JP7539136B2 - Method for site-specific introduction of cas9 gene using viral vector - Google Patents

Description

The present invention relates to a method for introducing the cas9 gene or a mutant gene thereof into a desired genomic region and an introduction kit for said method.

CRISPR (clustered regularly interspaced short palindromic repeats) and CRISPR-associated (Cas) proteins are known to act as a bacterial adaptive immune system by cleaving target DNA in a single-stranded guide RNA (sgRNA)- and protospacer adjacent motif (PAM)-dependent manner. Cas9 nuclease from Streptococcus pyogenes is widely used as a powerful genome editing tool in eukaryotes that have a DNA double-strand break (DSB) repair pathway (e.g., Non-Patent Documents 1 and 2). During DSB repair by the non-homologous end joining (NHEJ) pathway, small insertions and/or deletions (indels) are introduced into the target DNA, resulting in site-specific gene disruption (knock out). It is also possible to introduce genes into the target region by homology-directed repair (HDR) by providing donor DNA containing homology arms to the target region (knock in), although the efficiency depends on the host cell.

When performing knock-in using the CRISPR/Cas9 system, if the cas9 gene can be introduced into a genomic region where it can be stably expressed when introducing Cas9, sgRNA, and donor DNA into target cells, it will be possible to efficiently repair not only single gene mutations but also multiple gene mutations seen in cancer, etc., simply by supplying the appropriate sgRNA and donor DNA to the cells. In ex vivo, the cas9 gene can be introduced into the genome by transfection or electroporation, which have the characteristic that there is no limit to the size of the DNA contained in the vector, but these methods cannot be applied in vivo.

A typical method for introducing genes into genomes in vivo is the use of viral vectors. Examples of viral vectors used in the in vivo method include lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors. However, the use of these viral vectors has traditionally had both advantages and disadvantages. For example, lentiviral vectors can easily incorporate desired genes into the genome of a host, but they have the disadvantage that the site of incorporation cannot be selected, which raises the concern that unpredictable destruction of endogenous genes may occur. Adenoviral vectors can carry even large-sized DNA, and since DNA is not inserted into the genome of the host, endogenous genes are not destroyed, but they are highly immunogenic to the living body and are not suitable for frequent administration. On the other hand, adeno-associated viral vectors are preferable in that they have low immunogenicity and can introduce genes selectively into tissues. However, the adeno-associated viral vector has the disadvantage that the DNA size that can be carried is small, and the cas9 gene, sgRNA, and donor DNA cannot be carried at the same time. Therefore, the method for introducing the cas9 gene into a safe site of the host genome in vivo has still not been solved.

Mali, P. et al., Science 339, 823-827 (2013).

Cong, L. et al., Science 339, 819-823 (2013).

The objective of the present invention is to provide a site-specific introduction method of the cas9 gene using a viral vector to achieve stable expression of the cas9 gene not only ex vivo but also in vivo, and an introduction kit for said method.

The present inventors randomly integrated the wild-type cas9 (Cas9v1) gene into the genome of human cultured cells using a lentivirus vector. In addition, an adeno-associated virus vector carrying an N-terminal 1.9 kb fragment of the wild-type cas9 (Cas9v2) gene sandwiched between a base sequence (sg1 sequence) complementary to the base sequence of the aavs1 gene, a homologous sequence of the upstream base sequence of the cleavage site targeted by the sg1 sequence, and a homologous sequence of the downstream base sequence was introduced into the cells, and the N-terminal 1.9 kb fragment of the cas9v2 gene was integrated into the aavs1 gene using the activity of Cas9v1. Furthermore, an adeno-associated virus vector carrying a 2.3 kb C-terminal fragment of the cas9v2 gene sandwiched between a base sequence (sg2 sequence) complementary to the base sequence at the 3' end of the N-terminal 1.9 kb fragment of the cas9v2 gene, a homologous sequence of the upstream base sequence of the cleavage site targeted by the sg2 sequence, and a homologous sequence of the downstream base sequence was introduced into the cells, and the cas9v2 gene was reconstructed at the aavs1 locus. Finally, an adeno-associated virus vector containing a base sequence complementary to the LTR of the lentivirus vector was introduced into the cells, and the cas9v1 gene was removed. It was confirmed that the Cas9v2 expressed in the cells thus obtained was functional. In addition, instead of the cas9v2 gene, Cas9 nickase with a D10A mutation and inactive Cas9 (dCas9) with D10A and N876A mutations were incorporated into the aavs1 locus, and it was confirmed that they were functional.
Based on these findings, the present inventors conducted further investigations and have completed the present invention.

That is, the present invention comprises the following.
[1] A method for introducing a cas9 gene or a mutated gene thereof into a desired genomic region of a cell, comprising the steps of:
(1) expressing the cas9 gene or a mutated version thereof in a cell;
(2) cleaving a desired genomic region of the cell with the Cas9 protein or its mutant protein expressed in step (1) and introducing a partial base sequence contained in the cas9 gene or its mutant gene into the cleavage site; and (3) cleaving the partial base sequence introduced in step (2) with the Cas9 protein or its mutant protein expressed in step (1) and introducing a partial base sequence contained in the cas9 gene or its mutant gene other than the partial base sequence introduced in step (2) into the cleavage site, thereby constructing the cas9 gene or its mutant gene in the desired genomic region of the cell.
[2] The method according to [1], wherein step (1) is carried out by infecting a cell with a viral vector containing the base sequence of the cas9 gene or a mutated gene thereof.
[3] The method according to [2], wherein step (1) is a step of expressing the cas9 gene or a mutated gene thereof from the genome of the cell.
[4] The method according to [3], wherein the viral vector in step (1) is a lentiviral vector.
[5] The method according to any one of [1] to [4], wherein the step (2) is carried out by infecting a cell with a viral vector comprising the following base sequence:
(i) a nucleotide sequence complementary to a nucleotide sequence of a desired genomic region of the cell; and
(ii) A partial base sequence contained in the cas9 gene or a mutant gene thereof, the partial base sequence being sandwiched between a homologous sequence of the upstream base sequence and a homologous sequence of the downstream base sequence of the cleavage site targeted by the base sequence of (i).
[6] The method according to [5], wherein the viral vector in step (2) is an adeno-associated viral vector.
[7] The method according to any one of [1] to [6], wherein the step (3) is carried out by infecting a cell with a viral vector comprising the following base sequence:
(iii) a base sequence complementary to the partial base sequence introduced in step (2), and
(iv) A partial base sequence contained in the cas9 gene or a mutant gene thereof other than the partial base sequence introduced in step (2), the partial base sequence being sandwiched between a homologous sequence of the upstream base sequence and a homologous sequence of the downstream base sequence of the cleavage site targeted by the base sequence of (iii).
[8] The method according to [7], wherein the viral vector in step (3) is an adeno-associated viral vector.
[9] (4) The method according to any one of [2] to [8], further comprising a step of removing the cas9 gene or a mutated gene thereof of step (1) from the genome of the cell.
[10] The method according to [9], wherein step (4) is carried out by infecting a cell with a viral vector comprising a base sequence complementary to the upstream base sequence and a base sequence complementary to the downstream base sequence of the cas9 gene or its mutated gene in step (1).
[11] The method according to [10], wherein the viral vector in step (4) is an adeno-associated viral vector.
[12] The method described in [2], wherein step (1) is a step of expressing the cas9 gene or a mutated gene thereof from the viral genome.
[13] The method according to [12], wherein the viral vector in step (1) is an adenoviral vector.
[14] The method according to any one of [1] to [13], wherein the mutant gene in step (1) is a gene encoding a Cas9 protein having a mutation selected from the group consisting of a D10A mutation, an H840A mutation, and an N863A mutation.
[15] The method according to any one of [1] to [14], wherein the mutant gene constructed in step (3) is a gene encoding a Cas9 protein having at least one mutation selected from the group consisting of a D10A mutation, an H840A mutation, and an N863A mutation.
[16] The method according to any one of [1] to [15], wherein the desired genomic region is the aavs1 locus.
[17] The method according to any one of [1] to [16], wherein the partial base sequence introduced in step (2) comprises the base sequence represented by SEQ ID NO: 7 or 31.
[18] The method according to any one of [1] to [17], wherein the partial base sequence introduced in step (3) comprises the base sequence represented by SEQ ID NO: 17 or 32.
[19] A kit for introducing a Cas9 gene or a mutant gene thereof into a desired genomic region of a cell, comprising the following viral vector:
(1) a viral vector containing the base sequence of the cas9 gene or a mutant gene thereof;
(2) a viral vector comprising the following nucleotide sequence:
(i) a nucleotide sequence complementary to a nucleotide sequence of a desired genomic region of the cell; and
(ii) A partial base sequence contained in the cas9 gene or a mutant gene thereof, the partial base sequence being sandwiched between a homologous sequence of an upstream base sequence and a homologous sequence of a downstream base sequence of a cleavage site targeted by the base sequence of (2) (i);
(3) A viral vector comprising the following nucleotide sequence:
(i) a base sequence complementary to the partial base sequence of (2)(ii) above; and
(ii) A partial base sequence contained in the cas9 gene or a mutant gene thereof other than the partial base sequence of (2)(ii) above, the partial base sequence being sandwiched between the upstream base sequence and the downstream base sequence of the cleavage site targeted by the base sequence of (3)(i).
[20] The kit according to [19], wherein the viral vector of (1) is a lentiviral vector or an adenoviral vector.
[21] The kit according to [19] or [20], wherein the viral vector of (2) is an adeno-associated viral vector.
[22] The kit according to any one of [19] to [21], wherein the viral vector of (3) is an adeno-associated viral vector.
[23] (4) The kit according to any one of [19] to [22], further comprising a viral vector comprising a base sequence complementary to the upstream base sequence and a base sequence complementary to the downstream base sequence of the cas9 gene or a mutant gene thereof of (1) above.
[24] The kit according to [23], wherein the viral vector of (4) is an adeno-associated viral vector.
[25] The kit according to any one of [19] to [24], wherein the mutant gene contained in the viral vector of (1) is a gene encoding a Cas9 protein having a mutation selected from the group consisting of a D10A mutation, an H840A mutation, and an N863A mutation.
[26] The kit according to any one of [19] to [25], wherein the mutant gene contained in the viral vector of (2) or (3) is a gene encoding a Cas9 protein having at least one mutation selected from the group consisting of a D10A mutation, an H840A mutation, and an N863A mutation.
[27] The kit according to any one of [19] to [26], wherein the desired genomic region is the aavs1 locus.
[28] The kit according to any one of [19] to [27], wherein the partial base sequence contained in the viral vector of (2) includes the base sequence represented by SEQ ID NO: 7 or 31.
[29] The kit according to any one of [19] to [28], wherein the partial base sequence contained in the viral vector of (3) includes the base sequence represented by SEQ ID NO: 17 or 32.

