Nucleic acid cleavage enzyme, nucleic acid, vector, nucleic acid modification kit, method for modifying nucleic acid, method for producing variant, method for expressing gene, eukaryotic cell, vector or DNA fragment, kit, and method for producing genetically engineered eukaryotic cell

Abstract

Provided are: a nucleic acid cleavage enzyme which comprises a nuclease domain having a specific amino acid sequence and a nucleic acid binding domain; a use of the nucleic acid cleavage enzyme; a method for producing a variant of a eukaryotic cell, the method comprising deleting a centromere region of a genome on a chromosome in the eukaryotic cell; a use of the method; a vector or a DNA fragment for use in engineering of a gene of a eukaryotic organism, the vector or the DNA fragment including a telomeric repeat sequence; and a use of the vector or the DNA fragment.

Description

Nucleic acid cleaving enzyme, nucleic acid, vector, nucleic acid modification kit, nucleic acid modification method, method for producing mutant, gene expression method, eukaryotic cell, vector or DNA fragment, kit, and method for producing genetically engineered eukaryotic cell

[0001] The present disclosure relates to nucleic acid cleaving enzymes, nucleic acids, vectors, kits for modifying nucleic acids, methods for modifying nucleic acids, methods for producing mutants, gene expression methods, eukaryotic cells, vectors or DNA fragments, kits, and methods for producing genetically engineered eukaryotic cells.

[0002] In recent years, genome editing tools such as Zinc Finger Nuclease (ZFN), Transcription Activator-like Effector Nuclease (TALEN), and CRISPR / Cas systems have been widely used. These genome editing tools contain a nuclease domain that cleaves nucleic acid and a nucleic acid binding domain that binds to a target nucleic acid.

[0003] A typical ZFN is an artificial restriction enzyme in which a zinc finger array (a nucleic acid binding domain) is linked to a nuclease domain, FokI. Each zinc finger in the zinc finger array recognizes and binds to three bases. When the ZFN translocates into the nucleus and the zinc finger array binds to the target base sequence, FokI dimerizes and cleaves the target nucleic acid. A TALEN is an artificial restriction enzyme in which a TALE (a nucleic acid binding domain) is linked to a nuclease domain, FokI. The TALE has a repeating structure called a TALE repeat, and each repeat recognizes and binds to one base. In this case, too, when the TALE binds to the target base sequence, FokI dimerizes and cleaves the target nucleic acid. While ZFNs and TALENs recognize target sequences using a nucleic acid-binding domain of a protein, the CRISPR / Cas system recognizes the target sequence using a guide RNA and cleaves the target nucleic acid using a Cas nuclease complexed with the guide RNA.

[0004] Various improvements to nuclease domains in genome editing tools have been attempted. For example, International Publication No. 2020 / 045281 describes an artificial nucleic acid cleaving enzyme containing a nuclease domain that is an improved version of the FokI nuclease domain. Blair B. Madison et al., Cas-CLOVER is a novel high-fidelity nuclease for safe and robust generation of TSCM-enriched allogeneic CAR-T cells. Molecular Therapy: Nucleic Acids, Vol. 29, 13 September 2022, pp. 979-995, describes an artificial nucleic acid cleaving enzyme that combines inactivated Cas9 with Clo051, a nuclease similar to FokI, to cleave a target nucleic acid together with a guide RNA.

[0005] JP 2020-524489 A proposes a platform for manipulating the genome of eukaryotes. This document describes a technique for transforming multiple protoplasts with two types of constructs containing sequences homologous to a target gene locus, and then obtaining, purifying, and propagating the transformants.

[0006] In the field of genetic engineering, various gene transfer techniques using vectors and DNA fragments are known. For example, transformation techniques that introduce plasmid vectors or DNA fragments into host cells using polyethylene glycol, electrical techniques, etc. are widely used. Also known are RNA silencing techniques that reduce the expression of specific genes by expressing RNA within cells, and genome editing techniques that use site-specific nucleases such as ZFN (Zinc Finger Nuclease), TALEN (Transcription Activator-Like Effector Nuclease), and CRISPR-Cas9.

[0007] JP 2017-538425 A describes a method for homologous recombination between a genomic locus and donor DNA in filamentous fungal cells using a guide RNA / Cas endonuclease system. This document describes inserting a telomere sequence into an expression vector for Cas9 and guide RNA, and transforming Trichoderma reesei with the constructed vector. It also describes that, as a result, expression of Cas9 and guide RNA from the vector enabled targeting of Cas9 to a specific locus.

[0008] On the other hand, a technique for adding a telomere sequence to exogenous DNA and introducing the DNA into a cell is also known. For example, JP-A-2002-519057 describes a method for adding one or more telomere repeat sequences to exogenous DNA by introducing the exogenous DNA into a Pestalotiopsis cell.

[0009] While the development of various artificial nucleic acid cleaving enzymes is progressing, the types of artificial nucleic acid cleaving enzymes that have excellent nucleic acid modification efficiency are still limited. In view of this situation, a first embodiment of the present disclosure relates to providing novel nucleic acid cleaving enzymes, nucleic acids, vectors, and nucleic acid modification kits that can be used in nucleic acid modification techniques, as well as methods for modifying nucleic acids using these.

[0010] There is also a need for the application of gene modification techniques to create useful mutants of eukaryotes. The inventors attempted various modifications of eukaryotic chromosomes and found that it is possible to create mutants that can increase the chromosome copy number of eukaryotes. A second embodiment of the present disclosure relates to a method for creating a mutant that can increase the chromosome copy number of eukaryotes, a gene expression method using the same, and a eukaryotic cell with an increased chromosome copy number.

[0011] Furthermore, in gene transfer techniques, high gene transfer efficiency may not be achieved depending on the host and other conditions, and therefore improvement of gene transfer efficiency is desired. In view of such circumstances, a third embodiment of the present disclosure provides a vector or DNA fragment and kit that enable improvement of gene transfer efficiency, as well as a method for producing a genetically engineered eukaryotic cell using the vector or DNA fragment.

[0012] The first embodiment includes the following aspects. <1> A nucleic acid cleaving enzyme comprising a nuclease domain having an amino acid sequence having 90% or more identity to the amino acid sequence represented by any one of SEQ ID NOs: 2 to 5, 82, and 83, and a nucleic acid binding domain. <2> The nucleic acid cleaving enzyme according to <1>, wherein the nucleic acid binding domain comprises a zinc finger, a TALE, a Cas protein, or a PPR. <3> The nucleic acid cleaving enzyme according to <1> or <2>, further comprising a linker between the nuclease domain and the nucleic acid binding domain. <4> The nucleic acid cleaving enzyme according to any one of <1> to <3>, which binds to a target nucleic acid and dimerizes. <5> A nucleic acid encoding the nucleic acid cleaving enzyme according to any one of <1> to <4>. <6> A vector comprising the nucleic acid according to <5>, or a transcription product or translation product thereof. <7> A kit for modifying nucleic acid, comprising: the nucleic acid cleaving enzyme according to any one of <1> to <4>; a nucleic acid encoding the nucleic acid cleaving enzyme; or a vector containing the nucleic acid encoding the nucleic acid cleaving enzyme or a transcription product or translation product thereof, all contained in a container. <8> A method for modifying nucleic acid, comprising introducing the nucleic acid cleaving enzyme according to any one of <1> to <4>; the nucleic acid encoding the nucleic acid cleaving enzyme; or a vector containing the nucleic acid encoding the nucleic acid cleaving enzyme or a transcription product or translation product thereof into a cell (excluding cells present in the human body, human germ cells, and human embryonic cells).

[0013] The second embodiment includes the following aspects: <1> A method for producing a mutant of a eukaryotic cell, comprising deleting a centromere region of a genome on a chromosome in the eukaryotic cell. <2> The method for producing a mutant according to <1>, further comprising introducing a marker gene into the chromosome so as to replace the deleted centromere region. <3> The method for producing a mutant according to <2>, in which the marker gene is a drug resistance gene. <4> The method for producing a mutant according to any one of <1> to <3>, in which the deletion of the centromere region is achieved by inducing a DNA repair mechanism. <5> The method for producing a mutant according to <4>, in which the deletion of the centromere region is achieved by DNA cleavage using ZFN, TALEN, a CRISPR / Cas system, or I-SceI, and cleavage repair by a DNA repair mechanism. <6> The method for producing a mutant according to any one of <1> to <5>, in which the length of the centromere region to be deleted is 40 to 500 kb. <7> The method for producing a mutant according to any one of <1> to <6>, wherein the eukaryotic cell is a fungal cell. <8> The method for producing a mutant according to any one of <1> to <7>, wherein the eukaryotic cell is a cell of a fungus belonging to the genus Fusarium. <9> The method for producing a mutant according to any one of <1> to <8>, wherein the chromosome is an accessory chromosome. <10> A gene expression method comprising expressing a gene on the chromosome in a mutant produced by the method according to any one of <1> to <9>. <11> The gene expression method according to <10>, wherein the number of copies of the chromosome per cell of the mutant is increased compared to before the deletion of the centromere region. <12> A eukaryotic cell in which the centromere region of the genome on a chromosome is deleted. <13> The eukaryotic cell according to <12>, wherein the genome on the chromosome contains a marker gene. <14> The eukaryotic cell according to <13>, wherein the marker gene is a drug resistance gene. <15> The eukaryotic cell according to any one of <12> to <14>, which is a fungal cell. <16> The eukaryotic cell according to <15>, which is a cell of the genus Fusarium.

[0014] The third embodiment includes the following aspects. <1> A vector or DNA fragment (excluding artificial chromosomes) for eukaryotic genetic manipulation, comprising a telomere repeat sequence of three or more, the telomere repeat sequence being contained in only one direction. <2> A vector or DNA fragment (excluding artificial chromosomes) for eukaryotic genetic manipulation, comprising a telomere repeat sequence of 3 to 26 times. <3> A vector or DNA fragment (excluding artificial chromosomes) for eukaryotic genetic manipulation, comprising a telomere repeat sequence of three or more times and a sequence of 21 bases or more in length, with an AT content of 60% or more. <4> The vector or DNA fragment according to <3>, wherein the sequence of 21 bases or more is at least a part of a centromere region. <5> The vector or DNA fragment according to <3> or <4>, wherein the AT content of the sequence of 21 bases or more is 75% or more. <6> The vector or DNA fragment according to any one of <3> to <5>, wherein the sequence of 21 bases or more is 1000 bases or more in length. <7> A vector or DNA fragment (excluding artificial chromosomes) for eukaryotic genetic manipulation, comprising three or more repeats of a telomere sequence, which is artificially synthesized DNA. <8> The vector or DNA fragment according to any one of <1> to <7>, wherein the telomere sequence is a telomere sequence in fungi. <9> The vector or DNA fragment according to any one of <1> to <8>, wherein the telomere sequence is a telomere sequence in bacteria of the genus Fusarium or Pyricularia. <10> The vector or DNA fragment according to any one of <1> to <9>, further comprising a base sequence encoding ZFN, TALEN, CRISPR / Cas system, or I-SceI. <11> A kit for eukaryotic genetic manipulation, comprising the vector or DNA fragment according to any one of <1> to <10> stored in a container. <12> A method for producing a genetically engineered eukaryotic cell, comprising introducing the vector or DNA fragment according to any one of <1> to <10> into a eukaryotic cell. <13> The method for producing a genetically engineered eukaryotic cell according to <12>, wherein the vector or DNA fragment is produced by adding an artificially synthesized DNA fragment that is a telomeric repeat sequence to a vector or DNA fragment that does not contain a telomeric repeat sequence.<14> The method for producing a genetically engineered eukaryotic cell according to <12> or <13>, wherein the eukaryotic cell is a fungal cell. <15> The method for producing a genetically engineered eukaryotic cell according to any one of <12> to <14>, wherein the eukaryotic cell is a cell of a fungus belonging to the genus Fusarium or Pyricularia.

[0015] According to the first embodiment, there are provided a novel nucleic acid cleaving enzyme, nucleic acid, vector, and nucleic acid modification kit that can be used in nucleic acid modification technology, as well as a nucleic acid modification method using these.

[0016] According to the second embodiment, there are provided a method for producing a mutant capable of increasing the chromosome copy number of a eukaryote, a gene expression method using the same, and a eukaryotic cell with an increased chromosome copy number.

[0017] According to the third embodiment, a vector or DNA fragment and kit capable of improving gene transfer efficiency, as well as a method for producing genetically engineered eukaryotic cells using the vector or DNA fragment, are provided.

[0018] FIG. 1 is a schematic diagram showing a genome editing method used to evaluate the DNA cleavage activity of a nucleic acid cleaving enzyme in an example according to the first embodiment.

[0019] FIG. 1 is a schematic diagram of a genome editing method for the tomato wilt fungus carried out in an example according to the second embodiment. The centromere region of chromosome 14 (accessory chromosome) was replaced with a hygromycin B resistance gene (Hph). A PCR band of approximately 4,500 bp is shown, indicating that the centromere region was removed in the transformant of the example according to the second embodiment. A schematic diagram showing a method for evaluating the retention rate of the accessory chromosome in the transformant from which the centromere region was removed and in the negative control (ΔABHL strain) in the example according to the second embodiment. A graph showing the number of colonies in a hygromycin B-containing medium and a hygromycin B-free medium for the transformant from which the centromere region was removed (Δcentromere #1) and the negative control (ΔABHL strain) in the example according to the second embodiment. (Seeded cells: 1 x 10 １1 is a graph showing the number of colonies in a hygromycin B-containing medium and a hygromycin B-free medium for a transformant (Δcentromere #1) in which the centromere region was deleted and a negative control (ΔABHL strain) in an example according to the second embodiment (seeded cells: 1×10 ２ 1 is a graph showing the number of colonies in a hygromycin B-containing medium and a hygromycin B-free medium for a transformant (Δcentromere #1) in which the centromere region was deleted and a negative control (ΔABHL strain) in an example according to the second embodiment (seeded cells: 5×10 ２ 10 is a graph showing the gene copy number present in the cell nucleus of a negative control (ΔABHL strain) estimated from the relative value of the DNA amplification level of the accessory chromosome-located genes (14-1 and 14-2) to the amplification level of the core chromosome-located Ste3 gene in an example according to the second embodiment. 11 is a graph showing the gene copy number present in the cell nucleus of a centromere-removed transformant (Δcentromere #1) estimated from the relative value of the DNA amplification level of the accessory chromosome-located genes (14-1 and 14-2) to the amplification level of the core chromosome-located Ste3 gene in an example according to the second embodiment.

[0020] 1 shows a schematic diagram of the plasmid vector and negative control constructed in Example 1 according to the third embodiment, and the results of evaluation of the transformation efficiency of each vector. A schematic diagram of the DNA fragment constructed in Example 2 according to the third embodiment is shown. A result of evaluation of the transformation efficiency of the DNA fragment constructed in Example 2 according to the third embodiment is shown. A result of evaluation of the transformation efficiency of the DNA fragment constructed in Example 3 according to the third embodiment is shown. A graph showing the retention rate of the DNA fragment and the plasmid in the transformant introduced with the DNA fragment or the plasmid vector in Example 4 according to the third embodiment is shown. A schematic diagram of the plasmid vector constructed in Example 5 according to the third embodiment is shown. A result of culturing the transformant with the plasmid vector constructed in Example 5 according to the third embodiment in hygromycin B-containing YG medium and 5-FOA-containing PDA medium, and a result of subsequent culturing of the colony grown in 5-FOA-containing PDA medium in hygromycin B-containing YG medium.

[0021] Hereinafter, modes for carrying out embodiments of the present disclosure will be described in detail. However, the embodiments of the present disclosure are not limited to the following embodiments. In the following embodiments, components (including element steps, etc.) are not essential unless otherwise specified. The same applies to numerical values ​​and their ranges, and they do not limit the embodiments of the present disclosure.

[0022] In the present disclosure, numerical ranges indicated using "to" include the numerical values ​​before and after "to" as the minimum and maximum values, respectively. In numerical ranges described in stages in the present disclosure, the upper or lower limit value described in one numerical range may be replaced with the upper or lower limit value of another numerical range described in stages. Furthermore, in numerical ranges described in the present disclosure, the upper or lower limit value of that numerical range may be replaced with a value shown in the examples. In the present disclosure, a combination of two or more preferred embodiments is a more preferred embodiment.

