WO2020059708A1 - METHOD FOR MODULATING ACTIVITY OF Cas PROTEIN - Google Patents

Abstract

It was discovered that the endonuclease activity of a Cas protein can be modulated in a cell-cycle-dependent manner by causing a fusion protein of AcrIIA4 and Cdt1 to be expressed intracellularly together with the Cas protein.

Description

Method for regulating the activity of Cas protein

The present invention relates to a method for regulating the endonuclease activity of Cas protein in a cell cycle-dependent manner, and a kit for use in the method.

Site-specific nuclease (SSN) recognizes a DNA sequence of about 20 bp and selectively cleaves the target sequence. However, site-specific nucleases can cleave non-target DNA sequences and introduce mutations (off-target mutations). Off-target mutations often occur at non-target sequences that are similar to the target sequence and can result in unexpected side effects or phenotypic changes. Thus, off-target effects need to be carefully addressed, especially when using genome editing tools for clinical applications.

Off-target mutations can occur due to the presence of higher concentrations and longer duration of site-specific nucleases in cells and the higher affinity of site-specific nucleases. Both events can increase the likelihood that the site-specific nuclease will bind to the off-target site. Similarly, the accuracy of repair after an unexpected double-strand break (DSB) at off-target sites is affected.

Many studies have so far developed methods to reduce off-target effects of CRISPR-Cas9 and other genomic editing platforms. For example, it has been reported that direct delivery of a site-specific nuclease can reduce off-target effects without inhibiting on-target editing efficiency (Non-Patent Document 1). In another approach, it has been reported that nuclease activity is controlled by an activity-inducible domain (Non-patent Document 2). These techniques can reduce the duration of active Cas9 and control the amount of Cas9 in cells, as compared to methods that use plasmid DNA for Cas9 expression.

In recent years, new methods using nanomaterials for delivering macromolecules directly into cells have been developed. These methods can also help reduce off-target effects (Non-Patent Document 3). To reduce the affinity for the target DNA, a high-fidelity Cas9 mutant has been developed (Non-Patent Document 4). The use of truncated sgRNAs is also an effective approach for this purpose [5]. These methods suppress the off-target cleavage by reducing the stability of the tertiary complex of Cas9-sgRNA-target DNA. The use of nickases [6] may reduce uncertain errors in the repair process. Other effective approaches include predicting possible off-target sites and highly specific sgRNA sequences with bioinformatics tools (7). Combinations of these approaches can be powerful tools for reducing off-target effects, but it is difficult to completely suppress off-target mutations.

相同 By the way, homologous recombination repair (HDR) is a genome editing mechanism including a homologous recombination DNA repair pathway. Homologous recombination repair requires a template DNA having a sequence homologous to the sequence around the cleavage site. The homologous recombination repair allows precise editing of the target genomic sequence. Furthermore, off-target cleavage can be precisely repaired via homologous recombination. Therefore, for precise genome editing, it is important to increase the rate of DNA repair via homologous recombination repair.

Homologous recombination occurs in S and G2 phases. Another pathway, non-homologous end joining (NHEJ), operates throughout the cell cycle [8]. The ratio of homologous recombination to non-homologous end joining reaches a peak in mid-S phase (Non-Patent Document 9). The efficiency of genome editing using homologous recombination repair is affected by chemical or genetic disruption of the non-homologous end joining pathway (10). The efficiency of homologous recombination repair can also be increased by controlling the timing of the delivery of the SpCas9-sgRNA complex to chemically synchronized cells (Non-Patent Document 11). If chemicals are used to block non-homologous end-joining pathways or synchronize cells, the cytotoxicity of these chemicals on cells must be considered. Cas9, which is fused to the N-terminus of geminin (1/110) protein and can be specifically activated in S phase and G2 phase, has been reported (Non-Patent Document 12). Geminin-fused Cas9 is more preferred for cells because it can control Cas9 activity without chemicals. However, the homologous recombination repair activity is only slightly increased. This is probably because it takes time to recover the amount of Cas9 that has deteriorated in the G1 phase, and the activity of Cas9 in the S phase decreases.

Gaj, T. et al. Nat Methods 9, 805-807 (2012) Liu, K.I.etal.Nat Chem Biol 12, 980-987 (2016) Matsumoto, D. et al. Sci Rep-Uk 5 (2015) Kleinstiver, B.P. et al. Nature 529, 490-495 (2016) Fu, Y. et al. Nat Biotechnol 32, 279-284 (2014) Shen, B. et al. Nat Methods 11, 399-402 (2014) Cradick, T.J.etal.BMC Bioinformatics 12, 152 (2011) Mao, Z. et al. Cell Cycle 7, 2902-2906 (2008) Karanam, K. et al. Molecular Cell 47, 320-329 (2012) Vasquez, K.M. et al. Proc Natl Acad Sci U S A 98, 8403-8410 (2001) Lin, S., Staahl, B.T., Elife 3, e04766 (2014) Howden, S.E.etal.Stem Cell Rep 7, 508-517 (2016) Yang, H. & Patel, D.J.Mol Cell 67, 117-127 e115 (2017) Kim, I. et al. Sci Rep 8, 3883 (2018) Shin, J. et al. Sci Adv 3, e1701620 (2017) Sakaue-Sawano, A. et al. Cell 132, 487-498 (2008)

The present invention has been made in view of such a situation, and an object of the present invention is to provide a new method for increasing the efficiency of repair of homologous recombination in cells.

In recent years, an anti-CRISPR (Acr) inhibitor of the CRISPR-Cas9 system has been reported (Non-Patent Document 13). Among the Acr inhibitors, AcrIIA4 binds strongly to the Cas9-sgRNA complex, but has a lower binding affinity for Cas9 (Non-Patent Document 14). AcrIIA4 also efficiently inhibits Cas9 activity in mammalian cells (Non-Patent Document 15). Furthermore, inhibition of Cas9 activity by AcrIIA4 reduces off-target editing (15). Focusing on these characteristics of AcrIIA4, the present inventors first considered adopting AcrIIA4 as a switch for controlling Cas9 activity.

Here, based on the finding that non-homologous end joining operates throughout the cell cycle, but homologous recombination does not operate in the G1 phase of the cell cycle, AcrIIA4 functions specifically in the G1 phase to allow non-homologous end joining by Cas9. Is considered to be able to relatively enhance the efficiency of repair of homologous recombination. The present inventors have reported that a fluorescent protein fused to a license factor called Cdt1 is degraded by SCF ^Skp2 E3 ubiquitin ligase-mediated proteolysis in the S, G2, and M phases of the cell cycle. In view of this (Non-Patent Document 16), Cdt1 was adopted as a means for degrading AcrIIA4 in a cell cycle-dependent manner.

Based on this concept, the present inventors fused AcrIIA4 with Cdt1, expressed this fusion protein together with Cas9 in cells, and evaluated Cas9 activity in the cell cycle. As a result, they found that Cas9 activity was suppressed during the cell cycle when homologous recombination did not operate, while Cas9 activity was exerted during the cell cycle when homologous recombination operates (FIG. 1). This fact indicates that AcrIIA4 fused with Cdt1 succeeded in exhibiting the inhibitory activity against Cas9, and that the function of AcrIIA4 was also lost with cell cycle-dependent degradation of Cdt1 by ubiquitin ligase. means.

In addition, the present inventor aims to improve the homologous recombination efficiency and reduce non-homologous end joining and off-target by expressing a protein obtained by fusing the fusion protein and Cas9 via a self-cleaving peptide. I found that I can do it. Furthermore, the present inventors have found that a system using a single-stranded donor DNA or a shortened guide RNA can further increase the rate of precise editing of a target by homologous recombination and further reduce the off-target, The present invention has been completed.