According to the present invention, stable expression of the cas9 gene is possible not only ex vivo but also in vivo, and by simply supplying cells with the appropriate sgRNA and donor DNA, it is possible to efficiently repair not only single gene mutations but also multiple gene mutations seen in cancer and other conditions.

FIG. 1 shows a schematic diagram of the four-step strategy for integration of Cas9 into the AAVS1 locus. FIG. 2 shows the AAV vector #1 construct for introducing the N-terminal 1.9 kb Cas9v2 into the AAVS1 locus. FIG. 3 shows PCR and immunoblot to confirm genomic structure and N-terminal Cas9v2 expression. FIG. 4 shows the AAV vector #2 construct for reconstituting full-length Cas9v2-FLAG in the AAVS1 locus. FIG. 5 shows PCR and immunoblot to confirm the reconstruction of full-length Cas9v2-FLAG at the AAVS1 locus. FIG. 6 shows the AAV vector #3 construct targeting the lentiviral LTR integrated into the genome. FIG. 7 shows the removal of lentivirus containing Cas9v1-Myc from the genome. FIG. 8 shows knockout of IFNAR1 using AAV vector #4 to verify the function of the reconstructed Cas9v2-FLAG. FIG. 9 shows a schematic diagram of the four-step strategy using Cas9(Nick)-Myc. FIG. 10 shows a method for sequential infection of AAV vectors. FIG. 11 shows expression of Cas9v1-Myc in HEK 293T. FIG. 12 shows the structure of two alleles of N-terminal Cas9v2 clone #8. FIG. 13 shows the removal of randomly integrated Cas9v1-Myc-containing lentivirus genomes without drug selection. FIG. 14 shows the removal of randomly integrated Cas9v1-Myc-containing lentivirus genomes without drug selection. FIG. 15 shows the assessment of elimination of genomically integrated lentivirus without drug selection. FIG. 16 shows DNA sequence analysis of the IFNAR1 gene after sgRNA treatment. FIG. 17 shows the AAV vector #5 construct for introducing the N-terminal 1.9 kb Cas9v2 nickase into the AAVS1 locus. FIG. 18 shows PCR and immunoblot to confirm genomic structure and N-terminal Cas9v2 nickase expression. FIG. 19 shows reconstruction of full-length Cas9v2-FLAG nickase at the AAVS1 locus. FIG. 20 shows PCR and immunoblot to confirm reconstitution of full-length Cas9v2-FLAG nickase. FIG. 21 shows PCR and immunoblot to confirm that full-length Cas9v2-FLAG nickase could be reconstituted without drug selection. FIG. 22 shows the removal of lentivirus containing Cas9v1-Myc. FIG. 23 shows the elimination of lentivirus containing Cas9v1-Myc without drug selection. FIG. 24 shows knockout of IFNAR1 using AAV vector #4 to verify the function of Cas9v2-FLAG nickase. FIG. 25 shows sequence analysis of the IFNAR1 gene after sgRNA treatment. FIG. 26 shows the AAV vector #6 construct for reconstituting full-length dCas9v2-VP64 at the AAVS1 locus. FIG. 27 shows PCR and immunoblot to confirm the reconstruction of full-length dCas9v2-VP64 at the AAVS1 locus. FIG. 28 shows PCR and immunoblot to confirm reconstitution of full-length dCas9v2-VP64 at the AAVS1 locus without drug selection. FIG. 29 shows the removal of lentivirus containing Cas9v1-Myc. FIG. 30 shows activation of the endogenous IFNβ gene promoter via reconstituted dCas9v2-VP64. FIG. 31 shows immunoblot analysis of two independent clones at each step of the entire process. FIG. 32 shows immunoblot analysis of two independent clones at each step of the entire process. FIG. 33 shows a lentiviral construct for expressing Myc-tagged Cas9 nickase (Cas9(Nick)-Myc). FIG. 34 shows the emergence of HEK 293T cells expressing lentiviral Cas9(Nick)-Myc without drug selection. FIG. 35 shows the AAV vector #7 construct for introducing the N-terminal 1.9 kb Cas9v2 nickase into the AAVS1 locus. FIG. 36 shows introduction of the N-terminal 1.9 kb Cas9v2 nickase into the AAVS1 locus. FIG. 37 shows the AAV vector #8 construct for reconstituting full-length Cas9v2-FLAG nickase at the AAVS1 locus. FIG. 38 shows reconstruction of full-length Cas9v2-FLAG nickase at the AAVS1 locus. FIG. 39 shows the AAV vector #9 construct for reconstituting full-length dCas9v2-VP64 at the AAVS1 locus. FIG. 40 shows reconstruction of full-length dCas9v2-VP64 at the AAVS1 locus. FIG. 41 shows PCR and immunoblot to confirm the reconstitution of full-length Cas9v2-FLAG nickase at the AAVS1 locus by sequential infection with AAV vectors #7 and #8. FIG. 42 shows PCR and immunoblot to confirm the reconstruction of full-length dCas9v2-VP64 at the AAVS1 locus by sequential infection with AAV vectors #7 and #9. FIG. 43 shows a comparison of the genomic structures of 293T cells (clones #1-#6) carrying the full-length Cas9v2 nickase-FLAG gene reconstructed in FIG. 41. Figure 44 shows that integration of the Cas9(Nick)-Myc lentiviral genome remained at the gene level in all clones, even in 293T cells, where it was not detectable by the immunoblots in Figures 41D and 42D.

The present invention provides a method for introducing the cas9 gene or a mutant gene thereof into a desired genomic region of a cell (hereinafter, the method of the present invention).

The protein encoded by the cas9 gene introduced in the method of the present invention is a member of the Cas protein family that constitutes the adaptive immune system that provides acquired resistance to foreign genes in bacteria and archaea. The Cas9 protein functions as an endonuclease that recognizes the PAM sequence in DNA and cleaves it upstream. The Cas9 protein contains two functional domains with endonuclease activity, and is therefore capable of cleaving double-stranded DNA to produce blunt ends. Therefore, the cas9 gene introduced in the method of the present invention refers to a gene that forms a complex with a guide RNA and encodes a protein that has double-stranded DNA cleavage activity.

Examples of the cas9 gene to be introduced in the method of the present invention include, but are not limited to, a gene encoding SpCas9 derived from Streptococcus pyogenes, a gene encoding StCas9 derived from Streptococcus thermophilus, and a gene encoding NmCas9 derived from Neisseria meningitidis.

In addition, the cas9 gene introduced in the method of the present invention may be a mutant cas9 gene. The mutant cas9 gene is not particularly limited as long as it maintains the ability to form a complex with the guide RNA and has a mutation in which one or two functional domains having endonuclease activity are inactivated. In one embodiment, such a mutant cas9 gene may be, for example, a cas9 gene having a mutation selected from the group consisting of a mutation in which the 10th aspartic acid of the cas9 protein is replaced with alanine (D10A mutation), a mutation in which the 840th histidine is replaced with alanine (H840A mutation), and a mutation in which the 863rd asparagine is replaced with alanine (N863A mutation). In another embodiment, the mutated cas9 gene may be, for example, a cas9 gene having at least one mutation selected from the group consisting of a mutation in which the 10th aspartic acid of the cas9 protein is replaced with alanine (D10A mutation), a mutation in which the 840th histidine is replaced with alanine (H840A mutation), and a mutation in which the 863rd asparagine is replaced with alanine (N863A mutation). A protein encoded by a cas9 gene having a mutation selected from the group consisting of D10A mutation, H840A mutation, and N863A mutation has one of the two endonuclease domains of the Cas9 protein inactivated, and acts as a nickase that cuts only one strand of double-stranded DNA, so that off-target effects can be expected to be suppressed. In addition, the protein encoded by the cas9 gene having the D10A and H840A mutations, the D10A and N863A mutations, or the D10A, H840A, and N863A mutations has two endonuclease domains inactivated and cannot cleave DNA, but the transcription of the target gene can be activated by forming a fusion protein with a transcription activator using a Cas9 protein having this mutation.

In the method of the present invention, the cells into which the cas9 gene or its mutant gene is introduced may be cells in a living body or cultured cells. The living body may be, but is not limited to, a human or other warm-blooded animal (e.g., mouse, rat, rabbit, sheep, pig, cow, cat, dog, monkey, bird, etc.). The cells in the body may be any cells into which the cas9 gene or its mutant gene can be introduced by the method of the present invention, such as hepatocytes, spleen cells, nerve cells, glial cells, pancreatic β cells, bone marrow cells, mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fibrocytes, muscle cells, adipocytes, immune cells (e.g., macrophages, T cells, B cells, natural killer cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synovial cells, chondrocytes, osteocytes, osteoblasts, osteoclasts, mammary cells, or stromal cells, or precursor cells, stem cells, or cancer cells of these cells, and are preferably cells that cause some disease. The cultured cells are not particularly limited as long as they are cultured cells of the above-mentioned cells.

In the method of the present invention, the desired genomic region of the cell into which the cas9 gene or its mutant gene is introduced is not particularly limited as long as it is a genomic region in which the cas9 gene or its mutant gene can be stably expressed. Examples of such genomic regions include the AAVS1 locus, the hROSA26 locus, and the CCR5 locus, and the AAVS1 locus, which has been most extensively analyzed and is used frequently, is preferred.

