WO2017131150A1 - Method for producing cas9-grna complex, method for introducing cas9-grna complex into cell nucleus, and method for modifying target gene in cell - Google Patents

Abstract

This method for producing a Cas9-gRNA complex is provided with a step for mixing Cas9 and gRNA at 20°C or higher to form a Cas9-gRNA complex, the gRNA including a polynucleotide that comprises a base sequence complementary to a base sequence from 1 base upstream to 20-24 bases upstream of the PAM sequence in the target gene. This method for introducing a Cas9-gRNA complex into a cell nucleus is provided with: a step for causing a streptococcal enzyme-sensitive toxin streptolysin O to act on the plasma membrane of a cell to form pores in at least part of the plasma membrane; a step for introducing a Cas9-gRNA complex obtained by the above method for producing a Cas9-gRNA complex into the cell in which pores have been formed in at least part of the plasma membrane; and a step for adding a solution that includes calcium ions to the cell in which pores have been formed in at least part of the plasma membrane and into which the Cas9-gRNA complex has been introduced, and resealing the pores formed in at least part of the plasma membrane.

Description

Method for producing Cas9-gRNA complex, method for introducing Cas9-gRNA complex into cell nucleus, and method for modifying target gene in cell

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.
This application claims priority based on US Patent No. 62 / 288,449, 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 competition for new genome editing technology is intensifying. The features of the CRISPR-Cas9 system are that the guide RNA determines the target site, and that genome editing can be performed quickly, simply, and inexpensively in just the time required for guide RNA design, and that multiple guide RNAs can act. Thus, it is possible to destroy a plurality of genes at the same time, and to add various protein engineering modifications to Cas9 so that it can be used for transcriptional control and induction of epigenetic dynamic changes. Recently, as a method for visualizing a specific gene locus, attention has also been paid as a new visualization method that replaces the conventional FISH method (see, for example, Non-Patent Document 1).

Chen, Baohui, et al., “19.Dynamic19Imaging of Genomic Loci in Living Human Cells by an Optimized CRISPR / Cas System”, Cell, vol.155, p1479-1491, 2013.

However, the application of the CRISPR-Cas9 system as a genome editing technique for medical purposes has the following problems.
(1) Problem of off-target: CRISPR has a problem because it is known that non-specific DNA cleavage (off-target cleavage) exists in addition to specific DNA cleavage (on-target cleavage). Basically, the site on which Cas9 cleaves depends on the protospacer sequence of the guide RNA (gRNA). However, even if this sequence and the target DNA strand do not completely match, cleavage by Cas9 occurs. It 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. However, there is a need for a more effective solution that not only eliminates the cleavage of DNA that does not perfectly match gRNA, but also allows for temporal and spatial functional control of the CRISPR-Cas9 system.

(2) Problem of genome damage: The current CRISPR-Cas9 system is mainly implemented using an expression system using a viral vector such as a retrovirus or a lentivirus. By using expression systems utilizing these viral vectors, the damage of the target cell genome due to the constant expression of Cas9 is a major problem in genome editing for human gene therapy.
(3) Problem of introducing Cas9 into the nucleus (intracellular): In the current CRISPR-Cas9 system, when not introduced into the cell using the above-described viral vector expression system, Cas9 and gRNA expressed in advance are introduced. There is a need. At this time, Cas9 is highly hydrophobic, and Cas9 expressed in vitro tends to form an aggregate. In addition, since Cas9 has a large molecular weight, it is difficult to permeate the cell membrane by electroporation or the like. In particular, in the introduction using the electroporation method, damage to cells due to electric shocks in the cell membrane is a problem. In addition, the microinjection method can be applied only to cells that are somewhat large in size, and further, it is necessary to introduce the cells one by one, resulting in poor work efficiency.
(4) Problem that genome editing of multiple genes is difficult: 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, since the CRISPR-Cas9 system based on the current expression system relies on the expression system for both Cas9 and gRNA, a combination of Cas9 and gRNA having different functions in the cell is created by co-translation. Difficult to function. 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 of different colors, in Co-translation, a specific gRNA and a fluorescent label are labeled in the cell. Since complex formation with Cas9 occurs randomly, it is difficult to visualize specific genomic sites with specific fluorescence.

(5) Problem of optimal niche for genome editing: When considering the medical application of genome editing, the objective is to make efficient homologous recombination and epigenetic modification targeting specific genes. In order to improve the efficiency of homologous recombination and epigenetic modification, activation of various induced proteins in the nucleus (in the cytoplasm) and inactivation or removal of inhibitors are indispensable. In other words, it is necessary to optimize the environment (niche) for homologous recombination and epigenetic modification.
(6) Problems when handling a large amount of cells: When gene correction cells using genome editing are prepared and gene therapy is performed, it is necessary to simultaneously apply a large number of cells to genome editing. In an expression system using a viral vector, since it is difficult to control the expression level, it is difficult to uniformly process a large amount of cell genomes at once.

The present invention has been made in view of the above circumstances, and provides a method for producing a Cas9-gRNA complex stably solubilized in vitro. Also provided is a method of introducing a Cas9-gRNA complex into a cell nucleus in which a nuclear niche for genome editing is optimized without introducing a viral vector into the cell. Furthermore, off-target is reduced, viral vectors are not introduced into cells, the nuclear niche for genome editing is optimized, and modification of target genes in cells that can simultaneously edit multiple loci in large numbers of cells Provide a method.

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

That is, the present invention includes the following aspects.
[1] A Cas9-gRNA complex forming step of forming a Cas9-gRNA complex by mixing Cas9 (Clustered Regularly Interspaced Short Palindromatic Repeats (CRISPR) -associated 9) and a guide RNA (gRNA) at 20 ° C. or higher. And the gRNA includes a polynucleotide having a base sequence complementary to a base sequence from 1 base upstream to 20 bases to 24 bases upstream of a PAM (Proto-spacer Adjacent Motif) sequence in a target gene. A method for producing a Cas9-gRNA complex.
[2] The method for producing a Cas9-gRNA complex according to [1], wherein, in the Cas9-gRNA complex formation step, the Cas9 and the gRNA are mixed at 30 ° C. or higher and 65 ° C. or lower.
[3] The method for producing a Cas9-gRNA complex according to [1] or [2], wherein the Cas9 is bound to a nuclear localization signal (NLS) peptide at the N-terminus or C-terminus. .
[4] 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 each N-terminal or C9- A protein that forms a dimer in a light-dependent or chemical substance-dependent manner is bound to the terminal, and Cas9 restores RNA-induced DNA endonuclease activity in the presence of light or a chemical substance [1] to [ 3] The method for producing a Cas9-gRNA complex according to any one of 3).
[5] The method for producing a Cas9-gRNA complex 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.
[6] A perforation step in which a perforating substance is allowed to act on the plasma membrane of the cell to form a hole in at least a part of the plasma membrane, and a cell in which a hole is formed in at least a part of the plasma membrane is [1 ] An introduction step of introducing the Cas9-gRNA complex obtained by the method for producing a Cas9-gRNA complex according to any one of [5] to [5], and a plasma membrane on which the Cas9-gRNA complex has been introduced. A re-encapsulation step of adding a solution containing calcium ions to cells having pores formed in at least a part thereof, and re-encapsulating the pores formed in at least a part on the plasma membrane. A method for introducing a gRNA complex into a cell nucleus.
[7] Further, before the introduction step, the Cas9-gRNA complex is mixed with the cytoplasm collected from cells derived from the same species as the cells having pores formed on at least a part of the plasma membrane. The method for introducing a Cas9-gRNA complex according to [6] into a cell nucleus, comprising a first mixing step.
[8] The method further comprises a second mixing step of mixing the Cas9-gRNA complex with the inhibitor of homologous recombination inhibitor or the activator of homologous recombination before the introducing step. [6] A method for introducing the Cas9-gRNA complex according to [7] into a cell nucleus.
[9] The Cas9-gRNA complex was introduced into the cell nucleus obtained by the method for introducing the Cas9-gRNA complex according to any one of [6] to [8] into the cell nucleus and re-encapsulated. In a cell having a target gene, a region where the 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 of the gRNA and the target gene And a modification step of obtaining a cell having the modified target gene. A method for modifying a target gene in a cell, comprising:

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

FIG. 2 is a schematic process diagram showing one embodiment of a method for producing a Cas9-gRNA complex of the present invention. FIG. 3 is a schematic process diagram showing one embodiment of a method for introducing a Cas9-gRNA complex of the present invention into a cell nucleus. It is the image which observed the HeLa cell which introduce | transduced cytoplasm of dCas9 only in test example 1, only dCas9-gRNA complex, or dCas9-gRNA complex, and L5178Y cell using the fluorescence microscope. FIG. 7 is an image showing the results of a T7E1 assay using HeLa cell genomic DNA into which a complex of NLS-SpCas9-NLS and sgAAVS1 and the cytoplasm of L5178Y cells were introduced in Test Example 2. FIG. Image showing results of T7E1 assay using the genomic DNA of HeLa cells into which HA-NLS-SaCas9 and sgTET1 complex or HA-NLS-SaCas9 and sgTET2 complex and L5178Y cell cytoplasm were introduced in Test Example 3 It is. FIG. 10 is a schematic diagram showing a configuration of an N-terminal side and a C-terminal side of split-type SpCas9 (paCas9) that is a light induction type in Test Example 4. N-terminal side of split-type SpCas9 (paCas9) and sgAAVS1 complex that are light-induced in Test Example 4, or C-terminal side of split-type SpCas9 (paCas9) that is light-induced and sgAAVS1 complex and L5178Y cells It is the image which observed the HeLa cell which introduce | transduced cytoplasm using the fluorescence microscope. FIG. 6 is a schematic diagram showing the configuration of the N-terminal side and the C-terminal side of split-type SpCas9 (rpCas9), which is a rapamycin-derived type in Test Example 5. Results of T7E1 assay using genomic DNA of HeLa cells into which N-terminal side and C-terminal side of split-type SpCas9 (rpCas9), which is rapamycin-derived type, and sgAAVS1 complex and L5178Y cell cytoplasm were introduced in Test Example 5 It is an image which shows.

<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 more to form a Cas9-gRNA complex, wherein the gRNA is a PAM (Proto) in a target gene. A method for producing a Cas9-gRNA complex comprising a polynucleotide having a base sequence complementary to a base sequence from 1 base upstream to 20 bases to 24 bases upstream of the sequence (Spacer Adjust Motif).

According to the method for producing a Cas9-gRNA complex of this embodiment, a Cas9-gRNA complex that is stably solubilized in vitro can be obtained.
The present inventors form an aggregate 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, stable Cas9-gRNA complex It was found that the body was formed and solubilized in the solution, and the present invention was completed.
The method for producing the Cas9-gRNA complex of this 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 one embodiment of a method for producing a Cas9-gRNA complex of the present invention. First, a Cas9-gRNA complex (10) is formed by mixing and incubating Cas9 (1) and gRNA (2) that have been expressed in advance at 20 ° C. or higher.
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, further preferably 30 ° C or higher and 37 ° C or lower, 32 ° C. or more and 37 ° C. or less is particularly preferable.
Usually, when a protein is handled, a low temperature such as 4 ° C. is generally used. On the other hand, in this embodiment, when the temperature is in the above range, the aggregation of Cas9 can be prevented, and a stable Cas9-gRNA complex can be formed and solubilized in the solution.

Examples of the medium used when mixing Cas9 and gRNA include an aqueous solvent, such as water, physiological saline, phosphate buffered saline (PBS), Tris buffered saline [PBS] Examples include, but are not limited to, 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. .
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 depending on the type of cells used in <Method of introducing Cas9-gRNA complex into cell nucleus> described later.
Further, the medium may include an ATP reproduction 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 family that constitutes the adaptive immune system that provides acquired resistance to invading foreign nucleic acids in bacteria and archaea, and recognizes the PAM sequence in the invading DNA. Thus, it is an endonuclease that cleaves double-stranded DNA upstream or downstream thereof. In this specification, Cas9 means a substance that forms a complex with gRNA and has DNA cleavage activity.

In this specification, 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, Csx17, Csx3, Csx17, Csx , Csx15, Csf1, Csf2, Csf3, Csf4, homologs thereof, or modified ones thereof. Cas9 used in this embodiment may be Cas9, a homologue thereof, or a modified one thereof.

Examples of the bacterial species from which Cas9 is derived include Streptococcus pyogenes (S. pyogenes), Staphylococcus aureus, Franciscilla novocida, Streptococcus thermophilus thermophilus, S. pyogenes,・ Dasson Bey, Streptomyces pristina espiralis, Streptomyces viridochromogenes, Streptosporangium roseum, Alicyclobacillus acidocardarius, Bacillus pseudomycedes Exigobacteriu m. Aeruginosa (Microcystis aeruginosa), Synecococcus spp., Acethalobium arabaticum, Ammonifex degensii, Calgicellulosylptor bedisdulcidulcide Botulinum, Clostridium difficile, Finegordia magna, Natranaerobius thermophilum, Perotomaculum thermopropionicum, Acidithiobacillus caldas, Acidithiobacillus ferrochidan Binosum, Marinobacter genus, Nitrosococcus halophyllus, Nitrosococcus watsoni, Pseudoalteromonas haloplanktis, Kutedobacter racefifer albeum evestigatum), Anabena variabilis, Nodularia spumigena, Nostock genus, Arsulospira maxima, Arsulospira platensis, Arsulospira spp. Examples include, but are not limited to, Nus, Acariochloris Marina, etc. Among them, the bacterial species from which Cas9 used in the present embodiment is preferably S. pyogenes or S. aureus.

Cas9 is, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, upstream or downstream from the first or last nucleotide of the PAM sequence in the target gene. Directs cleavage of one or both strands within 50, 100, 200, 500 or more base pairs. The number of bps 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.

Further, Cas9 used in this embodiment may be a variant lacking the ability to cleave one or both strands of the target gene containing the target sequence. For example, S.M. The substitution of aspartate to alanine (D10A) in the RuvC I 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 a 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 cleavage activity. In this case, a chimeric enzyme fused with an enzyme having other DNA cleavage activity is preferable. The D10A mutation may be combined with one or more of the H840A, N854A or N863A mutations to produce Cas9 that substantially lacks all DNA cleavage activity. All DNA-cleaving activities when the activity of the mutated Cas9 DNA is less than about 25%, 10%, 5%, 1%, 0.1% or 0.01% relative to its unmutated form. Considered substantially lacking.

In this embodiment, the base sequence encoding Cas9 may be codon-optimized for expression in specific cells such as eukaryotic cells. Examples of 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 of the native sequence with a codon that is used more frequently or most frequently in the gene of the introduced species while maintaining the native amino acid sequence. , Refers to the process of modifying a nucleic acid sequence for enhanced expression in an introduced species. Different species exhibit specific biases for specific codons of specific amino acids. Codon bias (difference in codon usage between organisms) often correlates with the translation efficiency of mRNA, which is thought to depend inter alia on the properties of the codon being translated and the availability of a particular tRNA. Yes. The predominance of selected tRNAs in cells is generally a reflection of the most frequently used codons in peptide synthesis. Thus, genes can be individualized for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the “Codon Usage Database” published at www.kazusa.or.jp/codon/ (visited on 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, at Gene Forge (Aptagen; Jacobus, PA). In this embodiment, the one or more codons in the sequence encoding Cas9 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-terminus or C-terminus.

The NLS includes, for example, a polypeptide consisting of the amino acid sequence PKKKKRKV (SEQ ID NO: 1), an NLS of the SV40 virus large T antigen having the amino acid sequence consisting of the SEQ ID NO: 1; an NLS derived from nucleoplasmin (for example, the sequence KRPAATKKAGQAKKKKK (sequence Nucleoplasmin bipartite NLS having number 2); c-myc NLS having amino acid sequence PAAKRVKLD (SEQ ID NO: 3) or RQRRNELKRSP (SEQ ID NO: 4); Sequence of the IBB domain from RRMRIZFKNKKGKDTAELRRRRVEVSVVERRKAKKDEQILKRR V (SEQ ID NO: 6); myoma T protein sequences VSRKRPRP (SEQ ID NO: 7) and PPKKARED (SEQ ID NO: 8); human p53 sequence PQPKKKKPL (SEQ ID NO: 9); mouse c-abl IV sequence SALIKKKKKKMAP (SEQ ID NO: 10) Sequences of influenza virus NS1 DRLRR (SEQ ID NO: 11) and PKQKKR (SEQ ID NO: 12); sequence of hepatitis virus delta antigen RKLKKKIKKL (SEQ ID NO: 13); mouse Mx1 protein sequence REKKKKLKRR (SEQ ID NO: 14); human poly (ADP-ribose) ) Polymerase sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 15); Steroid hormone receptor (human) glucocorticoid sequence RKCLQAGMNLEARKTKK (SEQ ID NO: 16) But it is not limited to these.

