KR20050021366A - Method of transferring mutation into target nucleic acid - Google Patents

Abstract

Preparing a DNA having a reverse repeat sequence, wherein the nucleotide sequence of the DNA having a reverse repeat sequence is homologous to the target nucleic acid and comprises a mutation introduced into the target nucleic acid; And introducing into the cell a DNA having a reverse repeat sequence; And a kit for said method.

Description

METHOOD OF TRANSFERRING MUTATION INTO TARGET NUCLEIC ACID

A method of introducing a mutation into a target nucleic acid useful for introduction of the mutation into a gene and repair of the mutation in the gene.

According to the method currently used for gene therapy using a viral vector or the like, the gene is introduced into the cell into a cell whose function is abnormal due to deletion or mutation of the gene in the cell. This restores the normal function of the cell and allows the cell to play its original role. This may be called gene replacement therapy.

Gene introduction methods using viral vectors have the following problems. The mutant gene is actually on the chromosome. Mutant proteins derived from mutant genes have structures similar to those of normal proteins derived from normal genes, and mutant proteins may interfere with the function of normal proteins.

Recently, attention has been paid to gene targeting methods for converting nucleotides of genes of interest into cells other than virus-mediated DNA introduction. The chimeric formation method developed by Eric B. Kmiec (Thomas Jefferson University (Proc. Natl. Acad. Sci. USA, Vol. 93, p.2071-2076 (1996))) is a specific example of a gene targeting method. The portion containing the nucleotide to be changed constitutes DNA, and the RNA stretch uses chimeric oligonucleotides located at both ends for positioning, RNA-DNA binding is stronger than DNA-DNA binding. When a chimeric oligonucleotide is introduced into a cell, the RNA nucleotide sequences at both ends form a double strand by looking for the corresponding nucleotide sequence, and mutations are introduced at the site of interest as a result of inconsistent repair. Oligonucleotides have a complex cyclic structure, in particular, it is necessary to use an additional eight thymine residues to maintain the structure. , Which leads to side effects in significant levels in the binding capacity of the target DNA.

O. Igoucheva et al. Developed a method of forming single stranded oligonucleotides (also referred to as ss oligos below) (Gene Therapy, Vol. 8, p. 391-399 (2001)). This method uses oligonucleotides of dozens of nucleotides. The nucleotide corresponding to the nucleotide in the target nucleic acid to be mutated is located in the center of the oligonucleotide, and several methylated uracil residues that are not readily degraded by intracellular nucleases bind at both ends.

The repair efficiency of the two above mentioned mutagenesis methods is still low. The method of forming single stranded oligonucleotides (ss oligos), which leads to the better repair efficiency of the two, still has a clear drawback. Specifically, it is not possible to simultaneously repair the mutant nucleotides of both the sense and antisense strands of double-stranded DNA during inconsistent repair after binding to the target nucleic acid using this method. Only one of the mutant nucleotides is repaired, and the remaining mutant nucleotides on the complementary strand must be further repaired by the cell's mismatch repair mechanism.

In order to maintain the cyclic structure according to Kmiec et al., Sequences not related to the target DNA are included in the chimeric oligonucleotides (chimeric oligos). Unrelated sequences occupy at least 10% of the oligo sequences. This reduces the activity of targeting the target DNA of the chimeric oligos.

Another important point is the inability to simultaneously repair two or more nucleotides located at a distance relative to the repair mechanism using chimeric oligos or ss oligos and the structure of the oligonucleotides used. If one wants to repair two or more nucleotides located at a distance, or introduces a mutation into two or more sites located at a distance, cloning should be performed several times after oligonucleotide introduction. The process naturally takes a lot of time and labor. In addition, current techniques for introducing DNA into cells significantly damage cells. Thus, it is difficult to obtain viable cells that retain the desired function. In order to repair mutant nucleotides on a chromosome intracellularly, a repair DNA is introduced into the cytoplasm. Restorative DNA migrates to the nucleus as a result of free diffusion with a concentration gradient. In this case a number of repaired DNA molecules have to be introduced into the cell. However, finally only a few molecules enter the nucleus, which results in unsatisfactory mutation repair efficiency.

With gene therapy development, a highly effective method capable of simultaneously repairing multiple nucleotides and active transport of repair DNA into the nucleus has been required as an alternative to the above mentioned less effective repair method based on a complex mechanism.

Summary of the Invention

As a result of intensive research to achieve the above-mentioned object, the inventors have found that DNA having a reverse repeat sequence can be used to efficiently mutate nucleotides in a target nucleic acid. Thus, the present invention has been completed.

The first aspect of the invention

(1) preparing a DNA having a reverse repeat sequence, wherein the nucleotide sequence of the DNA having a reverse repeat sequence is homologous to the target nucleic acid and comprises a mutation introduced into the target nucleic acid;

(2) a method for introducing a mutation into a nucleotide sequence of a target nucleic acid, the method comprising introducing into the cell a DNA having a reverse repeat sequence.

According to the first aspect, a DNA having a reverse repeat sequence may have a binding motif sequence for a protein having a nuclear transport signal, such as a binding motif sequence for a transcription factor. DNA can have suitably modified nucleotides.

According to the first aspect, the target nucleic acid may be a nucleic acid located in the cytoplasm or a nucleic acid located in the nucleus. DNA having a reverse repeat sequence may be double-stranded DNA or single-stranded DNA.

According to the method of the first aspect, a plurality of mutations can be introduced simultaneously into a target nucleic acid. Mutations introduced into the target nucleic acid are exemplified by substitution, deletion and / or insertion of nucleotides.

The second aspect of the present invention provides a target nucleic acid by a method of the first aspect, wherein the nucleotide sequence of the DNA having a reverse repeat sequence comprises DNA having a reverse repeat sequence, wherein the nucleotide sequence is homologous to the target nucleic acid and comprises a mutation introduced into the target nucleic acid. It relates to a kit for introducing a mutation into.

DNA having a reverse repeat sequence included in the kit of the second aspect may have a binding motif sequence for a transcription factor or an appropriately modified nucleotide. DNA having a reverse repeat sequence may be double-stranded DNA or single-stranded DNA.

Brief description of the drawings

1 is a schematic of a plasmid with each reverse repeat sequence prepared according to the present invention.