The method of the present invention comprises the following steps.
(1) A process for expressing the cas9 gene or a mutated gene thereof in a cell.
(2) A step of cleaving a desired genomic region of the cell using the Cas9 protein or a mutant protein thereof expressed in step (1) and introducing a partial base sequence contained in the Cas9 gene or a mutant gene thereof into the cleavage site.
(3) A process of cleaving the partial base sequence introduced in step (2) by the Cas9 protein or its mutant protein expressed in step (1), and introducing a partial base sequence contained in the cas9 gene or its mutant gene other than the partial base sequence introduced in step (2) into the cleavage site, thereby constructing the cas9 gene or its mutant gene in a desired genomic region of the cell.

The method of the present invention includes a step (step (1)) of expressing the cas9 gene or a mutant gene thereof (hereinafter, the cas9 gene of step (1)) in a cell. The cas9 gene to be introduced in step (1) is not limited as long as it is a gene that forms a complex with a guide RNA and encodes a protein having double-stranded DNA cleavage activity, and for example, the above-mentioned cas9 gene is preferably used. In addition, the mutant gene of the cas9 gene to be introduced in step (1) includes a cas9 gene having a mutation selected from the group consisting of a mutation in which the 10th aspartic acid of the Cas9 protein is replaced with alanine (D10A mutation), a mutation in which the 840th histidine is replaced with alanine (H840A mutation), and a mutation in which the 863rd asparagine is replaced with alanine (N863A mutation). As the cas9 gene in step (1), a mutant gene is more preferable because it is expected to suppress off-target effects.

In step (1), the expression of the cas9 gene of step (1) in the cells is not particularly limited as long as the Cas9 protein or a mutant protein thereof (hereinafter, the Cas9 protein of step (1)) is expressed in the cells. For example, step (1) is carried out by infecting the cells with a viral vector containing the base sequence of the cas9 gene of step (1). Examples of viral vectors used to infect the cells include viral vectors that express the cas9 gene of step (1) by introducing it into the genome of the cells, and viral vectors that directly express the gene from the viral vector without introducing it into the genome of the cells. However, as described below, a viral vector that directly expresses the cas9 gene of step (1) from the viral vector is more preferred because of the advantage that it is not necessary to remove the cas9 gene of step (1) introduced into the genome of the cells.

In step (1), when a viral vector for introducing the cas9 gene of step (1) into the genome of a cell is used as the viral vector used for infecting the cell, a viral vector containing the base sequence of the cas9 gene of step (1) sandwiched between LTRs is used. For example, the base sequence of the cas9 gene of step (1) inserted between the 5'LTR and 3'LTR is inserted into an appropriate plasmid for preparing a viral vector, which is then introduced into a packaging cell, packaged into a virus, and the recovered virus is administered orally or parenterally to a living body or added to the medium of a cultured cell according to a conventional method, thereby randomly introducing the cas9 gene of step (1) into the genome of the cell. The site of the genome to which the cas9 gene of step (1) is introduced is not particularly limited as long as it can be expressed. In addition, the number of cas9 genes of step (1) introduced into the genome may be one or more.

The viral vector is not particularly limited as long as it is capable of expressing the cas9 gene of step (1) in the genome of the cell, but examples include retroviral vectors, lentiviral vectors, and adeno-associated viral vectors. Lentiviral vectors are preferred because they can carry large-sized genes and are less likely to cause cancer in cells.

On the other hand, in the case where a viral vector capable of directly expressing the cas9 gene of step (1) from the viral vector is used as the viral vector used to infect the cells in step (1) without introducing the gene into the genome of the cells, for example, the base sequence of the cas9 gene of step (1) is inserted into an appropriate plasmid for preparing a viral vector, and then introduced into a packaging cell, packaged into a virus, and the recovered virus is administered orally or parenterally to a living body or added to the medium of a cultured cell according to a conventional method, thereby introducing the viral vector containing the cas9 gene of step (1) into a cell and expressing the gene. In addition, the number of cas9 genes of step (1) introduced into the viral vector may be one or more.

The viral vector is not particularly limited as long as it is possible to directly express the cas9 gene from the viral vector without introducing the gene from step (1) into the genome of the cell, but from the viewpoint of enabling transient expression, it is also possible to use a non-replicating adenoviral vector or a lentiviral vector lacking integrase activity.

The method of the present invention includes a step (step (2)) of cleaving a desired genomic region of a cell with the Cas9 protein or a mutant protein thereof expressed in step (1) and introducing a partial base sequence contained in the cas9 gene or a mutant gene thereof into the cleavage site. In step (2), first, the desired genomic region of the cell is cleaved by the Cas9 protein of step (1). The Cas9 protein forms a complex with a single-stranded nucleic acid (sgRNA) containing a base sequence complementary to the base sequence of the desired genomic region of the cell, and cleaves the double-stranded DNA on the 5' side of the protospacer adjacent motif (PAM).

The base sequence (CRISPR-RNA (crRNA) (a)) complementary to the base sequence of the desired genomic region is not particularly limited as long as it is adjacent to the 5' side of the PAM in the desired genomic region. The length of the base sequence of the crRNA (a) is not particularly limited as long as it can ensure specificity for the desired genomic region, but is usually 10 to 30 bases long, preferably 15 to 25 bases long, and more preferably 20 bases long.

The PAM varies depending on the type of Cas9 protein in step (1), but examples include NGG (N is A, G, T, or C; the same applies below) when a Cas9 (SpCas9) protein derived from Streptococcus pyogenes or a mutant protein thereof is used, NNAGAAW (W is A or T; the same applies below) when a Cas9 (StCas9) protein derived from Streptococcus thermophilus or a mutant protein thereof is used, and NNNNGATT when a Cas9 (NmCas9) protein derived from Neisseria meningitidis or a mutant protein thereof is used.

The single-stranded nucleic acid (sgRNA(a)) containing a base sequence complementary to the base sequence of the desired genomic region of the cell may further contain a base sequence (trans-activating RNA (tracrRNA)) necessary for recruiting the Cas9 protein in step (1). The base sequence of the tracrRNA may be a known base sequence, such as the base sequence represented by SEQ ID NO: 2. The tracrRNA may be linked directly to the 3' end of the crRNA(a), or a spacer sequence may be inserted between them.

Secondly, in step (2), a partial base sequence contained in the cas9 gene or its mutant gene (the partial base sequence of the cas9 gene in step (2)) is introduced into the cleavage site targeted by a base sequence complementary to the base sequence of the desired genomic region of the cell. In the present invention, the partial base sequence of the cas9 gene in step (2) refers to a partial base sequence starting from the start codon of the cas9 gene or its mutant gene. The cas9 gene introduced in step (2) is not limited as long as it is a gene that forms a complex with a guide RNA and codes for a protein having double-stranded DNA cleavage activity, similar to the cas9 gene introduced in step (1), and for example, the above-mentioned cas9 gene is preferably used. In addition, the mutant gene of the cas9 gene introduced in step (2) includes a cas9 gene having at least one mutation selected from the group consisting of a mutation in which the 10th aspartic acid of the cas9 protein is replaced with alanine (D10A mutation), a mutation in which the 840th histidine is replaced with alanine (H840A mutation), and a mutation in which the 863rd asparagine is replaced with alanine (N863A mutation).

The length of the partial base sequence of the cas9 gene in step (2) is not particularly limited as long as it is within the range of the length of the base sequence that can be incorporated into the viral vector containing the partial base sequence of the cas9 gene in step (2), but is usually several tens of bp to several kbp, preferably 200 bp to 500 bp. Examples of the partial base sequence of the cas9 gene in step (2) include a base sequence containing the base sequence represented by SEQ ID NO: 7 or 37.

The partial base sequence of the cas9 gene in step (2) is inserted into the cleavage site of the desired genomic region of the cell cleaved by the Cas9 protein in step (1). Here, the insertion of the partial base sequence of the cas9 gene in step (2) into the cleavage site is achieved by homologous recombination repair (HDR). Therefore, the partial base sequence of the cas9 gene in step (2) may be further sandwiched between a homologous sequence of the upstream base sequence and a homologous sequence of the downstream base sequence of the cleavage site in the desired genomic region of the cell cleaved by the Cas9 protein in step (1).

The degree of identity of the above-mentioned homologous sequences is not particularly limited as long as it allows homologous recombination. The degree of identity that allows homologous recombination varies depending on the length of the polynucleotide, but may be, for example, at least 80%, preferably at least 85%, more preferably at least 90%, even more preferably 95%, and most preferably 100%. The degree of identity can be determined by a method known per se, for example, by using BLASTN available at NCBI in its default settings. The degree of identity may also be determined by performing an affine gap search using the Smith-Waterman algorithm with a gap open penalty of 12 and a gap extension penalty of 1.

The length of the homologous sequence is not particularly limited as long as it is long enough for homologous recombination to occur at the cleavage site. Generally speaking, however, the longer the homologous region, the better for efficient homologous recombination. On the other hand, the length of the homologous sequence is limited to a certain extent by the length of the base sequence that can be incorporated by the viral vector. Therefore, taking these factors into consideration, the length of the homologous sequence may usually be 200 bp to 500 bp, preferably 250 bp to 400 bp.

In step (2), a method of cleaving a desired genomic region of a cell using the Cas9 protein in step (1) and introducing a partial base sequence of the cas9 gene in step (2) into the cleavage site can be carried out, for example, by infecting a cell with a viral vector containing the following base sequence:
(i) A base sequence (base sequence (i)) that is complementary to a base sequence of a desired genomic region of a cell.
(ii) A partial base sequence contained in the cas9 gene or a mutant gene thereof, the partial base sequence being sandwiched between a homologous sequence of the upstream base sequence and a homologous sequence of the downstream base sequence of the cleavage site targeted by the base sequence of (i) (base sequence (ii)).
The base sequences (i) and (ii) may be identical to the base sequences described above.

In step (2), the cells can be infected by, for example, inserting the base sequences (i) and (ii) into an appropriate viral vector, introducing the vector into a packaging cell, packaging the vector into a virus, and then administering the recovered virus orally or parenterally to a living body or adding the virus to the medium of a cultured cell in a conventional manner, thereby cleaving the desired genomic region of the cell with the Cas9 protein in step (1) and introducing the partial base sequence of the cas9 gene in step (2) into the cleavage site.