In the present 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 analogues or modified derivatives of the corresponding naturally occurring amino acids.

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 each N A protein that forms a dimer in a light-dependent or chemical-dependent manner is bound to the terminal or the C-terminal, and Cas9 is a substance that restores RNA-induced DNA endonuclease activity in the presence of light or a chemical substance. There may be.
By using the bisected Cas9, the activity of Cas9 in the cell can be controlled, and the off-target can be further reduced.
Specifically, the polypeptide (paCas9) consisting of the amino acid sequence represented by SEQ ID NOs: 17 and 18 which is a light-induced type and the rapamycin-derived type represented by SEQ ID NOs: 19 and 20 shown in the Examples described later. And a polypeptide comprising an amino acid sequence (rpCas9).

In this specification, “a protein that forms a dimer in a light-dependent manner” (photoswitch protein) was developed by a research group such as Associate Professor Moritoshi Sato of the University of Tokyo Graduate School of Arts and Sciences ( Nat. Commun. 6, 6256 (2015). Doi: 10.1038 / ncomms 7256), a pair of proteins engineered multifaceted to the small photoreceptor Vivid possessed by Neurospora crassa Means. The photoswitch protein pair exists as a monomer in the dark and forms a heterodimer when it receives blue light. Various photoactivatable tools can be designed and developed using the conversion of monomer and dimer by light.

Further, in this specification, “a protein that forms a dimer in a chemical substance-dependent manner” is a protein used in a chemically induced dimerization system, and FK1012 was changed in 1993. A model for dimer formation was first proposed (Reference 2: Tamsir, A., et al., “Controlling signal transduction with synthetic ligands”, Science, vol.262, p1019-1024, 1993). Thereafter, a method using FRB, which is a binding protein for a complex of FK506-binding protein (FKBP) and FK506-binding protein / rapamycin, using rapamycin, a macrolide antibiotic, as a dimer forming substance (reference) Reference 3: Bayle, J. H., 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, J. L. et al., “Conditional glycosylation in eukaryotic cells using a biocompatible chemical inducer of dimerization”, J. Am. Chem. Soc., Vol.130, p13186- 13187, 2008 .; Reference 5: Liang, F. S., et al, “Engineering the ABA plant stress pathway for regulation of induced proximit y ”, Sci. Signal., vol.4, rs2, 2011 .; Reference 6: Liberles, S. D., 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 medium composition, culture temperature, time, addition of inducer, etc. can be determined by those skilled in the art according to known methods so that transformants grow and Cas9 is efficiently produced. 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. Then, Cas9 is obtained by purifying Cas9 expressed by the host by an appropriate method.

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

(GRNA)
As used herein, “gRNA” refers to 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. Mimics the hairpin structure of a tracrRNA-crRNA chimera in which the crRNA is fused with a partially complementary RNA (trans-activating crRNA: tracrRNA).
The gRNA used in the present embodiment may be a tracrRNA-crRNA chimera synthesized in a state where crRNA and tracrRNA containing a guide sequence are fused. The crRNA and tracrRNA containing a guide sequence are separately prepared and introduced. It may be one that has been annealed before to form a tracrRNA-crRNA chimera.

The gRNA in the present embodiment is a polynuclear sequence consisting of a base sequence complementary to a base sequence preferably from 20 to 24 bases, more preferably from 22 to 24 bases from one base upstream of the PAM sequence in the target gene. Nucleotides are included in the 5 ′ terminal region. Furthermore, it may contain one or more polynucleotides comprising a base sequence that is non-complementary to the target gene, arranged so as to be symmetrically complementary with one point as an axis, and that can have a hairpin structure. .

In the present specification, the “PAM sequence” is a sequence that exists in the target gene and can be recognized by Cas9. The length and base sequence of the PAM sequence vary depending on the bacterial species from which Cas9 is derived. For example, S.M. In pyogenes, 3 bases “NGG” (N represents an arbitrary base) are recognized.
S. thermophilus has two Cas9s, and recognizes 5 to 6 bases of “NGGNG” or “NNAGAA” (N represents an arbitrary base), respectively, as a PAM sequence. Since the recognized PAM sequence is short and it is difficult to limit the target gene that can be edited, the PAM sequence is preferably “NGG”.

As a method for producing gRNA, it can be produced using a known method (for example, in vitro transcription method or the like). Specifically, for example, first, a DNA double strand arranged so as to be 5′-T7 promoter sequence-gRNA (hereinafter sometimes referred to as “sequence 1”) is designed. Next, two long primers that anneal at the center of sequence 1 are designed so that the PCR product is sequence 1 above. Next, a DNA double strand consisting of sequence 1 is obtained as a PCR product by PCR amplification. Then, T7 RNA polymerase, ATP, UTP, GTP, CTP, buffer, RNase inhibitor, and pyrophosphate-degrading enzyme are mixed with the DNA obtained as a PCR product, and incubated at 37 ° C. for 2 hours to 4 hours. Subsequently, 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. Two or more types of Cas9-gRNA complexes can be obtained by mixing two or more types of gRNA. By using these two or more types of Cas9-gRNA complexes in <Intracellular target gene modification method> described later, a plurality of loci can be edited simultaneously.

<Method of introducing Cas9-gRNA complex into cell nucleus>
In one embodiment, the present invention comprises a perforating step in which a perforating substance is allowed to act on a plasma membrane of a cell to form a hole in at least a part of the plasma membrane, and a hole is formed in at least a part of the plasma membrane. And introducing a Cas9-gRNA complex obtained by the above-described method for producing Cas9-gRNA complex into a cell, and forming a pore in at least a part of the plasma membrane into which the Cas9-gRNA complex has been introduced. And a re-encapsulation step of re-encapsulating pores formed in at least a part of the plasma membrane by adding a solution containing calcium ions to the formed cells, and providing a method for introducing the Cas9-gRNA complex into the cell nucleus .

According to the introduction method of the present embodiment, the Cas9-gRNA complex can be introduced into the cell nucleus while the viral vector is not introduced into the cell, and the nuclear niche for genome editing is optimized.
The present inventors have so far developed a reversible membrane perforation method using 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 Patent Laid-Open No. 2013-213688), in semi-intact cells, the structure and function of organelles and cytoskeleton, and their relative spatial arrangements are retained intact, and the original cytoplasm flows out. Can be “exchanged” for cytoplasm prepared from other cells and organs. For example, a pathological environment can be constructed in a cell by exchanging the cytoplasm of a semi-intact cell with the cytoplasm obtained from the pathological cell.
The introduction method of this embodiment will be described in detail below with reference to the drawings.

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

(cell)
In the present specification, the “semi-intact cell” means a cell that has been made partially permeable by exfoliating the plasma membrane or the like, and the cytoplasm has flowed out of the cell, or another cytoplasm or other substance from the outside ( It means a cell in which a compound or the like) can be introduced into a cell.
In addition, examples of organisms that are derived from cells to which the introduction method of the present embodiment is applied include prokaryotes, yeasts, animals, plants, insects, and the like. There is no special limitation as said animal, For example, a human, a monkey, a dog, a cat, a rabbit, a pig, a cow, a mouse, a rat etc. are mentioned, It is not limited to these.

Examples of animal-derived cells to which the introduction method of the present embodiment is applied include, for example, germ cells (sperm, ova, etc.), somatic cells constituting the living body, stem cells, progenitor cells, cancer cells separated from living bodies, living bodies Cells that have been isolated from the body and have acquired immortalization and are stably maintained outside the body (cell lines), cells that have been isolated from the living body and have been artificially genetically modified, and cells that have been isolated from the living body and have been artificially exchanged in the nucleus However, it is not limited to these.

