JP6867689B2 - Method of introducing Cas9-gRNA complex into cell nucleus and method of modifying target gene in cell - Google Patents

Description

The present invention relates to a method for producing a Cas9-gRNA complex, a method for introducing a Cas9-gRNA complex into a cell nucleus, and a method for modifying a target gene in a cell.
The present application claims priority based on US Pat. No. 62 / 288,449, which was provisionally filed in the United States on January 29, 2016, the contents of which are incorporated herein by reference.

The CRISPR-Cas9 system is attracting attention as a next-generation genome editing technology, and the competition for new genome editing technology is intensifying. The features of the CRISPR-Cas9 system are that the guide RNA determines the target site, so that genome editing can be performed quickly, easily, and inexpensively only in the time required for designing the guide RNA, and that multiple guide RNAs are allowed to act. Therefore, it is possible to disrupt a plurality of genes at the same time, and it can be used for transcriptional regulation and induction of epigenetic dynamic changes by adding various protein engineering modifications to Cas9. Recently, as a method for visualizing a specific locus, it has been attracting attention as a novel visualization method instead of the conventional FISH method (see, for example, Non-Patent Document 1).

Chen, Baohui, et al., “19. Dynamic Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR / Cas System”, Cell, vol.155, p1479-1491, 2013.

However, applying this CRISPR-Cas9 system as a genome editing technology for medical purposes has the following problems.
(1) Off-target problem: It has been found that CRISPR has non-specific DNA cleavage (off-target cleavage) in addition to specific DNA cleavage (on-target cleavage), which is a problem. Basically, which site on the DNA Cas9 cleaves depends on the protospacer sequence of the guide RNA (gRNA), but even if this sequence and the target DNA strand do not completely match, Cas9 cleaves. Has been reported. Since this is due to an essential process in which Cas9 cleaves DNA, for example, a method using double nickase, a method using Fok1-dCas9 (fCas9), and the like are used as a method for eliminating it. However, there is a need for a more effective solution that not only eliminates cleavage of DNA that does not exactly match the gRNA, but also allows temporal and spatial functional control of the CRISPR-Cas9 system.

(2) Problems of genomic damage: The current CRISPR-Cas9 system is mainly carried out using an expression system using a viral vector such as a retrovirus or a lentivirus. By using an expression system using these viral vectors, damage to the target cell genome due to constitutive expression of Cas9 becomes a major problem in genome editing for the purpose of human gene therapy.
(3) Problem of intranuclear (intracellular) introduction of Cas9: In the current CRISPR-Cas9 system, if the above-mentioned viral vector expression system is not used to introduce Cas9 into cells, pre-expressed Cas9 and gRNA are introduced. There is a need. At this time, Cas9 is highly hydrophobic, and Cas9 expressed in vitro tends to form aggregates. Further, since Cas9 has a large molecular weight, it is difficult to permeate the cell membrane by an electroporation method or the like. Further, in particular, in the introduction using the electroporation method, damage to cells due to puncturing the cell membrane by electric shock becomes a problem. In addition, the microinjection method can be applied only to cells having a certain size, and it is necessary to introduce it into each cell, resulting in poor work efficiency.
(4) Difficulty in genome editing of multiple genes: The CRISPR-Cas9 system has a great advantage that it can function with a simple system set consisting only of gRNA targeting Cas9 and a specific gene. .. However, in the CRISPR-Cas9 system based on the current expression system, both Cas9 and gRNA depend on the expression system. Therefore, by Co-transfection, a combination of Cas9 and gRNA having different functions is created in the cell. Difficult to make it work. Specifically, for example, when it is desired to visualize the genome sequence of various parts of the genome with a fusion protein such as GFP, CFP, or YFP having a different color, in Co-transfection, a specific gRNA and a fluorescent label are used in the cell. Since the formation of a complex with Cas9 occurs randomly, it is difficult to visualize a specific genomic site with a specific fluorescence.

(5) Problem of optimal niche of genome editing: When considering the medical application of genome editing, the purpose is to require efficient homologous recombination and epigenetic modification targeting a specific gene. In order to improve the efficiency of homologous recombination and epigenetic modification, activation of various inducible proteins in the nucleus (in the cytoplasm) and inactivation or removal of inhibitory factors are indispensable. That is, it is necessary to optimize the environment (niche) for homologous recombination and epigenetic modification.
(6) Problems when handling a large number of cells: When producing gene-corrected cells using genome editing and performing gene therapy, it is necessary to simultaneously use a large number of cells for genome editing. In an expression system using a viral vector, it is difficult to control the expression level, so that it is difficult to uniformly process the genome of a large number of cells at one time.

The present invention has been made in view of the above circumstances, and provides a method for producing a Cas9-gRNA complex that is stably solubilized in vitro. It also provides a method for introducing a Cas9-gRNA complex into the cell nucleus with an optimized nuclear niche for genome editing without introducing the viral vector into the cell. Intracellular modification that also reduces off-targeting, does not introduce viral vectors into cells, optimizes the nuclear niche for genome editing, and allows simultaneous editing of multiple loci in large numbers of cells. Provide a method.

As a result of intensive studies to achieve the above object, the present inventors stably form and solubilize the Cas9-gRNA complex at a specific temperature, and further, the present invention uses the semi-intact cell reseal method. We have found that the Cas9-gRNA complex can be introduced into the nucleus with high efficiency, and have completed the present invention.

That is, the present invention includes the following aspects.
[1] Cas9 and (Clustered Regularly Interspaced Short Palindromic Repeats ( CRISPR) -associated 9) guides the RNA (gRNA) were mixed at 20 ° C. or higher, and Cas9-gRNA complex formation step of forming a Cas9-gRNA complex , A perforation step of allowing a perforating substance to act on the plasma membrane of a cell to form pores in at least a part of the plasma membrane, and a pore is formed in at least a part of the Cas9-gRNA complex and the plasma membrane. The Cas9-gRNA complex and the Cas9-gRNA complex were added to the first mixing step of mixing the cells and the cytoplasm collected from cells derived from the same or different species, and the cells having pores formed at least partially on the plasma membrane. In the introduction step of introducing the cytoplasm, a solution containing calcium ions is added to cells having pores formed in at least a part of the plasma membrane into which the Cas9-gRNA complex has been introduced, and the solution containing calcium ions is added to at least a part of the plasma membrane. A re-encapsulation step of re-encapsulating the formed pores is provided, and the gRNA complements the base sequence from 1 base upstream to 20 bases or more and 24 bases or less upstream of the PAM (Proto-spacer Adjacent Motif) sequence in the target gene. specific polynucleotide seen including consisting of the nucleotide sequence, wherein Cas9 is, N-terminal or C-terminal nuclear localization signal (nuclear localization signal: NLS) peptide is bound, to Cas9-gRNA complexes in the cell nucleus Introduction method .
[2] The method for introducing a Cas9-gRNA complex into a cell nucleus according to [1], wherein in the Cas9-gRNA complex forming step, the Cas9 and the gRNA are mixed at 30 ° C. or higher and 65 ° C. or lower.
[ 3 ] The Cas9 is divided into an N-terminal Cas9 (Cas9-N) and a C-terminal Cas9 (Cas9-C), and the Cas9-N and Cas9-C are N-terminal or C, respectively. A protein that forms a dimer in a photo-dependent or chemical-dependent manner is bound to the end, and Cas9 recovers RNA-induced DNA endonuclease activity in the presence of light or chemical [1] or [ 2] The method for introducing the Cas9-gRNA complex into the cell nucleus .
[ 4 ] The method for introducing a Cas9-gRNA complex into a cell nucleus according to any one of [1] to [3 ], wherein two or more types of the gRNA are mixed in the Cas9-gRNA complex forming step.
[ 5 ] Further, before the introduction step and after the first mixing step , the Cas9-gRNA complex and the cytoplasm and the inhibitor of the homologous recombination inhibitor or the activity of the homologous recombination The method for introducing a Cas9-gRNA complex into a cell nucleus according to any one of [1 ] to [4], which comprises a second mixing step of mixing with an agent.
[ 6 ] The Cas9-gRNA complex was introduced into the cell nucleus and reencapsulated using the method for introducing the Cas9-gRNA complex into the cell nucleus according to any one of [1 ] to [ 5]. In a cell having a target gene, a region determined by a cleavage step in which Cas9 cleaves the target gene at a cleavage site located upstream or downstream of a predetermined base of the PAM sequence and complementary binding between the gRNA and the target gene. in comprises a modification step of obtaining a cell having the target gene is modified, the modified method of a target gene in a cell.

According to the present invention, it is possible to provide a method for producing a Cas9-gRNA complex that is stably solubilized in vitro. Further, according to the present invention, it is possible to provide a method for introducing a Cas9-gRNA complex into the cell nucleus in which the nuclear niche of genome editing is optimized without introducing the viral vector into the cell. Further, according to the present invention, off-targeting is reduced, the nuclear niche of genome editing is optimized without the need to introduce a viral vector into cells, and a plurality of loci in a large number of cells can be edited simultaneously. It is possible to provide a method for modifying a target gene in a cell.

It is a schematic process diagram which shows one Embodiment of the manufacturing method of Cas9-gRNA complex of this invention. It is a schematic process diagram which shows one Embodiment of the method of introducing the Cas9-gRNA complex of this invention into a cell nucleus. It is an image which observed the HeLa cell which introduced the cytoplasm of dCas9 only, dCas9-gRNA complex only, or dCas9-gRNA complex and L5178Y cell in Test Example 1 using a fluorescence microscope. It is an image which shows the result of the T7E1 assay using the genomic DNA of the HeLa cell which introduced the complex of NLS-SpCas9-NLS and sgAAVS1 and the cytoplasm of L5178Y cell in Test Example 2. Image showing the result of T7E1 assay using the genomic DNA of HeLa cells into which the complex of HA-NLS-SaCas9 and sgTET1 or the complex of HA-NLS-SaCas9 and sgTET2 and the cytoplasm of L5178Y cells in Test Example 3 was introduced. Is. It is a schematic diagram which shows the structure of the N-terminal side and the C-terminal side of the split type SpCas9 (paCas9) which is a light induction type in Test Example 4. In Test Example 4, the photo-induced split-type SpCas9 (paCas9) N-terminal side and sgAAVS1 complex, or the photo-induced split-type SpCas9 (paCas9) C-terminal side and sgAAVS1 complex and L5178Y cells It is an image which observed the HeLa cell which introduced the cytoplasm with a fluorescence microscope. It is a schematic diagram which shows the structure of the N-terminal side and the C-terminal side of the split type SpCas9 (rpCas9) which is a rapamycin-induced type in Test Example 5. Results of T7E1 assay using the N-terminal side and C-terminal side of the rapamycin-induced split-type SpCas9 (rpCas9) in Test Example 5 and the genomic DNA of HeLa cells into which the sgAAVS1 complex and the cytoplasm of L5178Y cells were introduced. It is an image showing.

