Polynucleotides containing site-specific nuclease expression cassettes

Abstract

The present invention addresses the problem of providing plant genome editing technology with higher genome editing efficiency. The problem addressed by the present invention is solved by a polynucleotide including a site-specific nuclease expression cassette and a sequence that binds to a nuclear translocation factor and / or an intranuclear structure, wherein the site-specific nuclease expression cassette includes (b) a site-specific nuclease coding sequence and (c) a 3'UTR including a terminator.

Description

Technical Field

[0001] The present invention relates to a polynucleotide containing a site-specific nuclease expression cassette, etc.

Background Art

[0002] In plants, improvements and modifications have conventionally been made by traditional breeding, molecular breeding, genetic recombination, etc. As a result, for example, it is possible to improve and modify plant traits such as yield, environmental adaptability, disease resistance, insect resistance, growth rate, etc.

[0003] In recent years, genome editing techniques using site-specific nucleases have attracted attention as new plant breeding techniques. As one such technique, the CRISPR / Cas system is known. Typically, by introducing a Cas protein and a guide RNA, target genes can be mutated in various animals and plants.

[0004] In genome editing techniques, improvement of their efficiency is desired. In particular, when trying to mutate multiple genes simultaneously, if the efficiency is low, mutations cannot be introduced into all the target genes simultaneously.

[0005] Patent Document 1 discloses a technique for improving expression by arranging a terminator and a scaffold / matrix attachment region in an expression cassette. However, Patent Document 1 does not describe or suggest expressing a site-specific nuclease.

Prior Art Documents

Patent Documents

[0006]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0007] The objective of this invention is to provide a plant genome editing technology with higher genome editing efficiency. [Means for solving the problem]

[0008] In view of the above problems, the inventors conducted diligent research and found that the above problems can be solved if the polynucleotide comprises a site-specific nuclease expression cassette and a sequence that binds to a nuclear localization factor and / or an intranuclear structure, wherein the site-specific nuclease expression cassette comprises (b) a site-specific nuclease coding sequence and (c) a 3'UTR including a terminator. Based on this finding, the inventors furthered their research and completed the present invention. That is, the present invention encompasses the following aspects.

[0009] Item 1. A polynucleotide comprising a site-specific nuclease expression cassette and a sequence that binds to a nuclear localization factor and / or nuclear structure, wherein the site-specific nuclease expression cassette is (b) Site-specific nuclease sequences, and (c) Terminator and other 3'UTR Polynucleotides, including [specifically, polynucleotides].

[0010] Item 2. The polynucleotide according to Item 1, wherein the sequence that binds to the nuclear translocation factor and / or nuclear structure is an insulator.

[0011] Item 3. The polynucleotide according to item 1 or 2, wherein the sequence that binds to the nuclear translocation factor and / or nuclear structure is included in the 3'UTR.

[0012] Item 4. The polynucleotide according to any one of items 1 to 3, wherein the sequence that binds to the nuclear translocation factor and / or nuclear structure is an insulator other than a scaffold / matrix binding region.

[0013] Item 5. The polynucleotide according to Item 4, wherein the insulator is at least one selected from the group consisting of Ars insulator, Gypsy insulator, Bead1 insulator, TEF insulator, and TEF2 insulator.

[0014] Item 6. The polynucleotide according to item 5, wherein the insulator is an Ars insulator.

[0015] Item 7. The polynucleotide according to any one of items 1 to 3, wherein the sequence that binds to the nuclear translocation factor and / or nuclear structure is a scaffold / matrix binding region.

[0016] Item 8. The polynucleotide according to any one of items 1 to 7, wherein the terminator is a conjugate of at least two terminators.

[0017] Item 9. The polynucleotide according to any one of items 1 to 8, wherein a sequence that binds to the nuclear localization factor and / or nuclear structure is located within the 3'UTR and downstream of the terminator.

[0018] Item 10. The polynucleotide according to any one of items 1 to 9, wherein the terminator is at least one selected from the group consisting of a 35S terminator, an ACT3 terminator, and an extensin terminator.

[0019] Item 11. The site-specific nuclease expression cassette is (a) Promoter A polynucleotide as described in any of items 1 to 10, including the polynucleotides described in any of items 1 to 10.

[0020] Item 12. The polynucleotide according to item 11, wherein the promoter is a shoot apical expression promoter.

[0021] Item 13. The polynucleotide according to Item 11 or 12, wherein the promoter is a CmYLCV promoter, a UBQ promoter, an EF-1α promoter, or a RPS5A promoter.

[0022] Item 14. The polynucleotide according to any one of Items 1 to 13, comprising a viral vector sequence.

[0023] Item 15. The polynucleotide according to Item 14, wherein the viral vector sequence is a geminivirus vector sequence.

[0024] Item 16. The polynucleotide according to Item 14 or 15, wherein the viral vector sequence is a BeYDV sequence.

[0025] Item 17. The polynucleotide according to any one of Items 1 to 16, further comprising a guide RNA expression cassette.

[0026] Item 18. A vector for plant genome editing, comprising the polynucleotide according to any one of Items 1 to 17.

[0027] Item 19. A kit for plant genome editing, comprising the polynucleotide according to any one of Items 1 to 17.

[0028] Item 20. A method for plant genome editing, comprising introducing the polynucleotide according to any one of Items 1 to 17 into a plant.

[0029] Item 21. The method for plant genome editing according to Item 20, further comprising introducing a mitotic cell activating factor into the introduction site of the polynucleotide.

[0030] Item 22. A method for producing a genome-edited plant, comprising introducing the polynucleotide according to any one of Items 1 to 17 into a plant.

[0031] Item A. A composition for plant genome editing, containing a polynucleotide comprising a mitotic cell activating factor expression cassette.

[0032] Item B. A plant genome editing efficiency enhancer containing polynucleotides including a cell division activator expression cassette.

[0033] Section C. A method for editing the plant genome or a method for producing a genome-edited plant, comprising introducing a site-specific nuclease expression cassette and a cell-dividing activator expression cassette into a plant.

[0034] Item D. A method for improving the efficiency of plant genome editing, including the introduction of a cell division activator expression cassette into plants. [Effects of the Invention]

[0035] According to the present invention, it is possible to provide a plant genome editing technology with higher genome editing efficiency. [Brief explanation of the drawing]

[0036] [Figure 1] The vector map of the Cas expression vector prepared in Example 1 is shown. The arrows indicate the promoter. [Figure 2] The vector maps of the Cas expression vectors prepared in Comparative Examples 1 and 2 are shown. The arrows indicate the promoters. [Figure 3] The results (genome editing efficiency) for Test Example 1 are shown. The top of the bar above the column indicates the maximum value, the top of the column indicates the third quartile, the horizontal line within the column indicates the median, the × within the column indicates the mean, the bottom of the column indicates the first quartile, and the bottom of the bar below the column indicates the minimum value. Gray circles indicate outliers. [Figure 4] The results of Test Example 2-2 are shown. (a) shows the chromatogram containing the target sequence obtained by Sanger sequencing, and (b) shows the results of mutation analysis using the analysis tool "DECODR". [Figure 5] The results for test example 2-3 are shown. (a) and (b) show the results for different guide RNAs. Uncleaved indicates an uncleaved band, WT indicates the case without Cas expression vector introduction, and #33 indicates the case with Cas expression vector introduction. Genome editing efficiency is shown below the lane. [Figure 6] The vector map of the Cas expression vector prepared in Example 4 is shown. The arrows indicate the promoter. [Figure 7] The vector maps for Comparative Example 4 and the Cas expression vectors prepared in Examples 3 and 5-7 are shown. The arrows indicate the promoter. [Figure 8] The results (genome editing efficiency) for Test Example 3 are shown. The top of the bar above the column indicates the maximum value, the top of the column indicates the third quartile, the horizontal line within the column indicates the median, the × within the column indicates the mean value, the bottom of the column indicates the first quartile, and the bottom of the bar below the column indicates the minimum value. NtEU represents Comparative Example 4, and NtEU_Ars represents Example 4. [Figure 9] The results for Test Example 4 are shown. "Uncleaved" indicates the uncleaved band. MAD7 shows the case where the effector expression vector from Example 8 was introduced, and WT shows the case where the vector was not introduced. Genome editing efficiency is shown below the lane. [Modes for carrying out the invention]

[0037] 1.Definition In this specification, the terms “contains” and “includes” include the concepts of “contains,” “includes,” “substantially consist of,” and “consist solely of.”

[0038] In this specification, "identity" of amino acid sequences refers to the degree of agreement between two or more comparable amino acid sequences. Therefore, the higher the agreement between two amino acid sequences, the higher their identity or similarity. The level of amino acid sequence identity can be determined, for example, using the sequence analysis tool FASTA and its default parameters. Alternatively, it can be determined using the BLAST algorithm by Karlin and Altschul (KarlinS, Altschul SF. "Methods for assessing the statistical significance of molecular sequence features by using general scoring schemes" Proc Natl Acad Sci USA. 87:2264-2268 (1990), KarlinS, Altschul SF. "Applications and statistics for multiple high-scoring segments in molecular sequences." Proc Natl Acad Sci USA. 90:5873-7 (1993)). Programs called blastp and tblastn have been developed based on this BLAST algorithm. The specific methods for these analyses are publicly known and can be found on the National Center of Biotechnology Information (NCBI) website (http: / / www.ncbi.nlm.nih.gov / ). Furthermore, the "identity" of the base sequence is defined in accordance with the above.

[0039] In this specification, "conservative substitution" means that an amino acid residue is substituted for an amino acid residue having a similar side chain. For example, substitutions between amino acid residues having basic side chains, such as lysine, arginine, and histidine, are considered conservative substitutions. Other examples of conservative substitutions include: amino acid residues with acidic side chains, such as aspartic acid and glutamic acid; amino acid residues with non-charged polar side chains, such as asparagine, glutamine, serine, threonine, tyrosine, and cysteine; amino acid residues with non-polar side chains, such as glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan; amino acid residues with branched chains, such as valine, isoleucine, and leucine; and amino acid residues with aromatic side chains, such as tyrosine, phenylalanine, and tryptophan.

