-repeat repeats in the DNA binding domain to study the binding activity of DNA and GFP fusion proteins. Specifically, the prepared expression vector was introduced into HeLa cells using a liposome transfection reagent to express the GFP fusion protein.

[0111] Figure 2A Fluorescent images of cells expressing GFP fusion proteins are shown. In addition, Table 2 shows the results of the tendency of nuclear staining unevenness (coefficient of variation or "variation coefficient"). According to the results, GFP fusion proteins with one or two non-selective binding TAL-repeat repeats in the DNA binding domain are positioned in a nearly uniform state in the nucleus, and there is no difference in positioning between the GFP fusion proteins that do not contain non-selective binding TAL-repeat. In contrast, in the case of GFP fusion proteins with a DNA binding domain with three or more non-selective binding TAL-repeat repeats, fluorescent images of binding to specific genomic structures were obtained in the nucleus.

[0112] The nuclei of the expressing cells were irradiated with laser light, and the time for the fluorescence of the area faded by the laser light to recover was measured. The results of FRAP measurement are shown in Table 2 and Figure 2B The time required for fluorescence recovery in FRAP (T) is shown in Figure 2 for GFP fusion proteins with a non-selective TAL-repeat DNA binding domain with a repeat number of 3 or more. 1 / 2) becomes significantly longer, and the time required for recovery is significantly longer when the number of tandem repeats is more than 8. Furthermore, in the case of 12 and 15 tandem repeats, it was confirmed that the time required for recovery is significantly prolonged. On the other hand, the unevenness of nuclear staining is similar to that of cells without non-selective binding TAL-repeat when the number of repeats of non-selective binding TAL-repeat is 1 or 2, and increases significantly when the number of repeats is more than 3. However, the unevenness of nuclear staining is roughly the same when the number of repeats is 3 to 15. From these results, it can be confirmed that the DNA binding domain of non-selective binding TAL-repeat with a repeat number of more than 3 can bind to nucleic acids in chromosomes; and the stronger the binding force with nucleic acids, the specificity of binding to nucleic acids is basically saturated when the number of repeats is 3, and there is little change even if the number of repeats is increased to 15.

[0113] [Table 2]

[0114]

[0115] [Example 2]

[0116] In order to study which structure in the nucleus is marked by the DNA binding domain composed of multiple repeats of non-selective binding TAL-repeat, a comparison was made with the labeling methods used to label various genomic structures in previous epigenome analysis. As previous labeling methods, nuclear staining, immunostaining methods for various modified histones, and the ATAC-see method (Non-Patent Document 6) were used.

[0117] As a fluorescent protein containing a DNA binding domain, a GFP fusion protein having a DNA binding domain in which 15 non-selective binding TAL-repeat repeats were linked (hereinafter sometimes referred to as "ChrocodiLE") prepared in Example 1 was used.

[0118] First, for HeLa cells expressing ChrocodiLE, the cells were kept alive and stained with Hoechst fused to silicon rhodamine (SiR-Hoechst), and the intracellular localization of ChrocodiLE and SiR-Hoechst was compared. In addition, the ChrocodiLE expression vector was introduced into HeLa cells that stably expressed the red fluorescent protein (RFP) fused to histone 2B (H2B) protein, so that the two were co-expressed, and the cells were kept alive to compare the intracellular localization of ChrocodiLE and H2B. As a result, the areas that reflect the local density of nucleosomes and are stained with SiR-Hoechst, the areas around the nuclear membrane stained with FRB-H2B, and the heterochromatin aggregation areas around the nucleosomes were hardly stained with ChrocodiLE. From these results, it can be confirmed that ChrocodiLE does not bind to areas with dense heterochromatin and areas of heterochromatin aggregation in living cells.

[0119] Next, HeLa cells expressing ChrocodiLE were fixed, and the genome accessible to the transposase (open chromatin) was labeled with a red fluorescent substance using the ATAC (Assay for Transposase-Accessible Chromatin)-see method (in situ hybridization using fluorescently labeled Tn5 transposase).

