JP7210028B2 - Gene mutation introduction method - Google Patents

Description

The present invention relates to a gene mutation introduction method. More specifically, it relates to gene mutagenesis methods, fusion proteins, nucleic acids, vectors and cells. This application claims priority based on Japanese Patent Application No. 2017-139268 filed in Japan on July 18, 2017, the content of which is incorporated herein.

In order to understand the pathology of diseases caused by genetic mutations including single nucleotide polymorphisms (SNPs) and to reproduce genomic mutations, efficient genome sequence modification technology is required. Recent advances in genome editing technology have made it possible to delete or insert a genome sequence at a target site.

Genome editing utilizes a repair mechanism that forms a double-stranded DNA break (DSB) at the target site and via the non-homologous end joining (NHEJ) pathway or the homologous recombination (HR) pathway. Repair of double-stranded DNA breaks in genome editing is mainly performed by the NHEJ pathway, and deletion and insertion of bases can occur at target sites.

On the other hand, the HR pathway mainly occurs only in the S and G2 phases of the cell cycle, and occurs less frequently than the NHEJ pathway. Therefore, in genome editing, the efficiency of accurate base substitution by the HR pathway using donor DNA as a template is very low. However, in order to introduce the desired base substitution at the target site, it is necessary to induce the correct base substitution by the HR pathway.

Various methods have been investigated as methods for utilizing the HR pathway in genome editing. Classically, plasmid DNA with a homologous arm of about 10 kb or BAC DNA with a homologous arm of up to several hundred kb is used as a donor (template), and genome editing is used to induce DNA damage in the vicinity of the site. A method of introducing gene mutation while introducing a drug selection cassette can be mentioned. However, this method leaves the drug selection cassette in the vicinity of the gene mutation introduction site.

Therefore, the drug selection cassette is sandwiched between loxP sequences in advance, and after the gene mutation is introduced at the target site by homologous recombination, Cre recombinase is reacted to remove it. It is necessary to cut and delete the transposon terminal repeat (TR) sequences, or put the terminal repeat (TR) sequences of the transposon on both sides of the drug selection cassette in advance and remove the drug selection cassette by the action of transposase. For this reason, these methods require at least two subcloning processes, and require time and effort to establish target cells.

As another method using the HR pathway in genome editing, a method using single-stranded DNA as a donor DNA has been investigated (see, for example, Non-Patent Document 1). A single-stranded DNA of about 200 bases can be easily produced by chemical DNA synthesis technology, and there is less risk of random insertion onto the chromosome than double-stranded DNA.

However, it is known that homologous recombination efficiency is low when single-stranded DNA is used as donor DNA. Therefore, in order to obtain a cell line into which a desired gene mutation has been introduced by homologous recombination, a cell population containing as many recombinants as possible is selected from a cell population containing low-frequency recombinants by the droplet digital PCR method. By detecting and extracting by a highly sensitive screening method such as the Sib-selection method and repeating this operation multiple times, an additional step such as the Sib-selection method to enrich a very small number of recombinants is required.

In addition, by inhibiting the NHEJ pathway with an inhibitor such as L-77705, the HR pathway is activated to increase the efficiency of homologous recombination; A method of increasing the HR efficiency by increasing the number of cells in the stage is also being studied. In addition, a method of increasing HR efficiency by optimizing the length and orientation of single-stranded DNA, the distance from the DSB formation site in genome editing, and the like has also been reported.

Radecke F., et al., Targeted Chromosomal Gene Modification in Human Cells by Single-Stranded Oligodeoxynucleotides in the Presence of a DNA Double-Strand Break., MOLECULAR THERAPY, vol. 14 (6), 798-808, 2006.

By the way, pluripotent stem cells, including ES cells and iPS cells, are being applied to various fields such as reproduction of pathological conditions, drug discovery, cell therapy, etc. due to their vigorous proliferation ability and differentiation diversity. Under such circumstances, there is a demand for a technique for accurately introducing target gene mutations into cells including pluripotent stem cells.

However, conventional genome editing techniques sometimes pose a problem of so-called off-target effects, in which gene mutation occurs at positions other than the target sequence. Accordingly, an object of the present invention is to provide a technique for improving the specificity of a sequence-specific DNA cleaving enzyme for a target sequence.

The present invention includes the following aspects.
[1] expressing a fusion protein of a sequence-specific DNA-cleavage enzyme and a nuclear receptor under the control of an expression-inducible promoter; Sequence-specific cleavage of genomic DNA to form a double-stranded DNA break, repair of the double-stranded DNA break using a DNA break repair mechanism, and a gene in the vicinity of the double-stranded DNA break A method of gene mutagenesis, comprising: introducing a mutation.
[2] In introducing the genetic mutation, a donor DNA is allowed to coexist, the donor DNA has sequence identity with a region containing the position of the double-stranded DNA break in the genomic DNA, and the genomic DNA The method for introducing gene mutation according to [1], wherein the nucleotide sequence of has one to several desired nucleotide mutations.
[3] The method of introducing gene mutation according to [2], wherein the desired base mutation includes random mutation. [4] The method for introducing gene mutation according to any one of [1] to [3], wherein the sequence-specific DNA-cleaving enzyme is an RNA-guided nuclease or an artificial nuclease.
[5] The method of introducing gene mutation according to any one of [1] to [4], wherein the nuclear receptor is an estrogen receptor or a glucocorticoid receptor.
[6] The method for introducing gene mutation according to any one of [1] to [5], wherein the expression-inducible promoter is a doxycycline-inducible promoter.
[7] A fusion protein between a sequence-specific DNA-cleaving enzyme and a nuclear receptor.
[8] The fusion protein of [7], wherein the sequence-specific DNA-cleaving enzyme is an RNA-guided nuclease or an artificial nuclease.
[9] The fusion protein of [7] or [8], wherein the nuclear receptor is an estrogen receptor or a glucocorticoid receptor.
[10] A nucleic acid encoding the fusion protein of any one of [7] to [9].
[11] A vector comprising an expression-inducible promoter and the nucleic acid of [10] linked downstream of the expression-inducible promoter.
[12] The vector of [11], wherein the expression-inducible promoter is a doxycycline-inducible promoter.
[13] A cell introduced with the vector of [11] or [12].
[14] The cell of [13], which is a pluripotent stem cell, tumor cell or established cell line.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a technique for improving the specificity of a sequence-specific DNA cleaving enzyme for a target sequence.

(a) is a diagram showing the structure of a transposon vector produced in Experimental Example 1. FIG. (b) is a diagram illustrating the structure of a reporter vector used in a single strand annealing (SSA) assay in Experimental Example 1. FIG. (c) is a graph showing the results of an SSA assay in Experimental Example 1; (a) is a diagram showing the structure of a transposon vector produced in Experimental Example 2. FIG. (b) is a graph showing the results of an SSA assay in Experimental Example 2; (a) is a diagram showing the structure of a transposon vector produced in Experimental Example 3. FIG. (b) is a graph showing the results of SSA assay in Experimental Example 3; 10 is a graph showing the results of SSA assay in Experimental Example 4. FIG. It is a graph which shows the result of having measured the expression level of Cas9 mRNA in Experimental example 5. FIG. 10 shows the concentrations of doxycycline (Dox) and dexamethasone (Dex) added to cell culture media and the measurement results of genome editing efficiency by T7 endonuclease I (T7EI) assay in Experimental Example 6. FIG. 10 is a graph showing the results of measurement of changes in genome editing efficiency over time after adding Dox and Dex to the medium in Experimental Example 7. FIG. 10 is a photograph showing the results of T7EI assay in Experimental Example 8. FIG. 10 is a graph showing the results of knocking down KU80, KU70 and LIG4 in Experimental Example 9 and measuring the expression of mRNA of each gene by reverse transcription quantitative PCR. 10 is a graph showing the results of allele copy number quantitative PCR in Experimental Example 9. FIG. 10 is a graph showing the results of allele copy number quantitative PCR in Experimental Example 10. FIG. 11 is a graph showing the results of allele copy number quantitative PCR in Experimental Example 11. FIG. (a) is a photograph showing the results of a Restriction Fragment Length Polymorphism (RFLP) assay in Experimental Example 12. FIG. (b) is a graph summarizing the results of base sequence analysis in Experimental Example 12. FIG. (c) is a diagram showing Sanger sequence waveform data of representative homologous recombination clones in Experimental Example 12. FIG. (a) is a diagram showing the timing of addition of Dox and Dex in Experimental Example 13. FIG. (b) is a photograph showing the results of RFLP assay in Experimental Example 13; FIG. 10 is a photograph showing the results of T7EI assay in Experimental Example 14. FIG. FIG. 10 is a photograph showing the results of RFLP assay in Experimental Example 15. FIG. 16 is a photograph showing the results of RFLP assay in Experimental Example 16. FIG. (a) is a graph summarizing the results of base sequence analysis in Experimental Example 17. FIG. (b) is a diagram showing amino acids encoded by codons consisting of a base sequence of 'NNC' (where N represents adenine (A), thymine (T), guanine (G) or cytosine (C)); is. (c) is a diagram showing the nucleotide sequences of clones into which random mutations were introduced in Experimental Example 17 and the amino acid sequences encoded by the nucleotide sequences. (a) is a graph summarizing the results of base sequence analysis in Experimental Example 18. FIG. (b) is a diagram showing amino acids encoded by codons consisting of a base sequence of 'NNC' (where N represents A, T, G or C). (c) shows the base sequences of clones into which random mutations were introduced in Experimental Example 18 and the amino acid sequences encoded by the base sequences. 10 is a graph summarizing the results of base sequence analysis in Experimental Example 19. FIG. FIG. 10 shows the base sequences of clones into which random mutations were introduced in Experimental Example 19 and the amino acid sequences encoded by the base sequences. (a) is a graph summarizing the results of base sequence analysis in Experimental Example 20. FIG. (b) is a diagram showing amino acids encoded by codons consisting of a base sequence of 'NNC' (where N represents A, T, G or C). (c) shows the base sequences of clones into which random mutations were introduced in Experimental Example 20 and the amino acid sequences encoded by the base sequences.