The present invention relates to a method for regulating Cas activity in a cell cycle-dependent manner using a fusion protein of AcrIIA4 and Cdt1, a method for editing DNA in a cell in a cell cycle-dependent manner using the method, and these methods. More specifically, the present invention provides the following.

[1] A method for regulating the endonuclease activity of a Cas protein in a cell in a cell cycle-dependent manner,
(I) providing a fusion protein of AcrIIA4 protein and Cdt1 protein; and (ii) providing a cell containing Cas protein.
A method in which the fusion protein suppresses the endonuclease activity of the Cas protein in the cell in a cell cycle-dependent manner.

[2] The method according to [1], wherein the Cdt1 protein in the fusion protein is a partial peptide containing an amino acid sequence that undergoes E3 ubiquitin ligase-mediated proteolysis.

[3] The method according to [1] or [2], wherein the cell comprises a protein in which the fusion protein and the Cas protein are fused via a self-cleaving peptide.

[4] The method according to any one of [1] to [3], wherein the Cas protein is a Cas9 protein.

[5] A method for producing a cell in which DNA has been edited,
(I) providing a cell comprising a fusion protein of AcrIIA4 protein and Cdt1 protein; and (ii) a cell comprising a CRISPR-Cas system,
A method in which the fusion protein suppresses the endonuclease activity of the Cas protein in the CRISPR-Cas system in a cell cycle-dependent manner in the cell, whereby the DNA in the cell is edited in a cell cycle-dependent manner.

[6] The method according to [5], wherein the Cdt1 protein in the fusion protein is a partial peptide containing an amino acid sequence that undergoes E3 ubiquitin ligase-mediated proteolysis.

[7] The method according to [5] or [6], wherein the cell comprises a protein in which the fusion protein and the Cas protein are fused via a self-cleaving peptide.

[8] The method according to any one of [5] to [7], wherein the cells further contain a donor DNA.

[9] The method according to [8], wherein the donor DNA is a single-stranded donor DNA.

[10] The method according to any one of [5] to [9], wherein in the guide RNA constituting the CRISPR-Cas system, the base sequence complementary to the base sequence of the target DNA region is less than 20 bases in length. .

[11] The method according to any one of [5] to [10], wherein the Cas protein is a Cas9 protein.

[12] A kit for use in the method according to [1] to [11], which comprises a fusion protein of an AcrIIA4 protein and a Cdt1 protein, a polynucleotide encoding the fusion protein, an expression vector containing the polynucleotide, or A kit comprising cells into which the expression vector has been introduced.

[13] The kit according to [12], further comprising at least one of the following (i) to (iii):

(I) Cas protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide (ii) guide RNA, polynucleotide encoding the guide RNA, or expression vector containing the polynucleotide (iii) donor DNA

According to the present invention, it has become possible to regulate the endonuclease activity of Cas protein in a cell cycle-dependent manner. In particular, according to the present invention, the endonuclease activity of the Cas protein can be exerted at a stage in the cell cycle in which homologous recombination repair operates, and at other times, the endonuclease activity can be suppressed. Therefore, in the present invention, if the CRISPR-Cas system is used together with the donor DNA, the efficiency of accurate genome editing by repair of homologous recombination can be increased, and the off-target effect due to non-homologous end joining can be suppressed.

It is a conceptual diagram of the CRISPR-Cas9 system activated in a cell cycle dependent manner. 4 is a photograph showing a change in the expression level of mKO2-Cdt1. 1 is a photograph showing the results of confirming the expression and localization of AcrIIA4-Cdt1 in 293A cells. FIG. 2 is a photograph showing the dose-dependent inhibition of mutagenesis by AcrIIA4 or AcrIIA4-Cdt1. FIG. 4 is a photograph showing inhibition of mutagenesis by AcrIIA4 or AcrIIA4-Cdt1 when the molar ratio of CRISPR-Cas system to AcrIIA4 or AcrIIA4-Cdt1 is 1: 5. The CRISPR-Cas system was verified with or without guide RNA. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 shows a vector for co-expressing AcrIIA4-Cdt1 and Cas9 using a self-cleaving 2A peptide, and a graph showing the expression levels of AcrIIA4-Cdt1 and Cas9 in the cell cycle when the vector is used. 7 is a graph showing changes in the expression level of AcrIIA4 or AcrIIA4-Cdt1 in the cell cycle when the vector shown in FIG. 6 is used. FIG. 7 is a photograph showing the results of testing genome editing via homologous recombination repair (HDR) using the vector and the donor DNA shown in FIG. 6. FIG. 7 is a photograph showing the results of a test of genome editing at a target site by non-homologous end joining (NHEJ) using the vector shown in FIG. 6. FIG. 7 is a photograph showing the results of testing genome editing at off-target sites by non-homologous end joining (NHEJ) using the vector shown in FIG. FIG. 7 shows the results of testing genome editing at a target site or off-target site via homologous recombination repair (HDR) or non-homologous end joining (NHEJ) using the vector described in FIG. 6 and single-stranded donor DNA. It is a photograph. The AAVS1 gene was used as a target gene. 12 is a graph showing the results of performing the same experiment as in FIG. 11 using the EMX1 gene as a target gene. 12 is a graph showing the results of performing the same experiment as in FIG. 11 using the VEGFA gene as a target gene. 13 is a graph showing the results of performing the same experiment as in FIG. 12 using the shortened single-stranded guide RNA. 14 is a graph showing the results of performing the same experiment as in FIG. 13 using the shortened single-stranded guide RNA.

-Cas protein activity regulation method-
The present invention provides a method for regulating the endonuclease activity of Cas protein in a cell in a cell cycle-dependent manner. The method of the present invention comprises providing a cell comprising (i) a fusion protein of AcrIIA4 protein and Cdt1 protein, and (ii) a Cas protein, wherein the fusion protein is dependent on the cell cycle in the cell. Suppresses endonuclease activity of Cas protein.

「“ AcrIIA4 protein ”in the present invention is one of the proteins known as an anti-CRISPR (Acr) inhibitor. The amino acid sequence of a typical Listeria @ monocytogenes-derived AcrIIA4 protein is shown in SEQ ID NO: 1, and the nucleotide sequence of the DNA encoded by the protein is shown in SEQ ID NO: 2. The “AcrIIA4 protein” used in the present invention may be a homolog, a mutant, or a partial peptide of the AcrIIA4 protein derived from Listeria @ monocytogenes as long as it has an ability to inhibit the endonuclease activity of Cas protein. As the homolog, for example, the amino acid sequence of the target AcrIIA4 protein (for example, SEQ ID NO: 1) and 85% or more, preferably 90% or more, more preferably 95% or more (for example, 96% or more, 97% or more, (98% or more, 99% or more), and a protein having the ability to inhibit the endonuclease activity of Cas protein. The identity of the base sequence can be evaluated by a numerical value calculated using BLAST or the like (for example, default, that is, a parameter of initial setting) (the same applies hereinafter). In addition, the mutant includes an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted with respect to the amino acid sequence (for example, SEQ ID NO: 1) of the native AcrIIA4 protein, And a protein having an ability to inhibit the endonuclease activity. Here, the “plurality” means, for example, 2 to 15, preferably 2 to 10, more preferably 2 to 8 (eg, 2 to 7, 2 to 6, 2 to 5, 2 ~ 4, 2 ~ 3, 2). "Inhibition of endonuclease activity of Cas protein" by AcrIIA4 protein includes complete inhibition and partial inhibition (for example, 50% or more inhibition, 70% or more inhibition, 90% or more inhibition). Is preferably complete inhibition. As described in this example, the endonuclease activity of Cas protein can be evaluated using the target site cleavage activity as an index when introduced into cells in combination with a guide RNA.