[0120] Separately, HeLa cells expressing ChrocodiLE were fixed, and fluorescent immunostaining of each modified histone was performed using anti-H3K4me 3 antibody, anti-H3K27ac antibody, anti-H3K9me 3 antibody, or anti-H3K27me 3 antibody as a primary antibody and a secondary antibody labeled with a red fluorescent substance as a secondary antibody. H3K4me 3 and H3K27ac are modified forms of histones that are mainly present in open chromatin, and H3K9me 3 and H3K27me 3 are modified forms of histones that are mainly present in condensed chromatin.

[0121] The fluorescence images of each cell after fluorescence staining are shown in Figure 2. Figure 3A In addition, for each cell after fluorescence staining, the ratio (correlation coefficient) of co-localization (co-localization) of green fluorescence and red fluorescence of ChrocodiLE was studied, and the results are as follows Figure 3BAs shown. As a result, the correlation coefficients of ChrocodiLE with ATAC, H3K4me 3 and H3K27ac were high, especially the correlation coefficient with ATAC was very high, reaching more than 0.5. On the other hand, the correlation coefficients of ChrocodiLE with H3K9me 3 and H3K27me 3 were low, especially with H3K9me 3, there was no co-localization. These results indicate that ChrocodiLE specifically recognizes open chromatin structure.

[0122] Next, the binding region of ChrocodiLE in the genome was studied using the chromatin immunoprecipitation (ChIP) method and the DNA sequencing method using the next generation sequencer. Specifically, HeLa cells expressing ChrocodiLE were fixed with formaldehyde to cross-link the genomic DNA with ChrocodiLE, and then the genomic DNA was recovered and fragmented by enzyme digestion. The chopped genomic DNA was immunoprecipitated using an antibody against the peptide tag at the N-terminus of ChrocodiLE, and the base sequence of the recovered DNA was analyzed using a next generation sequencer. Thus, the base sequence of the genomic region bound by ChrocodiLE was identified.

[0123] For HeLa cells that do not express ChrocodiLE, ChIP was also performed using anti-H3K4me 3 antibodies, anti-H3K27 ac antibodies, anti-H3K9me 3 antibodies, or anti-H3K27me 3 antibodies, and the base sequence of the DNA recovered by immunoprecipitation was analyzed using a next-generation sequencer to identify the base sequence of the genomic region bound by each modified histone. In addition, ATAC-seq was performed to identify the base sequence of the genomic region with an open chromatin structure.

[0124] Figure 4 The binding regions of ChrocodiLE and the transcription start site (TSS) identified in the regions of 36, 100 to 36, and 270 kb of chromosome 19 are shown in the figure. In addition, the genomic regions of open chromatin structure identified by ATAC-seq (the top section in the figure) and the genomic regions bound by four modified histones are also shown. As a result, it can be seen that ChrocodiLE binds to regions roughly the same as the regions identified by ATAC-seq. In addition, ChrocodiLE also binds to the vicinity of the transcription start site of UPK1A, which was not identified by ATAC-seq. Since there is a high possibility that there is an open chromatin structure near the transcription start site, it can be considered that ChrocodiLE can identify open chromatin structure in the same way as ATAC-seq, and can also identify open chromatin structure that cannot be identified by ATAC-seq. These results show that ChrocodiLE is very useful as a probe for marking open chromatin structure.

[0125] [Example 3]

[0126] Transgenic mice (hereinafter sometimes referred to as "ChrocodiLE-Tg mice") were prepared, wherein the transgenic mice were obtained by knocking in a gene encoding ChrocodiLE-EGFP into the ROSA26 locus, and the ChrocodiLE-EGFP was obtained by fusing the ChrocodiLE prepared in Example 1 (a protein having a DNA binding domain obtained by repeatedly connecting 15 non-selective binding TAL-repeat) with the fluorescent protein EGFP. ChrocodiLE-EGFP was prepared by connecting a base sequence encoding ChrocodiLE (sequence number 4) with a base sequence encoding EGFP. The preparation of transgenic mice was carried out according to the method of Abe et al. (Non-patent document 7).

[0127] Thin slices were made from each tissue of the ChrocodiLE-Tg mouse, and fluorescence images were obtained. Fluorescence images of the cerebral cortex, hippocampus, and cerebellar cortex in the brain and fluorescence images of skin tissue are shown in Figure 1. Figure 5 As shown, ChrocodiLE-EGFP expression was found in all tissues.

