Methods for producing fusion proteins, nucleic acids, cells, and animals

Abstract

To provide a modified Cas9 that improves the knockout efficiency by disabling only specific genes and the knockin efficiency by introducing foreign genes into mice or specific gene loci.SOLUTION: A fusion protein comprising a Cas9 protein and a modified peptide that modifies the Cas9 protein, where the modified peptide is a peptide consisting of a specific amino acid sequence, or a peptide consisting of the specific amino acid sequence in which one or several amino acids are deleted, substituted, or added therefrom, and by forming the fusion protein from the Cas9 protein a peptide is formed that has the activity of localizing Cas9 protein to the nucleus.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to methods for producing fusion proteins, nucleic acids, cells and animals. [Background technology]

[0002] Knockout (hereinafter sometimes referred to as "KO") mice, in which only specific genes have been disabled, and knockin (hereinafter sometimes referred to as "KI") mice, in which an exogenous gene has been introduced into a specific gene locus, are used in many medical and life science research projects because they allow direct evaluation of gene function in vivo.

[0003] Traditionally, KO and KI mice have been produced by gene targeting using embryonic stem (ES) cells. However, the production of these mice was problematic in that it was very expensive and took years to complete. Genome editing technology has solved this problem.

[0004] Genome editing can knock out a target gene by introducing into cells an artificial restriction enzyme that recognizes and cuts only a specific DNA sequence. It is also possible to knock out an exogenous gene at the target locus by simultaneously introducing donor DNA containing the exogenous gene flanked by sequences adjacent to the cutting site into cells together with the artificial restriction enzyme.

[0005] Currently, the most promising artificial restriction enzyme that can be used for genome editing is the Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR Associated Protein 9 (hereinafter referred to as "CRISPR-Cas9") system.

[0006] Among CRISPR-Cas9, CRISPR-Cas9 (hereinafter sometimes referred to as "SpCas9") derived from Streptococcus pyogenes (hereinafter sometimes referred to as "Sp") is used in many studies because of its ability to cut DNA simply and efficiently.

[0007] The Sp-CRISPR-Cas9 system is an RNA-protein complex consisting of a single guide RNA (hereinafter sometimes referred to as "sgRNA") that recognizes a target sequence and the Cas9 protein, which primarily functions to cleave that target sequence. KO mice can be obtained by simultaneously introducing these sgRNA and Cas9 protein into mouse fertilized eggs.

[0008] In addition, two different sgRNAs can be used to simultaneously cut upstream and downstream of a target gene region, resulting in excision of the region from the chromosome (excision-type knockout). This process utilizes non-homologous end-joining (NHEJ), which is prone to errors. This method makes it possible to excise millions of base pairs, which was previously difficult to achieve with conventional gene targeting methods.

[0009] Furthermore, by simultaneously introducing sgRNA, Cas9 protein, and donor DNA into fertilized eggs, the break site can be repaired by homology-directed repair (HDR) using the donor DNA as a template, and KI mice can be obtained.

[0010] Previous studies have shown that HDR activity is not observed in the G1 phase, increases rapidly in the S phase, and decreases in the G2 / M phase. Furthermore, NHEJ activity has been shown to occur throughout the cell cycle. Therefore, efforts are being made to control the presence of the Cas9 protein in a cell cycle-specific manner and to suppress the introduction of unintended insertion or deletion mutations (indels) by NHEJ.

[0011] For example, human Geminin protein is known to be degraded in the G1 phase. Human Cdt1 protein is also known to be degraded in the S / G2 phase. Non-Patent Document 1 describes the production of a fusion protein in which a portion of human Geminin protein is linked to Cas9 (hereinafter, sometimes referred to as "SpCas9-hGem"), and a fusion protein in which a portion of human Cdt1 protein is linked to Cas9 (hereinafter, sometimes referred to as "SpCas9-hCdt1").

[0012] Non-Patent Document 1 also describes that SpCas9-hGem was degraded in the G1 phase, and SpCas9-hCdt1 was degraded in the S / G2 phase (Non-Patent Document 1, Fig. 1C, etc.).

[0013] Non-Patent Document 1 also describes that the KI efficiency at the DNMT3B locus using conventional SpCas9 was 17.1%, the KI efficiency using SpCas9-hGem was 16.5%, and the KI efficiency using SpCas9-hCdt1 was 9.9% (Non-Patent Document 1, Fig. 2C, etc.). [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Howden SE, et, al., A Cas9 Variant for Efficient Generation of Indel-Free Knockin or Gene-Corrected Human Pluripotent Stem Cells, Stem Cell Reports, Vol. 7, 508-517, 2016. Summary of the Invention [Problem to be solved by the invention]

[0015] The generation of KO and KI mice by genome editing of fertilized eggs is widely used in research facilities because it significantly reduces costs and time compared to methods using ES cells. However, the efficiency of generating excision-type KO mice and HDR-dependent KI mice is low. Therefore, the present invention aims to provide a modified Cas9 that improves KO and KI efficiency. [Means for solving the problem]

[0016] The present invention includes the following aspects. [1] A fusion protein comprising a Cas9 protein and a modifying peptide that modifies the Cas9 protein, wherein the modifying peptide is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 29, or an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 29, and which has the activity of localizing the Cas9 protein to the nucleus by forming a fusion protein with the Cas9 protein. [2] The fusion protein according to [1], wherein the modified peptide comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted, or added, and which has the activity of localizing the Cas9 protein to the nucleus by forming a fusion protein with the Cas9 protein. [3] The fusion protein according to [1] or [2], wherein the modified peptide further comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, or a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted, or added, and which has the activity of degrading the Cas9 protein in the G1 phase by forming a fusion protein with the Cas9 protein. [4] The fusion protein according to any one of [1] to [3], wherein the modified peptide is located at the N-terminus, C-terminus, or a site other than the N-terminus or C-terminus of the Cas9 protein. [5] A nucleic acid encoding the fusion protein according to any one of [1] to [4]. [6] A method for producing a cell whose genomic DNA has been edited, comprising contacting the fusion protein according to any one of [1] to [4] with genomic DNA in the cell. [7] The method of manufacturing described in [6], wherein the cell is a fertilized egg. [8] A method for producing an animal whose genomic DNA has been edited, comprising: contacting the fusion protein according to any one of [1] to [4] with genomic DNA in a fertilized egg to obtain a fertilized egg whose genomic DNA has been edited; and growing the fertilized egg whose genomic DNA has been edited into an individual to obtain an animal whose genomic DNA has been edited. [Effects of the Invention]

[0017] The present invention provides modified Cas9 that improves KO and KI efficiency. [Brief explanation of the drawings]