The “Cdt1 protein” in the present invention is known as a protein that prevents excessive replication in higher eukaryotes, and its function is known to be inhibited by ubiquitin-mediated proteolysis and degradation by geminin binding (Nishitani). J. Biol Chem 276, 44905-44911 (2001), Kim, Y. Y. & Kipreos, ET Cell Div. 2 (2007), Wohlschlegel, J.A., 230, ec. 2312 (2000), Lee, C. et al. Nature 430, 913-917 (2004)). The amino acid sequence of a typical human Cdt1 protein is shown in SEQ ID NO: 3, and the nucleotide sequence of the DNA encoded by the protein is shown in SEQ ID NO: 4. The “Cdt1 protein” used in the present invention may be a homologue, a mutant, or a partial peptide of the above-mentioned human Cdt1 protein as long as it is degraded in a cell cycle-dependent manner. As the homolog, for example, the amino acid sequence of the target Cdt1 protein (for example, SEQ ID NO: 3) and 85% or more, preferably 90% or more, more preferably 95% or more (for example, 96% or more, 97% or more, (98% or more, 99% or more), and includes proteins that are degraded in a cell cycle-dependent manner. In addition, the mutant comprises an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted with respect to the amino acid sequence of the natural Cas protein (eg, SEQ ID NO: 3). Independently degraded proteins are included. Here, the term “plurality” means, for example, 2 to 80, preferably 2 to 40, more preferably 2 to 20 (eg, 2 to 10, 2 to 5, 2 to 3, 2 ).

The partial peptide of Cdt1 used in the present invention is preferably a partial peptide containing an amino acid sequence that undergoes E3 ubiquitin ligase-mediated proteolysis. The N-terminus of Cdt1 is a ubiquitination domain that is targeted by two types of E3 ubiquitin ligases: SCF ^Skp2 E3 ligase and CUL4 ^Ddb1 (Cullin4, damage-specific DNA binding protein 1) E3 ligase (Nishitani, H. et al. EMBO J 25, 1126-1136 (2006)).

SCF ^Skp2 E3 ligase targets phosphorylated amino acids (Ser31 and / or Thr29) in the S and G2 phases. Cyclin A-dependent kinase catalyzes these phosphorylation reactions, and this reaction requires Arg68-Arg69-Leu70, which is a cyclin-binding motif (Cy motif) of Cdt1. Therefore, one embodiment of the partial peptide of Cdt1 used in the present invention is a partial peptide containing a phosphorylated amino acid (Ser31 and / or Thr29) and a Cy motif (Arg68-Arg69-Leu70) in the full-length amino acid sequence of the Cdt1 protein. is there. Examples of the partial peptide of the present embodiment include a partial peptide consisting of the amino acid sequence at positions 30 to 120 in the full-length amino acid sequence of the Cdt1 protein used in the present example. The Cas peptide can be activated in the S phase and G2 phase of the cell cycle by fusing the partial peptide of this embodiment with the AcrIIA4 protein and introducing it into the cell, or expressing it in the cell.

On the other hand, CUL4 ^Ddb1 E3 ligase recognizes six amino acids that are highly conserved in the proliferating cell nuclear antigen-interacting protein motif (PIP box) at the N-terminus of Cdt1 (about 1-10 amino acid residues). CUL4 ^Ddb1 E3 ligase-mediated proteolysis of Cdt1 occurs only during S phase or after DNA damage (Havens, CG & Walter, JC Mol Cell 35, 93-104 (2009), Ishii, T. et al. et al. J Biol Chem 285, 41993-42000 (2010), Roukos, V. et al. J Cell Sci 124, 422-434 (2011)). Therefore, another embodiment of the partial peptide of Cdt1 used in the present invention is a partial peptide containing the N-terminus of Cdt1 (about 1 to 10 amino acid residues). Here, a new Fucci system called Fucci (CA) applying CUL4 ^Ddb1 E3 ligase-mediated proteolysis has been reported (Sakaue-Sawano, A. et al. Mol Cell 68, 626-640 e625 (2017)). Fucci (CA) is a fusion protein of mKO and amino acid residues 1 to 100 in the absence of a Cy motif derived from Cdt1. Therefore, the Cy motif is not essential in the partial peptide of this embodiment. Therefore, examples of the partial peptide of the present embodiment include a partial peptide consisting of the amino acid sequence at positions 1 to 100 in the full-length amino acid sequence of the Cdt1 protein, in which the Cy motif is mutated. The Cas peptide can be activated in the S phase of the cell cycle by fusing the partial peptide of this embodiment with the AcrIIA4 protein and introducing it into the cell, or expressing it in the cell.

The “fusion protein of AcrIIA4 protein and Cdt1 protein” in the present invention can be prepared by ligating a DNA encoding AcrIIA4 protein and a DNA encoding Cdt1 protein, inserting the DNA into an appropriate vector, and expressing it. There is no particular limitation on the way in which the DNAs encoding both proteins can be ligated, as long as the expressed fusion protein can exert the above-mentioned desired function. Further, another functional protein may be further fused to the fusion protein. Other functional proteins are directly or indirectly fused to one or both of the N-terminal and C-terminal of the fusion protein, or between the two proteins, as long as they can exert their functions. be able to. The other functional protein is not particularly limited and is appropriately selected depending on the function to be imparted to the fusion protein. For example, if the function to be imparted to the fusion protein is intracellular localization, a nuclear transport signal (NLS ), And if the function to be imparted to the fusion protein is detection or purification of the fusion protein, examples thereof include green fluorescent protein (GFP) and FLAG-tag protein. Further, a linker peptide may be interposed between the proteins constituting the fusion protein.

「The“ Cas protein ”used in the present invention is not particularly limited as long as the AcrIIA4 protein can inhibit its endonuclease activity. As such “Cas protein”, Cas9 protein is preferable, and Cas9 (SpCas9) protein derived from Streptococcus @ pyogenes is particularly preferable. Amino acid sequences and base sequences of various Cas proteins are published in published databases such as literature and NCBI (for example, WO2013 / 176772, WO2014 / 093712, WO2013 / 142578, WO2014 / 131833, Accession No .: Q99ZW2. 1, WP_0217373622). The amino acid sequence of a typical SpCas9 protein is shown in SEQ ID NO: 5, and the nucleotide sequence of the DNA encoded by the protein is shown in SEQ ID NO: 6.

「The“ Cas protein ”used in the present invention may be a homolog, a mutant, or a partial peptide of a known Cas protein. As the homolog, for example, the amino acid sequence of the subject Cas protein (for example, SEQ ID NO: 5) and 85% or more, preferably 90% or more, more preferably 95% or more (for example, 96% or more, 97% or more, (98% or more, 99% or more), and a protein having an endonuclease activity. In addition, the mutant includes an amino acid sequence in which one or more amino acids have been substituted, deleted, added, or inserted with respect to the amino acid sequence (for example, SEQ ID NO: 3) of a naturally-occurring Cas protein. Active proteins. Here, the term “plurality” means, for example, 2 to 200, preferably 2 to 100, more preferably 2 to 50 (eg, 2 to 30, 2 to 10, 2 to 5, 2 ~ 3, 2). Examples of the mutant include, for example, a nickase-type Cas protein (nCas) in which a part of the endonuclease activity is lost by introducing a mutation into a specific amino acid residue, or a mutation into a specific amino acid residue. A Cas protein (Benjamin, P. et al., Nature 523, 481-485 (2015), Hirano, S. et al., Molecular Cell 61, 886-894 (2016)), which has altered the recognition specificity of PAM.