There is no particular limitation with respect to the "target nucleic acid" according to the invention. Any nucleic acid for which mutational introduction is desired may be included. For example, the present invention can be used to introduce mutations into intracellular DNA. DNA located within the cytoplasm (episome DNA, plasmid, mitochondrial DNA, etc.) or DNA located within the nucleus (chromosomal DNA) serves as a target nucleic acid.

There is no particular limitation regarding the mutations introduced into the target nucleic acid according to the present invention. Mutations can be introduced such as base substitutions, deletions or insertions. In this case, the nucleotide sequence having the mutation may be included in the DNA having the reverse repeat sequence to be used.

Indeed, introducing a mutation in accordance with the present invention is not limited to introducing the mutation into normal normal nucleotides, but also includes an example of restoring a nucleotide sequence with naturally occurring mutations to a normal sequence.

DNA having a reverse repeat sequence (hereinafter referred to as a reverse repeat sequence) used according to the present invention is a DNA having a nucleotide sequence homologous to the target nucleic acid and containing a mutation introduced into the target nucleic acid. As used herein, “homology” refers to a nucleotide sequence capable of forming a double chain with a target nucleic acid or a strand complementary thereto, and does not mean that the nucleotide sequence is a complete match. That is, sufficient nucleotide sequence to form a double strand with a target nucleic acid via base pairs.

There is no particular limitation as regards the method for preparing DNA having a reverse repeat sequence. Chemical synthesis, enzymatic synthesis (nucleic acid amplification reaction, etc.) or biological synthesis (methods using self-replicating nucleic acids such as plasmids) can be used. DNA having a reverse repeating sequence is one in which the sense strand sequence and the antisense strand sequence of the target nucleic acid are arranged in tandem. Any sequence (spacer) can be inserted between these sequences where the resulting DNA can be used to introduce mutation into the target nucleic acid.

Double-stranded DNA with reverse repeat sequences can be used in accordance with the present invention. Single stranded DNA obtained by denaturing double-stranded DNA can also be used. Since the sense strand region and antisense strand region of the single stranded DNA have complementary nucleotide sequences from each other, the DNA is considered to be a hairpin structure as a result of base pairing of these sites.

For example, DNA having a reverse repeat sequence and forming a hairpin structure is disclosed in United States Patent Nos. It can be prepared according to the method for preparing single stranded DNA named slDNA described in 5,643,762, 5,714,323 and 6,043,028.

According to the present invention, mutations can be introduced simultaneously into two or more sites in a target nucleic acid. It is contemplated that DNA having a reverse sequence as far as possible is preferably used for this purpose. DNA homologous recombination repair occurs during the DNA synthesizer (S phase) in the cell cycle. While replicating daughter DNA from the parent DNA, the helix of the parent DNA is released and a replication fork is formed. At this time, the target activity of the DNA capable of simultaneously binding to both the sense and antisense strand of the DNA of the fork region should be high. Thus, we have produced a single strand having the sense and antisense strand sequences of the target DNA, ie single strand reverse repeat sequence DNA capable of binding to both the sense and antisense strand of the target DNA.

Plasmids comprising reverse repeating sequence DNA in which two identical genes or fragments thereof are arranged in opposite directions are first constructed in this production method. The plasmid construction method described in Example 2 illustrates the method. Plasmids can be obtained in large quantities by culturing Escherichia coli cells transformed with plasmids. Reverse repeat sequence DNA can be obtained by cleaving a reverse repeat sequence DNA insert in a plasmid from a vector using sites for restriction enzymes at both ends of the insert. Alternatively, reverse repeat sequence DNA can be amplified by PCR using a plasmid as a template.

Although not intending to limit the invention, it is generally preferred that the length of the gene or fragment thereof inserted into the reverse repeat sequence DNA for plasmid construction is between 500 bp and 1500 bp. If the length is less than 500 bp, it may be difficult to insert two identical DNA fragments in opposite directions in the vector plasmid. In such cases, the DNA of interest can be synthesized using different methods (eg, chemical or enzymatic methods).

The following reverse repeat sequence DNA can be used in accordance with the present invention. The DNA has at least 10 nucleotides, preferably at least 100 nucleotide sequences, located upstream and downstream of the target nucleic acid into which the mutations are introduced. Results Homologous recombination occurs easily.

Mutant nucleotides can be repaired as follows when mutations are introduced into the target nucleic acid using the reverse repeat sequence DNA according to the present invention (eg, to correct for mutant nucleotides in the gene). Reverse repeat sequence DNA is prepared having the sequence of a wild type gene comprising a normal nucleotide at a site complementary to a mutant nucleotide. Subsequently, they are introduced into cells using known DNA introduction methods such as, for example, calcium phosphate methods, electroporation or lipid-mediated hemotransfection methods.

On the other hand, when the mutation is to be introduced into the wild type gene, the mutation can be introduced into the target gene as follows. Reverse repeat sequence DNA comprising nucleotides (nucleotide sequences) introduced into the gene is prepared, and then introduced into cells using the above-mentioned DNA introduction method.

In order to prevent degradation of the DNA by the nuclease, the 5 'end and 3' end of the DNA having the reverse repeat sequence according to the present invention can be protected from the action of the nuclease. Without wishing to limit the invention, for example, terminal protection can be accomplished by physically or chemically closing the DNA. Alternatively, modified nucleotides may be included, such as modified deoxynucleotides, modified ribonucleotides or LNAs (WO 99/14226). Preferably, methylated ribonucleotides, sulfided deoxynucleotides, and the like are introduced to protect the termini. Nucleotides can be introduced into the terminal region of the DNA having a reverse repeat sequence by chemical or enzymatic means as described in the Examples below.

DNA having a reverse repeat sequence according to the present invention may comprise a binding motif sequence for a protein having a nuclear transport signal, ie, a DNA sequence capable of binding to a protein.

There is no particular limitation with regard to a protein having a nuclear transport signal which can be used according to the present invention. Naturally generated or artificial ones can be used. Examples thereof include transcription factors, SV40 large T antigen, histones, nucleoplasmin and double-stranded RNA-binding protein NF90. In addition, Biochim. Biophys. Acta, Vol. 1071, p. 83-101 (1991) can be used with a protein having a nuclear transport signal.

If a protein having a nuclear transport signal is capable of binding a specific DNA sequence, the binding motif for the protein may be included in the DNA having a reverse repeat sequence according to the present invention.

There is no particular limitation with regard to the binding motif sequence that can be used according to the present invention. For example, sequences capable of binding to proteins with nuclear transport signals expressed in the cells into which the mutations are introduced can be used in accordance with the present invention. Although a protein having a nuclear transport signal does not have a DNA binding site or is known to have a binding site, a chimeric protein having a suitable DNA binding sequence in addition to the nuclear transport signal may be prepared and used according to the present invention.