[0023] In this disclosure, amino acids are represented by the single-letter abbreviations established by the IUPAC-IUBMB JCBN (IUPAC-IUBMB Joint Commission on Biochemical Nomenclature). Unless otherwise specified, amino acids referred to in this disclosure are L-amino acids. In this disclosure, unless otherwise specified, nucleic acids include all nucleic acids (DNA, RNA, their analogs, natural products, artificial products, etc.), as well as all nucleic acids linked to low molecular weight compounds, groups, non-nucleic acid molecules, structures, etc. Nucleic acids may be single-stranded or double-stranded. Thus, examples of nucleic acids include single-stranded DNA, double-stranded DNA, single-stranded RNA, and DNA-RNA hybrid duplexes. In this disclosure, "identity" of amino acid sequences refers to the percentage of matching amino acid residues or matching nucleic acid residues among all amino acid residues or bases when the two sequences being compared are aligned, with gaps inserted as needed in one or both sequences to maximize the number of matches. Alignment can be performed using well-known alignment tools such as BLAST, FASTA, CLUSTAL W, etc. For example, alignment can be evaluated using the default parameters of BLAST. In this disclosure, even if an element is referred to in the singular, this does not exclude the presence of a plurality unless a technical contradiction arises, unless expressly stated otherwise.

[0024] 1. First Embodiment <Nucleic Acid Cleaving Enzyme, Nucleic Acid, and Vector> [Nucleic Acid Cleaving Enzyme] The nucleic acid cleaving enzyme of the first embodiment comprises a nuclease domain having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by any one of SEQ ID NOs: 2 to 5, 82, and 83, and a nucleic acid-binding domain. The nucleic acid cleaving enzyme binds to a target sequence on a nucleic acid via the nucleic acid-binding domain, and cleaves the nucleic acid at the target site via the nuclease domain. Therefore, the nucleic acid cleaving enzyme of the first embodiment has nuclease activity. The nucleic acid cleaving enzyme may be an artificial nucleic acid cleaving enzyme in which the nuclease domain and the nucleic acid-binding domain are linked.

[0025] (Nuclease domain) The nuclease domain has an amino acid sequence that has 90% or more identity to the amino acid sequence represented by any one of SEQ ID NOs: 2 to 5, 82, and 83. The amino acid sequences of SEQ ID NOs: 2 to 5, 82, and 83 are amino acid sequences of novel nuclease domains that have been discovered as nuclease domains that can replace FokI, and correspond to the following nuclease domains, respectively: SEQ ID NO: 2: EsFokI (TUS1) SEQ ID NO: 3: CsaFokI (TUS2) SEQ ID NO: 4: IbFokI (TUS3) SEQ ID NO: 5: RsFokI (TUS4) SEQ ID NO: 82: EsFokI-DDD (TUS1-DDD) SEQ ID NO: 83: EsFokI-RRR (TUS1-RRR)

[0026] The nuclease domain preferably has an amino acid sequence that is 90% or more identical to the amino acid sequence represented by any one of SEQ ID NOs: 2 to 4, 82, and 83; from the viewpoint of excellent nucleic acid modification efficiency, it is more preferable that it has an amino acid sequence that is 90% or more identical to the amino acid sequence represented by any one of SEQ ID NOs: 2, 3, 82, and 83; and it is even more preferable that it has an amino acid sequence that is 90% or more identical to the amino acid sequence represented by SEQ ID NO: 82 or 83.

[0027] The identity of the amino acid sequence is 90% or more, preferably 93% or more, more preferably 95% or more, even more preferably 97% or more, particularly preferably 99% or more, and may be 100%. That is, the nuclease domain may have the amino acid sequence of any one of SEQ ID NOs: 2 to 5, 82, and 83.

[0028] The nuclease domain may have an amino acid sequence in which 1 to 20, 1 to 15, 1 to 10, 1 to 5, or 1 or 2 amino acids are deleted, substituted, or added relative to the amino acid sequence of any one of SEQ ID NOs: 2 to 5, 82, and 83.

[0029] In the nuclease domain, the amino acids at positions 59 to 72 and 78 to 122 are preferably highly conserved in the amino acid sequence of any one of SEQ ID NOS: 2 to 5, 82, and 83. The amino acids at positions 59 to 72 and 78 to 122 in SEQ ID NO: 1 (FokI) are highly conserved among various FokI variants, and it is desirable that the corresponding positions are also highly conserved in the nuclease domain of the nucleic acid-cleaving enzyme of the first embodiment. For example, the amino acid sequence at positions 59 to 72 and 78 to 122 in the nuclease domain preferably has 93% or more, 95% or more, or 97% or more identity to the amino acid sequence at positions 59 to 72 and 78 to 122 in the amino acid sequence of any one of SEQ ID NOS: 2 to 5, 82, and 83 (here, the combined sequence of the amino acid sequences at positions 59 to 72 and 78 to 122 is used as the reference). Furthermore, the amino acid sequences of positions 59 to 72 and 78 to 122 in the nuclease domain may be those in which a total of 1 to 4, 1 to 3, 1 to 2, or 1 amino acid residue has been deleted, substituted, or added, or any combination thereof, in the amino acid sequences of positions 59 to 72 and 78 to 122 in any one of the amino acid sequences of SEQ ID NOs: 2 to 5, 82, and 83 (here, the combined sequence of the amino acid sequences of positions 59 to 72 and 78 to 122 is used as the reference).

[0030] The amino acid length of the nuclease domain may be, for example, 175 amino acids or more, 180 amino acids or more, or 190 amino acids or more. The amino acid length of the nuclease domain may be, for example, 220 amino acids or less, 210 amino acids or less, or 200 amino acids or less. Thus, the amino acid length of the nuclease domain may be 175 to 220 amino acids, 180 to 210 amino acids, or 190 to 200 amino acids.

[0031] As the nuclease domain, one type (i.e., a nuclease domain having one type of amino acid sequence) may be used, or two or more types (i.e., nuclease domains having two or more types of amino acid sequences) may be used in combination.

[0032] (Nucleic acid binding domain) A nucleic acid binding domain is a domain capable of binding to a sequence in the vicinity of a target site of a nucleic acid. Examples of nucleic acid binding domains include zinc fingers, TALEs (Transcription Activator-like Effectors), Cas proteins, and PPRs (Pentatricopeptide Repeats). One type of nucleic acid binding domain may be used alone, or two or more types may be used in combination.

[0033] Cas protein is a nuclease in the CRISPR / Cas system. Cas proteins include Cas3, Cas9, Cas10, Cas12, Cas13, Cas14, etc., with Cas9 being widely used. As the Cas protein, it is preferable to use Cas (dCas) whose nuclease activity has been inactivated. Cas protein has the ability to bind guide RNA, and by binding to the guide RNA, it becomes possible for the guide RNA to recognize the target sequence.

[0034] Each zinc finger recognizes a three-base sequence. Linking zinc fingers to form a zinc finger array allows recognition of longer base sequences, thereby enhancing specificity for the target sequence. When the nucleic acid-binding domain contains zinc fingers, the number of zinc fingers contained in the nucleic acid-binding domain is preferably two or more, and may be three to nine, or may be three to six.

[0035] TALEs have a repeating structure called a TALE repeat, and each repeat recognizes and binds to one base. Linking TALE repeats allows recognition and binding to a base sequence of multiple bases.

[0036] PPRs have a repeat structure of a 35-amino acid sequence motif (PPR motif), and each PPR motif binds to a single base on RNA, so PPRs can be used as nucleic acid binding domains for RNA editing tools.

[0037] (Other Components) The nucleic acid cleaving enzyme may contain other components in addition to the nuclease domain and the nucleic acid binding domain. For example, the nucleic acid cleaving enzyme may further contain a linker between the nuclease domain and the nucleic acid binding domain. Examples of linkers include linkers of 2 to 20 amino acids in length or 2 to 10 amino acids in length. The linker is not particularly limited as long as it allows the nuclease domain and the nucleic acid binding domain to perform their functions, and examples include TGAAARA (SEQ ID NO: 14), GS, RPGEKP (SEQ ID NO: 15), TGPGAAARA (SEQ ID NO: 16), LRGS (SEQ ID NO: 17), and LRSSVIPNRGVTKQLVKG (SEQ ID NO: 18). The nuclease domain and the nucleic acid binding domain may be directly linked without a linker.

[0038] The nucleic acid cleaving enzyme may contain other components used for cleaving a target nucleic acid in addition to the nuclease domain, the nucleic acid binding domain, and an optional linker. For example, the nucleic acid cleaving enzyme may contain a nuclear localization signal (NLS) for translocating the nucleic acid cleaving enzyme into the nucleus. When the nucleic acid cleaving enzyme contains an NLS, the NLS is preferably located at the N-terminus.

[0039] The arrangement of each component in the nucleic acid cleaving enzyme is not particularly limited as long as it is capable of site-specific cleavage of the target nucleic acid. For example, when the nucleic acid binding domain is a zinc finger array, typically, an NLS, a zinc finger array, and a nuclease domain are arranged in order from the N-terminus. When the nucleic acid binding domain is a TALE, typically, an NLS, a TALE, and a nuclease domain are arranged in order from the N-terminus.

[0040] The nucleic acid cleaving enzyme can bind to a target nucleic acid and dimerize. Each nuclease domain in the dimer can be the same or different. For example, the nucleic acid cleaving enzyme can include a nuclease domain having the amino acid sequence of SEQ ID NO: 82 and a nuclease domain having the amino acid sequence of SEQ ID NO: 83.

[0041] (Method for producing nucleic acid cleaving enzymes) Nucleic acid cleaving enzymes can be produced using various techniques in the field of genetic engineering. For example, nucleic acid cleaving enzymes may be artificially synthesized based on amino acid sequence information. Alternatively, nucleic acid encoding the nucleic acid cleaving enzyme may be artificially synthesized, introduced into an expression vector, and introduced into a suitable host cell to express the nucleic acid cleaving enzyme. Nucleic acids encoding the nuclease domain and nucleic acid binding domain may be artificially synthesized, introduced into an expression vector, and introduced into a suitable host cell to express each domain, and these may be linked. Nucleic acid encoding the nucleic acid cleaving enzyme may be synthesized by in vitro or in vivo translation. RNA encoding the nucleic acid cleaving enzyme may be synthesized by in vitro transcription, and introduced into a suitable host cell to express the nucleic acid cleaving enzyme.

[0042] When a nucleic acid cleaving enzyme is expressed in a host cell, the nucleic acid cleaving enzyme may be allowed to act in the host cell to modify the nucleic acid, or the expressed nucleic acid cleaving enzyme may be allowed to act in another host cell to modify the nucleic acid.

[0043] Expression vectors used for expressing nucleic acid cleaving enzymes include various vectors used in the field of genetic engineering, such as plasmid vectors, viral vectors (lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, retroviral vectors, etc.), phage vectors, phagemid vectors, BAC vectors, YAC vectors, MAC vectors, and HAC vectors.

[0044] [Nucleic Acid and Vector] In one aspect, there is provided a nucleic acid encoding the nucleic acid cleaving enzyme of the first embodiment. In a further aspect, there is provided a vector comprising the nucleic acid encoding the nucleic acid cleaving enzyme of the first embodiment, or a transcription product or translation product thereof.

[0045] In one aspect, the nucleic acid encoding the nuclease of the first embodiment is an isolated nucleic acid. The nucleic acid may be synthesized based on the amino acid sequence of the nuclease. The nucleic acid may be codon-optimized according to the host organism species.

[0046] The type of vector that serves as the basis for the vector containing the nucleic acid encoding the nucleic acid cleaving enzyme of the first embodiment, or its transcription product or translation product, is not particularly limited, and examples include various vectors used in the field of genetic engineering. Examples include plasmid vectors, viral vectors (lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, retroviral vectors, etc.), phage vectors, phagemid vectors, BAC vectors, YAC vectors, MAC vectors, and HAC vectors. Lentiviral vectors may also be used to introduce transcription products. Here, the term "transcription product" refers to a nucleic acid having the base sequence of mRNA corresponding to the nucleic acid encoding the nucleic acid cleaving enzyme, and does not necessarily refer to the product obtained by transcription. Liposomes, peptide vectors (cell membrane-permeable peptides, etc.), and the like may also be used as vectors.

[0047] A vector can contain various components such as regulatory sequences such as a promoter, enhancer, and polyadenylation signal; restriction enzyme cleavage sites; replication origins; drug resistance genes; and nucleic acids encoding markers such as fluorescent proteins.

[0048] Examples of promoters include a TEF promoter, a TRPC promoter, a CMV promoter, an EF1 promoter, an SV40 promoter, an MSCV promoter, an hTERT promoter, a β-actin promoter, a CAG promoter, mouse and human U6-snRNA promoters, a human H1-RNase P RNA promoter, a human valine-tRNA promoter, a GPD promoter, and a TET on / off promoter.

[0049] Examples of the enhancer include an SV40 enhancer, a cytomegalovirus early promoter enhancer, a polyoma enhancer, and an adenovirus enhancer.

[0050] Examples of restriction enzyme cleavage sites include various restriction enzyme cleavage sites such as AccI, BamHI, EcoRI, HincII, HindIII, I-SceI, KpnI, PstI, SacI, SalI, SmaI, SphI, Sse8387I, TaqI, XbaI, Esp3I, BsaI, SpeI, AscI, PacI, ApaI, NdeI, NcoI, and XhoI. The vector or DNA fragment may contain a multicloning site containing multiple restriction enzyme cleavage sites.

[0051] Examples of drug resistance genes include a chloramphenicol resistance gene, a tetracycline resistance gene, a neomycin resistance gene, an erythromycin resistance gene, a spectinomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a bialaphos resistance gene, a blasticidin S resistance gene, a zeocin resistance gene, and a pyrithiamine resistance gene.

[0052] Examples of fluorescent proteins include green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and orange fluorescent protein (YFP).

[0053] As a method for adding a nucleic acid encoding a nucleic acid cleaving enzyme or its transcription product or translation product to a base vector, various techniques used in the field of genetic engineering, such as restriction enzyme digestion and ligation, can be used.

[0054] <Nucleic Acid Modification Kit> In one aspect, a nucleic acid modification kit is provided, which includes the nucleic acid cleaving enzyme of the first embodiment described above, a nucleic acid encoding the nucleic acid cleaving enzyme, or a vector containing the nucleic acid encoding the nucleic acid cleaving enzyme or its transcription product or translation product, all stored in a container. Details of the nucleic acid cleaving enzyme, nucleic acid, and vector are as described above. The kit may further include various other reagents (dilution buffer, reconstitution solution, washing buffer, nucleic acid introduction reagent, protein introduction reagent, control reagent, etc.) stored in containers. The kit may also include an instruction manual.

[0055] <Method for modifying nucleic acid> In one aspect, a method for modifying nucleic acid is provided, comprising introducing into a cell the nucleic acid cleaving enzyme of the first embodiment described above; a nucleic acid encoding the nucleic acid cleaving enzyme; or a vector comprising a nucleic acid encoding the nucleic acid cleaving enzyme or a transcription product or translation product thereof. The details of the nucleic acid cleaving enzyme, nucleic acid, and vector are as described above. When the nucleic acid cleaving enzyme of the first embodiment is introduced into a cell, the target site of the nucleic acid is cleaved by the nucleic acid cleaving enzyme. The cleaved nucleic acid is then repaired by non-homologous end joining repair (NHEJ), homologous recombination repair (HR), or the like. During this repair, a mutation is introduced into a portion of the cleavage site, thereby modifying the nucleic acid. The modification of the nucleic acid may be any of deletion, insertion, and substitution of nucleotides.

[0056] Cells into which the nucleic acid cleaving enzyme, nucleic acid, or vector can be introduced include prokaryotic cells and eukaryotic cells such as plants, fungi, and animals. Examples of prokaryotes include bacteria (Escherichia coli, actinomycetes, etc.) and archaea. Examples of plants include rice, wheat, tobacco, tomato, potato, and Arabidopsis. Examples of animals include mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; birds such as chickens; amphibians; reptiles; fish; chordates; arthropods; and insects. Examples of animal cells include somatic cells, germ cells, fertilized eggs, germ cells, and stem cells (embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), hematopoietic stem cells, etc.). Examples of fungi include filamentous fungi such as ascomycetes, basidiomycetes, and zygomycetes; and yeast. In one embodiment, the cell may be a non-human cell. In one embodiment, the cell is not a cell present in the human body, a human germ cell, or a human germ cell. The cells may be in vivo cells or isolated cells, and may be primary cells or cultured cells.