In the present invention, a cell containing the fusion protein and Cas protein is provided. In preparing such cells, the fusion protein and Cas protein may be directly introduced into cells, or the fusion protein and Cas protein may be expressed in cells. Alternatively, one of the proteins may be directly introduced into a cell, and the other may be expressed in the cell.

方法 The method for expressing the protein in the cells is not particularly limited, and a known method can be appropriately selected and used. For example, a fusion protein expression vector and a Cas protein expression vector may be introduced into cells, or a vector expressing both the fusion protein and Cas protein may be introduced into cells.

When employing the form of an expression vector, it contains one or more regulatory elements operably linked to the DNA to be expressed. Here, “operably linked” means that the DNA is operably linked to the regulatory element. "Regulatory elements" include promoters, enhancers, internal ribosome entry sites (IRES), and other expression control elements such as transcription termination signals, such as polyadenylation signals and poly-U sequences. The expression vector is preferably one that can stably express the encoded protein without being integrated into the host genome. Examples of the form of such an expression vector include an episomal vector.

In the present invention, the fusion protein and the Cas protein may be expressed by introducing them into cells in the form of mRNA instead of in the form of an expression vector.

に お い て In the present invention, it is preferable to introduce a self-cleaving peptide between the fusion protein and the Cas protein. Thereby, the expression levels of the fusion protein and Cas protein can be strictly controlled (FIG. 6). Examples of the self-cleaving peptide include, but are not limited to, T2A, P2A, E2A, F2A, and the like.

The “cell” used in the present invention is a cell in which the above-mentioned fusion protein and Cas protein function, and a cell having a mechanism for specifically degrading Cdt1 in the above-mentioned fusion protein in a cell cycle-specific manner. No restrictions. The cell is preferably an eukaryotic cell such as an animal cell, a plant cell, an algal cell, a fungal cell, and particularly preferably an animal cell.

"Animal cells" include, for example, cells constituting an individual animal, cells constituting an organ / tissue extracted from an animal, cultured cells derived from an animal tissue, and the like. Specifically, for example, embryo cells of each stage embryo (eg, 1-cell stage embryo, 2-cell stage embryo, 4-cell stage embryo, 8-cell stage embryo, 16-cell stage embryo, morula embryo, etc.); Stem cells such as pluripotent stem (iPS) cells, embryonic stem (ES) cells, and hematopoietic stem cells; fibroblasts, hematopoietic cells, neurons, muscle cells, bone cells, hepatocytes, hepatocytes, pancreatic cells, brain cells, kidney cells, etc. Somatic cells. A fertilized egg can be used to create a genome-edited animal.

Animals include, for example, mammals, fish, birds, reptiles, amphibians, and insects. “Mammal” is a concept that includes humans and non-human mammals. Examples of non-human mammals include artiodactyls such as cows, boars, pigs, sheep and goats, artichokes such as horses, rodents such as mice, rats, guinea pigs, hamsters, squirrels, and lagomorphs such as rabbits. And meat such as dogs, cats, and ferrets. The non-human mammal described above may be a domestic animal, a companion animal, or a wild animal.

"Plant cells" include, for example, cells of cereals, oil crops, feed crops, fruits and vegetables. “Plant cells” include, for example, cells constituting a plant individual, cells constituting organs and tissues separated from plants, cultured cells derived from plant tissues, and the like. Examples of plant organs and tissues include leaves, stems, shoot apices (growth points), roots, tubers, tubers, seeds, calli, and the like. Examples of plants include rice, corn, banana, peanut, sunflower, tomato, rape, tobacco, wheat, barley, potato, soybean, cotton, carnation, and the like.

The fusion protein and Cas protein, and the introduction of mRNA or vector for expressing the same into cells include, for example, electroporation, microinjection, DEAE-dextran method, lipofection method, nanoparticle-mediated transfection method, Known methods such as a virus-mediated nucleic acid delivery method can be appropriately selected and performed.

細胞 In the cell containing the fusion protein and the Cas protein thus prepared, the endonuclease activity of the Cas protein is regulated in a cell cycle-dependent manner. Here, "cell cycle dependent" means that the endonuclease activity of Cas protein changes depending on the stage of the cell cycle. In a preferred embodiment of the invention, the endonuclease activity of the Cas protein is high in the S / G2 / M phase of the cell cycle and low in the G1 phase. Such cell cycle dependence can be achieved by degrading the fusion protein of the present invention containing AcrIIA4, which is an anti-CRISPR inhibitor, at the S / G2 / M phase of the cell cycle. The degradation of such a fusion protein can be induced by the cell cycle-dependent degradation of Cdt1, another domain contained in the fusion protein of the present invention. The cell cycle-dependent degradation of Cdt1 is caused by, for example, the action of endogenous E3 ubiquitin ligase.

-Genome editing-
In the above method of the present invention, by combining Cas protein with guide RNA and allowing it to function as a CRISPR-Cas system, it is possible to edit the intracellular DNA targeted by the CRISPR-Cas system. Accordingly, the present invention provides a method for producing a cell having a DNA edited, comprising: providing a cell comprising (i) a fusion protein of AcrIIA4 protein and Cdt1 protein, and (ii) a CRISPR-Cas system, In the cell, the fusion protein suppresses the endonuclease activity of the Cas protein in the CRISPR-Cas system in a cell cycle-dependent manner, thereby providing a method in which intracellular DNA is edited in a cell cycle-dependent manner.

The guide RNA is composed of a base sequence interacting with the Cas protein (hereinafter referred to as “Cas interacting base sequence”) and a base sequence complementary to the base sequence of the target DNA region (hereinafter referred to as “targeting base sequence”). RNA). For this reason, the guide RNA forms a complex with the Cas protein and induces the complex to the target DNA region. The Cas protein induced by the target DNA region is converted into the target site in the target DNA region by its endonuclease activity. Disconnect. When the cleavage of the target site is repaired by non-homologous terminal joining, insertion or deletion of nucleotides mainly occurs, and gene knockout can be performed by frame shift or the like. On the other hand, when a donor DNA serving as a repair template is introduced from outside the cell, a gene can be knocked in the target DNA region by homologous recombination between the region and the donor DNA.

When the CRISPR-Cas system includes crRNA and tracrRNA (eg, in the case of the CRISPR-Cas9 system), the guide RNA may be a single-molecule guide RNA including crRNA and tracrRNA, or a bimolecular guide composed of a crRNA fragment and a tracrRNA fragment. It may be RNA.

The targeting base sequence in crRNA is usually a base sequence consisting of 12 to 50 bases, preferably 17 to 30 bases, more preferably 17 to 25 bases, and is adjacent to a PAM (proto-spacer adjent motif) sequence. It is selected to target a region. In the present invention, it has been found that the off-target effect can be reduced by using a short targeted base sequence in the guide RNA (FIGS. 14 and 15). Therefore, the chain length of the targeting base sequence is preferably less than 20 bases (eg, 19 bases, 18 bases, 17 bases) for this purpose.

に お い て In many CRISPR-Cas systems, crRNA further includes a base sequence capable of interacting (hybridizing) with tracrRNA on the 3 ′ side. On the other hand, tracrRNA contains on the 5 'side a base sequence capable of interacting (hybridizing) with a part of the base sequence of crRNA. The double-stranded RNA formed by the interaction of these nucleotide sequences interacts with the Cas protein.