When a protein with a nuclear transport signal is expressed in a cell into which a mutation is introduced, DNA having a reverse repeat sequence with a binding motif for the protein is introduced into the cell. The introduced DNA then binds to the protein and is transported to the nucleus and the mutation of interest can also be introduced into the chromosomal DNA. When a protein having a nuclear transport signal is not expressed in a cell into which a mutation is introduced or is artificially produced, the method of the present invention may be performed by introducing a protein or a gene encoding the protein into a cell. There is no particular limitation regarding the method of introducing a protein or a gene encoding the protein into a cell. Known methods can be used, such as methods using liposomes or the like, or methods using plasmid vectors, viral vectors, and the like. The protein or gene encoding the protein can be introduced into the cell simultaneously with DNA having a reverse repeat sequence having a binding motif sequence for the protein.

A sequence capable of binding to a protein having a nuclear transport signal preferably used in the present invention is exemplified by a DNA sequence capable of binding to a transcription factor. As used herein, transcription factors refer to factors that affect transcriptional efficiency from DNA or RNA by RNA polymerase. Examples of binding motif sequences for transcription factors that may be used in the present invention include, but are not limited to, sequences capable of binding transcription factors, such as NF-κB, Sp1, AP1, NF-IL6 (C / EBPβ), AP2. , Oct-1, SRF and Ets-1. For example, binding motif sequences for transcription factors are described in Eur. J. Biochem, Vol. 268.p 1828-1836 (2001); Biochem, Vol. 263.p 3372-3379 (1998); Cell, Vol. 49, p 741-752 (1987); EMBO J. Vol. 9, p. 1987-1906 (1990); Proc. Natl, Acad. Sci. USA. Vol 88.p 7948-7952 (1991); Genes Dev. Vol 2, p1582-1599 (1988); Nucleic Acids Res., Vol 20 p. 3297-3303 (1992); Genes., Vol 4.p 1451-1453 (1990).

There is no particular limitation regarding the number of copies of the binding motifs included in the DNA having the reverse repeat sequence according to the present invention. One copy or a plurality of sequence copies may be used. There is no particular limitation with regard to the position of the sequence. It may be located at the 5'- or 3'- end of the DNA. For example, two or more copy motif binding sequences can be included at both 5'- and 3'-terminal ends. Preferably, the binding motif sequence is incorporated at the center of the reverse repeat sequence site, ie between the repeat sequences.

DNA having a reverse repeat sequence comprising a binding motif for a protein with a nuclear transport signal can be actively transported into the nucleus. Thus, it is highly effective for introducing mutations into the chromosome or repairing mutations.

The methods of the invention can be used to knock out genes. In this case, for example, one or two nucleotides in the start codon "ATG" can be substituted to prevent translation into a protein. Alternatively, stop codons can be introduced at appropriate locations in the target gene to inhibit the production of full-length detoxification products. Additionally, reverse repeat sequence DNA is prepared in which one or two nucleotide (s) are inserted into (or deleted from) the nucleotide sequence of a target gene and used to produce frame shifts in the nucleotide sequence of the protein-coding site in the target gene. By triggering gene knockout can be achieved. For example, in a gene, a nucleotide in the start codon, 2-30 nucleotides from the start codon, a nucleotide encoding an amino acid residue constituting the active site of the enzyme (if it is an enzyme protein), or (a fluorescence-emitting protein) It is preferred to mutate the nucleotides encoding amino acid residues which are the determinants of fluorescence.

The most important feature of the present invention is that it can be used to simultaneously repair two or more nucleotides located at a distance. The plurality of mutations can be repaired in a manner similar to that for repair of a single mutant nucleotide using reverse repeat sequence DNA. Such DNA consists of hundreds to thousands of nucleotides and includes nucleotides for repairing a plurality of mutant nucleotides in a target nucleic acid among sites of hundreds to thousands of nucleotides. For example, two mutant nucleotides located at a distance of about 200 nucleotides in a gene can be repaired at a time point as described in Example 5.

In the above-mentioned example where a mutation is introduced at two or more sites, there is no particular limitation with regard to the distance of that site. The sites can be hundreds of nucleotides apart. In terms of mutagenesis efficiency, the site is preferably located within 200 nucleotides, more preferably within 100 nucleotides and most preferably within 30 nucleotides.

The present invention provides a kit used for introducing a mutation according to the present invention. In one example, the kit comprises DNA having a reverse repeat sequence for introducing a mutation into the target nucleic acid. The kit also includes reagents for introducing DNA into cells.

In addition, cells into which a mutation is introduced into a gene according to the method of the present invention may be transplanted in vivo. Thus, gene therapy can be performed in which the mutant gene or the mutated repaired gene is expressed in vivo.

Examples of cells include, but are not limited to, blood cells (hematopoietic cells, hematopoietic stem cells, bone marrow cells, umbilical cord blood cells, peripheral blood stem cells, monocytes, lymphocytes, B cells, T cells, etc.), fibroblasts, neuroblasts, Nerve cells, endothelial cells, vascular endothelial cells, liver cells, myoblasts, skeletal muscle cells, smooth muscle cells, cancer cells and leukemia cells.

For example, gene therapy using hematopoietic stem cells as target cells can be performed as follows. First, hematopoietic stem cell-containing material (eg bone marrow tissue, peripheral blood or umbilical cord blood) is taken from the donor. The material can actually be used in gene transduction methods. Generally, however, mononuclear cell fractions containing hematopoietic stem cells are prepared by density gradient centrifugation or the like, or hematopoietic stem cells are further purified using appropriate wash surface labeling molecules. Hematopoietic stem cell-containing materials are then used for mutagenesis introduction or mutation repair using the methods of the invention. The cells thus obtained can be transplanted to the recipient by methods such as, for example, intravenous administration. The beneficiary is preferably the donor himself, but allogeneic transplants can be performed. For example, if cord blood is used as the material, allogeneic transplants are performed.

The gene therapy of the present invention is exemplified for genes whose actions have been reduced or lost due to mutations or whose expression has been enhanced in patients. For example, genetic diseases resulting from single nucleotide substitutions (eg sickle cell anemia) are preferably treated according to the methods of the invention.

The above mentioned gene therapy can be applied not only to humans but also to non-human vertebrates and invertebrates.