<Method for producing Cas9-gRNA complex>
In one embodiment, the present invention comprises a Cas9-gRNA complex forming step of mixing Cas9 and gRNA at 20 ° C. or higher to form a Cas9-gRNA complex, wherein the gRNA is a PAM (Proto) in a target gene. Provided is a method for producing a Cas9-gRNA complex containing a polynucleotide having a base sequence complementary to a base sequence from 1 base upstream to 20 bases or more and 24 bases or less upstream of the −sperer Adjacent Motif) sequence.

According to the method for producing a Cas9-gRNA complex of the present embodiment, a Cas9-gRNA complex that is stably solubilized in vitro can be obtained.
The present inventors form aggregates at a low temperature of about 4 ° C. with Cas9 alone, but by mixing Cas9 and gRNA expressed in advance under a temperature condition of 20 ° C. or higher, a stable Cas9-gRNA complex is formed. They have found that they form a body and solubilize in solution, completing the present invention.
The method for producing the Cas9-gRNA complex of the present embodiment will be described in detail below with reference to the drawings.

[Cas9-gRNA complex formation step]
FIG. 1 is a schematic process diagram showing an embodiment of the method for producing a Cas9-gRNA complex of the present invention. First, Cas9 (1) and gRNA (2) that have been expressed in advance are mixed and incubated at 20 ° C. or higher to form a Cas9-gRNA complex (10).
At this time, the temperature is 20 ° C. or higher, preferably 30 ° C. or higher and 65 ° C. or lower, more preferably 30 ° C. or higher and 40 ° C. or lower, and further preferably 30 ° C. or higher and 37 ° C. or lower. 32 ° C. or higher and 37 ° C. or lower is particularly preferable.
Generally, when handling a protein, it is generally at a low temperature such as 4 ° C. On the other hand, in the present embodiment, when the temperature is in the above range, Cas9 can be prevented from aggregating, a stable Cas9-gRNA complex can be formed, and solubilization can be performed in the solution.

Examples of the medium used when mixing Cas9 and gRNA include aqueous solvents, water, physiological saline, phosphate buffered saline [Phosphate buffered saline; PBS], and Tris buffered saline [PBS]. Tris Buffered Saline; TBS], HEPES buffered saline, TB (Transport buffer: 25 mM Hepes, 1.15 mM KOAC, 250 μM MgCl2, pH 7.2), glucose aqueous solution, serum-free medium, and the like, but not limited to these. ..
Examples of the serum-free medium include, but are not limited to, DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199 and the like. The serum-free medium may be appropriately selected according to the type of cells used in <Method for introducing Cas9-gRNA complex into cell nucleus> described later.
Further, the medium may include an ATP regeneration system. Examples of the ATP regeneration system include a combination of ATP, creatine kinase, and creatine phosphate.

(Cas9)
As used herein, "Cas9" is one of the Cas protein families constituting an adaptive immune system that provides acquisition resistance to invasive exogenous nucleic acids in bacteria and archaea, and recognizes PAM sequences in exogenous DNA. It is an endonuclease that cleaves double-stranded DNA upstream or downstream of it. As used herein, Cas9 means one that forms a complex with gRNA and has DNA cleaving activity.

In the present specification, the Cas protein family includes, for example, Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csy1, Csy2, Csy3, Cse1. , Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx1 , Csx15, Csf1, Csf2, Csf3, Csf4, their homologues, or their modifications. Cas9 used in this embodiment may be Cas9, their homologues, or a modified version thereof.

Bacterial species from which Cas9 is derived include, for example, Streptococcus pyogenes (S. pyogenes), S. aureus, Franciscilla novicida, Streptococcus vircoccus. Dassonbiei, Streptomyces pristina espillaris, Streptomyces viridochromogenes, Streptococcus roseum, Aricyclobatylus acidcardarius, Bacillus pseudomycoides, Bacillus pseudomycoides , Exigobacterium sibilicum, Lactobacillus delbricky, Lactobacillus salivarius, Microsila Marina, Burkholderia bacterium, Polaromonas naphthalenivolance, Polaromonas species, Crocosfaera sato , Cyanoseis, Microcystis aeruginosa, Cinecococcus, Acethalobium alabaticum, Ammonifex degensi, Cardicellosylptor dicystidisci , Crostridium botulinum, Crostridium difficile, Finegordia Magna, Natranaerobius thermophilium, Perotomaculum thermopropionicum, Acidiciobacillus cardas, Acidiciobacillus Arochromatium binosum, genus Marinobacta, nitrosococcus halophyrus, nitrosococcus wattsoni, Pseudoalteromonas haloplanctis, cutedonovactera Ghatam (Methanohalbium evestigatum), Anabaena Variabilis, Nodularia spmigena, Nostock, Arslospyra maxima, Arslospyra platensis, Arsulospira, Lyngbya, Microcholes xonoplasmotes, Microcoreus sto・ Africanus, Acario chloris marina, etc. are mentioned, but not limited to these. Among them, the bacterial species from which Cas9 used in the present embodiment is derived is preferably Streptococcus pyogenes (S. pyogenes) or Staphylococcus aureus (S. aureus).

Cas9 is upstream or downstream from the first or last nucleotide of the PAM sequence in the target gene, eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, Orients the cleavage of one or both strands within 50, 100, 200, 500 or more base pairs. The number of bp to be cleaved upstream or downstream of the PAM sequence depends on the bacterial species from which Cas9 is derived. Most Cas9, including pyogenes, cleaves 3 bases upstream of the PAM sequence.

In addition, Cas9 used in this embodiment may be a variant lacking the ability to cleave one or both strands of a target gene containing a target sequence. For example, S. Substitution of aspartic acid for alanine (D10A) in the RuvCI catalytic domain of Cas9 from pyogenes converts Cas9 from a nuclease that cleaves both strands to a nickase (which cleaves a single strand). Other examples of mutations that make Cas9 nickase include, but are not limited to, H840A, N854A and N863A. Also, two or more catalytic domains of Cas9 (RuvC I, RuvC II and RuvC III) can produce mutated Cas9 that substantially lacks all DNA-cleaving activity. In this case, it is preferably a chimeric enzyme fused with another enzyme having DNA cleaving activity. The D10A mutation may be combined with one or more of the H840A, N854A or N863A mutations to produce Cas9, which substantially lacks all DNA cleavage activity. When the DNA cleavage activity of the mutated Cas9 is less than about 25%, 10%, 5%, 1%, 0.1% or 0.01% with respect to its non-mutated form, all DNA cleavage activity is exhibited. Considered to be virtually lacking.

In this embodiment, the base sequence encoding Cas9 may be codon-optimized for expression in a particular cell, such as a eukaryotic cell. Eukaryotic cells include, but are not limited to, specific organisms such as humans, mice, rats, rabbits, dogs, pigs or non-human primates. In general, codon optimization is by replacing at least one codon in the native sequence with a codon that is more frequently or most frequently used in the gene of the organism to be introduced, while preserving the native amino acid sequence. Refers to the process of modifying a nucleic acid sequence for enhanced expression in the introduced organism. Various species exhibit a particular bias for a particular codon of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the translation efficiency of mRNA, which is believed to be particularly dependent on the properties of the codon being translated and the availability of a particular tRNA. There is. The predominance of selected tRNAs in cells is generally a reflection of the most frequently used codons in peptide synthesis. Thus, genes can be personalized for optimal gene expression in a given organism, based on codon optimization. Codon usage frequency tables are readily available, for example, in the "Codon Usage Database" posted at www.kazusa.or.jp/codon/ (visited December 16, 2015). Can be used to optimize codons (see, eg, Nakamura, Y. et al., Codon usage tabulated from the international DNA sequence databases: status for the year 2000, Nucl. Acids Res. 28: 292 (2000)). .. Computer algorithms for codon-optimizing specific sequences for expression in specific species are also available, for example, in Gene Forge (Aptagen; Jacobus, PA). In this embodiment, one or more codons in the Cas9 encoding sequence correspond to the most frequently used codons for a particular amino acid.

Cas9 in the present embodiment may have a nuclear localization signal (NLS) peptide bound to the N-terminal or C-terminal.

Examples of the NLS include a polypeptide consisting of the amino acid sequence PKKKRKV (SEQ ID NO: 1), the NLS of the SV40 virus large T antigen having the amino acid sequence consisting of the above SEQ ID NO: 1, and the NLS derived from the Nucleoplastin (for example, the sequence KRPAATKKA GQAKKK (sequence)). Nucleoplasmin dichotomy with No. 2) (NLS); c-myc NLS with amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4); Sequence of origin IBB domain RMRIZFKKKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 6); Sequence of myoma T protein VSRKRPRP (SEQ ID NO: 8) and PPKKARED (SEQ ID NO: 8); Sequence of human p53 PQPKKKPL (SEQ ID NO: 9) SALIKKKKKKMAP (SEQ ID NO: 10); Influenza virus NS1 sequence DRLRRR (SEQ ID NO: 11) and PKQKKR (SEQ ID NO: 12); Hepatitis virus delta antigen sequence RKLKKKIKKL (SEQ ID NO: 13); Mouse Mx1 protein sequence REKKKFLKRR (SEQ ID NO: 14) The sequence of human poly (ADP-ribose) polymerase KRKGDEVDGVDEVAKKKKSK (SEQ ID NO: 15); the sequence of steroid hormone receptor (human) glucocorticoid, RKCLQAGMNLEARKTKK (SEQ ID NO: 16), and the like are included, but not limited to these.

In addition, in this specification, "polypeptide", "peptide" and "protein" mean a polymer of amino acid residues and are used interchangeably. It also means an amino acid polymer in which one or more amino acids are chemical analogs or modified derivatives of the corresponding naturally occurring amino acids.

Further, in the present embodiment, Cas9 is divided into N-terminal Cas9 (Cas9-N) and C-terminal Cas9 (Cas9-C), and Cas9-N and Cas9-C are N, respectively. A protein that forms a dimer is bound to the terminal or C terminal in a photo-dependent or chemical-dependent manner, and Cas9 restores RNA-induced DNA endonuclease activity in the presence of light or a chemical substance. There may be.
By using the above-mentioned two-divided Cas9, the activity of Cas9 in the cell can be controlled, and the off-target can be further reduced.
Specifically, it is represented by the polypeptide (paCas9) consisting of the amino acid sequences represented by the photo-induced SEQ ID NOs: 17 and 18 and the rapamycin-induced SEQ ID NOs: 19 and 20 shown in Examples described later. Examples thereof include a polypeptide having an amino acid sequence (rpCas9).

In this specification, "protein that forms a dimer in a photo-dependent manner" (optical switch protein) was developed by a research group of Associate Professor Moritoshi Sato of the Graduate School of Arts and Sciences, University of Tokyo (). Nat.Commun. 6,6256 (2015) .doi: 10.1038 / ncomms7256), a pair of proteins that have undergone multifaceted protein engineering on the small photoreceptor Vivid of Neurospora crassa. Means. Pairs of photoswitch proteins exist as monomers in the dark and form heterodimers when they receive blue light. The conversion of monomers and dimers by light can be used to design and develop a variety of photoactivated tools.