The viral vector is not particularly limited as long as it is possible to cleave the desired genomic region of the cell and introduce the partial base sequence of the cas9 gene of step (2) into the cleavage site. For example, an adeno-associated viral vector is preferred because it has the advantages of not randomly inserting into the genome and having low antigenicity.

The method of the present invention includes a step (step (3)) of cleaving the partial base sequence introduced in step (2) by the Cas9 protein or mutant protein expressed in step (1), introducing a partial base sequence contained in the cas9 gene or mutant gene other than the partial base sequence introduced in step (2) into the cleavage site, and constructing the cas9 gene or mutant gene in a desired genomic region of the cell. In step (3), first, the partial base sequence introduced in step (2) is cleaved by the Cas9 protein of step (1). The Cas9 protein forms a complex with a single-stranded nucleic acid (sgRNA) containing a base sequence complementary to the partial base sequence introduced in step (2), and cleaves the double-stranded DNA on the 5' side of the protospacer adjacent motif (PAM).

The base sequence (crRNA(b)) complementary to the partial base sequence introduced in step (2) is not particularly limited as long as it is adjacent to the 5' side of the PAM in the partial base sequence introduced in step (2). The length of the base sequence of crRNA(b) is not particularly limited as long as it can ensure specificity for the desired genomic region, but is usually 10 to 30 bases long, preferably 15 to 25 bases long, and more preferably 20 bases long. The PAM may be the same as the PAM described in step (2).

The single-stranded nucleic acid (sgRNA(b)) containing a base sequence complementary to the partial base sequence introduced in step (2) may further contain a base sequence (trans-activating RNA (tracrRNA)) required for recruiting the Cas9 protein in step (1). The base sequence of the tracrRNA may be identical to the tracrRNA described in step (2). The tracrRNA may be linked directly to the 3' end of the crRNA(b) or may be linked via a spacer sequence.

Secondly, in step (3), a partial base sequence (partial base sequence of the cas9 gene in step (3)) contained in the cas9 gene or its mutant gene other than the partial base sequence introduced in step (2) is introduced into the cleavage site targeted by a base sequence complementary to the partial base sequence introduced in step (2). In the present invention, the partial base sequence of the cas9 gene in step (3) refers to the partial base sequence of the cas9 gene or its mutant gene downstream of the cleavage site in the partial base sequence introduced in step (2). The length of the partial base sequence of the cas9 gene in step (3) may be the length obtained by subtracting the partial base sequence of the cas9 gene in step (2) from the entire base sequence of the cas9 gene. The partial base sequence of the cas9 gene in step (3) may be, for example, a base sequence containing the base sequence represented by SEQ ID NO: 17 or 47.

The partial base sequence of the cas9 gene in step (3) is inserted into the cleavage site of the partial base sequence introduced in step (2) cleaved by the Cas9 protein in step (1). Here, the insertion of the partial base sequence of the cas9 gene in step (3) into the cleavage site is achieved by homologous recombination repair (HDR). Therefore, the partial base sequence of the cas9 gene in step (3) may be further sandwiched between a homologous sequence of the upstream base sequence and a homologous sequence of the downstream base sequence of the cleavage site in the partial base sequence of the cas9 gene in step (3) cleaved by the Cas9 protein in step (1). The degree of identity and length of the above homologous sequences may be the same as the degree of identity and length of the homologous sequence in step (2).

In step (3), the partial base sequence introduced in step (2) is cleaved by the Cas9 protein in step (1) and the partial base sequence of the cas9 gene in step (3) is introduced into the cleavage site. This can be carried out, for example, by infecting a cell with a viral vector containing the following base sequence:
(iii) a nucleotide sequence complementary to the partial nucleotide sequence introduced in step (2).
(iv) A partial base sequence contained in the cas9 gene or a mutant gene thereof other than the partial base sequence introduced in step (2), the partial base sequence being sandwiched between a homologous sequence of the upstream base sequence and a homologous sequence of the downstream base sequence of the cleavage site targeted by the base sequence of (iii).
The base sequences (iii) and (iv) may be identical to the base sequences described above.

In step (3), the cells can be infected by, for example, inserting the base sequences (i) and (ii) into an appropriate viral vector, introducing the vector into a packaging cell, packaging the vector into a virus, and recovering the virus by a conventional method, orally or parenterally administering the virus to a living body or adding the virus to the medium of a cultured cell, thereby cleaving the partial base sequence introduced in step (2) by the Cas9 protein in step (1), and introducing the partial base sequence of the cas9 gene in step (3) into the cleavage site.

The viral vector is not particularly limited as long as it is possible to cleave the partial base sequence introduced in step (2) and introduce the partial base sequence of the cas9 gene in step (3) into the cleavage site. For example, an adeno-associated viral vector is preferred because it has the advantages of not randomly inserting into the genome and having low antigenicity.

As described above, in step (3), the downstream partial base sequence contained in the cas9 gene or its mutant gene is linked immediately after the upstream partial base sequence contained in the cas9 gene or its mutant gene in the desired genomic region of the cell, and the full-length cas9 gene or its mutant gene is reconstructed in the desired genomic region of the cell. The reconstructed full-length cas9 gene or its mutant gene can play an alternative or different role to the cas9 gene or its mutant gene expressed in step (1).

In step (1), if a viral vector that introduces the cas9 gene of step (1) into the genome of a cell is used as the viral vector used to infect the cell, the cas9 gene introduced into the genome of the cell may destroy endogenous genes and may cause abnormal proliferation of normal cells. Therefore, it is preferable that the cas9 gene introduced into the genome of the cell in step (1) is removed. Therefore, the method of the present invention may further include a step (step (4)) of removing the cas9 gene or a mutated gene thereof of step (1) from the genome of the cell.

In step (4), the upstream and downstream base sequences of the cas9 gene or its mutant gene in step (1) are cleaved by the Cas9 protein or its mutant protein. The Cas9 protein or its mutant protein for cleaving the upstream and downstream base sequences of the cas9 gene may be the Cas9 protein or its mutant protein expressed from the genome of the cell in step (1), or may be the Cas9 protein or its mutant protein expressed from the full-length cas9 gene or its mutant gene reconstructed in steps (2) and (3). The Cas9 protein forms a complex with single-stranded nucleic acids (sgRNA(c1) and sgRNA(c2)) containing a base sequence complementary to the upstream base sequence and a base sequence complementary to the downstream base sequence of the cas9 gene or its mutant gene in step (1), respectively, and cleaves the double-stranded DNA on the 5' side of the protospacer adjacent motif (PAM).

The base sequence (CRISPR-RNA (crRNA) (c1)) complementary to the upstream base sequence of the cas9 gene or its mutant gene in step (1) and the base sequence (CRISPR-RNA (crRNA) (c2)) complementary to the downstream base sequence are not particularly limited as long as they are adjacent to the 5' side of the PAM in the upstream base sequence of the cas9 gene or its mutant gene in step (1) and adjacent to the 5' side of the PAM in the downstream base sequence, respectively. The length of the base sequence of crRNA (c1) and crRNA (c2) is not particularly limited as long as specificity to the upstream base sequence or downstream base sequence of the cas9 gene can be ensured, but is usually 10 to 30 bases long, preferably 15 to 25 bases long, and more preferably 20 bases long. The PAM may be the same as the PAM described in step (2).

The single-stranded nucleic acid (sgRNA(c1) or sgRNA(c2)) containing a base sequence complementary to the upstream base sequence or the downstream base sequence of the cas9 gene or its mutant gene in step (1) may further contain a base sequence required for Cas9 recruitment (trans-activating RNA (tracrRNA)). The base sequence of the tracrRNA may be identical to the tracrRNA described in step (2). The tracrRNA may be directly linked to the 3' ends of the crRNA(c1) and sgRNA(c2), or a spacer sequence may be inserted between them.

In step (4), the method of cleaving the upstream and downstream base sequences of the cas9 gene or its mutant gene in step (1) with the Cas9 protein or its mutant protein can be carried out in the same manner as the method of infecting cells with a viral vector in step (2) or (3). Examples of viral vectors used to infect cells include viral vectors that are directly expressed from the viral vector without being introduced into the genome, and adeno-associated viral vectors are more preferred.

In step (4), the upstream and downstream nucleotide sequences of the cas9 gene or its mutant gene in step (1) may be cleaved by the Cas9 protein or its mutant protein using a viral vector that expresses sgRNAs that target a nucleotide sequence complementary to the 5'LTR and a nucleotide sequence complementary to the 3'LTR.

The present invention also provides a kit for introducing a Cas9 gene or a mutant gene thereof into a desired genomic region (hereinafter, the kit of the present invention). The Cas9 gene or a mutant gene thereof introduced using the kit of the present invention, and the desired genomic region to which they are introduced, may be the same as the Cas9 gene or a mutant gene thereof, and the desired genomic region described in the method of the present invention.

The kit of the present invention includes the following viral vectors:
(1) a viral vector containing the base sequence of the cas9 gene or a mutant gene thereof;
(2) a viral vector comprising the following nucleotide sequence:
(i) a nucleotide sequence complementary to a nucleotide sequence of a desired genomic region of the cell; and
(ii) A partial base sequence contained in the cas9 gene or a mutant gene thereof, the partial base sequence being sandwiched between a homologous sequence of an upstream base sequence and a homologous sequence of a downstream base sequence of a cleavage site targeted by the base sequence of (2) (i);
(3) A viral vector comprising the following nucleotide sequence:
(i) a base sequence complementary to the partial base sequence of (2)(ii) above; and
(ii) A partial base sequence contained in the cas9 gene or a mutant gene thereof other than the partial base sequence of (2)(ii) above, the partial base sequence being sandwiched between the upstream base sequence and the downstream base sequence of the cleavage site targeted by the base sequence of (3)(i).
In addition, the kit of the present invention may further include (4) a viral vector containing a base sequence complementary to the upstream base sequence and the downstream base sequence of the cas9 gene or a mutated gene thereof.
The viral vector and the base sequence contained in the viral vector contained in the kit of the present invention may be the same as the viral vector and the base sequence contained in the viral vector described in the method of the present invention.