[0018] [Figure 1] (a) is a diagram aligning the amino acid sequence of positions 1 to 110 of human Geminin protein with the amino acid sequence of positions 1 to 107 of mouse Geminin protein. (b) is a diagram aligning the amino acid sequence of positions 30 to 120 of human Cdt1 protein with the amino acid sequence of positions 29 to 132 of mouse Cdt1 protein. (c) The top diagram is a schematic diagram showing the structure of the pX330 vector, the middle diagram is a schematic diagram showing the structure of the pX330-mG vector, and the bottom diagram is a schematic diagram showing the structure of the pX330-mC vector. [Figure 2] 1(a) to 1(d) are fluorescent micrographs showing the results of immunostaining in Experimental Example 2. [Figure 3] 1(a) is a schematic diagram illustrating the method for excising the Tyr gene in Experimental Example 3. FIG. 1(b) and FIG. 1(c) are photographs showing representative mice obtained in Experimental Example 3. [Figure 4] FIG. 10 is a schematic diagram illustrating the method for excising the Dmd gene in Experimental Example 4. [Figure 5](a) is a schematic diagram showing the structure of the wild-type ROSA26 locus. (b) is a schematic diagram showing the structure of the pRosa-CAG-fEGFP-Cables1 donor DNA plasmid used in Experimental Example 5. (c) is a schematic diagram showing the structure of the ROSA26 locus when the desired knock-in occurred in Experimental Example 5. [Figure 6] (a) is a schematic diagram showing the structure of the wild-type Prdm14 locus, (b) is a schematic diagram showing the structure of the pflox-Prdm14 donor DNA plasmid used in Experimental Example 6, and (c) is a schematic diagram showing the structure of the Prdm14 locus when the desired knock-in has occurred in Experimental Example 6. [Figure 7] 10(a) to 10(c) are fluorescence micrographs showing the results of immunostaining in Experimental Example 7. [Figure 8] (a) is a schematic diagram showing the structure of the pX330-mC vector. (b) is a schematic diagram showing the structure of the pX330-pFmC vector. (c) is a schematic diagram showing the structure of the pX330-pCmC vector. (d) is a schematic diagram showing the structure of the pX330-pMmC vector. (e) is a schematic diagram showing the structure of the pX330-pNmC vector. (f) is a schematic diagram showing the structure of the pX330-pNNmC vector. (g) is a schematic diagram showing the structure of the pX330-pNCmC vector. [Figure 9] 10 is a fluorescence microscope photograph showing the results of immunostaining in Experimental Example 9. [Figure 10] (a) is a photograph showing the results of detecting each Cas9 fusion protein with an anti-FLAG antibody in Experimental Example 10. (b) is a photograph showing the results of detecting each Cas9 fusion protein with an anti-Cas9 antibody in Experimental Example 10. (c) is a photograph showing the results of detecting PARP1, a nuclear protein, with an anti-PARP1 antibody in Experimental Example 10. (d) is a photograph showing the results of detecting GAPDH, a cytoplasmic protein, with an anti-GAPDH antibody in Experimental Example 10. [Figure 11] This figure summarizes the structure and nuclear transport ability of each Cas9 fusion protein based on the results of Experimental Examples 9 and 10. [Figure 12]10 is a fluorescence microscope photograph showing the results of immunostaining in Experimental Example 11. DETAILED DESCRIPTION OF THE INVENTION

[0019] [Fusion protein] In one embodiment, the present invention provides a fusion protein comprising a Cas9 protein and a modifying peptide that modifies the Cas9 protein, wherein the modified peptide is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 or an amino acid sequence in which one or more amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1, and which forms a fusion protein with the Cas9 protein and has the activity of localizing the Cas9 protein to the nucleus.

[0020] Examples of Cas9 proteins include those derived from Streptococcus pyogenes, Staphylococcus aureus, Streptococcus thermophilus, Geobacillus stearothermophilus, etc. Among these, Cas9 derived from Streptococcus pyogenes (SpCas9) can be preferably used.

[0021] An example of a modifying peptide that modifies a Cas9 protein is a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1. The peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 is a peptide consisting of the amino acid sequence from positions 29 to 132 of mouse Cdt1 protein.

[0022] Cdt1 is one of the licensing regulators that ensures that chromosomes are replicated exactly once during the cell cycle. It is known that Cdt1 expression is high during the G1 phase and decreases during the S phase due to ubiquitin-dependent degradation.

[0023] As described in Non-Patent Document 1, the abundance of a fusion protein (SpCas9-Cdt1) linking Cas9 with a portion of the human Cdt1 protein is known to be cell cycle dependent and to be degraded during the S / G2 phase.

[0024] However, as described later in the Examples, the fusion protein of this embodiment, in which Cas9 is fused with a portion of the mouse Cdt1 protein (hereinafter sometimes referred to as "Cas9-mC"), was found to be unexpectedly not cell cycle dependent and to be localized to the nucleus in all cell cycles. Nuclear localization was confirmed not only in mouse cells but also in human cells.

[0025] Furthermore, as described later in the Examples, it has been demonstrated that genome editing using Cas9-mC can efficiently produce mice with large-scale genome deletions ranging from tens of kilobase pairs to several megabase pairs, and that knock-in mice and floxed mice, which are difficult to produce, can also be produced efficiently.

[0026] The modified peptide is not limited to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, and may contain mutations as long as it has the activity of localizing the Cas9 protein to the nucleus by forming a fusion protein with the Cas9 protein.

[0027] Specifically, it may be a peptide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1. Here, "one or several" may be, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acids.

[0028] As described later in the Examples, the inventors have demonstrated that a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 29, which is used as a modifying peptide for modifying the Cas9 protein, also maintains the activity of localizing the Cas9 protein to the nucleus. The peptide consisting of the amino acid sequence set forth in SEQ ID NO: 29 is a peptide consisting of the amino acid sequence from positions 29 to 80 of the mouse Cdt1 protein.

[0029] The peptide consisting of the amino acid sequence set forth in SEQ ID NO: 29 is shorter than the peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1. This allows the molecular weight of the fusion protein to be reduced. It also allows the size of the expression vector for the fusion protein to be reduced. This makes it possible to provide a fusion protein or a nucleic acid encoding the fusion protein that is easier to use.

[0030] The modified peptide is not limited to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 29, and may contain mutations as long as it has the activity of localizing the Cas9 protein to the nucleus by forming a fusion protein with the Cas9 protein.

[0031] Specifically, it may be a peptide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 29. Here, "one or several" may be, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acids.

[0032] Hereinafter, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1 in which one or several amino acids have been deleted, substituted or added, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 29, and a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 29 in which one or several amino acids have been deleted, substituted or added may be referred to as a "mouse Cdt1-derived peptide."

[0033] In the fusion protein of this embodiment, the modified peptide may further comprise a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2. The amino acid sequence set forth in SEQ ID NO: 2 is a peptide consisting of the amino acid sequence of positions 1 to 107 of mouse Geminin protein.

[0034] Geminin is a licensing inhibitor that inhibits the binding of licensing factors to the origin of genome replication once replication has initiated during the S phase. Geminin expression is known to be high during the S, G2, and M phases, but decreases during the G1 phase due to ubiquitin-dependent degradation.

[0035] As described below in the Examples, a fusion protein formed by fusing Cas9 with a portion of the mouse Geminin protein (hereinafter sometimes referred to as "Cas9-mG") exhibits cell cycle dependence, being present in the cytoplasm from the S phase to the early G2 phase, and being degraded from the late G1 phase to the early S phase.

[0036] Therefore, when the modified peptide further contains a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, it can be degraded during the G1 phase by ubiquitin-dependent degradation, thereby suppressing NHEJ activity during the G1 phase and preventing the introduction of unintended indel mutations during knock-in by genome editing.