One application of the invention is a non-human transgenic animal (eg, mouse, using germ cells (eg embryonic stem cells, primitive seed cells, oocytes, ovary cells, eggs, sperm cells or sperm) as target cells. Rats, dogs, pigs, cattle, chickens, frogs, or medaka. For example, gene knockout animals in which only expression of the gene of interest is specifically inhibited can be produced according to the methods of the invention. Knockout animals are very useful for analyzing gene function, selecting reagents related to gene function, and the like.

The following examples illustrate the invention in detail and are not intended to limit its scope.

Example 1

Mutant nucleotide introduction into the red-shift green fluorescent protein gene

A single nucleotide in the nucleotide sequence of the red-shift green fluorescent protein (hereinafter referred to as GFP) -coding gene (SEQ ID NO: 1) inserted into plasmid pQBI25 (Quantum Biotechnologies Inc.) was substituted to prevent the gene from being expressed. Amino acid residues at positions 66-68 are important for fluorescence of GFP. PCR was used in an in vitro mutation kit (Takara Bio) to change the codon "TAT" encoding tyrosine at position 67 to the stop codon "TAG". Mutant GFP (mGFP) genes prepared as described above and GFP-coding genes containing no mutations were independently inserted into plasmid pDON-AI (Takara Bio) to construct pDON-mGFP and pDON-GFP, respectively. 50 μl of TE buffer containing 1 μg of one of these plasmids was mixed with 50 μl of Optime medium (Gibco BRL) containing 1 μg of Lipofectamine 2000 (LF2000, Gibo BRL). The mixture was used to transfect 293 cells. After 4 days the cells were observed under fluorescence microscopy. Strong green fluorescence was observed for cells added with pDON-GFP, while no fluorescence was observed for cells comprising pDON-mGFP. Based on these results, a mutant gene that does not fluoresce in cells was constructed. mGFP gene was used in the following test.

Example 2

To prepare reverse repeat sequence DNA (hereinafter referred to as irDNA) having the nucleotide sequence of the GFP gene, a set of two GFP gene fragments in opposite directions was first introduced into the plasmid as a template for the amplification of irDNA. Fragments of the full length sequence of the GFP gene inserted into plasmid pQBI25 were amplified using primers Us-EcoRI (SEQ ID NO: 2) and DEND (SEQ ID NO: 3). 760-bp DNA fragments obtained by digesting fragments with EcoRI and BamHI (both Takara Bio) were introduced between the EcoRI and BamHI sites in plasmid pUC19 (Takara Bio). DNA fragments of the full length sequence of the GFP gene were obtained by amplification using primers Us-hindIII (SEQ ID NO: 4) and DEND. The 760-bp DNA fragment obtained by digesting the DNA fragment with HindIII (Takara Bio) and BamHI was introduced between the HindIII and BamHI sites in this plasmid. The plasmid constructed as described above was named pucGFP0-0.

In addition, primers Us-EcoRI and DEND were used to amplify DNA fragments of the full length sequence of the GFP gene. The 714-bp DNA fragment obtained by digesting the fragment with EcoRI and PvuII (Takara Bio) was introduced between the EcoRI and SmaI sites in plasmid pUC19. DNA fragments of the full length sequence of the GFP gene were obtained by amplification using primers Us-hindIII and DEND. A 760-bp DNA fragment obtained by digesting the DNA fragment with HindIII and BamHI was introduced between the HindIII and BamHI sites in this plasmid. The plasmid constructed as described above was named pucGFP0-2.

In addition, DNA fragments were amplified from the GFP gene using primers U100hindIII (SEQ ID NO: 5) and U100bamhl (SEQ ID NO: 6). 110-bp DNA fragments were prepared by digesting the DNA fragments with HindIII and BamHI. Plasmid pucGFP0-6 was constructed by replacing 760-bp HindIII-BamHI fragment with 110-bp DNA fragment in plasmid pucGFP0-0.

pucGFP0-0 has the reverse repeat sequence for the full length GFP-encoding gene. The insert DNA is 1518 bp in length. 38 nucleotide sites from the stop codon are deleted in one of the repeat sequences in pucGFP0-2. The insert DNA is 1479 bp in length. Plasmid pucGFP0-6 has a reverse repeat sequence consisting of the full length GFP-encoding gene and the nucleotides at positions 150-250 of the GFP gene. Insert DNA is 868 bp in length. 1A, 1B and 1C show pucGFP0-0, pucGFP0-2 and pucGFP0-6, respectively.

Reverse repeat sequence DNA, 0-0 irDNA, 0-2 irDNA and 0-6 irDNA were prepared by PCR using pucGFP0-0, pucGFP0-2 and pucGFP0-6 as templates. 0-0 irDNA and 0-2 irDNA were prepared using primers Us-ecoRI-1 (SEQ ID NO: 7) and Us-hindIII-1 (SEQ ID NO: 8). 0-6 irDNA was prepared using primers Us-ecoRI-1 and U100hindIII-1 (SEQ ID NO: 9). The nucleotides "T" and "A" of the wild-type GFP gene for restoring mutant nucleotides "G" and "C" introduced into the mGFP gene are respectively expressed in the sense strands at 0-0 irDNA, 0-2 irDNA and 0-6 irDNA, respectively. 237th and 1282th nucleotides, 237th and 1243th nucleotides, and 237th and 813th nucleotides from the 5 'end or from the 3' end of the antisense strand (EcoRI). The position of the nucleotides involved in mutant nucleotide repair in the sense or antisense of the mGFP gene is indicated by the marks "*" and "#" in FIG. 1.

Example 3

Repair of Single Mutant Nucleotides of the mGFP Gene in Episomal Experimental Models

Gene repair experiments were performed using reverse repeat sequence DNA and binding to the target nucleic acid and repair of the mutant nucleotides in the target nucleic acid were investigated. Toxic effects on cells due to several DNA introductions were achieved by introducing into the cytoplasm a plasmid with the mGFP gene (pcep-mGFP) introduced as the target nucleic acid, and the ss oligo or one of three reverse repeat sequence DNAs (irDNAs) as a control. Simultaneously introduced and prevented

The nucleotide sequence of the ss oligo is shown in SEQ ID NO: 10.