Examples of somatic cells constituting the living body include skin, kidney, spleen, adrenal gland, liver, lung, ovary, pancreas, uterus, stomach, colon, small intestine, large intestine, bladder, prostate, testis, thymus, muscle, connective tissue, Examples include, but are not limited to, cells collected from any tissue such as bone, cartilage, vascular tissue, blood, heart, eye, brain, and nerve tissue. More specifically, as somatic cells, for example, fibroblasts, bone marrow cells, immune cells (for example, B lymphocytes, T lymphocytes, neutrophils, macrophages, monocytes, etc.), erythrocytes, platelets, bone cells Bone marrow cells, pericytes, dendritic cells, keratinocytes, adipocytes, mesenchymal cells, epithelial cells, epidermal cells, endothelial cells, vascular endothelial cells, lymphatic endothelial cells, hepatocytes, islet cells (eg, α cells, β cells, δ cells, ε cells, PP cells, etc.), chondrocytes, cumulus cells, glial cells, neurons (neurons), oligodendrocytes, microglia, astrocytes, cardiomyocytes, esophageal cells, muscle cells (For example, smooth muscle cells, skeletal muscle cells, etc.), melanocytes, mononuclear cells, and the like, but are not limited thereto.

Stem cells are cells that have both the ability to replicate themselves and the ability to differentiate into other multiple cell lines. Stem cells include, for example, embryonic stem cells (ES cells), embryonic tumor cells, embryonic germ stem cells, induced pluripotent stem cells (iPS cells), neural stem cells, hematopoietic stem cells, mesenchymal stem cells, hepatic stem cells, pancreatic stem cells , Muscle stem cells, germ stem cells, intestinal stem cells, cancer stem cells, hair follicle stem cells, and the like, but are not limited thereto.

Cancer cells are cells that have been derived from somatic cells and have acquired unlimited proliferative capacity. Examples of cancers from which cancer cells are derived include breast cancer (eg, invasive breast cancer, non-invasive breast cancer, inflammatory breast cancer, etc.), prostate cancer (eg, hormone-dependent prostate). Cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (eg, pancreatic duct cancer, etc.), stomach cancer (eg, papillary adenocarcinoma, mucinous adenocarcinoma, adenosquamous carcinoma, etc.), lung cancer (eg, Non-small cell lung cancer, small cell lung cancer, malignant mesothelioma, etc.), colon cancer (eg, gastrointestinal stromal tumor), rectal cancer (eg, gastrointestinal stromal tumor), colorectal cancer (eg, Familial colorectal cancer, hereditary nonpolyposis colorectal cancer, gastrointestinal stromal tumor, etc.), small intestine cancer (eg, non-Hodgkin lymphoma, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue Cancer, pharyngeal cancer (eg, nasopharyngeal cancer, oropharyngeal cancer, hypopharyngeal cancer), head and neck cancer, saliva Cancer, brain tumor (eg, pineal astrocytoma, ciliary astrocytoma, diffuse astrocytoma, anaplastic astrocytoma), schwannoma, liver cancer (eg, primary liver) Cancer, extrahepatic bile duct cancer, etc.), kidney cancer (eg, renal cell carcinoma, transitional cell carcinoma of the renal pelvis and ureter), gallbladder cancer, bile duct cancer, pancreatic cancer, endometrium Cancer, cervical cancer, ovarian cancer (eg, epithelial ovarian cancer, extragonadal germ cell tumor, ovarian germ cell tumor, ovarian low-grade tumor, etc.), bladder cancer, urethral cancer, skin cancer (Eg, intraocular (eye) melanoma, Merkel cell carcinoma, etc.), hemangioma, malignant lymphoma (eg, reticulosarcoma, lymphosarcoma, Hodgkin's disease, etc.), melanoma (malignant melanoma), thyroid cancer (eg, Medullary thyroid cancer, parathyroid cancer, nasal cavity cancer, sinus cancer, bone tumor (eg, osteosarcoma, Ewing tumor, uterine sarcoma, soft) Tissue sarcoma, etc.), metastatic medulloblastoma, hemangiofibromas, elevated dermal fibrosarcoma, retinal sarcoma, penile cancer, testicular tumor, childhood solid cancer (eg Wilms tumor, childhood kidney tumor, etc.), Kaposi's sarcoma, AIDS Kaposi's sarcoma, maxillary sinus tumor, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, chronic myeloproliferative disease, leukemia (eg, acute myeloid leukemia, acute lymphoblastic leukemia, etc.) But are not limited to these.

A cell line is a cell that has acquired infinite proliferative capacity through artificial manipulation in vitro. Examples of cell lines include HCT116, Huh7, HEK293 (human embryonic kidney cells), HeLa (human cervical cancer cell line), HepG2 (human hepatoma cell line), UT7 / TPO (human leukemia cell line), 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. However, it is not limited to these.

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

As used herein, “cholesterol-dependent cytolytic toxin” refers to a substance that has membrane perforation activity that binds to cholesterol and forms pores in the cell membrane (perforates the cell membrane). A toxin produced by streptococci is known as a cholesterol-dependent cytolytic toxin. 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 a cholesterol-dependent cytolytic toxin is brought into contact with a cell, a pore is formed in the cell membrane, and a desired substance (Cas9-gRNA complex in this embodiment) can be introduced through the formed pore. 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), listerilysin O (LLO), and the like.

Cholesterol-dependent cytolytic toxin binds to cells at low temperatures (usually 4 ° C. or lower) but does not have perforation activity, and only has perforation activity at high temperatures (about 25 to 37 ° C.). By utilizing such temperature sensitivity, the perforated material is bound on the plasma membrane of the cell at a low temperature, and then the excess perforated material is washed away and subsequently heated to a high temperature (about 25 to 37 ° C.). It is possible to selectively perforate only a specific portion in which a perforating substance is bound to a lipid of the plasma membrane.
Further, the existence of an optimum pH is known for the activity of cholesterol-dependent cytolytic toxin. For example, LLO is said to have an optimum pH in the 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 more and 40 ° C. or less, for example, 33 ° C. or more and 38 ° C. or less, for example, 35 ° C. or more and 37 ° C. or less.
The optimum pH for 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, 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, a cholesterol-dependent cytolytic toxin is brought into contact with erythrocytes cultured at the above temperature and pH conditions, and the degree of hemolytic activity (hemolytic unit, HU) of blood cells in which the erythrocyte membrane is destroyed and hemoglobin is eluted. The membrane perforation activity can be measured as a reference, and the pH at which the membrane perforation activity is maximized can be determined as the optimum pH.

The perforated material may be used by adding it to a medium or the like with respect to the cell A (12) cultured in advance.
In this specification, “culture” means breeding or growing cells outside a living body (individual). The period of breeding or growth may be, for example, from 1 minute to 7 days, for example, from 5 minutes to 16 hours, for example, from 10 minutes to 1 hour.
In the present specification, the term “medium” is a concept that generally refers to anything that can cultivate cells. In the medium, the remaining components excluding the perforated substance may be any substance that can culture cells even for a very short period of time, and is not generally referred to as a medium, such as water or a buffer (for example, physiological saline, phosphate buffer). PBS (phosphate buffered saline; PBS), Tris buffered saline (Tris Buffered Saline; TBS), HEPES buffered saline, TB (Transport buffer: 25 mM Hepes, 1.15 mM KOAC, 250 μM MgCl2, pH 7). Etc.). Examples of the components that can be contained in the medium include components contained in ordinary cell culture media, such as nutrient components such as glucose, sodium chloride, vitamins, minerals, and amino acids; growth factors and cell proliferation. Factors, differentiation-inducing factors, antibacterial agents, antifungal agents and the like can be mentioned. More specifically, examples of the medium include, but are not limited to, DMEM, EMEM, RPMI-1640, α-MEM, F-12, F-10, M-199, and the like. What is necessary is just to select the composition of a culture medium suitably according to the kind of cell A to be used.

The content of the perforated substance contained in the medium may be, for example, 0.01 μg / mL to 1 μg / mL, for example, 0.025 μg / mL to 0.6 μg / mL, for example 0.05 μg. / 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 perforated substance is in the above range, the damage to the cells is small and the effect of introducing the substance into the cells can be made uniform.

Further, the pH of the medium containing the perforated substance 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 or less. . The pH is a measured value in the range of 30 ° C to 40 ° C.
When the pH of the medium is within the above range, the cells can be cultured well. Furthermore, the membrane perforation activity of a perforating substance (especially cholesterol-dependent cytolytic toxin) having an optimum pH in the range of 0 or more and less than 6 is preferable from the viewpoint that the membrane perforating activity is exerted gently.