[0057] The nucleic acid to be cleaved may be a nucleic acid within a genome, or may be a nucleic acid outside a genome, such as a plasmid DNA, a mitochondrial DNA, or a plastid DNA. Preferably, the nucleic acid cleaving enzyme cleaves a nucleic acid within a genome. The nucleic acid to be cleaved may be a single-stranded nucleic acid or a double-stranded nucleic acid.

[0058] The target sequence on the nucleic acid recognized by the nucleic acid recognition domain is appropriately set depending on the purpose. In one embodiment, the target sequence is a pair of sequences sandwiching a spacer sequence, and the target cleavage site is located within or near the spacer. The pair of sequences may be palindromic or non-palindromic. When the pair of sequences is non-palindromic, two types of nucleic acid cleavage enzymes are used that target each sequence. The spacer sequence may be, for example, 1 to 20 bp. When the nucleic acid binding domain includes a Cas protein, a sequence located near a protospacer adjacent motif (PAM) sequence is selected as the target sequence.

[0059] Methods for introducing the nucleic acid cleaving enzyme of the first embodiment, the nucleic acid encoding the nucleic acid cleaving enzyme, or a vector containing the nucleic acid encoding the nucleic acid cleaving enzyme or its transcription product or translation product into cells include various techniques used in the field of genetic engineering, such as chemical techniques such as the protoplast-PEG method, electrical or physical techniques such as electroporation, particle gun method, microinjection method, lipofection method, and protein transduction method, and methods using viruses or organisms such as viral infection, Agrobacterium method, phage infection, and conjugation.

[0060] A vector can be used for introduction into cells. Examples of vectors include plasmid vectors, viral vectors (lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, retroviral vectors, etc.), phage vectors, phagemid vectors, BAC vectors, YAC vectors, MAC vectors, and HAC vectors. Lentiviral vectors may also be used for introducing transcription products. Alternatively, liposomes, peptide vectors (cell membrane-permeable peptides, etc.), etc. may also be used. The vector may contain various components, such as regulatory sequences such as promoters, enhancers, and polyadenylation signals; restriction enzyme cleavage sites; replication origins; drug resistance genes; and nucleic acids encoding markers such as fluorescent proteins. Specific examples of each component are as described above.

[0061] During (before, after, or simultaneously with) the introduction into the cell, a donor nucleic acid may also be introduced into the cell. The donor nucleic acid is an exogenous nucleic acid that contains the nucleic acid to be introduced into the target nucleic acid. The donor nucleic acid may be used for knock-in or knock-out.

[0062] The donor nucleic acid may be a vector in which the nucleic acid to be introduced is linked to any vector. Examples of the vector include a plasmid vector, a viral vector (lentiviral vector, adenoviral vector, adeno-associated viral vector, herpes viral vector, Sendai viral vector, retroviral vector, etc.), a phage vector, a phagemid vector, a BAC vector, a YAC vector, a MAC vector, and a HAC vector. The vector may contain various components, such as regulatory sequences such as a promoter, an enhancer, and a polyadenylation signal; a restriction enzyme cleavage site; a replication origin; a drug resistance gene; and a nucleic acid encoding a marker such as a fluorescent protein. Specific examples of each component are as described above.

[0063] In one embodiment, the donor nucleic acid may contain homology arms for targeting nucleic acid transfer. The homology arms consist of a 5' homology arm having a sequence homologous to a nucleic acid sequence upstream (5' side) of the target site on the nucleic acid (i.e., the site where nucleic acid transfer is intended) and a 3' homology arm having a sequence homologous to a nucleic acid sequence downstream (3' side) of the target site on the nucleic acid. The number of bases in each of the 5' and 3' homology arms may be, for example, 5 to 10,000 or 100 to 1,000. When the donor nucleic acid contains homology arms, the transferred nucleic acid is positioned between both homology arms. Furthermore, if the donor nucleic acid itself has sufficient homology to the target site on the nucleic acid, it can undergo homologous recombination with a sequence on the nucleic acid even without homology arms. Therefore, the donor nucleic acid may be a nucleic acid that differs by several bases from the target site or that contains an insertion or deletion for sequence modification. In this case, it is not necessary to use a combination of a 5' homology arm having a sequence homologous to a nucleic acid sequence upstream (5' side) of the target site and a 3' homology arm having a sequence homologous to a nucleic acid sequence downstream (3' side) of the target site on the nucleic acid, but rather it is sufficient to use a nucleic acid that is different from the target sequence. The number of identical bases between the donor nucleic acid and the target site may be, for example, 5 to 10,000, or may be 100 to 1,000.

[0064] In the nucleic acid modification method of the first embodiment, one type of nucleic acid cleaving enzyme may be used, or two or more types of nucleic acid cleaving enzymes may be used. The two or more types of nucleic acid cleaving enzymes may be two or more types of nucleic acid cleaving enzymes with different nuclease domains, two or more types of nucleic acid cleaving enzymes with different nucleic acid binding domains, or two or more types of nucleic acid cleaving enzymes with different nuclease domains and nucleic acid binding domains. Furthermore, the two or more types of nucleic acid cleaving enzymes may be two or more types of nucleic acid cleaving enzymes with the same nuclease domain and nucleic acid binding domain but with different other components (such as linkers). In a preferred aspect, the nucleic acid modification method of the first embodiment uses at least two types of nucleic acid cleaving enzymes with different nucleic acid binding domains. For example, when the target sequences on a nucleic acid are a pair of sequences that are non-palindromic, two types of nucleic acid cleaving enzymes containing nucleic acid binding domains corresponding to each target sequence may be used.

[0065] 2. Second Embodiment <Method for Producing a Mutant> In one aspect, a method for producing a mutant of a eukaryotic cell is provided, comprising deleting a centromere region of a genome on a chromosome in the eukaryotic cell. The inventors discovered that when a mutant in which the centromere region of a chromosome in a eukaryotic cell has been deleted and replaced with a drug resistance gene is cultured on a non-selective medium, the retention rate of the chromosome decreases. While the reason for this is unclear, it is speculated that removal of the centromere region destabilizes the chromosome in the cell nucleus, reducing the efficiency of chromosome segregation during cell division, making the chromosome more likely to be lost from the cell. On the other hand, it was found that when the mutant is cultured on a selective medium, the relative chromosome copy number per cell increases. It is speculated that this is because, under selective conditions, copy number control is released in cells that retain chromosomes lacking the centromere region, resulting in multiple copies of the chromosome in the cell nucleus.

[0066] Eukaryotic cells include plant, fungal, and animal cells. Examples of plants include rice, wheat, tobacco, tomato, potato, Arabidopsis, etc. Examples of animals include mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; birds such as chickens; amphibians; reptiles; fish; chordates; arthropods; and insects. Examples of animal cells include somatic cells, germ cells, fertilized eggs, germ cells, and stem cells (embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), hematopoietic stem cells, etc.). Examples of fungi include basidiomycetes, ascomycetes, and zygomycetes. In one embodiment, the cell may be a non-human cell. In one embodiment, the cell is not a cell present in the human body, a human germ cell, or a human embryonic cell. The cell may be an in vivo cell or an isolated cell. Furthermore, the cell may be a primary cell or a cultured cell.

[0067] Among these, eukaryotic cells are preferably eukaryotic cells having accessory chromosomes. Accessory chromosomes are chromosomes that are not essential for survival and are known to be present in the genus Fusarium (Fusarium oxysporum, Fusarium commune, Fusarium poae, Fusarium vanettenii, etc.), the genus Zymoseptoria, the genus Alternaria, the genus Colletotrichum, the genus Pyricularia, etc. Examples of Fusarium oxysporum include the tomato wilt fungus, the radish wilt fungus, the cabbage wilt fungus, the turnip wilt fungus, the lettuce root rot fungus, the lettuce root rot fungus, the spinach wilt fungus, and the chive dry rot fungus.

[0068] A centromere region is a region on a chromosome where the kinetochore to which the spindle attaches during cell division is formed, and is an AT-rich non-coding region. A centromere region can be identified by those skilled in the art as a continuous AT-rich region, and in the present disclosure, a centromere region is identified as a continuous region with an AT content of 75% or more (i.e., a GC content of 25% or less). Here, the AT content refers to the ratio of the number of A (adenine) and T (thymine) to the total number of bases in the sequence.

[0069] In the method for producing a mutant of the second embodiment, the entire centromere region may be deleted, or only a portion of the centromere region may be deleted. In addition to the entire or a portion of the centromere region, other adjacent regions may also be deleted. The base length of the centromere region to be deleted may be, for example, 40 kb or more, 100 kb or more, or 500 kb or more. The base length of the centromere region to be deleted may also be 500 kb or less, 100 kb or less, or 40 kb or less. Therefore, the base length of the centromere region to be deleted may be 10 to 500 kb, 10 to 100 kb, or 40 to 100 kb. In one aspect, the base length of the centromere region to be deleted may be 50 to 100%, 70 to 100%, 80 to 100%, or 90 to 100% of the base length of the estimated centromere region identified by the above-mentioned method, based on the number of bases. When other adjacent regions are deleted in addition to the entire or part of the centromere region, the base length of the other regions is preferably 1 to 10 kb, more preferably 1 to 5 kb.

[0070] In one embodiment, the chromosome in which the centromere region is deleted is preferably an accessory chromosome. In accessory chromosomes, deletion of the centromere region is unlikely to impair growth, and centromere-deleted living cells can be easily obtained by selection using an appropriate method, such as a selective medium. On the other hand, even if a chromosome other than an accessory chromosome (i.e., a core chromosome) is used, mutants can be similarly obtained, and similar effects can be expected, provided that chromosome instability due to deletion of the centromere region is not lethal. Furthermore, when a mutant is obtained in which the centromere of a chromosome carrying an essential gene required for survival is deleted, it is believed that the chromosome in which the centromere region is deleted can be maintained without subsequent drug selection.

[0071] In one embodiment, the deletion of the centromere region is achieved by inducing a DNA repair mechanism. Specifically, the centromere region to be deleted is identified, and both ends (i.e., two locations) of the centromere region are used as target sites, and each target site is cleaved using a site-specific nuclease. The cleaved DNA is repaired by non-homologous end joining repair (NHEJ), homologous recombination repair (HR), or the like. This allows the centromere region to be deleted. For example, the deletion of the centromere region is achieved by cleaving the DNA with a nuclease such as ZFN, TALEN, CRISPR / Cas system, or I-SceI, and repairing the cleavage using a DNA repair mechanism.

[0072] ZFNs are artificial restriction enzymes that link a DNA cleavage domain to a linkage of zinc fingers (zinc finger array) that recognize and bind to DNA. Each zinc finger recognizes a three-base sequence. Linking zinc fingers to form a zinc finger array allows recognition of longer base sequences, thereby increasing the specificity for the target sequence. When the nucleic acid binding domain contains zinc fingers, the number of zinc fingers contained in the nucleic acid binding domain is preferably two or more, and may be three to nine, or may be three to six. Typically, FokI is used as the DNA cleavage domain, but other nucleases, such as modified forms of FokI, may also be used.

[0073] A TALEN is an artificial nuclease that combines a TALE (Transcription Activator-Like Effector), which recognizes and binds to DNA, with a DNA cleavage domain. TALEs have a repeating structure called a TALE repeat, with each repeat recognizing and binding to one base. Linking TALE repeats allows recognition and binding to a multi-base sequence. The number of bases recognized by a TALE is not particularly limited as long as it is within a range of base lengths that can be specifically selected within a genome, and may be 5 to 10, 10 to 20, or 20 to 30. FokI is typically used as the DNA cleavage domain, but other nucleases, such as modified versions of FokI, may also be used.

[0074] CRISPR / Cas systems include Class 1 (including Types I, III, and IV) and Class 2 (including Types II, V, and VI) systems. CRISPR / Cas systems include CRISPR / Cas3, CRISPR / Cas9, CRISPR / Cas10, CRISPR / Cas12, CRISPR / Cas13, CRISPR / Cas14, and the like. Of these, CRISPR / Cas9, which is classified as Type II of Class 2, is widely used.

[0075] ZFN, TALEN, CRISPR / Cas system, site-specific nucleases such as I-SceI may be used to delete a centromere region by any method used in the art. For example, in the case of ZFN, DNA encoding a zinc finger array and a nuclease may be introduced into an appropriate vector and expressed in a eukaryotic cell, thereby deleting a centromere region of a chromosome in the eukaryotic cell. In the case of TALEN, DNA encoding a TALE and a nuclease may be introduced into an appropriate vector and expressed in a eukaryotic cell, thereby deleting a centromere region of a chromosome in the eukaryotic cell. In the case of the CRISPR / Cas system, DNA encoding a guide RNA and a Cas protein may be introduced into an appropriate vector and expressed in a eukaryotic cell, thereby deleting a centromere region of a chromosome in the eukaryotic cell. Alternatively, mRNA encoding a guide RNA and a Cas protein may be introduced into a eukaryotic cell and the Cas protein may be expressed, thereby deleting the centromere region of a chromosome in the eukaryotic cell. Alternatively, a complex of a guide RNA and a Cas protein may be introduced into a eukaryotic cell and used to deleting the centromere region of a chromosome in the eukaryotic cell. Of these, a simple method is to introduce DNA encoding a guide RNA and a Cas protein into an appropriate vector and express it in the eukaryotic cell. I-SceI is a nuclease that recognizes 18 bases. When using I-SceI, first, the recognition sequence is knocked into a genomic region. Then, DNA encoding I-SceI is introduced into an appropriate vector and expressed in the eukaryotic cell, or I-SceI is introduced into the eukaryotic cell using an appropriate method, thereby inducing cleavage within the recognition sequence. This may be used to deleting a centromere region.

[0076] The type of vector for intracellularly expressing a site-specific nuclease such as ZFN, TALEN, or CRISPR / Cas system is not particularly limited as long as it can be used for gene transfer, and examples thereof include plasmid vectors, viral vectors (retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, etc.), phage vectors, phagemid vectors, BAC vectors, YAC vectors, MAC vectors, and HAC vectors.

[0077] A vector can contain various components such as regulatory sequences such as a promoter, enhancer, and polyadenylation signal; restriction enzyme cleavage sites; replication origins; drug resistance genes; and nucleic acids encoding markers such as fluorescent proteins.

[0078] Examples of promoters include a TEF promoter, a TRPC promoter, a CMV promoter, an EF1 promoter, an SV40 promoter, an MSCV promoter, an hTERT promoter, a β-actin promoter, a CAG promoter, a U6-snRNA promoter, a human H1-RNase P RNA promoter, a human valine-tRNA promoter, a GPD promoter, and a TET on / off promoter.

[0079] Examples of the enhancer include an SV40 enhancer, a cytomegalovirus early promoter enhancer, a polyoma enhancer, and an adenovirus enhancer.

[0080] Examples of restriction enzyme cleavage sites include various restriction enzyme cleavage sites such as AccI, BamHI, EcoRI, HincII, HindIII, I-SceI, KpnI, PstI, SacI, SalI, SmaI, SphI, Sse8387I, TaqI, XbaI, Esp3I, BsaI, SpeI, AscI, PacI, ApaI, NdeI, NcoI, and XhoI. The vector or DNA fragment may contain a multicloning site containing multiple restriction enzyme cleavage sites.

[0081] Examples of drug resistance genes include a chloramphenicol resistance gene, a tetracycline resistance gene, a neomycin resistance gene, an erythromycin resistance gene, a spectinomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a bialaphos resistance gene, a blasticidin S resistance gene, a zeocin resistance gene, and a pyrithiamine resistance gene.

[0082] Examples of fluorescent proteins include green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and orange fluorescent protein (YFP).

[0083] When a site-specific nuclease such as a ZFN, TALEN, or CRISPR / Cas system is introduced into a cell (before, after, or simultaneously with the introduction; including when the nuclease is expressed in the cell), a donor nucleic acid may also be introduced into the cell. The donor nucleic acid is an exogenous nucleic acid that contains the nucleic acid to be introduced into the target nucleic acid.