[0040] In this specification, "coding sequence" means a nucleotide sequence that codes for the amino acid sequence of a protein, and is not particularly limited to that extent.

[0041] In this specification, the 5' side refers to the 5' side of the sense strand of the site-specific nuclease coding sequence, and the 3' side refers to the 3' side of the sense strand of the site-specific nuclease coding sequence. The 5' side may also be referred to as "upstream," and the 3' side as "downstream."

[0042] In this specification, the term "plant" is not particularly limited. Examples of plants include a wide range of plants, such as mosses, ferns, gymnosperms, magnolias of the angiosperms, monocots, and eudicots (roses I, roses II, chrysanthemums I, chrysanthemums II and their outgroups). More specific examples of plants include: tomatoes, bell peppers, chili peppers, eggplants, tobacco, torbams and other eggplant species; cucumbers, pumpkins, melons, watermelons and other gourd species; cabbage, broccoli, Chinese cabbage and other leafy greens; celery, parsley, lettuce and other raw and spicy vegetables; onions, leeks, garlic and other allium species; strawberries, melons and other fruit vegetables; taproots such as radishes, turnips, carrots, and burdock; tubers such as taro, cassava, potatoes, sweet potatoes, and yams; grains such as rice, corn, wheat, sorghum, barley, rye, buckwheat and other grains; and legumes such as soybeans, adzuki beans, mung beans, cowpeas, green beans, peanuts, peas, and broad beans. Examples include: tender vegetables such as asparagus, spinach, and Japanese parsley; flowers such as lisianthus, stock, carnation, and chrysanthemum; grasses such as bentgrass and Korean lawn grass; oil crops such as rapeseed, peanut, rapeseed, tung tree, sesame, and perilla; fiber crops such as cotton and rush; fodder crops such as clover, dent corn, and alfalfa; deciduous fruit trees such as apples, pears, grapes, and peaches; citrus fruits such as Satsuma mandarins, oranges, lemons, and grapefruits; and woody plants such as azaleas, rhododendrons, cedars, poplars, and rubber trees.

[0043] 2. Polynucleotides In one aspect, the present invention relates to a polynucleotide comprising a site-specific nuclease expression cassette and a sequence that binds to a nuclear localization factor and / or an intranuclear structure, wherein the site-specific nuclease expression cassette comprises (b) a site-specific nuclease coding sequence and (c) a 3'UTR including a terminator (which may be referred to herein as "the polynucleotide of the present invention"). This will be described below.

[0044] The polynucleotide of the present invention comprises a site-specific nuclease expression cassette. The site-specific nuclease expression cassette is not particularly limited, as long as it contains the base sequence necessary to transcribe mRNA that expresses a site-specific nuclease, specifically mRNA capable of producing a site-specific nuclease.

[0045] In the polynucleotide of the present invention, the site-specific nuclease expression cassette includes (b) a site-specific nuclease coding sequence. The site-specific nuclease coding sequence is not particularly limited as long as it is a base sequence that codes for the amino acid sequence of a site-specific nuclease.

[0046] Site-specific nucleases are not particularly limited as long as they can specifically cleave a target site on genomic DNA and produce a cleavage fragment. Examples of site-specific nucleases include Cas proteins, zinc finger nucleases (ZFNs), or TAL effector nucleases (TALENs). ZFNs are fusion proteins of several zinc finger motifs that recognize specific bases and a DNA cleavage effector domain. TALENs are fusion proteins of a Transcription Activator Like (TAL) effector and a DNA cleavage effector domain. Site-specific nucleases can also be composed of other additional targeting techniques, such as meganucleases or leucine zippers. Cas proteins are particularly preferred as site-specific nucleases.

[0047] The Cas protein is not particularly limited as long as it is used in the CRISPR / Cas system. For example, various types of Cas proteins can be used that can bind to a target site on genomic DNA while in a complex with guide RNA and cleave that target site. Cas proteins are known to originate from various organisms, including, for example, Cas9 protein (Type II) from S. pyogenes, Cas protein (Type IA) from S. solfataricus, Cas protein (Type IB) from H. walsbyi, Cas protein (Type ID) from Microcystis aeruginosa, Cas protein (Type IE) from E. coli, Cas protein (Type IF) from E. coli, Cas protein (Type IF) from P. aeruginosa, Cas9 protein (Type II) from S. thermophilus, Cas9 protein (Type II) from S. agalactiae, Cas9 protein from S. aureus, Cas9 protein from N. meningitidis, Cas9 protein from T. denticola, Cpf1 protein (Type VA) from F. novicida, and MAD7 protein (Type VA) from Eubacterium rectale. Among these, Cas9 protein is preferred, and Cas9 protein endogenously present in bacteria belonging to the genus Streptococcus is preferred. Information on the amino acid sequences and coding sequences of various Cas proteins can be easily obtained from various databases such as NCBI.

[0048] The Cas protein may be a wild-type double-strand break Cas protein or a nickase-type Cas protein. Double-strand break Cas proteins typically contain a domain involved in target strand cleavage (HNH domain) and a domain involved in non-target strand cleavage (RuvC domain). Examples of nickase-type Cas proteins include proteins that have mutations in one of these two domains of a double-strand break Cas protein that impair its cleavage activity (for example, reducing its cleavage activity to 1 / 2, 1 / 5, 1 / 10, 1 / 100, or 1 / 1000 or less). Examples of such mutations include, for instance, if the double-strand break type Cas protein is a Cas9 protein derived from S. pyogenes, mutations such as a mutation in the 10th amino acid from the N-terminus (aspartic acid) to alanine (D10A: mutation within the RuvCI domain), a mutation in the 840th amino acid from the N-terminus (histidine) to alanine (H840A: mutation within the HNH domain), a mutation in the 863rd amino acid from the N-terminus (asparagine) to alanine (N863A: mutation within the HNH domain), a mutation in the 762nd amino acid from the N-terminus (glutamic acid) to alanine (E762A: mutation within the RuvCII domain), and a mutation in the 986th amino acid from the N-terminus (aspartic acid) to alanine (D986A: mutation within the RuvCIII domain).

[0049] Cas proteins may have amino acid sequence mutations (e.g., substitutions, deletions, insertions, additions, etc.) as long as their activity is not impaired. From this viewpoint, Cas proteins may consist of an amino acid sequence that has, for example, 85% or more, preferably 90% or more, more preferably 95% or more, and even more preferably 98% or more identity with the amino acid sequence of a wild-type double-strand break Cas protein or a nickase-type Cas protein based on the wild-type double-strand break Cas protein, and may also be proteins that have activity (activity to bind to a target site on genomic DNA in a complex with guide RNA and cleave the target site). Alternatively, from a similar viewpoint, the Cas protein may consist of an amino acid sequence in which one or more amino acids (e.g., 2 to 100, preferably 2 to 50, more preferably 2 to 20, even more preferably 2 to 10, even more preferably 2 to 5, particularly preferably 2) are substituted, deleted, added, or inserted (preferably conservative substitutions) from the amino acid sequence of a wild-type double-strand break Cas protein or a nickase-type Cas protein based on the wild-type double-strand break Cas protein, and is a protein having activity (activity to bind to a target site of genomic DNA in a complex with guide RNA and cleave the target site). The above "activity" can be evaluated in vitro or in vivo according to or in accordance with known methods.

[0050] Site-directed nucleases may have known protein tags, signal sequences, enzyme proteins, or other proteins attached to them, as long as they possess the "activity" described above. Examples of protein tags include biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, and PA tags. Examples of signal sequences include nuclear localization signals. Examples of enzyme proteins include various histone modifying enzymes and deaminonases.

[0051] In the polynucleotide of the present invention, the site-specific nuclease expression cassette includes (c) a 3'UTR containing a terminator. The 3'UTR is located on the 3' side of the site-specific nuclease coding sequence.

[0052] A terminator is a base sequence that terminates the transcription of DNA into mRNA, and is not particularly limited in this respect.

[0053] Preferably, a terminator derived from a plant gene or plant virus can be used. The terminator is located on the 3' side of the coding region of the gene and can be easily identified by sequence analysis. Preferably, the terminator is a 35S terminator (preferably 35S of cauliflower mosaic virus) (specifically, for example, SEQ ID NO: 4 or its variant sequence), an ACT3 terminator (preferably ACT3 of Nicotiana benthamiana) (specifically, for example, SEQ ID NO: 5 or its variant sequence), or an extensin terminator (preferably extensin of Nicotiana tabacum) (specifically, for example, SEQ ID NO: 14 or its variant sequence).

[0054] The terminator may have mutations in its base sequence (e.g., substitutions, deletions, insertions, additions, etc.) as long as its function is not significantly impaired. The terminator has, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity with the base sequence of the wild-type terminator. The terminator also includes those that have been modified to enhance the function of the terminator, such as modifications that link multiple elements together, or modifications that increase the base length of a specific sequence.

[0055] The presence or absence of terminator function can be determined by expressing mRNA from an expression cassette containing the target base sequence in the 3'UTR, and using the fact that the mRNA generated when the target base sequence functions as a terminator (determinable by base length) is, for example, 50 mol% or more, preferably 70 mol% or more, more preferably 80 mol% or more, and even more preferably 90 mol% or more, relative to 100 mol% of the total mRNA generated from the expression cassette.

[0056] Preferably, the terminators include a terminator containing a nucleotide sequence that has 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 4, a terminator containing a nucleotide sequence that has 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 5, and a terminator containing a nucleotide sequence that has 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 14.

[0057] There may be one terminator or multiple terminators. In a preferred embodiment of the present invention, a terminator may be a combination of at least two terminators. In such a combination, the terminators may be directly connected or indirectly connected through other arrangements. If there are multiple terminators, the multiple terminators may be the same terminator or may be different terminators from one another.

[0058] The polynucleotide of the present invention includes a sequence that binds to nuclear localization factors and / or nuclear structures. The position of the sequence that binds to nuclear localization factors and / or nuclear structures may be anywhere on the polynucleotide of the present invention. The sequence that binds to nuclear localization factors and / or nuclear structures is preferably located within a site-directed nuclease expression cassette, more preferably within the 3'UTR of the expression cassette, and particularly preferably within the 3'UTR and downstream of the terminator.