[Gene mutation introduction method]
In one embodiment, the present invention comprises the step (a) of expressing a fusion protein of a sequence-specific DNA-cleaving enzyme and a nuclear receptor under the control of an expression-inducible promoter, and translocating the fusion protein into the nucleus. a step (b) in which the nuclear translocated fusion protein sequences-specifically cleaves genomic DNA to form a double-stranded DNA break; and repairing the double-stranded DNA break using a DNA break repair mechanism. and a step (c) of introducing a gene mutation in the vicinity of the double-stranded DNA break.

As will be described later in Examples, the gene mutation introduction method (base substitution method) of the present embodiment can improve the specificity of a sequence-specific DNA cleaving enzyme for a target sequence.

As used herein, improving the specificity of a sequence-specific DNA cleaving enzyme for a target sequence means, for example, in genome editing, suppressing the so-called off-target effect that causes gene mutation at a position other than the target sequence. means Alternatively, improving the specificity of a sequence-specific DNA cleaving enzyme for a target sequence means that genomic DNA is cleaved at unintended times, ie, reducing the background of genomic DNA cleavage.

As will be described later in Examples, the gene mutation introduction method of the present embodiment can suppress the off-target effect in genome editing and increase the efficiency of introducing a desired gene mutation. Therefore, it can be said that the method for introducing gene mutation of the present embodiment is a method for suppressing cleavage of base sequences other than the target sequence by a sequence-specific DNA cleaving enzyme.

In addition, as described later in Examples, according to the method for introducing gene mutation of the present embodiment, cleavage of genomic DNA can be strictly controlled, and the background of genomic DNA cleavage can be reduced. Therefore, it can be said that the method for introducing gene mutation of this embodiment is a method for strictly controlling the activity of a sequence-specific DNA-cleaving enzyme.

The gene mutation introduction method of this embodiment can be performed on cells. Examples of cells include eukaryotic cells such as animal cells and yeast. Animal cells may be human cells or non-human animal cells. Animal cells may also be pluripotent stem cells. Pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like.

(Sequence-specific DNA cleaving enzyme)
As used herein, the sequence-specific DNA cleaving enzyme is not particularly limited as long as it cleaves genomic DNA in a target sequence-specific manner to form a double-stranded DNA break. The length of the target sequence recognized by the sequence-specific DNA cleaving enzyme may be, for example, about 10 to 40 bases.

In general, sequence-specific DNA cleaving enzymes used for genome editing are roughly classified into RNA-guided nucleases and artificial nucleases. In the gene mutagenesis method of this embodiment, the sequence-specific DNA-cleaving enzyme may be an RNA-guided nuclease or an artificial nuclease.

Also, the sequence-specific DNA cleaving enzyme may be in a mode of cleaving DNA by combining, for example, a plurality of nickases. Here, nickase means an enzyme that forms a nick in a single strand of double-stranded DNA. For example, a double-stranded DNA break can be created by nicking both strands of the double-stranded DNA at close locations on the genomic DNA.

An RNA-guided nuclease is an enzyme that binds a guide short-stranded RNA to a target sequence and recruits a nuclease with two DNA-cleaving domains (nuclease domains) to induce sequence-specific cleavage. RNA-guided nucleases include Cas family proteins.

Cas family proteins include, for example, Cas9, Cpf1, CasX, CasY and the like. The RNA-guided nuclease may be a homologue of a Cas family protein or a modified Cas family protein. For example, it may be a nickase-modified nuclease in which one of two existing wild-type nuclease domains is modified to be inactive.

An artificial nuclease is an artificial restriction enzyme in which a DNA-binding domain designed and produced to specifically bind to a target sequence and a DNA-cleaving domain (nuclease domain) of a restriction enzyme FokI are linked. Artificial nucleases include, but are not limited to, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the like.

(nuclear receptor)
In the gene mutagenesis method of this embodiment, the sequence-specific DNA cleaving enzyme forms a fusion protein with the nuclear receptor. As will be described later in Examples, the inventors have investigated the fusion protein of a sequence-specific DNA cleaving enzyme and a nuclear receptor by allowing the ligand (substrate) of the nuclear receptor to act on the fusion protein. I found out that it can be transferred. Furthermore, it was clarified that the fusion protein described above maintains the activity of a sequence-specific DNA-cleaving enzyme.

Therefore, the intracellular localization of the sequence-specific DNA-cleaving enzyme can be controlled by the fusion protein of the sequence-specific DNA-cleaving enzyme and the nuclear receptor.

The nuclear receptor is not particularly limited as long as it enables control of localization to the nucleus by preparing a fusion protein with a sequence-specific DNA-cleavage enzyme. Examples include, but are not limited to, corticoid receptors. Nuclear receptors may be modified. For example, modified estrogen receptors include ERT2 and the like.

Examples of estrogen receptor ligands (substances that translocate estrogen receptors into the nucleus) include estrogen and estrogen derivatives. Estrogen derivatives include tamoxifen, 4-hydroxy tamoxifen and the like. Further, glucocorticoid receptor ligands (substances that translocate glucocorticoid receptors into the nucleus) include dexamethasone and the like.

(expression-inducible promoter)
In the gene mutagenesis method of this embodiment, a fusion protein between a sequence-specific DNA-cleavage enzyme and a nuclear receptor is expressed under the control of an expression-inducible promoter. As the expression-inducible promoter, for example, a promoter whose expression can be induced by addition or removal of an expression control substance to the medium, light irradiation, temperature change, or the like can be used.

The expression-inducible promoter may induce the expression of the fusion protein by adding an expression control substance to the medium, or induce the expression of the fusion protein by removing the expression control substance from the medium. It may be Examples of more specific expression-inducible promoters include, but are not limited to, doxycycline-inducible promoters (TetO promoters).

By expressing a fusion protein of a sequence-specific DNA-cleavage enzyme and a nuclear receptor under the control of an expression-inducible promoter, it becomes possible to control the expression of the fusion protein. That is, in the gene mutation introduction method of this embodiment, the sequence-specific DNA-cleaving enzyme is doubly regulated both at the expression stage (transcription) and at the post-translational stage.

As will be described later in Examples, the inventors found that, in genome editing, the specificity of a sequence-specific DNA-cleaving enzyme to a target sequence was achieved by controlling both the expression induction and the intracellular localization of the sequence-specific DNA-cleaving enzyme. demonstrated that it can improve performance.

(Step (a))
In this step, a fusion protein of a sequence-specific DNA-cleaving enzyme and a nuclear receptor is expressed under the control of an expression-inducible promoter. The expressed fusion protein should be localized in a location other than the nucleus, eg, in the cytoplasm.

In this step, for example, a construct in which a gene encoding a fusion protein gene is linked downstream of an expression-inducible promoter is introduced into a cell, and expression of the fusion protein is induced by a method corresponding to the expression-inducible promoter used. It can be carried out.

For example, if the expression-inducible promoter is a doxycycline-inducible promoter and expression is induced when doxycycline is added to the medium, the inducible protein can be expressed by adding doxycycline to the cell medium. This allows the fusion protein to be expressed only when needed and improves the specificity of the sequence-specific DNA-cleaving enzyme for the target sequence.

The above constructs may be transiently introduced into cells, or may be pre-integrated into cell chromosomes. The constructs described above may be incorporated into, for example, transposon vectors, adeno-associated virus vectors, adenovirus vectors, episomal vectors, plasmid vectors, and the like.

For example, when the above construct is incorporated into a transposon vector, it can be easily incorporated into the chromosome of cells by introducing it into cells and allowing transposase to act. In addition, the construct integrated into the chromosome can be excised from the chromosome and removed without leaving a trace by the action of transposase.

(Step (b))
In this step, the fusion protein between the sequence-specific DNA-cleaving enzyme and the nuclear receptor expressed in step (a) is translocated into the nucleus.

This step can be performed, for example, by adding a nuclear receptor ligand (a substance that translocates the nuclear receptor into the nucleus) to the culture medium of the cell.

For example, if the nuclear receptor is ERT2, addition of 4-hydroxy tamoxifen to the medium can translocate the fusion protein into the nucleus. In addition, when the nuclear receptor is the glucocorticoid receptor, addition of dexamethasone to the medium can translocate the fusion protein into the nucleus.

This allows the fusion protein to be translocated into the nucleus only when needed. As a result, the specificity of the sequence-specific DNA cleaving enzyme for the target sequence can be further improved. That is, in the gene mutation introduction method of this embodiment, the sequence-specific DNA-cleaving enzyme is doubly regulated both at the expression stage (transcription) and at the post-translational stage.

The nuclear translocated fusion protein cleaves genomic DNA in a target sequence-specific manner to form double-stranded DNA breaks. When the sequence-specific DNA cleaving enzyme is an RNA-guided nuclease, the coexistence of a short-strand RNA as a guide enables sequence-specific double-stranded DNA cleavage to occur. As a guide short-chain RNA, one corresponding to the target sequence is used.

As a guide short RNA, for example, CRISPR-sgRNA can be used. The sgRNA may be transiently introduced into cells, or may be expressed intracellularly using an expression vector. When sgRNA is expressed using an expression vector, the sgRNA may be expressed constitutively, or may be expressed under the control of an expression-inducible promoter similar to those described above.

The sgRNA expression vector may be, for example, a transposon vector, an adeno-associated virus vector, an adenovirus vector, an episomal vector, a plasmid vector, or the like.

(Step (c))
In this step, the double-stranded DNA break formed in step (b) is repaired by the DNA break repair mechanism of the cell itself. In this process, a genetic mutation is introduced near the double-stranded DNA break. Here, the vicinity generally means a double-stranded DNA cleavage site or within 100 bases, for example, within 50 bases, for example, within 20 bases from the double-stranded DNA cleavage site. Genetic mutations include insertion mutations or deletion mutations (Indel).