(1) Repair of episomal mGFP using heat-modified irDNA

To prepare an experimental model, a 760-bp HindIII-BamHI fragment comprising the mGFP gene was isolated from pDON-mGFP constructed in Example 1 and sub-divided between HindIII and BamHI in the episomal expression vector pCEP4 (Invitrogen) of mammals. Cloning to prepare plasmid pcep-mGFP. 293 cells (8 × 10 ⁴ ) were seeded in 48-well plates and incubated overnight in DEME medium containing 10% FBS. 2 μg of each 0-0 irDNA, 0-2 irDNA, and 0-6 irDNA were thermally denatured at 94 ° C. for 5 minutes and rapidly cooled in ice water. 2 μg ss oligo, diluted with 0-0 irDNA, 0-2 irDNA, or 0-6 irDNA, 37.5 μl Optimen medium of 1.5 μg prep-mGFP or pUC19 (negative control). The mixture was mixed with an equal amount of Optimen medium containing 2 μg of LF2000. A mixture prepared by mixing with 37.5 μl Optimen medium containing only 3.5 μg prep-mGFP and the same amount of Optimen medium containing 2 μg LF2000 was used as negative control 2.

After incubation for 20 minutes, each DNA-LF2000 reagent complex was added to the cells. The mixture was left at room temperature for 30 minutes. 150 μl of Optimen medium was further added. After 6 hours of transfection, 1 ml of DEME medium containing 10% FBS was added. After 48 hours, no green fluorescence-emitting cells (GFP-positive cells) were observed in negative controls 1 and 2. GFP-positive cells, on the other hand, were observed in wells that used one of three irDNAs for transfection for repair of ss oligos or pcep-mGFP containing the target DNA and mutant nucleotides. Maximum GFP-positive cells were observed on day 4. Trypsinized the cells from the plate and GFP-positive cells were measured using FACS. Table 1 shows the results obtained by subtracting the values for negative control 1 from the measurements as background.

The maximum for the number of GFP-positive cells per 10 ⁴ 293 cells as the repair index of mutant nucleotides in the mGFP gene was observed at 0-0 irDNA. The repair effect was about three times higher than that observed with ss oligo. A significant difference (p <0.05) was observed between the repair efficiency of 0-0 irDNA and ss oligos as a result of the Mann-Whitney U test during four independent experiments.

Table 1

DNA 10 Number of GFP-positive cells per ⁴ cells 0-0 irDNA 18.21 ± 4.22 0-2 irDNA 11.79 ± 2.84 0-6 irDNA 5.26 ± 0.74 ss oligo 5.40 ± 2.64

(2) Repair of episomal mGFP gene using irDNA without thermal denaturation

MGFP gene repair was investigated as described in Example 3- (1) except that irDNAs prepared by PCR amplification were used without thermal denaturation. The number of GFP-positive cells that appeared in 10 ⁴ 293 cells is shown in Table 2. Higher repair efficiency was observed than with ss oligos using irDNA without thermal denaturation. A significant difference (p <0.05) was observed between the repair efficiency of 0-0 irDNA or 0-2 irDNA and ss oligos as a result of the Mann-Whitney U test during four independent experiments.

TABLE 2

DNA 10 Number of GFP-positive cells per ⁴ cells 0-0 irDNA 39.27 ± 9.21 0-2 irDNA 20.78 ± 2.80 0-6 irDNA 13.12 ± 2.15 ss oligo 6.54 ± 2.64

Repair efficiency was observed higher than that observed with pcep-mGFP and 0-0 irDNA for mutation repair and ss oligos as target nucleic acids for transfection after 6 h time intervals.

The test described in (2) above showed that no plasmid was introduced into all cells used as target nucleic acids. pcep-GFP and 0-0 irDNA were introduced into 293 cells under the same conditions and the cells of GFP-positive cells were measured. As a result, 21.22 ± 4.67% of the cells harbored the plasmid. The results shown in Table 2 were converted by removing cells not containing plasmids.

Table 3 shows the number of GFP-positive cells repaired with mutant nucleotides per 10 ⁴ cells with introduced pcep-mGFP. A significant difference (p <0.05) was observed between the repair efficiency of 0-0 irDNA and ss oligos as a result of the Mann-Whitney U test during four independent experiments.

TABLE 3

DNA Number of GFP-positive cells per 10 ⁴ cells with introduced pcep-mGFP 0-0 irDNA 201.42 ± 58.42 0-2 irDNA 109.12 ± 27.48 0-6 irDNA 72.24 ± 23.97 ss oligo 39.76 ± 20.94

The amount of 3 irDNAs and ss oligos (2 μg) used for transfection in the above-mentioned experiments was 529 nmol (ss oligo), 9.5 nmol (0-0 irDNA), 10.1mnol (0-2irDNA) and 16.6 nmol (0-). 6ir DNA). The repair rate of mutant nucleotides by three irDNAs was higher than that by ss oligos. Even when transfection without denaturation, a high recovery rate similar to that observed when transfected with heat-denatured irDNA was observed using 0-0 irDNA containing the full-length GFP gene region. The recovery rate was five times higher than with ss oligos. Even when the number of 0-0 irDNA molecules was tens of times smaller than the number of ss oligo molecules, the repair rate by 0-0 irDNA was several times higher than that by ss oligo. This experiment showed that the activity of targeting the target nucleic acid of irDNA is significantly higher than that of ss oligo, which increases the repair rate.

Example 4

Introduction of mGFP gene into the chromosome of cells

Retroviral particles for introducing the mGFP gene into the chromosome of the cells were prepared using the retrovirus packing kit ampho (Takara Bio). The recombinant retroviral vector pDON-mGFP described in Example 1 was simultaneously introduced into 293 cells with a packing vector in a kit according to the calcium phosphate method. After incubation for 48 hours, the culture supernatant was collected and filtered. Culture supernatants (retroviral suspensions) were diluted and added to the medium for 293 cells. PCR-amplified DNA fragment sequencing confirmed that the cloning cells (293-10 cells) contained the mGFP gene. Genomic DNA was extracted from 293-10 cells and analyzed for the sequence of integration sites and retrospective cellular chromosomal DNA for retroviral vectors. It was confirmed that one copy of the GFP gene was introduced intracellularly. This cloning cell was used in the following experiment.