Further, in the present specification, the "protein that forms a dimer in a chemical substance-dependent manner" is a protein used in a chemically induced dimerization system, and FK1012 was used in 1993. A model using a dimer-forming substance was proposed for the first time (Reference 2: Tamsir, A., et al., “Controlling signal transduction with synthetic ligands”, Science, vol.262, p1019-1024, 1993). Then, using rapamycin, which is a macrolide antibiotic, as a dimer-forming substance, a method using FK506-binding protein (FKBP) and FRB, which is a binding protein to a complex of FK506-binding protein and rapamycin, was used (reference). Reference 3: Various methods have been developed, including Bayle, JH, et al, “Rapamycin analogs with differential binding specificity permit orthogonal control of protein activity”, Chem. Biol., Vol.13, p 99-107, 2006). (Reference 4: Czlapinski, JL et al., “Conditional glycosylation in eukaryotic cells using a biocompatible chemical inducer of dimerization”, J. Am. Chem. Soc., Vol.130, p13186-13187, 2008 .; Reference Reference 5: Liang, FS, et al, “Engineering the ABA plant stress pathway for regulation of induced proximity”, Sci. Signal., Vol.4, rs2, 2011 .; Reference 6: Liberles, SD, et al., “Inducible gene expression and protein translocation using nontoxic ligands identified by a mammalian three-hybrid screen”, Proc. Natl. Acad. Sci. USA, vol.94, p7825-7830, 1997.).

As a method for producing Cas9 in the present embodiment, for example, first, a host is transformed using an expression vector containing a gene encoding Cas9. Conditions such as the composition of the medium, the temperature and time of the culture, and the addition of the inducer can be determined by those skilled in the art according to a known method so that the transformant grows and Cas9 is efficiently produced. Further, for example, when an antibiotic resistance gene is incorporated into an expression vector as a selection marker, a transformant can be selected by adding an antibiotic to the medium. Subsequently, Cas9 expressed by the host is purified by an appropriate method to obtain Cas9.

As used herein, a "vector" is a tool that allows or facilitates the migration of an entity from one environment to another. As an example, some vectors used in recombinant DNA technology allow entities such as DNA segments (eg, heterologous DNA segments, eg, heterologous cDNA segments) to be translocated into cells of non-human organisms. To. In this embodiment, the vector may include a viral vector (eg, lentivirus vector, baculovirus vector, adenovirus / adeno-associated virus vector, etc.), bacterial vector, protozoan vector, DNA vector or recombination thereof. Includes vector.

(GRNA)
As used herein, "gRNA" is a small RNA fragment (CRISPR-RNA: crRNA) containing a foreign sequence (guide sequence) that constitutes an adaptive immune system that provides acquired resistance to invading foreign nucleic acids in bacteria and archaea. It mimics the hairpin structure of a tracrRNA-crRNA chimera in which the crRNA and a partially complementary RNA (trans-activating crRNA: tracrRNA) are fused.
The gRNA used in the present embodiment may be a tracrRNA-crRNA chimera synthesized in a state where the crRNA containing the guide sequence and the tracrRNA are fused, and the crRNA containing the guide sequence and the tracrRNA are separately prepared and introduced. It may be previously annealed into a tracrRNA-crRNA chimera.

The gRNA in the present embodiment is a poly composed of a base sequence complementary to a base sequence of preferably 20 bases or more and 24 bases or less, more preferably 22 bases or more and 24 bases or less, from one base upstream of the PAM sequence in the target gene. It contains a nucleotide in the 5'terminal region. Further, it may contain one or more polynucleotides having a base sequence that is non-complementary to the target gene, arranged so as to be symmetrically complementary to one point as an axis, and having a hairpin structure. ..

In the present specification, the "PAM sequence" is a sequence that is present in a target gene and can be recognized by Cas9, and the length and base sequence of the PAM sequence vary depending on the bacterial species from which Cas9 is derived. For example, S. In pyogenes, 3 bases of "NGG" (N represents an arbitrary base) are recognized.
S. The thermophilus has two Cas9s, and recognizes 5 to 6 bases of "NGGNG" or "NNAGAA" (N represents an arbitrary base) as a PAM sequence, respectively. The PAM sequence is preferably "NGG" because the PAM sequence to be recognized is short and the editable target gene is not easily restricted.

As a method for producing gRNA, a known method (for example, in vitro translation method or the like) can be used for production. Specifically, for example, first, a DNA double strand arranged so as to be a 5'-T7 promoter sequence-gRNA (hereinafter, may be referred to as "sequence 1") is designed. Two long primers are then designed to anneal in the center of sequence 1 so that the PCR product is in sequence 1 above. The DNA double strand consisting of sequence 1 is then obtained as a PCR product by PCR amplification. Next, T7 RNA polymerase, ATP, UTP, GTP, CTP, buffer, RNase inhibitor, and pyrophosphate degrading enzyme are mixed with DNA obtained as a PCR product, and incubated at 37 ° C. for 2 hours or more and 4 hours or less. Then, gRNA is purified using RNeasy mini kit or the like.

In the Cas9-gRNA complex formation step of the present embodiment, two or more types of gRNA may be mixed. By mixing two or more types of gRNA, two or more types of Cas9-gRNA complexes can be obtained. By using these two or more types of Cas9-gRNA complexes in the <method for modifying a target gene in a cell> described later, a plurality of loci can be edited at the same time.

<Method of introducing Cas9-gRNA complex into cell nucleus>
In one embodiment, the present invention comprises a perforation step in which a perforating substance is allowed to act on the plasma membrane of a cell to form a pore in at least a part of the plasma membrane, and a pore is formed in at least a part of the plasma membrane. The introduction step of introducing the Cas9-gRNA complex obtained by the above-mentioned method for producing the Cas9-gRNA complex into the cells, and the formation of pores in at least a part of the plasma membrane into which the Cas9-gRNA complex was introduced. Provided is a method for introducing a Cas9-gRNA complex into a cell nucleus, comprising a reencapsulation step of adding a solution containing calcium ions to the cells to reencapsulate pores formed in at least a part of the plasma membrane. ..

According to the introduction method of the present embodiment, the Cas9-gRNA complex can be introduced into the cell nucleus while the intracellular vector for genome editing is optimized without introducing the viral vector into the cell.
The present inventors have so far developed a reversible membrane perforation method using the streptococcal toxin streptolysin O (SLO). By using the reversible membrane perforation method, semi-intact cells in which the plasma membrane is partially permeable can be obtained.
As shown in Reference 1 (Japanese Unexamined Patent Publication No. 2013-213688), in semi-intact cells, the original cytoplasm flows out while maintaining the structures and functions of organelles and cytoskeletons and their relative spatial arrangements almost intact. It can be "exchanged" with cytoplasms prepared from other cells and organs. For example, by exchanging the cytoplasm of semi-intact cells with the cytoplasm obtained from pathological cells, a pathological environment can be constructed inside the cells.
The introduction method of the present embodiment will be described in detail below with reference to the drawings.

[Punching process]
FIG. 2 is a schematic process diagram showing an embodiment of the method for introducing the Cas9-gRNA complex of the present invention into the cell nucleus. First, a perforating substance is allowed to act on the plasma membrane of cell A (12) to form pores in at least a part of the plasma membrane to obtain semi-intact cell A (12a).

(cell)
As used herein, the term "semi-intact cell" refers to a cell that has been partially permeable by exfoliating the plasma membrane and has the cytoplasm drained to the outside, or another cytoplasm or other substance from the outside ( It means a cell in which a compound, etc.) can be introduced into the cell.
In addition, examples of organisms from which cells to which the introduction method of the present embodiment is applied include prokaryotes, yeasts, animals, plants, insects and the like. The animal is not particularly limited, and examples thereof include, but are not limited to, humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, rats, and the like.

The animal-derived cells to which the introduction method of the present embodiment is applied include, for example, germ cells (sperm, egg, etc.), somatic cells constituting the living body, stem cells, precursor cells, cancer cells isolated from the living body, and living body. Cells (cell lines) that are isolated from the body and acquire immortalization ability and are stably maintained in vitro, cells that are isolated from the living body and artificially genetically modified, and cells that are separated from the living body and artificially exchanged nuclei. Etc., and are not limited to these.

The body cells that make up the living body include, for example, skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thoracic gland, muscle, connective tissue, etc. Examples include, but are not limited to, cells collected from arbitrary tissues such as bone, cartilage, vascular tissue, blood, heart, eye, brain, and nervous tissue. More specifically, as somatic cells, for example, fibroblasts, bone marrow cells, immune cells (eg, B lymphocytes, T lymphocytes, neutrophils, macrophages, monospheres, etc.), erythrocytes, platelets, bone cells, etc. , Marrow cells, pericutaneous cells, dendritic cells, keratinocytes, fat cells, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, lymphatic endothelial cells, hepatocytes, pancreatic islet cells (eg, α cells, β cells, δ cells, ε cells, PP cells, etc.), chondrocytes, ocher cells, glial cells, nerve cells (neurons), oligodendrocytes, microglia, stellate glial cells, myocardial cells, esophageal cells, muscle cells (For example, smooth muscle cells, skeletal muscle cells, etc.), melanin cells, mononuclear cells, etc., and are not limited thereto.

Stem cells are cells that have the ability to replicate themselves and differentiate into other cells of multiple lineages. Examples of stem cells include embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, artificial pluripotent stem cells (iPS cells), nerve stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, and pancreatic stem cells. , Muscle stem cells, reproductive stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells and the like, and are not limited thereto.