[0084] The donor nucleic acid may be a vector in which the nucleic acid to be introduced is linked to any vector. Examples of the vector include a plasmid vector, a viral vector (lentiviral vector, adenoviral vector, adeno-associated viral vector, herpes viral vector, Sendai viral vector, retroviral vector, etc.), a phage vector, a phagemid vector, a BAC vector, a YAC vector, a MAC vector, and a HAC vector. The vector may contain various components, such as regulatory sequences such as a promoter, an enhancer, and a polyadenylation signal; a restriction enzyme cleavage site; a replication origin; a drug resistance gene; and a nucleic acid encoding a marker such as a fluorescent protein. Specific examples of each component are as described above.

[0085] In one embodiment, the donor nucleic acid may contain homology arms for targeting nucleic acid transfer. The homology arms consist of a 5' homology arm having a sequence homologous to a nucleic acid sequence upstream (5' side) of the target site on the nucleic acid (i.e., the site where nucleic acid transfer is intended) and a 3' homology arm having a sequence homologous to a nucleic acid sequence downstream (3' side) of the target site on the nucleic acid. The number of bases in each of the 5' and 3' homology arms may be, for example, 5 to 10,000 or 100 to 1,000. When the donor nucleic acid contains homology arms, the transferred nucleic acid is positioned between both homology arms. Furthermore, if the donor nucleic acid itself has sufficient homology to the target site on the nucleic acid, it can undergo homologous recombination with a sequence on the nucleic acid even without homology arms. Therefore, the donor nucleic acid may be a nucleic acid that differs by several bases from the target site or that contains an insertion or deletion for sequence modification. In this case, it is not necessary to use a combination of a 5' homology arm having a sequence homologous to a nucleic acid sequence upstream (5' side) of the target site and a 3' homology arm having a sequence homologous to a nucleic acid sequence downstream (3' side) of the target site on the nucleic acid, but rather it is sufficient to use a nucleic acid that is different from the target sequence. The number of identical bases between the donor nucleic acid and the target site may be, for example, 5 to 10,000, or may be 100 to 1,000.

[0086] In one embodiment, another gene may be inserted into the deleted centromere region. For example, by inserting a marker gene into the chromosome so as to replace the deleted centromere region, centromere-deficient strains can be suitably selected. Examples of marker genes include drug resistance genes, fluorescent protein genes, genes for enzymes that catalyze color reactions, and genes involved in auxotrophy. Examples of drug resistance genes include those described above. Examples of fluorescent protein genes include genes for the fluorescent proteins described above. Examples of genes for enzymes that catalyze color reactions include luciferase genes, β-glucuronidase genes, and β-galactosidase genes. Examples of genes involved in auxotrophy include uracil biosynthesis genes, histidine biosynthesis genes, and adenine biosynthesis genes.

[0087] In one embodiment, a mutant having a chromosome lacking a centromere region has an increased chromosome copy number per cell compared to before the centromere region was lost. In one embodiment, the mutant has a chromosome copy number that is 1.1 times or more, preferably 1.2 times or more, 1.3 times or more, 1.4 times or more, or 1.5 times or more, compared to a control having no centromere region lost. The chromosome copy number may be 3.0 times or less, or 2.0 times or less, compared to the control. The relative amount of chromosome copy number is calculated as follows: Genomic DNA is extracted from a mutant having a chromosome lacking a centromere region and a negative control cell. A region of a gene (e.g., Ste3 gene) located on a core chromosome and a region of a gene (e.g., 14-1, 14-2, etc.) located on a chromosome lacking a centromere region are amplified by real-time RT-PCR, and the relative amount of chromosome copy number is estimated from the ratio of the amplification levels.

[0088] <Gene Expression Method> In one aspect, there is provided a gene expression method comprising expressing a gene on a chromosome in which a centromere region has been deleted in a mutant prepared by the method for preparing a mutant of the second embodiment. The method for preparing a mutant of the second embodiment can increase the chromosome copy number per cell of a eukaryotic organism, thereby enabling efficient gene expression on the chromosome.

[0089] The gene can be expressed by culturing the mutant in an appropriate medium. If necessary, the transcription or translation product may be isolated or purified.

[0090] The mutant preferably has a drug resistance gene on a chromosome in which the centromere region has been deleted. It has been found that when the centromere region of a chromosome is deleted in a mutant, the chromosome is lost over time. On the other hand, if the drug resistance gene is on a chromosome in which the centromere region has been deleted, mutants that retain the chromosome can be suitably obtained by drug selection. Examples of drug resistance genes include those mentioned above.

[0091] The expression of a gene on a chromosome may be the expression of an endogenous gene originally possessed by the chromosome, or the expression of a gene (exogenous gene) that is not originally possessed by the chromosome and has been exogenously introduced. Introduction of an exogenous gene into a genome can be carried out by gene insertion utilizing a DNA repair mechanism including homologous recombination and non-homologous end joining using site-specific nucleases such as the ZFN, TALEN, and CRISPR / Cas system described above.

[0092] <Eukaryotic Cell> In one aspect, a eukaryotic cell is provided in which the centromere region of the genome on a chromosome is deleted. The eukaryotic cell may be one produced by the method for producing a mutant of the second embodiment described above. Details of the deletion of the centromere region and the eukaryotic cell are as described above.

[0093] In one embodiment, the genome on the chromosome contains a marker gene (e.g., a drug resistance gene). Examples of marker genes include those described above. The marker gene may be an endogenous gene that was originally present on the chromosome, or may be a gene that was not originally present on the chromosome but was introduced exogenously (an exogenous gene). Furthermore, if the chromosome can be prepared without using a marker gene, the marker gene is not necessary.

[0094] In one embodiment, the eukaryotic cell is a fungal cell. Examples of fungi include basidiomycetes, ascomycetes, and zygomycetes. Among these, fungi of the genus Fusarium (Fusarium oxysporum, Fusarium commune, Fusarium poae, Fusarium vanettenii, etc.) are preferred.

[0095] 3. Third Embodiment <Vector or DNA Fragment> In one aspect of the third embodiment, there is provided a vector or DNA fragment for eukaryotic genetic manipulation, which comprises three or more telomeric repeat sequences, with the telomeric repeat sequences being contained in only one direction. (Hereinafter, this embodiment will be referred to as "Embodiment 3-1.") In one aspect of the third embodiment, there is provided a vector or DNA fragment for eukaryotic genetic manipulation, which comprises 3 to 26 telomeric repeat sequences. (Hereinafter, (this embodiment will be referred to as "Embodiment 3-2.")) In one aspect of the third embodiment, there is provided a vector or DNA fragment for eukaryotic genetic manipulation, which comprises three or more telomeric repeat sequences and a sequence of 21 bases or more in length with an AT content of 60% or more (hereinafter also referred to as "AT-rich sequence"). (Hereinafter, this embodiment will be referred to as "Embodiment 3-3.") In one aspect of the third embodiment, there is provided a vector or DNA fragment for eukaryotic genetic manipulation, which comprises three or more telomeric repeat sequences that are artificially synthesized DNA. (Hereinafter, this embodiment will be referred to as "Embodiment 3-4.") However, in Embodiments 3-1 to 3-4, the vector or DNA fragment is not an artificial chromosome. In the present disclosure, a telomeric repeat sequence refers to a sequence in which the unit sequence of telomeric DNA constituting the telomere of a eukaryote is repeated. The vector or DNA fragment of the third embodiment will be described in detail below. In the following description of the vector or DNA fragment of the third embodiment, unless otherwise specified, it applies to any of Embodiments 3-1 to 3-4.

[0096] The inventors have found that the use of a vector or DNA fragment containing a telomere repeat sequence improves the efficiency of gene transfer into eukaryotes. Although the mechanism behind this is unclear, it is believed that the inclusion of a telomere repeat sequence in a vector or DNA fragment gives the vector or DNA fragment the ability to self-replicate within a cell nucleus, thereby improving gene transfer efficiency. Telomeres are naturally present at both ends of chromosomes in eukaryotes, and known techniques that utilize telomeres place telomere sequences in opposite directions at both ends. For example, in the expression vector containing telomeres described in JP 2017-538425 A, telomere repeat sequences are placed in opposite directions at both ends of a kanamycin resistance gene. Meanwhile, the inventors' studies have found that the above-mentioned improvement in gene transfer efficiency is observed whether the telomere repeat sequence is contained in only one direction or in both directions (i.e., in opposite directions). Furthermore, when the number of telomere repeats was increased, the gene transfer efficiency was significantly improved when the number of repeats was three or more, and a correlation between the number of repeats and the gene transfer efficiency was found. This suggests that an increase in the number of telomere repeats enhances the replication ability of vectors or DNA fragments in the nucleus.

[0097] It was also found that gene transfer efficiency was sufficiently improved even when the number of repeats of the telomere repeat sequence was about 3 to 20. In particular, a significant improvement in transfer efficiency was observed even when the number of repeats was about 3 to 7. Therefore, by artificially synthesizing a telomere repeat sequence and adding it to a vector or DNA fragment, the vector or DNA fragment of the third embodiment can be easily produced and used in gene transfer techniques.

[0098] Typically, vectors or DNA fragments used for gene transfer are lost at a certain rate over time after being introduced into a host. For example, it has been found that when transgenic colonies obtained by drug selection are cultured in a non-selective medium that does not contain the drug, a certain rate of the colonies loses drug resistance. Meanwhile, the inventors have discovered that using a vector or DNA fragment containing an AT-rich sequence in addition to a telomeric repeat sequence suppresses the loss of the vector or DNA fragment introduced into the host and increases its retention rate. This AT-rich sequence may be at least a portion of the centromere region. The centromere region is a region on a chromosome where the kinetochore to which the spindle binds during cell division is formed, and is an AT-rich region with an AT content of approximately 60% or more. Although the mechanism is unclear, it is speculated that this AT-rich region contributes to the stability of the introduced vector or DNA fragment.

[0099] The type of vector is not particularly limited as long as it can be used for gene transfer, and examples thereof include plasmid vectors, viral vectors (retroviral vectors, lentiviral vectors, adenoviral vectors, adeno-associated viral vectors, herpes viral vectors, Sendai viral vectors, etc.), phage vectors, phagemid vectors, BAC vectors, YAC vectors, MAC vectors, and HAC vectors.

[0100] The DNA fragment used in the third embodiment is typically a double-stranded DNA fragment. In the third embodiment, the DNA fragment refers to non-circular DNA. The size of the DNA fragment is not particularly limited as long as it allows gene transfer, and may be, for example, 200 to 100,000 bases, 500 to 50,000 bases, or 1,000 to 10,000 bases.

[0101] A vector or DNA fragment may contain various components, such as regulatory sequences such as a promoter, enhancer, and polyadenylation signal; restriction enzyme cleavage sites; replication origins; drug resistance genes; and nucleic acids encoding markers such as fluorescent proteins.

[0102] Examples of promoters include a TEF promoter, a TRPC promoter, a CMV promoter, an EF1 promoter, an SV40 promoter, an MSCV promoter, an hTERT promoter, a β-actin promoter, a CAG promoter, a U6-snRNA promoter, a human H1-RNase P RNA promoter, a human valine-tRNA promoter, a GPD promoter, and a TET on / off promoter.

[0103] Examples of the enhancer include an SV40 enhancer, a cytomegalovirus early promoter enhancer, a polyoma enhancer, and an adenovirus enhancer.

[0104] Examples of restriction enzyme cleavage sites include various restriction enzyme cleavage sites such as AccI, BamHI, EcoRI, HincII, HindIII, I-SceI, KpnI, PstI, SacI, SalI, SmaI, SphI, Sse8387I, TaqI, XbaI, Esp3I, BsaI, SpeI, AscI, PacI, ApaI, NdeI, NcoI, and XhoI. The vector or DNA fragment may contain a multicloning site containing multiple restriction enzyme cleavage sites.

[0105] Examples of drug resistance genes include a chloramphenicol resistance gene, a tetracycline resistance gene, a neomycin resistance gene, an erythromycin resistance gene, a spectinomycin resistance gene, a kanamycin resistance gene, a hygromycin resistance gene, a puromycin resistance gene, a bialaphos resistance gene, a blasticidin S resistance gene, a zeocin resistance gene, and a pyrithiamine resistance gene.

[0106] Examples of fluorescent proteins include green fluorescent protein (GFP), red fluorescent protein (RFP), cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), and orange fluorescent protein (YFP).

[0107] The vector or DNA fragment may contain a base sequence encoding a site-specific endonuclease, or may contain a base sequence encoding a ZFN, a TALEN, a CRISPR / Cas system, or I-SceI.

[0108] ZFNs are artificial restriction enzymes that link a DNA cleavage domain to a linkage of zinc fingers (zinc finger array) that recognize and bind to DNA. Each zinc finger recognizes a three-base sequence. Linking zinc fingers to form a zinc finger array allows recognition of longer base sequences, thereby increasing the specificity for the target sequence. When the nucleic acid binding domain contains zinc fingers, the number of zinc fingers contained in the nucleic acid binding domain is preferably two or more, and may be three to nine, or may be three to six. Typically, FokI is used as the DNA cleavage domain, but other nucleases, such as modified forms of FokI, may also be used.

[0109] TALEN is an artificial nuclease that links a TALE (Transcription Activator-Like Effector), which recognizes and binds to DNA, with a DNA cleavage domain. TALE has a repeating structure called a TALE repeat, with each repeat recognizing and binding to one base. Linking TALE repeats makes it possible to recognize and bind to a base sequence of multiple bases. FokI is typically used as the DNA cleavage domain, but other nucleases, such as modified versions of FokI, may also be used.

[0110] CRISPR / Cas systems include class 1 (including types I, III, and IV) and class 2 (including types II, V, and VI) systems. CRISPR / Cas systems include CRISPR / Cas3, CRISPR / Cas9, CRISPR / Cas10, CRISPR / Cas12, CRISPR / Cas13, CRISPR / Cas14, and the like. Of these, CRISPR / Cas9, which is classified as type II of class 2, is widely used. Note that the "nucleotide sequence encoding the CRISPR / Cas system" refers to a nucleotide sequence encoding a Cas protein and a guide RNA.

[0111] The vector or DNA fragment of the third embodiment may be prepared, for example, by adding artificially synthesized DNA that is a telomere repeat sequence to a vector or DNA fragment that does not contain a telomere repeat sequence. Furthermore, a vector or DNA fragment containing a telomere repeat sequence and an AT-rich sequence may be prepared by adding an AT-rich sequence. The addition of a telomere repeat sequence and / or an AT-rich sequence to a vector or DNA fragment can be performed using various techniques used in the field of genetic engineering. For example, a telomere repeat sequence can be prepared by artificial synthesis and added to a vector or DNA fragment using techniques such as nuclease cleavage and ligation. Alternatively, an AT-rich sequence can be prepared by artificial synthesis and similarly added to a vector or DNA fragment. Alternatively, at least a portion of a centromere region derived from a eukaryote may be obtained and similarly added to a vector or DNA fragment.

[0112] Vectors or DNA fragments that do not contain telomeric repeat sequences may be commercially available or may be prepared by genetic engineering techniques such as PCR, restriction enzyme digestion, DNA ligation, and in vitro transcription / translation.

[0113] [Telomere repeat sequence] The telomere sequence in the telomere repeat sequence may be the telomere sequence of any eukaryote. Eukaryotes include plants, fungi, and animals. Plants include rice, wheat, tobacco, tomato, potato, Arabidopsis, etc. Animals include mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; birds such as chickens; amphibians; reptiles; fish; chordates; arthropods, and insects. Fungi include basidiomycetes, ascomycetes, and zygomycetes. Note that the above-mentioned "telomere sequence of a eukaryote" does not mean that the telomere sequence is derived from the eukaryote, but rather that the telomere sequence has the same sequence as that possessed by the eukaryote.

[0114] In one embodiment, the telomere sequence is preferably a telomere sequence of a fungus, more preferably a telomere sequence of a basidiomycete or an ascomycete. In particular, the telomere sequence is preferably a telomere sequence of a fungus of the genus Fusarium or Pyricularia. The telomere sequence may be the telomere sequence of a eukaryote that serves as a host upon gene introduction, or may be different from the telomere sequence of the eukaryote that serves as a host.