PAM differs depending on the type and origin of Cas protein. Typical PAM sequences are described, for example, in In the Cas9 protein (type II) derived from P. pyogenes, it is "5'-NGG". In the Cas9 protein (type I-A1) derived from S. solfataricus, it is “5′-CCN”, In the case of Cas9 protein (type I-A2) derived from S. solfataricus, it is "5'-TCN". In the Cas9 protein (type IB) derived from Walsbyl, it is "5'-TTC", For the Cas9 protein (IE type) derived from E. coli, it is "5'-AWG", In the case of the Cas9 protein (type IF) derived from E. coli, it is "5'-CC", In the Cas9 protein (type IF) from Aeruginosa, it is “5′-CC”, In the case of Thermophilus-derived Cas9 protein (type II-A), it is "5'-NNAGAA". In the Cas9 protein (type II-A) derived from S. agalactiae, it is “5′-NGG”, aureus-derived Cas9 protein is "5'-NGRRT" or "5'-NGRRN"; In the Cas9 protein derived from P. meningitidis, it is "5'-NNNNNGATT", In Cas9 protein derived from denticola, it is "5'-NAAAAC".

As described above, it is also possible to modify PAM recognition by modifying the Cas protein (for example, introducing a mutation) (Benjamin, P. et al., Nature 523, 481-485 (2015), Hirano, S. et al., Molecular Cell 61, 886-894 (2016)). As a result, the choice of the target DNA region of the CRISPR-Cas system can be expanded.

に お い て In the present invention, by using a plurality of guide RNAs targeting different target DNA regions, the DNAs of different target DNA regions can be simultaneously edited.

The “CRISPR-Cas system” to be introduced into a cell may be, for example, in the form of a combination of a guide RNA and a Cas protein, or in the form of a combination of a guide RNA and a messenger RNA translated into a Cas protein. They may be in the form of vectors expressing them. The method for introducing the CRISPR-Cas system into cells is the same as the above-described method for introducing the fusion protein and Cas protein.

In the method of the present invention, the use of the donor DNA allows the desired DNA region to be exploited by utilizing the homologous recombination repair (HDR) generated in the target DNA region of the CRISPR-Cas system (the region around the cleavage site by the Cas protein). DNA can be inserted. In the process of homologous recombination repair, homology between the nucleotide sequence of the target DNA region and the nucleotide sequence of the donor DNA is required, and the donor DNA is used for template repair of the target DNA region including the cleavage site by the Cas protein. Resulting in the transfer of genetic information from the DNA to the target DNA region. Thereby, the base sequence of the target DNA region can be changed (for example, insertion, deletion, substitution, etc.). Therefore, the donor DNA is composed of two base sequences (homology arms) having a high degree of identity with the base sequence in the target DNA region and a desired DNA (a DNA to be inserted into the target DNA region) located between them. including.

The homology arm only needs to be large enough to carry out homologous recombination, and may vary depending on the form and chain length of the donor DNA. It is 1000 base pairs, and in the case of a single-stranded donor DNA, for example, 30 to 100 bases. The homology arm may not have 100% identity as long as it has an identity with the base sequence in the target DNA region to a degree sufficient for performing homologous recombination. For example, each has an identity of 95% or more, preferably 97% or more, more preferably 99% or more, and still more preferably 99.9% or more.

In addition, the length of the desired DNA existing between the homology arms is not particularly limited, and various sizes can be used. If the desired DNA contains a base sequence to be subsequently removed, for example, a recognition sequence of a recombinase (eg, a loxP sequence or an FRT sequence) may be added to both ends of the base sequence. . The base sequence sandwiched between the recognition sequences can be removed by the action of a recombinant enzyme (for example, a Cre recombinase or a FLP recombinase). For the purpose of confirming successful knock-in of DNA, for example, a selection marker sequence (for example, a fluorescent protein or a drug resistance gene) can be incorporated into desired DNA. Also, as the desired DNA, a gene operably linked to one or more regulatory elements can be used.

ド ナ ー The donor DNA used in the present invention may be a linear DNA or a circular DNA. Further, it may be single-stranded DNA or double-stranded DNA. Single-stranded DNA is preferred from the viewpoints of easy production and low cost, rapid reaction, increased efficiency of homologous recombination, and less unexpected integration.

-kit-
The present invention also provides a kit for use in the method of the present invention, wherein the fusion protein comprises an AcrIIA4 protein and a Cdt1 protein, a polynucleotide encoding the fusion protein, an expression vector containing the polynucleotide, or the expression vector. And a kit containing cells into which is introduced.

The fusion protein of the AcrIIA4 protein and the Cdt1 protein in the kit may be, for example, a form fused to the Cas protein via a self-cleaving peptide. That is, the fusion protein and Cas protein can be introduced into a cell or expressed as the same molecule.

When the fusion protein and the Cas protein are used as separate molecules, the kit of the present invention may include (i) a Cas protein, a polynucleotide encoding the protein, or an expression vector containing the polynucleotide. .

The kit of the present invention further comprises at least one of (ii) a guide RNA, a polynucleotide encoding the guide RNA, or an expression vector containing the polynucleotide, and (iii) a donor DNA, depending on the purpose. May be.

The kit of the present invention may further include one or more additional reagents. Examples of the additional reagents include a dilution buffer, a reconstitution solution, a washing buffer, a nucleic acid introduction reagent, and a protein introduction reagent. , A control reagent (eg, a control guide RNA), but is not limited thereto. The kit may include instructions for performing the method of the invention.

Hereinafter, the present invention will be described more specifically with reference to examples, but the present invention is not limited to the following examples.

A. Materials and Methods (1) Plasmid Construction Plasmid DNAs encoding AcrIIA4 and the FLAG tag gene were synthesized by Eurofin Genomics. The pFucci-G1 orange expression vector was purchased from the Institute for Medical Biology. The gRNA cloning vector and hCas9 were provided by George Church (Addgene plasmids # 41824 and 41815). AcrIIA4 fragment was produced by digesting the amplified DNA with BamHI and BstXI. This fragment was ligated to the pFucci-G1 orange expression vector at the N-terminus of hCdt1 (30/120) to construct AcrIIA4-hCdt1 (30/120) plasmid DNA. The AcrIIA4 fragment was amplified with CMV primers and Acr-REsite_XbaI_Rv and digested with BamHI and XbaI. This fragment was ligated to the pFucci-G1 orange vector, which was digested with BamHI and XbaI to construct AcrIIA4 plasmid DNA. To introduce NLS, primers BamHI_NLS-AcrIIA4_Fw and primer Acr-REsite_XbaI_Rv were used. The DNA fragment was amplified, digested with BamHI and XbaI, and inserted into the original vector, which was digested with BamHI and XbaI. A new plasmid DNA encoding AcrIIA4-2A-Cas9 or AcrIIA4-Cdt1-2A-Cas9 was constructed using Gibson assembly. AcrIIA4, AcrIIA4-Cdt1, and Cas9 fragments were amplified by PCR. Using a Gibson Assembly Master Mix (NEB), Notl-treated pEBMulti-Hyg (Fuji Film) and each fragment were inserted into a pEB vector.

ア ミ ノ 酸 The amino acid sequence of the fusion protein expressed by the constructed AcrIIA4-Cdt1 vector is shown in SEQ ID NO: 7, and the DNA encoded by this fusion protein is shown in SEQ ID NO: 8. The amino acid sequence of the fusion protein expressed by the constructed AcrIIA4-Cdt1-2A-Cas9 vector is shown in SEQ ID NO: 9, and the DNA encoded by the fusion protein is shown in SEQ ID NO: 10.