The amount ratio of the perforated substance (particularly cholesterol-dependent cytolytic toxin) contained in the medium and the amount of cells can be determined as appropriate according to the type of cells. For example, it is considered that the greater the cholesterol content in the cell membrane of the cell, the smaller the amount (concentration) of the perforated substance required for membrane perforation. If the cholesterol content in the cell membrane is high, the amount (concentration) of the perforated substance contained in the medium may be adjusted to decrease. If the cholesterol content in the cell membrane is low, the amount of the perforated substance contained in the medium (concentration) ) Should be adjusted in the direction of increasing.

When cultured in a medium containing a perforating substance (especially 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 culture medium may be controlled by placing a culture container including a culture medium containing the perforated substance and the cells A on ice. In this temperature range, the membrane perforation activity of the perforating substance (particularly cholesterol-dependent cytolytic toxin) is suppressed. Therefore, when the medium containing the perforated substance is brought into contact with the cell A, the perforated substance binds to the cell membrane of the cell A, but the effect of perforation is hardly 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 a medium not containing the perforating substance, the cells are cultured in the medium not containing the perforating substance, and the cell membrane of the cell A is perforated.
By exchanging the medium containing the perforated substance with the medium not containing the perforated substance, the perforated substances other than those bound to the cells A are removed from the reaction system in the culture vessel.

When cultured in a medium that does not contain a perforating substance, the culture temperature of cell A may be, for example, 30 ° C. or higher and 40 ° C. or lower, such as 33 ° C. or higher and 38 ° C. or lower, such as 35 ° C. or higher and 37 ° C. or lower. If it is. By culturing in the temperature range, the membrane perforation activity of the perforating substance (particularly cholesterol-dependent cytolytic toxin) is exerted, the cell membrane of the cell A (12) is perforated, and the cell A (where a pore is formed in the cell membrane ( 12a). Since the perforation substance other than the perforation substance bound to the cell A is removed from the medium not containing the perforation substance, it is possible to prevent the perforation substance from entering the cells further from the pores and perforating the organelle in the cytoplasm.

The culture time for culturing the cells A in a medium not containing a perforating substance may be appropriately determined according to the type of the perforating substance and the cell type. For example, about 1 minute or more and 30 minutes or less is mentioned.

Examples of the medium not containing the perforating substance include the same medium as exemplified in the medium containing the perforating substance. The medium not containing the perforating substance and the medium containing the perforating substance may be the same type of medium or different types of medium.

When culturing in a medium not containing a perforating substance, the optimum pH of the medium is, for example, from 6 to 10, for example, from 6.5 to 8, for example, from 7.0 to 7.5. The following is sufficient. The pH is a measured value in the range of 30 ° C to 40 ° C.
When the optimum pH of the medium is within the above range, the cells can be cultured well, and the membrane perforation activity of the perforated substance is preferably exhibited.

When culturing in a medium not containing a perforating substance, the optimum temperature of the medium may be, for example, 30 ° C. or higher and 40 ° C. or lower, such as 33 ° C. or higher and 38 ° C. or lower, such as 35 ° C. or higher and 37 ° C. The following is sufficient.

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

In the introduction step, the Cas9-gRNA complex may be in a state solubilized in a medium or the like. Examples of the medium include the same media as those exemplified in the above [Punching step].

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

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

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

In the mixing step, the temperature may be adjusted to the same temperature as in the subsequent introduction step, for example, 30 ° C. or more and 40 ° C. or less, for example, 30 ° C. or more and 38 ° C. or less, eg 32 ° C. or more and 37 ° C. It should just be below ℃.

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 cell membrane impermeability” means a substance that cannot be dissolved in the lipid bilayer of the cell membrane and cannot permeate the lipid bilayer. According to the introduction method of the present embodiment, even a cell membrane impermeable substance can be introduced into cells through the pores with high efficiency.
In addition, the substance may be a compound or an organic compound. The substance may contain a nucleic acid, and examples include antisense nucleic acid, miRNA, siRNA, shRNA, ribozyme, aptamer and the like. These compounds can be active ingredients of nucleic acid drugs. 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.
In the case where the substance to be introduced together with the Cas9-gRNA complex is a cytoplasm, or a suppressor of a homologous recombination inhibitor or an activator of homologous recombination, the following [first mixing step], [second The mixing step] is exemplified.

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

Examples of the cell-derived organism that is the origin of the cytoplasm and the types of cells include those exemplified in (Cell) of [Punching step] described above. Further, the cell type may be the same as or different from the cell A.
Further, when the cells A and B are the same type of somatic cell, the cell A is a normal cell, and the cell B is a cancer cell, the cell B is introduced by introducing the cytoplasm of the cell B into the cell A. It is possible to visualize and reconstruct life phenomena in a cytoplasm-dependent manner, and to analyze cell traits (phenotype), gene expression, and epigenetic dynamics changes after the subsequent [resealing step] can do.

(Second mixing step)
In the case of introducing an inhibitor of homologous recombination inhibitor or an activator of homologous recombination in addition to the Cas9-gRNA complex, the Cas9-gRNA complex (10) and homologous recombination are introduced before the introducing step. What is necessary is just to mix the inhibitor or activator of this inhibitory substance.

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

In addition, examples of the homologous recombination activator include, but are not limited to, Rad51 and Rad54 which are DNA homologous recombination enzymes.

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

In the present specification, “re-encapsulation (reseal)” means that the opening of the hole formed in the cell membrane in the perforation step is completely or partially closed.
Reencapsulation is believed to occur by removal of the perforated material from the cell membrane by endocytosis and / or exocytosis and is facilitated by the presence of calcium ions. 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) have been introduced has resealed cells. A (12c). The Cas9-gRNA complex introduced into the cell in the introduction step is efficiently retained in the resealed cell A (12c) by re-encapsulation.

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

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

In the re-encapsulation step, the pore may be re-encapsulated without contacting the solution containing the foreign cytoplasm with the cell in which the pore is formed, and the solution containing the foreign cytoplasm is added to the cell in which the pore is formed. The holes may be re-encapsulated by contact.

In this specification, “foreign cytoplasm” means the cytoplasm of cells other than the cells to be introduced that have been perforated in the perforation process. For example, the cytoplasm of the cell A (12a) in which pores are formed in the cell membrane does not correspond to the foreign cytoplasm. In the example shown in FIG. 2, the foreign cytoplasm is a cytoplasm obtained from cells other than the cell A, that is, the cytoplasm of the cell B (11a). The type of foreign cytoplasm may be a cytoplasm of a different type of cell from the introduction target cell, 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 performed as independent steps, but the drilling and the introduction may occur almost simultaneously. In this case, in the introduction method of this embodiment, the Cas9-gRNA complex is exemplified to be added to the medium in the introduction process after the perforation process. However, the Cas9-gRNA complex is added to the medium in the perforation process. May be.

<Method of modifying target gene in cell>
In one embodiment, the present invention provides a method in which a Cas9-gRNA complex is introduced into a cell nucleus obtained by the above-described method for introducing a Cas9-gRNA complex into a cell nucleus and has a re-encapsulated target gene. The target modified in the region determined by the cleavage step in which the Cas9 cleaves the target gene at a cleavage site located upstream or downstream of a predetermined base of the PAM sequence and the complementary binding of the gRNA and the target gene And a modification step for obtaining a cell having a gene, and a method for modifying a target gene in a cell.

According to the method for modifying a target gene in a cell of the present embodiment, off-target is reduced, and the target gene can be modified easily without introducing a viral vector into the cell. Moreover, the nuclear niche for genome editing is optimized, and multiple loci in a large number of cells can be edited simultaneously.
Details of the method for modifying a target gene in a cell according to this embodiment will be described 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, and starting from the PAM sequence, the double helix structure of the target gene is peeled off and annealed with a base sequence complementary to the target gene in the guide RNA. The double helix structure of the target gene is partially loosened. At this time, Cas9 cleaves the phosphodiester 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.

In the present specification, “modification” means that the base sequence of a target gene is changed. For example, cleavage of the target gene, change of the base sequence of the target double-stranded polynucleotide by insertion of exogenous sequence after cleavage (physical insertion or insertion by replication through homologous directed repair), non-homologous end ligation after cleavage ( NHEJ: changes in the base sequence of the target gene due to re-binding of DNA ends generated by cleavage). By modifying the target gene in the present embodiment, it is possible to introduce a mutation into the target gene or destroy the function of the target gene.
Therefore, in this embodiment, a cell in which the function of the target gene is destroyed (knocked out) or replaced (knocked in), or an organism having the cell can be easily produced.