[0115] The length of the telomere sequence per unit varies depending on the species, and is, for example, 4 to 30 bases. In one embodiment, the telomere sequence is TTAGGG, T(G) ２－３ T (G) １－６ , G ２－８ TTAC(A), or ACGGATGTCTAACTTCTTGGTGT. TTAGGG is a telomeric sequence in, for example, Fusarium, Pyricularia, and humans. T(G) ２－３ T (G) １－６ is the telomere sequence in Saccharomyces cerevisiae. ２－８ TTAC(A) is the telomere sequence in fission yeast ( Schizosaccharomyces pombe ). ACGGATGTCTAACTTCTTGGTGT is the telomere sequence in Candida albicans.

[0116] The number of repeats of the telomere repeat sequence is 3 or more. From the viewpoint of improving gene transfer efficiency, the number of repeats is preferably 4 or more, and more preferably 5 or more. From the viewpoint of ease of preparation of the vector or DNA fragment, the number of repeats is preferably 50 or less, more preferably 40 or less, even more preferably 30 or less, more preferably 26 or less, and may be 20 or less. From such viewpoints, the number of repeats is preferably 3 to 50, more preferably 3 to 40, even more preferably 3 to 30, particularly preferably 3 to 26, extremely preferably 3 to 20, even more preferably 4 to 20, and even more preferably 5 to 20. In embodiment 3-2, the number of repeats of the telomere is 3 to 26.

[0117] The telomere repeat sequence in a vector or DNA fragment may be present in one direction or in two directions. In embodiment 3-1, the telomere repeat sequence is present in one direction. Even if the telomere repeat sequence is present in one direction, gene transfer efficiency can be improved. Therefore, the vector or DNA fragment of the third embodiment can be prepared by a simple method and applied to gene transfer technology. Note that, when the telomere sequence is TTAGGG, for example, the telomere repeat sequence present in one direction means that the 5'-TTAGGG-3' repeat sequence is present only in one direction and not in the opposite direction. Furthermore, when the telomere sequence is TTAGGG, the telomere repeat sequence present in two directions means that the 5'-TTAGGG-3' repeat sequence is present in both directions (i.e., when the vector or DNA fragment is double-stranded DNA, one of the DNA strands contains the 5'-TTAGGG-3' repeat sequence and the 5'-CCCTAA-3' repeat sequence). The telomere repeat sequence is preferably contained in a vector or DNA fragment at least upstream and / or downstream of a gene expression cassette to be expressed, in the same orientation as the gene expression cassette.

[0118] The position of the telomere repeat sequence in a vector or DNA fragment is appropriately designed to allow for the desired genetic manipulation. For example, the telomere repeat sequence is placed upstream, downstream, or both upstream and downstream of the desired gene expression cassette. For example, it is preferable to place the telomere repeat sequence upstream, downstream, or both upstream and downstream of an appropriate selection marker (e.g., drug selection marker) and the gene expression cassette, and more preferably at least downstream. While not limiting the embodiments of the present disclosure by theory, it is believed that the gene expression cassette may be lost from a vector or DNA fragment, particularly a DNA fragment, containing a selection marker (e.g., drug selection marker) and a gene expression cassette. Here, it is believed that placing the telomere repeat sequence at least downstream of the gene expression cassette increases the likelihood of selecting cells that maintain the desired gene expression cassette.

[0119] [AT-Rich Sequence] The vector or DNA fragment may contain a sequence of 21 bases or more in length with an AT content of 60% or more (i.e., an AT-rich sequence). In embodiment 3-3, the vector or DNA fragment contains an AT-rich sequence. From the viewpoint of enhancing the stability of the gene introduced into the host, the AT content in the AT-rich sequence is 60% or more, preferably 70% or more, and more preferably 75% or more. The AT content may be 90% or less, or may be 85% or less. From this viewpoint, the AT content is preferably 60 to 90%, more preferably 70 to 85%, and even more preferably 75 to 85%. From the same viewpoint, the GC content in the AT-rich sequence is preferably 40% or less, more preferably 30% or less, and even more preferably 25% or less. The GC content may be 10% or more, or may be 15% or more. From this viewpoint, the GC content may be 10 to 40%, 15 to 30%, or 15 to 25%. The AT content represents the ratio of A (adenine) and T (thymine) to the total number of bases in a sequence. The GC content represents the ratio of G (guanine) and C (cytosine) to the total number of bases in a sequence. Whether a vector or DNA fragment contains an AT-rich sequence is determined by whether the vector or DNA fragment has a sequence of 21 bases or more in length with an AT content of 60% or more at any position.

[0120] The AT-rich sequence may be at least a part of the sequence of the centromere region. The centromere region is known to be an AT-rich region with an AT content of about 60% or more. The centromere region may be the centromere region of any eukaryote. Examples of eukaryotes include those exemplified in the section on telomere repeat sequences. The centromere region may be the centromere region of a eukaryote that serves as a host during gene introduction, or may be different from the centromere region of the host eukaryote.

[0121] The length of the AT-rich sequence is 21 bases or more, and from the viewpoint of enhancing the stability of the gene introduced into the host, it is preferably 500 bases or more, more preferably 1000 bases or more, and may be 2000 bases or more, 3000 bases or more, or 4000 bases or more. It has been found that even if the length of the AT-rich sequence is longer than 1000 bases, the stability of the introduced gene does not change significantly. Therefore, the length of the AT-rich sequence may be 4000 bases or less, 3000 bases or less, or 2000 bases or less. From this viewpoint, the length of the AT-rich sequence is preferably 21 to 4000 bases, more preferably 500 to 4000 bases, even more preferably 1000 to 4000 bases, particularly preferably 1000 to 3000 bases, and extremely preferably 1000 to 2000 bases.

[0122] The position of the AT-rich sequence in a vector or DNA fragment is appropriately designed within the range that allows the desired genetic manipulation. In the case of a vector or DNA fragment containing a telomere repeat sequence and a desired gene expression cassette, from the viewpoint of ease of preparation, it is preferable to arrange the telomere repeat sequence so that it is the outermost of the telomere repeat sequence, the gene expression cassette, and the AT-rich sequence. For example, it is preferable to arrange the telomere repeat sequence, the gene expression cassette, and the AT-rich sequence in any of the following orders. Here, the AT-rich sequence and the gene expression cassette may each independently be one or more. (A) Telomere repeat sequence, AT-rich sequence, gene expression cassette (B) Telomere repeat sequence, gene expression cassette, AT-rich sequence (C) AT-rich sequence, gene expression cassette, telomere repeat sequence (D) Gene expression cassette, AT-rich sequence, telomere repeat sequence (E) Telomere repeat sequence, AT-rich sequence, gene expression cassette, telomere repeat sequence (F) Telomere repeat sequence, gene expression cassette, AT-rich sequence, telomere repeat sequence In the case of a DNA fragment, the telomere repeat sequence is preferably located at the end.

[0123] [Uses of vectors or DNA fragments] Vectors or DNA fragments are used for genetic engineering of eukaryotes, details of which will be described later in the section "Method for producing genetically engineered eukaryotic cells."

[0124] In one aspect, the vector or DNA fragment is preferably detachable after being introduced into the host eukaryotic cell. For example, when genome editing is performed using the CRISPR / Cas system, conventional technologies have low cell introduction efficiency, so transformation is generally performed by incorporating a selection marker gene into the donor nucleic acid. In this case, the selection marker gene is also incorporated into the genome in the genome-edited strain, making it a genetically modified organism, and the same selection marker cannot be used repeatedly. Similarly, when transformation is performed by incorporating a selection marker gene into a plasmid containing a CRISPR / Cas system, the genome-edited strain also incorporates the CRISPR / Cas system and selection marker gene into the genome, making it a genetically modified organism, and the same selection marker cannot be used repeatedly. On the other hand, if the donor nucleic acid does not contain a selection marker gene, but the vector or DNA fragment of the third embodiment contains a selection marker gene, and then the vector or DNA fragment is detached after genome editing, a genome-edited organism without a selection marker can be produced. This makes it possible to produce a genome-edited strain that does not fall under genetic modification, and the same selection marker can be used repeatedly.

[0125] <Kit> In one aspect, a kit for eukaryotic genetic manipulation is provided, comprising the vector or DNA fragment of the third embodiment stored in a container. Details of the vector or DNA fragment of the third embodiment are as described above. The kit may further comprise various reagents that can be used for genetic manipulation (dilution buffer, reconstitution solution, wash buffer, nucleic acid introduction reagent, protein introduction reagent, control reagent, etc.). The kit may also comprise an instruction manual.

[0126] <Method for Producing Genetically Engineered Eukaryotic Cells> In one aspect, a method for producing a genetically engineered eukaryotic cell is provided, comprising introducing the vector or DNA fragment of the third embodiment into a eukaryotic cell. Genetic engineering in the third embodiment encompasses both modifying a host's genomic gene and expressing a gene from an extragenomic nucleic acid construct (e.g., an introduced vector or DNA fragment). Examples of genetic engineering include genome editing and gene expression via transformation. Genome editing may be knock-in or knock-out. Genome editing may be, for example, genome editing using a nuclease, such as ZFN, TALEN, CRISPR / Cas system, or I-SceI, encoded in the introduced vector or DNA fragment. Gene expression via transformation may be expression of a polypeptide (including a protein) or RNA encoded in the introduced vector or DNA fragment. Silencing via expression of RNA, such as siRNA or miRNA, is also encompassed. Genetic manipulation may be to newly express or enhance a specific gene within or outside the genome, or to eliminate or reduce the expression of a specific gene within or outside the genome.

[0127] Host eukaryotic cells include plant, fungal, and animal cells. Examples of plants include rice, wheat, tobacco, tomato, potato, Arabidopsis, etc. Examples of animals include mammals such as humans, monkeys, mice, rats, dogs, cats, and rabbits; birds such as chickens; amphibians; reptiles; fish; chordates; arthropods, and insects. Examples of animal cells include somatic cells, germ cells, fertilized eggs, germ cells, and stem cells (embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), hematopoietic stem cells, etc.). Examples of fungi include basidiomycetes, ascomycetes, and zygomycetes. In one embodiment, the eukaryotic cell may be a non-human cell. In one embodiment, the eukaryotic cell is not a cell present in the human body, a human germ cell, or a human embryonic cell. The eukaryotic cell may be an in vivo cell or an isolated cell. Furthermore, the eukaryotic cell may be a primary cell or a cultured cell.

[0128] In one embodiment, the host eukaryotic cell is preferably a fungal cell, more preferably a Basidiomycete or Ascomycete cell, and particularly preferably a Fusarium or Pyricularia fungal cell.

[0129] In one aspect, a method for producing a genetically engineered eukaryotic cell may comprise producing the vector or DNA fragment of the third embodiment. The method for producing the vector or DNA fragment is as described above.

[0130] In genome editing, a donor nucleic acid may be introduced into a eukaryotic cell together with the vector or DNA fragment of the third embodiment. The donor nucleic acid is a foreign nucleic acid that contains a nucleic acid to be introduced into the genome of the cell. The donor nucleic acid may be used for knock-in or knock-out.

[0131] The donor nucleic acid may be a nucleic acid to be introduced that has been linked to any vector. Examples of vectors include plasmid vectors; viral vectors such as retroviruses, lentiviruses, adenoviruses, adeno-associated viruses, herpes viruses, and Sendai viruses; and Agrobacterium vectors. The vector may contain various components, such as regulatory sequences such as promoters, enhancers, and polyadenylation signals; restriction enzyme cleavage sites; replication origins; drug resistance genes; and nucleic acids encoding markers such as fluorescent proteins. Specific examples of each component are described above.

[0132] In one embodiment, the donor nucleic acid may contain homology arms for targeting nucleic acid transfer. The homology arms consist of a 5' homology arm having a sequence homologous to a nucleic acid sequence upstream (5' side) of the target site on the genome (i.e., the site where nucleic acid transfer is intended) and a 3' homology arm having a sequence homologous to a nucleic acid sequence downstream (3' side) of the target site on the genome. The number of bases in each of the 5' and 3' homology arms may be, for example, 5 to 10,000 or 100 to 1,000. When the donor nucleic acid contains homology arms, the transferred nucleic acid is positioned between both homology arms. Furthermore, if the donor nucleic acid itself has sufficient homology to the target site on the genome, it can undergo homologous recombination with a sequence on the genome even without homology arms. Therefore, the donor nucleic acid may be a nucleic acid that differs by several bases from the target site or that contains an insertion or deletion for sequence manipulation. In this case, it is not necessary to use a combination of a 5' homology arm having a sequence homologous to a nucleic acid sequence upstream (5' side) of the target site and a 3' homology arm having a sequence homologous to a nucleic acid sequence downstream (3' side) of the target site on the genome, but rather it is sufficient to use a nucleic acid that is different from the target sequence. The number of identical bases in the donor nucleic acid to the target site may be, for example, 5 to 10,000, or may be 100 to 1,000.

[0133] Any gene transfer technique may be used to introduce a vector or DNA fragment into a eukaryotic cell. Examples include chemical techniques such as the protoplast PEG method; electrical or physical techniques such as electroporation, particle bombardment, microinjection, lipofection, and protein transduction; and methods using viruses or organisms such as viral infection, Agrobacterium, phage infection, and conjugation. Among these, methods using polyethylene glycol, viral infection, and electrical techniques can be easily used for animal cells. For plant cells, contact with polyethylene glycol (protoplast PEG method), electrical techniques, and the like can be easily used. For fungal cells, the protoplast PEG method, electrical techniques, and Agrobacterium method can be easily used.

[0134] Next, embodiments of the present disclosure will be described in detail using examples, but the embodiments of the present disclosure are not limited to these examples.

[0135] 1. Examples of the First Embodiment <Search for Novel Nuclease Domains> A search for homologous natural proteins was performed by BLASTP using the amino acid sequence of the FokI nuclease domain (SEQ ID NO: 1) as a query, and eight types (IbFokI, RsFokI, CsaFokI, EsFokI, ElFokI, TkFokI, ObFokI, and AsFokI) with homology of 48 to 56% were selected as candidate nuclease domains. The amino acid sequence identities with the FokI nuclease domain were 52% for EsFokI (SEQ ID NO: 2), 55% for CsaFokI (SEQ ID NO: 3), 56% for IbFokI (SEQ ID NO: 4), 54% for RsFokI (SEQ ID NO: 5), 51% for ElFokI (SEQ ID NO: 6), 50% for TkFokI (SEQ ID NO: 7), 49% for ObFokI (SEQ ID NO: 8), and 48% for AsFokI (SEQ ID NO: 9). The amino acid sequence identities between each candidate protein were 90% for EsFokI and ElFokI, but less than 80% for the others, and they were presumed to be different proteins.