[0037] The peptide further contained in the modified peptide is not limited to a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, and may contain a mutation as long as it has the activity of degrading the Cas9 protein during the G1 phase by forming a fusion protein with the Cas9 protein.

[0038] Specifically, it may be a peptide consisting of an amino acid sequence in which one or several amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 2. Here, "one or several" may be, for example, 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 to 3 amino acids.

[0039] Hereinafter, a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, or a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 with one or several amino acids deleted, substituted, or added, may be referred to as a "Geminin-derived peptide." A Geminin-derived peptide may have a partial amino acid sequence of human Geminin protein.

[0040] In the fusion protein of this embodiment, the position of the modified peptide is not particularly limited. For example, the modified peptide may be located at the N-terminus or C-terminus of the Cas9 protein, or at a site other than the N-terminus or C-terminus.

[0041] Furthermore, in the fusion protein of this embodiment, the mouse Cdt1-derived peptide and the Geminin-derived peptide may be adjacent to each other or may be separated from each other. Furthermore, either the mouse Cdt1-derived peptide or the Geminin-derived peptide may be located at the N-terminus. That is, the Geminin-derived peptide may be located at the C-terminus of the mouse Cdt1-derived peptide, or the mouse Cdt1-derived peptide may be located at the C-terminus of the Geminin-derived peptide.

[0042] Furthermore, when the mouse Cdt1-derived peptide and the Geminin-derived peptide are positioned apart, the positions of the mouse Cdt1-derived peptide and the Geminin-derived peptide may, independently, be the N-terminus, the C-terminus, or a site other than the N-terminus or C-terminus of the Cas9 protein.

[0043] For example, a Geminin-derived peptide may be placed at the N-terminus of the Cas9 protein, and a mouse Cdt1-derived peptide may be placed at the C-terminus of the Cas9 protein, or alternatively, a mouse Cdt1-derived peptide may be placed at the N-terminus of the Cas9 protein, and a Geminin-derived peptide may be placed at the C-terminus of the Cas9 protein.

[0044] Alternatively, one of the mouse Cdt1-derived peptide and the Geminin-derived peptide may be located at the N-terminus or C-terminus of the Cas9 protein, and the other may be located in the central portion.

[0045] As used herein, the term "N-terminus" may include the vicinity of the N-terminus. Furthermore, the term "C-terminus" may include the vicinity of the C-terminus. In other words, even if a modified peptide is not adjacent to the first amino acid from the N-terminus of the Cas9 protein, if the modified peptide is located near the N-terminus of the Cas9 protein, the position of the modified peptide may be referred to as the N-terminus of the Cas9 protein.

[0046] Similarly, even if the modified peptide is not adjacent to the first amino acid from the C-terminus of the Cas9 protein, if the modified peptide is located near the C-terminus of the Cas9 protein, the position of the modified peptide may be said to be the C-terminus of the Cas9 protein.

[0047] Here, "nearby" may mean, for example, a distance of 1 to 100 amino acids, a distance of 1 to 50 amino acids, a distance of 1 to 30 amino acids, or a distance of 1 to 20 amino acids.

[0048] The location of the modified peptide may be at a site other than the N-terminus or C-terminus of the Cas9 protein, for example, in the central portion of the Cas9 protein.

[0049] The fusion protein of this embodiment may contain an additional peptide in addition to the Cas9 protein and modified peptide, as long as the effects of the present invention are achieved. Examples of additional peptides include tag peptides such as FLAG tag, Hisx6 tag, MYC tag, HA tag, and V5 tag; fluorescent proteins such as green fluorescent protein (GFP) and GFP derivatives; peptides introduced during genetic recombination, such as peptides derived from the multicloning site of a vector or peptides derived from part of a primer; and nuclear localization signals (NLS) originally incorporated into the vector.

[0050] In this specification, the terms "protein" and "peptide" are used without strict distinction, and may be referred to as either a peptide or a protein depending on the number of amino acids.

[0051] [Nucleic acid] In one embodiment, the present invention provides a nucleic acid encoding the above-described fusion protein. By introducing the nucleic acid of this embodiment into cells, the above-described fusion protein can be expressed. Furthermore, by transcribing the nucleic acid of this embodiment in an in vitro transcription reaction system, mRNA encoding the above-described fusion protein can be obtained.

[0052] The nucleic acid of this embodiment may be an expression vector. The expression vector is not particularly limited, and examples thereof include transposon vectors, viral vectors, episomal vectors, and plasmid vectors.

[0053] [Method for producing cells with edited genomic DNA] In one embodiment, the present invention provides a method for producing a cell having edited genomic DNA, comprising contacting the fusion protein described above with genomic DNA in the cell.

[0054] The fusion protein described above is contacted with genomic DNA together with a gRNA corresponding to the target sequence. More specifically, the fusion protein described above is introduced into a cell together with the gRNA, allowing it to contact with genomic DNA in the cell. As a result, a DNA double-strand break (DSB) can be formed at the target sequence.

[0055] Examples of cells include ES cells, iPS cells, fertilized eggs, cultured cells, etc. Examples of cultured cells include animal cells, insect cells, plant cells, yeast cells, etc.

[0056] When the cell is a fertilized egg, a fertilized egg with edited genomic DNA can be obtained. By growing this fertilized egg into an individual, an animal with edited genomic DNA can be obtained. The animal is not particularly limited, and examples thereof include mammals such as mice, rats, sheep, pigs, monkeys, and humans, birds such as chickens, amphibians such as African clawed frogs, reptiles such as geckos, fish such as zebrafish and medaka, and insects such as silkworms.

[0057] The fusion protein and gRNA can be introduced into cells by lipofection, microinjection, electroporation, or the like.

[0058] The gRNA may be a complex of CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA), or a single gRNA (sgRNA) that combines tracrRNA and crRNA.

[0059] The gRNA may be introduced into cells in the form of RNA or in the form of an expression vector. Methods for preparing the gRNA in the form of RNA include synthesizing the gRNA by in vitro transcription reaction using a construct in which a promoter such as T7 is added upstream of the nucleic acid fragment encoding the gRNA, and chemically synthesizing the gRNA. When chemically synthesizing the gRNA, chemically modified RNA may be used.

[0060] When preparing gRNA in the form of an expression vector, a plasmid vector or a viral vector that transcribes gRNA from a Pol III promoter such as the H1 promoter or U6 promoter can be used. When expressing gRNA using an expression vector, gRNA may be expressed constitutively or under the control of an inducible promoter.

[0061] The fusion protein may be introduced into donor cells in the form of an expression vector that is expressed from a Pol II promoter, or in the form of a purified protein. Examples of expression vectors for fusion proteins include transposon vectors, viral vectors, episomal vectors, and plasmid vectors.

[0062] Indel mutations can be introduced during the NHEJ repair process of the DSBs. Alternatively, two different sgRNAs can be used to simultaneously cut upstream and downstream of the target gene region, thereby excising the region from the chromosome.