Example 5

Preparation of 0-0 irDNA for Repair Modified with Methylated Ribonucleotides and Repair of Mutant Nucleotides in mGFP

Nucleotide sequences of 5 'primer RNA-ecoRI and 3' primer RNA-hindIII synthesized using 2'-0-methyl RNA phosphoramidite and CE-phosphoamidite according to the phosphoramidite method SEQ ID NOs: 11 and 12, respectively. In each primer the first six nucleotides are methylated. PCR was performed using these two primers and pcuGFP0-0 as a template to bind 6 methylated ribonucleotides to the 5 'end of the sense strand and the antisense strand of 0-0 irDNA obtained. 0-0 irDNA, which is difficult to be degraded by nucleases in cells, was named R0-0 irDNA.

4 x 10 ⁵ 293-10 cells or 293 cells (negative control) were seeded into each well of a 24-well plate and incubated overnight in DEME medium containing 10% FBS. 4 μg of ss oligo and 0-0 irDNA or R0-0 irDNA were diluted with 75 μl Optimen medium. The mixture was mixed with an equal amount of Optimen medium containing 4 μg of LF2000. After incubation for 20 minutes, each DNA-LF2000 reagent complex was added directly to the cells. An additional 90 μl DEME medium containing 10% FBS was added for transfection. The moles of ss oligo, 0-0 irDNA and R0-0 irDNA in the medium were 992.00 nmol, 17.83 nmol and 17.82 nmol. After 6 hours, 1.5 ml of DEME medium containing 10% FBS was added. After 16-18 hours, the medium was exchanged with DEME medium containing 3% FBS. Incubation was continued at 32 ° C. for 2 and 1/2 days.

To accurately count GFP-positive cells, cells are washed with PBS, trypsinized to separate and transferred to wells of new well plates containing medium and the number of GFP-positive cells in each well (number of cells 1.6 to 1.9 x 10 ^6). ) Was counted under a fluorescence microscope. As a control, the background value for GFP-positive cells in 293 cells was subtracted from the value for GFP-positive cells in 293-10 cells with repair DNA added. Results The average number of GFP-positive cells per well containing ss oligos, 0-0 irDNA and R0-0 irDNA was about 54, 2300 and 3800. The number of GFP-positive cells per 293-10 cells of 10 ⁴ is shown in Table 4.

A significant difference (p <0.05) was observed between the repair efficiencies of the two irDNA and ss oligos as a result of the Mann-Whitney U test during four independent experiments.

Table 4

DNA 10 Number of GFP-positive cells per ⁴ cells 0-0 irDNA 12.32 ± 4.08 R0-0 irDNA 24.20 ± 3.84 ss oligo 0.31 ± 0.08

As described above, 293-10 cells containing R0-0 irDNA had higher repair rates of mutant nucleotides in the chromosome than those containing ss oligos or 0-0 irDNA. Experimental results suggest that the mutation repair rate was increased due to the resistance of methylated ribonucleotides to degradation by nucleases and the like, and that the R0-0 irDNA retention time was prolonged in the nucleus or cytoplasm compared with the inclusion of 0-0 irDNA. do.

Example 6

Mutant nucleotide repair using irDNA comprising a sequence capable of binding to a transcription factor

Transcription factors are important factors involved in nuclear signaling and regulate gene expression. Many transcription factors are synthesized in the cytoplasm and are always waiting for their turn in the cytoplasm. When a transcription factor receives an activation signal, it is transported and acts on the nucleus.

I-κB, which inhibits the activity of the transcription factors NF-κB and NF-κB, is mainly associated with each other and is present in the cytoplasm. Stimulated by cytokines such as TNF-α and phosphorylated I-κB, NF-κB is activated and transported into the nucleus (Proc. Natl. Acad. Sci. USA., Vol, Vol 91, p 11884-11888 ( 1994). NF-κB binds to a single chain DNA sequence named the NF-κB motif located upstream of the gene.

The presence of NF-κB in 293 cells and DNA sequences capable of binding to them have been reported (Eur. J. Biochem. Vol 268, p. 1828-1836 (2001)). Three primers GFP-κB1 (SEQ ID NO: 14), GFP-κB2 (SEQ ID NO: 15), and GFP-κB3 (SEQ ID NO: 16) were synthesized to introduce this sequence into the reverse repeat sequence in the center of irDNA. First, PCR reactions were performed using GFP-κB1 and the above-mentioned primers Us-EcoRI and EcoRI-BamHI fragment cleaved from pucGFP 0-0 containing the GFP gene as template DNA to obtain amplified DNA fragments. Subsequently, PCR was performed using this amplified fragment and GFP-κB2 and Us-EcoRI as templates to obtain amplified fragments. In addition, PCR was performed using these amplified fragments and GFP-κB3 and Us-EcoRI as templates to obtain amplified fragments. The resulting amplified fragment was digested with EcoRI and BamHI and the EcoRI-BamHI fragment in pucGFR0-0 was replaced with the fragment generated from the digestion. The plasmid constructed as described above was named pucGFP0nf-0.

pucGFP0nf-0 includes an insert comprising a reverse repeat sequence for GFP and an NF-κB-binding motif sequence. The length of the insert is 1548 bp. PCR was performed using this plasmid and primers RNA-ecoRI and RNA-hindIII, or S-ecoRI and S-hindIII as templates. S-ecoRI and S-hindIII are primers whose sequences match the primers RNA-ecoRI and RNA-hindIII, respectively, and from the 5 'end of each primer 6 nucleotides are replaced with deoxyribonucleotides, and the phosphate group between them Is modified with sulfur molecules. The amplified fragments were named R0nf-0 irDNA and S0nf-0 irDNA, respectively.

Preculture conditions for 293-10 cells and 293 cells (negative control) and the method of transfection of the repair DNA were obtained by raising TNF-α, introducing R0-0 irDNA, R0nf-0 irDNA or S0nf-0 irDNA. It was the same as described in Example 5 except for incubating for 6 hours at a concentration of 8 ng / ml in the medium added at 6 hours.

Cells were treated, counted under GFP-positive cell surrogate fluorescence microscopy in each well, and the background values for GFP-positive cells in 293 cells as controls were cells added with repair DNA as described in Example 5. Subtracted from the values for GFP-positive cells in. As a result, the average number of GFP-positive cells comprising ss oligo, R0-0 irDNA, R0nf-0 irDNA, and S0nf-0 irDNA was 88, about 6000, 10,000 or more and 10,000, respectively. The number of GFP-positive cells per 10 ⁴ 293-10 cells comprising the ss oligo, R0-0 irDNA, R0nf-0 irDNA or S0nf-0 irDNA is shown in Table 5. The highest recovery rate observed using S0nf-0 irDNA was 138 times higher than that observed using ss oligos.