[0059] Preferably, an insulator is used as a sequence that binds to nuclear translocation factors and / or intranuclear structures.

[0060] An insulator is a nucleotide sequence that can cleave transcriptional regulatory mechanisms even between adjacent genes, and is not particularly limited in this respect.

[0061] Examples of insulators include scaffold / matrix binding regions. These regions are areas to which the nuclear matrix attaches and have the function of defining the boundaries of individual chromosomal regions by inducing the formation of chromatin loops.

[0062] Preferably, a scaffold / matrix binding region from a plant can be used as the scaffold / matrix binding region. The scaffold / matrix binding region can be easily identified by publicly available information and sequence analysis. Preferably, the scaffold / matrix binding region is the Rb7 (preferably Rb7 of Nicotiana tabacum) scaffold / matrix binding region (specifically, for example, SEQ ID NO: 6 or its variant sequence).

[0063] The scaffold / matrix binding region may have mutations in its base sequence (e.g., substitutions, deletions, insertions, additions, etc.) as long as its function is not significantly impaired. The scaffold / matrix binding region has, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more of the base sequence of the wild-type scaffold / matrix binding region. Furthermore, the scaffold / matrix binding region also includes those that have been modified to enhance the function of the scaffold / matrix binding region, such as modifications that link multiple elements together or modifications that increase the base length of a specific sequence.

[0064] Preferably, the scaffold / matrix binding region includes a scaffold / matrix binding region that has 70% or more identity with the base sequence shown in SEQ ID NO: 6.

[0065] Examples of insulators other than the scaffold / matrix-binding region include enhancer-blocking insulators that form chromatin loop domains in the nucleus and separate enhancers from the promoters of target genes, and barrier insulators that inhibit specific processes in the heterochromatin formation pathway. Insulators other than the scaffold / matrix-binding region can be easily identified through publicly available information and sequence analysis. Other preferred insulators besides the scaffold / matrix binding region include the Drosophila Gypsy insulator (SEQ ID NO: 15 or its variant), the Human Bead1 (blocking element alpha / delta-1 of the human T-cell receptor α / δ locus) insulator (SEQ ID NO: 16 or its variant), the Sea urchin Ars (arylsulfatase) insulator (SEQ ID NO: 17 or its variant), the A. gossypii TEF (translation elongation factor) insulator (SEQ ID NO: 18 or its variant), and the Yeast TEF2 (translation elongation factor 2) insulator (SEQ ID NO: 19 or its variant). Among these, the Ars insulator is particularly preferred from the viewpoint of significantly improving genome editing efficiency.

[0066] Insulators other than the scaffold / matrix binding region may have mutations in their base sequence (e.g., substitutions, deletions, insertions, additions, etc.) as long as their function is not significantly impaired. Insulators other than the scaffold / matrix binding region have, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more in identity with the base sequence of wild-type insulators other than the scaffold / matrix binding region. Furthermore, insulators other than the scaffold / matrix binding region also include those that have undergone modifications to enhance their function, such as modifications that link multiple elements together or modifications that increase the base length of a specific sequence.

[0067] Preferably, insulators other than the scaffold / matrix binding region include insulators containing a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 15, insulators containing a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 16, insulators containing a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 17, insulators containing a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 18, and insulators containing a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 19. Among these, insulators containing a nucleotide sequence having 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 17 are particularly preferred from the viewpoint of significantly improving genome editing efficiency.

[0068] In a preferred embodiment of the present invention, a sequence that binds to nuclear translocation factors and / or intranuclear structures is located downstream (3' side) of the terminator.

[0069] In the polynucleotide of the present invention, the site-specific nuclease expression cassette preferably includes (a) a promoter. The promoter is positioned at 5' of the site-specific nuclease coding sequence.

[0070] A promoter is an upstream region of a gene involved in the initiation and promotion of gene transcription, and is not particularly limited in this respect. A promoter typically includes a transcription start site, a sequence upstream (5' side) thereof, and optionally a sequence downstream (3' side) thereof. Examples of a promoter include any DNA region containing a transcription start site within a DNA region of, for example, -10000 to +500, preferably -5000 to +200, more preferably -2000 to +150, where the base of the transcription start site of the gene is +1, the downstream (3' side) is a positive value, and the upstream (5' side) is 0 or a negative value. If there are multiple transcription start sites, the transcription start site with the highest transcription rate can be selected. The length of the promoter (base pairs: bp) is, for example, in the range of 100 to 10000 bp, preferably 200 to 5000 bp, more preferably 500 to 3000 bp.

[0071] Preferably, a promoter derived from a plant gene or plant virus can be used. The promoter is located on the 5' side of the gene's coding region and can be easily identified by sequence analysis. Preferred promoters include the CmYLCV promoter (specifically, for example, SEQ ID NO: 20 (=SEQ ID NO: 1) or its variant sequence), the UBQ promoter (specifically, for example, the lettuce ubiquitin promoter (SEQ ID NO: 21 or its variant sequence), the soybean ubiquitin promoter (SEQ ID NO: 22 or its variant sequence), the chrysanthemum ubiquitin promoter (SEQ ID NO: 23, 24, 25 or their variant sequences), the parsley ubiquitin promoter (SEQ ID NO: 26 or its variant sequence), the perilla ubiquitin promoter (SEQ ID NO: 27 or its variant sequence)), the EF-1α (preferably EF-1α of Nicotiana benthamiana) promoter (specifically, for example, SEQ ID NO: 28 or its variant sequence), the RPS5A (preferably RPS5A of Arabidopsis thaliana) promoter (specifically, for example, SEQ ID NO: 29 or its variant sequence), and so on.

[0072] The promoter may have mutations in its base sequence (e.g., substitutions, deletions, insertions, additions, etc.) as long as its function is not significantly impaired. The promoter has, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity with the base sequence of the wild-type promoter. The promoter also includes those that have been modified to enhance its function, such as modifications that link multiple elements together or modifications that increase the base length of a specific sequence.

[0073] Preferably, the promoters include a promoter containing a nucleotide sequence that has 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 20 (=SEQ ID NO: 1), a promoter containing a nucleotide sequence that has 70% or more identity with the nucleotide sequences shown in SEQ ID NOs: 21 to 27, a promoter containing a nucleotide sequence that has 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 28, and a promoter containing a nucleotide sequence that has 70% or more identity with the nucleotide sequence shown in SEQ ID NO: 29.

[0074] In one embodiment of the present invention, the promoter is preferably a shoot apical expression promoter. The shoot apical expression promoter is a promoter that can be expressed in shoot apical cells, and is not particularly limited to that extent.

[0075] The polynucleotide of the present invention preferably contains a viral vector sequence. The viral vector sequence is not particularly limited as long as it contains a sequence involved in viral replication.

[0076] Geminiviruses are particularly preferred as the source viruses for the viral vector sequences. Geminiviruses are not particularly limited as long as they have a rolling circle DNA replication mechanism, and examples include bean yellow spot dwarf virus (BeYDV), maize stripe virus (MSV), wheat dwarf virus (WDV), tobacco yellow wilt virus (TYDV), chickpea chlorosis dwarf virus (CpCDV) of the Mastrevirus genus in the Geminiviridae family, tomato golden mosaic virus (TGMV), African cassava mosaic virus (ACMV), cabbage leaf curl virus (CaLCuV), tomato leaf curl virus (ToLCV), cuckoo thistle leaf vein yellowing virus (AYVV), and beet severe curly top virus (BCTV) of the Kurtvirus genus in the Geminiviridae family.

[0077] In addition to the above, viral vector sequences derived from various plant viruses can also be used, such as tobacco mosaic virus, cucumber mosaic virus, apple microglobulin latent virus, barley leaf mosaic virus, bean pod mottle virus, beet curly top virus, brome mosaic virus, cabbage leaf curl virus, cotton leaf crumple virus, cymbidium mosaic virus, grape A virus, pea early browning virus, poplar mosaic virus, potato X virus, rice tungro bacilliform virus, satellite tobacco mosaic virus, tobacco curly shoot virus, and tobacco stem necrosis virus.

[0078] When the site-specific nuclease is a Cas protein, the polynucleotide of the present invention preferably further includes a guide RNA expression cassette. The presence of both the Cas protein expression cassette and the guide RNA expression cassette within the same molecule (polynucleotide) allows for efficient expression of both the Cas protein and the guide RNA within the same cell, thereby further enhancing genome editing efficiency.

[0079] The guide RNA expression cassette contains the guide RNA coding sequence.

[0080] The guide RNA coding sequence is not particularly limited as long as it is a nucleotide sequence that codes for guide RNA.

[0081] The guide RNA is not particularly limited as long as it is used in the CRISPR / Cas system. For example, various guide RNAs can be used that can bind to a target site on genomic DNA and, by binding to the Cas protein, guide the Cas protein to the target site on genomic DNA.

[0082] In this specification, the target site is a region on genomic DNA consisting of a DNA strand (non-target strand) consisting of a PAM (Protospacer Adjacent Motif) sequence and a sequence of about 17 to 30 base pairs (preferably 18 to 25 base pairs, more preferably 19 to 22 base pairs, and particularly preferably 20 base pairs) adjacent to its 5' or 3' side, and its complementary DNA strand (target strand).

[0083] The PAM sequence varies depending on the type of Cas protein used. For example, the PAM sequence corresponding to the Cas9 protein (Type II) from S. pyogenes is 5'-NGG, the PAM sequence corresponding to the Cas protein (Type IA) from S. solfataricus is 5'-CCN / 5'-TCN, the PAM sequence corresponding to the Cas protein (Type IB) from H. walsbyi is 5'-TTC, the PAM sequence corresponding to the Cas protein (Type IE) from E. coli is 5'-ARG, the PAM sequence corresponding to the Cas protein (Type IF) from E. coli is 5'-CC, the PAM sequence corresponding to the Cas protein (Type IF) from P. aeruginosa is 5'-CC, the PAM sequence corresponding to the Cas9 protein (Type II) from S. thermophilus is 5'-NNAGAA, the PAM sequence corresponding to the Cas9 protein (Type II) from S. agalactiae is 5'-NGG, the PAM sequence corresponding to the Cas9 protein (Type II) from S. aureus is 5'-NNGRRT, and N. The PAM sequence corresponding to the Cas9 protein from meningitidis is 5'-NNNNGATT, and the PAM sequence corresponding to the Cas9 protein from T. denticola is 5'-NAAAAC. In the above PAM sequences, N represents any base.