[0063] Alternatively, donor DNA may be introduced into cells together with the fusion protein and gRNA. The donor DNA preferably contains a 5' homology arm region, a target nucleotide sequence region, and a 3' homology arm region. In this case, DSBs are repaired by homology-directed repair (HDR) using the donor DNA as a template, allowing the target nucleotide sequence region in the donor DNA to be knocked in. Alternatively, donor DNA can be knocked in by methods that do not rely on HDR, such as the PITCh method or the HITI method.

[0064] [Method for producing animals with edited genomic DNA] In one embodiment, the present invention provides a method for producing an animal whose genomic DNA has been edited, comprising: contacting the fusion protein described above with genomic DNA in a fertilized egg to obtain a fertilized egg whose genomic DNA has been edited; and growing the fertilized egg whose genomic DNA has been edited into an individual to obtain an animal whose genomic DNA has been edited.

[0065] The process for obtaining a fertilized egg with edited genomic DNA is the same as that for the case where a fertilized egg is used as the cell in the above-mentioned method for producing a cell with edited genomic DNA. By growing this fertilized egg into an individual, an animal with edited genomic DNA can be obtained. Examples of animals include those mentioned above.

[0066] The method for growing a genomic DNA-edited fertilized egg into an individual can be selected appropriately depending on the animal species. For example, in the case of mammals, the genomic DNA-edited fertilized egg can be transplanted into the oviduct or uterus of a surrogate mother and allowed to develop, allowing it to grow into an individual. In addition, in the case of birds, amphibians, reptiles, fish, insects, etc., the genomic DNA-edited fertilized egg can be cultured and developed in an appropriate environment, allowing it to grow into an individual. [Example]

[0067] The present invention will now be described in more detail with reference to examples, but the present invention is not limited to the following examples.

[0068] Materials and Methods (mouse) C57BL / J mice were purchased from Charles River Japan. ICR mice (Charles River Japan) were used as pseudopregnant mice for mouse generation. Mice were housed under a room temperature of 23.5°C ± 2.5°C, humidity of 52.5°C ± 12.5%, and a light / dark cycle of 14 hours light and 10 hours dark.

[0069] (Creation of mice) Female C57BL / 6J mice aged 12–20 weeks were subcutaneously injected with 5 units of pregnant mare serum gonadotropin, followed 48 hours later by intraperitoneal injection of 5 units of human chorionic gonadotropin, and mated with male C57BL / 6J mice. Mating was confirmed by the presence of a vaginal plug, and fertilized eggs (pronuclear stage) were collected from the oviduct. Frozen fertilized eggs and in vitro fertilized eggs were not used. The pX330, pX330-mG, and pX330-mC vectors were diluted to 5 ng / μL with sterile distilled water and filtered through a 0.22 μm pore PVDF membrane before use. Various donor vectors were diluted to 10 ng / μL with sterile distilled water and filtered through a 0.22 μm pore PVDF membrane before use. Each vector was microinjected into the male pronucleus of pronuclear stage embryos. Fertilized eggs that survived 15 minutes to 2 hours after microinjection were transferred into the oviducts of pseudopregnant mice and allowed to develop into individuals.

[0070] (Genotype analysis) Genomic DNA was extracted and purified from the tip of mouse tails (lengths of 0.5 mm or less) using a PI-200 genome extraction instrument and used for genotyping. KI and floxed alleles were confirmed by PCR using PrimeSTAR GXL DNA Polymerase (TAKARA). KO alleles were confirmed by PCR using AmpliTaq Gold 360 Master Mix (Thermo Fisher Scientific).

[0071] [Experimental Example 1] (Construction of Cas9 fusion protein expression vector 1) pX330 (Plasmid #42230, Addgene) is a vector capable of expressing both sgRNA and Cas9. The structure of the pX330 vector is shown in the upper part of Figure 1(c). In the pX330 vector, sgRNA is expressed by the U6 promoter. SpCas9 protein is expressed by the CBh promoter. The SpCas9 protein contains nuclear localization signals (NLS) derived from the SV40 virus at its N- and C-termini. Furthermore, a 3xFLAG tag peptide is linked to the N-terminal side of the N-terminal NLS.

[0072] We constructed an expression vector for a Cas9 fusion protein based on the pX330 vector. More specifically, we constructed a vector (SEQ ID NO: 5, hereinafter referred to as "pX330-mG") that expresses a fusion protein (hereinafter referred to as "Cas9-mG") in which a peptide consisting of residues 1 to 107 of the mouse Geminin protein, not human, is linked to the C-terminus of the SpCas9 protein.

[0073] Figure 1(a) shows the amino acid sequence of amino acids 1 to 110 of human Geminin protein (SEQ ID NO: 4) aligned with the amino acid sequence of amino acids 1 to 107 of mouse Geminin protein (SEQ ID NO: 2). The middle section of Figure 1(c) shows the structure of the pX330-mG vector.

[0074] We also constructed a vector (sequence number 6, hereinafter sometimes referred to as "pX330-mC") that expresses a fusion protein (hereinafter sometimes referred to as "Cas9-mC") in which a peptide consisting of residues 29 to 132 of the Cdt1 protein derived from mouse, rather than human, is linked to the C-terminus of the SpCas9 protein.

[0075] Figure 1(b) shows the amino acid sequence of amino acids 30 to 120 of the human Cdt1 protein (SEQ ID NO: 3) aligned with the amino acid sequence of amino acids 29 to 132 of the mouse Cdt1 protein (SEQ ID NO: 1). The structure of the pX330-mC vector is shown in the lower part of Figure 1(c).

[0076] [Experimental Example 2] (Investigation of cell cycle dependence and intracellular localization of Cas9, Cas9-mG, and Cas9-mC) Using mouse fertilized eggs, the cell cycle dependency and intracellular localization of the Cas9-mG protein and Cas9-mC protein expressed by introducing the expression vector prepared in Experimental Example 1 were examined.

[0077] First, each expression vector prepared in Experimental Example 1 was transcribed in an in vitro transcription reaction system to prepare Cas9-mG mRNA, Cas9-mC mRNA, and Cas9 mRNA.

[0078] Next, we injected Cas9-mG mRNA and Cas9-mC mRNA into the cytoplasm of mouse fertilized eggs obtained by ICSI. For comparison, we also performed similar experiments on mouse fertilized eggs injected with normal Cas9 mRNA and uninjected mouse fertilized eggs.

[0079] As described above, the 3xFLAG tag peptide is linked to the Cas9-mG protein, Cas9-mC protein, and normal Cas9 protein encoded by each vector prepared in Experimental Example 1. Therefore, the 3xFLAG tag peptide is also linked to the Cas9-mG mRNA, Cas9-mC mRNA, and Cas9-mG protein, Cas9-mC protein, and normal Cas9 protein translated from the Cas9 mRNA injected into mouse fertilized eggs in this Experimental Example. Therefore, each Cas9 protein can be detected by immunostaining using an anti-FLAG antibody.

[0080] The fertilized eggs were then cultured in vitro, and two-cell embryos from the late G1 to early S phase, and two-cell embryos from the late S to early G2 phase, were immunostained with anti-FLAG antibody and observed under a fluorescent microscope.

[0081] Figures 2(a) to 2(d) are fluorescence micrographs showing the results of immunostaining. Figure 2(a) shows the results for the Cas9-mG protein, Figure 2(b) shows the results for the Cas9-mC protein, Figure 2(c) shows the results for the regular Cas9 protein, and Figure 2(d) shows the control (no transfection).