[0128] [Example 4]

[0129] Transgenic nematodes (hereinafter sometimes referred to as "ChrocodiLE-Tg nematodes") were prepared, wherein the gene encoding ChrocodiLE-GFP was inserted into the transgenic nematodes, and the ChrocodiLE-GFP was obtained by fusing the ChrocodiLE (a protein having a DNA binding domain formed by repeatedly connecting 15 non-selective binding TAL-repeat) prepared in Example 1 with the fluorescent protein GFP. The preparation of ChrocodiLE-Tg nematodes was carried out by the MosSCI method. The expression vector was prepared by inserting the following fragment into pCFJ 150-pDESTttTi 5605[R4-R3] (Plasmid#19329, manufactured by Addgene), wherein the gene encoding ChrocodiLE-GFP was inserted between mex5 (promoter) from pJA252 (Plasmid#21512, manufactured by Addgene) and tbb2 (3'UTR) from pCM1.36 (Plasmid#17249).

[0130] Thin sections were made from the gonads and developing embryos of the ChrocodiLE-Tg nematodes, and fluorescence images were obtained. Figure 6As shown. Regarding the gonad, a transmitted light image of the same field of view is also shown. In addition, in the fluorescent image of the embryo, the right image is an enlarged image of the left image. ChrocodiLE-EGFP expression was also found in the gonad and developing embryo.