When modifying two or more parts of one target gene, or when using two or more genes as a target gene, a polynucleotide having a base sequence complementary to the base sequence of each gene is converted to a 5 ′ end region. Two or more types of gRNAs included in the above are designed, and two or more types of Cas9-gRNA complexes are produced in the above <Method for producing Cas9-gRNA complex>, and then <Cas9-gRNA complex in the cell nucleus described above> The cells into which two or more types of Cas9-gRNA complexes are introduced may be used. Thereby, a plurality of different genes or a plurality of locations within the same gene can be edited simultaneously with high efficiency.

In the method for modifying a target gene in a cell according to this embodiment, a cell into which a foreign gene is introduced together with a Cas9-gRNA complex may be used by using <Method of introducing Cas9-gRNA complex into cell nucleus>. By introducing the foreign gene together, 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 can be obtained.

Moreover, it is preferable that a 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, for example, single base substitution without changing the amino acid sequence, the foreign gene can be inserted into the nuclear genome by homologous recombination. 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. Moreover, it is preferable that the vector which contains a foreign gene has DNA which has a sequence | arrangement homologous to the site | part which inserts a target gene in the 5 'terminal and 3' terminal of a foreign gene. The number of bases of DNA having a sequence homologous to the site where the target gene is inserted is preferably from 100 to 300 bases. In a vector containing a foreign gene, various promoters, polyadenylation signals, NLS, fluorescent protein marker genes, and the like may be operably linked to the 5 'end or 3' end of the foreign gene.

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

In one embodiment, the present invention also provides a method of performing targeted DNA insertion or targeted DNA deletion. This method involves transforming a cell with a nucleic acid construct comprising donor DNA. A scheme relating to DNA insertion and DNA deletion after target gene cleavage can be determined by those skilled in the art according to known methods.

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

In one embodiment, the present invention also provides a method for disrupting a gene in somatic cells. Here, a “gene” overexpresses a product that is harmful to the cell or organism and expresses a product that is harmful to the cell or organism. Such genes can be overexpressed in one or more cell types that occur in the disease. Disruption of the overexpressed gene according to the method of the present invention may lead to better health for an individual suffering from a disease caused by the overexpressed gene.
That is, the disruption of only a small percentage of the cells in the cells can work, reducing the expression level and producing a therapeutic effect.

Also, in one embodiment, the present 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 the particular gene. In cells where the gene has been disrupted, the gene can be completely knocked out. This loss of function in a particular cell can have a therapeutic effect.

In one embodiment, the present invention further provides insertion of donor DNA encoding a gene product. This gene product has a therapeutic effect when constitutively expressed.
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 cause 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 and treat individuals suffering from diabetes.

Also, the donor DNA can be inserted into crops and cause the production of pharmacologically related gene products. Protein product genes (eg, insulin, lipase, hemoglobin, etc.) can be inserted into plants along with regulatory elements (constitutively active promoters or inducible promoters) to produce large quantities of pharmaceuticals in plants. Such protein products can then be isolated from the plant. The genetically modified plant or the genetically modified organism uses a nucleic acid transfer technique (McCreath, KJ et al. (2000) Nature 405: 1066-1069; Polejaeva, IA et al. (2000) Nature 407: 86-90). Can be made by a method. Tissue type specific cells or cell type specific vectors can be utilized to provide gene expression only in selected cells.

Also, in one embodiment, the present invention selects cells that are utilized in germ cells and in which insertion occurs in a planned manner and all subsequent cell divisions produce cells with the engineered genetic alterations. Can do.

In one embodiment, the present invention also includes all organisms, cultured cells, cultured tissues, cultured nuclei (including cells, tissues or nuclei that can be used to regenerate intact organisms), gametes (eg, those At different stages of development).
Also, in one embodiment, the present invention provides for any organism (insects, fungi, rodents, cattle, sheep, goats, chickens, and other agriculturally important animals, as well as other mammals (dogs, cats and Can be applied to cells derived from, including but not limited to humans).
In one embodiment, the present invention can be applied to the generation of a disease state model animal (for example, a disease state model mouse).

Also, in one embodiment, the present invention can be used in plants. There is no special limitation as a plant, It can be used in arbitrary various plant species (For example, a monocotyledon or a dicotyledon etc.).

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

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

The sequence numbers of Cas9 and gRNA used are shown in Table 1 below. In Table 1, SaCas9 means Cas9 derived from S. aureus, and SpCas9 means Cas9 derived from S. pyogenes or derived. Moreover, dCas9 means SpCas9 in which endonuclease activity is inactivated, and NLS means a nuclear localization signal (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 32 ° C heat block to prepare sgRNA. did.

(2) Preparation of Cas9 Expression vector having constructs of His6-SUMO-tev protease site-HA-NLS-SaCas9, NLS-SpCas9-NLS, SpCas9-NLS, and His6-NLS-GFP-dCas9-NLS (Tokyo University Graduate School) (Transferd from the research group of Prof. Rizuki Nukiki, Graduate School of Science) was transformed into E. coli. Then, it culture | cultivated on 20 degreeC conditions with 0.1 mM IPTG containing an antibiotic for 18 hours. After culturing, E. coli was recovered. Next, E. coli was disrupted by sonication.
Next, SpCas9-NLS and NLS-SpCas9-NLS are purified with a P20.1 antibody column that recognizes the target tag added to the C-terminus, and then subjected to HiTrapSP (GE) to produce SpCas9-NLS and NLS-SpCas9-NLS. Obtained.
In addition, His6-NLS-GFP-dCas9-NLS was purified with NiNTA and subjected to HiTrapSP (GE) to obtain His6-NLS-GFP-dCas9-NLS.
In addition, His6-SUMO-tv protease site-HA-NLS-SaCas9 was purified with NiNTA and cleaved with tv protease. It was purified again with NiNTA to obtain HA-NLS-SaCas9.

(3) Production of Cas9-gRNA Complex Next, sgRNA prepared in (1) and Cas9 prepared in (2) were each 20 μL TB (Transport buffer: 25 mM Hepes, 1.15 mM KOAC, 250 μM MgCl ₂ , pH 7.2). ) 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 allowed to stand on ice.

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

[Test Example 1] Introduction of dCas9-gRNA complex into 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. Subsequently, a 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. Next, the mixture was centrifuged at 1500 rpm for 5 minutes, the supernatant was removed, and the resulting cell pellet was transferred to a Dounce homogenizer, and protease inhibitors (antipain, chymostatin, pepstatin A, and leupeptin, 25 μg / mL each) were 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 disrupted by further stroke 10 times or more. After crushing, the sample was centrifuged for 16 minutes at 12000 rpm and 4 ° C. After centrifugation, the supernatant was collected and centrifuged for 90 minutes at 100,000 g and 4 ° C. Subsequently, TB containing ATP regeneration system (ATP, creatine kinase (CK), creatine phosphate (CP), GTP, and glucose) was mixed with the obtained supernatant (cytoplasm), For 10 minutes.

(2) Preparation of Mixed Solution of Cas9-gRNA Complex and Cytoplasm Next, a complex of His6-NLS-GFP-dCas9-NLS and sgAAVS1 obtained in “1. Production of Cas9-gRNA Complex” , And may be referred to as “dCas9-gRNA complex”), and the cytoplasm of the L5178Y cells obtained in (1) was mixed to a total of 100 μL and stored at 32 ° C. until use. The composition of the mixture of dCas9-gRNA complex and 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. The HeLa cells were then transferred to ice and washed twice with pre-chilled PBS. Then, 1 mL of D-MEM (without fetal calf serum (FCS)) containing SLO (0.125 μg / mL) was added, and incubated on ice for 5 minutes. Subsequently, it was washed 3 times with PBS that had been cooled in advance. Then, 1 mL of 37 ° C. TB (without EGTA) was added and incubated at 37 ° C. for 10 minutes. Subsequently, it was washed twice with TB (without EGTA).