Cancer cells are cells that are derived from somatic cells and have acquired infinite proliferative potential. Cancers from which cancer cells are derived include, for example, breast cancer (eg, invasive breast cancer, non-invasive breast cancer, inflammatory breast cancer, etc.) and prostate cancer (eg, hormone-dependent prostate). , Hormone-independent prostate cancer, etc.), pancreatic cancer (eg, pancreatic duct cancer, etc.), gastric cancer (eg, papillary adenocarcinoma, mucinous adenocarcinoma, adenoflat epithelial cancer, etc.), lung cancer (eg, squamous cell carcinoma, etc.) Non-small cell lung cancer, small cell lung cancer, malignant mesoderma, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.), rectal cancer (eg, gastrointestinal stromal tumor, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.) Familial colon cancer, hereditary non-polyposis colon cancer, gastrointestinal stromal tumor, etc.), small intestinal cancer (eg, non-hodgkin lymphoma, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue Hmm, pharyngeal cancer (eg, nasopharyngeal cancer, mesopharyngeal cancer, hypopharyngeal cancer, etc.), head and neck cancer, salivary adenocarcinoma, brain tumor (eg, pine fruit stellate cell tumor, hairy cell star) Celloma, diffuse stellate cell tumor, degenerative stellate cell tumor, etc.), nerve sheath tumor, liver cancer (eg, primary liver cancer, extrahepatic bile duct cancer, etc.), kidney cancer (eg, renal cells) Cancer, metastatic epithelial cancer of the renal pelvis and urinary tract, etc.), bile sac cancer, bile duct cancer, pancreatic cancer, endometrial cancer, cervical cancer, ovarian cancer (eg, epithelial ovarian cancer, Extragonal embryonic cell tumor, ovarian embryonic cell tumor, low-grade ovarian tumor, etc.), bladder cancer, urinary tract cancer, skin cancer (for example, intraocular (eye) melanoma, Mercel cell carcinoma, etc.), blood vessels Tumor, malignant lymphoma (eg, reticular sarcoma, lymphosarcoma, Hodgkin's disease, etc.), melanoma (malignant melanoma), thyroid cancer (eg, thyroid medullary cancer, etc.), parathyroid cancer, nasal cavity cancer, sinus cavity Cancer, bone tumors (eg, osteosarcoma, Ewing tumor, uterine sarcoma, soft tissue sarcoma, etc.), metastatic myeloma, vascular fibroma, elevated cutaneous fibrosarcoma, retinal sarcoma, penile cancer, testicular tumor, pediatric solid Cancer (eg Wilms tumor, pediatric renal tumor, etc.), Kaposi sarcoma, Kaposi sarcoma caused by AIDS, maxillary sinus tumor, fibrous histiocytoma, smooth myoma, rhizome myoma, chronic myeloproliferative disease, leukemia (For example, acute myeloid leukemia, acute lymphoblastic leukemia, etc.) and the like, but are not limited thereto.

A cell line is a cell that has acquired infinite proliferative capacity by artificial manipulation in vitro. Examples of cell lines include HCT116, Huh7, HEK293 (human fetal kidney cells), HeLa (human cervical cancer cell line), HepG2 (human liver cancer cell line), UT7 / TPO (human leukemia cell line), and the like. CHO (Chinese hamster ovary cell line), MDCK, MDBK, BHK, C-33A, HT-29, AE-1, 3D9, Ns0 / 1, Jurkat, NIH3T3, PC12, S2, Sf9, Sf21, High Five, Vero, etc. , But not limited to these.

(Perforated substance)
Examples of the perforating substance include, but are not limited to, cytotoxins such as dichitonin, atoxin, streptolysin, and cholesterol-dependent cytolytic toxins. Among them, the perforating substance in the present embodiment is preferably a cholesterol-dependent cytolytic toxin when animal cells are used.

As used herein, the term "cholesterol-dependent cytolytic toxin" refers to one having a membrane perforating activity that binds to cholesterol and forms pores in the cell membrane (perforates the cell membrane). As a cholesterol-dependent cytolytic toxin, a toxin produced by streptococcus is known. The toxin is a protein that binds to cholesterol on the cell membrane and then self-assembles on the cell membrane to form pores in the cell membrane.
When cells are brought into contact with a cholesterol-dependent cytolytic toxin, pores are formed in the cell membrane, and a desired substance (Cas9-gRNA complex in this embodiment) can be introduced through the formed pores. The molecular weight of the substance that can be introduced by the pores formed by the cholesterol-dependent cytolytic toxin is, for example, 1 k or more and 200 k or less.
More specific examples of the cholesterol-dependent cytolytic toxin include, but are not limited to, streptolysin O (SLO), listeriolysin O (LLO), and the like.

In addition, cholesterol-dependent cytolytic toxin binds to cells at low temperature (usually 4 ° C. or lower) but does not have perforation activity, and becomes perforation activity only at high temperature (about 25 to 37 ° C.). Utilizing such temperature sensitivity, the perforating substance is bound to the plasma membrane of the cell at a low temperature, and then the excess perforating substance is washed away, and then the temperature is raised to a high temperature (about 25 to 37 ° C.). , Only a specific part in which the perforating substance is bound to the lipid of the plasma membrane can be selectively perforated.
In addition, the existence of an optimum pH is known for the activity of cholesterol-dependent cytolytic toxin. For example, LLO is said to have optimal pH in the pH range of less than 6 (Schuerch, DW, Wilson-Kubalek, EM and Tweten, RK (2005) Molecular basis of listeriolysin O pH dependence. PNAS, 102, 12537-12542.).

The optimum temperature for the membrane perforation activity may be, for example, 30 ° C. or higher and 40 ° C. or lower, 33 ° C. or higher and 38 ° C. or lower, or 35 ° C. or higher and 37 ° C. or lower.
The optimum pH of the membrane perforation activity may be, for example, pH 1 or more and less than 6, for example, pH 3 or more and 5.5 or less, and for example, pH 4.5 or more and less than 5.5.

The optimum pH of the membrane perforation activity can be measured by a known method. For example, the degree of hemolytic activity (hemolytic unit, HU) of blood cells in which hemoglobin is eluted by contacting erythrocytes cultured under the above temperature and pH conditions with a cholesterol-dependent cytolytic toxin to destroy the erythrocyte membrane. The membrane perforation activity can be measured as a reference, and the pH at which the membrane perforation activity is maximized can be determined to be the optimum pH.

The perforating substance may be used by adding it to a medium or the like with respect to the cells A (12) cultured in advance.
As used herein, the term "culture" means breeding or growing cells outside a living body (individual). The breeding or growing period may be, for example, 1 minute or more and 7 days or less, for example, 5 minutes or more and 16 hours or less, and for example, 10 minutes or more and 1 hour or less.
As used herein, the term "medium" is a concept that refers to all cells that can be cultured. In the medium, the remaining components excluding the perforating substance need only be those capable of culturing cells even in a very short period of time, and water or a buffer solution (for example, physiological saline or phosphate buffer) which is not generally called a medium is used. Phosphate buffered saline [PBS], Tris Buffered Saline; TBS, HEPES buffered saline, TB (Transport buffer: 25 mM Hepes, 1.15 mM KOAC, 250 μM MgCl2, pH Etc.) etc. Examples of the components that can be contained in the medium include components contained in a normal cell culture medium, for example, nutritional components such as glucose, sodium chloride, vitamins, minerals, amino acids, etc .; growth factors, cell proliferation, etc. Factors, differentiation-inducing factors, antibacterial agents, antifungal agents and the like can be mentioned. More specifically, the medium includes, and is not limited to, for example, DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199 and the like. The composition of the medium may be appropriately selected according to the type of cell A to be used.

The content of the perforating substance contained in the medium may be, for example, 0.01 μg / mL or more and 1 μg / mL or less, for example, 0.025 μg / mL or more and 0.6 μg / mL or less, for example, 0.05 μg. It may be / mL or more and 0.3 μg / mL or less, for example, 0.08 μg / mL or more and 0.1 μg / mL or less.
When the content of the perforating substance is in the above range, the damage given to the cells is small, and the effect of introducing the substance into the cells can be made uniform.

The pH of the medium containing the perforating substance may be, for example, pH 6 or more and 10 or less, for example, 6.5 or more and 8 or less, and for example, 7.0 or more and 7.5 or less. .. The pH shall be a measured value in the range of 30 ° C. or higher and 40 ° C. or lower.
When the pH of the medium is within the above range, the cells can be cultivated well. Furthermore, it is preferable from the viewpoint that the membrane perforating activity of a perforating substance (particularly, cholesterol-dependent cytolytic toxin) having an optimum pH in the range of 0 or more and less than 6 is gently exerted.

The amount ratio of the amount of perforating substance (particularly cholesterol-dependent cytolytic toxin) contained in the medium to the cells can be appropriately determined according to the type of cells. For example, it is considered that the higher the cholesterol content in the cell membrane of a cell, the smaller the amount (concentration) of perforating substance required for perforating the membrane. If the cholesterol content in the cell membrane is high, the amount (concentration) of the perforating substance contained in the medium should be adjusted to decrease, and if the cholesterol content in the cell membrane is low, the amount (concentration) of the perforating substance contained in the medium should be adjusted. ) May increase.

When culturing in a medium containing a perforating substance (particularly, cholesterol-dependent cytolytic toxin), the culture temperature of cell A may be, for example, 0 ° C. or higher and 10 ° C. or lower, for example, 2 ° C. or higher and 5 ° C. or lower. Good. Specifically, the temperature of the medium may be controlled by placing the culture vessel containing the medium containing the perforating substance and the cells A on ice. In this temperature range, the membrane perforation activity of perforating substances (particularly cholesterol-dependent cytolytic toxins) is suppressed. Therefore, when the medium containing the perforating substance is brought into contact with the cell A, the perforating substance binds to the cell membrane of the cell A, but the perforating action is not easily exerted, and the degree of perforation of the cell membrane can be easily controlled.

After culturing the cells in the medium containing the perforating substance, the medium containing the perforating substance is replaced with the medium containing no perforating substance, and the cells are cultured in the medium containing no perforating substance to perforate the cell membrane of the cell A.
By exchanging the medium containing the perforating substance with the medium containing no perforating substance, the perforating substances other than those bound to the cell A are removed from the reaction system in the culture vessel.

When culturing in a medium containing no perforating substance, the culture temperature of cell A may be, for example, 30 ° C. or higher and 40 ° C. or lower, for example, 33 ° C. or higher and 38 ° C. or lower, for example, 35 ° C. or higher and 37 ° C. or lower. It should be. By culturing in the temperature range, the membrane perforating activity of perforating substances (particularly cholesterol-dependent cytolytic toxins) is exhibited, the cell membrane of cell A (12) is perforated, and the cell membrane is perforated. 12a). Since the perforating substance other than the perforating substance bound to the cell A is removed from the medium containing no perforating substance, it is possible to prevent the perforating substance from further entering the cell from the pores and perforating the organelle in the cytoplasm.

The culture time for culturing the cell A in a medium containing no perforating substance may be appropriately determined according to the type and cell type of the perforating substance. For example, about 1 minute or more and 30 minutes or less can be mentioned.

Examples of the medium containing no perforating substance include the same medium as the medium exemplified for the medium containing the perforating substance. The medium containing no perforating substance and the medium containing a perforating substance may be the same type of medium or different types of medium.

When culturing in a medium containing no perforating substance, the optimum pH of the medium may be, for example, pH 6 or more and 10 or less, for example, 6.5 or more and 8 or less, for example, 7.0 or more and 7.5. It may be as follows. The pH shall be a measured value in the range of 30 ° C. or higher and 40 ° C. or lower.
When the optimum pH of the medium is within the above range, cells can be cultivated well, and the membrane perforation activity of the perforating substance is gently exhibited, which is also preferable.

When culturing in a medium containing no perforating substance, the optimum temperature of the medium may be, for example, 30 ° C. or higher and 40 ° C. or lower, for example, 33 ° C. or higher and 38 ° C. or lower, for example, 35 ° C. or higher and 37 ° C. It may be as follows.

[Introduction process]
Next, the Cas9-gRNA complex (10) described above is introduced into cells (semi-intact cells) A (12a) having pores formed at least partially on the plasma membrane.

In the introduction step, the Cas9-gRNA complex may be in a state of being solubilized in a medium or the like. Examples of the medium include those similar to those exemplified in the above-mentioned [perforation step].