[0131] Industrial feasibility

[0132] The nucleic acid binding protein of the present invention strongly binds to nucleic acids independently of the base sequence. Therefore, the nucleic acid binding protein is useful as a probe that specifically recognizes open chromatin structure, and is particularly useful for epigenomic studies in living cells. By using the nucleic acid binding protein, unknown gene activation sites can be detected, and structural changes in chromatin can be tracked by following the passage of time in living cells, and it becomes an effective tool for clarifying the effects of drugs and the relationship between diseases and epigenomic changes. Sequence Listing <110> Japan Science and Technology Agency <120> PC-29324 <130> Nucleic acid binding protein <160> 4 <210> 1 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> TAL-repeat in which RVD is HT <400> 1 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Thr Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly <210> 2 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> TAL-repeat in which RVD is RS <400> 2 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser Arg Ser Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly <210> 3 <211> 34 <212> PRT <213> Artificial Sequence <220> <223> TAL-repeat in which RVD is HS <400> 3 Leu Thr Pro Glu Gln Val Val Ala Ile Ala Ser His Ser Gly Gly Lys 1 5 10 15 Gln Ala Leu Glu Thr Val Gln Arg Leu Leu Pro Val Leu Cys Gln Ala 20 25 30 His Gly <210> 4 <211> 10617 <212> DNA <213> Artificial Sequence <220> <223> Gene coding ChrocodiLE <400> 4 ctaaattgta agcgttaata ttttgttaaa attcgcgtta aatttttgtt aaatcagctc 60 attttttaac caataggccg aaatcggcaa aatcccttat aaatcaaaag aatagaccga 120 gatagggtttg agtgttgttc cagtttggaa caagagtcca ctattaaaga acgtggactc 180 caacgtcaaa gggcgaaaaa ccgtctatca gggcgatggc ccactacgtg aaccatcacc 240 ctaatcaagt tttttggggt cgaggtgccg taaagcacta aatcggaacc ctaaagggag 300 cccccgattt agagcttgac ggggaaagcc ggcgaacgtg gcgagaaagg aagggagaa 360 aggaaagga gcgggcgcta gggcgctggc aagtgtagcg gtcacgctgc gcgtaaccac 420 cacaccccgcc gcgcttaatg cgccgctaca gggcgcgtcc cattcgccat tcaggctgcg 480 caactgttgg gaagggcgat cggtgcgggc ctcttcgcta ttacgccagc tggcgaaagg 540 gggatgtgct gcaaggcgat taagttgggt aacgccaggg ttttcccagt caggacgttg 600 taaacgacg gccagtgagc gcgcgtaata cgactcacta tagggcgaat tgggtaccac 660 gcgtgtagtc ttatgcaata ctcttgtagt cttgcaacat ggtaacgatg agttagcaac 720 atgccttaca aggagagaaa aagcaccgtg catgccgatt ggtggaagta aggtggtacg 780 atcgtgcctt attack caacagacgg gtctgacatg gattggacga accactgaat tgccgcattg cagagatatt gtatttaagt gcctagctcg attack cgggtctctc tggttagacc agatctgagc ctggggagctc tctggctaac tagggacc actgcttaag cctcaataaa gcttgccttg agtgcttcaa gtagtgtgtg cccgtctgtt gtgtgactct ggtaactaga gatccctcag acccttttag tcagtgtgga aaatctctag cagtggcgcc cgaacaggga cctgaaagcg aaagggaaac cagagctctc tcgacgcagg actcggcttg ctgaagcgcg cacggcaaga ggcgaggggc ggcgactggt gagtacgcca aaaatttga ctagcggagg ctagaaggag agagatgggt gcgagagcgt cgtattaag cgggggagaa 1260 ttagatcgcg atgggaaaa attcggttaa ggccaggggg aaagaaaaaa tataattaa aacatatagt atgggcaagc agggagctag aacgattcgc agttaatcct ggcctgttag 1380. aaacatcaga aggctgtaga caatactgg accatccctt cagacaggat splitter tagatcatta tatatacag tagcaaccct ctattgtgtg catchaagga 1500 windowgaaaaaaaaaagtaagaaaaaagta 1560 agaccaccgc acagcaagcg gccgctgatc ttcagacctg gaggaggaga tatgagggac 1620 aattggagaa gtgaattata taaattaaa gtagtaaaa ttgaaccatt aggagtagca 1680 cccaccaagg aagagag agtggtgcag aggaaaaaa gagcagtggg ataggagct 1740 ttgttccttg gttcttggg agcagcagga agcactatgg gcgcagccctc atgacgctg 1800 acggtacagg ccagacaatt attgtctggt atagtgcagc agcagaaca ttgctgagg 