The kit of the present invention may also contain, in addition to the viral vector, cells into which the cas9 gene or its mutant gene is introduced, a buffer solution, a medium, etc., as appropriate. Furthermore, the viral vectors contained in the kit of the present invention can be dissolved separately (or mixed together, if possible) in water or an appropriate buffer solution (e.g., TE buffer, etc.) to an appropriate concentration and stored at approximately -20°C.

The present invention will be explained in more detail below with reference to examples, but it is clear that the present invention is not limited to these.

Materials and Methods Cell Lines and Cell Culture Human Embryonic Kidney HEK293T and Lenti-X ^™ 293T cells (Takara Bio Inc. #632180) were cultured in Dulbecco's Modified Eagle Medium containing 10% fetal bovine serum. To generate stable cell lines, unless otherwise stated, we used 0.3 mg/ml hygromycin B (Wako #084-07681), 2.5 μg/ml puromycin (Nacalai #14861-71) or 32 μg/ml blasticidin S (Wako #026-18711) for at least 2 weeks.

Viral constructs
The SIN (self-inactivating) lentiviral vector pLVSIN-EF1a Hyg (#6185) and the AAV2 serotype vector pAAV-CMV (#6230) were purchased from Takara Bio Inc. pCas-Guide (#GE10002) was purchased from OriGene Tech. Inc. lentiCRISPR v2 was kindly provided by Feng Zhang (Addgene plasmid # 52961; http://n2t.net/addgene:52961; RRID:Addgene_52961). lentiSAMv2 was also kindly provided by Feng Zhang (Addgene plasmid # 75112; http://n2t.net/addgene:75112; RRID:Addgene_75112). The lentivirus pLVSIN was modified to express Cas9v1 (SpCas9 from pCas-Guide)-Myc and the hygromycin resistance gene (Hygro) linked to the self-cleaving peptide T2A under the control of the CMV promoter (Figure 6). Cas9(Nick)-Myc was constructed by replacing the N-terminal side of the only BspHI restriction enzyme site in this Cas9v1-Myc with Cas9v2 nickase (Figure 33B). After infection of 293T cells with serial dilutions of the lentivirus, the virus titer (introduced titer/ml) was determined by the number of colonies that survived for 10 days in the presence of hygromycin B.
Adeno-associated virus serotype 2 (Takara-Bio #6230) was purchased to construct the following vectors. AAV vector #1 was generated targeting the AAVS1 locus (T1) by flanking the AAVS1 homology arms and placing it between the viral ITRs (inverted repeats) so that the AAVS1-specific sgRNA1 (sg1) is transcribed by the U6 promoter and several elements are transcribed together with the AAVS1 gene (Figure 2). The elements are SA (splice acceptor)-T2A (self-cleaving peptide)-1.9 kb N-terminal Cas9v2 fragment-T2A (self-cleaving peptide)-puromycin resistance gene and polyadenylation sequence (pA). A target sequence (T1) was added adjacent to the homology arms to promote homology-directed repair (HDR) by forming a double-cut donor. See Table 1 for the sequence.

The AAV vector #2 sequence is also listed in Table 2. sgRNA2 (sg2) targets the junction (T2) between the 3' end of the 1.9 kb N-terminal Cas9v2 and the 5' end of P2A (self-cleaving peptide). To reconstruct the full-length Cas9v2, we overlapped a 0.3 kb region between the 1.9 kb N-terminal and 2.3 kb C-terminal parts of Cas9v2 (Figure 4). The 0.3 kb overlap serves as the left arm in HDR. As the right arm, the N-terminal 0.3 kb fragment of P2A and puromycin is flanked by the T2 target sequence and ITR to provide a double-cut donor.

As described in Table 3, AAV vector #3 targets the LTR of the lentiviral vector with a pair of sgRNAs (sg3-1 and sg3-2) with a 16 bp offset for DNA double-strand cleavage by wild-type Cas9 and nickase Cas9 (Figure 6).

AAV vector #4 (Table 4) was constructed targeting exon 1 of IFNAR1 (T4), and a pair of sgRNAs (sg4-1 and sg4-2) with a 7-bp offset and hygromycin were placed between the ITRs for DNA double-strand cleavage by wild-type Cas9 and nickase Cas9 (Figure 8).

AAV#5 (Table 5) is identical to AAV#1 except for the D10A (GAC→GCC) mutation in the 1.7 kb N-terminal Cas9v2.

AAV#6 (Table 6) is identical to AAV#2 except for the N863A (AAC→GCC) mutation in the 2.3 kb C-terminal Cas9v2 and the replacement of FLAG with VP641.

AAV vector #7 was generated by adding another sgRNA (sg7) to AAV vector #5 and replacing the target sequence T1 with T1/7 (Figure 35). The exact sequence of AAV vector #7 is shown in Table 7.

AAV vector #8 and AAV vector #9 were also constructed by adding another sgRNA (sg8) to AAV vector #2 and #6, respectively, and replacing the target sequence T2 with T8/2 (Figures 37, 39). The exact sequences of AAV vector #8 and #9 are listed in Tables 8 and 9.

After infecting 293T cells with serially diluted virus solutions, the AAV virus titers (induction titers/ml) of vectors #3 and #4 were directly determined by the number of colonies that survived for 10 days in the presence of 2.5 μg/ml puromycin or 0.3 mg/ml hygromycin B. For AAV vectors #1, #2, #5, #6, #7, #8, and #9, the inventors first measured the amount of puromycin gene using real-time PCR with specific primers P11-P12 (Table 10) after removing excess DNA fragments from the virus solution through incubation with anion exchange resin in 0.3 M NaCl, 20 mM Tris, pH 7.0 (Q-Fast Flow, Pharmacia). Next, the virus titers (induction titers/ml) of the above AAV vectors were estimated from the relationship between the biological virus titers and the amount of puromycin gene of AAV vector #3.

Intracellular delivery
The SIN lentiviral vector was co-transfected with ViraPower ^™ Lentiviral Packaging Mix (Invitrogen #K497500) into Lenti-X™ 293T cells (Takara #632180) using Lipofectamine ^™ 2000 Transfection Reagent (Thermo Fisher Scientific #11668027). After 3 days, the supernatant containing the recombinant viral particles was concentrated with Lenti-X™ Concentration Reagent (Takara Bio #631231) and used for cell infection (MOI=0.1-2) in the presence of 10μg/ml Polybrene (Nakalai #12996-81).
For sequential infections, 293T-Cas9(Nick)-Myc cells (500/well) seeded in 24-well plates were incubated for 36 h with a titer of 10,000 for AAV#7, delivering sgRNA targeting the AAVS1 site and the Cas9v2 nickase NT 1.9 kb fragment. After aspirating the medium, cells were sequentially incubated for 24 h with a titer of 10,000 for AAV#8 or AAV#9, reconstituting full-length Cas9(v2) nickase-FLAG or dCas9(v2)-VP64, respectively. Cells were transferred to 6-well plates and blasticidin was added 2 days later. Colonies were collected after 10 days in the presence of 32 μg/ml blasticidin S and analyzed by PCR and immunoblot.

Genomic DNA isolation and PCR
Genomic DNA was extracted from semi-confluent cells in 24-well plates. After 1% Triton-X extraction of cytoplasmic components, the cell residues were treated with 180 μg/ml Proteinase K (Nakalai #15679-06) in 0.5% SDS-containing buffer for 24 h. They were treated with phenol/chloroform/isoamyl alcohol (25:24:1), precipitated with ethanol, washed with 70% ethanol, and dissolved in TE. Genomic DNA (100 ng each) was subjected to PCR on a LightCycler 1.5 (ABI) using specific primers listed in Table 10 and KOD-FX-neo DNA Polymerase (TOYOBO #KFX-201). Unless otherwise stated, typical reaction conditions were 94°C for 2 min, followed by 35 cycles of denaturation at 98°C for 10 s, annealing at 62°C for 10 s, and extension at 68°C for 1 min. PCR products were analyzed on a 1% agarose gel. Several DNA fragments were purified and sequenced either directly or after subcloning into plasmids.

Immunoblot analysis
Cell lysates were extracted from semi-confluent cells in 24-well plates with Glo lysis buffer (Promega #E2661). Cell lysates (10 μg each) were separated by 5-12.5% SDS-PAGE and transferred to PVDF membranes (Millipore). To detect specific proteins, anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc tag (9B11) mouse monoclonal antibody (Cell Signaling Tech #2276S), anti-FLAG M2 mouse monoclonal antibody (Sigma-Aldrich #F1804) or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) were used at 1:500-3,000 dilutions. They were visualized with HRP-conjugated anti-rabbit or anti-mouse IgG antibodies and ECL reagents.

Luciferase assay
HEK293T cells ( ^2x104 /well) were seeded in 96-well plates and transfected with 10 ng of firefly luciferase reporter plasmid pISRE(Interferon-stimulated response element)-Luc 24 hours later. Cells were stimulated with 40 ng/ml IFNα 24 hours after transfection, and luciferase activity was measured by adding Steady-Glo® Luciferase Substrate (Promega #E2510) 12 hours after stimulation.

Cell transduction without drug selection. HEK293T cells (500/well) seeded in 24-well plates were treated with a titer of 10,000 AAV vectors (MOI=20) the day before infection. After 24 h of incubation, infected cells were transferred to 6-well plates at three different dilutions (1/30, 1/10, and 1/3). After approximately 10 days of incubation without antibiotics for selection, fully isolated colonies were collected, transferred to 24-well plates, and grown sufficiently for the preparation of genomic DNA and cell lysates.

Cell Lines To evaluate the removal of lentivirus integrated into the host genome, we generated a stable cell line expressing 5ng/ml TNFα-stimulated luciferase reporter via 3 x κB (NF-κB binding site) containing a minimal promoter and a blasticidin resistance gene constitutively by lentivirus infection. The cells resistant to 32μg/ml blasticidin were further transfected with a plasmid for constitutively expressing Cas9 and puromycin resistance gene under the CMV promoter. As shown in Figure 15, the resulting 2.5μg/ml puromycin-resistant cells were confirmed to express TNFα-responsive κB-Luc and CMV-driven Cas9 (HEK 293T-lentiviral-κB-Luc+CMV-Cas9). When the lentivirus integrated by infection with AAV vector #3 (sg-Lenti) targeting the lentivirus LTR is eliminated from the cells, the cells lose TNFα-responsive luciferase activity, allowing the elimination rate of the lentivirus to be quantified.