[0084] Guide RNA has a sequence (sometimes called a crRNA (CRISPR RNA) sequence) that is involved in binding to a target site on genomic DNA. This crRNA sequence binds complementaryly (preferably complementary and specific) to a sequence of the target strand excluding the complementary sequence of the PAM sequence, thereby enabling the guide RNA to bind to the target site on genomic DNA.

[0085] Furthermore, "complementary" binding includes not only binding based on perfect complementarity (A and T, and G and C), but also binding based on a degree of complementarity that allows for hybridization under stringent conditions. Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the complex or probe is bound, as taught in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). For example, typical washing conditions after hybridization include conditions of approximately "1×SSC, 0.1%SDS, 37°C". It is preferable that the hybridized state is maintained even after washing under such conditions. While not particularly limited, more stringent hybridization conditions include washing conditions of approximately "0.5×SSC, 0.1%SDS, 42°C", and even more stringent hybridization conditions include washing conditions of approximately "0.1×SSC, 0.1%SDS, 65°C".

[0086] Specifically, the crRNA sequence that binds to the target sequence has, for example, 90% or more, preferably 95% or more, more preferably 98% or more, even more preferably 99% or more, and especially preferably 100% identity with the non-target strand.

[0087] Regarding guide RNA, if it contains a sequence involved in binding to the Cas protein (sometimes called a tracrRNA (trans-activating crRNA) sequence), this tracrRNA sequence can bind to the Cas protein, thereby guiding the Cas protein to its target site in the genomic DNA.

[0088] The tracrRNA sequence is not particularly restricted. Typically, a tracrRNA sequence is an RNA sequence of about 50-100 nucleotides in length that can form multiple (usually three) stem-loops, and its sequence varies depending on the type of Cas protein used. Various known sequences can be used as tracrRNA sequences, depending on the type of Cas protein used.

[0089] Guide RNA typically contains the crRNA sequence and tracr RNA sequence described above. The guide RNA may be a single-stranded RNA (sgRNA) containing both the crRNA and tracr RNA sequences, or it may be an RNA complex formed by the complementary binding of RNA containing the crRNA sequence and RNA containing the tracr RNA sequence.

[0090] The polynucleotides of the present invention may include other base sequences besides those described above. Examples of other base sequences include drug resistance genes, reporter protein coding sequences, their expression cassettes; protein tag coding sequences; signal sequence coding sequences; origins of replication; and elemental sequences of binary vectors used in the Agrobacterium method (e.g., left boundary region, right boundary region, etc.).

[0091] Examples of drug resistance genes include chloramphenicol resistance genes, tetracycline resistance genes, neomycin resistance genes, erythromycin resistance genes, spectinomycin resistance genes, kanamycin resistance genes, hygromycin resistance genes, and puromycin resistance genes.

[0092] Reporter proteins are not particularly limited and include, for example, luminescent proteins that emit light (produce color) in reaction with a specific substrate, or fluorescent proteins that emit fluorescence when excited by light. Examples of luminescent proteins include luciferase, β-galactosidase, chloramphenicol acetyltransferase, and β-glucuronidase, while examples of fluorescent proteins include GFP, Azami-Green, ZsGreen, GFP2, HyPer, Sirius, BFP, CFP, Turquoise, Cyan, TFP1, YFP, Venus, ZsYellow, Banana, KusabiraOrange, RFP, DsRed, AsRed, Strawberry, Jred, KillerRed, Cherry, HcRed, and mPlum. Furthermore, reporter proteins also include fusion proteins of luminescent (chromogenic) proteins or fluorescent proteins with other proteins (e.g., seed-specific proteins), as well as proteins to which known protein tags, known signal sequences, etc., have been added to luminescent (chromogenic) proteins or fluorescent proteins.

[0093] Examples of protein tags include biotin, His tags, FLAG tags, Halo tags, MBP tags, HA tags, Myc tags, V5 tags, and PA tags.

[0094] The polynucleotides of the present invention may be single-stranded or double-stranded. They may also be linear or cyclic. Furthermore, the polynucleotides of the present invention include not only DNA and RNA, but also those that have been chemically modified as shown below. To prevent degradation by hydrolytic enzymes such as nucleases, the phosphate residue of each nucleotide may be replaced with a chemically modified phosphate residue such as phosphorothioate (PS), methylphosphonate, or phosphorodithionate. In addition, the hydroxyl group at position 2 of the sugar (ribose) of each ribonucleotide may be replaced with -OR (where R represents, for example, CH3(2'-O-Me), CH2CH2OCH3(2'-O-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.). Furthermore, the base portion (pyrimidine, purine) may be chemically modified, for example, by introducing a methyl group or cationic functional group at the 5th position of the pyrimidine base, or by substituting the carbonyl group at the 2nd position with a thiocarbonyl group. In addition, the phosphate portion or hydroxyl portion may be modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group, but is not limited to these. Also, the term "polynucleotide" includes not only natural nucleic acids but also BNA (Bridged Nucleic Acid), LNA (Locked Nucleic Acid), PNA (Peptide Nucleic Acid), etc.

[0095] In one embodiment, the polynucleotide of the present invention is a single-stranded polynucleotide (e.g., RNA). In another embodiment, the polynucleotide of the present invention is a double-stranded polynucleotide (e.g., a vector such as a binary vector).

[0096] The polynucleotides of the present invention may constitute a vector. The type of vector is not particularly limited and includes, for example, plasmid vectors; Agrobacterium vectors; plant virus vectors, etc. In addition to vectors suitable for introduction into plants or plant cells, as described above, a vector for transferring the polynucleotides of the present invention into such a vector (for example, an entry clone vector for a gateway®) can also be given as an example. In one embodiment, the present invention relates to a vector (preferably a plant genome editing vector) comprising the polynucleotides of the present invention.

[0097] The polynucleotides of the present invention can be easily produced according to known genetic engineering techniques. For example, they can be produced using PCR, restriction enzyme digestion, DNA ligation, in vitro transcription, etc. They can also be easily produced by incorporating the necessary sequences (e.g., site-specific nuclease coding sequences) into various commercially available plant virus vectors (binary vectors, etc.).

[0098] 3. Compositions for plant genome editing, kits for plant genome editing, methods for plant genome editing, and methods for producing plants. The polynucleotides of the present invention can be used as a composition for plant genome editing, or as a kit for plant genome editing. Furthermore, a method for plant genome editing and a method for producing genome-edited plants can be provided using the polynucleotides of the present invention.

[0099] The plant genome editing composition is not particularly limited as long as it contains the polynucleotide of the present invention, and may further contain other components as needed. Other components are not particularly limited, but include, for example, bases, carriers, solvents, dispersants, emulsifiers, buffers, stabilizers, excipients, binders, disintegrants, lubricants, thickeners, humectants, colorants, fragrances, chelating agents, etc. The plant genome editing composition may optionally contain a polynucleotide containing a guide RNA expression cassette. It may also optionally contain a donor polynucleotide.

[0100] The plant genome editing kit is not particularly limited as long as it contains the polynucleotide of the present invention, and may optionally include nucleic acid introduction reagents, buffers, and other materials, reagents, and equipment necessary for carrying out the plant genome editing method of the present invention. As other materials necessary for carrying out the plant genome editing method of the present invention, the plant genome editing kit may optionally include polynucleotides containing a guide RNA expression cassette. It may also optionally include donor polynucleotides.

[0101] The plant genome editing method and the method for producing plants include introducing the polynucleotide of the present invention into a plant.

[0102] Furthermore, the plant genome editing method and the plant production method preferably include introducing a cell-dividing activator at the polynucleotide introduction site of the present invention, or controlling the expression of an endogenous cell-dividing activator at the polynucleotide introduction site of the present invention. The cell-dividing activator is not particularly limited as long as it can promote the proliferation of cells at the polynucleotide introduction site of the present invention, promote the growth of the introduced tissue, or alter the cell division plane at the introduction site. When the introduction site is the shoot apex, among the cell-activating factors, those that can promote cell proliferation and the growth of the introduced tissue include, for example, WUSCHEL-Related Homeobox transcription factor (preferably WUS2 of Zea mays, hereinafter ZmWUS2) (specifically, the amino acid sequence is, for example, SEQ ID NO: 11 or its variant), isopentenyl transferase (ipt) (specifically, the amino acid sequence is, for example, SEQ ID NO: 13 or its variant), NAC transcription factor (e.g., CUC1 of Arabidopsis thaliana) (specifically, the amino acid sequence is, for example, SEQ ID NO: 30 or its variant), Response Regulator with deleted N-terminal signal receiver domain (e.g., ARR1 of Arabidopsis thaliana with deleted DDK signal receiver domain at the N-terminus) (specifically, the amino acid sequence is, for example, SEQ ID NO: 31 or its variant), and Cyclin (e.g., CycD2 of Arabidopsis thaliana).1) Examples include (specifically, the amino acid sequence is, for example, SEQ ID NO: 32 or its variant sequence), MYB3R transcription factor lacking the Negative regulation domain (e.g., myb A2 of Nicotiana tabacum with amino acids 631-1042 deleted) (specifically, the amino acid sequence is, for example, SEQ ID NO: 33 or its variant sequence), E2F (e.g., E2F3 of Arabidopsis thaliana) (specifically, the amino acid sequence is, for example, SEQ ID NO: 34 or its variant sequence), DP (e.g., DPa of Arabidopsis thaliana) (specifically, the amino acid sequence is, for example, SEQ ID NO: 35 or its variant sequence), and bHLH transcription factor (e.g., HEC1 of Arabidopsis thaliana) (specifically, the amino acid sequence is, for example, SEQ ID NO: 36 or its variant sequence). Examples of those that can alter the cell division plane at the introduction site include, for example, protein phosphatase 2 inhibitors (e.g., Arabidopsis Examples include inhibitors of thaliana PP2AC1-5 (e.g., polypeptides that dominantly inhibit the function of the protein, specifically, for example, SEQ ID NOs. 37-41 or their variant sequences), and TONNEAU 2 inhibitors (e.g., inhibitors of Solanum lycopersicum TON2 (specifically, amino acid sequences, for example, SEQ ID NOs. 42 or its variant sequences) (e.g., RNAi vectors that target and suppress the expression of the protein)), as well as combinations thereof. Among these, from the viewpoint of significantly improving genome editing efficiency, the combination of WUSCHEL-Related Homeobox transcription factor (preferably ZmWUS2) and ipt, NAC transcription factor (e.g., CUC1 of Arabidopsis thaliana), Cyclin (e.g., CycD2;1 of Arabidopsis thaliana), E2F (e.g., E2F3 of Arabidopsis thaliana) and DP (e.g., DPa of Arabidopsis thaliana) is preferred, and among these, the combination of WUSCHEL-Related Homeobox transcription factor (preferably ZmWUS2) and ipt, Cyclin (e.g., CycD2;1 of Arabidopsis thaliana) is preferred.1) A combination of E2F (e.g., E2F3 of Arabidopsis thaliana) and DP (e.g., DPa of Arabidopsis thaliana) is more preferred, and a combination of a WUSCHEL-Related Homeobox transcription factor (preferably ZmWUS2) and ipt is particularly preferred.