1860 gctattgagg cgcacagca tctgttgcaa ctcacagtct ggggcatca gcagctcag 1920 gcaagaatcc tggctgtgga aagataccta aaggatcac agctcctggg gatttggggt 1980 tgctctggaa aactcatttg caccactgct gtgccttgga atgctagttg gagtaataaa 2040 tctctggaac agattggaat cacacgacct ggatggagtg ggacagagaa attackacatt 2100 accacaagctt atacactcc ttaattgaag atcgcaaaa ccacaagct aagaatgaac 2160 aagaattatt ggaattagat aaatgggcaa gttgtgga ttggtttaac atacaatt 2220 ggctgtgta tataaaatta ttcataatga tagtaggagg cttggtaggt ttagaatag 2280 ttttgctgt actttctata gtgaatagag ttaggcagg atattcacca ttatcgtttc 2340 agacccacct cccaaccccg agggaccg acaggcccga aggaatagaa gaagaaggtg 2400 gagagaga cagagacaga tccattcgat tagtgaacgg atctcgacgg ttaactttta 2460 aaagaaaagg ggggattggg gggtacagtg cagggaaag atagtagac atatagcaa 2520 cagacataca aactaagaa ttacaaaaac aaattacaa attcaaatt ttgggccctt 2580 ccaaccttcc aaccggtacc tagtaatcaa ttacggggtc attagttcat agcccatata 2640 tggagttccg cgttacataa cttacggtaa atggcccgcc tggctgaccg cccaacgacc 2700 cccgcccatt gacgtcaata atgacgtag ttcccatagt aacgccaata gggactttcc 2760 attgacgtca atgggtggac tatttacggt aactgccca cttggcagta catcaagtgt 2820 atcatatgcc aagtacgccc cctattgacg tcaatgacgg taatggccc gcctggcatt 2880 atgcccagta catgacctta tgggactttc ctacttggca gtacatctac gtattagtca 2940 tcgctattac catgggtcga ggtgagcccc acgttctgct tcactctccc catctccccc 3000 ccctccccac ccccaatttt gtatttattt attttttaat tattttgtgc agcgatgggg 3060 gcgggggggg ggggggcgcg cgccaggcgg ggcggggcgg ggcgaggggc ggggcggggc 3120 gaggcggaga ggtgcggcgg cagccaatca gagcggcgcg ctccgaaagt ttccttttat 3180 ggcgaggcgg cggcggcggc ggccctataa aaagcgaagc gcgcggcggg cgggagtcgc 3240 tgcgttgcct tcgccccgtg ccccgctccg cgccgcctcg cgccgcccgc cccggctctg 3300 actgaccgcg ttactcccac aggtgagcgg gcgggacggc ccttctcctc cgggctgtaa 3360 ttagcgcttg gtttaatgac ggctcgtttc ttttctgtgg ctgcgtgaaa gccttaaagg 3420 gctccgggag ggccctttgt gcggggggga gcggctcggg gggtgcgtgc gtgtgtgtgt 3480 gcgtggggag cgccgcgtgc ggcccgcgct gcccggcggc tgtgagcgct gcgggcgcgg 3540 cgcggggctt tgtgcgctcc gcgtgtgcgc gaggggagcg cggccggggg cggtgccccg 3600 cggtgcgggg gggctgcgag gggaacaaag gctgcgtgcg gggtgtgtgc gtgggggggt 3660 gagcaggggg tgtgggcgcg gcggtcgggc tgtaaccccc ccctgcaccc ccctccccga 3720 gttgctgagc acggcccggc ttcgggtgcg gggctccgtg cggggcgtgg cgcggggctc 3780 gccgtgccgg gcggggggtg gcggcaggtg ggggtgccgg gcggggcggg gccgcctcgg 3840 gccggggagg gctcggggga ggggcgcggc ggccccggag cgccggcggc tgtcgaggcg 3900 cggcgagccg cagccattgc cttttatggt aatcgtgcga gagggcgcag ggacttcctt 3960 tgtcccaaat ctggcggagc cgaaatctgg gaggcgccgc cgcaccccct ctagcgggcg 4020 cgggcgaagc ggtgcggcgc cggcaggaag gaaatgggcg gggagggcct tcgtgcgtcg 4080 ccgcgccgcc gtccccttct ccatctccag cctcggggct gccgcagggg gacggctgcc 4140 ttcggggggg acggggcagg gcggggttcg gcttctggcg tgtgaccggc ggctctagag 4200 cctctgctaa ccatgttcat gccttcttct ttttcctaca gctcctgggc aacgtgctgg 4260 ttgttgtgct gtctcatcat tttggcaaag gattcctcga ggtcgacacc atggctccaa 4320 agaagaagcg taaggtagac tacaaagacc atgacggtga ttaaagat catgacatcg attacaagga tgacgatgac aagggtaccg tggatctgag aaccctgggc tacagccagc 4500. 4500. 4500. 4500. 4500. 4500. 4500. 4500. 4500. 4560. tcgtgggaca cggcttcacc cacgcccata tcgtggccct gtctcagcat cctgccgccc 4620. tgggaacagt ggccgtgacc taccagcaca tcatcaccgc cctgcctgag gccacccacg aggatattgt gggagtgggc aagcagtgga gcggcgccag agcactgga gccctgctga 4680. cagatgccgg cgagctgaga ggacctcccc tgcagctgga tacaggccag ctcgtgaaga 4740 tcgccaagcg gggaggcgtg acagccatgg aagccgtgca cgccagcaga aacgctctga caggcgctcc cctgaacctg acaccagagc aagtggtggc aatcgcctcc cacactggcg 4920. actcgaaact gtccaaaggc ttcttcccgt gctctgccag gctcatgggc tgacaccaga gcaagtggtg gctattgcat ctcataccgg aggcaaacaa gctctcgaaa 5040. ccgtccagag acttctgcca