Luciferase assay (2)
To evaluate the removal of lentivirus from the genome, we used HEK 293T-lentiviral-κB-Luc+CMV-Cas9 cells in Figure 15. The cells (1,000/well) were seeded in 48-well plates and 24 hours later, infected with a titer of 500-5,000 AAV vector #3 (MOI = 0.5-5) targeting the lentiviral LTR or control sgRNA. 24 hours after transfection, cells were stimulated with 5 ng/ml TNFα, and luciferase activity was measured by adding Steady-Glo® Luciferase Substrate (Promega #E2510) 12 hours after stimulation.
In Figure 29, to examine dCas9v2-VP64 function in lentivirus-cleared sgLenti #1 and #2 cells, we examined endogenous IFNβ gene promoter-specific activation in the cells ( ^1x104 /well) on 48-well plates. Cells were transfected with 10ng 4xISRE (Interferon Response Element)-Luciferase plasmid DNA, 10ng MS2-P65-HSF1 plasmid DNA, and 10ng MS2-sgRNA expression plasmid targeting the IFNβ gene promoter site (sg-IFNβ) or control plasmid (sg-control) in 0.2μl Lipofectamine® 2000 transfection reagent (Thermo Fisher Scientific #11668027). Luciferase activity was measured by adding Steady-Glo® Luciferase Substrate 36 hours after transfection. IFNβ specifically activated by dCas9v2-VP64, MS2-P65-HSF1, and MS2-sgIFNβ significantly increased luciferase activity via ISRE. The IFNβ gene promoter target site and control sequence of the MS2-sgRNA expression plasmid are listed in Table 11.

DNA sequence analysis The precise genomic structures of several established cells were analyzed by direct sequencing of PCR fragments amplified from purified genomic DNA. For the IFNAR1 gene, the region surrounding exon 1 of IFNAR1 was amplified with P9-P10 primers using genomic DNA from each clone as a PCR template (Figure 8). The primer sequences are listed in Table 10. The amplified DNA fragments (which were mostly heterogeneous, approximately 0.4 kb) were excised from agarose gel, purified with GeneClean kit (Funakoshi #1102-200), and cloned into pcDNA vector plasmid. Plasmid DNA sequences (more than four clones from one cell line) were examined for structural analysis.

Statistical treatment Quantitative data were analyzed using Student's t test with two-tailed P values. A value of p<0.02 was considered statistically significant.

Example 1 Integration of wild-type Cas9 into the AAVS1 locus
To directly manipulate human somatic cells in vivo, we took advantage of the advantages of lentivirus and AAV viral vectors and developed a method to introduce Cas9 into the AASV1 locus using only viral vectors. Stable expression of Cas9 allows precise gene editing after infection with AAV vectors that contain specific sgRNAs and homology arms and can promote HDR. As shown in Figure 1, we used two kinds of Cas: Cas9v1 (original SpCas9: Streptococcus pyogenes) and Cas9v2 (optimized to human codons from SpCas9). They code for the same protein but differ in terms of DNA sequence, making sgRNA #2 capable of recognizing only Cas9v2 but not Cas9v1. To monitor the expression of both Cas9s at the protein level, we added Myc and Flag tags to Cas9v1 and Cas9v2, respectively (Figure 1). We also adopted a double-cut donor method with short homology arms of 0.3 kb to enable HDR and minimize the size of the inserted DNA into the AAV vector. This method included the following four steps.
1) We randomly introduced Cas9v1 (derived from Streptococcus pyogenes) into the genome of host cells using a lentiviral vector, which was able to accept the large size of Cas9v1 and ensure its long-term and stable expression even when the integration site could not be controlled.
2) Because the carrying capacity of an AAV vector for a foreign gene is 2.5 kb or less, the Cas9v1 nuclease activity was used to introduce a 1.9 kb Cas9v2 N-terminal fragment (optimized for human codons) into the AAVS1 locus via AAV vector #1.
3) Full-length Cas9v2 was reconstructed using HDR in AAV vector #2, providing a 0.3 kb region overlapping with a 2.3 kb Cas9v2 C-terminal fragment and a 1.9 kb N-terminal fragment.
4) The lentivirus containing Csa9v1 was removed from the genome using AAV vector #3, which contained an sgRNA targeting the long terminal repeat (LTR) of the lentiviral vector.

1) Random integration of Cas9v1-Myc into genome First, we introduced Cas9v1-Myc into the host genome of human embryonic kidney (HEK) 293T cells by lentiviral vector, although the integration was random. For long-term and stable expression in many cells, both Cas9v1-Myc transcripts and hygromycin resistance gene linked with self-cleaving peptide T2A were driven via cytomegalovirus (CMV) promoter. Infected HEK293T cells were selected with hygromycin B, and all five clones examined 2 weeks after selection (100%) expressed Cas9v1-Myc (Figure 11). Here, as shown in Cas9(Nick)-Myc infection, we used a relatively low concentration of lentivirus (multiplicity of infection (MOI) = 0.1), and the transduction efficiency was low, but the transduction efficiency could be improved by using a high concentration of lentivirus (Figure 34).

2) Integration of the 1.9 kb Cas9v2 N-terminal fragment into the AAVS1 locus
Using Cas9v1-Myc nuclease activity, the 1.9 kb Cas9v2 N-terminal fragment was introduced into the AAVS1 locus using an AAV vector (Figure 2). To enable HDR, AAV vector #1 was designed to express AAVS1-specific sgRNA1 (sg1) and provide a T1 target sequence containing a double-cut donor with a short homology arm of 0.3 kb and a protospacer adjacent motif (PAM). The sequence is listed in Table 1. When HDR occurs, both the 1.9 kb Cas9v2 N-terminus and the puromycin resistance gene are expressed from the stable PPP1R12C transcript via the splice acceptor (SA), T2A and P2A self-cleaving peptides. HEK293T cells expressing Cas9v1-Myc (#2 clone) were infected with AAV vector #1 and selected with puromycin for 2 weeks. Genomic DNA prepared from puromycin-resistant cells (clones #1-#15) was analyzed by PCR with primers P1-P2 set outside the homology arms (Figure 3). The primers are listed in Table 10. The 4.5 kb and 1.5 kb PCR products were derived from the allele with N-terminal Cas9v2 integrated and the original allele without integration, respectively. Eight of the 15 clones examined (53%) had two bands, indicating that only one allele had N-terminal Cas9v2, and only clone #8 carried inserts (4.5 kb and 4.2 kb) in both alleles. Clones with possible Cas9v2 integration (PCR 4.5 kb product) were further analyzed with primers P1-P3 and P4-P2. All eight candidates carried the Cas9v2 gene (Fig. 3B: 1.2 kb using P1-P3 primers) and the puromycin resistance gene (Fig. 3C: 1.1 kb using P4-P2 primers). Cell lysates from the same cells were also analyzed by 6% SDS-PAGE followed by immunoblotting using an antibody that recognizes both Cas9s. All eight cells expressed Cas9v1-Myc (160 kDa) and the 1.9 kb N-terminus of Cas9v2 (70 kDa) (Fig. 3D). The 4.5 kb sequence of the N-terminal (NT) clone #4 fragment was as designed (Fig. 2B). The 4.5 kb fragment from clone #8 was identical to that from clone #4, but the 4.2 kb fragment from clone #8 contained both an insertion and a deletion around the right arm (Fig. 12). The sequences are listed in Table 12.

3) Reconstruction of the full-length Cas9v2-FLAG gene at the AAVS1 locus
To reconstruct full-length Cas9v2-FLAG by HDR, we introduced a 0.3 kb region overlapping the C-terminal 2.3 kb fragment and the N-terminal 1.9 kb fragment of Cas9v2-FLAG via AAV vector #2 (Figure 4). sgRNA2 (sg2) targets the junction sequence (T2) between the 3' end of the 1.9 kb N-terminal Cas9v2 and the 5' end of the P2A self-cleaving peptide. A 0.3 kb N-terminal fragment of P2A and puromycin was used as the right arm to provide a double-cut donor flanked by the T2 target sequence and ITRs (Table 2). P2A expresses the reconstructed full-length Cas9v2-FLAG and blasticidin resistance gene from a transcript driven by the PPP1R12C promoter present in the AAVS1 site. The puromycin resistance gene was also reconstructed using HDR, but its transcript was not translated due to the stop codon of the blasticidin resistance gene inserted before it. HEK293T cells expressing Cas9v1-Myc and the 1.9 kb N-terminus of Cas9v2 (NT clone #4) were infected with AAV vector #2 and selected with blasticidin for 2 weeks. The blasticidin-resistant cells (clones #1-#9) became sensitive to puromycin, and their genomic DNA was prepared and analyzed by PCR with P5-P6 primers (Figure 5). Integration of the donor element was indicated by a 1.2 kb PCR product detected in all 9 clones (100%). Cell lysates from the same cells were analyzed by 6% SDS-PAGE followed by immunoblotting with antibodies that recognize both. In all cells infected with AAV vector #2, the 70-kDa band representing the 1.9 kb N-terminus of Cas9v2 disappeared, and the intensity of the 160-kDa band increased. Anti-FLAG and anti-Myc antibodies confirmed that these bands contained the reconstructed full-length Cas9v2-FLAG and Cas9v1-Myc, respectively. The sequence of Cas9v2 full-length clone #7 containing the AAVS1 integration site was confirmed (Table 13).