[0103] The cell-forming activator expression cassette contains a coding sequence in a state capable of expressing it or in a state capable of suppressing the function of the target protein, and is not particularly limited in this respect.

[0104] Cell-activating factors and the factors targeted by cell-activating factors may have amino acid sequence mutations (e.g., substitutions, deletions, insertions, additions, etc.) as long as their function is not significantly impaired. The amino acid sequence of a cell-activating factor or its target factor has, for example, 70% or more, preferably 80% or more, more preferably 90% or more, even more preferably 95% or more, even more preferably 97% or more, and particularly preferably 99% or more identity with the wild-type amino acid sequence.

[0105] The cell division activator expression cassette preferably includes a promoter, a terminator, etc. In one embodiment of the present invention, these expression cassettes may have a configuration similar to that of the site-specific nuclease expression cassette of the polynucleotide of the present invention. These expression cassettes may be incorporated into the polynucleotide of the present invention, or they may be incorporated into a polynucleotide that is a separate molecule from the polynucleotide of the present invention.

[0106] By using the above-mentioned cell-dividing activator in combination with the CRISPR / Cas system, genome editing efficiency can be improved. From this perspective, in one aspect of the present invention, the present invention relates to a plant genome editing method or a method for producing a genome-edited plant, comprising introducing a site-specific nuclease expression cassette and a cell-dividing activator expression cassette into a plant. Furthermore, in one aspect of the present invention, the present invention relates to a plant genome editing composition, a plant genome editing efficiency enhancer, etc., containing a polynucleotide including a cell-dividing activator expression cassette. The composition of these compositions and agents can be based on the composition of the plant genome editing composition containing the polynucleotide of the present invention.

[0107] The method for introducing the polynucleotide and cell division activator of the present invention into plants is not particularly limited and can be carried out in accordance with or in accordance with known methods.

[0108] In plants, examples of entry points include the stem apex, flowers (especially egg cells, pollen, etc., within the flower), leaves, and roots.

[0109] The method of introduction is not particularly limited and can be appropriately selected depending on the type of object to be introduced and the target of introduction. Examples of introduction methods include Agrobacterium methods such as the floral dip method and the floral spray method; particle gun method; and virus-mediated nucleic acid delivery. Among these, the particle gun method is preferred from the viewpoint of simplicity and safety.

[0110] A more specific example of the implementation method is shown below.

[0111] One example of an introduction method (Introduction Example 1) is a method that includes the step of preparing a plasmid containing a promoter (e.g., T7 promoter, T3 promoter, 35S promoter, etc.) and the sequence of the polynucleotide of the present invention downstream thereof (step a1). If the plasmid obtained in step a1 is a Ti plasmid containing a promoter that has transcriptional activation ability in plant cells, such as the 35S promoter, then the method can be carried out by introducing the plasmid obtained in step a1 into Agrobacterium and culturing it (step b1), and inoculating the culture solution obtained in step b1 into plants (e.g., infiltration method, toothpick inoculation method, aspiration injection method, etc.) (step c1). Alternatively, instead of steps b1 and c1, the method can be carried out by including a step of inoculating the plasmid obtained in step a1 into plants (e.g., grinding inoculation method, particle gun method, etc.) (step c2). Alternatively, instead of step c1, the method can be carried out by including a step of performing a step of performing a leaf disc method, inflorescence infiltration method, vacuum filtration method, etc. (step c3). These methods allow the target protein to be produced from plasmids or T-DNA introduced into the plant.

[0112] In particle gun therapy, nucleic acids and proteins encoding nuclease genes or cell activators that target the target gene can be coated onto the surface of microparticles and injected into target cells. As microparticles, metal microparticles are preferably used because they have a high specific gravity, are chemically inert, and are less likely to harm living organisms, thus increasing their penetration power into cells. Among metal microparticles, gold particles and tungsten particles are particularly preferred.

[0113] The particle gun method allows for the introduction of target genes into plant cells as follows: First, microparticles such as gold or tungsten particles are washed and sterilized. These microparticles, nucleic acids (recombinant vectors, linear DNA, RNA, etc.) or proteins, CaCl2, and spermidine are added while stirring with a vortex mixer or similar device to coat the gold or tungsten particles with DNA, RNA, or protein, and then washed with ethanol or phosphate-buffered saline.

[0114] The particle size (diameter) of the aforementioned microparticles can be appropriately determined to an optimal value depending on the type of target cell, the type of microparticle, the gas pressure, etc.

[0115] Gold or tungsten particles are applied as uniformly as possible to a macrocarrier film using a pipette or similar tool, and then dried in a sterile environment such as a clean bench. The macrocarrier film and a plate containing target cells are then placed in a particle gun device, and high-pressure helium gas is fired from a gas accelerating tube towards the macrocarrier film. The macrocarrier film is stopped by a stopping plate, but the particles pass through the stopping plate and penetrate the target cells placed beneath it, introducing the target gene.

[0116] The distance between the stopping plate and the target cells can be appropriately determined to an optimal value depending on the type of target cells, the type of microparticles, particle size, gas pressure, etc.

[0117] The optimal gas pressure can be determined as needed, depending on factors such as the type of target cells, the type of microparticles, and the distance between the target and the stopping plate.

[0118] In cells injected with microparticles, nucleic acids and / or proteins are released from the microparticles. In the case of nucleic acids, expression begins after translocation to the nucleus, and the translated nuclease translocates to the nucleus and cleaves the genomic DNA. Subsequently, mutations occur during the repair process, resulting in genome-edited cells. In the case of proteins, they translocate to the nucleus (organelle) via a nuclear localization signal (which may also be an organelle localization signal), and genome editing occurs by cleaving the DNA. Genome editing is applicable not only to nuclear genomic genes but also to genes in organelles (intracellular organelles such as chloroplasts and mitochondria). In this case, the localization signal for each organelle may be linked to the nuclease and introduced, or a promoter that is expressed only in each organelle may be used to link to the nuclease.

[0119] After introduction, culturing the plants for a certain period of time induces the expression of site-specific nucleases within the plants, leading to genome editing. The culturing period is, for example, 1 to 30 days.

[0120] The method of cultivation is not particularly limited. For example, if the plant is a plant body or a part thereof growing in a growing environment, it can simply continue growing as is. If the plant is a plant body or a part thereof separated from the growing environment, or if it has been separated from a plant body, the object should be cultured in an appropriate environment (for example, a humid environment or an environment where a constant temperature is maintained).

[0121] The presence or absence of genome editing can be confirmed using nucleic acids obtained from the target material, according to known methods, such as the T7 Endonuclease I assay, CAPS (cleaved amplified polymorphic sequence) analysis, or methods for detecting mutations in the base sequence around the genome editing site (around the site-specific nuclease cleavage site) (e.g., methods using primers designed for predicted mutation locations, sequencing analysis, etc.).

[0122] The plant genome editing method of the present invention makes it possible to produce genome-edited plants.

[0123] Furthermore, the genome editing technology of the present invention is expected to enable the acquisition of genome-edited germ cells. By obtaining the next generation of plants from these germ cells, it is possible to obtain plants in which all cells are genome-edited. [Examples]

[0124] The present invention will be described in detail below based on examples, but the present invention is not limited to these examples.

[0125] Example 1. Preparation of Cas expression vector 1 A Cas expression vector (Figure 1) was obtained by inserting a Cas protein expression cassette and a guide RNA expression cassette into a pTTK352 binary vector containing the sequence of a geminivirus vector (bean yellow dwarf virus (BeYDV)).

[0126] The Cas expression vector was prepared as follows: An artificially synthesized BeYDV-derived sequence was seamlessly cloning inserted between the left and right borders of the pKIR vector (binary) described in a previous report (Tsutsui et al. (2016) pKAMA-ITACHI Vectors for Highly Efficient CRISPR / Cas9-Mediated Gene Knockout in Arabidopsis thaliana, Plant and Cell Physiology, Vol.58 (Issue 1), pp.46-56). The BeYDV-derived MP and CP were deleted, and the sequences encoding Replicase proteins, the long intergenic region (LIR), and the small intergenic region (SIR) were used.

[0127] The inserted Cas protein expression cassette is arranged in order from the 5' end: (1) Cestrum yellow leaf curling virus (CmYLCV) promoter (SEQ ID NO: 1), (2) dMac3 sequence (sequence number 2) or NtADH 5'UTR sequence (sequence number 66) (3) Streptococcus pyogenes-derived Cas9 (SpCas9) protein coding sequence (SEQ ID NO: 3) with added epitope tag and nuclear localization signal, (4) Cauliflower mosaic virus 35S terminator (SEQ ID NO: 4), (5) ACT3 (ACTIN3) (NbACT3) terminator of Nicotiana benthamiana (sequence number 5), (6) Rb7 scaffold / matrix binding region (MAR) of Nicotiana tabacum (SEQ ID NO: 6), It is DNA, which is made up of these elements arranged in a specific way.