In the introduction step, the temperature may be, for example, 30 ° C. or higher and 40 ° C. or lower, for example, 30 ° C. or higher and 38 ° C. or lower, and for example, 32 ° C. or higher and 37 ° C. or lower.

[Mixing process]
Further, when there is a substance to be introduced together with the Cas9-gRNA complex (10) described above, a mixing step of mixing the Cas9-gRNA complex and the substance may be provided before the introduction step.

In the mixing step, the medium used for mixing the Cas9-gRNA complex and the substance is the same as that exemplified in the [Cas9-gRNA complex forming step] of the above-mentioned <Method for producing Cas9-gRNA complex>. Things can be mentioned.

Further, in the mixing step, the temperature may be adjusted to the same temperature as the subsequent introduction step, for example, 30 ° C. or higher and 40 ° C. or lower, for example, 30 ° C. or higher and 38 ° C. or lower, for example, 32 ° C. or higher and 37 ° C. It may be ℃ or less.

The molecular weight of the substance may be, for example, 1 k or more and 200 k or less.
Further, the substance may have a cell membrane impermeable property. The "substance having impermeable cell membrane" means a substance having a property of not being soluble in the lipid bilayer membrane of the cell membrane and not being able to permeate the lipid bilayer membrane. According to the introduction method of the present embodiment, even a substance that is impermeable to cell membranes can be introduced into cells with high efficiency through the pores.
Further, the substance may be a compound or an organic compound. The substance may contain nucleic acids, and examples thereof include antisense nucleic acids, miRNA, siRNA, shRNA, ribozymes, aptamers and the like. These compounds can be active ingredients in nucleic acid medicines. Compounds containing these nucleic acids are usually impermeable to cell membranes, but according to the substance introduction method of the embodiment, they can be efficiently introduced into cells.
Regarding the case where the substance to be introduced together with the Cas9-gRNA complex is the cytoplasm, an inhibitor of an inhibitor of homologous recombination, or an activator of homologous recombination, the following [first mixing step], [second Mixing process].

(First mixing step)
In addition to the Cas9-gRNA complex, cytoplasm (11a) collected from cells B (11) derived from an organism of the same species as cells (semi-intact cells) A (12a) having pores formed in at least a part of the plasma membrane was obtained. In the case of introduction, the Cas9-gRNA complex (10) and the cytoplasm (11a) collected from the semi-intact cell A (12a) and the cell B (11) derived from the same organism may be mixed before the introduction step. ..

Examples of the cell-derived organism from which the cytoplasm is derived and the type of cell include those exemplified in (cell) of the above-mentioned [perforation step]. Further, the type of cell may be the same as or different from that of cell A.
When cell A and cell B are somatic cells of the same type, cell A is a normal cell, and cell B is a cancer cell, cell B is introduced by introducing the cytoplasm of cell B into cell A. It is possible to visualize and reconstruct biological phenomena for the purpose in a cytoplasmic manner, and further analyze cell traits (phenotype), gene expression, epigenotic dynamic changes, etc. after the subsequent [re-encapsulation step]. can do.

(Second mixing step)
When introducing an inhibitor of an inhibitor of homologous recombination or an activator of homologous recombination in addition to the Cas9-gRNA complex, homologous recombination with the Cas9-gRNA complex (10) is performed before the introduction step. It may be mixed with an inhibitor or activator of the inhibitor of the above.

Examples of the homologous recombination inhibitor include, but are not limited to, CUL4A / B, which is a family of Cullin E3 ligase, and CUL3, which is a ubiquitinizing enzyme that ubiquitinates PALB2. Therefore, examples of the inhibitor of these inhibitors include antisense nucleic acids, siRNA, shRNA, aptamers, antibodies and the like against these inhibitors.

Further, examples of the homologous recombination activator include, and are not limited to, the DNA homologous recombination enzymes Rad51 and Rad54.

[Re-encapsulation process]
Next, a solution containing calcium ions is added to cells having pores formed in at least a part of the plasma membrane into which the Cas9-gRNA complex has been introduced, and the pores formed in at least a part of the plasma membrane are re-encapsulated. To do.
In FIG. 2, a semi-intact cell A (12b) into which the Cas9-gRNA complex (10) and the cytoplasm (11a) of the cell B (11) have been introduced is illustrated.

As used herein, the term "resealing" means that the openings of the pores formed in the cell membrane in the perforation step are completely or partially closed.
Reencapsulation is attributed to the removal of perforating material from the cell membrane by endocytosis and / or exocytosis and is facilitated by the presence of calcium ions. The cell membrane of the semi-intact cell A (12b) into which the Cas9-gRNA complex (10) cultured in a solution (medium) containing calcium ions and the cytoplasm (11a) of the cell B (11) was introduced was resealed and the cell membrane was resealed. It becomes A (12c). The Cas9-gRNA complex introduced into the cells in the introduction step is efficiently retained in the resealed cells A (12c) by reencapsulation.

The concentration of calcium ions contained in the medium may be, for example, 0.1 mmol / L or more and 10 mmol / L or less, and may be 0.5 mmol / L or more and 5 mmol / L or less.

A solution containing calcium ions can be obtained, for example, by adding a salt of calcium to a medium for cell culture. Examples of the calcium salt to be added include, but are not limited to, calcium chloride.

In the reencapsulation step, the pores may be reencapsulated without bringing the solution containing the foreign cytoplasm into contact with the cells in which the pores are formed, and the solution containing the foreign cytoplasm is applied to the cells in which the pores are formed. The holes may be resealed by contacting them.

As used herein, the term "foreign cytoplasm" means the cytoplasm of cells other than the cells to be introduced that have been perforated in the perforation step. For example, the cytoplasm of cell A (12a) having pores formed in the cell membrane does not correspond to the foreign cytoplasm. Further, in the example shown in FIG. 2, the foreign cytoplasm is a cytoplasm obtained from a cell other than cell A, that is, the cytoplasm of cell B (11a). The type of foreign cytoplasm may be a cytoplasm of a cell of a type different from the cell to be introduced, a cytoplasm of the same type of cell, or a cytoplasm prepared from a tissue.

In the introduction method of the present embodiment, the drilling step and the introduction step are carried out as independent steps, but the drilling and the introduction may occur almost at the same time. In that case, in the introduction method of the present embodiment, it is exemplified that the Cas9-gRNA complex is added to the medium in the introduction step after the perforation step, but the Cas9-gRNA complex is added to the medium in the perforation step. You may.

<Method of modifying target gene in cells>
In one embodiment, the present invention presents the present invention in a cell in which the Cas9-gRNA complex is introduced into the cell nucleus obtained by the method for introducing the Cas9-gRNA complex into the cell nucleus and has a resealed target gene. The modified target in the region where Cas9 is determined by a cleavage step of cleaving the target gene at a cleavage site located upstream or downstream of a predetermined base of the PAM sequence and complementary binding between the gRNA and the target gene. Provided is a modification step for obtaining a cell having a gene, and a method for modifying a target gene in the cell.

According to the method for modifying the target gene in the cell of the present embodiment, the off-target is reduced, and the target gene can be easily modified without introducing the viral vector into the cell. In addition, the nuclear niche of genome editing is optimized, and multiple loci in a large number of cells can be edited at the same time.
The method for modifying the target gene in the cell of the present embodiment will be described in detail below.

[Cutting process]
First, in the Cas9-guide RNA complex, a part of the guide RNA binds to the target gene, and Cas9 recognizes the PAM sequence in the target gene. Subsequently, the target gene is cleaved at a cleavage site located upstream or downstream of a predetermined base (for example, 3 bases). More specifically, Cas9 recognizes the PAM sequence, the double helix structure of the target gene is stripped from the PAM sequence, and it is annealed with a base sequence complementary to the target gene in the guide RNA. The double helix structure of the target gene is partially unraveled. At this time, Cas9 cleaves the phosphate diester bond of the target gene at a cleavage site located upstream or downstream of a predetermined base (for example, 3 bases) of the PAM sequence.

[Modification process]
Subsequently, in the region determined by the complementary binding of the gRNA and the target gene, a target gene modified according to the purpose can be obtained.

As used herein, the term "modification" means that the base sequence of a target gene is changed. For example, cleavage of the target gene, change in the base sequence of the target double-stranded polynucleotide due to insertion of an exogenous sequence after cleavage (insertion by physical insertion or replication via homologous orientation repair), non-homologous end linkage after cleavage (for example) NHEJ: Changes in the base sequence of the target gene due to rebinding of DNA ends generated by cleavage) can be mentioned. By modifying the target gene in this embodiment, it is possible to introduce a mutation into the target gene or destroy the function of the target gene.
Therefore, in the present embodiment, it is possible to easily prepare a cell in which the function of the target gene is disrupted (knocked out) or substituted (knocked in), or an organism having the cell.

When modifying two or more parts of one target gene, or when targeting two or more genes, a polynucleotide consisting of a base sequence complementary to the base sequence of each gene is used in the 5'end region. Two or more types of gRNA contained in the above are designed, two or more types of Cas9-gRNA complexes are produced in the above-mentioned <Method for producing Cas9-gRNA complex>, and then into the cell nucleus of the above-mentioned <Cas9-gRNA complex. A cell into which two or more types of Cas9-gRNA complexes have been introduced may be used. This makes it possible to edit a plurality of different genes or a plurality of locations within the same gene at the same time with high efficiency.

In the method for modifying a target gene in a cell of the present embodiment, a cell into which a foreign gene has been introduced together with the Cas9-gRNA complex may be used by using <a method for introducing a Cas9-gRNA complex into the cell nucleus>. By introducing the foreign gene together, it is possible to obtain a genetically modified non-human organism having a nuclear genome in which the foreign gene is inserted into the target gene by homologous recombination after the target gene is cleaved.

Moreover, it is preferable that the foreign gene does not have a PAM sequence. In a foreign gene, for example, when the PAM sequence is changed to a nonPAM sequence by a single nucleotide substitution that does not change the amino acid sequence, the foreign gene can be inserted into the nuclear genome by homologous recombination, and the inserted foreign gene is further formed. It is possible to prevent the gene from being cleaved again by the endogenous Cas9.

The foreign gene is preferably introduced using a vector containing the foreign gene. Further, in the vector containing the foreign gene, it is preferable that DNA having a sequence homologous to the site where the target gene is inserted is linked to the 5'end and 3'end of the foreign gene. The number of bases of DNA having a sequence homologous to the site where the target gene is inserted is preferably 100 bases or more and 300 bases or less. In the vector containing the foreign gene, various promoters, polyadenylation signals, NLS, marker genes for fluorescent proteins and the like may be operably linked to the 5'end or 3'end of the foreign gene.

<Use and utilization method>
In one embodiment, the invention provides methods and compositions for carrying out genetic modification. In contrast to previously known methods of targeted genetic recombination, the present invention is efficient and inexpensive to carry out and is adaptable to any cell or organism. Any segment of the target gene of a cell or organism can be modified by the methods of the invention. This method utilizes both homologous and illegitimate recombination processes that are endogenous to all cells.