(4) Introduction of dCas9-gRNA complex and cytoplasm mixture into HeLa cells Next, the cover glass is taken out into parafilm, and 100 μL of the mixture of dCas9-gRNA complex and cytoplasm prepared in (1) is placed thereon. And incubated at 32 ° C. for 30 minutes. As a control group, i) a sample containing only His6-NLS-GFP-dCas9-NLS and no cytoplasm, and ii) a sample containing only dCas9-gRNA complex and no cytoplasm were tested.

(5) Re-encapsulation of HeLa cells Next, the mixed solution was collected, mixed with 1 μL of 100 mM calcium chloride, returned to 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 Subsequently, 2 mL of D-MEM (containing FCS) containing PI (Propidium Iodide) was added and cultured under conditions of 37 ° C. and 5% carbon dioxide. Each cell after __ hours from the culture was observed using a fluorescence microscope. The results are shown in FIG. In FIG. 3, i) is a His6-NLS-GFP-dCas9-NLS-only sample without cytoplasm, ii) is a dCas9-gRNA complex-only sample without cytoplasm, and iii) is a dCas9-gRNA complex-cytoplasmic sample. It means a sample using a mixed solution. “GFP-dCas9” means an image obtained by observing the localization of dCas9 inside and outside the cell using fluorescence of GFP, and “PI” means an image obtained by staining the nucleus using Propium Iodide, and “merge”. Means an image obtained by combining images of “GFP-dCas9” and “PI”.

From FIG. 3, in i), dCas9 formed a giant complex in the solution and aggregated, and thus was adsorbed nonspecifically on the plasma membrane and was not introduced into the cell. In ii), aggregate formation was significantly reduced, and dCas9-gRNA complex could be introduced into the cytoplasm, but no translocation into the nucleus was observed.
On the other hand, in iii), using the dCas9-gRNA complex and the cytoplasm, the translocation of the dCas9-gRNA complex into the nucleus was observed. It was speculated that this was because the transfer 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 can form a complex before cell introduction, thereby suppressing the formation of an aggregate of Cas9 and enabling cell introduction. Furthermore, the cytoplasm is the nucleus of 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 were introduced (1) Preparation of cytoplasm of L5178Y cells Using the same method as (1) of Test Example 1, the cytoplasm of L5178Y cells Was prepared.

(2) Preparation of a mixture of Cas9-gRNA complex and cytoplasm Next, a complex of NLS-SpCas9-NLS and sgAAVS1 obtained in “1. Production of Cas9-gRNA complex” (hereinafter referred to as “SpCas9- and the cytoplasm of L5178Y cells obtained in (1) were mixed to a total of 100 μL and stored at 32 ° C. until use. The composition of the mixture of the SpCas9-gRNA complex and the 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) HeLa cell perforation Using the same method as in Test Example 1, (3), pores were formed in at least part of the HeLa cells.

(4) Introduction of a mixed solution of SpCas9-gRNA complex and cytoplasm into HeLa cells Other than using a mixed solution of SpCas9-gRNA complex and cytoplasm instead of a mixed solution of dCas9-gRNA complex and cytoplasm In the same manner as in (4) of Test Example 1, a mixture of SpCas9-gRNA complex and cytoplasm was introduced into HeLa cells.

(5) Re-encapsulation of HeLa cells HeLa cells were re-encapsulated using the same method as in Test Example 1 (5). Next, 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) Genomic extraction from re-encapsulated HeLa cells Next, the re-encapsulated HeLa cells (hereinafter sometimes referred to as “reseal HeLa cells”) after 24 hours of culture were washed twice with PBS. Next, 500 μL of 1% SDS Lysate Buffer was added to Reseal HeLa cells and incubated at 37 ° C. under 5% carbon dioxide for 3 hours. Next, the solution on the petri dish was collected in a 1.5 mL tube, and 2.25 μL of 20 mg / mL proteinase K was added, mixed, and incubated at 50 ° C. overnight. Subsequently, 7.5 μL of 10 mg / mL RNase was added, and further 10 μL of 1% SDS Lysate Buffer was added, mixed, and incubated at 37 ° C. for 30 minutes. Subsequently, 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 similarly phenol / chloroform extraction was performed. Next, 750 μL of 2-propanol was added to the supernatant, mixed by inverting 10 times, and centrifuged (14,000 rpm, 4 ° C., 10 minutes). After centrifugation, the pellet was washed with 70% ethanol and then vacuum dried. The pellet was then dissolved in 20 μL of deionized-destiled H ₂ O (ddH ₂ O) to prepare a genomic solution.

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

The number of cycles was 20 for the first PCR and 35 for the second PCR. As the PCR template, the first PCR was performed using 10 ng of the genomic DNA prepared in (6), and the second PCR was performed using 1 μL of a 10-fold diluted solution of the first PCR product. After PCR amplification, the PCR product was dissolved in 20 μL of ddH ₂ O using Wizard SV Gel and PCR Clean-UP System (Promega). Next, the PCR product was diluted with ddH ₂ O to ₂ μg / 20 μL, and heat denaturation and reannealing were performed using a thermal cycler (95 ° C. 2 minutes / 85 ° C. → 35 ° C. over 10 minutes) Cooling / 16 ° C).

(8) T7E1 assay The T7E1 assay is a function in which the T7 endonuclease recognizes and cleaves DNA double-strand mismatch sites (sites that are not base pairs capable of forming hydrogen bonds such as AT and CG). It is a method for detecting and quantifying mutations in the genome by utilizing the possession.
Next, 1 μL of NE Buffer 2 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 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 the band was confirmed on an illuminator. The results are shown in FIG. In FIG. 4, lane 1 is a sample that was not subjected to reaction with T7 endonuclease 1 as a control group, lane 2 is a sample that was subjected to reaction with T7 endonuclease 1, and lane M is a marker.

FIG. 4 shows that in the HeLa cell (lane 2) into which the SpCas9-gRNA complex and cytoplasm have been introduced, a band cleaved by T7 endonuclease is generated, and the SpCas9-gRNA complex causes the intranuclear region of HeLa cells. It was confirmed that the mutation was introduced into the genome.

Test Example 3 T7E1 Assay Using HeLa Cells Introduced with SaCas9-gRNA Complex and Cytoplasm (1) Preparation of L5178Y Cell Cytoplasm 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, a complex of HA-NLS-SaCas9 and sgTET2 obtained in “1. Production of Cas9-gRNA complex” (hereinafter referred to as “SaCas9- sgTET2 complex ”) and the cytoplasm of L5178Y cells obtained in (1) were mixed to a total of 100 μL and stored at 32 ° C. until use. The composition of the mixture of the SaCas9-sgTET2 complex and the cytoplasm is the same as in Table 2 above. In Table 2, the content of SaCas9 is 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. Production of Cas9-gRNA complex” (hereinafter sometimes referred to as “SaCas9-sgTET1 complex”). A mixed solution with the cytoplasm of L5178Y cells obtained in (1) was also prepared.

(3) HeLa cell perforation Using the same method as in Test Example 1 (3), pores were formed in at least a portion of HeLa cells (adherent cells). Further, HeLa cells were peeled off with trypsin, and were used as floating cells to form pores at least partially using the same method as in Test Example 1 (3).

(4) Introduction of a mixed solution of SaCas9-sgTET1 complex or SaCas9-sgTET2 complex and cytoplasm into HeLa cells Instead of a mixed solution of dCas9-gRNA complex and cytoplasm, SaCas9-sgTET1 complex or SaCas9-sgTET2 HeLa cells (adherent cells) and HeLa cells (floating cells) were used except that a mixed solution of the complex and cytoplasm was used, and the same method as in Test Example 1 (4) was used. A mixture 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 (suspension cells) were re-encapsulated using the same method as in Test Example 1 (5). Next, 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) Genomic extraction from re-encapsulated HeLa cells A genome solution was prepared using the same method as in Test Example 2 (6).

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

The number of cycles was 20 for the first PCR and 35 for the second PCR. As the PCR template, the first PCR was performed using 10 ng of the genomic DNA prepared in (6), and the second PCR was performed using 1 μL of a 10-fold diluted solution of the first PCR product. After PCR amplification, the PCR product was dissolved in 20 μL of ddH ₂ O using Wizard SV Gel and PCR Clean-UP System (Promega). Next, the PCR product was diluted with ddH ₂ O to ₂ μg / 20 μL, and heat denaturation and reannealing were performed using a thermal cycler (95 ° C. 2 minutes / 85 ° C. → 35 ° C. over 10 minutes) Cooling / 16 ° C).