Moreover, homologous recombination (HR) can be induced by performing this step in the coexistence of donor DNA. Here, the donor DNA has sequence identity with a region including the position before and after the double-stranded DNA break in the genomic DNA, and has one to several desired nucleotide mutations in the nucleotide sequence of the genomic DNA. should be used. The donor DNA may be single-stranded DNA or double-stranded DNA. Also, the donor DNA may be a single sequence or a mixture of multiple sequences.

As will be described later in Examples, the gene mutation introduction method of the present embodiment can induce homologous recombination at a frequency as high as 10 times or more compared to the conventional method. Therefore, it can be said that the gene mutation introduction method of this embodiment is a method for increasing homologous recombination efficiency in genome editing.

In addition, as will be described later in Examples, increasing the amount of donor DNA introduced into cells tends to increase the efficiency of homologous recombination. Therefore, in order to achieve high homologous recombination efficiency, it is preferable to increase the amount of donor DNA introduced into the cells. For example, an expression vector for a fusion protein of a sequence-specific DNA-cleavage enzyme and a nuclear receptor, an expression vector for a short-chain RNA that serves as a guide, etc., is incorporated into the chromosome of the cell in advance, and then introduced into the cell. The amount of DNA can be maximized.

The donor DNA may be single-stranded DNA of about 50 to 5,000 base pairs, or double-stranded DNA of about 50 to 5,000 base pairs. If the donor DNA is single-stranded DNA, the donor DNA may have sequence identity with either strand of the genomic DNA double strand.

The donor DNA preferably has a sequence identity of 90% or more, preferably 95% or more, more preferably 99% or more, with the region containing the position of the double-stranded DNA break in the genomic DNA.

As used herein, the term “sequence identity” refers to a value that indicates the proportion of a base sequence of interest that matches a reference base sequence (reference base sequence). The sequence identity of the target base sequence with respect to the reference base sequence can be obtained, for example, as follows. First, the reference base sequence and the target base sequence are aligned. Here, each base sequence may include gaps to maximize sequence identity. Subsequently, the number of matching bases in the reference base sequence and the target base sequence is calculated, and the sequence identity can be obtained according to the following formula (1).
Sequence identity (%) = number of matched bases/total number of bases of target base sequence x 100 (1)

The donor DNA has one to several desired base mutations. As a result, the genome of the cell is replaced with the desired nucleotide sequence by introducing a gene mutation. Here, several is preferably 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1.

Also, the desired nucleotide mutation may contain random mutation. In this case, the donor DNA is preferably a mixture of multiple sequences. As used herein, random mutation refers to a mixture of two or more kinds of target bases in the donor DNA.

Random mutation is not particularly limited as long as the specific base is a mixture of two or more types of bases. (C), for example 'B', ie C, G or T, or for example 'K', ie G or T, but is not limited thereto. Also, the number of random mutations may be one, or two or more.

When chemically synthesizing the donor DNA, random mutations can be introduced by mixing two or more types of basic materials (amidite reagents) in equal amounts or in arbitrary ratios. When the donor DNA is enzymatically synthesized, random mutations can also be introduced by mixing two or more base materials (dATP, dCTP, dGTP, dTTP) in an arbitrary ratio. Alternatively, primers with random mutations can be synthesized by the chemical synthesis described above, and donor DNA can be prepared by reverse transcription reaction or PCR reaction from RNA. Alternatively, the donor DNA can be prepared by cleaving the DNA strand on one side of the double-stranded DNA at two locations with Nickase (single-stranded DNA cleaving enzyme) and isolating the single-stranded DNA by denaturing gel electrophoresis. can.

As described later in Examples, mutants having multiple mutations can be obtained at once by introducing random mutations into donor DNA.

[Fusion protein]
In one embodiment, the invention provides a fusion protein between a sequence-specific DNA-cleaving enzyme and a nuclear receptor. The fusion protein of this embodiment can be suitably used for the gene mutation introduction method described above.

In the fusion protein of this embodiment, the sequence-specific DNA-cleaving enzyme is the same as those described above, and may be an RNA-guided nuclease or an artificial nuclease.

In addition, in the fusion protein of this embodiment, the nuclear receptor is the same as those described above, and may be an estrogen receptor or a glucocorticoid receptor.

[Nucleic acid]
In one embodiment, the invention provides nucleic acids encoding the fusion proteins described above. A fusion protein between a sequence-specific DNA-cleavage enzyme and a nuclear receptor can be obtained by expressing the nucleic acid of this embodiment. Therefore, the nucleic acid of this embodiment can be suitably used for the gene mutation introduction method described above.

[vector]
In one embodiment, the present invention provides a vector comprising an expression-inducible promoter and a nucleic acid encoding the aforementioned fusion protein linked downstream of the expression-inducible promoter. The vector of this embodiment can be suitably used for the gene mutation introduction method described above.

The vector of this embodiment may be, for example, a transposon vector, a retroviral vector, a lentiviral vector, an adeno-associated viral vector, an adenoviral vector, an episomal vector, a plasmid vector, or the like.

In the vector of this embodiment, the expression-inducible promoter is the same as those described above, and examples thereof include doxycycline-inducible promoters, Cumate-inducible promoters, heat shock promoters, light-inducible promoters, and the like. Not limited. Alternatively, a system in which expression is induced upon action of Cre recombinase by a protein translation stop codon or polyadenin sequence flanked by loxP sites may also be used.

As described above, by introducing the vector of the present embodiment into cells, the sequence-specific DNA-cleaving enzyme can be doubly regulated at both the expression stage and the post-translational stage.

[cell]
In one embodiment, the present invention provides cells into which the above vectors have been introduced. Here, the vector is a vector in which an expression-inducible promoter and a nucleic acid encoding a fusion protein of a sequence-specific DNA-cleavage enzyme and a nuclear receptor are linked in this order. The cells of this embodiment can be suitably used for the gene mutation introduction method described above.

In the cells of this embodiment, vectors may be, for example, transposon vectors, retroviral vectors, lentiviral vectors, adeno-associated viral vectors, adenoviral vectors, episomal vectors, plasmid vectors, and the like. Among them, transposon vectors are preferred.

A transposon vector can easily integrate into the chromosome of a cell and can be removed if desired. The transposon may be, for example, piggyBac, Sleeping Beauty, Tol II, mariner, and the like.

Cells of the present embodiment include eukaryotic cells such as animal cells and yeast. Animal cells may be human cells or non-human animal cells. Animal cells may also be pluripotent stem cells or tumor cells. Animal cells may also be established cell lines. Pluripotent stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and the like. Moreover, the cell line is not particularly limited, and examples thereof include HEK293T cells, HeLa cells, and the like.

Any known means can be used to introduce the vector into cells. For example, an electroporation method using a commercially available device can be mentioned. Apparatus for carrying out the electroporation method includes, for example, NEPA21 (Neppagene), 4D-Nucleofector (Lonza), Neon (Thermo Fisher Scientific), Gene Pulser Xcell (Bio-Rad), ECM839 (BTX Harvard Apparatus). company) etc. can be used.

A transfection reagent may also be used as a method of introducing the vector into cells. Transfection reagents include, for example, Lipofectamine2000 (Thermo Fisher Scientific), StemFect (STEMGEN), FuGENE6/HD (Promega), CRISPRMAX (Thermo Fisher Scientific), jetPRIME Kit (Polyplus Transfection Co.), DreamFect (Oz Bioscience Co.), GenePorter 3000 (Oz Bioscience Co.), reagents for the calcium phosphate method, and the like can be used.

In addition, when the vector is a viral vector, a gene can be introduced into cells by utilizing the mechanism by which viruses enter cells. A vector may be introduced into a cell once or multiple times.

By the way, as of March 1, 2017, 197,952 human gene mutations are registered in the Human Gene Mutation Database (HGMD, http://www.hgmd.cf.ac.uk/).

Among these gene mutations, missense mutations (mutations that change amino acids) or nonsense mutations (mutations that result in stop codons) due to single-nucleotide mutations are the most common, and 111,135 mutations have been registered.

Homologous recombination technology is very convenient and effective for analyzing the action of these single nucleotide mutations or for repairing them. However, it has hitherto been difficult to induce homologous recombination with high efficiency, especially in human pluripotent stem cells.

On the other hand, as will be described later in Examples, insertion mutations, deletion mutations, base substitutions by homologous recombination, random mutations by homologous recombination, etc. can be introduced easily and efficiently by using the cells of the present embodiment. can do. This makes it possible to elucidate the pathology of various gene mutation diseases and apply them to treatment.

In addition, the cells of the present embodiment can be used not only for clarification and treatment of diseases, but also for production of industrially useful proteins, for example. Methods for introducing sequence-specific gene mutations are widely used for functional analysis and modification of proteins. By introducing sequence-specific genetic mutations, e.g., increasing protein stability, increasing protein solubility, enhancing enzymatic activity, inactivating enzymatic activity, changing substrate specificity, and promoting molecular evolution. etc. can be performed. For example, by introducing a sequence-specific gene mutation into an enzyme or the like, it is possible to produce an industrially useful enzyme.

EXAMPLES Next, the present invention will be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[Experimental example 1]
(Examination of expression-inducible DNA-cleaving enzyme)
A sequence-specific DNA-cleaving enzyme was expressed under the control of an expression-inducible promoter, and the DNA-cleaving activity was examined. CRISPR-Cas9 was used as a sequence-specific DNA-cleaving enzyme. In addition, as the Cas9 gene, those whose base sequences were optimized according to the human codon frequency were used. A doxycycline (Dox)-inducible promoter (TetO promoter) was used as an expression-inducible promoter. Specifically, first, a transposon vector having the structure shown in FIG. 1(a) was produced.

In FIG. 1(a), “3′TR” and “5′TR” represent the piggyBac transposon-specific terminal inverted sequences located on the 3′ and 5′ sides, respectively, and “TetO” represents the TetO promoter. , "Cas9" stands for Cas9 gene, "NLS" stands for nuclear localization signal sequence, "IRES" stands for Internal Ribosome Entry Site, "mCherry" stands for mCherry fluorescent protein gene, "pA" stands for poly-A addition stands for signal sequence, "EF1α pro" for EF1α promoter, "rtTA" for reverse tetracycline-regulated transactivator, "Puro ^R " for puromycin resistance gene, "Hygro ^R " for hygromycin resistance represents a gene.