[0136] SEQ ID NO: 1: Amino acid sequence of FokI QLVKSELEEKKSELRHKLKYVPHEYIELIEIARNSTQDRILEMKVMEFFMKVYGYRGKHLGGSRKPDGAIYTVGSPIDYGVIVDTKAYSGGYNLPIGQADEMQRYVEENQTRNKHINPNEWWKVYPSSVTEFKFLFVSGHFKGNYKAQLTRLNHITNCNGAVLSVEELLIGGEMIKAGTLTLEEVRRKFNNGEINFRS*

[0137] SEQ ID NO: 2: Amino acid sequence of EsFokI SLAKSDLAGTKEEVREKLLNLSHEYLALIDLAYDSKQNRLFEMKTLDLLTEECNYQGLHLGGSRKPDGIIYTSENKCKYGVIIDTKAYSKGYNLPISQADEMERYIGENQTRNEKVNPNKWWDNFGVDINEFYFMFVSGHFVGNFKAQIERISRNKSVNGVALAVTNLILLAEAYKAGRFTHETIKNEIFNNSEFILD*

[0138] SEQ ID NO: 3: Amino acid sequence of CsaFokI SFTKSDFEETKEQIRGKLLHLPHEYLSLIDLAYDSKQNRLFEMKTLGLLTEECGYQGLHLGGSRKPDGIIYTSSEKYNYGVIIDTKAYSRGYNLPISQADEMERYIGENQTRDAKINPNKWWKHFPEEVNEFYFMFVSGHFIGNFKAQIMRISRNKAINGTAIAVANLLLCVEAYKAGQLTHEVIKTKVFNNGEFELL*

[0139] SEQ ID NO: 4: Amino acid sequence of IbFokI QTIKSSIEELKSELRTQLNVISHDYLQLLDISQDSQQNRLFEMKVMDLFINEFGYNGSHLGGSRKPDGILYTEGLSKDYGIIVDTKAYKDGYNLPIAQADEMERYIRENIDRNEVVNQNRWWEVFPSKINDYKFLFVSAYFKGNFKEQLERISINTGILGGAISVEHLLLGAEYFKRGILSLEDVRDKFCNTEIEF* *

[0140] SEQ ID NO: 5: Amino acid sequence of RsFokI EASRSSVEELKSELRPQLRVLSHDYLQLVEIAQEPDQNRLFEMKVMELFVNELGFQGCHLGGSRRPDGILYTTDLEKNYGVIVDTKAYREGYSLPIGQADEMERYIRENIDRNPSVNPNKWWEAFPDDIENFTFLFVSGFFKGNFKDQLERISLNTGAAGGAISVEHLLLGADYYKRGVLTLEDFENHFCNGEIQF*

[0141] SEQ ID NO: 6: Amino acid sequence of ElFokI SLAKSDLAETKEEIREKMLNLSHEYLALIDLAYDSKQNRLFEMKTLDLLTEECNYQGLHLGGSRKPDGIIYTNDDKGKYGVIIDTKAYSKGYNLPISQADEMERYIGENQTRNEKVNPNKWWENFADGINEFYFMFVSGHFIGKFKEQIERISRNKSINGTALAVTNLILLAEEYKAGRFTHETIKNKMFNNSEFIL*

[0142] SEQ ID NO: 7: Amino acid sequence of TkFokI NLTKSSLTEIKEEIRPYITHISHEYLSLIDLAYDSKQNRLFEMKTLELLIDECEYNGLHLGGSRKPDGIIYTSEDAENYGVIIDTKAYSKGYSLPISQADEMERYIRENQTRDDEVNPNEWWNNFDNRISKHYFMFISGHFIGNYKSQIERISRNTNTRGTSLNVKNLLLCANLYKAGELTHHSIGEKINNSYEFVLQ*

[0143] SEQ ID NO: 8: Amino acid sequence of ObFokI EATRTPLEERKDELRAVLKNISHEYLSLIDLAYDSVQHRLFEAMTLKLLTDEYGYAGLHLGGGRKPDGVIYTEGLENDYGVIIDTKAYAEGYSVPIAQADEMQRYIQENQRRDALENPNKWWRHFAEPVRLFYFMFVSGHFKGQYSSQISRIIRITGVAGAAVEIFNLLLTAERVKGGEESLSGIARSFFPRDYEDETLS*

[0144] SEQ ID NO: 9: Amino acid sequence of AsFokI LSERSEIETIKEQMRGELTHLSHEYLGLLDLAYDSKQNRLFELKTLQLLTEECGFEGLHLGGSRKPDGIVYTSREDRLVDDVHYGIIIDTKAYSGGYSLPISQADEMERYIGENQTRDVRINPNEWWNNFGDDVTQFYYMFVAGHFKGKYQEQIERINCNKNIKGAAVSIQELLRIANAYKAGETTHEEIKAKLFLG*

[0145] <Evaluation of DNA cleavage activity of novel nuclease domains> [Materials] ・Cells: Rice blast fungus ・Vector: pZFN SDH1F, pZFN SDH1R (Arazoe et al., Journal of the Botanical Society of Japan, 79, 176-177, 2013), pTV-scytalone (Arazoe, et al., Biotechnol Bioeng, 112, 1335-1342, 2015), pMK412 Neo (Arazoe, et al., J Gen Plant Pathol, 79, 422-430, 2013) ・PDA medium

[0146] [Method] (Vector Construction) Various vectors were constructed by replacing the FokI nuclease domain of ZFN vectors (pZFN SDH1F and pZFN SDH1R) targeting the scytalone dehydratase (SDH) gene involved in melanin biosynthesis in the rice blast fungus (Arazoe, et al., Biotechnol Bioeng, 112, 1335-1342, 2015) with eight artificially synthesized novel nuclease domains.

[0147] (Transformation) Two corresponding ZFN vectors and pTV-scytalone (a donor vector containing the bialaphos resistance gene bar and its upstream and downstream homology arms) were introduced into rice blast fungus by the protoplast PEG method, and the SDH gene disruption efficiency (DNA cleavage activity) was calculated from the number of bialaphos-resistant colonies in which melanin biosynthesis was inhibited (Figure 1).

[0148] [Results] Introduction of vectors in which the FokI nuclease domain was replaced with EsFokI, IbFokI, RsFokI, or CsaFokI resulted in SDH gene disruption efficiencies comparable to those achieved with pZFN SDHF1 and pZFN SDHR1 (Table 1), demonstrating that nucleic acid cleavage enzymes containing the FokI nuclease domain have DNA cleavage activity comparable to that of FokI. EsFokI, which exhibited activity, was designated TUS1, CsaFokI TUS2, IbFokI TUS3, and RsFokI TUS4. An increase in the number of colonies obtained was observed with TUS1 and TUS2, suggesting that they have a different cleavage mode compared to FokI and that they have lower cytotoxicity and off-target activity.

[0149]

[0150] The base sequences of the DNAs encoding the polypeptides represented by the amino acid sequences of SEQ ID NOs: 2 to 5 used in the examples are shown below.

[0151] SEQ ID NO:10: Nucleotide sequence of DNA encoding EsFokI 5’-TCCCTCGCCAAGTCCGACCTCGCCGGCACCAAGGAGGAGGTCCGCGAGAAGCTCCTCAACCTCTCCCACGAGTACCTCGCCCTCATCGACCTCGCCTACGACTCCAAGCAGAACCGCCTCTTCGAGATGAAGACCCTCGACCTCCTCACCGAGGAGTGCAACTACCAGGGTCTCCACCTCGGTGGTTCCCGCAAGCCCGACGGTATCATCTACACCTCCGAGAACAAGTGCAAGTACGGTGTCATCATCGACACCAAGGCCTACTCCAAGGGTTACAACCTCCCCATCTCCCAGGCCGACGAGATGGAGCGCTACATCGGTGAGAACCAGACCCGCAACGAGAAGGTCAACCCCAACAAGTGGTGGGACAACTTCGGTGTCGATATCAACGAGTTCTACTTCATGTTCGTTTCCGGTCACTTCGTCGGTAACTTCAAGGCCCAGATCGAGCGCATCTCCCGCAACAAGTCCGTCAACGGTGTCGCCCTCGCCGTCACCAACCTCATCCTCCTCGCCGAGGCCTACAAGGCCGGTCGCTTCACCCACGAGACCATCAAGAACGAGATCTTCAACAACTCCGAGTTCATCCTCGACTAG-3’

[0152] SEQ ID NO: 11: Nucleotide sequence of DNA encoding CsaFokI 5’-TCCTTCACCAAGTCCGACTTCGAGGAGACCAAGGAGCAGATCCGCGGTAAGCTCCTCCACCTCCCCCACGAGTACCTCTCCCTCATCGACCTCGCCTACGACTCCAAGCAGAACCGCCTCTTCGAGATGAAGACCCTCGGTCTCCTCACCGAGGAGTGCGGTTACCAGGGTCTCCACCTCGGTGGTTCCCGCAAGCCCGACGGTATCATCTACACCTCCTCCGAGAAGTACAACTACGGTGTCATCATCGACACCAAGGCCTACTCCCGCGGTTACAACCTCCCCATCTCCCAGGCCGACGAGATGGAGCGCTACATCGGTGAGAACCAGACCCGCGACGCCAAGATCAACCCCAACAAGTGGTGGAAGCACTTCCCCGAGGAGGTCAACGAGTTCTACTTCATGTTCGTTTCCGGTCACTTCATCGGTAACTTCAAGGCCCAGATCATGCGCATCTCCCGCAACAAGGCCATCAACGGCACCGCCATCGCCGTCGCCAACCTCCTCCTCTGCGTCGAGGCCTACAAGGCCGGTCAGCTCACCCACGAGGTCATCAAGACCAAGGTCTTCAACAACGGTGAGTTCGAGCTGCTCTAG-3’

[0153] SEQ ID NO: 12: Base sequence of DNA encoding IbFokI 5’-CAGACCATCAAGTCCTCCATCGAGGAGCTGAAGTCCGAGCTGCGCACCCAGCTCAACGTCATCTCCCACGACTACCTCCAGCTCCTCGACATCTCCCAGGACTCCCAGCAGAACCGCCTCTTCGAGATGAAGGTCATGGACCTCTTCATCAACGAGTTCGGTTACAACGGTTCCCACCTCGGTGGTTCCCGCAAGCCCGACGGTATCCTCTACACCGAGGGTCTCTCCAAGGACTACGGTATCATCGTTGACACCAAGGCCTACAAGGACGGTTACAACCTCCCCATCGCCCAGGCCGACGAGATGGAGCGCTACATCCGCGAGAACATCGACCGCAACGAGGTCGTCAACCAGAACCGCTGGTGGGAGGTCTTCCCCTCCAAGATCAACGACTACAAGTTCCTCTTCGTTTCCGCCTACTTCAAGGGTAACTTCAAGGAGCAGCTCGAGCGCATCTCCATCAACACCGGTATCCTCGGTGGTGCCATCTCCGTCGAGCACCTCCTCCTCGGTGCCGAGTACTTCAAGCGCGGTATCCTCTCGCTCGAGGACGTCCGCGACAAGTTCTGCAACACCGAGATCGAGTTCTAG-3’

[0154] SEQ ID NO: 13: Base sequence of DNA encoding RsFokI 5’-GAGGCCTCCCGCTCCTCCGTCGAGGAGCTGAAGTCCGAGCTGCGCCCCCAGCTCCGCGTCCTCTCCCACGACTACCTCCAGCTCGTCGAGATCGCCCAGGAGCCCGACCAGAACCGCCTCTTCGAGATGAAGGTCATGGAGCTGTTCGTCAACGAGCTCGGCTTCCAGGGTTGTCATCTCGGTGGTTCCCGCCGCCCCGACGGTATCCTCTACACCACCGACCTCGAGAAGAACTACGGTGTCATCGTCGACACCAAGGCCTACCGCGAGGGTTACTCCCTCCCCATCGGTCAGGCCGACGAGATGGAGCGCTACATCCGCGAGAACATCGACCGCAACCCCTCCGTCAACCCCAACAAGTGGTGGGAGGCCTTCCCCGACGACATCGAGAACTTCACCTTCCTCTTCGTTTCCGGTTTCTTCAAGGGTAACTTCAAGGACCAGCTCGAGCGCATCTCCCTCAACACCGGTGCCGCCGGTGGTGCCATCTCCGTCGAGCACCTCCTCCTCGGTGCCGACTACTACAAGCGCGGTGTCCTCACCCTCGAGGACTTCGAGAACCACTTCTGCAACGGTGAGATCCAGTTCTAG-3’

[0155] <Evaluation of DNA cleavage activity of novel nuclease domains 2> Next, TUS1-DDD, which added amino acid mutations R104D and N113D to TUS1, and TUS1-RRR, which added amino acid mutations D100R and N155R, were prepared. Similarly, vectors were constructed in which the FokI nuclease domains of pZFN SDHF1 and pZFN SDHR1 were substituted, respectively. Nuclease activity was also evaluated by co-introduction of vectors substituted with TUS1-DDD and TUS1-RRR. As a result, higher SDH gene disruption efficiency was obtained compared to when pZFN SDHF1 and pZFN SDHR1 were introduced (Table 2). The introduction of these mutations inhibited homodimerization, forming only paired dimers, which is thought to have improved genome editing efficiency due to reduced cytotoxicity and off-target activity.

[0156] SEQ ID NO: 82: Amino acid sequence of TUS1-DDD SLAKSDLAGTKEEVREKLLNLSHEYLALIDLAYDSKQNRLFEMKTLDLLTEECNYQGLHLGGSRKPDGIIYTSENKCKYGVIIDTKAYSKGYNLPISQADEMEDYIGENQTRDEKVNPNKWWDNFGVDINEFYFMFVSGHFVGNFKAQIERISRNKSVNGVALAVTNLILLAEAYKAGRFTHETIKNEIFNNSEFILD*

[0157] SEQ ID NO: 83: Amino acid sequence of TUS1-RRR SLAKSDLAGTKEEVREKLLNLSHEYLALIDLAYDSKQNRLFEMKTLDLLTEECNYQGLHLGGSRKPDGIIYTSENKCKYGVIIDTKAYSKGYNLPISQAREMERYIGENQTRNEKVNPNKWWDNFGVDINEFYFMFVSGHFVGNFKAQIERISRRKSVNGVALAVTNLILLAEAYKAGRFTHETIKNEIFNNSEFILD*

[0158]

[0159] Furthermore, it has been confirmed that the above-mentioned nuclease activity can be widely used in fungi, and since FokI and other nucleases are used as genome editing methods in animals, plants, fungi, and bacteria, it is speculated that this technology may also be widely applicable to cells of other biological species.

[0160] 2. Example of the Second Embodiment <Removal of the Putative Centromere Region of the Accessory Chromosome> The plant pathogenic fungus, Fusarium oxysporum f. sp. tomato, has accessory chromosomes that are not essential for survival, and it has been shown that some of these chromosomes carry multiple factors involved in pathogenicity (Ma et al., Nature 464, 367-373, 2010). Below, we took advantage of the property of accessory chromosomes that they are not essential for survival to create strains lacking the putative centromere region of the accessory chromosome.

[0161] [Materials] - Cell: Tomato wilt fungus - Vector: pCRISPR / Cas9-FoU6-FoNLS (Shinkado et al., Sci Rep, 12, 16243, 2022), pMK412-dGFP (Arazoe, et al., J Gen Plant Pathol, 79, 422-430, 2013)・PDA medium ・YG medium

[0162] [Method] (Vector Construction) A 48,560-bp region with a GC content of 25% or less was designated the centromere region of chromosome 14 (accessory chromosome) of the tomato wilt fungus (Ma et al., Nature 464, 367-373, 2010). Two CRISPR target sequences were designed to cleave outside this region (Figure 2). Oligonucleotides (SEQ ID NOS: 19, 20, 21, and 22) were inserted into pCRISPR / Cas9-FoU6-FoNLS according to a previously reported method (Shinkado et al., Sci Rep, 12, 16243, 2022) to construct two CRISPR vectors. Furthermore, homologous regions 1,000 bp upstream and 1,000 bp downstream of the cleavage sequence were amplified using the respective primer sets (SEQ ID NOs: 23 and 24, and SEQ ID NOs: 25 and 26), and inserted into the multicloning sites on both sides of the hygromycin B resistance gene (Hph) of pMK412-dGFP, thereby constructing a donor vector for removing the putative centromere region (Figure 2).

[0163] SEQ ID NO: 19: CRISPR target sequence 1-1 5'-AAACAGCTAGGTATCCTCGAAGAC-3' SEQ ID NO: 20: CRISPR target sequence 1-2 5'-CTCGGTCTTCGAGGATACCTAGCT-3' SEQ ID NO: 21: CRISPR target sequence 2-1 5'-AAACCCGTACTGTAGCCCACTAAC-3' SEQ ID NO: 22: CRISPR target sequence 2-2 5'-CTCGGTTAGTGGGCTACAGTACGG-3'

[0164] SEQ ID NO: 23: Homologous region amplification Fw primer (upstream) 5'-CCGGGCCCGCTATCCAAGAGGTCTACAACTCAACA-3' SEQ ID NO: 24: Homologous region amplification Rev primer (upstream) 5'-TTCTCGAGCCTGACTCTTGGTCCCGAAGCGTGTCT-3' SEQ ID NO: 25: Homologous region amplification Fw primer (downstream) 5'-ATAAGCTTGCAACATGAGAACATTTTCACGCTGGA-3' SEQ ID NO: 26: Homologous region amplification Rev primer (downstream) 5'-TGGCGGCCGCCTGGCAAAGCATGGCCATTACTACG-3'

[0165] (Transformation) Two types of pCRISPR / Cas9-FoU6-NLS with target sequences inserted and a donor vector were co-introduced into tomato wilt fungus by the protoplast PEG method, and hygromycin B-resistant colonies were obtained.