Also, in one embodiment, the invention provides a method of performing targeted DNA insertion or targeted DNA deletion. This method involves transforming cells with a nucleic acid construct containing donor DNA. The scheme for DNA insertion and DNA deletion after cleavage of the target gene can be determined by those skilled in the art according to known methods.

Also, in one embodiment, the invention is utilized in both somatic and germ cells to provide genetic manipulation at specific loci.

Also, in one embodiment, the invention provides a method for disrupting a gene in somatic cells. Here, the "gene" overexpresses a product harmful to a cell or an organism and expresses a product harmful to the cell or an organism. Such genes can be overexpressed in one or more cell types that occur in the disease. Disruption of the overexpressed gene by the method of the present invention may result in better health for an individual suffering from a disease caused by the overexpressed gene.
That is, only a small percentage of the cells can be disrupted, reducing expression levels and producing therapeutic effects.

Also, in one embodiment, the invention provides a method for disrupting a gene in germ cells. Cells in which a particular gene has been disrupted can be selected to create an organism that does not have the function of a particular gene. In cells in which the gene has been disrupted, the gene can be completely knocked out. The loss of function in this particular cell can have a therapeutic effect.

Also, in one embodiment, the invention further provides insertion of donor DNA encoding a gene product. This gene product, when constitutively expressed, has a therapeutic effect.
For example, in a population of pancreatic cells, there is a method of inserting the donor DNA into an individual suffering from diabetes in order to induce insertion of a donor DNA encoding an active promoter and an insulin gene. The population of pancreatic cells containing exogenous DNA can then produce insulin to treat individuals suffering from diabetes.

In addition, the donor DNA can be inserted into crops and cause the production of drug-related gene products. Genes of protein products (eg, insulin, lipase, hemoglobin, etc.) can be inserted into plants together with regulatory elements (constitutive active promoters, or inducible promoters) to produce large amounts of pharmaceuticals in plants. Such protein products can then be isolated from the plant. Genetically modified plants or organisms use nucleic acid transfer techniques (McCreath, KJ et al. (2000) Nature 405: 1066-1069; Polejeva, IA et al., (2000) Nature 407: 86-90). Can be made by the method. Tissue-specific or cell-type-specific vectors can be utilized to provide gene expression only within selected cells.

Also, in one embodiment, the invention selects cells that are utilized within germ cells, where insertions occur in a planned manner and all subsequent cell division produces cells with the designed genetic alterations. Can be.

Also, in one embodiment, the invention relates to all organisms, cultured cells, cultured tissues, cultured nuclei (including cells, tissues or nuclei that can be used to regenerate intact organisms), gametes (eg, they). It can be applied in eggs or sperms at various stages of development.
Also, in one embodiment, the invention relates to any organism (insects, fungi, rodents, cows, sheep, goats, chickens, and other agriculturally important animals, as well as other mammals (dogs, cats and). It can be applied to cells derived from (but not limited to) humans).
Also, in one embodiment, the present invention can be applied in the production of pathological model animals (eg, pathological model mice).

Also, in one embodiment, the invention can be used in plants. The plant is not particularly limited and can be used in any various plant species (eg, monocotyledonous plant, dicotyledonous plant, etc.).

Hereinafter, the present invention will be described in more detail with reference to Examples and the like, but the present invention is not limited to these Examples and the like.

[Reagents]
The reagents used in this example are shown below.
-Streptolysin O (SLO): (manufactured by Bio-Academia, model number: 01-531)

The SEQ ID NOs of Cas9 and gRNA used are shown in Table 1 below. In Table 1, SaCas9 means Cas9 derived from Staphylococcus aureus (S. aureus), and SpCas9 means Cas9 derived from Streptococcus pyogenes (S. pyogenes). Further, dCas9 means SpCas9 in which endonuclease activity is inactivated, and NLS means a nuclear localization signal. The amino acid sequence of NLS is shown in SEQ ID NO: 1.

[Example 1] Preparation of Cas9-gRNA complex (1) Preparation of sgRNA Each gRNA (SEQ ID NOs: 25, 26, and 27) was incubated at 95 ° C. for 1 minute and cooled on a heat block at 32 ° C. to prepare sgRNA. did.

(2) Preparation of Cas9 An expression vector having a construct of His6-SUMO-tev protease site-HA-NLS-SaCas9, NLS-SpCas9-NLS, SpCas9-NLS, and His6-NLS-GFP-dCas9-NLS (Graduate School of Tokyo University). (Transferred from the research group of Professor Osamu Nureki of the Graduate School of Science) was transformed into Escherichia coli. Then, the cells were cultured in 0.1 mM IPTG containing antibiotics at 20 ° C. for 18 hours. After culturing, Escherichia coli was recovered. The E. coli was then crushed by sonication.
Next, SpCas9-NLS and NLS-SpCas9-NLS were purified with a P20.1 antibody column that recognizes the target tag added to the C-terminal, and SpCas9-NLS and NLS-SpCas9-NLS were subjected to HiTrapSP (GE). Obtained.
Further, His6-NLS-GFP-dCas9-NLS was purified by NiNTA and subjected to HiTrapSP (GE) to obtain His6-NLS-GFP-dCas9-NLS.
In addition, His6-SUMO-tev protease site-HA-NLS-SaCas9 was purified by NiNTA and cleaved using TEV protease. Purification with NiNTA was performed again to obtain HA-NLS-SaCas9.

(3) Preparation of Cas9-gRNA complex Next, 20 μL TB (Transport buffer: 25 mM Hepes, 1.15 mM KOAC, 250 μM MgCl ₂ , pH 7.2) was added to the sgRNA prepared in (1) and Cas9 prepared in (2), respectively. ), And incubated at 32 ° C. for 10 minutes. As a control group, only Cas9 was added to 20 μL TB without mixing with gRNA, and the mixture was allowed to stand on ice.

None of the Cas9-gRNA complexes produced Cas9 aggregates and were solubilized (not shown). On the other hand, in a sample in which only Cas9 was added to 20 μL TB and allowed to stand on ice, Cas9 aggregates were generated and precipitation was observed (not shown).
From the above, Cas9 and gRNA can suppress the formation of Cas9 aggregates by forming a complex before cell introduction.

[Test Example 1] Introduction of dCas9-gRNA complex into the cell nucleus (1) Preparation of cytoplasm of L5178Y cells L5178Y cells cultured in a 150 mm dish were collected in a 50 mL tube and centrifuged at 2000 rpm for 3 minutes. After centrifugation, the supernatant was removed and the cells were suspended in PBS and collected in a single 50 mL tube. Subsequently, the supernatant was removed after centrifugation at 2000 rpm for 3 minutes. Then, hypotonic buffer (1 mM EGTA, 1 mM MgCl ₂ , 45 mM Hepes-KOH (pH 7.4), 1 mM DTT, 1 μM cytochalasin D) was added and suspended, and then allowed to stand for 5 minutes to expand the cells. Then, the cells were centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and the obtained cell pellet was transferred to a downhomogenizer, and a protease inhibitor (antipain, hypostatin, pepstatin A, leupeptin, 25 μg / mL each) was added. , Stroke 5 times on ice. Subsequently, 1/10 amount of 10 × TB (250 mM HEPES-KOH (pH 7.4), 1150 mM potassium acetate, 25 mM MgCl ₂ ) was added to the pellet, and the cells were further stroked 10 times or more to disrupt the cells. After crushing, the sample was centrifuged at 12000 rpm and 4 ° C. for 16 minutes. After centrifugation, the supernatant was collected and centrifuged at 100,000 g at 4 ° C. for 90 minutes. Next, TB containing an ATP regeneration system (ATP, creatine kinase (CK), creatine phosphate (CP), GTP, and glucose) was mixed with the obtained supernatant (cytoplasm) at 32 ° C. Incubated for 10 minutes.

(2) Preparation of mixed solution of Cas9-gRNA complex and cytoplasm Next, the complex of His6-NLS-GFP-dCas9-NLS and sgAAVS1 obtained in "1. Preparation of Cas9-gRNA complex" (hereinafter referred to as "Cass9-gRNA complex"). , "DCas9-gRNA complex") and the cytoplasm of the L5178Y cells obtained in (1) were mixed to a total of 100 μL and stored at 32 ° C. until use. The composition of the mixed solution of the dCas9-gRNA complex and the cytoplasm is shown in Table 2 below.

(3) Perforation of HeLa cells Next, 80% confluent HeLa cells were prepared on a 3.5 cm dish and a square cover glass. HeLa cells were then transferred onto ice and washed twice with pre-chilled PBS. Then, 1 mL of D-MEM containing SLO (0.125 μg / mL) (without Fetal bovine serum (FCS)) was added, and the mixture was incubated on ice for 5 minutes. It was then washed 3 times with pre-chilled PBS. Then, 1 mL of TB (without EGTA) at 37 ° C. was added, and the mixture was incubated at 37 ° C. for 10 minutes. Then, it was washed twice with TB (without EGTA).

(4) Introduction of a mixed solution of dCas9-gRNA complex and cytoplasm into HeLa cells Next, the cover glass was taken out on a parafilm, and 100 μL of the mixed solution of dCas9-gRNA complex and cytoplasm prepared in (1) was placed. Then, it was incubated at 32 ° C. for 30 minutes. As a control group, i) a sample containing only His6-NLS-GFP-dCas9-NLS without cytoplasm, and ii) a sample containing only dCas9-gRNA complex without cytoplasm were placed on the test.

(5) Reencapsulation of HeLa cells Next, the mixed solution was collected, mixed with 1 μL of 100 mM calcium chloride, returned on the cover glass, and incubated at 32 ° C. for 10 minutes. The cover glass was then transferred to a new 3.5 cm dish and washed twice with PBS.

(6) Fluorescent Staining of HeLa Cells Next, 2 mL of D-MEM (containing FCS) containing PI (Propidium Iodide) was added, and the cells were cultured under the conditions of 37 ° C. and 5% carbon dioxide. Each cell _____ hours after culturing was observed using a fluorescence microscope. The results are shown in FIG. In FIG. 3, i) is a sample containing only His6-NLS-GFP-dCas9-NLS and no cytoplasm, ii) is a sample containing only dCas9-gRNA complex and no cytoplasm, and iii) is a sample of dCas9-gRNA complex and cytoplasm. Means a sample using a mixed solution. Further, "GFP-dCas9" means an image in which the localization of dCas9 is observed inside and outside the cell using fluorescence of GFP, and "PI" means an image in which the nucleus is stained using Propidium Iodide, and "merge". Means an image in which the images of "GFP-dCas9" and "PI" are combined.

From FIG. 3, in i), dCas9 formed a huge complex in the solution and aggregated, so that it was non-specifically adsorbed on the plasma membrane and was not introduced into the cell. In ii), aggregate formation was significantly reduced, and the dCas9-gRNA complex could be introduced into the cytoplasm, but translocation into the nucleus was not observed.
On the other hand, in iii), the translocation of the dCas9-gRNA complex into the nucleus was observed by using the dCas9-gRNA complex and the cytoplasm. It was speculated that this was because the translocation of the dCas9-gRNA complex into the nucleus was promoted by the influence of factors contained in the cytoplasm.