A significant difference (p <0.05) was observed between the repair efficiencies of three irDNA and ss oligos as a result of the Mann-Whitney U test during four independent experiments. In addition, a significant difference (p <0.05) was observed between the repair efficiencies of R0nf-0 irDNA and S0nf-0 irDNA with introduced binding motif sequences for R0-0 irDNAs and transcription factors.

Table 5

DNA 10 Number of GFP-positive cells per ⁴ cells R0-0 irDNA 28.19 ± 6.54 R0nf-0 irDNA 61.02 ± 13.72 S0nf-0 irDNA 67.58 ± 20.31 ss oligo 0.49 ± 0.14

The ss oligo or 0-0 irDNA introduced into the cytoplasm with lipofectamine migrates to the nucleus in a passive diffusion mode as a result of free diffusion with a concentration gradient. Because the concentration of 0-0 irDNA is several orders of magnitude lower than that of the ss oligo, and therefore fewer copies of 0-0 irDNA transported to the nucleus, the repair rate of the mutant nucleotides was expected to be lower than that of the ss oligo. However, the actual results showed that the repair rate including 0-0 irDNA was higher than that of the ss oligo. This result indicated that the repair rate of mutant nucleotides containing 0-0 irDNA was high due to the high target activity of 0-0 irDNA, despite the smaller number of copies transported into the cell nucleus. The effect is further increased by modifying irDNA at the 5 'end with methylated ribonucleotides or sulfided deoxynucleotides that are not readily degraded by nucleases.

The repair effect of DNA having the same sequence as the target DNA that does not include the reverse repeat sequence structure to which the NB-κB-binding motif sequence binds was several times lower than that of the reverse repeat sequence DNA (0-nf-0 irDNA). The effect of DNA in which two molecules of NB-κB-binding motif sequence were introduced in the opposite direction to the center of 0-0 irDNA was half the effect of 0nf-0 irDNA in which one molecule of NB-κB-binding motif sequence was introduced.

Example 7

Repair of the GFP Gene with a Double Mutation

PCR was applied in an in vitro mutagenesis method to replace residue "G" in the initiation codon "ATG" of the GFP gene with "T". The mutant GFP (m1GFP) gene was used for subcloning into pCEP4 and transfecting 293 cells, confirming that no fluorescence was observed. In addition, the HindIII-NheI fragment (-36-174) upstream of the mGFP gene prepared in Example 1 was replaced with the HindIII-NheI fragment (-36-174) of the m1GFP gene to replace the double mutant GFP (dmGFP) gene. It was constructed.

The dmGFP gene has two mutant nucleotides ("T" in position 3 and "G" in position 201) separated by 198 nucleotides. The 760-bp HindIII-BamHI fragment of the dmGFP gene was subcloned in the HindIII and BamHI sequences in pCEP4 to prepare the plasmid, pcep-dmGFP, to be used as a target nucleic acid.

The efficiency of repairing double mutations simultaneously using 0-0 irDNA was investigated under the experimental conditions described in Example 3. As a result, GFP-positive cells showing simultaneous repair of two mutant nucleotides were observed at a rate of 15.85 ± 2.00 per 10 ⁴ cells.

Industrial availability

The present invention provides a method for introducing a mutation into a nucleotide sequence on a gene with high efficiency. Introduction of artificial mutations into intracellular DNA or repair of nonfunctional genes resulting from mutations is possible according to the invention. The methods of the invention are useful for gene therapy, construction of knockout organisms, functional analysis of genes and the like.

Sequence Listing Free Text

SEQ ID NO: 1; Genes Encoding Red-Shift Green Fluorescent Proteins

SEQ ID NO: 2; PCR primers Us-EcoRI for amplifying genes encoding red-shift green fluorescent proteins

SEQ ID NO: 3; PCR primer DEND to amplify the gene encoding the red-shift green fluorescent protein

SEQ ID NO: 4; PCR primers Us-HindIII for amplifying genes encoding red-shift green fluorescent proteins

SEQ ID NO: 5; PCR primer U100HindIII for amplifying a gene region encoding a red-shift green fluorescent protein

SEQ ID NO: 6; PCR primer D100BamHI to amplify the locus encoding the red-shift green fluorescent protein

SEQ ID NO: 7; PCR primers Us-EcoRI-1 for amplifying genes encoding red-shift green fluorescent proteins

SEQ ID NO: 8; PCR primers for amplifying genes encoding red-shift green fluorescent proteins Us-HindIII-1.

SEQ ID NO: 9; PCR primer U100HindIII-1 for amplifying a gene region encoding a red-shift green fluorescent protein.

SEQ ID NO: 10; Chimeric oligonucleotide ss oligo. "Nucleotides 1-4 and 50-53 are 2'-0-methyluridines".

SEQ ID NO: 11; PCR primers RNA-ecoRI for amplifying a gene region encoding a red-shift green fluorescent protein. Nucleotides 1-6 are 2'-0-methylribonucleotides-other nucleotides are deoxynucleotides "

SEQ ID NO: 12; PCR primer RNA-hindIII for amplifying the gene region encoding the red-shift green fluorescent protein. "Nucleotides 1 to 6 are 2'-0-methyl ribonucleotides-other nucleotides are deoxynucleotides"

SEQ ID NO: 14; PCR primer GFP-κB1 for amplifying a gene region encoding a red-shift green fluorescent protein.

SEQ ID NO: 15; PCR primer GFP-κB2 for amplifying the gene region encoding the red-shift green fluorescent protein.

SEQ ID NO: 16; PCR primer GFP-κB3 for amplifying a gene region encoding a red-shift green fluorescent protein.