[0128] The inserted guide RNA expression cassette is arranged in order from the 5' end: (7) U6-26 promoter of Arabidopsis thaliana (SEQ ID NO: 7), (8) sgRNA sequences and polyT sequences (SEQ ID NO: 8) that target sequences within the phytoene desaturase (NbPDS) gene of Nicotiana benthamiana, It is DNA, which is made up of these elements arranged in a specific way.

[0129] The insertion method was as follows: A Golden Gate method, partially modified from the method of Lampropoulos et al. (2013), was used. Cassettes containing sequences 1-8 above were subjected to a Golden Gate reaction using the NEB Golden Gate Assembly Kit, and the sequences were inserted by assembling them between the LIR and SIR sequences derived from BeYDV. The reaction conditions were carried out according to the recommended protocol of the above kit. As nucleotide sequences (4 nucleotides) for linking between each cassette, ACCT was inserted directly above the cassette containing sequence 1, AACA between the cassette containing sequence 1 and the cassette containing sequence 2, GGCT between the cassette containing sequence 2 and the cassette containing sequence 3, ACTA between the cassettes containing sequences 4-6 and the cassettes containing sequences 7-8, and GTAT was inserted directly below the cassette containing sequences 7-8. Furthermore, a ligation sequence of TCAG + 46 bases + CTGC was inserted between the cassette containing sequence 3 above and the cassette containing sequences 4-6 above.

[0130] Comparative Example 1. Preparation of Cas expression vector 2 As the Cas protein expression cassette, instead of (4), (5), and (6), (4') NOS Terminator (Sequence ID 9), A Cas expression vector (Figure 2) was obtained in the same manner as in Example 1, except that a Cas protein expression cassette incorporating the Cas protein was inserted.

[0131] Comparative Example 2. Preparation of Cas expression vector 3 As the Cas protein expression cassette, instead of (4), (5), and (6), (4") UBQ Terminator of Arabidopsis thaliana (SEQ ID NO: 43), A Cas expression vector (Figure 2) was obtained in the same manner as in Example 1, except that a Cas protein expression cassette incorporating the Cas protein was inserted.

[0132] Example Test 1: Genome Editing Test 1 Genome editing was performed using the Cas expression vector from Example 1, and the Cas expression vectors from Comparative Examples 1 and 2, and the genome editing efficiency was compared. Specifically, the procedure was as follows.

[0133] <Preparing for Agrobacterium> The Cas expression vector from Example 1, and the Cas expression vectors from Comparative Examples 1 and 2 were introduced into Rhizobium radiobacter strain GV3101 by electroporation and plated onto LB plates containing antibiotics, then cultured at 28°C for 3 days. Single colonies were pre-cultured overnight in 3 mL of LB liquid medium containing antibiotics (28°C, 180 rpm), then 7 mL of LB liquid medium was added and cultured for 2 hours (28°C, 180 rpm), after which the cells were collected. The cells were resuspended in induction buffer (10 mM MES, 100 μM Acetosyringone, pH 5.5 with NaOH) and induced for 2-3 hours. After resuspending in infiltration buffer (5 mM MES, pH 5.5 with NaOH), each agrobacterial suspension was adjusted to an OD of 0.7 to prepare the inoculum.

[0134] <Infiltration of tobacco leaves> The Nicotiana benthamiana used was surface-sterilized with high heat, sown on a seeding medium, left standing in the dark at 4°C for 4 days, and then grown for 1 week in a chamber with a 25°C, 16-hour light period and 8-hour dark period cycle (16L8D). The germinated seedlings were transplanted into a culture medium mixed with flower soil (flower heart): vermiculite at a ratio of 1:1, grown under the conditions of 25°C and 16L8D, and individuals 2 - 3 weeks after transplantation were used for infiltration. Infiltration was performed from the back of the leaves using a 1 mL syringe. After inoculation, the plants were kept moist to prevent drying and grown in an environment of 26 - 30°C and 16L8D.

[0135] <Mutation detection by T7EI assay> Seven days after inoculation, the inoculation site was cut out using a punch with a diameter of 8 mm, crushed with a multi-bead shocker MB2200 (YASUI KIKAI), and total DNA was extracted using an automatic nucleic acid extraction device Maxwell (Promega). Using the extracted DNA as a template, the DNA region containing the target sequence was PCR-amplified using KOD Fx Neo (TOYOBO). The PCR product was treated with T7 Endonuclease I (NEB), and the presence or absence of mutagenesis was detected using a microchip electrophoresis device MultiNA (SHIMADZU) for DNA / RNA analysis. When a cleaved band occurred, it could be determined that genome editing was successful. The ratio (%) of the cleaved band was calculated with (non-cleaved band + cleaved band) as 100%, and this was taken as the genome editing efficiency. The results are shown in Figure 3 and Table 1.

[0136]

Table 1

[0137] [[ID=I7]] When 35S + NbACT3 + Rb7 (3×terminator in Figure 3 and Table 1) was used (the Cas expression vector of Example 1), mutations were detected at an efficiency of approximately 40 - 50%, and high-efficiency genome editing editing could editing could be induced in tobacco. Also, the genome editing efficiency tended to be higher than when UBQ and NOS were used as terminators.

[0138] Example 2-1. Preparation of Cas expression vector 4 A Cas expression vector was obtained in the same manner as in Example 1, except that a vector portion was inserted as a guide RNA expression cassette, consisting of eight expression cassettes linked in tandem (each expression cassette has a different target gene for its sgRNA: in order from the 5' end, SlGGP1, SlGAD2, SlGAD3, SlSGR, SlLCY-E, SlBIc, SlLCY-B1, SlLCY-B2), each containing an sgRNA sequence targeting a sequence within the Solanum lycopersicum gene under the control of the Arabidopsis thaliana U6-26 promoter.

[0139] Example 2-2. Preparation of Cas expression vector 5 A Cas expression vector was obtained in the same manner as in Example 2-1, except that a vector portion consisting of four tandemly linked expression cassettes, each containing an sgRNA sequence targeting a sequence within the sg-5 gene of Glycine max under the control of the U6-26 promoter of Arabidopsis thaliana, was inserted as a guide RNA expression cassette.

[0140] Examples 2-3. Preparation of Cas expression vectors 6 A Cas expression vector was obtained in the same manner as in Example 2-1, except that a vector portion consisting of six tandemly linked expression cassettes, each containing an sgRNA sequence targeting a sequence within the ADPG gene of Perilla frutescens under the control of the U6-26 promoter of Arabidopsis thaliana, and a sequence encoding a kanamycin resistance gene were inserted.

[0141] Comparative Example 3. Preparation of Cas expression vector 7 A Cas expression vector was obtained in the same manner as in Comparative Example 1, except that a vector portion was inserted in tandem, consisting of eight expression cassettes (each expression cassette targeting a sequence within the Solanum lycopersicum gene under the control of the Arabidopsis thaliana U6-26 promoter) linked together (each expression cassette targeting a different gene: from the 5' end, SlGGP1, SlGAD2, SlGAD3, SlSGR, SlLCY-E, SlBIc, SlLCY-B1, SlLCY-B2).

[0142] Example 1: Preparation of an expression vector In Test Example 2, the following expression vectors were prepared to be introduced into plants.

[0143] An expression vector for the fluorescent protein (tdTomato) was constructed.

[0144] Furthermore, as cell-dividing activators, we prepared expression vectors for the Zea mays WUS2 (WUSCHEL 2) gene (coding sequence: SEQ ID NO: 10, amino acid sequence: SEQ ID NO: 11) and the ipt (isopentenyl transferase) gene (cytokinin biosynthesis gene) (coding sequence: SEQ ID NO: 12, amino acid sequence: SEQ ID NO: 13). These two expression vectors were prepared in the same manner as the Cas expression vector in Example 1, except that (3) the ZmWUS2 coding sequence or the ipt coding sequence was used instead of the SpCas9 coding sequence, and (7) and (8) spacer sequences were used instead of guide RNA expression cassettes.

[0145] Example Test 2-1. Genome Editing Test 2-1 Genome editing was performed using the Cas expression vector from Example 2-1 and the Cas expression vector from Comparative Example 3, and the genome editing efficiency was compared. The genome editing efficiency with and without the cell-dividing activator from Reference Example 1 was also compared. Specifically, the following procedures were followed.

[0146] <Tomato sterile cultivation> The tomato variety used was "Home Momotaro" (Takii Seed Co., Ltd.), a commercially available variety. After washing the seeds with 70% ethanol, they were sterilized with bleach, soaked in water overnight, and then spread on a seeding medium (MS, 0.5% sucrose, 0.4% gellan gum).

[0147] <Removal of the stem tip> The procedure was performed aseptically using a table coach KOACH T500-F (KOKEN). The above-ground portion was cut to a length of approximately 2-3 cm with a scalpel and placed on filter paper (ADVANTEC) moistened with sterile water. Subsequent procedures were performed under a stereomicroscope (OLYMPUS SZX10). Using a 1 mL syringe (TERUMO) and a 26G needle (TERUMO), the leaf primordia were removed without damaging the shoot apex. The shoot apex was placed in a 6 cm dish (IWAKI) containing culture medium (MS basal salt, 3% Sucrose, 150 mg / L Cefotaxime, 0.01 μM gibberellic Acid 3, 0.01 μM Kinetin, 0.6% Phytagel) with the shoot apex facing directly upwards and left to stand overnight.

[0148] <Particle Bombardier Drink> Particle bombardment was performed using PDS-1000 / He(Bio-Rad) with a modified protocol. 0.6 μm diameter gold particles (InBio) were washed with 70% ethanol, rinsed three times with sterile water, and then suspended in 50% glycerol. 5 μg of plasmid was mixed with 0.1 M spermidine and 2.5 M CaCl2 for every 3 mg of gold particles. The plasmids used in each test group and their molar ratios are as follows. • Test group 1: Cas expression vector (Example 2-1): tdTomato expression vector = 1:1 • Test group 2: Cas expression vector (Comparative Example 3): tdTomato expression vector = 1:1 • Test group 3: Cas expression vector (Example 2-1): tdTomato expression vector: ZmWUS2 expression vector (Reference Example 1): ipt expression vector (Reference Example 1) = 1:1:1:1 Subsequently, after washing with 70% ethanol, it was suspended in 100% ethanol. While suspending the prepared gold particles, they were spread evenly at the center of the macrocarrier and dried. Two rupture disks of 650 psi were placed in the retaining cap, and the macrocarrier with the gold particles and the stopping screen were inserted into the Microcarrier Launch Assembly and installed on the PDS-1000 main body. After placing the dish with the shoot apical meristems in the PDS-1000 and performing the bombardment, it was wrapped with aluminum foil and left standing overnight.