From the above results, Cas9 and gRNA form a complex before cell introduction, thereby suppressing the formation of Cas9 aggregates and enabling cell introduction, and the cytoplasm is the nucleus of the Cas9-gRNA complex. It became clear that it promoted internal intentions.

[Test Example 2] T7E1 assay using HeLa cells into which SpCas9-gRNA complex and cytoplasm have been introduced (1) Preparation of cytoplasm of L5178Y cells Using the same method as in Test Example 1 (1), cytoplasm of L5178Y cells Was prepared.

(2) Preparation of mixed solution of Cas9-gRNA complex and cytoplasm Next, the complex of NLS-SpCas9-NLS and sgAAVS1 obtained in "1. Preparation of Cas9-gRNA complex" (hereinafter, "SpCas9-"). (Sometimes referred to as “gRNA complex”) and the cytoplasm of the L5178Y cells obtained in (1) were mixed to a total of 100 μL and stored at 32 ° C. until use. The composition of the mixed solution of SpCas9-gRNA complex and cytoplasm is the same as in Table 2 above. In Table 2, the content of SpCas9 is in 5 μg / 100 μL described as the content of dCas9.

(3) Perforation of HeLa cells Using the same method as in (3) of Test Example 1, pores were formed in at least a part of HeLa cells.

(4) Introduction of a mixed solution of SpCas9-gRNA complex and cytoplasm into HeLa cells Except that a mixed solution of SpCas9-gRNA complex and cytoplasm was used instead of the mixed solution of dCas9-gRNA complex and cytoplasm. Introduced a mixed solution of SpCas9-gRNA complex and cytoplasm into HeLa cells using the same method as in Test Example 1 (4).

(5) Re-encapsulation of HeLa cells HeLa cells were re-encapsulated using the same method as in (5) of Test Example 1. Then, 2 mL of D-MEM (containing FCS) was added, and the cells were cultured at 37 ° C. and 5% carbon dioxide for 24 hours.

(6) Genome Extraction from Re-encapsulated HeLa cells Next, the re-encapsulated HeLa cells (hereinafter, may be referred to as “Reseal HeLa cells”) after culturing for 24 hours were washed twice with PBS. Then, 500 μL of 1% SDS Lysate Buffer was added to the Reseal HeLa cells, and the cells were incubated at 37 ° C. and 5% carbon dioxide for 3 hours. The solution on the petri dish was then collected in 1.5 mL tubes, 20 mg / mL proteinase K 2.25 μL was added, mixed and incubated overnight at 50 ° C. Then, 7.5 μL of 10 mg / mL RNase was added, and 10 μL of 1% SDS Lysate Buffer was added and mixed, and the mixture was incubated at 37 ° C. for 30 minutes. Then, 750 μL of phenol / chloroform was added, vortexed, and centrifuged (14,000 rpm, room temperature, 10 minutes). After centrifugation, the supernatant was transferred to a new 1.5 mL tube and phenol / chloroform extraction was performed in the same manner. Then, 750 μL of 2-propanol was added to the supernatant, mixed by inversion for 10 rotations, and centrifuged (14,000 rpm, 4 ° C., 10 minutes). After centrifugation, the pellet was washed with 70% ethanol and vacuum dried. The pellet was then dissolved in 20 μL of deionized-destilled H ₂ O (ddH ₂ O) to prepare a genomic solution.

(7) PCR amplification of the AAVS1 region Next, using the genomic solution obtained in (6), nested PCR was performed so as to produce a product of about 1 kbp containing the Cas9 target sequence of the AAVS1 region. The primers used in the first PCR and the second PCT are shown in Table 3 below.

The number of cycles was 20 for the first PCR and 35 for the second PCR. Further, as a PCR template, 10 ng of the genomic DNA prepared in (6) was used in the first PCR, and 1 μL of a 10-fold diluted solution of the first PCR product was used in the second PCR. After PCR amplification, the PCR product was lysed in 20 μL of _{ddH 2} O using the Wizard SV Gel and PCR Clean-UP System (manufactured by Promega). The PCR product was then _{diluted with ddH 2} O to 2 μg / 20 μL and heat denatured and reannealed using a thermal cycler (95 ° C for 2 minutes / 85 ° C → 35 ° C for 10 minutes). Cooling / 16 ° C).

(8) T7E1 assay The T7E1 assay is an action in which T7 endonuclease recognizes and cleaves DNA double-stranded mismatch sites (sites that are not base pairs such as AT and CG that can form hydrogen bonds). It is a method of detecting and quantifying mutations in the genome by utilizing having.
Next, 1 μL of NE Buffer2 and 0.125 μL of T7 endonuclease 1 (10,000 U / mL) were added to 8.875 μL of the reannealed product obtained in (7), and the mixture was incubated at 37 ° C. for 20 minutes. After the reaction, 1 μL of 10 × Loading Dye was mixed and electrophoresed on a 2% agarose gel for 17 minutes at 135 V, and a band was confirmed on an illuminator. The results are shown in FIG. In FIG. 4, lane 1 shows a sample that did not react with T7 endonuclease 1 as a control group, lane 2 shows a sample that reacted with T7 endonuclease 1, and lane M shows a marker.

From FIG. 4, in the HeLa cells (lane 2) into which the SpCas9-gRNA complex and the cytoplasm were introduced, a band cleaved by T7 endonuclease was generated, and the SpCas9-gRNA complex generated a band in the nucleus of the HeLa cell. It was confirmed that the mutation was introduced into the genome of.

[Test Example 3] T7E1 assay using HeLa cells introduced with SaCas9-gRNA complex and cytoplasm (1) Preparation of cytoplasm of L5178Y cells Using the same method as in Test Example 1 (1), cytoplasm of L5178Y cells Was prepared.

(2) Preparation of mixed solution of Cas9-gRNA complex and cytoplasm Next, the complex of HA-NLS-SaCas9 and sgTET2 obtained in "1. Preparation of Cas9-gRNA complex" (hereinafter, "SaCas9-"). The cytoplasm of the L5178Y cells obtained in (1) was mixed so as to have a total of 100 μL, and stored at 32 ° C. until use. The composition of the mixed solution of the SaCas9-sgTET2 complex and the cytoplasm is the same as in Table 2 above. In Table 2, the content of SaCas9 is in 5 μg / 100 μL described as the content of dCas9.
In addition, as a control group, a complex of HA-NLS-SaCas9 and sgTET1 obtained in "1. Preparation of Cas9-gRNA complex" (hereinafter, may be referred to as "SaCas9-sgTET1 complex"). , A mixed solution of L5178Y cells obtained in (1) with the cytoplasm was also prepared.

(3) Perforation of HeLa cells Using the same method as in (3) of Test Example 1, pores were formed in at least a part of HeLa cells (adherent cells). In addition, HeLa cells were peeled off with trypsin and used as floating cells to form pores in at least a part by the same method as in (3) of Test Example 1.

(4) Introduction of SaCas9-sgTET1 complex or SaCas9-sgTET2 complex and cytoplasmic mixture into HeLa cells Instead of dCas9-gRNA complex and cytoplasmic mixture, SaCas9-sgTET1 complex or SaCas9-sgTET2 HeLa was used in the same manner as in Test Example 1 (4) except that a mixed solution of the complex and the cytoplasm was used, and HeLa cells (adherent cells) and HeLa cells (floating cells) were used. A mixed solution of SaCas9-sgTET1 complex or SaCas9-sgTET2 complex and cytoplasm was introduced into cells (adherent cells) and HeLa cells (floating cells). As a control group, only SaCas9 was introduced into HeLa cells.

(5) Re-encapsulation of HeLa cells HeLa cells (adherent cells) and HeLa cells (floating cells) were re-encapsulated using the same method as in (5) of Test Example 1. Then, 2 mL of D-MEM (containing FCS) was added, and the cells were cultured at 37 ° C. and 5% carbon dioxide for 24 hours.

(6) Genome extraction from reencapsulated HeLa cells A genome solution was prepared using the same method as in (6) of Test Example 2.

(7) PCR amplification of the TET2 region Next, using the genomic solution obtained in (6), nested PCR was performed so as to produce a product of about 1 kbp containing the Cas9 target sequence of the TET1 region or the TET2 region. The primers used in the first PCR and the second PCT are shown in Table 4 below.

The number of cycles was 20 for the first PCR and 35 for the second PCR. Further, as a PCR template, 10 ng of the genomic DNA prepared in (6) was used in the first PCR, and 1 μL of a 10-fold diluted solution of the first PCR product was used in the second PCR. After PCR amplification, the PCR product was lysed in 20 μL of _{ddH 2} O using the Wizard SV Gel and PCR Clean-UP System (manufactured by Promega). The PCR product was then _{diluted with ddH 2} O to 2 μg / 20 μL and heat denatured and reannealed using a thermal cycler (95 ° C for 2 minutes / 85 ° C → 35 ° C for 10 minutes). Cooling / 16 ° C).

(8) T7E1 Assay A T7E1 assay was performed using the same method as in (8) of Test Example 2. The results are shown in FIG. In FIG. 5, intact means a sample in which only SaCas9 is introduced into HeLa cells as a control group, adherent reseal means a sample using reseal HeLa cells which are adherent cells, and floating reseal means a sample using floating cells. It means a sample using Reseal HeLa cells, TET1 means a sample using sgTET1, and TET2 means a sample using sgTET2.

From FIG. 5, no mutation was observed in the sample using the SaCas9-sgTET1 complex, but the introduction of mutation was observed in the sample using the SaCas9-sgTET2 complex.
In addition, there was no difference in mutagenesis efficiency (3.5% and 2.6%) between adherent cells and floating cells.

[Test Example 4] Light-induced split-type SpCas9 (paCas9) and introduction of cytoplasm into cells (1) Preparation of cytoplasm of L5178Y cells Using the same method as in Test Example 1 (1), L5178Y cells The cytoplasm was prepared.

(2) Preparation of mixed solution of Cas9-gRNA complex and cytoplasm Next, PaCas9-N (nMag-High1-SpCas9 (N) -NLS) or PaCas9 obtained in "1. Preparation of Cas9-gRNA complex". -C (HisTag-NLS-SpCas9 (C) -pMag) and sgAAVS1 complex (hereinafter referred to as "nMag-SpCas9 (N) -sgAAVS1 complex" or "pMag-SpCas9 (C) -sgAAVS1 complex". In some cases) and the cytoplasm of the L5178Y cells obtained in (1) were mixed to a total of 100 μL and stored at 32 ° C. until use.
Note that FIG. 6A is a schematic diagram showing the configuration of PaCas9-N (nMag-High1-SpCas9 (N) -NLS) and PaCas9-C (HisTag-NLS-SpCas9 (C) -pMag). Further, SpCas9 (N) and SpCas9 (C) used in Test Example 4 are the N-terminal side and the C-terminal side of the inactive dCas.
The composition of the nMag-SpCas9 (N) -sgAAVS1 complex or the mixed solution of the pMag-SpCas9 (C) -sgAAVS1 complex and the cytoplasm is the same as in Table 2 above. In Table 2, the content of PaCas9-N or PaCas9-C is in 5 μg / 100 μL described as the content of dCas9.