4) Removal of lentivirus containing randomly integrated Cas9v1-Myc from the genome To remove lentivirus containing randomly integrated Cas9v1-Myc from the genome, we constructed AAV vector #3 that targets the LTR (T3) of the lentivirus vector using non-homologous end joining (NHEJ) (Figure 6). Because cells expressing Cas9v1-Myc and full-length Cas9v2-FLAG lose functional puromycin expression (Figures 4 and 5), we used a puromycin resistance gene to select cells expressing sgRNA. There are reports that sgRNAs targeting HIV LTRs have successfully removed genes between LTRs in many cases. We set a pair of sgRNAs (sg3-1 and sg3-2) with a 16bp offset for DNA double-strand cleavage by wild-type Cas9 and Cas9 nickase (Figure 6 and Table 3). Nickase requires a pair of sgRNAs complementary to opposite strands with an offset (usually 4 to 20 bp) as the target site. HEK293T cells expressing Cas9v1-Myc and full-length Cas9v2-FLAG (Cas9v2 full-length clone #7) were infected with AAV vector #3 and selected with puromycin. Puromycin-resistant cells (clones #1-#10) were analyzed by PCR using specific primers (P7-P8) for Cas9v1-Myc and immunoblotting for Cas9v1-Myc and Cas9v2-FLAG (Figure 7). In 5 of 10 clones (50%), the lentivirus containing Cas9v1-Myc was successfully removed from the genome using NHEJ, leaving a small portion of the LTR behind. The overall transduction efficiency was not very high when a low concentration of AAV (MOI=0.1) was used. However, by increasing the MOI to 20, the removal rate without drug selection increased to approximately 50% (3/6, 5/9, and 4/10 in three independent experiments) (Figures 13 and 14). The specific removal of Cas9v1 compared to Cas9v2 was confirmed by PCR and immunoblot. Achieving a high removal rate of lentiviral vectors without drug selection is important for in vivo use.
The sgRNA-mediated removal rate of lentivirus from the integrated genome was quantitatively evaluated by another method (Figure 15). Luciferase activity in the presence of TNFα depends on the amount of lentiviral κB-luciferase gene in cells that also stably express Cas9. The removal rate of integrated lentiviral κB-luciferase gene was measured via relative luciferase activity 36 hours after infection with AAV vectors expressing sgRNA (sg-Lenti (sg3-1 and sg3-2) to sg-Control). At a relatively low MOI of 5, lentivirus was removed in more than half of the cells (59%). This suggests that the removal rate of lentivirus was more than 50%.

5) Verification of reconstituted Cas9v2-FLAG function
To verify that the reconstructed Cas9v2-FLAG in the AAVS1 locus is functional in terms of nuclease activity, we investigated whether Cas9v2-FLAG, together with sgRNA targeting a specific site, generates a DNA double-strand break (DSB) at the site. We selected the interferon alpha receptor 1 (IFNAR1) gene as a target because it is easy to examine whether the gene is knocked out using a transient luciferase assay. Cells transfected with the interferon-stimulated response element-luciferase plasmid showed good response to IFNα stimulation, while cells lacking IFNAR1 did not respond to stimulation. AAV vector #4 was generated targeting exon 1 (T4) of IFNAR1 by placing a pair of sgRNAs (sg4-1 and sg4-2) with a 7 bp offset for DNA double-strand cleavage by Cas9 nickase and wild-type Cas9 (Table 4) (Figure 8). In cells expressing sgRNA3 targeting the lentiviral LTR (sgLenti clone #10), the hygromycin resistance gene was deleted along with Cas9v1-Myc, so a new hygromycin resistance gene was placed between the ITRs to select cells expressing this sgIFNAR1 (sgRNA4).
sgLenti clone #10 cells were infected with AAV vector #4 and the ISRE response was measured after transfection with ISRE-Luciferase plasmid. Hygromycin-resistant clones (#1-#6) expressing Cas9v2-FLAG and sgIFNAR1 were examined by luciferase assay to see whether IFNAR1 was actually knocked out. Five of the six clones (83%) lost IFNα response, indicating that NHEJ occurred in Cas9v2-FLAG and sgIFNAR1, specifically knocking out IFNAR1. We analyzed the genomic structure of IFNAR1 clones from #1 and #3 clones (Figure 16). The cells had heterozygous insertions and deletions in the IFNAR1 allele, resulting in loss of response to IFNα stimulation. Thus, we confirmed that the reconstructed Cas9v2-FLAG was functional and could be used to edit other genes.

Example 2 Introduction of Cas9 nickase into AAVS1 locus As in Example 1, the reconstructed Cas9v2-FLAG in the AAVS1 locus was functional, but Cas9 often causes genomic cleavage at non-target sites due to off-target effects. To reduce possible off-target effects in gene editing, we also introduced Cas9v2 nickase with D10A mutation into the AAVS1 locus. We used the 1.7 kb N-terminus of Cas9v2 with D10A mutation (AAV#5) (Table 5) instead of AAV#1 to infect HEK293T cells expressing Cas9v1-Myc (clone#2) (Figure 17). Three of the 10 puromycin-resistant clones (#1-#10) (30%) had a candidate 4.5 kb band in one allele (Figure 18). All three clones were confirmed to have the mutated 1.9 kb N-terminal Cas9v2 gene and puromycin gene by PCR, and the 70 kDa N-terminal Cas9v2 nickase and 160 kDa Cas9v1-Myc proteins by immunoblotting with an antibody against Cas9. The transduction efficiency of the N-terminal Cas9v2 nickase fragment was low, estimated to be about 3-6% based on data using a high concentration of vector (MOI=20) in sequential infections without drug selection (Figures 9, 10, 41 and 42).
To reconstruct the full-length Cas9v2 nickase, we used the same AAV vector #2 as in Figure 4 to infect cells expressing the mutated 1.9 kb N-terminus of Cas9v1 and Cas9v2 (nickase-NT clone #9) (Figure 19). PCR and immunoblot showed that all 10 cell lines (#1-#10) expressed Cas9v1-Myc and full-length Cas9v2-FLAG nickase (Figure 20). The sequence of the full-length Cas9v2-FLAG nickase clone #3, including the AAVS1 integration site, was confirmed (Table 14).

Because drug selection cannot be applied in vivo, we measured the reconstitution rate of full-length Cas9v2-FLAG nickase from cells containing the nickase N-terminal 1.9 kb fragment (clone #9) without drug selection. Three of six clones (50%) were successfully reconstituted without drug selection using high-amount AAV vector #2 (MOI=20) (Figure 21).
Finally, the inventors removed the lentivirus vector containing Cas9v1 from the genome using the same AAV vector #3 as in Figure 6 (Cas9v2nickase full-length #3 clone) (Figure 22). Three of the 10 clones (30%) successfully removed the lentivirus containing Cas9v1-Myc from the genome by NHEJ. By increasing the MOI to 20 (Figure 23), the removal rate without drug selection increased to 56% (5/9). Specific removal compared to Cas9v2 was confirmed by PCR.
To confirm the nuclease activity of the reconstructed full-length Cas9v2-FLAG nickase in cells expressing sgLenti#4, cells were infected with AAV vector#4 to express a pair of sgRNAs (sg4-1 and sg4-2) targeting the IFNAR1 gene (Figure 8). Five of the six resulting hygromycin-resistant clones (83%) completely lost IFNAR1-mediated IFNα response (Figure 24). We analyzed the genomic structure of IFNAR1 in clones#3 and #5 (Figure 25). The cells had heterologous insertions and deletions in the IFNAR1 allele, resulting in loss of response to IFN stimulation. Although one allele in clone#3 had an in-frame deletion, the loss of the signal sequence affected the proper expression of IFNAR1 on the cell surface. Thus, the inventors confirmed that the reconstituted Cas9v2-FLAG nickase is functional and can be used to edit other genes more precisely.

Example 3 Introduction of inactive Cas9 (dCas9) into the AAVS1 locus
The CRISPR/Cas9 system is used not only to knock out or knock in specific genes, but also to activate transcription of specific genes. A nuclease-free inactive Cas9 (dCas9-VP64) with D10A and N876A mutations and conjugated to the transcription activator VP64 was used together with a specifically modified sgRNA (MS2-sgRNA) that targets a specific promoter. MS2-sgRNA recruits transcription activators (P65 and HSF1) to the promoter via the MS2 protein together with dCas9-VP64. As a result, transcription from the promoter was activated. We reconstructed full-length dCas9v2-VP64 at the AAVS1 locus by infecting HEK293T cells (nickase-NT#9) (Figure 18) expressing Cas9v1 and D10A mutant Cas9v2 with AAV vector #6 (Figure 26). PCR and immunoblotting showed that all (100%) of the 10 blasticidin-resistant cell lines expressed the reconstituted full-length dCas9v2-VP64 and Cas9v1-Myc (Figure 27). The reconstitution rate of full-length Cas9v2-VP64 from cells (clone#9) containing the 1.9 kb N-terminal nickase fragment without drug selection was 3 out of 6 (50%) without drug selection with a high amount of AAV vector#26 (MOI=20) (Figure 28). The sequence of full-length dCas9v2-VP64 clone#1 containing the AAVS1 integration site was confirmed (Table 15).

Finally, we used the same AAV vector #3 as in Figure 6 to remove the lentivirus vector containing Cas9v1-Myc from cells expressing full-length dCas9v2-VP64#1 (Figure 29). Four of the 10 clones (40%) successfully removed the lentivirus containing Cas9v1-Myc. Given that the reconstructed full-length dCas9v2-VP64 does not have nuclease activity, it is likely that the residual Cas9v1-Myc protein transcribed and translated from the Cas9v1-Myc gene before the gene was removed acted to remove the lentivirus containing the Cas9v1-Myc gene itself. To confirm the function of dCas9v2-VP64 to activate a specific promoter, we tested whether modified sgRNA targeting the IFN β gene promoter site and MS2-P65-HSF1 could activate IFN β transcription (Figure 30). After using ISRE (interferon response element)-Luc plasmid to transfect the indicated plasmids for expressing MS2-sgRNA and MS2-P65-HSF1, the expression level of IFNβ transcription was monitored using ISRE-luciferase activity via IFNAR. ISRE-luciferase activity was significantly activated by IFNβ target (12.8 and 19.0-fold compared to the control in #1 and #2 clones, respectively). Therefore, it is reasonable that dCas9v2-VP64 activates any other genes later. Immunoblot analysis to confirm the protein structure of all constructs is shown in Figures 31 and 32.