[0149] <Introduction confirmation> On the day after bombardment, the number of tdTomato fluorescent spots on each shoot apical meristem was measured using a fluorescence microscope BZ-X810 (KEYENCE), and the shoot apical meristems were transferred to a new medium (MS basal salt, 3% Sucrose, 150 mg / L Cefotaxime, 0.01 μM gibberellic Acid3, 0.01 μM Kinetin, 0.3% Phytagel).

[0150] <Transplantation> Seven days after bombardment, the state of the shoot apical meristems was observed, and the shoot apical meristems with 5 or more fluorescent spots and surviving were transferred to a growth medium (MS, 3% Sucrose, 150 mg / L Cefotaxime, 0.01 μM gibberellic Acid3, 0.01 μM Kinetin, 0.3% Phytagel in ECO2box) and grown under the conditions of 25°C and 18L6D.

[0151] <Detection of genome editing by T7EI assay> A newly unfolded fifth lobe was sampled, disrupted using a multi-bead shocker MB2200 (YASUI KIKAI), and total DNA was extracted using an automated nucleic acid extraction system Maxwell (Promega). The extracted DNA was used as a template, and fragments containing the target sequence were PCR amplified using KOD Fx Neo (TOYOBO). The PCR products were treated with T7 Endonuclease I (NEB), and the presence or absence of mutations was detected using a DNA / RNA analysis microchip electrophoresis system MultiNA (SHIMADZU). The results are shown in Table 2.

[0152] [Table 2]

[0153] When using an NOS terminator (Cas expression vector in Comparative Example 3), mutations were detected at a rate of 2.4%, but when using a 35S terminator + NbACT3 terminator + Rb7 MAR (Cas expression vector in Example 2-1), the rate rose to 5.1%, which was 2.1 times higher than when using an NOS terminator. Furthermore, when a ZmWUS2 expression vector and an ipt expression vector (expression vector in Reference Example 1) were injected together with the genome editing vector, the mutation rate was 9.1%, which was 1.8 times higher than when ZmWUS2 and ipt were not included.

[0154] <Detection of genome editing in next-generation technologies> The mutant individuals obtained as described above were grown to obtain next-generation seeds. The obtained seeds were sown, and total DNA was extracted from the cotyledons of multiple individuals. Using the extracted DNA as a template, fragments containing the target sequence on the SGR gene were amplified by PCR using KOD Fx Neo (TOYOBO), and the presence or absence of mutations was analyzed by Sanger sequencing. As a result, "mutant homozygous" individuals with 100% mutation, "mutant heterozygous" individuals with 50% mutation, and "non-mutant" individuals with no mutation were separated and detected, confirming that the mutation was transmitted to the next generation of individuals.

[0155] Example 2-2: Genome Editing Experiment 2-2 Genome editing was performed using the Cas expression vector from Example 2-2. Specifically, it was carried out as follows.

[0156] <Soybean sterile cultivation> The soybeans used were the commercially available "Fukuyutaka" variety. The seeds, which had been sterilized using chlorine gas sterilization, were placed on a moistened Prowipe to absorb water.

[0157] <Removal of the stem tip> The procedure was carried out in the same manner as in Test Example 2-1.

[0158] <Particle Bombardier Drink> Particle bombardment was performed in the same manner as in Test Example 2-1 (Test Group 3).

[0159] <Implementation Confirmation> The day after injection, the number of bright spots of tdTomato at each shoot apex was measured using a fluorescence microscope BZ-X810 (KEYENCE), and the shoot apex was transferred to a new culture medium (MS basal salt, 3% Sucrose, 25 mg / L Meropenem, 0.01 μM gibberellic Acid 3, 0.01 μM Kinetin, 0.3% Phytagel).

[0160] <Transplant> Seven days after planting, the shoot apex was observed, and shoot apex with one or more bright spots and still viable was transferred to a growth medium (MS, 3% Sucrose, 25 mg / L Meropeneme, 0.01 μM gibberellic Acid 3, 0.01 μM Kinetin, 0.3% Phytagel in ECO2box) and grown under conditions of 25°C and 18L6D.

[0161] <Mutation detection by Sanger sequencing> Total DNA was extracted from the newly unfolded fifth lobe. Using the extracted DNA as a template, the fragment containing the target sequence was amplified by PCR using KOD Fx Neo (TOYOBO), and sequence information was obtained by Sanger sequencing (Figure 4a). Based on the obtained sequence information, the presence or absence of mutations was analyzed using the analysis software "DECODR," and a four-base deletion was detected (Figure 4b). <Detection of genome editing in next-generation technologies> Individuals in which mutations were confirmed to have been introduced in the current generation were grown, and the resulting next-generation seeds were analyzed in the same manner as in Experiment 2-1 to see if the mutations had been introduced. As a result, the same mutation as the 4-base deletion detected in the current generation was found, confirming that the mutations had been transmitted to the next generation of individuals.

[0162] Example 2-3: Genome Editing Experiment 2-3 Genome editing was performed using the Cas expression vectors from Example 2-3. Specifically, it was carried out as follows.

[0163] <Preparing the Perilla Plant> We used the "Shirakawa Late-Blooming" variety of perilla, soaking the seeds in water overnight before sowing them in soil for cultivation.

[0164] <Removal of the stem tip> The procedure was carried out in the same manner as in Test Example 2-1.

[0165] <Particle Bombardier Drink> The procedure was carried out in the same manner as in Test Example 2-1 (Test Group 3).

[0166] <Implementation Confirmation> The day after injection, the number of bright spots of tdTomato at each shoot apex was measured using a fluorescence microscope BZ-X810 (KEYENCE), and the shoot apex was transferred to a new culture medium (MS basal salt, 3% Sucrose, 25 mg / L Meropeneme, 0.01 μM gibberellic Acid 3, 0.01 μM Kinetin, 0.3% Phytagel).

[0167] <Transplant> Seven days after injection, the shoot apical meristems were observed, and those with five or more bright spots and still alive were transferred to a growth medium (MS, 3% sucrose, 25 mg / L meropenem, 0.01 μM gibberellic acid 3, 0.01 μM kinetin, 0.3% phytagel in an ECO2 box).

[0168] <Mutation detection by CAPS (cleaved amplified polymorphic sequence)> Total DNA was extracted from the newly developed fifth leaf. Using the extracted DNA as a template, the fragment containing the target sequence was PCR-amplified using KOD Fx Neo (TOYOBO). After column purification of the PCR product, it was treated with BsrGI (NEB) and SspI (NEB), and the presence or absence of mutagenesis was analyzed using a microchip electrophoresis apparatus for DNA / RNA analysis, MultiNA (SHIMADZU). When an uncleaved band occurred, it could be determined that genome editing had occurred. The ratio (%) of the uncleaved band when (uncleaved band + cleaved band) was set to 100% was calculated as the genome editing efficiency. The results of BsrGI treatment are shown in Fig. 5(a), and the results of SspI treatment are shown in Fig. 5(b). As a result of CAPS analysis, mutagenesis of about 2% was detected.

[0169] Comparative Example 4. Preparation of Cas expression vector 8 In the Cas protein expression cassette, a Cas protein expression cassette in which the terminator of extensin of Nicotiana tabacum (SEQ ID NO: 14) was incorporated instead of (4), (5), and (6) was inserted, and a Cas expression vector (Fig. 7) was obtained in the same manner as in Example 1.

[0170] Example 3. Preparation of Cas expression vector 9 A Cas expression vector (Figure 7) was obtained in the same manner as in Example 1, except that a Cas protein expression cassette was inserted in which the (4"") Nicotiana tabacum extensin terminator (SEQ ID NO: 14) was incorporated in place of (4) and (5), and the A. gossypii TEF (translation elongation factor) insulator (SEQ ID NO: 18) was incorporated in place of (6).

[0171] Example 4. Preparation of Cas expression vector 10 A Cas expression vector (Figure 6) was obtained in the same manner as in Example 1, except that a Cas protein expression cassette was inserted in which the (4"") Nicotiana tabacum extensin terminator (SEQ ID NO: 14) was incorporated in place of (4) and (5), and the Sea urchin Ars (arylsulfatase) insulator (SEQ ID NO: 17) was incorporated in place of (6).

[0172] Example 5. Preparation of Cas expression vector 11 A Cas expression vector (Figure 7) was obtained in the same manner as in Example 1, except that a Cas protein expression cassette was inserted in which the (4"") Nicotiana tabacum extensin terminator (SEQ ID NO: 14) was incorporated in place of (4) and (5), and the Human Bead1 (blocking element alpha / delta-1 of the human T-cell receptor α / δ locus) insulator (SEQ ID NO: 16) was incorporated in place of (6).

[0173] Example 6. Preparation of Cas expression vector 12 A Cas expression vector (Figure 7) was obtained in the same manner as in Example 1, except that a Cas protein expression cassette was inserted in which the (4"") Nicotiana tabacum extensin terminator (SEQ ID NO: 14) was incorporated in place of (4) and (5), and the Drosophila gypsy insulator (SEQ ID NO: 15) was incorporated in place of (6).

[0174] Example 7. Preparation of Cas expression vector 13 A Cas expression vector (Figure 7) was obtained in the same manner as in Example 1, except that a Cas protein expression cassette was inserted in which the (4"") Nicotiana tabacum extensin terminator (SEQ ID NO: 14) was incorporated in place of (4) and (5), and the Yeast TEF2 (translation elongation factor 2) insulator (SEQ ID NO: 19) was incorporated in place of (6).

[0175] Example Test 3. Genome Editing Test 3 Genome editing was performed using the Cas expression vectors from Examples 3-7 and the Cas expression vector from Comparative Example 4, and the mutation rates were compared. The method was the same as in Experimental Example 1.