(2) Cell culture and transfection 293A cells (Invitrogen) were maintained at 37 ° C. in DMEM supplemented with 10% FBS and penicillin / streptomycin in a 5% CO ₂ atmosphere. After introducing episomal vectors encoding Cas9, AcrIIA4-2A-Cas9 or AcrIIA4-Cdt1-2A-Cas9 by Lipofectamine 3000, cells are selected using a 350 μg / mL hygromycin solution (Fuji Film) for 3 to 7 days. did. Lipofectamine 3000 (ThermoFisher Scientific) was used for Western blots and evaluation of plasmid content. Endogenous homologous recombination repair activity was assessed using a Neon® Transfection System 10 μL kit (Thermo Fisher Scientific). In Western blot analysis and time-lapse observation, 500 ng of plasmid DNA was transfected into 293A cells that had grown to 80-90% confluence by lipofection. To demonstrate inhibition at the target site, the repair template plasmid and the sgRNA plasmid (250 ng each) were transfected into 293A cells grown to 80-90% confluence by lipofection. In a homologous recombination repair assay using ssODN as a template, 50 pmol of ssODN and 250 ng of sgRNA plasmid were combined with 5 × 10 ⁴ using a pulse voltage of 1,245 V, a pulse width of 10 ms, and three pulses. Cells were transfected.

(3) Time-lapse observation of mKO-Cdt1 (30/120) 293A cells were seeded at a density of 4 × 10 ⁴ cells / well in a well of a 24-well plate (Greiner Bio One), and 10% FBS and penicillin / streptomycin were added. The cells were cultured for 24 hours at 37 ° C. in an atmosphere of 5% CO _{2 in} the contained high glucose DMEM (Wako). PFucci-G1 orange expression vector (500 ng) was transfected into 293A cells using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. Twenty-four hours after transfection, the transfected cells are plated in 35 mm glass bottom dishes (Greiner BioOne) using phenol red-free high glucose DMEM (Thermo Fisher Scientific) containing 10% FBS and penicillin / streptomycin. Seeded. mLO expression was monitored hourly using a FLUOVIEW FV10i microscope (Olympus) for 25 hours.

(4) Immunocytochemistry 293A cells were seeded in a well of a 24-well plate (Greiner Bio One) at a density of 4 × 10 ⁴ cells / well, and high glucose DMEM containing 10% FBS and penicillin / streptomycin (Wako) And cultured for 24 hours at 37 ° C. in an atmosphere of 5% CO ₂ . Cells were transfected with plasmid (500 ng) using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer's protocol. Twenty-four hours after transfection, the growth medium was replaced with fresh medium and the cells were cultured for another 24 hours. Transfected cells were seeded in 35 mm glass bottom dishes 24 hours prior to observation. Cells were fixed with a 4% formaldehyde solution of a 16% formaldehyde solution (Thermo Fisher Scientific) diluted in phosphate buffered saline (PBS) for 10 minutes at room temperature. Cells were permeabilized with 0.1% Triton X-100 (Calbiochem) at room temperature for 10 minutes and blocked with Blocking One (Nacalai Tesque) for 1 hour at room temperature. Thereafter, the cells were reacted with an anti-FRAG tag antibody (Sigma-Aldrich) for 1 hour at room temperature, and then reacted with an anti-mouse IgG (H + L) Cross-Adsorbed secondary antibody labeled with AlexaFluor 594 (Thermo Fisher Scientific) at room temperature for 30 hours. Allowed to react for minutes. Cell nuclei were stained with Hoechst 33258 (Dojindo Laboratories) at room temperature for 15 minutes. Observation of the stained cells was performed using the aforementioned FLUOVIEW FV10i microscope.

(5) Fluorescence-activated cell sorting (FACS) analysis 293A cells were seeded at a density of 1 × 10 ⁵ cells / well in a well of a 6 × well plate. Double thymidine block and nocodazole treatment was performed. For the double thymidine block, 5 μM thymidine was added and incubated for 18 hours. Thereafter, the medium was replaced with a fresh medium and cultured for 8 hours, followed by addition of 5 μM thymidine and cultured for 16 hours. For nocodazole treatment, 200 ng / mL of nocodazole was added and cultured for 18 hours. After both treatments, the cells were washed 3 times with PBS and the cells were released from the drug by adding fresh medium after the last wash. Cells were harvested every 3 hours (0 hour, 3 hours, 6 hours, 9 hours, 12 hours, 15 hours) and fixed on ice with 70% ethanol for 30 minutes. The fixed cells were washed twice with PBS, and stained with a propidium iodide solution (Funakoshi) containing RNase A (10 μg / mL) at room temperature for 30 minutes. Stained cells were analyzed by NovoCyte (ACEA Bioscience) and NovoExpress software.

(6) Western blot analysis 293A cells were seeded at a density of 4 × 10 ⁴ cells / well in wells of a 24-well plate. After 24 hours, cells were transfected with 500 ng of plasmid DNA. Twenty-four hours later, transfected cells were seeded in 10 cm ² dishes containing DMEM containing 350 μg / mL hygromycin B, and cells were selected during one week of culture. Cells were seeded at a density of 1 × 10 ⁵ cells / well in wells of a 6-well plate and treated with double thymidine block and nocodazole as described above. After these treatments, the cells were released from the drug by washing the cells three times with PBS and replacing the medium with fresh drug-free medium. Cells were collected every 3 hours after recovery (0 hour, 3 hours, 6 hours, 9 hours, 12 hours, 15 hours). The collected cells were lysed by culturing on ice for 30 minutes using RIPA buffer (Nacalai Tesque). Protein concentration was calculated using a commercially available protein assay (Bio-Rad) and an iMark microplate reader (Bio-Rad). Standard curves were generated using a pre-dilution protein assay standard (Thermo Fisher Scientific). The same amount of protein was loaded on a well of a Bolt 4-12% Bis-Tris Plus gel (Thermo Fisher Scientific) and electrophoresis was performed at 100 V for 1 hour. Degraded proteins were transferred to a PVDF membrane (Thermo Fisher Scientific) using an iBlot gel transfer device (Thermo Fisher Scientific) according to the manufacturer's protocol. The transfer film was treated with a blocking one solution (Nacalai Tesque) at room temperature for 1 hour. Next, a primary antibody against β-actin (Wako), FRAG-tag (Wako) and Cas9 (Clontech) (all diluted 1: 10,000) were combined with 0.05% Tween20 (TBS-T, TAKARA BIO INC.). ), And rinsed three times using Tris-buffered saline, followed by reaction at room temperature for 1 hour. Thereafter, a horseradish peroxidase-conjugated secondary antibody against anti-mouse IgG (Wako) and anti-rabbit IgG (Wako) were rinsed three times using TBS-T, and reacted at room temperature for 1 hour. After rinsing horseradish peroxidase three times, it was reacted with Clarity Western ECL substrate (Bio-Rad) for 1 hour at room temperature. Luminescence was detected using the ChemiDoc XRS + system (Bio-Rad).