gtcctctgtc aggctcacgg gctcacacct gaacaggtgg tcgcaatcgc atctcataca ggaggaaaac aggccctcga aacagtgcaa aggctgctgc 5100 ctgtgctgtg tcaggctcat ggactgacac ctgagcaagt cgtggccatt gcctctcata 5160 cagggggaaa gcaagccctc gaaactgtgc agagactgct tcctgtcctg tgccaggctc 5220 atggacttac acccgaacaa gtggtggcca ttgcatctca taccggaggc aagcaagccc 5280 tcgaaactgt ccagaggctg cttcctgtgc tgtgtcaggc acatggcctt acaccagagc 5340 aggtggtggc tattgcatcc cacactgggg ggaagcaggc tctcgaaact gtccagaggc 5400 ttcttcctgt gctgtgccag gcacatggac tgacacctga acaggtcgtg gctatcgctt 5460 ctcatactgg ggggaaacag gctctcgaaa cagtgcagag actgctgcca gtgctttgtc 5520 aagctcatgg ccttacacca gagcaagtcg tcgccatcgc ttctcacact ggcggcaagc 5580 aggccctcga aactgtgcag aggcttcttc ctgtgctttg tcaagctcat ggactgacac 5640 ctgagcaggt cgtcgctatt gcctcccata ctgggggaaa gcaggctctc gaaactgtcc 5700 agaggctcct tcctgtcctt tgtcaagcac atggacttac acccgaacaa gtggtggcca 5760 ttgcatccca taccggcggc aagcaagctc tcgaaaccgt gcaaaggctt cttcccgtgc 5820 tctgccaagc tcatgggctg acacctgaac aggtcgtggc aattgcatcc catactggag 5880 gcaagcaagc actcgaaaca gtgcaaagac tgcttcccgt cctttgtcag gcacacgggc 5940 ttacacctga acaggtggtg gcaattgcct cccataccgg gggaaagcag gctctcgaaa 6000 cagtgcaaag acttctgcct gtgctgtgcc aggcacacgg cctgacaccc gaacaggtgg 6060 tggctatcgc ctcccatacc ggaggcaagc aggctctcga aactgtgcaa agactcctgc 6120 ctgtgctgtg ccaggcacac gggcttacac ctgagcaagt cgtggctatc gcttcccata 6180 cagggggaaa gcaggctctc gaaacagtgc aaaggcttct ccctgtgctt tgtcaggctc 6240 acgggctgac acccgagcaa gtggtcgcca tcgcttctca tacaggcggc aaacaggctc 6300 tcgaaagcat cgtggcccag ctgagcagac ccgaccccgc cctggccgcc ctgaccaacg 6360 accatctggt ggccctggcc tgtctgggag gcagacctgc tatggacgcc gtgaagaagg 6420 gactgcctca cgcccccgag ctgatcagaa gagtgaacag acggatcggc gagcggacca 6480 gccatagagt ggctggatcc atggtgagca agggcgagga gctgttcacc ggggtggtgc 6540 ccatcctggt cgagctggac ggcgacgtaa acggccacaa gttcagcgtg tccggcgagg 6600 gcgagggcga tgccacctac ggcaagctga ccctgaagtt catctgcacc accggcaagc 6660 tgcccgtgcc ctggcccacc ctcgtgacca ccctgaccta cggcgtgcag tgcttcagcc 6720 gctaccccga ccacatgaag cagcacgact tcttcaagtc cgccatgccc gaaggctacg 6780 tccaggagcg caccatcttc ttcaaggacg acggcaacta caagacccgc gccgaggtga 6840 agttcgaggg cgacaccctg gtgaaccgca tcgagctgaa gggcatcgac ttcaaggagg 6900 acggcaacat cctggggcac aagctggagt acaactacaa cagccacaac gtctatatca 6960 tggccgacaa gcagaagaac ggcatcaagg tgaacttcaa gatccgccac aacatcgagg 7020 acggcagcgt gcagctcgcc gaccactacc agcagaacac ccccatcggc gacggccccg 7080 tgctgctgcc cgacaaccac tacctgagca cccagtccgc cctgagcaaa gaccccaacg 7140 agaagcgcga tcacatggtc ctgctggagt tcgtgaccgc cgccgggatc actctcggca 7200 tggacgagct gtacaaggga aagcccatcc ctaacccact gctcggcctg gacagcacct 7260 aacgatccac tagtctcgac tcgacaatca acctctggat tacaaaattt gtgaaagatt 7320 gactggtatt cttaactatg ttgctccttt tacgctatgt ggatacgctg ctttaatgcc 7380 tttgtatcat gctattgctt cccgtatggc tttcattttc tcctccttgt ataaatcctg 7440 gttgctgtct ctttatgagg agttgtggcc cgttgtcagg caacgtggcg tggtgtgcac 7500 tgtgtttgct gacgcaaccc ccactggttg gggcattgcc accacctgtc agctcctttc 7560 cgggactttc gctttccccc tccctattgc cacggcggaa ctcatcgccg cctgccttgc 7620 ccgctgctgg acaggggctc ggctgttggg cactgacaat tccgtggtgt tgtcggggaa 7680 gctgacgtcc tttccatggc tgctcgcctg tgttgccacc tggattctgc gcgggacgtc 7740 cttctgctac gtcccttcgg ccctcaatcc agcggacctt ccttcccgcg gcctgctgcc 7800 ggctctgcgg cctcttccgc atcttcgcct tcgccctcag acgagtcgga tctccctttg 7860 ggccgcctcc ccgcctggaa ttaattcgag ctcggtacct ttaagaccaa tgacttacaa 7920 ggcagctgta gatcttagcc actttttaaa agaaaagggg ggactggaag ggctaattca 7980 ctcccaacga aaataagatc tgctttttgc ttgtactggg tctctctggt tagaccagat 8040 ctgagcctgg gagctctctg gctaactagg gaacccactg cttaagcctc