[0082] As a result, the normal Cas9 protein was detected as a dot-like signal in the cytoplasm in both cell cycles. As mentioned above, the Cas9 protein used in this experiment had the SV40 nuclear localization signal attached to its N- and C-termini, but the Cas9 protein did not translocate to the nucleus.

[0083] On the other hand, the Cas9-mG protein was not detected from the late G1 phase to the early S phase, confirming its degradation. Furthermore, the Cas9-mG protein was detected as a dot-like signal in the cytoplasm from the S phase to the early G2 phase. As mentioned above, the SV40 virus nuclear localization signal was attached to the N-terminus and C-terminus of the Cas9-mG protein used in this experiment, but the Cas9 protein was not observed to translocate to the nucleus.

[0084] Contrary to our expectations, the Cas9-mC protein was not cell cycle dependent, and signals were detected throughout all cell cycle stages. Furthermore, strong nuclear staining was observed throughout all cell cycle stages. These results demonstrate that the Cas9-mC protein is highly localized in the nucleus.

[0085] [Experimental Example 3] (Generation of large-scale genome-deleted mice using Cas9-mC 1) We investigated whether Cas9-mC could increase the efficiency of excision of large genomic regions.

[0086] Specifically, we used Cas9-mC to generate mice in which the Tyr gene was completely ablated (ablation-type KO mice). For comparison, we also generated mice in which the Tyr gene was completely ablated using a similar method, except that we used standard Cas9. The Tyr gene controls coat color in mice, and mice lacking both Tyr alleles exhibit albino traits. Mice lacking one allele do not exhibit albino traits.

[0087] Figure 3(a) is a schematic diagram illustrating the method for excising the Tyr gene. In Figure 3(a), "Tyr-G5F primer" (SEQ ID NO: 7) and "Tyr-G3R primer" (SEQ ID NO: 8) represent the primers used to confirm the genotype of the mice.

[0088] Specifically, we first created the pX330-mC-Tyr-L vector, which expresses an sgRNA targeting the genomic sequence upstream of the Tyr gene (Left CRISPR Target, SEQ ID NO: 9) and the Cas9-mC protein, and the pX330-mC-Tyr-R vector, which expresses an sgRNA targeting the genomic sequence downstream of the Tyr gene (Right CRISPR Target, SEQ ID NO: 10) and the Cas9-mC protein.

[0089] We also constructed two vectors: the pX330-Tyr-L vector, which expresses an sgRNA targeting the genomic sequence upstream of the Tyr gene (Left CRISPR Target, SEQ ID NO: 9) and the standard Cas9 protein; and the pX330-Tyr-R vector, which expresses an sgRNA targeting the genomic sequence downstream of the Tyr gene (Right CRISPR Target, SEQ ID NO: 10) and the standard Cas9 protein. The distance between the Left CRISPR Target (SEQ ID NO: 9) and the Right CRISPR Target (SEQ ID NO: 10) was 72,172 base pairs.

[0090] Next, a mixture of pX330-mC-Tyr-L vector (5 ng / μL) and pX330-mC-Tyr-R vector (5 ng / μL), and a mixture of pX330-Tyr-L vector (5 ng / μL) and pX330-Tyr-R vector (5 ng / μL), were microinjected into the male pronuclei of fertilized eggs (pronuclear stage) from black-haired C57BL6 / J mice obtained by natural mating. The vector-injected fertilized eggs were then transplanted into the oviducts of pseudopregnant mice and allowed to develop into mice, and their coat color was observed.

[0091] Figures 3(b) and (c) are photographs showing representative mice obtained. Figure 3(b) is a photograph of a mouse developed from a fertilized egg into which Cas9-mC was introduced, and Figure 3(c) is a photograph of a mouse developed from a fertilized egg into which conventional Cas9 was introduced. In Figures 3(b) and (c), "*" indicates a mouse showing complete albino traits, and "#" indicates a mouse showing mosaic albino traits.

[0092] The complete albino trait is probably the result of the deletion of the entire Tyr gene in both alleles at the one-cell stage of the fertilized egg, while the mosaic albino trait is thought to be the result of the deletion of the entire Tyr gene in both alleles in any cell at the two-cell stage or later.

[0093] As a result, a total of 34 mice were obtained from Cas9-mC-introduced fertilized eggs, of which 5 showed complete albino traits and 3 showed mosaic albino traits. Furthermore, PCR genotyping revealed that 17 of the 34 mice (50.0%) developed from Cas9-mC-introduced fertilized eggs had an allele in which the Tyr gene was completely excised.

[0094] In addition, a total of 21 mice were obtained from fertilized eggs transfected with standard Cas9, of which one exhibited mosaic albino traits, and no mice exhibiting complete albino traits were obtained. Furthermore, PCR analysis of the genotype revealed that 6 of the 21 mice (28.6%) developed from fertilized eggs transfected with standard Cas9 possessed an allele in which the Tyr gene was completely excised. Table 1 below shows the results of generating mice with a large Tyr gene deletion.

[0095] These results demonstrate that Cas9-mC can be used to efficiently generate mice with large-scale genome deletions of tens of kilobase pairs.

[0096] [Table 1] a : Number of newborns with large deletion alleles / total number of newborns x 100

[0097] [Experimental Example 4] (Generation of large-scale genome-deleted mice using Cas9-mC 2) We investigated whether Cas9-mC could increase the efficiency of excision of large genomic regions.

[0098] Specifically, we used Cas9-mC to generate mice in which the Dmd gene was completely ablated (ablation-type KO mice). The Dmd gene is known to be the longest protein-coding gene. For comparison, we also generated mice in which the Dmd gene was completely ablated using a similar method but with standard Cas9. Because the Dmd gene is located on the X chromosome, males have one copy and females have two copies.

[0099] Figure 4 is a schematic diagram illustrating the method for excising the Dmd gene. In Figure 4, "Dmd-G5F" (SEQ ID NO: 11), "Dmd-GMF" (SEQ ID NO: 12), "Dmd-GMR" (SEQ ID NO: 13), and "Dmd-G3R" (SEQ ID NO: 14) represent primers used to confirm the genotype of the mice.

[0100] Specifically, we first created the pX330-mC-Dmd-L vector, which expresses an sgRNA targeting the genomic sequence upstream of the Dmd gene (Left CRISPR Target, SEQ ID NO: 15) and the Cas9-mC protein, and the pX330-mC-Dmd-R vector, which expresses an sgRNA targeting the genomic sequence downstream of the Dmd gene (Right CRISPR Target, SEQ ID NO: 16) and the Cas9-mC protein.

[0101] We also constructed two vectors: pX330-Dmd-L, which expresses an sgRNA targeting the genomic sequence upstream of the Dmd gene (Left CRISPR Target, SEQ ID NO: 15) and a standard Cas9 protein; and pX330-Dmd-R, which expresses an sgRNA targeting the genomic sequence downstream of the Dmd gene (Right CRISPR Target, SEQ ID NO: 16) and a standard Cas9 protein. The distance between the Left CRISPR Target (SEQ ID NO: 15) and the Right CRISPR Target (SEQ ID NO: 16) was 2,265,855 base pairs.