(7) T7E1 Assay and Restriction Enzyme Assay 293A cells were seeded in 24-well plates at a density of 4 × 10 ⁴ cells / well. After 24 hours, cells were transfected with 500 ng of plasmid DNA. Twenty-four hours after transfection, transfected cells were seeded into wells of a 6-well plate with DMEM containing 350 μg / mL hygromycin B, and cells were selected during one week of culture. Transfected cells were seeded at a density of 4 × 10 ⁴ cells / well in wells of a 24-well plate. Cells were cultured for 24 hours and transfected with two types of plasmid DNA encoding sgRNA and template DNA. Forty-eight hours after transfection, genomic DNA was extracted using the QIAamp DNA mini kit (Qiagen). Genomic DNA (100 ng) was amplified using Hercules II fusion DNA polymerase (Agilent) with the T7E1 primer for each target. PCR conditions for several genes, AAVS1 target, EMX1 target and off-target, and vascular endothelial growth factor A (VEGFA) target and off-target, were at 95 ° C. for 3 minutes for initial denaturation and 98 ° C. for pre-amplification. 10 seconds, 72 ° C. to 62 ° C. (reduced by 1 degree per cycle) for 20 seconds, and 10 cycles of 72 ° C. for 30 seconds, amplification at 98 ° C. for 10 seconds, 62 ° C. for 20 seconds, and 72 ° C. at 30 ° C. Twenty-five cycles per second, and a final extension at 72 ° C. for three minutes. The product was stored in 3% dimethyl sulfoxide at 4 ° C. until use. PCR conditions for other genes were according to the manufacturer's manual. The PCR fragment DNA was purified using the QIAquick PCR Purification Kit (Qiagen). Fragment DNA (200 ng) was placed in a 19 μL solution containing 2 μL of 10 × NE buffer 2 (NEB) at 95 ° C. for 10 minutes, and a ramp rate of 0.1 ° C./sec from 95 ° C. to 25 ° C. And annealed to 4 ° C. 1 μL of T7 endonuclease 1 was added to the annealed DNA, and the mixture was cultured at 37 ° C. for 1 hour. The sample after the reaction was purified using a QIAquick PCR purification kit (Qiagen). DNA fragments were analyzed using MultiNA (Shimadzu Corporation). Indel efficiency was calculated as follows.

100 * (1- (1- (a + b) / (a + b + c)) ＾ (1/2))
Here, a and b indicate the area of the cleaved fragment, and c indicates the area of the uncleaved fragment.

In a restriction enzyme assay, 200 ng of the amplified DNA was combined with 0.5 μL of XhoI or HindIII (NEB) in CutSmart Buffer (NEB) and 1 × Bovine Serum Albumin (NEB) for 1 hour at 37 ° C. (XhoI) or 3 hours (HindIII). The sample after the reaction was purified by ethanol precipitation. DNA fragments were analyzed using MultiNA (Shimadzu Corporation). The indel efficiency of indel was calculated as follows.

100 * ((a + b) / (a + b + c))
Here, a and b indicate the area of the cleavage fragment, and c indicates the area of the non-cleaved fragment.

(8) Primer list The lists of primers used in this example are shown in Tables 1 and 2 below.

B. Results (1) Cell cycle-dependent expression of AcrIIA4-Cdt1 Cell cycle-dependent expression of the Cdt1 domain fused with the mKO fluorescent protein was observed using a confocal laser scanning microscope (CLSM). The 293A cells were transiently transfected with the expression plasmid Fucci vector (Sakaue-Sawano, A. et al. Cell 132, 487-498 (2008)). mKO expression was monitored using CLSM. Time-lapse observation of mKO expression suggested that the expression level changed in a time-dependent manner (FIG. 2).

Plasmid DNA encoding AcrIIA4 or AcrIIA4-Cdt1 (30/120) with NLS was constructed and used to transiently transfect 293A cells. 48 hours after transfection, the localization of AcrIIA4 and AcrIIA4-Cdt1 in the cell nuclei was confirmed by CLSM (FIG. 3).

(2) Effect of AcrIIA4-Cdt1 on indel introduction In order to demonstrate whether the Cdt1 (30/120) domain affects AcrIIA4 inhibitory activity, Cas9, sgRNA targeting the H2B gene, and AcrIIA4 / AcrIIA4-Cdt1 was co-expressed. Inhibitory activity was assessed by mutation analysis in the T7E1 assay. A decrease in the mutagenesis rate was observed when the molar ratio of AcrIIA4 / AcrIIA4-Cdt1 to Cas9 was increased (FIG. 4). When the molar ratio of AcrIIA4 or AcrIIA4-Cdt1 to Cas9 was 5: 1, the rate of mutagenesis inhibited by AcrAII and AcrIIA4-Cdt1 was 0.9%, respectively, from 8.0% when Cas9 was expressed alone. % And 3.7% (FIG. 5). The observed decrease in the rate of mutagenesis due to non-homologous end joining suggested that AcrIIA4-Cdt1 expression could be limited by cell cycle-dependent degradation of Cdt1.

(3) Effect of AcrIIA4-Cdt1 on genome editing using donor plasmid for precision editing Mutagenesis by non-homologous end joining was reduced by AcrIIA4-Cdt1, and AcrIIA4 alone could almost completely suppress mutagenesis It has been shown. In order to control the amount of Cas9 and AcrIIA4 DNA, a plasmid DNA encoding AcrIIA4-Cdt1 and Cas9 separated by a self-cleaving peptide sequence (T2A) was constructed (FIG. 6, left). Using this expression system, the amounts of AcrIIA4-Cdt1 and Cas9 could be strictly adjusted (FIG. 6, right). After cleavage of T2A, a proline residue was added to the N-terminus of Cas9, but some of Cas9 was exposed on the surface in the form of apotype or a complex with gRNA and target DNA, which affected the activity of Cas9. Had no effect. Since the plasmid is based on the episomal vector, the encoded protein can be stably expressed without being integrated into the host genome.

Cell cycle dependent expression of の AcrIIA4-Cdt1 fusion protein was analyzed by Western blot. After transient transfection of the plasmid encoding AcrIIA4-Cdt1-2A-Cas9, cells were selected with hygromycin. Surviving cells were treated with thymidine or nocodazole for synchronization. Expression of AcrIIA4 and Cas9 in cells obtained after release from drug treatment was confirmed by Western blot (FIG. 7). Decreased expression of AcrIIA4 fused to the Cdt1 domain was observed in S / G2 / M phase, but expression increased in G1 phase. The expression level of AcrIIA4 without the Cdt1 domain did not change over the cell cycle. Note that, contrary to AcrIIA4-Cdt1, Cas9 expressed from the same gene cassette did not show a correlation between the expression level and the cell cycle.

細胞 After transient transfection of episomal vectors for Cas9 and AcrIIA4 / AcrIIA4-Cdt1, cells were selected with hygromycin for 3-7 days. Surviving cells were transfected by lipofection with an AAVS1 site encoding plasmid and sgRNA targeting the donor template DNA. Since the donor template DNA encoded two recognition sites for sgRNA-Cas9, the plasmid appeared to have been cleaved intracellularly to form a double-stranded repair fragment. Insertion of the repair DNA sequence was confirmed by XhoI digestion 72 hours after transfection (FIG. 8). In the absence of sgRNA, no specific XhoI digestion indicating insertion of the repair sequence was observed. When AcrIIA4 and Cas9 were co-expressed, no XhoI digested band was observed. This means that sgRNA-Cas9 cleavage was completely suppressed. In the presence of Cas9 alone, an XhoI digested band was observed, and the efficiency of fine editing was estimated to be 1.6%. For co-expression of AcrIIA4-Cdt1 and Cas9, the efficiency increased to 2.0%. These results indicated that cell cycle-dependent activation of Cas9 increased the efficiency of homologous recombination repair.