When a transposon vector having the structure shown in FIG. 1(a) and piggyBac transposase are co-expressed in a cell, the transposase excises the region sandwiched between "3'TR" and "5'TR", and the region in the host cell genome is excised. is integrated into the TATA site of the cell line to establish a stable expression cell line. In addition, cells into which a transposon vector has been introduced can be drug-selected using puromycin or hygromycin.

Cells into which the transposon vector has been introduced constitutively express rtTA under the control of the EF1α promoter. Also, rtTA binds to the TetO promoter and expresses the Cas9 gene when Dox is added to the cell culture medium.

Using these vectors, DNA cleavage activity in exon 45 of the human dystrophin (DMD) gene was examined by single strand annealing (SSA) assay using a luciferase reporter.

《SSA Assay》
FIG. 1(b) is a diagram explaining the structure of the reporter vector used for the SSA assay. As shown in FIG. 1(b), the reporter vector has a 5'-side fragment and a 3'-side fragment of the Firefly luciferase (Firefly Luc) gene downstream of the CMV promoter, and has a CRISPR-sgRNA target sequence in between. are doing. The 5' and 3' fragments of the Firefly luciferase gene each have an overlapping sequence of approximately 700 bp. The Firefly luciferase expressed by the reporter vector shown on the left side of FIG. 1(b) is in an inactive form.

When the target sequence in the reporter vector is cleaved by Cas9, it is repaired by the HR or SSA pathway. As a result, the structure of the reporter vector changes as shown on the right side of FIG. 1(b), and active Firefly luciferase is expressed. Therefore, the DNA-cleaving activity of Cas9 can be measured by measuring the activity of Firefly luciferase.

In this experimental example, a base sequence (SEQ ID NO: 1) present in exon 45 of the dystrophin gene was used as the target sequence. Further, as an sgRNA expression vector, an oligo DNA having the nucleotide sequence of SEQ ID NO: 4 and an oligo DNA having the nucleotide sequence of SEQ ID NO: 7 were mixed to perform a PCR reaction, and the resulting amplified fragment was transferred to a transposon vector. I used the built in.

First, 100 ng of the above reporter vector, 20 ng of phRL-TK vector expressing Renilla luciferase (Renilla Luc) as an internal standard, Cas9 expression vector 200 ng having the structure shown in FIG. Human embryonic kidney-derived HEK293T cells were transfected using a gene transfer reagent (Lipofectamine 2000, Thermo Fisher Scientific) and seeded in a 96-well plate at 50,000 cells/100 μL/well.

Dox was also added to the medium at final concentrations of 0, 0.0065, 0.065, 0.65 and 6.5 μM to induce Cas9 expression.

Subsequently, on the day after gene transfer, luciferase reporter activity was analyzed using a commercially available kit (“Dual-Glo Luciferase Assay system” Cat. No. E2920, Promega).

FIG. 1(c) is a graph showing the results of measuring Firefly luciferase activity based on Renilla luciferase activity. In FIG. 1 (c), "NC" represents the results of the negative control without the addition of the sgRNA expression vector, "No Cas9" represents the results of the negative control without the addition of the Cas9 expression vector, " EF1α-Cas9 'represents the result of introducing Cas9 expression vector that constitutively expresses Cas9 into cells, and 'TetO-Cas9-NLS (Puro ^R )' has a puromycin resistance gene shown in FIG. Represents the results of introducing the Cas9 expression vector into the cells, "TetO-Cas9-NLS (Hygro ^R )" represents the results of introducing the Cas9 expression vector having the hygromycin resistance gene shown in Figure 1 (a) into the cells. In addition, "*" indicates the existence of a statistically significant difference at p<0.05 by Student's t-test.

As a result, even when using any Cas9 expression vector of Puro ^R and Hygro ^R , it was revealed that it was possible to control the DNA cleavage activity by Cas9 in a Dox concentration-dependent manner.

However, significant DNA cleavage activity was also detected in Dox-free samples compared to negative controls (no Cas9 or no sgRNA). From this result, it became clear that the control of the DNA cleavage activity of Cas9 is insufficient only by controlling the transcription level by the expression-inducible promoter.

[Experimental example 2]
(Examination of intracellular localization control of sequence-specific DNA-cleaving enzyme)
Examination of whether or not the intracellular localization of sequence-specific DNA-cleaving enzymes can be controlled, and whether DNA-cleaving activity can be controlled by controlling the intracellular localization of sequence-specific DNA-cleaving enzymes bottom. CRISPR-Cas9 was used as a sequence-specific DNA-cleaving enzyme. In addition, as the Cas9 gene, those whose base sequences were optimized according to the human codon frequency were used.

Fusion proteins of Cas9 and modified estrogen receptor (ERT2) or glucocorticoid receptor (GR) were prepared as a method of controlling the subcellular localization of sequence-specific DNA-cleaving enzymes. The amino acid sequence of the fusion protein between Cas9 and ERT2 is shown in SEQ ID NO:75, and the amino acid sequence of the fusion protein between Cas9 and GR is shown in SEQ ID NO:76.

ERT2 and GR are known to localize to the cytoplasm in the absence of substrate, but translocate to the nucleus in the presence of substrate. The substrate for ERT2 is 4-hydroxytamoxifen (4-OHT) and for GR is dexamethasone (Dex).

Specifically, first, each transposon vector having the structure shown in FIG. 2(a) was constructed. In FIG. 2(a), “EF1α pro” represents the EF1α promoter, “attR1” and “attR2” represent DNA recombination sequences derived from phage λ, “Cm ^R ” represents the chloramphenicol resistance gene, "ccdB" represents the ccdB gene, "pA" represents the poly A addition signal sequence, "Cas9" represents the Cas9 gene, "NLS" represents the nuclear localization signal sequence, "ERT2" represents the ERT2 gene, "GR" represents the GR gene. In the completed transposon vector, “Cm ^R ” and “ccdB” in FIG. 2(a) are replaced with Cas9-NLS, Cas9-ERT2, or Cas9-GR, respectively.

Subsequently, in the same manner as in Experimental Example 1, these vectors were used to examine the DNA cleavage activity in exon 45 of the dystrophin gene by SSA assay using a luciferase reporter.

The SSA assay consisted of adding 4-OHT to the medium at final concentrations of 0, 0.0025, 0.025, 0.25 and 2.58 μM or Dex at 0, 0.0037, 0.015, 0 Experimental Example 1 was repeated, except that it was added to the medium at final concentrations of 0.06, 0.25 and 1 μM.

FIG. 2(b) is a graph showing the results of measuring Firefly luciferase activity based on Renilla luciferase activity. In FIG. 2 (b), "sgRNA only" represents the results of the negative control in which only the sgRNA expression vector was introduced into the cells, and "Cas9-NLS only" represents the negative control in which only the Cas-NLS expression vector was introduced into the cells. Represents the results, "Cas9-ERT2 only" represents the results of the negative control in which only the Cas-ERT2 expression vector was introduced into the cells, "Cas9-GR only" represents the Cas-GR expression vector only introduced into the cells negative Represents the control results, "Cas9-NLS + sgRNA" represents the result of introducing the Cas-NLS expression vector and sgRNA expression vector into cells, "EF1α-Cas9-ERT2 + sgRNA" represents the Cas-ERT2 expression vector and sgRNA Represents the results of introducing an expression vector into cells, "EF1α-Cas9-GR + sgRNA" represents the results of introducing Cas-GR expression vector and sgRNA expression vector into cells. In addition, "*" indicates the existence of a statistically significant difference at p<0.05 by Student's t-test.

As a result, it became clear that the DNA cleavage activity by Cas9 could be controlled in a substrate (4-OHT or Dex) concentration-dependent manner.

These results demonstrate that the intracellular localization of the sequence-specific DNA-cleaving enzyme can be controlled by adding a substrate to the fusion protein of the sequence-specific DNA-cleaving enzyme and the nuclear receptor.

Further, from this result, it was revealed that the fusion protein between Cas9 and GR exhibits higher DNA cleavage activity than the fusion protein between Cas9 and ERT2 in the above substrate concentration range.

On the other hand, significant DNA cleavage activity was detected for Cas9-GR compared to the negative controls (no Cas9 or no sgRNA) even in samples to which no substrate was added. From this result, it became clear that the control of the DNA-cleaving activity of Cas9 is insufficient only by controlling the intracellular localization by the fusion protein of the sequence-specific DNA-cleaving enzyme and the nuclear receptor.

[Experimental example 3]
(Examination of dual control of sequence-specific DNA-cleavage enzyme expression induction and intracellular localization)
We examined whether or not the DNA-cleaving activity can be controlled by controlling both the expression induction and intracellular localization of the sequence-specific DNA-cleaving enzyme.

Specifically, first, a transposon vector having the structure shown in FIG. 3(a) was constructed. In FIG. 3 (a), "3'TR", "5'TR", "TetO", "Cas9", "GR", "IRES", "mCherry", "pA", "EF1α pro", "rtTA ”, “Puro ^R ”, and “Hygro ^R ” have the same meanings as described above. Hereinafter, as shown in FIG. 3 (a), a Cas9 expression vector capable of controlling both expression induction and intracellular localization is referred to as a "dual control type Cas9 expression vector", expression induction and intracellular localization Cas9 that can control both of the existing is sometimes referred to as "dual control type Cas9".

Subsequently, in the same manner as in Experimental Example 1, these vectors were used to examine the DNA cleavage activity in exon 45 of the dystrophin gene by SSA assay using a luciferase reporter.

The SSA assay consisted of experiments except that Dex was added to the medium at final concentrations of 0, 0.001, 0.1 and 10 μM and Dox was either not added or added to the medium at a final concentration of 2 μM. It was carried out in the same way as in Example 1.