(8) T7E1 assay The T7E1 assay was performed using the same method as in Test Example 2 (8). 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 that are adherent cells, and suspended reseal is suspended cells. A sample using Reseal HeLa cells is meant, TET1 means a sample using sgTET1, and TET2 means a sample using sgTET2.

From FIG. 5, no introduction of 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 suspension cells.

[Test Example 4] Light-induced split-type SpCas9 (paCas9) and introduction into cytoplasmic 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. Production 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") And the cytoplasm of L5178Y cells obtained in (1) was mixed to a total of 100 μL and stored at 32 ° C. until use.
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). Moreover, SpCas9 (N) and SpCas9 (C) used in Test Example 4 are the N-terminal side and the C-terminal side of dCas that are inactive.
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) HeLa cell perforation Using the same method as in Test Example 1, (3), pores were formed in at least part of the HeLa cells.

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

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

(6) Fluorescent staining of HeLa cells Subsequently, 2 mL of D-MEM (containing FCS) containing PI (Propidium Iodide) was added and cultured under conditions of 37 ° C. and 5% carbon dioxide. Each cell after __ hours of culture was fixed with 4% paraformaldehyde. Next, fluorescent immunostaining was performed on the immobilized HeLa cells using an anti-His tag antibody, and observed using a fluorescence microscope. The result is shown in FIG. 6B. In FIG. 6B, “nMag-dCas9 / sgRNA” means a HeLa cell into which an nMag-SpCas9 (N) -sgAAVS1 complex and cytoplasm have been introduced, and “pMag-dCas9 / sgRNA” means pMag-SpCas9 (C ) —Means a HeLa cell into which sgAAVS1 complex and cytoplasm have been introduced, and “without Cas9” means a HeLa cell 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, a signal was detected in the cell nucleus for both the HeLa cell into which the nMag-SpCas9 (N) -sgAAVS1 complex and cytoplasm were introduced, and the HeLa cell into which the pMag-SpCas9 (C) -sgAAVS1 complex and cytoplasm were introduced. It was done.
From this, it was confirmed that Cas9 of the light induction system can be introduced into the cell nucleus using the introduction method of the present invention.

[Test Example 5] T7E1 assay using rapamycin-inducible split-type SpCas9 (rpCas9) and HeLa cells into which cytoplasm was introduced (1) Preparation of cytoplasm of L5178Y cells The same method as in Test Example 1 (1) was used. 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. Production of Cas9-gRNA complex” (SpCas9 (C) -FKBP-Histag) and sgAAVS1 complex (hereinafter sometimes referred to as “rpCas9 (N) -rpCas9 (C) -gRNA complex”) and (1) The cytoplasm of L5178Y cells was mixed to a total of 100 μL and stored at 32 ° C. until use.
FIG. 7A is a schematic diagram showing the configuration of rpCas9-N (HisTag-SpCas9 (N) -FRB) and rpCas9-C (SpCas9 (C) -FKBP-Histag).
The composition of the mixture of rpCas9 (N) -rpCas9 (C) -gRNA complex and cytoplasm is the same as in Table 2 above. In Table 2, 5 μg / 100 μL described as the content of dCas9 is the total content of rpCas9-N and rpCas9-C.

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

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

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

(6) Genomic extraction from re-encapsulated HeLa cells Next, a genomic solution was prepared using the same method as in Test Example 2 except that the re-encapsulated HeLa cells after 18 hours of culture were used.

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

(8) T7E1 assay Next, T7E1 assay was performed using the same method as in Test Example 2 (7). The result is shown in FIG. 7B. In FIG. 7B, “T7E1 (+)” means a sample that has undergone a T7E1 assay, and “T7E1 (−)” means a sample that has not undergone a T7E1 assay. “Inducible Cas9 sgRNA + rapamycin” means a sample obtained by adding rapamycin to a riceal HeLa cell into which rpCas9 (N) -rpCas9 (C) -gRNA complex and cytoplasm have been introduced, and cultivating “Inducible Cas9 sgRNA × 2 + rapamycin "introduces rpCas9 (N) -rpCas9 (C) -gRNA complex and cytoplasm in which the amount of gRNA added is twice that of" Inductable Cas9 sgRNA + rapamycin "in the Cas9-gRNA complex formation step It means a sample obtained by adding rapamycin to cultured Reseal HeLa cells. “Inducible Cas9-rapamycin” is a reseal HeLa cell into which only rpCas9 (N) -rpCas9 (C) has been introduced, and is a sample cultured without addition of rapamycin, and “Negative control (without Cas9)” Means a sample cultured without addition of rapamycin.

From FIG. 7B, in the sample cultured without rapamycin (including the negative control), no cleavage occurred and no cleaved DNA band was observed. On the other hand, in the sample cultured with rapamycin added, a cleaved DNA band 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 stably solubilized in vitro can be obtained. In addition, according to the method of introducing the Cas9-gRNA complex of the present invention into the cell nucleus, the Cas9-gRNA complex is prepared in a state in which the nuclear niche for genome editing is optimized without introducing the viral vector into the cell. It can be introduced into the cell nucleus. Furthermore, according to the method for modifying a target gene in a cell of the present invention, off-target is reduced, the nuclear niche for genome editing is optimized without the need to introduce a viral vector into the cell, and in a large number of cells. Multiple loci can be edited simultaneously.

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

Claims (9)

A Cas9-gRNA complex forming step is provided in which Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) -associated 9) and guide RNA (gRNA) are mixed at 20 ° C. or more to form a Cas9-gRNA complex.
Cas9, wherein the gRNA comprises a polynucleotide having a base sequence complementary to a base sequence from 1 base upstream to 20 bases to 24 bases upstream of a PAM (Proto-spacer Adjacent Motif) sequence in a target gene A method for producing a gRNA complex. The method for producing a Cas9-gRNA complex according to claim 1, wherein, in the Cas9-gRNA complex formation step, the Cas9 and the gRNA are mixed at 30 ° C to 65 ° C. The method for producing a Cas9-gRNA complex according to claim 1 or 2, wherein the Cas9 has a nuclear localization signal (NLS) peptide bound to the N-terminus or C-terminus. Cas9 is divided into N-terminal Cas9 (Cas9-N) and C-terminal Cas9 (Cas9-C),
The Cas9-N and the Cas9-C each have a protein that forms a dimer in a light-dependent or chemical-dependent manner at the N-terminus or C-terminus,
The method for producing a Cas9-gRNA complex according to any one of claims 1 to 3, wherein the Cas9 recovers RNA-induced DNA endonuclease activity in the presence of light or a chemical substance. The method for producing a Cas9-gRNA complex according to any one of claims 1 to 4, wherein two or more types of the gRNA are mixed in the Cas9-gRNA complex formation step. A perforating step in which a perforating substance is allowed to act on the plasma membrane of the cell to form pores in at least a part of the plasma membrane;
The Cas9-gRNA complex obtained by the method for producing a Cas9-gRNA complex according to any one of claims 1 to 5 is introduced into a cell having a pore formed at least in part on the plasma membrane. Introduction process;
A solution containing calcium ions is added to cells in which pores are formed in at least a part of the plasma membrane into which the Cas9-gRNA complex has been introduced, and re-encapsulation of the pores formed in at least a part of the plasma membrane. A method for introducing a Cas9-gRNA complex into a cell nucleus, comprising an encapsulation step. Further, before the introducing step, the Cas9-gRNA complex is mixed with a cytoplasm collected from cells derived from the same species as cells having pores formed on at least a part of the plasma membrane. The method for introducing a Cas9-gRNA complex according to claim 6 into a cell nucleus, comprising a mixing step. The method further comprises a second mixing step of mixing the Cas9-gRNA complex with an inhibitor of a homologous recombination inhibitor or an activator of homologous recombination before the introducing step. 8. A method for introducing the Cas9-gRNA complex according to 7 into a cell nucleus. A cell having a re-encapsulated target gene, wherein the Cas9-gRNA complex is introduced into the cell nucleus obtained by the method for introducing the Cas9-gRNA complex according to any one of claims 6 to 8 into the cell nucleus. Wherein the Cas9 cleaves the target gene at a cleavage site located upstream or downstream of a predetermined base of the PAM sequence;
And a modification step of obtaining a cell having the modified target gene in a region determined by complementary binding between the gRNA and the target gene.

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