aataaagctt 8100 gccttgagtg cttcaagtag tgtgtgcccg tctgttgtgt gactctggta actagagatc 8160 cctcagaccc ttttagtcag tgtggaaaat ctctagcagt agtagttcat gtcatcttat 8220 tattcagtat ttataacttg caaagaaatg aatatcagag agtgagagga acttgtttat 8280 tgcagcttat aatggttaca aataaagcaa tagcatcaca aatttcacaa ataaagcatt 8340 tttttcactg cattctagtt gtggtttgtc caaactcatc aatgtatcta gagcggccgc 8400 caccgcggtg gagctccagc ttttgttccc tttagtgagg gttaattgcg cgcttggcgt 8460 aatcatggtc atagctgttt cctgtgtgaa attgttatcc gctcacaatt ccacacaaca 8520 tacgagccgg aagcataaag tgtaaagcct ggggtgccta atgagtgagc taactcacat 8580 taattgcgtt gcgctcactg cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt 8640 aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct tccgcttcct 8700 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca gctcactcaa 8760 aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac atgtgagcaa 8820 aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 8880 tccgcccccc tgacgagcat cacaaaaaatc gacgctcaag tcagaggtgg cgaaacccga 8940 caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc 9000 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc gtggcgcttt 9060 ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc aagctggggct 9120 gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac tatcgtcttg 9180 agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt aacaggatta 9240 gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 9300 acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa 9360 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt 9420 gcaagcagca gattacgc agaaaaaaag gatctcaaga agatcctttg atcttttcta 9480 cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc atgagattat 9540 caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa tcaatctaaa 9600 gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 9660 cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta 9720 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga gacccacgct 9780 caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag cgcagaagtg 9840 gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa gctagagtaa 9900 gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 9960 cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 10020 catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca 10080 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta aaagtgctca tcattggaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaca ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac attttgaat gtatttaga aataaacaa ataggggttc cgcgcacatt tccccgaaaa gtgccac

Claims

1. A method for fluorescent labeling of open chromatin in living cells, the method being a non-diagnostic method, characterized in that: The method comprises the steps of introducing a gene encoding a nucleic acid binding protein into a living cell, The nucleic acid binding protein is a protein comprising a DNA binding domain formed by three or more non-selective binding TAL-repeat repeats and a fluorescent protein. The non-selective binding TAL-repeat consists of the amino acid sequence shown in any one of SEQ ID NOs. 1 to 3, The DNA binding domain binds to nucleic acid independently of the base sequence.

2. The method of claim 1, wherein: The number of repeats of the TAL-repeat in the DNA binding domain is 3 or more and less than 35.

3. The method according to claim 1 or 2, wherein: The TAL-repeat consisting of the same amino acid sequence is repeatedly connected to the DNA binding domain.

4. The method according to claim 1 or 2, wherein: The nucleic acid binding protein also has a nuclear localization signal sequence.

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