FIG. 3(b) is a graph showing the results of measuring Firefly luciferase activity based on Renilla luciferase activity. In FIG. 3 (b), "EF1α-Cas9-NLS" represents the result of introducing only the expression vector that constitutively expresses Cas9-NLS into cells, and "TetO-Cas9-GR (Puro ^R )" is shown in FIG. Represents the result of introducing only the Cas9-GR expression vector having the puromycin resistance gene shown in (a) into cells, "TetO-Cas9-GR (Hygro ^R )" is the hygromycin resistance gene shown in FIG. Represents the result of introducing into cells only the Cas9-GR expression vector having, "EF1α-Cas9-NLS + sgRNA" represents the result of introducing an expression vector and sgRNA expression vector that constitutively expresses Cas-NLS into cells , "TetO-Cas9-GR (Puro ^R ) + sgRNA" represents the result of introducing the Cas9-GR expression vector and sgRNA expression vector having the puromycin resistance gene shown in FIG. 3 (a) into cells, "TetO- Cas9-GR (Hygro ^R ) + sgRNA” represents the result of introducing the Cas9-GR expression vector and sgRNA expression vector having the hygromycin resistance gene shown in FIG. 3(a) into cells. "N.S." indicates that there is no statistically significant difference.

As a result, it was revealed that DNA cleavage activity was induced when the double-regulatory Cas9 expression vector was introduced into cells and treated simultaneously with two agents, Dox and Dex. On the other hand, under substrate-free conditions, the DNA cleavage activity was suppressed to the same extent as the negative control (no sgRNA).

[Experimental example 4]
(Time course of DNA cleavage activity by dual control type Cas9 expression vector)
A double regulatory Cas9 expression vector and an sgRNA expression vector were introduced into cells, and the time course of DNA cleavage activity when treated simultaneously with two agents, Dox and Dex, was examined. Specifically, DNA cleavage activity in exon 45 of the dystrophin gene was measured in the same manner as in Experimental Example 1 by SSA assay using a luciferase reporter.

“TetO-Cas9-GR (Puro ^R )” was used as the double-regulated Cas9 expression vector. Also, as a control, instead of the double-regulated Cas9 expression vector, an expression vector that constitutively expresses Cas9-GR "EF1α-Cas9-GR", an expression vector that expresses Cas9-NLS in the presence of Dox The same measurement was performed for the sample introduced with "TetO-Cas9-NLS (Puro ^R )". In addition, the same measurement was performed for the sample "No Cas9" in which the Cas9 expression vector was not introduced.

After introducing each expression vector into the cells, Dox and Dex were added to the medium at final concentrations of 2 μM and 1 μM, respectively, and the DNA cleavage activity was measured over time.

FIG. 4 is a graph showing the results of measurement of Firefly luciferase activity for each sample based on Renilla luciferase activity. As a result, when the EF1α-Cas9-GR expression vector and the TetO-Cas9-NLS (Puro ^R ) expression vector were introduced into the cells, both DNA cleavage activity was observed even when Dox and Dex were not added (0 hours). was detected. On the other hand, when TetO-Cas9-GR (Puro ^R ) is introduced into the cells is a dual control type Cas9 expression vector, DNA cleavage activity in the absence of Dox and Dex (0 hours), Cas9 not added (No Cas9) It became clear that it can be suppressed to the same extent.

In addition, when TetO-Cas9-GR (Puro ^R ), a double-regulated Cas9 expression vector, was introduced into the cells, the DNA cleavage activity was induced clearly from 8 hours to 24 hours after the addition of Dox and Dex. It became clear.

[Experimental example 5]
(Preparation of iPS cells introduced with double regulatory Cas9 expression vector and sgRNA expression vector)
Both of which are piggyBac transposon vectors, a double-regulated Cas9 expression vector and an sgRNA expression vector are introduced into iPS cells, and puromycin (Cat. No. 29455-54, Nacalai Tesque) at a final concentration of 1 to 15 μg / mL, or A population of stably transfected chromosomes was obtained by drug selection for about 5 days with hygromycin B (Cat. No. 10687-010, Thermo Fisher Scientific) at a final concentration of 100-200 μg/mL. As the sgRNA expression vector, the same vector as used in Experimental Example 1, whose target sequence is the nucleotide sequence present in exon 45 of the dystrophin gene, was used. The 1383D2 strain was used as iPS cells and cultured according to a standard method.

Subsequently, Dox was added to the culture medium of the obtained iPS cell population at a final concentration of 2 μM, and the time course of Cas9 mRNA expression level was examined by reverse transcription quantitative PCR. A sense primer of SEQ ID NO: 73 and an antisense primer of SEQ ID NO: 74 were used for PCR of Cas9 cDNA.

FIG. 5 is a graph showing the results of measuring the expression level of Cas9 mRNA. In FIG. 5, "Puro" represents puromycin. The expression level of Cas9 mRNA is expressed as a relative value based on the expression level of ACTB mRNA.

Consequently, in either puromycin 5 μg/mL or 15 μg/mL drug-selected cell populations, the addition of Dox induced Cas9 mRNA transcription, and mRNA expression continued for at least 72 hours after induction. became clear.

[Experimental example 6]
(Examination of optimal concentrations of Dox and Dex in genome editing)
Optimal concentrations of Dox and Dex when performing genome editing in iPS cells introduced double regulatory Cas9 expression vector and sgRNA expression vector were examined. Dox and Dex were added at various concentrations to the iPS cell culture medium prepared in Experimental Example 5, and gene mutation introduction activity at the target position present in exon 45 of the dystrophin gene was measured by T7 endonuclease I (T7EI) assay. .

<<T7EI Assay>>
The T7EI assay was performed as follows. First, the target genomic site of CRISPR-sgRNA was amplified by PCR reaction. In this experimental example, a sense primer (SEQ ID NO: 14) and an antisense primer (SEQ ID NO: 15) that amplify a region containing the target position in exon 45 of the dystrophin gene were used.

The resulting PCR product was then purified. Subsequently, 1/10 volume of 10×NEBuffer2 (NEB) buffer was added to the purified PCR product (400 ng) and heated at 95° C. for 5 minutes to heat denature the double-stranded DNA. was re-annealed by lowering the More specifically, it was cooled from 95°C to 85°C at -2°C/sec, and from 85°C to 25°C at -0.1°C/sec.

Subsequently, 10 units of T7 endonuclease I (T7EI, Cat. No. M0302S, NEB) was added to the re-annealed PCR product and treated at 37° C. for 15 minutes. Subsequently, the activity of T7EI was stopped by adding 1/10 volume of 0.25 M EDTA solution to the reaction volume, after which the sample was kept cold (on ice).

Subsequently, the T7EI-treated PCR products were subjected to 2% agarose gel electrophoresis, and the DNA signal intensities of the cleaved and uncleaved bands were quantified using ImageJ software, or using a fully automated electrophoresis system ("TapeStation 2200", Agilent Technologies). ) was used to analyze the size and band intensity of DNA fragments.

FIG. 6 shows the concentrations of Dox and Dex added to the medium and the measurement results of genome editing efficiency (Indel, insertion mutation or deletion mutation) by T7EI assay. As a result, it was revealed that genome editing efficiency (DNA cutting activity) increases in a concentration-dependent manner for both Dox and Dex.

[Experimental example 7]
(Examination of temporal changes in genome editing)
Subsequently, in the same manner as in Experimental Example 6, changes in genome editing over time were examined. Specifically, Dox and Dex were added to the iPS cell culture medium prepared in Experimental Example 5 at final concentrations of 2 μM and 1 μM, respectively, and T7EI for the gene mutation introduction activity at the target position present in exon 45 of the dystrophin gene. Measured by assay.

FIG. 7 is a graph showing the results of measurement of changes over time in genome editing efficiency (Indel) after addition of Dox and Dex. As a result, it was revealed that genome editing began to be detected 6 hours after adding Dox and Dex at final concentrations of 2 μM and 1 μM, respectively, and reached a plateau after 24 hours.

[Experimental example 8]
(Consideration of off-target)
From the results of the above-described Experimental Examples, on-target cleavage activity was confirmed with high efficiency in double-regulatory Cas9-expressing iPS cells. Therefore, the effect of off-targets in dual control Cas9-expressing iPS cells was examined.

All potential off-targets with up to 3 mismatches to the target sequence in exon 45 of the human dystrophin gene were assayed for insertion or deletion mutations by T7EI assay as in Example 6.

Table 1 below shows the target sequence (on-target) in exon 45 of the dystrophin gene and the positions, base sequences, sequence numbers, and number of mismatches to the on-target of off-targets (OT1 to OT5) examined. In Table 1, lower case letters in base sequences represent base sequences different from the on-target.

In addition, Table 2 below shows the sequence numbers of the sense primers and antisense primers used in the PCR reaction in the T7EI assay for on-target and each off-target.

Specifically, Dox and Dex were added to the iPS cell culture medium prepared in Experimental Example 5 at final concentrations of 2 μM and 1 μM, respectively. Subsequently, after 24 hours or 48 hours, the on-target and each off-target shown in Table 1 were examined for gene mutation introduction by T7EI assay. The T7EI assay was performed in the same manner as in Experimental Example 6, except that the primers shown in Table 2 were used as the sense primer and antisense primer.

FIG. 8 is a photograph showing the results of the T7EI assay. In FIG. 8, "DMD on-target" represents the on-target results in exon 45 of the dystrophin gene, and "OT1" to "OT5" represent the respective off-target results. Also, "Alu polyA artifact" in OT1 indicates that it is an artifact.

As a result, it was revealed that no gene mutation was observed in the off-target. From this result, in the induction of genome editing by Dox and Dex in double-regulatory Cas9-expressing iPS cells, it became clear that high sequence recognition specificity is maintained if the induction is within at least 48 hours.

[Experimental example 9]
(Examination of base substitution efficiency by HR pathway 1)
It was examined whether the base substitution efficiency (gene mutation introduction efficiency) by the HR pathway is increased by knocking down the expression of KU80, KU70, and LIG4, which are DNA repair factors in the NHEJ pathway, using siRNA.