[0102] Next, a mixture of pX330-mC-Dmd-L vector (5 ng / μL) and pX330-mC-Dmd-R vector (5 ng / μL), and a mixture of pX330-Dmd-L vector (5 ng / μL) and pX330-Dmd-R vector (5 ng / μL), were microinjected into the male pronuclei of fertilized eggs (pronuclear stage) from C57BL6 / J mice obtained by natural mating. The vector-injected fertilized eggs were then implanted into the oviducts of pseudopregnant mice and allowed to develop into mice. Genotypes were confirmed by PCR.

[0103] As a result, a total of 68 mice were obtained from the Cas9-mC-transfected fertilized eggs. Genotyping by PCR revealed that 11 (16.2%) of the 68 mice developed from the Cas9-mC-transfected fertilized eggs had an allele in which the Dmd gene was completely ablated.

[0104] A total of 70 mice were obtained from fertilized eggs transfected with standard Cas9. Genotyping by PCR revealed that 6 of the 70 mice (8.6%) developed from the fertilized eggs transfected with standard Cas9 had an allele in which the Dmd gene was completely ablated.

[0105] It is also noteworthy that the six mice with the Dmd excision KO allele obtained by conventional Cas9 transfection were mosaic or heterozygous mutants that also possessed other Dmd alleles, whereas the five male mice and one female mouse with the Dmd excision KO allele obtained by Cas9-mC transfection were Dmd-deficient individuals that possessed only the Dmd excision KO allele. Table 2 below shows the results of generating mice with a large Dmd gene deletion.

[0106] These results demonstrate that Cas9-mC can be used to efficiently generate mice with large genome deletions of several megabase pairs.

[0107] [Table 2] a : Number of newborns with large deletion alleles / total number of newborns x 100

[0108] [Experimental Example 5] (Generation of knock-in mice using Cas9-mC 1) It is known that genome editing of fertilized eggs is difficult to produce mice with knock-in gene fragments consisting of base sequences with a high GC content, or floxed mice, which require simultaneous introduction of LoxP sequences at two sites. We investigated whether Cas9-mC is useful for producing knock-in or floxed mice, which are difficult to produce.

[0109] First, we performed a knock-in experiment using the expression cassette CAG-flox EGFP-Cables1, which contains a CAG promoter with a high GC content and the Cables1 cDNA with a high GC content, at the ROSA26 locus, whose 5' homologous arm region also contains a high GC content.

[0110] Figures 5(a) to 5(c) are schematic diagrams illustrating a method for knocking in a CAG-flox EGFP-Cables1 gene fragment into the ROSA26 locus. Figure 5(a) is a schematic diagram showing the structure of the wild-type ROSA26 locus. Figure 5(a) also shows the target sequence of the sgRNA (SEQ ID NO: 17) and the locations of the 5' homology arm region and the 3' homology arm region.

[0111] Figure 5(b) is a schematic diagram showing the structure of the pRosa-CAG-fEGFP-Cables1 donor DNA plasmid. Figure 5(c) is a schematic diagram showing the structure of the ROSA26 locus when the desired knock-in occurred. In Figure 5(c), "ROSA-G5F" (SEQ ID NO: 18), "CAG-G5R" (SEQ ID NO: 19), "pA-G3F" (SEQ ID NO: 20), "ROSA-G3R Nested" (SEQ ID NO: 21), and "ROSA-G3R 1st" (SEQ ID NO: 22) represent primers used to confirm the genotype of the mice.

[0112] Specifically, we first constructed the pX330-mC-ROSA vector, which expresses an sgRNA targeted to the first intron of the ROSA26 locus and the Cas9-mC protein, and the pX330-ROSA vector, which expresses an sgRNA targeted to the first intron of the ROSA26 locus and the normal Cas9 protein.

[0113] Next, we microinjected a mixture of pX330-mC-ROSA vector (5 ng / μL) and pRosa-CAG-fEGFP-Cables1 vector (10 ng / μL) into the male pronuclei of C57BL6 / J zygotes (pronuclear stage) obtained by natural mating. The vector-injected zygotes were then transferred into the oviducts of pseudopregnant mice and allowed to develop. Genotypes were confirmed by PCR.

[0114] As a result, a total of 19 mice were obtained from the Cas9-mC-introduced fertilized eggs. Genotyping by PCR revealed that 4 of the 19 mice (21.1%) developed from the Cas9-mC-introduced fertilized eggs had the desired knock-in allele.

[0115] In addition, a total of 44 mice were obtained from fertilized eggs transfected with standard Cas9, but genotyping by PCR revealed that none of the mice had the desired knock-in allele. Table 3 below shows the results of knock-in mouse production.

[0116] These results demonstrate that the use of Cas9-mC makes it possible to generate highly challenging KI mice and floxed mice.

[0117] [Table 3] a: Number of newborns or fetuses with knock-in alleles / Total number of newborns or fetuses analyzed for genotype × 100 b :Embryo 18.5 days after fertilization

[0118] [Experimental Example 6] (Generation of knock-in mice using Cas9-mC 2) Next, LoxP sequences were introduced upstream and downstream of the sixth exon of the Prdm14 gene to generate Prdm14 flox mice.

[0119] Figures 6(a) to 6(c) are schematic diagrams illustrating a method for knocking in a LoxP sequence into the Prdm14 locus: Figure 6(a) is a schematic diagram showing the structure of the wild-type Prdm14 locus.

[0120] Figure 6(a) also shows the locations of the sgRNA targeting the genomic sequence upstream of the 6th exon of the Prdm14 gene (Left CRISPR Target, SEQ ID NO: 23), the sgRNA targeting the genomic sequence downstream of the 6th exon of the Prdm14 gene (Right CRISPR Target, SEQ ID NO: 24), the 5' homology arm region, and the 3' homology arm region.

[0121] Figure 6(b) is a schematic diagram showing the structure of the pflox-Prdm14 donor DNA plasmid. Figure 6(c) is a schematic diagram showing the structure of the Prdm14 locus when the desired knock-in has occurred. In Figure 6(c), "Prdm14-GF" (SEQ ID NO: 25) and "Prdm14-GR" (SEQ ID NO: 26) represent the primers used to confirm the genotype of the mice, and "LoxP" represents the knocked-in LoxP sequence.

[0122] Specifically, we first created the pX330-mC-Prdm14-L vector, which expresses an sgRNA targeting the genomic sequence upstream of the Prdm14 gene (Left CRISPR Target, SEQ ID NO: 23) and the Cas9-mC protein, and the pX330-mC-Prdm14-R vector, which expresses an sgRNA targeting the genomic sequence downstream of the Prdm14 gene (Right CRISPR Target, SEQ ID NO: 24) and the Cas9-mC protein.

[0123] We also constructed the pX330-Prdm14-L vector, which expresses an sgRNA targeting the genomic sequence upstream of the Prdm14 gene (Left CRISPR Target, SEQ ID NO: 23) and a conventional Cas9 protein, and the pX330-Prdm14-R vector, which expresses an sgRNA targeting the genomic sequence downstream of the Prdm14 gene (Right CRISPR Target, SEQ ID NO: 24) and a conventional Cas9 protein.