(3) Perforation of HeLa cells Using the same method as in (3) of Test Example 1, pores were formed in at least a part of HeLa cells.

(4) Introduction of a mixed solution of nMag-SpCas9 (N) -sgAAVS1 complex or pMag-SpCas9 (C) -sgAAVS1 complex and cytoplasm into HeLa cells Instead of a mixed solution of dCas9-gRNA complex and cytoplasm. HeLa using the same method as in Test Example 1 (4) except that a mixed solution of nMag-SpCas9 (N) -sgAAVS1 complex or pMag-SpCas9 (C) -sgAAVS1 complex and cytoplasm was used. A mixed solution of nMag-SpCas9 (N) -sgAAVS1 complex or pMag-SpCas9 (C) -sgAAVS1 complex and cytoplasm was introduced into cells.

(5) Re-encapsulation of HeLa cells HeLa cells were re-encapsulated using the same method as in (5) of Test Example 1.

(6) Fluorescent Staining of HeLa Cells Next, 2 mL of D-MEM (containing FCS) containing PI (Propidium Iodide) was added, and the cells were cultured under the conditions of 37 ° C. and 5% carbon dioxide. Each cell _____ hours after culturing was immobilized with 4% paraformaldehyde. Next, the immobilized HeLa cells were subjected to fluorescent immunostaining using an anti-His tag antibody, and observed using a fluorescence microscope. The results are shown in FIG. 6B. In FIG. 6B, "nMag-dCas9 / sgRNA" means HeLa cells into which the nMag-SpCas9 (N) -sgAAVS1 complex and the cytoplasm have been introduced, and "pMag-dCas9 / sgRNA" means pMag-SpCas9 (C). ) -SgAAVS1 complex and cytoplasm are introduced into HeLa cells, and "without Cas9" means HeLa cells into which nothing has been introduced as a control group. In each group of FIG. 6B, the left side is an image in which nMag-SpCas9 (N) or pMag-SpCas9 (C) is detected using an anti-His tag antibody, and the right side is an image in which the nucleus is stained with PI. is there.

From FIG. 6B, signals were detected in the cell nucleus of both HeLa cells into which the nMag-SpCas9 (N) -sgAAVS1 complex and the cytoplasm were introduced and HeLa cells into which the pMag-SpCas9 (C) -sgAAVS1 complex and the cytoplasm were introduced. Was done.
From this, it was confirmed that the light-inducing system Cas9 can be introduced into the cell nucleus by using the introduction method of the present invention.

[Test Example 5] T7E1 assay using split-type SpCas9 (rpCas9) which is a rapamycin-induced type and HeLa cells into which cytoplasm has been introduced (1) Preparation of cytoplasm of L5178Y cells Using the same method as in Test Example 1 (1). The cytoplasm of L5178Y cells was prepared.

(2) Preparation of mixed solution of Cas9-gRNA complex and cytoplasm Next, rpCas9-N (HisTag-SpCas9 (N) -FRB) and rpCas9-C obtained in "1. Preparation of Cas9-gRNA complex" (Complex of SpCas9 (C) -FKBP-Histag) and sgAAVS1 (hereinafter, may be referred to as "rpCas9 (N) -rpCas9 (C) -gRNA complex") and obtained in (1). The cytoplasm of L5178Y cells was mixed to a total of 100 μL and stored at 32 ° C. until use.
Note that FIG. 7A is a schematic diagram showing the configurations of rpCas9-N (HisTag-SpCas9 (N) -FRB) and rpCas9-C (SpCas9 (C) -FKBP-Histag).
The composition of the mixed solution of the rpCas9 (N) -rpCas9 (C) -gRNA complex and the cytoplasm is the same as in Table 2 above. In Table 2, the total content of rpCas9-N and rpCas9-C is in 5 μg / 100 μL described as the content of dCas9.

(3) Perforation of HeLa cells Using the same method as in (3) of Test Example 1, pores were formed in at least a part of HeLa cells.

(4) Introduction of a mixed solution of rpCas9 (N) -rpCas9 (C) -gRNA complex and cytoplasm into HeLa cells Instead of a mixed solution of dCas9-gRNA complex and cytoplasm, rpCas9 (N) -rpCas9 ( C) Using the same method as in Test Example 1 (4) except that a mixed solution of the -gRNA complex and the cytoplasm was used, the rpCas9 (N) -rpCas9 (C) -gRNA complex was added to the HeLa cells. A mixed solution with cytoplasm was introduced.

(5) Re-encapsulation of HeLa cells HeLa cells were re-encapsulated using the same method as in (5) of Test Example 1. Then, 2 mL of D-MEM (containing FCS) containing 200 nM rapamycin was added, and the cells were cultured at 37 ° C. and 5% carbon dioxide for 18 hours.

(6) Genome Extraction from Re-Encapsulated HeLa Cells Next, a genome solution was prepared using the same method as in Test Example 2 except that the re-encapsulated HeLa cells after culturing for 18 hours were used.

(7) PCR amplification of the AAVS1 region Then, using the genomic solution obtained in (6), using the same method as in (7) of Test Example 2, about 1 kbp containing the Cas9 target sequence of the AAVS1 region. Nested PCR was performed to produce the product.

(8) T7E1 Assay Next, the T7E1 assay was performed using the same method as in (7) of Test Example 2. The results are shown in FIG. 7B. In FIG. 7B, "T7E1 (+)" means a sample that has undergone the T7E1 assay, and "T7E1 (-)" means a sample that has not undergone the T7E1 assay. In addition, "Inducible Cas9 sgRNA + rapamycin" means a sample in which rapamycin was added and cultured to a reseal HeLa cell into which an rpCas9 (N) -rpCas9 (C) -gRNA complex and a cytoplasm were introduced, and "Inducible Cas9 sgRNA""2 + rapamycin" introduces rpCas9 (N) -rpCas9 (C) -gRNA complex and cytoplasm in which the amount of gRNA added is twice as much as that of "Inducible Cas9 sgRNA + rapamycin" in the Cas9-gRNA complex formation step. It means a sample obtained by adding rapamycin to the obtained RISEAL HeLa cells and culturing it. In addition, "Industrial Cas9-rapamycin" means a reseal HeLa cell into which only rpCas9 (N) -rpCas9 (C) has been introduced, and is a sample cultured without addition of rapamycin. What is "Negative control (without Cas9)"? Means a sample cultured without rapamycin added without introducing.

From FIG. 7B, in the sample cultured without addition of rapamycin (including the negative control), no cleavage occurred and no band of the cleaved DNA was observed. On the other hand, in the sample cultured with the addition of rapamycin, a band of cleaved DNA was detected, and the amount of cleaved DNA increased depending on the amount of sgRNA added.

According to the method for producing a Cas9-gRNA complex of the present invention, a Cas9-gRNA complex that is stably solubilized in vitro can be obtained. Further, according to the method for introducing the Cas9-gRNA complex into the cell nucleus of the present invention, the Cas9-gRNA complex is introduced in a state where the nuclear niche of genome editing is optimized without introducing the viral vector into the cell. It can be introduced in the cell nucleus. In addition, according to the method for modifying a target gene in a cell of the present invention, off-targeting is reduced, the nuclear niche of genome editing is optimized without the need to introduce a viral vector into the cell, and in a large amount of cells. Multiple loci can be edited at the same time.

1 ... Cas9, 2 ... gRNA, 10 ... Cas9-gRNA complex, 11 ... cell B, 11a ... cell B cytoplasm, 12 ... cell A, 12a ... semi-intact cell A, 12b ... Cas9-gRNA complex and cell B Semi-intact cell A with cytoplasm introduced, 12c ... Reseal cell A

Claims (6)

A Cas9-gRNA complex forming step of mixing Cas9 (Crustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated 9) and guide RNA (gRNA) at 20 ° C. or higher to form a Cas9-gRNA complex .
A perforation step in which a perforating substance acts on the plasma membrane of a cell to form a pore in at least a part of the plasma membrane.
A first mixing step of mixing the Cas9-gRNA complex with a cytoplasm collected from cells derived from the same or different organisms as cells having pores formed at least partially on the plasma membrane.
An introduction step of introducing the Cas9-gRNA complex and the cytoplasm into cells having pores formed in at least a part of the plasma membrane.
A solution containing calcium ions is added to cells having pores formed in at least a part of the plasma membrane into which the Cas9-gRNA complex has been introduced, and the pores formed in at least a part of the plasma membrane are re-encapsulated. With an encapsulation process ,
The gRNA is seen containing a PAM (Proto-spacer Adjacent Motif) the nucleotide sequence from one base upstream of the sequence to the upstream 20 nucleotides or more 24 bases or less comprising a nucleotide sequence complementary polynucleotide in the target gene,
Cas9 is a method for introducing a Cas9-gRNA complex into the cell nucleus, in which a nuclear localization signal (NLS) peptide is bound to the N-terminal or C-terminal . The method for introducing a Cas9-gRNA complex into a cell nucleus according to claim 1, wherein in the Cas9-gRNA complex forming step, the Cas9 and the gRNA are mixed at 30 ° C. or higher and 65 ° C. or lower. The Cas9 is divided into two parts, an N-terminal side Cas9 (Cas9-N) and a C-terminal side Cas9 (Cas9-C).
The Cas9-N and the Cas9-C have proteins that form dimers photo-dependently or chemical-dependently bound to the N-terminal or C-terminal, respectively.
The method for introducing Cas9-gRNA complex into the cell nucleus according to claim 1 or 2 , wherein the RNA-induced DNA endonuclease activity is restored in the presence of light or a chemical substance. The method for introducing a Cas9-gRNA complex into a cell nucleus according to any one of claims 1 to 3 , wherein two or more types of the gRNA are mixed in the Cas9-gRNA complex forming step. Further, before the introduction step and after the first mixing step , the Cas9-gRNA complex and the cytoplasm are combined with an inhibitor of an inhibitor of homologous recombination or an activator of homologous recombination. The method for introducing a Cas9-gRNA complex into a cell nucleus according to any one of claims 1 to 4, further comprising a second mixing step of mixing the Cas9-gRNA complex. A cell in which the Cas9-gRNA complex is introduced into the cell nucleus and has a resealed target gene by using the method for introducing the Cas9-gRNA complex into the cell nucleus according to any one of claims 1 to 5. In the cleavage step, Cas9 cleaves the target gene at a cleavage site located upstream or downstream of a predetermined base of the PAM sequence.
In the region determined by the complementary binding of the target gene and the GRNA, comprising a modified step of obtaining a cell having the target gene is modified, the modified method of a target gene in a cell.

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