《Confirmation of knockdown efficiency》
First, siRNAs against KU80, KU70, and LIG4 were introduced into HEK293T cells, a human fetal kidney cell line. A commercially available siRNA was used. As siRNA against KU80, catalog number "s14953" (SEQ ID NO: 8, Applied Biosystems) and catalog number "s14952" (SEQ ID NO: 9, Applied Biosystems) were used. As siRNA against KU70, catalog number "s52594" (SEQ ID NO: 10, Applied Biosystems) and catalog number "s54155" (SEQ ID NO: 11, Applied Biosystems) were used. As siRNA against LIG4, catalog number "s8181" (SEQ ID NO: 12, Applied Biosystems) and catalog number "s8179" (SEQ ID NO: 13, Applied Biosystems) were used.

《Reverse transcription quantitative PCR》
Subsequently, expression levels of KU80, KU70, and LIG4 mRNAs were measured by reverse transcription quantitative PCR.

For PCR of KU80 cDNA, the sense primer of SEQ ID NO: 57 and the antisense primer of SEQ ID NO: 58 were used when siRNA of catalog number "s14953" was used. In addition, when the siRNA of catalog number "s14952" was used, the sense primer of SEQ ID NO: 59 and the antisense primer of SEQ ID NO: 60 were used.

For PCR of KU70 cDNA, the sense primer of SEQ ID NO: 61 and the antisense primer of SEQ ID NO: 62 were used when siRNA of catalog number "s52594" was used. In addition, when the siRNA of catalog number "s54155" was used, the sense primer of SEQ ID NO: 63 and the antisense primer of SEQ ID NO: 64 were used.

For LIG4 cDNA PCR, the sense primer of SEQ ID NO: 65 and the antisense primer of SEQ ID NO: 66 were used when siRNA of catalog number "s8181" was used. In addition, when the siRNA of catalog number "s8179" was used, the sense primer of SEQ ID NO: 67 and the antisense primer of SEQ ID NO: 68 were used.

For any gene, the samples were standardized using primers for amplifying the cDNA of the GAPDH gene or ACTB gene as an internal standard, and quantified by the comparative Ct method. A sense primer of SEQ ID NO: 69 and an antisense primer of SEQ ID NO: 70 were used for PCR of the GAPDH gene. A sense primer of SEQ ID NO: 71 and an antisense primer of SEQ ID NO: 72 were used for PCR of the ACTB gene.

FIG. 9 is a graph showing the results of measuring the expression of KU80, KU70, and LIG4 mRNAs by reverse transcription quantitative PCR. As a result, it was confirmed that KU80, KU70 or LIG4 could be knocked down using any siRNA.

Subsequently, KU80, KU70, HEK293T cells 48 hours after the introduction of siRNA against LIG4, EF1α-Cas9-NLS expression vector, sgRNA expression vector and single-stranded DNA donor was introduced, and allele copy number quantitative PCR Base substitution efficiency (homologous recombination efficiency) by HR pathway was measured.

A single-stranded DNA (DMD-ssODN1) having the base sequence shown in SEQ ID NO: 35 was used as the single-stranded DNA donor. As the sgRNA expression vector, the same vector as used in Experimental Example 1 was used, the base sequence of which is located in exon 45 of the dystrophin gene as the target sequence.

《Allelic copy number quantitative PCR》
Primers specifically recognizing the nucleotide sequence of the wild-type dystrophin gene before homologous recombination (sense primer: SEQ ID NO: 42, antisense primer: SEQ ID NO: 44) and specifically recognizing the nucleotide sequence after homologous recombination The DNA copy number of each base sequence was measured by quantitative PCR using primers (sense primer: SEQ ID NO: 43, antisense primer: SEQ ID NO: 44). As an internal standard for the allele copy number of the human genome, primers that amplify the NANOG gene region (sense primer: SEQ ID NO: 45, antisense primer: SEQ ID NO: 46) or primers that amplify the ACTB gene region (sense primer: SEQ ID NO: 47, anti Samples were standardized using the sense primer: SEQ ID NO: 48) and quantified by the comparative Ct method.

FIG. 10 is a graph showing the results of allele copy number quantitative PCR. In FIG. 10, "no siRNA" represents the results of cells into which siRNA was not introduced, "Ku80 KD" represents the results of cells into which siRNA against Ku80 was introduced, and "Ku70 KD" represents the results of Ku70. "LIG4 KD" represents the result of cells introduced with siRNA against LIG4, and "mix" represents the result of introducing a mixture of siRNA against Ku80, Ku70 and LIG4. "+ssODN" indicates that it was the result of introducing a single-stranded DNA donor into the cell, "ND" indicates that it was not detected, and "wild type". represents an allele having a wild-type base sequence, and "HR" represents an allele having a base sequence after homologous recombination.

As a result, it was revealed that no significant increase in homologous recombination efficiency was observed when KU80, KU70, and LIG4 were knocked down individually, and when KU80, KU70, and LIG4 were knocked down simultaneously. .

[Experimental example 10]
(Examination of base substitution efficiency by HR pathway 2)
Subsequently, instead of introducing siRNA into cells, L755507, Brefeldin A, or YM155, which are compounds reported to increase the efficiency of homologous recombination, were added to the cell culture medium in the same manner as in Experimental Example 9. Then, the base substitution efficiency by homologous recombination was examined using the base sequence present in exon 45 of the dystrophin gene as the target sequence.

FIG. 11 is a graph showing the results of allele copy number quantitative PCR. In FIG. 11, "L7" represents the results of cells with L755507 added to the medium, "BA" represents the results of cells with Brefeldin A added to the medium, and "YM" represents the results of YM155 in the medium. "ssODN(+)" represents the result of introducing the single-stranded DNA donor into the cells, and "ssODN(-)" represents the result of the single-stranded DNA donor introduced into the cells. "Wild type" represents an allele having a wild-type base sequence, and "HR" represents an allele having a base sequence after homologous recombination. .

As a result, it was found that no significant increase in homologous recombination efficiency was observed even when L755507, Brefeldin A or YM155 was applied to the cells.

[Experimental example 11]
(Examination of base substitution efficiency by HR pathway 3)
Subsequently, prepared in Experimental Example 5, the iPS cells introduced with the double-regulatory Cas9 expression vector and the sgRNA expression vector were introduced with a single-stranded DNA donor by electroporation, and homologous recombination efficiency was examined. A single-stranded DNA having the base sequence shown in SEQ ID NO: 35 was used as the single-stranded DNA donor. In the above iPS cells, since Cas9 and sgRNA have already been stably introduced into the chromosome, it is possible to introduce a single-stranded DNA donor to the maximum extent.

Single-stranded DNA donors of 1.5, 3, 6 and 12 μg were introduced per 0.5×10 ⁶ iPS cells, and Dox at a final concentration of 2 μM and Dex at a final concentration of 1 μM were added to the medium. Subsequently, after culturing for 24 to 48 hours, genomic DNA was extracted from each cell, and in the same manner as in Experimental Example 9, base substitution was performed by homologous recombination using the base sequence present in exon 45 of the dystrophin gene as the target sequence. Efficiency was considered.

FIG. 12 is a graph showing the results of allele copy number quantitative PCR. In FIG. 12, "ssODN" represents a single-stranded DNA donor, "wild-type" represents an allele having a wild-type base sequence, and "HR" represents an allele having a base sequence after homologous recombination. represents something.

As a result, it was found that the efficiency of homologous recombination increases with an increase in the amount of introduced single-stranded DNA donor. Moreover, the proportion of alleles into which gene mutation was introduced by homologous recombination reached 30% at maximum. This ratio was more than 10 times higher than the homologous recombination efficiency that the inventors usually observe in systems that do not use dual-regulatory Cas9.

[Experimental example 12]
(Examination of base substitution efficiency by HR pathway 4)
Subsequently, a single-stranded DNA donor having a different nucleotide sequence from that used in Experimental Example 11 was introduced into the iPS cells introduced with the double-regulatory Cas9 expression vector and the sgRNA expression vector prepared in Experimental Example 5, Homologous recombination efficiency was examined. As a single-stranded DNA donor, a single-stranded DNA (DMD-ssODN-AgeI) having the nucleotide sequence shown in SEQ ID NO:36 was used. When homologous recombination with this single-stranded DNA donor occurs, an AgeI restriction enzyme site is generated.

Specifically, 12 μg of single-stranded DNA donor was introduced per 0.5×10 ⁶ iPS cells, and Dox at a final concentration of 2 μM and Dex at a final concentration of 1 μM were added to the medium. Subsequently, after culturing for 24 to 48 hours, genomic DNA was extracted from each cell and subjected to homologous recombination using the nucleotide sequence present in exon 45 of the dystrophin gene as the target sequence by Restriction Fragment Length Polymorphism (RFLP) assay. Base substitution efficiency was examined.

<<RFLP assay>>
First, using the extracted genomic DNA as a template, a PCR reaction was performed using a sense primer (SEQ ID NO: 49) and an antisense primer (SEQ ID NO: 50) to amplify a region containing the target position present in exon 45 of the dystrophin gene. was performed to purify the PCR product.

Subsequently, the purified PCR product was digested with the restriction enzyme AgeI, subjected to 2% agarose gel electrophoresis, and the DNA signal intensity of the cleaved band and the uncleaved band was quantified using ImageJ software to quantify the alleles that had undergone homologous recombination. .

FIG. 13(a) is a photograph showing the results of agarose gel electrophoresis in RFLP assay. In FIG. 13(a), "DMD" represents the dystrophin gene, "ssODN" represents the single-stranded DNA donor, and "HR" represents homologous recombination. As a result, the proportion of alleles into which gene mutation was introduced by homologous recombination was determined to be 19.7±1.0%.

Using the genomic DNA used in the RFLP assay as a template, the region containing the target position was amplified by PCR and TA cloned, and each clone of the PCR product was analyzed by Sanger sequencing. For TA cloning, pGEM-T vector (Cat. No. A3600, Promega) or pGEM-T Easy vector (Cat. No. A1360, Promega) was used.