[0124] Next, a mixture of pX330-mC-Prdm14-L vector (5 ng / μL), pX330-mC-Prdm14-R vector (5 ng / μL), and pflox-Prdm14 vector (10 ng / μL) was microinjected into the male pronuclei of C57BL6 / J mouse fertilized eggs (pronuclear stage) obtained by natural mating. Alternatively, a mixture of pX330-Prdm14-L vector (5 ng / μL), pX330-Prdm14-R vector (5 ng / μL), and pflox-Prdm14 vector (10 ng / μL) was microinjected into the oviducts of pseudopregnant mice. The fertilized eggs were then developed into mice, and their genotypes were confirmed by PCR.

[0125] As a result, a total of 22 mice were obtained from the Cas9-mC-introduced fertilized eggs. Genotyping by PCR revealed that 4 of the 22 mice (18.2%) developed from the Cas9-mC-introduced fertilized eggs had the desired knock-in allele.

[0126] In addition, a total of 25 mice were obtained from fertilized eggs transfected with standard Cas9, but genotyping by PCR revealed that none of the mice had the desired knock-in allele. Table 4 below shows the results of knock-in mouse production.

[0127] [Table 4] a : Number of newborns with knock-in allele / Total number of newborns that grew to weaning × 100

[0128] [Experimental Example 7] (Investigation of the intracellular localization of Cas9, Cas9-mG, and Cas9-mC) Using HEK293T cells, which are cells derived from human fetal kidney, the intracellular localization of the Cas9-mG protein and Cas9-mC protein expressed by the introduction of the expression vector prepared in Experimental Example 1 was examined.

[0129] First, HEK293T cells were transfected with the pX330-mG vector and the pX330-mC vector, respectively. For comparison, HEK293T cells transfected with the pX330 vector were also prepared.

[0130] As mentioned above, the Cas9-mG protein, Cas9-mC protein, and standard Cas9 protein encoded by these vectors are linked to a 3xFLAG tag peptide, and each Cas9 protein can be detected by immunostaining with an anti-FLAG antibody.

[0131] Next, each cell was fixed with formaldehyde and immunostained with anti-FLAG antibody. The nuclei were stained with 4',6-diamidino-2-phenylindole (DAPI). The stained cells were then observed under a fluorescence microscope.

[0132] Figures 7(a) to (c) are fluorescence micrographs showing the results of immunostaining. Figure 7(a) shows the results for the normal Cas9 protein, Figure 7(b) shows the results for the Cas9-mG protein, and Figure 7(c) shows the results for the Cas9-mC protein. In Figure 7, "Overlay" indicates the result of combining the results of staining with the anti-FLAG antibody and the results of staining with DAPI.

[0133] As a result, the normal Cas9 protein was detected in the cytoplasm. As mentioned above, the Cas9 protein used in this experiment had the SV40 nuclear localization signal attached to its N- and C-termini, but the Cas9 protein did not translocate to the nucleus.

[0134] On the other hand, the Cas9-mG protein was found to be localized in the nucleus. The Cas9-mC protein was also found to be localized in the nucleus. These results demonstrate that the Cas9-mC protein exhibits high nuclear localization even in human cells.

[0135] [Experimental Example 8] (Construction of Cas9 fusion protein expression vector 2) The SpCas9 protein expressed from the pX330-mC vector prepared in Experimental Example 1 contained SV40-derived nuclear localization signals (NLS) at the N- and C-termini of the SpCas9 protein. Figure 8(a) shows the structure of the pX330-mC vector.

[0136] In this experimental example, the NLSs present at the N- and C-termini of the SpCas9 protein were removed, and a peptide consisting of residues 29 to 132 of the mouse Cdt1 protein was linked to the C-terminus of the SpCas9 protein (hereinafter referred to as "Cas9-pFmC") to express this fusion protein. Figure 8(b) shows the structure of the pX330-pFmC vector.

[0137] In this experimental example, we further constructed fusion proteins in which the position and length of the mouse Cdt1-derived peptide linked to the C-terminus of the SpCas9 protein were varied. Specifically, we constructed a vector (SEQ ID NO: 31, hereinafter sometimes referred to as "pX330-pCmC") that expresses a fusion protein (hereinafter sometimes referred to as "Cas9-pCmC") that does not contain the SV40 virus-derived nuclear localization signal and in which a peptide consisting of positions 81 to 132 of the mouse Cdt1 protein is linked to the C-terminus of the SpCas9 protein. The structure of the pX330-pCmC vector is shown in Figure 8(c).

[0138] We also constructed a vector (SEQ ID NO: 32, hereinafter referred to as "pX330-pMmC") that expresses a fusion protein (hereinafter referred to as "Cas9-pMmC") that lacks the SV40-derived nuclear localization signal and is constructed by linking a peptide consisting of the 55th to 106th residues of the mouse Cdt1 protein to the C-terminus of the SpCas9 protein. The structure of the pX330-pMmC vector is shown in Figure 8(d).

[0139] We also constructed a vector (SEQ ID NO: 33, hereinafter referred to as "pX330-pNmC") that expresses a fusion protein (hereinafter referred to as "Cas9-pNmC") that lacks the SV40-derived nuclear localization signal and is constructed by linking a peptide consisting of the 29th to 80th positions of the mouse Cdt1 protein to the C-terminus of the SpCas9 protein. The structure of the pX330-pNmC vector is shown in Figure 8(e).

[0140] We also constructed a vector (SEQ ID NO: 34, hereinafter referred to as "pX330-pNNmC") that expresses a fusion protein (hereinafter referred to as "Cas9-pNNmC") that lacks the SV40-derived nuclear localization signal and is constructed by linking a peptide consisting of the 29th to 54th positions of the mouse Cdt1 protein to the C-terminus of the SpCas9 protein. The structure of the pX330-pNNmC vector is shown in Figure 8(f).

[0141] We also constructed a vector (SEQ ID NO: 35, hereinafter referred to as "pX330-pNCmC") that expresses a fusion protein (hereinafter referred to as "Cas9-pNCmC") that lacks the SV40-derived nuclear localization signal and is constructed by linking a peptide consisting of the 54th to 80th positions of the mouse Cdt1 protein to the C-terminus of the SpCas9 protein. The structure of the pX330-pNCmC vector is shown in Figure 8(g).

[0142] [Experimental Example 9] (Investigation of intracellular localization of Cas9 fusion proteins 1) Using HEK293T cells, which are cells derived from human fetal kidney, the intracellular localization of each fusion protein expressed by the introduction of the expression vector prepared in Experimental Example 8 was examined.

[0143] First, HEK293T cells were transfected with the pX330-pFmC vector, pX330-pCmC vector, pX330-pMmC vector, pX330-pNmC vector, pX330-pNNmC vector, and pX330-pNCmC vector. For comparison, HEK293T cells transfected with the pX330 vector were also prepared.

[0144] Each fusion protein encoded by these vectors is linked to a 3xFLAG tag peptide, allowing the detection of each Cas9 protein by immunostaining with an anti-FLAG antibody.

[0145] Next, each cell was fixed with formaldehyde and immunostained with anti-FLAG antibody. The nuclei were stained with DAPI. The stained cells were then observed under a fluorescence microscope.