SEQUENCE LISTING <110> TAKARA BIO INC. <120> Method for introducing mutation into target nucleic acid <130> 663910 <150> JP 2002-204887 <151> 2002-07-12 <150> JP 2003-113534 <151> 2003-04-18 <160> 16 <170> Patent In Ver. 2.1 <210> 1 <211> 720 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: Gene encoding red-shifted green fluorescence protein. <400> 1 atggctagca aaggagaaga actcttcact ggagttgtcc caattcttgt tgaattagat 60 ggtgatgtta acggccacaa gttctctgtc agtggagagg gtgaaggtga tgcaacatac 120 ggaaaactta ccctgaagtt catctgcact actggcaaac tgcctgttcc atggccaaca 180 ctagtcacta ctctgtgcta tggtgttcaa tgcttttcaa gatacccgga tcatatgaaa 240 cggcatgact ttttcaagag tgccatgccc gaaggttatg tacaggaaag gaccatcttc 300 ttcaaagatg acggcaacta caagacacgt gctgaagtca agtttgaagg tgataccctt 360 gttaatagaa tcgagttaaa aggtattgac ttcaaggaag atggaaacat tctgggacac 420 aaattggaat acaactataa ctcacacaat gtatacatca tggcagacaa acaaaagaat 480 ggaatcaaag tgaacttcaa gacccgccac aacattgaag atggaagcgt tcaactagca 540 gaccattatc aacaaaatac tccaattggc gatggccctg tccttttacc agacaaccat 600 tacctgtcca cacaatctgc cctttcgaaa gatcccaacg aaaagagaga ccacatggtc 660 cttcttgagt ttgtaacagc tgctgggatt acacatggca tggatgaact gtacaactga 720 <210> 2 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer Us-EcoRI to amplify a gene encoding red-shifted green fluorescence protein. <400> 2 cttgaattcg gtaccgagct cggatcgggc gcgcaagaaa 40 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer DEND to amplify a gene encoding red-shifted green fluorescence protein. <400> 3 cactggcggc cgttactagt 20 <210> 4 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer Us-HindIII to amplify a gene encoding red-shifted green fluorescence protein. <400> 4 cttaagcttg gtaccgagct cggatcgggc gcgcaagaaa 40 <210> 5 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer U100HindIII to amplify a portion of gene encoding red-shifted green fluorescence protein. <400> 5 ctaagcttct ggcaaactgc ctgttccatg gccaacacta 40 <210> 6 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer D100BamHI to amplify a portion of gene encoding red-shifted green fluorescence protein. <400> 6 tcggatccaa gtcatgccgt ttcatatgat ccgggtatct 40 <210> 7 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer Us-EcoRI-1 to amplify a gene encoding red-shifted green fluorescence protein. <400> 7 gaattcggta ccgagctcgg atcgggcgcg caagaaa 37 <210> 8 <211> 37 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer Us-HindIII-1 to amplify a gene encoding red-shifted green fluorescence protein. <400> 8 aagcttggta ccgagctcgg atcgggcgcg caagaaa 37 <210> 9 <211> 38 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer U100HindIII-1 to amplify a portion of gene encoding red-shifted green fluorescence protein. <400> 9 aagcttctgg caaactgcct gttccatggc caacacta 38 <210> 10 <211> 54 <212> DNA <213> Artificial Sequence <220> <221> modified_base (222) (1) .. (4) <223> um <220> <221> modified_base (222) (50) .. (53) <223> um <220> <223> Description of Artificial Sequence: Chimeric oligonucleotide ss Oligo. <400> 10 uuuuatcttg aaaagcattg aacaccatag cacagagtag tgactagtgu uuut 54 <210> 11 <211> 35 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: PCR primer RNA-ecoRI to amplify a portion of gene encoding red-shifted green fluorescence protein. “nucleotides 1 to 6 are 2” -O-methyl ribonucleotides? other nucleotides are deoxyribonucleotides ” <400> 11 gaauucggta ccgagctcgg atcgggcgcg caaga 35 <210> 12 <211> 35 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer RNA-hindIII to amplify a portion of gene encoding red-shifted green fluorescence protein. “Nucleotides 1 to 6 are 2’-O-methyl ribonucleotides? other nucleotides are deoxyribonucleotides ” <400> 12 aagcuuggta ccgagctcgg atcgggagag caaga 35 <210> 13 <211> 30 <212> DNA <213> homo sapience <400> 13 gattgcttta gcttggaaat tccggagctg 30 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> Description of Artificial Sequence: PCR primer GFP-kB1 to amplify a portion of gene encoding red-shifted green fluorescence protein. <400> 14 agctaaagca atctcagttg tacagttcat ccatgccatg 40 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: PCR primer GFP-kB2 to amplify a portion of gene encoding red-shifted green fluorescence protein. <400> 15 tccggaattt ccaagctaaa gcaatctcag ttgtacagtt 40 <210> 16 <211> 40 <212> DNA <213> Artificial Sequence <223> Description of Artificial Sequence: PCR primer GFP-kB3 to amplify a portion of gene encoding red-shifted green fluorescence protein. <400> 16 ttttggatcc cagctccgga atttccaagc taaagcaatc 40 663910 10/9

Claims (16)

(1) preparing a DNA having a reverse repeat sequence, wherein the nucleotide sequence of the DNA having a reverse repeat sequence is homologous to the target nucleic acid and comprises a mutation introduced into the target nucleic acid; (2) introducing a mutation into the nucleotide sequence of the target nucleic acid, comprising introducing into the cell DNA having a reverse repeat sequence. The method of claim 1, wherein the DNA having a reverse repeat sequence has a binding motif sequence for a protein having a nuclear transport signal. The method of claim 2, wherein the binding motif sequence for the protein having a nuclear transport signal is a binding motif sequence for a transcription factor. The method of claim 1, wherein the DNA having the reverse repeat sequence has modified nucleotides. The method of claim 1, wherein the DNA having a reverse repeat sequence is double-stranded DNA. The method of claim 1, wherein the DNA having a reverse repeat sequence is single-stranded DNA. The method of claim 1, wherein the target nucleic acid is a nucleic acid located within the cytoplasm. The method of claim 1, wherein the target nucleic acid is a nucleic acid located in the nucleus. The method of claim 1, wherein the plurality of mutations are introduced simultaneously into the target nucleic acid. The method of claim 1, wherein the mutations introduced into the target nucleic acid are substitutions, deletions and / or insertions of nucleotides. A kit for introducing a mutation into a target nucleic acid by the method of claim 1, wherein the nucleotide sequence of the DNA having the reverse repeat sequence comprises a DNA having a reverse repeat sequence homologous to the target nucleic acid and comprising a mutation introduced into the target nucleic acid. 12. The kit of claim 11, wherein the DNA having the reverse repeat sequence has a binding motif sequence for a protein having a nuclear transport signal. The kit of claim 12, wherein the binding motif sequence for the protein having a nuclear transport signal is a binding motif sequence for a transcription factor. The kit of claim 11, wherein the DNA having the reverse repeat sequence has modified nucleotides. 12. The kit of claim 11, wherein the DNA having the reverse repeat sequence is double-stranded DNA. 12. The kit of claim 11, wherein the DNA having the reverse repeat sequence is single-stranded DNA.