FIG. 13(b) is a graph summarizing the analysis results. In FIG. 13(b), "Knock-in" represents homologous recombination by a single-stranded DNA donor, "Indel" represents insertion or deletion mutation, and "WT" represents wild-type sequence. FIG. 13(c) shows Sanger sequence data of representative homologous recombination clones. As a result, some indel mutation was observed in 47.2% of clones (34/72). In addition, homologous recombination with the single-stranded DNA donor was observed in 13.9% of clones (10/72).

[Experimental example 13]
(Examination of the influence of the timing of addition of Dox and Dex on the efficiency of base substitution by the HR pathway)
Examination of the influence of the timing of addition of Dox and Dex on the efficiency of base substitution by the HR pathway was examined. Specifically, the same single-stranded DNA donor used in Experimental Example 12 was introduced into the iPS cells introduced with the double-regulatory Cas9 expression vector and the sgRNA expression vector prepared in Experimental Example 5.

In addition, Dox and Dex were added to the medium at various timings, and RFLP assays were performed in the same manner as in Experimental Example 12, using the nucleotide sequence present in exon 45 of the dystrophin gene as the target sequence. Substitution efficiency was investigated. Final concentrations of Dox and Dex were 2 μM and 1 μM, respectively.

FIG. 14(a) is a diagram showing the timing of addition of Dox and Dex. In FIG. 14(a), "ssODN" represents a single-stranded DNA donor. As shown in Figure 14(a), 24 hours (-24), 12 hours (-12), 6 hours (-6), 2 hours ( -2), Dox and Dex were added at the time of introduction of the single-stranded DNA donor (0), 6 hours (6), 12 hours (12), and 24 hours (24).

FIG. 14(b) is a photograph showing the results of agarose gel electrophoresis in RFLP assay. In FIG. 14(b), "KI" means knock-in, and represents the ratio of alleles into which gene mutation is introduced by homologous recombination. As a result, it was revealed that the efficiency of homologous recombination increased when the addition of Dox and Dex was started between 12 hours before and 12 hours after the introduction of the single-stranded DNA donor into the cells.

[Experimental example 14]
(Examination of genome editing efficiency at the ILF3 (NF110) locus 1)
The dystrophin gene, which was the target sequence for genome editing in the above experimental examples, was a gene present on the X chromosome. Therefore, genome editing efficiency was examined at a position different from the above experimental example. Specifically, genome editing efficiency was examined using the nucleotide sequence in the ILF3 (NF110) gene on chromosome 19 as a target sequence.

First, an oligo DNA having the nucleotide sequence of SEQ ID NO: 5 and an oligo DNA having the nucleotide sequence of SEQ ID NO: 7 were mixed to carry out a PCR reaction, the resulting amplified fragment was incorporated into a transposon vector, and human ILF3 An sgRNA expression vector was constructed with the nucleotide sequence (SEQ ID NO: 2) present in the (NF110) gene as the target sequence.

Subsequently, in the same manner as in Experimental Example 5, a double-regulated Cas9 expression vector and an sgRNA expression vector were introduced into iPS cells, and a chromosome stably introduced cell population was obtained by drug selection.

Subsequently, Dox and Dex were added to the cell culture medium at final concentrations of 2 μM and 1 μM, respectively, and gene mutation introduction activity at the target position present in the ILF3 (NF110) gene was measured by the same T7EI assay as in Experimental Example 6. .

In this experimental example, a sense primer (SEQ ID NO: 16) and an antisense primer (SEQ ID NO: 17) were used to amplify a region containing the target position present in the ILF3 (NF110) gene.

FIG. 15 is a photograph showing the results of the T7EI assay. In FIG. 15, "Indel" represents insertion mutation or deletion mutation. As a result, it was found that the addition of Dox and Dex could introduce a gene mutation at a high efficiency of 35.0±0.9% in the ILF3 (NF110) gene as well.

[Experimental example 15]
(Examination of genome editing efficiency at the ILF3 (NF110) locus 2)
A single-stranded DNA donor was introduced into the iPS cells introduced with the double regulatory Cas9 expression vector and the sgRNA expression vector prepared in Experimental Example 14, and the homologous recombination efficiency was examined. A single-stranded DNA (NF110-ssODN) having the base sequence shown in SEQ ID NO: 37 was used as the single-stranded DNA donor. When homologous recombination with this single-stranded DNA donor occurs, a PstI restriction enzyme site is generated.

Specifically, 12 μg of single-stranded DNA donor was introduced per 0.5×10 ⁶ iPS cells, and Dox at a final concentration of 2 μM and Dex at a final concentration of 1 μM were added to the medium. Subsequently, after culturing for 24 to 48 hours, genomic DNA was extracted from each cell, and the nucleotide sequence present in the ILF3 (NF110) gene was used as the target sequence by RFLP assay to examine the base substitution efficiency by homologous recombination. bottom.

In the RFLP assay, amplification of the region containing the target position in the ILF3 (NF110) gene was performed by PCR reaction using sense primer (SEQ ID NO:51) and antisense primer (SEQ ID NO:52).

FIG. 16 is a photograph showing the results of agarose gel electrophoresis in RFLP assay. In Figure 16, "ssODN" represents single-stranded DNA donor and "HR" represents homologous recombination. As a result, the proportion of alleles into which gene mutation was introduced by homologous recombination was determined to be 14.0±1.0%.

[Experimental example 16]
(Examination of genome editing efficiency at HLA-A locus)
Subsequently, genome editing efficiency was examined using the nucleotide sequence in the HLA-A gene on chromosome 6 as the target sequence.

First, an oligo DNA having the base sequence of SEQ ID NO: 6 and an oligo DNA having the base sequence of SEQ ID NO: 7 are mixed to perform PCR reaction, the resulting amplified fragment is incorporated into a transposon vector, and human HLA. An sgRNA expression vector was constructed with the base sequence (SEQ ID NO: 3) present in the A gene as the target sequence.

Subsequently, in the same manner as in Experimental Example 5, a double-regulated Cas9 expression vector and an sgRNA expression vector were introduced into iPS cells, and a chromosome stably introduced cell population was obtained by drug selection.

Subsequently, a single-stranded DNA donor was introduced into the obtained iPS cells, and homologous recombination efficiency was examined. Single-stranded DNA (HLA-ssODN1) having the nucleotide sequence shown in SEQ ID NO: 38 and single-stranded DNA (HLA-ssODN2) having the nucleotide sequence shown in SEQ ID NO: 39 were used as single-stranded DNA donors.

When homologous recombination occurs with these single-stranded DNA donors, an AvrII restriction enzyme site is generated. In addition, among these single-stranded DNA donors, HLA-ssODN2 has a point mutation (single nucleotide mutation) in the PAM sequence and was designed so that it would not be re-cut by the same gRNA after homologous recombination.

Subsequently, 3 μg of single-stranded DNA donor was introduced per 0.5×10 ⁶ iPS cells, and Dox at a final concentration of 2 μM and Dex at a final concentration of 1 μM were added to the medium. Subsequently, after culturing for 24 to 48 hours, genomic DNA was extracted from each cell, and the base substitution efficiency by homologous recombination was examined by RFLP assay using the base sequence present in the HLA-A gene as the target sequence. .

In the RFLP assay, PCR amplification of the region containing the target position in the HLA-A gene was performed by Nested-PCR to enhance specificity. In nested-PCR, first, a PCR reaction was performed using a sense primer (SEQ ID NO: 53) and an antisense primer (SEQ ID NO: 54), and then the resulting PCR product was used as a template for the sense primer (SEQ ID NO: 55) and A PCR reaction was performed using an antisense primer (SEQ ID NO:56).

FIG. 17 is a photograph showing the results of agarose gel electrophoresis in RFLP assay. In FIG. 17, "ssODN1" and "ssODN2" represent the single-stranded DNA donors described above, respectively, and "HR" represents homologous recombination. As a result, the ratio of alleles into which gene mutation was introduced by homologous recombination was 39.8±2.6% and 31.3±2.2%, indicating that gene mutation was introduced by homologous recombination with high efficiency. It became clear that

Subsequently, PCR products of samples introduced with HLA-ssODN1 as a single-stranded DNA donor were TA cloned, and each clone was confirmed by Sanger sequencing. As a result, insertion deletion mutations were observed in 10/18 clones, but desired homologous recombination (knock-in) was observed in 7/18 strains.

[Experimental example 17]
(Introduction of random mutation 1)
From the results of the above-described Examples, by performing genome editing by introducing a single-stranded DNA donor into the iPS cells introduced with the double-regulatory Cas9 expression vector and the sgRNA expression vector, the drug of cells after gene mutation introduction It was found that a gene mutation-introduced strain can be obtained by homologous recombination with high efficiency without selection or sorting. Therefore, the inventors thought that it would be possible to introduce random mutations at specific sites in the genome sequence by applying this technique, and conducted studies.

First, a single-stranded DNA donor (DMD-ssODN-NN1, SEQ ID NO: 40) was prepared by introducing two random bases near the dystrophin gene cleavage site of CRISPR-sgRNA.

Subsequently, 12 μg of this single-stranded DNA donor was introduced per 0.5×10 ⁶ iPS cells produced in Experimental Example 5. Subsequently, Dox and Dex were added to the culture medium of the cells at final concentrations of 2 μM and 1 μM, respectively, to induce homologous recombination. Subsequently, after culturing for 24 to 48 hours, genomic DNA was extracted from each cell, the region containing the target position was amplified by PCR, subjected to TA cloning, and each clone of the PCR product was analyzed by Sanger sequencing.

FIG. 18(a) is a graph summarizing the analysis results. In FIG. 18(a), "DMD locus" represents the dystrophin locus, "Randomized" represents random mutation, "Indel" represents insertion mutation or deletion mutation, and "WT" represents the wild-type base sequence. show.

As a result, although 15.9% of clones (24/151) had some kind of insertion deletion, 35.1% of clones (53/151) had random base substitutions and other insertion deletions were present. confirmed not to.