[0146] 9 shows fluorescent micrographs showing the results of immunostaining. In FIG. 9, "pX330" indicates the results obtained by introducing the pX330 vector, "pX330-pFmC" indicates the results obtained by introducing the pX330-pFmC vector, "pX330-pCmC" indicates the results obtained by introducing the pX330-pCmC vector, "pX330-pMmC" indicates the results obtained by introducing the pX330-pMmC vector, "pX330-pNmC" indicates the results obtained by introducing the pX330-pNmC vector, "pX330-pNNmC" indicates the results obtained by introducing the pX330-pNNmC vector, and "pX330-pNCmC" indicates the results obtained by introducing the pX330-pNCmC vector. In addition, "DAPI" indicates the result of nuclear staining with DAPI, "FLAG" indicates the result of detecting each Cas9 protein by immunostaining using anti-FLAG antibody, and "Overlay" indicates the result of combining the staining results with DAPI and anti-FLAG antibody.

[0147] The results showed that the Cas9-pFmC and Cas9-pNmC fusion proteins translocated to the nucleus, whereas the SpCas9, Cas9-pCmC, Cas9-pMmC, Cas9-pNNmC, and Cas9-pNCmC fusion proteins expressed using the pX330 vector did not translocate to the nucleus.

[0148] [Experimental Example 10] (Investigation of intracellular localization of Cas9 fusion proteins 2) From HEK293T cells expressing each fusion protein in Experimental Example 9, soluble nuclear protein fractions and cytoplasmic protein fractions were extracted using a commercially available kit (code number "295-73901", Wako Pure Chemical Industries, Ltd.), and each Cas9 fusion protein was detected by Western blotting.

[0149] Figure 10(a) is a photograph showing the results of detecting each Cas9 fusion protein with an anti-FLAG antibody. Figure 10(b) is a photograph showing the results of detecting each Cas9 fusion protein with an anti-Cas9 antibody. As a result, similar to the results of Experimental Example 9, the Cas9-pFmC fusion protein and the Cas9-pNmC fusion protein were detected in greater amounts in the nucleus than in the cytoplasm, confirming their nuclear translocation.

[0150] Figure 10(c) is a photograph showing the results of detecting PARP1, a nuclear protein, as a control, using an anti-PARP1 antibody. Figure 10(d) is a photograph showing the results of detecting GAPDH, a cytoplasmic protein, as a control, using an anti-GAPDH antibody. As a result, it was confirmed that each protein fraction and cytoplasmic fraction were successfully extracted.

[0151] Figure 11 is a diagram summarizing the structure and nuclear localization of each Cas9 fusion protein based on the results of Experimental Examples 9 and 10. In Figure 11, "◯" indicates high nuclear localization, and "×" indicates no nuclear localization was observed.

[0152] [Experimental Example 11] (Investigation of degradability of Cas9 fusion proteins) The degradability of the Cas9-pFmC fusion protein and the Cas9-pNmC fusion protein prepared in Experimental Example 8 was examined using HEK293T cells, which are cells derived from human fetal kidney.

[0153] First, HEK293T cells were transfected with the pX330-pFmC vector and the pX330-pNmC vector. For comparison, HEK293T cells transfected with the pX330 vector were also prepared.

[0154] Next, each cell was fixed with formaldehyde and immunostained with anti-FLAG antibody. The nuclei were stained with DAPI. The stained cells were then observed under a fluorescence microscope.

[0155] Figure 12 is a fluorescence micrograph showing the results of immunostaining. In Figure 9, "pX330" indicates the results of introducing the pX330 vector, "pX330-pFmC" indicates the results of introducing the pX330-pFmC vector, and "pX330-pNmC" indicates the results of introducing the pX330-pNmC vector. Furthermore, "DAPI" indicates the results of nuclear staining with DAPI, and "FLAG" indicates the results of detecting each Cas9 protein by immunostaining using an anti-FLAG antibody.

[0156] The Cas9-pNmC fusion protein was detected with higher fluorescence intensity of the anti-FLAG antibody than the Cas9-pFmC fusion protein, indicating that the Cas9-pNmC fusion protein is less susceptible to degradation than the Cas9-pFmC fusion protein. [Industrial Applicability]

[0157] The present invention provides modified Cas9 that improves KO and KI efficiency.

Claims

1. A fusion protein comprising a Cas9 protein and a modifying peptide that modifies the Cas9 protein, The modified peptide comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 1, or a peptide consisting of an amino acid sequence set forth in SEQ ID NO: 1 in which 1 to 10 amino acids have been deleted, substituted or added, and which has the activity of localizing the Cas9 protein to the nucleus in any cell cycle when a fusion protein in which the amino acid sequence set forth in SEQ ID NO: 1 has been deleted, substituted or added at the C-terminus of the Cas9 protein is introduced into a fertilized egg; or a peptide consisting of the amino acid sequence of positions 1 to 52 of SEQ ID NO: 1, or a peptide consisting of an amino acid sequence in which 1 to 5 amino acids have been deleted, substituted or added at the C-terminus of the Cas9 protein is introduced into a fertilized egg, which has the activity of localizing the Cas9 protein to the nucleus in any cell cycle the modified peptide is located at the C-terminus of the Cas9 protein; A fusion protein, wherein the Cas9 protein is an SpCas9 protein derived from Streptococcus pyogenes.

2. The fusion protein according to claim 1, wherein the modified peptide comprises a peptide consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted, or added in the amino acid sequence set forth in SEQ ID NO: 1, and which has the activity of localizing the Cas9 protein to the nucleus in any cell cycle when a fusion protein in which the modified peptide is arranged at the C-terminus of the Cas9 protein is introduced into a fertilized egg, or comprises a peptide consisting of an amino acid sequence in which 1 to 3 amino acids have been deleted, substituted, or added in the amino acid sequence at positions 1 to 52 of SEQ ID NO: 1, and which has the activity of localizing the Cas9 protein to the nucleus in any cell cycle when a fusion protein in which the modified peptide is arranged at the C-terminus of the Cas9 protein is introduced into a fertilized egg.

3. The fusion protein of claim 1 or 2, wherein the modified peptide further comprises a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2, or a peptide consisting of the amino acid sequence set forth in SEQ ID NO: 2 in which one or several amino acids have been deleted, substituted, or added, and which has the activity of degrading the Cas9 protein in the G1 phase by forming a fusion protein with the Cas9 protein.

4. A nucleic acid encoding the fusion protein according to any one of claims 1 to 3.

5. A method for producing a fusion protein according to any one of claims 1 to 3 in vitro in a cell (excluding a human fertilized egg). A method for producing a cell having edited genomic DNA, comprising contacting genomic DNA in a cell.

6. The method of claim 5 , wherein the cell is a fertilized egg.

7. contacting the fusion protein according to any one of claims 1 to 3 with genomic DNA in a fertilized egg (excluding human fertilized eggs) to obtain a fertilized egg whose genomic DNA has been edited; growing the fertilized egg whose genomic DNA has been edited into an individual to obtain an animal whose genomic DNA has been edited; A method for producing an animal whose genomic DNA has been edited, comprising:

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