[0166] [Results] We assumed that co-introduction of CRISPR and the donor vector would induce simultaneous cleavage of two chromosomal regions and homologous recombination (Figure 2). Therefore, we examined the resulting hygromycin B-resistant colonies to determine whether a PCR band of approximately 4,500 bp, indicating centromere removal, could be detected using two primers (SEQ ID NOs: 27 and 28) designed outside the centromere region. As a result, a PCR band of the desired size was detected (Figure 3), successfully obtaining transformants in which the centromere region had been removed.

[0167] SEQ ID NO: 27: Centromere deletion detection Fw primer 5'-CCACGCCGAAGCTGAGCATTATTGGCAGAGTGATG-3' SEQ ID NO: 28: Centromere deletion detection Rev primer 5'-AGAGCATGAATGGGTCCGTTCTCCGATAGGTAACG-3'

[0168] <Verification of Chromosome Stability Due to Centromere Region Removal> Using the obtained centromere region-removed strains, it was verified whether or not there was a change in the stability (retention or non-retention) of accessory chromosomes.

[0169] [Materials] Cells: Tomato wilt fungus transformants [Δcentromere strain and Δbacterial alpha / beta hydrolase-like protein (ABHL) strain] PDB medium YG medium

[0170] [Method] (Bud cell formation and fluorescence observation) Each transformant was cultured in PDB medium for 3 to 4 days, and the resulting culture solution was filtered through Kimtowel to recover bud cells. The recovered bud cells were collected in a volume of 5 × 10 ２ cells / mL, 1×10 ２ cells / mL, 1×10 １After adjusting the concentration to 100 μL / mL, 100 μL of each solution was plated on YG medium containing and not containing hygromycin B, and the accessory chromosome retention rate was calculated from the number of colonies obtained ( FIG. 4 ).

[0171] [Results] The ΔABHL strain, in which the ABHL gene on chromosome 14 was replaced with an Hph cassette (Shinkado et al., Sci Rep, 12, 16243, 2022), was used as a negative control. The colony formation numbers were compared with those of the Δcentromere strain, in which the centromere region of chromosome 14 had been deleted. The ABHL gene is located near the centromere of chromosome 14, and no significant phenotypes have been observed to date when the ABHL gene was deleted. The comparison revealed that the ΔABHL strain showed no difference in colony numbers between hygromycin B-containing and -free media when plated with various concentrations of cell suspension. However, the Δcentromere strain showed a decrease in colony numbers in hygromycin B-containing media, indicating the loss of accessory chromosomes (Figures 5A-C). These results suggest that the deletion of the centromere region destabilizes chromosomes within the cell nucleus.

[0172] The above experiments demonstrated that centromere loss causes chromosome instability and sheds them from the cell nucleus. Therefore, we investigated the copy number of centromere-deficient chromosomes in the cell nucleus under drug selection conditions.

[0173] [Materials] Cells: tomato wilt fungus transformants (Δcentromere strain and ΔABHL strain) YG liquid medium

[0174] [Method] (Extraction of genomic DNA and real-time RT-PCR) Bacteria cultured for 5 days in hygromycin B-containing YG liquid medium were harvested, and genomic DNA was extracted according to a previously published method (Fulton et al., Plant Mol Biol Rep, 13, 207-209, 1995). Genomic DNA was extracted using primers (SEQ ID NOs: 29, 30, 31, 32, 33, and 34) targeting the Ste3 gene located on the core chromosome and two genes (14-1 and 14-2) located specifically on chromosome 14. TM SYBRTM Real-time RT-PCR was performed using genomic DNA as a template with Green Master Mix (Thermo Fisher) (95°C, 5 minutes hold → 95°C, 5 seconds → 60°C, 10 seconds → 72°C, 10 seconds 6 cycles → 72°C to 95°C (0.3°C / second) melt).

[0175] SEQ ID NO: 29: Fw (Ste3) primer sequence 5'-cgtaatacgactcactatagggcgaattgg-3' SEQ ID NO: 30: Rev (Ste3) primer sequence 5'-cggagcattcactaggcaaccatgg-3' SEQ ID NO: 31: Fw (14-1) primer sequence 5'-atctgcttgatctcgtctcccgaaaatgaa-3' SEQ ID NO: 32: Rev (14-1) primer sequence 5'-gcctgaaggcgttactaggttgcagtcaatgcatt-3' SEQ ID NO: 33: Fw (14-2) primer sequence 5'-atggagctattaaatcactagaaggcactctttgc-3' SEQ ID NO: 34: Rev (14-2) primer sequence 5'-catcgacaccaacgatcttatatccagattcgtca-3'

[0176] [Results] The gene copy number present in the cell nucleus was estimated from the relative DNA amplification levels of the accessory chromosome-located genes (14-1 and 14-2) when the amplification level of the Ste3 gene located on the core chromosome was set to 1. In the ΔABHL strain, the relative copy numbers of both 14-1 and 14-2 tended to decrease, which is thought to be due to the fact that accessory chromosomes are not essential for viability. On the other hand, the Δcentromere strain exhibited an increase in relative copy number, approximately 1.8-fold higher than the ΔABHL strain (Figures 6A and 6B). These results suggest that the loss of the centromere region leads to chromosome instability, but that the loss of copy number control in chromosomes carrying genes essential for viability, such as under drug selection, leads to the generation of multiple copies of the chromosome in the cell nucleus. It is anticipated that the generation of multiple copies of the chromosome can increase the expression level of all genes located on the chromosome while maintaining the chromosome structure. Since chromosome maintenance is essential for survival in core chromosomes, it is thought that a similar phenomenon can be induced without the use of selectable markers, etc., if chromosomal instability is not lethal.

[0177] 3. Examples of the Third Embodiment Example 1: Verification of the autonomous replication ability of a vector containing a telomere repeat sequence In the following test, it was verified that an artificially synthesized telomere repeat sequence inserted into a foreign DNA vector enables autonomous replication within a cell nucleus.

[0178] [Materials] Cells: tomato wilt fungus, rice blast fungus Vectors: pMK412 Neo (Arazoe, et al., J Gen Plant Pathol, 79, 422-430, 2013), pMK RFP (based on pMK412 Neo, the GFP expression cassette was replaced with an RFP expression cassette) PDA medium YG medium

[0179] [Method] (Vector Construction) - pMK-telx20 was constructed by inserting an artificially synthesized telomere repeat sequence (20 repeats of 5'-TTAGGG-3': 120 bp; SEQ ID NO: 35) into the HindIII and SpeI sites of the pMK412 Neo vector, which contains a hygromycin B resistance (Hph) gene and a green fluorescent protein (GFP) gene expression cassette. Similarly, pMK-tel rev and pMK-tel random were constructed by inserting a repeat sequence in which the 6 bases constituting the telomere repeat sequence were arranged in the reverse direction (20 repeats of 5'-GGGATT-3': 120 bp; SEQ ID NO: 36) and a randomly arranged repeat sequence (20 repeats of 5'-TGTAGG-3': 120 bp; SEQ ID NO: 37) (Figure 7). The underlined parts in lowercase letters correspond to the HindIII sequence (5'-aagctt-3') and the SpeI sequence (5'-actagt-3').

[0180] SEQ ID NO: 35: Artificial synthetic telomere repeat sequence 5'-gataagcttcgtctcgTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGTTAGGGtgagacgactagttct-3'

[0181] SEQ ID NO: 36: Artificial synthetic repeat sequence (reverse) 5'-gataagcttcgtctcgGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTGGGATTtgagacgactagttct-3'

[0182] SEQ ID NO: 37: Artificial synthetic repeat sequence (random) 5'-gataagcttcgtctcgTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGTGTAGGtgagacgactagttct-3'

[0183] (Transformation) pMK RFP (negative control), pMK-tel x 20, pMK-tel rev, or pMK-tel random was introduced into tomato wilt fungus and rice blast fungus by the protoplast PEG method, and the autonomous replication ability of each vector was evaluated based on the number of hygromycin B-resistant colonies.

[0184] [Results] A significant increase in the number of hygromycin B-resistant colonies was observed only when pMK-telx20 was introduced, and when cultured on PDA medium without hygromycin B, hygromycin B resistance was lost within 3 to 9 days. These results suggest that foreign DNA is autonomously replicating within the cell nucleus in a manner dependent on telomere repeat sequences (Figure 7). Similar results were also observed in the rice blast fungus.

[0185] Example 2: Verification of shortening of telomere repeat sequence and replication ability To investigate the effect of the number of repeats (length) of telomere sequence, a double-stranded DNA fragment containing an Hph gene and a GFP gene expression cassette with a telomere repeat sequence added was introduced into tomato wilt fungus, and the replication ability within the cell nucleus was evaluated based on the number of resistant colonies obtained and the intensity of GFP expression.

[0186] [Materials] ・Cell: Tomato wilt fungus ・Vector: pMK412 Neo (Arazoe, et al., J Gen Plant Pathol, 79, 422-430, 2013) ・YG medium

[0187] [Method] (Preparation of DNA Fragments) Using the pMK412 Neo vector as a template, a total of 22 DNA fragments were prepared by PCR using primers with or without telomere sequences (SEQ ID NOS: 38-53; telomere sequences are underlined) ( FIG. 8A ). Specifically, the following DNA fragments were prepared: a DNA fragment without telomere sequences (negative control; GFP-Hph), a DNA fragment containing one to seven telomere sequences upstream of the GFP gene expression cassette, a DNA fragment containing one to seven telomere sequences downstream of the Hph gene expression cassette, and a DNA fragment containing one to seven telomere sequences upstream of the GFP gene expression cassette and downstream of the Hph gene expression cassette.

[0188] SEQ ID NO: 38: Fw (GFP side) primer sequence (no telomere) 5'-ctgtctggtcttctacacgaaggaaagac-3' SEQ ID NO: 39: Fw (GFP side) primer sequence (telomere x 1) 5'-ccctaactgtctggtcttctacacgaaggaaagac-3' SEQ ID NO: 40: Fw (GFP side) primer sequence (telomere x 2) 5'-ccctaaccctaactgtctggtcttctacacgaaggaaagac-3' SEQ ID NO: 41: Fw (GFP side) primer sequence (telomere x 3) 5'-ccctaaccctaaccctaactgtctggtcttctacacgaaggaaagac-3' SEQ ID NO: 42: Fw (GFP side) primer sequence (telomere x 4) SEQ ID NO: 43: Fw (GFP side) primer sequence (telomere × 5) 5'-ccctaaccctaaccctaaccctaaccctaaccctaactgtctggtcttctacacgaaggaaagac-3' SEQ ID NO: 44: Fw (GFP side) primer sequence (telomere × 6) 5'-ccctaaccctaaccctaaccctaaccctaaccctaaccctaactgtctggtcttctacacgaaggaaagac-3' SEQ ID NO: 45: Fw (GFP side) primer sequence (telomere × 7) 5'-ccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaactgtctggtcttctacacgaaggaaagac-3' SEQ ID NO: 46: Rev (Hph side) primer sequence (no telomere) 5'-gatcctctagaaagaaggattacctctaa-3' SEQ ID NO: 47: Rev (Hph side) primer sequence (telomere x 1) 5'-ccctaagatcctctagaaagaaggattacctctaa-3' SEQ ID NO: 48: Rev (Hph side) primer sequence (telomere x 2) 5'-ccctaaccctaagatcctctagaaagaaggattacctctaa-3' SEQ ID NO: 49: Rev (Hph side) primer sequence (telomere x 3)5'-ccctaaccctaaccctaagatcctctagaaagaaggattacctctaa-3' SEQ ID NO: 50: Rev (Hph side) primer sequence (telomere x 4) 5'-ccctaaccctaaccctaaccctaagatcctctagaaagaaggattacctctaa-3' SEQ ID NO: 51: Rev (Hph side) primer sequence (telomere x 5) 5'-ccctaaccctaaccctaaccctaaccctaagatcctctagaaagaaggattacctctaa-3' SEQ ID NO: 52: Rev (Hph side) primer sequence (telomere x 6) 5'-ccctaaccctaaccctaaccctaaccctaaccctaagatcctctagaaagaaggattacctctaa-3' SEQ ID NO: 53: Rev (Hph side) primer sequence (telomere x 7) 5'-ccctaaccctaaccctaaccctaaccctaaccctaaccctaagatcctctagaaagaaggattacctctaa-3'

[0189] (Transformation) GFP-Hph (negative control) amplified DNA fragments containing no telomere sequences, or amplified DNA fragments containing telomere sequences on one side (1 to 7 times) or both sides (1 to 7 times each) were introduced by the protoplast PEG method. The autonomous replication ability of each DNA fragment was evaluated based on the number of hygromycin B-resistant colonies obtained and the GFP fluorescence intensity.

[0190] [Results] The number of transformants increased when DNA amplified fragments containing three or more telomere repeats on one side or three or more on each side were introduced (Fig. 8B). GFP fluorescence intensity also increased depending on the number of telomere repeats (Fig. 8C). These results suggest that changing the number of telomere repeats can control the copy number in the cell nucleus and the expression of the gene locus.

[0191] Example 3: Verification of gene sequences involved in autonomous replication ability The above experiments confirmed that adding unidirectional telomere repeat sequences to one side of the amplified DNA fragments also enabled autonomous replication within the cell nucleus. Therefore, we investigated whether there were any sequences other than telomere sequences that were involved in replication within the cell nucleus.

[0192] [Materials] Cells: Tomato wilt fungus Vector: pMK-tel x 20 YG medium

[0193] [Method] (Preparation of DNA fragments and transformation) Using pMK-telx20 as a template, a total of 13 TrpC terminator-truncated DNA fragments (i.e., DNA fragments extending from the telomeric repeat sequence to any of points a to m in Figure 9) were prepared by combining primers (SEQ ID NOs: 54 to 66) that anneal to the TrpC terminator region of the Hph expression cassette with a primer (SEQ ID NO: 67) that anneals near the telomere (Figure 9). The obtained DNA fragments were introduced into protoplasts using the PEG method, and the number of transformed colonies was counted.

[0194] Array number 54: Fw (annealing to a in Fig. 9) primer sequence 5'-cgtaatacgactcactatagggcgaattgg-3' Array number 55: Fw (annealing to b in Fig. 9) primer sequence 5'-cggagcattcactaggcaaccatgg-3' Array number 56: Fw (annealing to c in Fig. 9) primer sequence 5'-atctgcttgatctcgtctcccgaaaatgaa-3' Array number 57: Fw (annealing to d in Fig. 9) primer sequence 5'-gcctgaaggcgttactaggttgcagtcaatgcatt-3' Array number 58: Fw (annealing to e in Fig. 9) primer sequence 5'-atggagctattaaatcactagaaggcactctttgc-3' Array number 59: Fw (annealing to f in Fig. 9) primer sequence 5'-catcgacaccaacgatcttatatccagattcgtca-3' Array number 60: Fw (annealing to g in Fig. 9) primer sequence 5'-agctgtttgatgatttcagtaacgttaagtggatc-3' Array number 61: Fw (annealing to h in Fig. 9) primer sequence 5'-gatttcagtaacgttaagtggatcctattcctttg-3' Array number 62: Fw (annealing to i in Fig. 9) primer sequence 5'-cgttaagtggatcctattcctttgccctcggacg-3' Array number 63: Fw (annealing to j in Fig. 9) primer sequence 5'-taagtggatcctattcctttgccctcggacgagtg-3' Array number 64: Fw (annealing to k in Fig. 9) primer sequence 5'-gtggatcctattcctttgccctcggacgagtgct-3' Array number 65: Fw (annealing to l in Fig. 9) primer sequence 5'-gatcctattcctttgccctcggacgagtgctggg-3' Array number 66: Fw (annealing to m in Fig. 9) primer sequence 5'-ctattcctttgccctcggacgagtgctggg-3' Array number 67: Rev (telomere side) primer sequence 5'-gcggccgctctagaactagtcgtctca-3'

[0195] [Results] The number of transformants increased when a DNA fragment (i.e., a DNA fragment extending from the telomeric repeat sequence to any of points a to i in Figure 9) was prepared using a combination of a primer of any of SEQ ID NOs: 54 to 62 and a primer of SEQ ID NO: 67 (Figure 9). Of the agctgtttgatgatttcagtaacgttaagtgg (SEQ ID NO: 81) in Figure 9, the region i to m is a non-coding sequence of 21 AT-rich bases (5'-gatttcagtaacgttaagtgg-3') (SEQ ID NO: 80) with a GC content of approximately 30%, suggesting that the region is involved in the autonomous replication of foreign DNA within the cell nucleus via the telomeric repeat sequence.