FIG. 18(b) shows amino acids encoded by codons consisting of a base sequence of 'NNC' (where N represents adenine (A), thymine (T), guanine (G) or cytosine (C)). It is a figure which shows. As shown in FIG. 18(b), as a result of introducing random mutations into the first two bases of the codon consisting of the base sequence of 'NNC', it is possible to change the codon sequence encoding amino acids in 4 ² =16 ways. is.

FIG. 18(c) shows the base sequences of clones into which random mutations have been introduced and the amino acid sequences encoded by the base sequences. The size of letters corresponds to the abundance ratio of bases or amino acids. As a result, it was revealed that all possible mutations were introduced by introducing random mutations.

[Experimental example 18]
(Introduction of random mutation 2)
An experiment similar to Experimental Example 17 was performed for the HLA-A gene. First, a single-stranded DNA donor (HLA-ssODN-NN, SEQ ID NO: 41) was prepared by introducing two random bases near the HLA-A gene cleavage site of CRISPR-sgRNA.

Subsequently, 6 μg of this single-stranded DNA donor was introduced per 0.5×10 ⁶ iPS cells produced in Experimental Example 16. Subsequently, Dox and Dex were added to the culture medium of the cells at final concentrations of 2 μM and 1 μM, respectively, to induce homologous recombination. Subsequently, after culturing for 24 to 48 hours, genomic DNA was extracted from each cell, the region containing the target position was amplified by PCR, subjected to TA cloning, and each clone of the PCR product was analyzed by Sanger sequencing.

PCR amplification of the region containing the target position in the HLA-A gene is performed by first performing a PCR reaction using a sense primer (SEQ ID NO: 53) and an antisense primer (SEQ ID NO: 54), followed by the resulting PCR product. was performed by Nested-PCR using a sense primer (SEQ ID NO: 55) and an antisense primer (SEQ ID NO: 56) as templates.

FIG. 19A is a graph summarizing the analysis results. In FIG. 19 (a), "HLA-A locus" represents the HLA-A locus, "Randomized" represents random mutation, "Indel" represents insertion or deletion mutation, and "WT" represents wild type. represents the base sequence of

As a result, 15.8% of clones (24/152) had some insertion deletions, but 40.1% of clones (61/152) had random base substitutions and other insertion deletions were present. confirmed not to.

FIG. 19(b) is a diagram showing amino acids encoded by codons consisting of a nucleotide sequence of 'NNC' (where N represents A, T, G or C). As shown in FIG. 19(b), as a result of introducing random mutations into the first two bases of the codon consisting of the base sequence of 'NNC', it is possible to change the codon sequence encoding amino acids in 4 ² =16 ways. is.

FIG. 19(c) is a diagram showing the base sequences of clones into which random mutations have been introduced and the amino acid sequences encoded by the base sequences. The size of letters corresponds to the abundance ratio of bases or amino acids. As a result, it was revealed that all possible mutations were introduced by introducing random mutations.

[Experimental example 19]
(Introduction of random mutation 3)
An experiment similar to Example 17 was performed using single-stranded DNA donors with more 'NN' sequences. First, a single-stranded DNA donor (DMD-ssODN-NN1 to 5, SEQ ID NO: 40, 77 ~80) were prepared.

Subsequently, 6 μg of each of these single-stranded DNA donors was introduced per 0.5×10 ⁶ iPS cells produced in Experimental Example 16. Subsequently, Dox and Dex were added to the culture medium of the cells at final concentrations of 2 μM and 1 μM, respectively, to induce homologous recombination.

Subsequently, after culturing for 48 to 72 hours, genomic DNA was extracted from each cell, the region containing the target position was amplified by PCR, and the base sequence of each DNA molecule of the PCR product was analyzed using an Illumina HiSeq sequencer.

FIG. 20 is a graph summarizing the analysis results. In FIG. 20, "Randomized" represents random mutation, "Indel" represents insertion mutation or deletion mutation, and "WT" represents a wild-type nucleotide sequence. As a result, it was confirmed that random base substitutions occurred in 5.3% to 13.25% of the sequences in the cells introduced with the single-stranded DNA donor, and no other insertion defects were present.

FIG. 21 is a diagram showing the base sequences of clones into which random mutations have been introduced and the amino acid sequences encoded by the base sequences. The size of letters corresponds to the abundance ratio of bases or amino acids. As a result, introduction of a single-stranded DNA donor having a random base sequence introduces random mutations at 2, 4, 6 or 8 positions in the base sequence, resulting in the introduction of random mutations at 1, 2, 3 or 4 positions in the amino acid sequence. It became clear that random mutations had been introduced.

[Experimental example 20]
(Introduction of random mutation 4)
An experiment similar to Experimental Example 17 was performed on HEK293T cells, which are human embryonic kidney-derived cell lines. First, a dual regulatory Cas9 expression vector and an sgRNA expression vector, both piggyBac transposon vectors, were introduced into HEK293T cells. HEK293T cells were cultured according to standard methods. As the sgRNA expression vector, the same vector as used in Experimental Example 1, whose target sequence is the nucleotide sequence present in exon 45 of the dystrophin gene, was used.

followed by puromycin at a final concentration of 1-15 μg/mL (Cat. No. 29455-54, Nacalai Tesque) or hygromycin B at a final concentration of 100-200 μg/mL (Cat. No. 10687-010, Thermo Fisher Scientific). A population of cells stably transfected with chromosomes was obtained by drug selection for about 5 days.

Subsequently, a single-stranded DNA donor (DMD-ssODN-NN1, SEQ ID NO: 40) in which two random bases were introduced near the DMD gene cleavage site of CRISPR-sgRNA was added to HEK293T cells prepared above at 0.5 × 12 μg was introduced per 10 ⁶ cells.

Subsequently, Dox and Dex were added to the culture medium of the cells at final concentrations of 2 μM and 1 μM, respectively, to induce homologous recombination. Subsequently, after culturing for 3 to 6 days, genomic DNA was extracted from each cell, the region containing the target position was amplified by PCR, subjected to TA cloning, and each clone of the PCR product was analyzed by Sanger sequencing.

FIG. 22(a) is a graph summarizing the analysis results. In FIG. 22(a), "Randomized" represents random mutation, "Indel" represents insertion mutation or deletion mutation, and "WT" represents a wild-type nucleotide sequence. As a result, 42.4% of the clones (28/66) had some insertion deletions, but 47.0% of the clones (31/66) had random base substitutions and other insertion deletions were present. confirmed not to.

FIG. 22(b) is a diagram showing amino acids encoded by codons consisting of a nucleotide sequence of 'NNC' (where N represents A, T, G or C). As shown in FIG. 22(b), as a result of introducing random mutations into the first two bases of the codon consisting of the base sequence of 'NNC', it is possible to change the codon sequence encoding amino acids in 4 ² =16 ways. is.

FIG. 22(c) is a diagram showing the base sequences of clones into which random mutations have been introduced and the amino acid sequences encoded by the base sequences. The size of letters corresponds to the abundance ratio of bases or amino acids. As a result, it was found that even when HEK293T cells are used, random mutations can be introduced into the amino acid sequence by introducing a single-stranded DNA donor having a random base sequence.

INDUSTRIAL APPLICABILITY According to the present invention, it is possible to provide a technique for improving the specificity of a sequence-specific DNA cleaving enzyme for a target sequence.
Moreover, according to the present invention, it is possible to provide techniques for deleting, inserting, or substituting arbitrary sequences into target sequences. The arbitrary sequence referred to herein may be a sequence that exists in nature or a sequence that does not exist in nature.
Further, according to the present invention, the cause of a disease can be elucidated by introducing a gene sequence presumed to be the cause of the disease, or by repairing the gene sequence presumed to be the cause of the disease and converting it into a healthy sequence. We can provide the technology to Here, target diseases include, but are not limited to, gene mutations and diseases registered in the aforementioned Human Gene Mutation Database (HGMD).
Moreover, according to the present invention, it is possible to provide a technique for inserting or substituting different arbitrary sequences into a plurality of cells. Then, by utilizing the fact that a plurality of cells have different sequences, it is possible to provide a technique for individually distinguishing cells. For example, single-cell RNA-seq analysis is performed after inserting or substituting a cell-discriminating sequence into a gene expressed in a cell to determine which cell the single-cell RNA-seq data originates from. Sequence data can be retroactively analyzed.
Moreover, according to the present invention, a technique for treating a disease can be provided by restoring a gene sequence that is thought to be the cause of the disease and converting it into a healthy sequence.

Claims (5)

expressing a fusion protein of a sequence-specific DNA-cleaving enzyme and a nuclear receptor under the control of an expression-inducible promoter;
translocating the fusion protein into the nucleus, wherein the nuclear translocated fusion protein sequence-specifically cleaves genomic DNA to form double-stranded DNA breaks;
utilizing a DNA break repair mechanism to repair the double-stranded DNA break and introducing a genetic mutation in the vicinity of the double-stranded DNA break ;
In introducing the genetic mutation, a donor DNA is allowed to coexist, the donor DNA has sequence identity with a region containing the position of the double-stranded DNA break in the genomic DNA, and the nucleotide sequence of the genomic DNA. has 1 to several desired base mutations for
The expression induction time of the fusion protein is within 48 hours,
A method for introducing gene mutation , wherein prophase expression induction is initiated between 12 hours before and 12 hours after coexistence of the donor DNA . 2. The method for introducing gene mutation according to claim 1 , wherein the desired base mutation includes random mutation. 3. The method for introducing gene mutation according to claim 1 or 2 , wherein the sequence-specific DNA-cleaving enzyme is an RNA-guided nuclease or an artificial nuclease. 4. The method for introducing gene mutation according to any one of claims 1 to 3 , wherein the nuclear receptor is an estrogen receptor or a glucocorticoid receptor. The method for introducing gene mutation according to any one of claims 1 to 4 , wherein the expression-inducible promoter is a doxycycline-inducible promoter.

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