標的 To assess the ratio of efficiency between homologous recombination repair and non-homologous end joining, targeted mutagenesis via non-homologous end joining was assessed in a T7E1 assay (FIG. 9). The target mutagenesis rates were estimated to be 1.5% and 0.8% for Cas9 alone and for co-expression of Cas9 with AcrIIA4-CdtI, respectively. Co-expression of AcrIIA4 and Cas9 completely suppressed mutagenesis due to non-homologous end joining. The increase in homologous recombination repair efficiency and the decrease in mutagenesis due to non-homologous end joining for the combination of AcrIIA4-Cdt1 and Cas9 compared to the case using Cas9 alone indicate that Cas9 has cell-cycle activation. It can be seen that fine genome editing was achieved using 293 cells.

に 関 し て Regarding the accuracy of genome editing, the reduction of off-target effects is another important issue, especially for clinical use or animal and plant breeding and cultivation. Several methods have been developed and reported to reduce off-target effects. These include direct delivery of the sgRNA-Cas9 complex and the shortened seed sequence of the sgRNA. These results indicated the importance of limiting Cas9 activity in cells. Furthermore, in addition to cleavage at off-target sites similar to the target, the uncertain repair process due to non-homologous end joining may be another key, as it leads to the repair of mutant sequences. We hypothesized that suppression of Cas9 activity in the cell cycle where heterologous end joining is dominant could suppress off-target effects. Off-target effects were also addressed by the T7E1 assay using the AAVS1 target site (FIG. 10). As an off-target candidate, the MYBPC2 gene having a 2-base mutation compared to the AAVS1 target sequence was selected. At this site, expression with Cas9 alone showed an off-target rate of 8.3%. Surprisingly, no band showing off-target mutations due to T7E1 digestion was observed in co-expression of AcrIIA4-Cdt1 and Cas9. Furthermore, co-expression of AcrIIA4 and Cas9 showed no on-target and off-target mutations.

(4) Precise genome editing by homologous recombination repair using ssODN The use of ssODN for improving homologous recombination repair efficiency in cell cycle-dependent Cas9 activation was verified. Cells were transfected with a plasmid encoding Cas9, AcrIIA4-2A-Cas9, or AcrIIA4-Cdt1-2A-Cas9. Following selection with hygromycin, further transfection of the sgRNA encoding plasmid and ssODN template DNA was performed by electroporation. At 72 hours after the second transfection, on-target and off-target mutations via non-homologous end joining and fine editing via homologous recombination repair were analyzed by HindIII or T7E1 digestion. Three sgRNA target sites were examined for three genes, AAVS1, EMX1, and VEGFA. For AAVS1, when AcrIIA4-Cdt1 was expressed with Cas9 compared to Cas9 alone expression, the precision editing efficiency was increased about 1.4-fold. These results were similar to those of the previous experiment using the plasmid DNA template, but the efficiency values increased to 10.8% and 7.8%, respectively (FIG. 11). Similarly, the efficiency of mutagenesis at the target sequence via non-homologous end joining also increased to 36.6% and 27.1% for Cas9 alone and co-expression of AcrIIA4-Cdt1 and Cas9, respectively. AcrIIA4 almost completely suppressed targeted mutagenesis, up to 0.6%. Co-expression of AcrIIA4-Cdt1 and Cas9 suppressed target mutation by 74%. When Cas9 was expressed alone, off-target mutagenesis in the MYBPC2 gene was 3.4%, but was efficiently reduced to 0.6% when AcrIIA4-Cdt1 was used.

Another sgRNA targeting the EMX1 gene or VEGFA gene was used to determine if the results were similar at other target sites (FIGS. 12, 13). As a result, as in the case of the AAVS1 gene, accurate editing of the target was significantly more pronounced when AcrIIA4-Cdt1 was expressed with Cas9 compared to when only Cas9 was expressed, while the mutation at the off-target site was increased. The rate decreased significantly.

(5) Reduction of off-target effects by combining with truncated sgRNA To reduce off-target mutations, truncated sgRNA was applied to AcrIIA4-Cdt1. Two new truncated sgRNAs targeting the EMX1 and VEGFA genes were constructed and the effect of reducing off-target mutations was evaluated using the T7E1 assay. Regardless of whether the EMX1 gene or the VEGFA gene was targeted, the use of truncated sgRNA significantly reduced off-target mutations, but did not reduce the efficiency of accurate editing of the target (FIGS. 14 and 15). .

As described above, according to the present invention, it is possible to regulate the endonuclease activity of Cas protein in a cell cycle-dependent manner. Further, in the present invention, if the CRISPR-Cas system is used together with the donor DNA, the efficiency of accurate genome editing by repair of homologous recombination can be increased, and the off-target effect due to non-homologous end joining can be suppressed. The present invention relates to genome editing techniques including medical fields such as regenerative medicine, agricultural fields such as creation of crops having useful traits, industrial fields such as production of useful substances using microorganisms, and research fields such as creation of experimental animals. Can contribute to a wide range of available fields.

SEQ ID NO: 7: amino acid sequence of the fusion protein SEQ ID NO: 8: base sequence encoding the fusion protein SEQ ID NO: 9: amino acid sequence of the fusion protein SEQ ID NO: 10: nucleotide sequence encoding the fusion protein SEQ ID NOS: 11 to 53: primer

Claims (13)

A method for regulating the endonuclease activity of a Cas protein in a cell cycle-dependent manner in a cell,
(I) providing a fusion protein of AcrIIA4 protein and Cdt1 protein; and (ii) providing a cell containing Cas protein.
A method in which the fusion protein suppresses the endonuclease activity of the Cas protein in the cell in a cell cycle-dependent manner. The method according to claim 1, wherein the Cdt1 protein in the fusion protein is a partial peptide containing an amino acid sequence that undergoes E3 ubiquitin ligase-mediated proteolysis. The method according to claim 1 or 2, wherein the cell comprises a protein in which the fusion protein and the Cas protein are fused via a self-cleaving peptide. 4. The method according to claim 1, wherein the Cas protein is a Cas9 protein. A method for producing a cell in which DNA has been edited,
(I) providing a cell comprising a fusion protein of AcrIIA4 protein and Cdt1 protein; and (ii) a cell comprising a CRISPR-Cas system,
A method in which the fusion protein suppresses the endonuclease activity of the Cas protein in the CRISPR-Cas system in a cell cycle-dependent manner in the cell, whereby the DNA in the cell is edited in a cell cycle-dependent manner. The method according to claim 5, wherein the Cdt1 protein in the fusion protein is a partial peptide containing an amino acid sequence that undergoes E3 ubiquitin ligase-mediated proteolysis. The method according to claim 5 or 6, wherein the cell comprises a protein in which the fusion protein and the Cas protein are fused via a self-cleaving peptide. The method according to any one of claims 5 to 7, wherein the cell further comprises a donor DNA. 9. The method according to claim 8, wherein the donor DNA is a single-stranded donor DNA. 10. The method according to any one of claims 5 to 9, wherein in the guide RNA constituting the CRISPR-Cas system, the base sequence complementary to the base sequence of the target DNA region is less than 20 bases in length. The method according to any one of claims 5 to 10, wherein the Cas protein is a Cas9 protein. A kit for use in the method according to any one of claims 1 to 11, wherein the fusion protein of the AcrIIA4 protein and the Cdt1 protein, a polynucleotide encoding the fusion protein, an expression vector containing the polynucleotide, or the expression vector is introduced. Kit containing the isolated cells. The kit according to claim 12, further comprising at least one of the following (i) to (iii):
(I) Cas protein, polynucleotide encoding the protein, or expression vector containing the polynucleotide (ii) guide RNA, polynucleotide encoding the guide RNA, or expression vector containing the polynucleotide (iii) donor DNA

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