[0196] Example 4: Verification of autonomous replication ability by centromere addition The above results demonstrate that the AT-rich non-coding region is involved in autonomous replication by telomere repeat sequences. Since such characteristics are observed in the centromere region, we obtained the putative centromere sequence of Fusarium oxysporum f. sp. tomato and investigated its effect on autonomous replication ability.

[0197] [Materials] ・Cell: Tomato wilt fungus ・Vector: pMK412 Neo (Arazoe, et al., J Gen Plant Pathol, 79, 422-430, 2013) ・PDA medium ・YG medium ・NO ３ Liquid medium (0.17% yeast nitrogen base, 3% sucrose, 100 mM KNO ３ )

[0198] [Method] (Preparation of DNA fragments and plasmids, transformation) Vectors were constructed by inserting a putative centromere sequence of 1000 bp (SEQ ID NO: 68; GC content 16%), 2000 bp (SEQ ID NO: 69; GC content 17%), 3000 bp (SEQ ID NO: 70; GC content 18%), or 4267 bp (SEQ ID NO: 71; GC content 19%) into the pMK412 Neo vector. Using these vectors as templates, eight types of DNA fragments were prepared using primers (SEQ ID NOs: 72, 73, 74, and 75) containing 7 or 23 telomere repeats. Additionally, four types of plasmid vectors were constructed by inserting the above-mentioned putative centromere sequence into pMK-telx20. The prepared DNA fragments and plasmids were introduced into the protoplast using the PEG method, and transformants with hygromycin B resistance and GFP fluorescence were obtained.

[0199] Array number 68: Putative centromere sequence (1000 bp) 5’-ataattattaatagtattataattatattattataatacttatctaaagaagcttttaattacttaataaggccttttatattgaaatacttaatttaacttcttattttataatagctcttaatagctataaatatatgaataacctttatattataataatagagattaagaatatacaaaataaaaaggatactattacccttattaaataaactgcttttagctataaggtcttcttaattatataaccttatattatattaattaaaaagctaagtaggtataataaaaatgtattatattaaagcttaaagaaaaaagaatgttattataagtttttattaagagattataagaatatcaaaaggataatatgatatattaaaaatatggctaactatatataatagctgggttattattacagttatcttttttctttttaaatagttatctttagttagtaatcttgagataatattacatctttaaatataaatataaataatttttaattatttataatataaagtagttttaattcttagtacgtattatttatttagtaaaaactattaaagacttacttatagccttattataattacttataattatatatattttttaatttaataaattattattaatattattccctaatttaaattattgcctttttactaattttattctatttattcttatatataagattaatttattatataccctgttatatatatttaattaataaagctataactctgagttaatttaatctttataacctaatttatataaaagttaacttaatttctgtaatttaatttttaaatcttacctttatcttatatttatttaatattatatataaactccctttaattttaaataaaaaggaggataataaagtacctatttagccttaataggttatattcttaagttactttttatattaggctaagttatttaaaaatataataagaaaagctg-3'

[0200]

[0201]

[0202]

[0203] SEQ ID NO: 72: Fw primer sequence (telomere x 7). The underlined sequence indicates the telomere repeat. 5'-ccctaaccctaaccctaaccctaaccctaaccctaaccctaaagcaaacggtggtcaaag-3' SEQ ID NO: 73: Rev primer sequence (telomere x 7). The underlined sequence indicates the telomere repeat. 5'-ccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaatcgacagaagatgatatt-3' SEQ ID NO: 74: Fw primer sequence (telomere x 23). The underlined sequence indicates the telomere repeat. 5'-ccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaagcaaacggtggtcaaaggatg-3' SEQ ID NO: 75: Rev primer sequence (telomere x 23). The underlined portion indicates the telomere repeat. 5'-ccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaaccctaatcgacagaagatgatattgaag-3'

[0204] (Subculture) Ten strains of each of the transformants obtained above were inoculated onto PDA medium. After three days, each colony was inoculated onto hygromycin B-containing YG medium and PDA medium, respectively. For colonies confirmed to have grown on hygromycin B-containing YG medium, the corresponding colony on PDA medium was used to inoculate again onto hygromycin B-containing YG medium and PDA medium, respectively. This procedure was repeated until no growth was confirmed on hygromycin B-containing YG medium.

[0205] (Bud-cell formation) Each transformant was cultured in NO ３After culturing in liquid medium for 7 days, the observed bud cells were observed for fluorescence, and the number of bud cells in which fluorescence was confirmed was counted to calculate the percentage.

[0206] [Results] Transformants containing DNA fragments and plasmids lacking the predicted centromere sequence (negative control), transformants containing pMK412 Neo (positive control), and transformants containing eight DNA fragments or four plasmid DNAs containing the predicted centromere and telomere repeat sequences were evaluated for the stability (retention rate) of each foreign DNA in the cell nucleus by subculture and bud-cell observation. The results showed that the addition of the predicted centromere sequence to both the DNA fragments and the plasmids increased drug resistance and the number of cells retaining GFP fluorescence. This suggests that the DNA fragments or plasmids replicate and distribute more stably in the cell nucleus (Table 2, Figure 10). Table 2 shows the number of foreign DNAs retained per 10 colonies, and Figure 10 shows the retention rate of foreign DNA. The meanings of the labels in Figure 10 are as follows:

[0207] Ect: positive control Tel x 7: DNA fragment containing 7 telomere repeat sequences Tel x 23: DNA fragment containing 23 telomere repeat sequences Circular: plasmid containing 20 telomere repeat sequences (pMK-tel x 20) Cen-1kbp-Tel x 7: DNA fragment containing 7 telomere repeat sequences and a 1000bp centromere sequence Cen-1kbp-Tel x 23: DNA fragment containing 23 telomere repeat sequences and a 1000bp centromere sequence Cen-1kbp-circular: plasmid containing 20 telomere repeat sequences and a 1000bp centromere sequence Cen-2kbp-Tel x 7: DNA fragment containing 7 telomere repeat sequences and a 2000bp centromere sequence・Cen-2kbp-Tel x 23: A DNA fragment comprising 23 telomere repeat sequences and a 2000 bp centromere sequence. ・Cen-2kbp-circular: A plasmid comprising 20 telomere repeat sequences and a 2000 bp centromere sequence. ・Cen-3kbp-Tel x 7: A DNA fragment comprising 7 telomere repeat sequences and a 3000 bp centromere sequence. ・Cen-3kbp-Tel x 23: A DNA fragment comprising 23 telomere repeat sequences and a 3000 bp centromere sequence. ・Cen-3kbp-circular: A plasmid comprising 20 telomere repeat sequences and a 3000 bp centromere sequence. ・Cen-4kbp-Tel x 7: A DNA fragment comprising 7 telomere repeat sequences and a 4267 bp centromere sequence. Cen-4kbp-Tel x 23: A DNA fragment containing 23 telomere repeat sequences and a 4267 bp centromere sequence. Cen-4kbp-circular: A plasmid containing 20 telomere repeat sequences and a 4267 bp centromere sequence.

[0208]

[0209] Example 5: Marker-free genome editing using a telomere repeat sequence insertion vector. Based on the above, it was thought that a plasmid with a telomere repeat sequence inserted would be temporarily retained in the cell nucleus and would be lost during culture without drug selection. Therefore, we investigated whether marker-free, non-recombinant genome editing is possible using a CRISPR / Cas9 vector with a telomere repeat sequence inserted, by introducing mutations via transient intracellular nuclease expression and removing the CRISPR vector without drug selection.

[0210] [Materials] Cells: Fusarium wilt fungus Vectors: pCRISPR / Cas9-FoU6-FoNLS (Shinkado, et al., Sci Rep, 12, 16243, 2022), pMK-tel x 20 PDA medium YG medium

[0211] [Method] (Plasmid Preparation, Transformation, and Culturing) Using pMK-telx20 as a template, a DNA fragment containing the Hph expression cassette and telomere repeat sequence was amplified with two primers (SEQ ID NOs: 76 and 77). This DNA fragment was inserted into the PacI and SacI sites of the pCRISPR / Cas9-FoU6-FoNLS vector incorporating the CRISPR / Cas9 system to construct the pCRISPR-tel vector (Figure 11A). Oligonucleotides (SEQ ID NOs: 78 and 79) were annealed and then inserted into pCRISPR-tel to construct a pCRISPR-tel vector targeting the Ura3 gene involved in uracil biosynthesis (Shinkado, et al., Sci Rep, 12, 16243, 2022). The constructed vector was introduced into protoplasts using the PEG method, and hygromycin B-resistant colonies were obtained. The resulting transformants were inoculated onto PDA medium containing uridine, uracil, and 5-FOA, and colonies that exhibited 5-FOA resistance were counted as strains with successful genome editing. The 5-FOA-resistant strains were cultured in medium not containing hygromycin B, and those that had lost hygromycin B resistance were defined as non-recombinant genome-edited strains in which the plasmid had been removed.

[0212] SEQ ID NO: 76: Fw primer sequence 5'-cgcttaattaagatcctctagaaagaaggattacctctaaa-3' SEQ ID NO: 77: Rev primer sequence 5'-caattaaccctcactaaagggaacaaaagc-3' SEQ ID NO: 78: Ura3 target oligo DNA sequence 5'-ctcggctcgagcgataccactacg-3' SEQ ID NO: 79: Ura3 target oligo DNA sequence 5'-aaaccgtagtggtatcgctcgagc-3'

[0213] [Results] Eighty hygromycin B-resistant transformants obtained by introducing the pCRISPR-tel vector were inoculated into 5-FOA-containing PDA medium. Seven strains exhibited 5-FOA resistance, indicating that functional mutations due to cleavage of the Ura3 gene had been introduced. These 5-FOA-resistant strains were cultured in PDA medium and then inoculated again into YG medium containing hygromycin B. These strains exhibited hygromycin B sensitivity (Figure 11B). These results demonstrate that non-recombinant genome editing is possible by using a CRISPR vector with a telomere repeat sequence inserted, resulting in transient nuclease expression and subsequent plasmid removal.

[0214] These results demonstrate that vectors incorporating telomere repeat sequences enhance gene transfer efficiency. Additionally, vectors incorporating AT-rich sequences, especially putative centromere regions, enhance the stability of the introduced gene.

[0215] Furthermore, the above findings have been observed to have similar properties in multiple fungi, and are presumed to be widely applicable to cells of other species.

[0216] The disclosures of Japanese Patent Application Nos. 2024-057389, 2024-057393, and 2024-057399 are incorporated herein by reference in their entirety. All documents, patent applications, and technical standards mentioned herein are incorporated by reference to the same extent as if each individual document, patent application, and technical standard was specifically and individually indicated to be incorporated by reference.

Claims

1. A nucleic acid cleaving enzyme comprising a nuclease domain having an amino acid sequence that is 90% or more identical to the amino acid sequence represented by any one of SEQ ID NOs: 2 to 5, 82, and 83, and a nucleic acid binding domain.

2. The nucleic acid cleaving enzyme according to claim 1, wherein the nucleic acid binding domain comprises a zinc finger, a TALE, a Cas protein, or a PPR.

3. The nucleic acid cleaving enzyme according to claim 1, further comprising a linker between the nuclease domain and the nucleic acid binding domain.

4. The nucleic acid cleaving enzyme according to claim 1, which binds to a target nucleic acid and dimerizes.

5. A nucleic acid encoding the nucleic acid-cleaving enzyme according to any one of claims 1 to 4.

6. A vector comprising the nucleic acid of claim 5, or its transcription or translation product.

7. A nucleic acid modification kit comprising: a nucleic acid cleaving enzyme according to any one of claims 1 to 4; a nucleic acid encoding said nucleic acid cleaving enzyme; or a vector containing the nucleic acid encoding said nucleic acid cleaving enzyme or its transcription product or translation product, all stored in a container.

8. A method for modifying nucleic acids, comprising introducing into cells (excluding cells present in the human body, human germ cells, and human embryonic cells) the nucleic acid cleaving enzyme according to any one of claims 1 to 4; a nucleic acid encoding said nucleic acid cleaving enzyme; or a vector comprising the nucleic acid encoding said nucleic acid cleaving enzyme or its transcription product or translation product.

9. A method for producing a mutant of a eukaryotic cell, which comprises deleting a centromere region of the genome on a chromosome in the eukaryotic cell.

10. The method for producing a mutant according to claim 9, further comprising introducing a marker gene into the chromosome so as to replace the deleted centromere region.

11. The method for producing a mutant according to claim 10, wherein the marker gene is a drug resistance gene.

12. The method for producing a mutant according to claim 9, wherein the centromere region is deleted by inducing a DNA repair mechanism.

13. The method for producing a mutant according to claim 12, wherein the centromere region is deleted by DNA cleavage using ZFN, TALEN, the CRISPR / Cas system, or I-SceI, and the cleavage repair is performed by a DNA repair mechanism.

14. The method for producing a mutant according to claim 9, wherein the length of the centromere region to be deleted is 40 to 500 kb.

15. The method for producing a mutant according to claim 9, wherein the eukaryotic cell is a fungal cell.

16. The method for producing a mutant according to claim 9, wherein the eukaryotic cell is a cell of the genus Fusarium.

17. The method for producing a mutant according to claim 9, wherein the chromosome is an accessory chromosome.

18. A method for gene expression, comprising expressing a gene on the chromosome in a mutant prepared by the method according to any one of claims 9 to 17.

19. The gene expression method according to claim 18, wherein the number of copies of the chromosome per cell of the mutant is increased compared to before the deletion of the centromere region.

20. A eukaryotic cell that lacks the centromeric region of the genome on a chromosome.

21. The eukaryotic cell of claim 20, wherein the genome on the chromosome comprises a marker gene.

22. The eukaryotic cell of claim 21, wherein the marker gene is a drug resistance gene.

23. A eukaryotic cell according to any one of claims 20 to 22, which is a fungal cell.

24. The eukaryotic cell of claim 23, which is a cell of the genus Fusarium.

25. A vector or DNA fragment (excluding artificial chromosomes) for eukaryotic genetic engineering that contains three or more telomeric repeat sequences, and the telomeric repeat sequences are contained in only one orientation.

26. A vector or DNA fragment (excluding artificial chromosomes) for eukaryotic genetic engineering containing 3 to 26 telomeric repeat sequences.

27. A vector or DNA fragment (excluding artificial chromosomes) for eukaryotic genetic engineering, which contains three or more telomeric repeat sequences and a sequence of 21 bases or more in length with an AT content of 60% or more.

28. The vector or DNA fragment according to claim 27, wherein the sequence of 21 or more bases is at least a part of the sequence of a centromere region.

29. The vector or DNA fragment according to claim 27, wherein the AT content of the sequence of 21 or more bases in length is 75% or more.

30. The vector or DNA fragment of claim 27, wherein the sequence of 21 or more bases in length is 1,000 or more bases in length.

31. Vectors or DNA fragments (excluding artificial chromosomes) for eukaryotic genetic engineering that contain three or more repeats of a telomere sequence, which is an artificially synthesized DNA.

32. The vector or DNA fragment according to any one of claims 25 to 31, wherein the telomere sequence is a telomere sequence in a fungus.

33. The vector or DNA fragment according to any one of claims 25 to 31, wherein the telomere sequence is a telomere sequence in a fungus of the genus Fusarium or Pyricularia.

34. The vector or DNA fragment according to any one of claims 25 to 31, further comprising a base sequence encoding a ZFN, a TALEN, a CRISPR / Cas system, or I-SceI.

35. A kit for eukaryotic genetic manipulation, comprising the vector or DNA fragment according to any one of claims 25 to 31 stored in a container.

36. A method for producing a genetically engineered eukaryotic cell, comprising introducing into a eukaryotic cell a vector or DNA fragment according to any one of claims 25 to 31.

37. A method for producing a genetically engineered eukaryotic cell as described in claim 36, wherein the vector or DNA fragment is produced by adding an artificially synthesized DNA fragment that is a telomeric repeat sequence to a vector or DNA fragment that does not contain a telomeric repeat sequence.

38. The method for producing a genetically engineered eukaryotic cell of claim 36, wherein the eukaryotic cell is a fungal cell.

39. The method for producing a genetically engineered eukaryotic cell of claim 36, wherein the eukaryotic cell is a cell of the genus Fusarium or Pyricularia.

Images