Example 4 Use of lentiviral Cas9 nickase in the first step to reduce off-target effects The use of lentiviral Cas9 in the first step of the method of the present invention has potential off-target effects as well as genetic risks due to its genomic integration. To reduce the off-target effects caused by lentiviral Cas9v1 during the reconstruction of Csa9v2-nickase or dCas9v2-VP64 in the AAVS1 locus, the inventors used lentiviral Cas9 nickase (Cas9(Nick)-Myc) instead of wild-type Cas9v1 in the first step of the method of the present invention. As shown in Figure 33, in order to distinguish between lentiviral Cas9(Nick)-Myc randomly integrated into the host genome and Csa9v2-FLAG nickase or dCas9v2-VP64 integrated into the AAVS1 locus, Cas9(Nick)-Myc was constructed by replacing N-terminal Cas9v1 with Cas9v2 nickase at the only BspHI restriction enzyme site. After hygromycin selection, immunoblot confirmed Cas9(Nick)-Myc expression (Fig. 33C). Also, about 10% of infected cells (1/10 and 1/10 in two independent experiments) expressed Cas9(Nick)-Myc without drug selection with a high amount of lentivirus (MOI=2) (Fig. 34). Fortunately, there were possible cleavage nodes by Cas9 nickase at both the AAVS1 integration site and the junction between N-terminal Cas9v2 and T2A (Figs. 35 and 37). For the introduction of N-terminal Cas9v2 nickase with D10A mutation into the AAVS1 locus, we generated AAV vector#7 by adding another sgRNA (sg7) to AAV vector#5 for DNA double-strand break (DSB) by Cas9(Nick) (Figs. 35 and 36). To provide a double-cut donor, the target sequence (T1) was also replaced with T7/1. The exact sequence of AAV vector #7 is shown in Table 7. AAV vector #8 (Figures 37 and 38) and AAV vector #9 (Figures 39 and 40) were constructed to reconstitute full-length Cas9v2-FLAG nickase and dCas9v2-VP64, respectively, and their sequences are also listed in Tables 7-9.

Example 5 Sequential infection of AAV vectors without drug selection The data in Figure 15 showed that about 24 hours was sufficient to complete genome reconstitution. We considered that reconstitution of Cas9 from two vectors at the AAVS1 locus could be achieved by sequential infection of two AAV vectors at a short interval (24h _to 36h) without drug selection during infection. As shown in Figure 10, sequential infection of AAV vectors #7 and #8 or #9 at a short interval of 36h efficiently and successfully reconstituted Cas9v2 (Figures 41-44). For the reconstitution of full-length Cas9v2-FLAG nickase at the AAVS1 locus, immunological and PCR analysis revealed that 83% (5/6) of the blasticidin-resistant cells had full-length Cas9v2-FLAG nickase at the AAVS1 locus (Figure 41). All clones expressed full-length Cas9v2-FLAG nickase protein, but clone #6 may have a deletion in the 3' region of the AAVS1 integration site. Meanwhile, clones #1, #2 and #3 all had Cas9(Nick)-Myc genes in their genomes, but lentiviral Cas9(Nick)-Myc proteins were not detected in the clones (Figure 44). We have not yet reached a conclusion as to the reason for this discrepancy. Full-length dCas9v2-VP64 was also successfully reconstructed in all six clones (100%), but clone #4 had a deletion around the right arm (Figures 42 and 43). In addition, all clones had Cas9(Nick)-Myc genes in their genomes, but lentiviral Cas9(Nick)-Myc proteins were not detected in clones #1, #3 and #6 (Figure 44). The reason is unclear. An average of 15 colonies emerged from the first 500 HEK 293T-Cas9(Nick)-Myc cells in both experiments, so the overall transduction efficiency of these successive infections was approximately 3% (Figure 10).

According to the present invention, stable expression of the cas9 gene is possible in cells not only ex vivo but also in vivo, and by simply supplying cells with the appropriate sgRNA and donor DNA, it is possible to efficiently repair not only single gene mutations but also multiple gene mutations seen in cancer and other diseases. By stably expressing the Cas9 protein in the genome, and combining sgRNA targeting mutated genes or virus-derived genes with donor DNA as necessary, it is expected that effective treatments can be achieved for cancer caused by multiple gene mutations and viral diseases caused by the incorporation of causative viral genes into the genome.

Claims (29)

A method for introducing a cas9 gene or a mutated gene thereof into a desired genomic region of a cell ex vivo , comprising the steps of:
(1) expressing the cas9 gene or a mutated version thereof in a cell;
(2) cleaving a desired genomic region of the cell with the Cas9 protein or its mutant protein expressed in step (1) and introducing a partial base sequence contained in the cas9 gene or its mutant gene into the cleavage site; and (3) cleaving the partial base sequence introduced in step (2) with the Cas9 protein or its mutant protein expressed in step (1) and introducing a partial base sequence contained in the cas9 gene or its mutant gene other than the partial base sequence introduced in step (2) into the cleavage site, thereby constructing the cas9 gene or its mutant gene in the desired genomic region of the cell. The method according to claim 1, wherein step (1) is carried out by infecting a cell with a viral vector containing a base sequence of the cas9 gene or a mutant gene thereof. The method according to claim 2, wherein step (1) is a step of expressing the cas9 gene or a mutant gene thereof from the genome of the cell. The method according to claim 3, wherein the viral vector in step (1) is a lentiviral vector. The method according to any one of claims 1 to 4, wherein the step (2) is carried out by infecting a cell with a viral vector comprising the following base sequence:
(i) a nucleotide sequence complementary to a nucleotide sequence of a desired genomic region of the cell; and
(ii) A partial base sequence contained in the cas9 gene or a mutant gene thereof, the partial base sequence being sandwiched between a homologous sequence of the upstream base sequence and a homologous sequence of the downstream base sequence of the cleavage site targeted by the base sequence of (i). The method according to claim 5, wherein the viral vector in step (2) is an adeno-associated viral vector. The method according to any one of claims 1 to 6, wherein the step (3) is carried out by infecting a cell with a viral vector comprising the following base sequence:
(iii) a base sequence complementary to the partial base sequence introduced in step (2), and
(iv) A partial base sequence contained in the cas9 gene or a mutant gene thereof other than the partial base sequence introduced in step (2), the partial base sequence being sandwiched between a homologous sequence of the upstream base sequence and a homologous sequence of the downstream base sequence of the cleavage site targeted by the base sequence of (iii). The method according to claim 7, wherein the viral vector in step (3) is an adeno-associated viral vector. (4) The method according to any one of claims 2 to 8, further comprising a step of removing the cas9 gene or a mutant gene thereof from the genome of the cell. The method according to claim 9, wherein step (4) is carried out by infecting a cell with a viral vector comprising a base sequence complementary to the upstream base sequence and a base sequence complementary to the downstream base sequence of the cas9 gene or a mutant gene thereof in step (1). The method according to claim 10, wherein the viral vector in step (4) is an adeno-associated viral vector. The method according to claim 2, wherein step (1) is a step of expressing the cas9 gene or a mutant gene thereof from the viral genome. The method according to claim 12, wherein the viral vector in step (1) is an adenoviral vector. The method according to any one of claims 1 to 13, wherein the mutated gene in step (1) is a gene encoding a Cas9 protein having a mutation selected from the group consisting of a D10A mutation, an H840A mutation, and an N863A mutation. The method according to any one of claims 1 to 14, wherein the mutant gene constructed in step (3) is a gene encoding a Cas9 protein having at least one mutation selected from the group consisting of a D10A mutation, an H840A mutation, and an N863A mutation. The method according to any one of claims 1 to 15, wherein the desired genomic region is the aavs1 locus. The method according to any one of claims 1 to 16, wherein the partial base sequence introduced in step (2) includes the base sequence represented by SEQ ID NO: 7 or 31. The method according to any one of claims 1 to 17, wherein the partial base sequence introduced in step (3) includes the base sequence represented by SEQ ID NO: 17 or 32. A kit for introducing a Cas9 gene or a mutant gene thereof into a desired genomic region of a cell, comprising the following viral vector:
(1) a viral vector containing the base sequence of the cas9 gene or a mutant gene thereof;
(2) a viral vector comprising the following nucleotide sequence:
(i) a nucleotide sequence complementary to a nucleotide sequence of a desired genomic region of the cell; and
(ii) a partial base sequence contained in the cas9 gene or a mutant gene thereof, the partial base sequence being sandwiched between a homologous sequence of the upstream base sequence and a homologous sequence of the downstream base sequence of the cleavage site targeted by the base sequence of (2) (i); and
(3) A viral vector comprising the following nucleotide sequence :
( i) a nucleotide sequence complementary to the partial nucleotide sequence of (2)(ii) above; and
(ii) A partial base sequence contained in the cas9 gene or a mutant gene thereof other than the partial base sequence of (2)(ii) above, the partial base sequence being sandwiched between the upstream base sequence and the downstream base sequence of the cleavage site targeted by the base sequence of (3)(i). The kit according to claim 19, wherein the viral vector (1) is a lentiviral vector or an adenoviral vector. The kit according to claim 19 or 20, wherein the viral vector (2) is an adeno-associated viral vector. The kit according to any one of claims 19 to 21, wherein the viral vector (3) is an adeno-associated viral vector. (4) The kit according to any one of claims 19 to 22, further comprising a viral vector comprising a base sequence complementary to the upstream base sequence and the downstream base sequence of the cas9 gene or a mutant gene thereof in (1). The kit according to claim 23, wherein the viral vector of (4) is an adeno-associated viral vector. The kit according to any one of claims 19 to 24, wherein the mutant gene contained in the viral vector of (1) is a gene encoding a Cas9 protein having a mutation selected from the group consisting of a D10A mutation, an H840A mutation, and an N863A mutation. The kit according to any one of claims 19 to 25, wherein the mutant gene contained in the viral vector of (2) or (3) is a gene encoding a Cas9 protein having at least one mutation selected from the group consisting of a D10A mutation, an H840A mutation, and an N863A mutation. The kit according to any one of claims 19 to 26, wherein the desired genomic region is the aavs1 locus. The kit according to any one of claims 19 to 27, wherein the partial base sequence contained in the viral vector (2) includes the base sequence represented by SEQ ID NO: 7 or 31. The kit according to any one of claims 19 to 28, wherein the partial base sequence contained in the viral vector of (3) includes the base sequence represented by SEQ ID NO: 17 or 32.