[0176] As representative examples, the results of Example 4 and Comparative Example 4 are shown in Figure 8. When the Sea urchin Ars insulator was fused to the extensin terminator of Nicotiana tabacum, a mutation rate of over 44% was detected, demonstrating highly efficient genome editing in tobacco. The genome editing efficiency was significantly higher when using the Sea urchin Ars insulator (Example 4) compared to Examples 3 and 5-7, which used other insulators.

[0177] Example 8. Preparation of MAD7 expression vector A MAD7 expression vector was obtained by inserting a MAD7 protein expression cassette and a guide RNA expression cassette into a pTTK352 binary vector containing the sequence of a geminivirus vector (bean yellow dwarf virus (BeYDV)).

[0178] The MAD7 expression vector was prepared as follows: An artificially synthesized BeYDV-derived sequence was seamlessly inserted between the left and right borders of the pKIR vector (binary) described in a previous report (Tsutsui et al. (2016) pKAMA-ITACHI Vectors for Highly Efficient CRISPR / Cas9-Mediated Gene Knockout in Arabidopsis thaliana, Plant and Cell Physiology, Vol.58 (Issue 1), pp.46-56) using seamless cloning. The BeYDV-derived MP and CP were deleted, and the sequences encoding replicase proteins, the long intergenic region (LIR), and the small intergenic region (SIR) were used.

[0179] The inserted MAD7 protein expression cassette is arranged from the 5' end in order: (1) Cestrum yellow leaf curling virus (CmYLCV) promoter (SEQ ID NO: 1), (2) dMac3 sequence (sequence number 2), (3) MAD7 protein coding sequence in a form with epitope tag and nuclear localization signal added (SEQ ID NO: 44), (4) Cauliflower mosaic virus 35S terminator (SEQ ID NO: 4), (5) ACT3 (ACTIN3) (NbACT3) terminator of Nicotiana benthamiana (SEQ ID NO: 5), (6) Rb7 scaffold / matrix binding region (MAR) of Nicotiana tabacum (SEQ ID NO: 6), It is DNA, which is made up of these elements arranged in a specific way. The inserted guide RNA expression cassette is arranged in order from the 5' end: (7) The 35S promoter of cauliflower mosaic virus (SEQ ID NO: 45), (8) hammerhead (HH) ribozyme (Sequence No. 46) (9) Direct repeat sequence (sequence number 47) (10) A sequence derived from the Benthamiana tobacco XT1 gene (SEQ ID NO: 48) as a spacer, (11) Hepatitis delta virus (HDV) ribozyme (SEQ ID NO: 49) (12) Nos Terminator (Sequence ID 50) It is DNA, which is made up of these elements arranged in a specific way.

[0180] The insertion method was as follows: A Golden Gate method, partially modified from the method of Lampropoulos et al. (2013), was used. The cassettes containing the above sequences were subjected to a Golden Gate reaction using the NEB Golden Gate Assembly Kit, and the sequences were inserted by assembling them between the LIR and SIR sequences derived from BeYDV. The reaction conditions were carried out according to the recommended protocol of the above kit. As nucleotide sequences (4 bases) for linking between each cassette, ACCT was inserted directly above the cassette containing sequence 1 above, AACA between the cassette containing sequence 1 above and the cassette containing sequence 2 above, GGCT between the cassette containing sequence 2 above and the cassette containing sequence 3 above, ACTA between the cassettes containing sequences 4-6 above and the cassettes containing sequences 7-12 above, and GTAT was inserted directly below the cassette containing sequences 7-12 above. In addition, TCAG + 46 bases + CTGC was inserted as a nucleotide sequence for linking between the cassette containing sequence 3 above and the cassettes containing sequences 4-6 above.

[0181] Example Test 4. Genome Editing Test 4 Genome editing experiments were conducted in plants using the effector expression vector and silencing suppressor expression vector (pDGB3alpha2_35S:P19:Tnos, reference Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D. (2013) GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162(3):1618-1631) from Example 8, and the mutation rate was analyzed. Specifically, the procedure was as follows.

[0182] <Preparing for Agrobacterium> The effector expression vector and silencing suppressor expression vector from Example 8 were introduced into Rhizobium radiobacter strain GV3101 by electroporation and plated onto LB plates containing antibiotics, then cultured at 28°C for 2-3 days. Single colonies were pre-cultured overnight in 3 mL of LB liquid medium containing antibiotics (28°C, 180 rpm), then 7 mL of LB liquid medium was added and cultured for 2 hours (28°C, 180 rpm), after which the cells were collected. The cells were resuspended in induction buffer (10 mM MES, 100 μM Acetosyringone, pH 5.5 with NaOH) and induced for 2-3 hours. After resuspending in infiltration buffer (5 mM MES, pH 5.5 with NaOH), each agro-bacterial solution was adjusted to an OD of 0.7, and equal volumes were mixed to prepare the inoculum.

[0183] <Infiltration of tobacco leaves> The Nicotiana benthamiana used was sterilized by high-temperature after seeds were sown on the seeding medium, left standing in the dark at 4°C for 4 days, and then grown in a chamber with a 25°C, 16-hour light period and 8-hour dark period cycle (16L8D) for 1 week. The germinated seedlings were transplanted into a culture soil mixed with flower compost (flower heart) and vermiculite at a ratio of 1:1, and grown under the conditions of 26 - 30°C and 16L8D. The plants 2 - 3 weeks after transplantation were used for infiltration. Infiltration was performed from the back of the leaves using a 1 mL syringe. After inoculation, the plants were moisturized to prevent drying and grown under the environment of 26 - 30°C and 16L8D. [[ID=​​​​​​​​​​​​​​​In Experimental Example 5, the following expression vectors were prepared to be introduced into plants.

[0187] The Cas expression vector was prepared in the same manner as in Example 1, except that a vector portion consisting of seven tandem expression cassettes, each containing an sgRNA sequence targeting the FAD7 gene sequence of Solanum lycopersicum under the control of the Arabidopsis thaliana U6-26 promoter, was inserted as a guide RNA expression cassette.

[0188] As cell-dividing activators, the following are expression vectors: Arabidopsis thaliana CUC1 (CUP-SHAPED COTYLEDON 1) gene (NAC transcription factor) (coded sequence: SEQ ID NO: 51, amino acid sequence: SEQ ID NO: 30), Arabidopsis thaliana ARR1 (Arabidopsis Response Regulator 1) gene (coded sequence: SEQ ID NO: 52, amino acid sequence: SEQ ID NO: 31) with the N-terminal DDK signal receiver domain deleted, Arabidopsis thaliana CycD2;1 gene (coded sequence: SEQ ID NO: 53, amino acid sequence: SEQ ID NO: 32), Nicotiana tabacum myb A2 gene (MYB3R transcription factor) (coded sequence: SEQ ID NO: 54, amino acid sequence: SEQ ID NO: 33) with amino acids 631-1042 deleted, Arabidopsis thaliana E2F3 gene (coded sequence: SEQ ID NO: 55, amino acid sequence: SEQ ID NO: 34), Arabidopsis Expression vectors were prepared for the DPa gene of *thaliana* (coding sequence: SEQ ID NO: 56, amino acid sequence: SEQ ID NO: 35), the HEC1 gene (bHLH transcription factor) of *Arabidopsis thaliana* (coding sequence: SEQ ID NO: 57, amino acid sequence: SEQ ID NO: 36), and the PP2AC1-5 gene (protein phosphatase 2) of *Arabidopsis thaliana* (coding sequence: SEQ ID NO: 58-62, amino acid sequence: SEQ ID NO: 37-41) with amino acid mutations introduced to make it dominant-negative.

[0189] The 12 expression vectors described above were prepared in the same manner as the Cas expression vector in Example 1, except that the coding sequences of each cell-dividing activator were inserted instead of the SpCas9 coding sequence, and a spacer sequence was inserted instead of the guide RNA expression cassette.

[0190] We also prepared an RNAi (RNAi) vector to suppress the expression of the TON2 (TONNEAU 2) gene (coding sequence: SEQ ID NO: 63, amino acid sequence: SEQ ID NO: 42) of Solanum lycopersicum.

[0191] The TON2 expression repression vector was prepared in the same manner as the Cas expression vector in Example 1, except that instead of the SpCas9 coding sequence, the target sequence of the TON2 gene (SEQ ID NO: 64), the ligation sequence (ACGA), the pdk intron (SEQ ID NO: 65), the ligation sequence (TCGT), and the reverse complementary sequence of the TON2 gene target sequence were inserted in order from the 5' end to form an ihpRNA (Intron-containing hairpin RNA) targeting the TON2 coding sequence, and a spacer sequence was inserted instead of the guide RNA expression cassette.

[0192] Example Test 5. Genome Editing Test 5 We compared the genome editing efficiency with and without the cell division activator in Reference Example 2. Specifically, we performed the following procedure.

[0193] <Tomato sterile cultivation> The procedure was carried out in the same manner as in Test Example 2-1.

[0194] <Removing the stem tip> The procedure was carried out in the same manner as in Test Example 2-1.

[0195] <Particle Bombardment> The procedure was carried out in the same manner as in Test Example 2-1.

[0196] The plasmids used in each test group and their molar ratios are as follows: • Test group 1: Cas expression vector: tdTomato expression vector = 1:1 • Test group 2: Cas expression vector: tdTomato expression vector: ZmWUS2 expression vector (Reference Example 1): ipt expression vector (Reference Example 1) = 1:1:1:1 • Test group 3: Cas expression vector: tdTomato expression vector: AtCUC1 expression vector = 1:1:1 • Test group 4: Cas expression vector: tdTomato expression vector: AtARR1ΔDDK expression vector = 1:1:1 • Test group 5: Cas expression vector: tdTomato expression vector: AtCycD2;1 expression vector = 1:1:1 • Test group 6: Cas expression vector: tdTomato expression vector: NtMYBA2Δ631-1042 expression vector = 1:1:1 • Test group 7: Cas expression vector: tdTomato expression vector: AtE2F3 expression vector: Dpa expression vector = 1:1:1:1 • Test group 8: Cas expression vector: tdTomato expression vector: AtHEC1 expression vector = 1:1:1 • Test group 9: Cas expression vector: tdTomato expression vector: ZmWUS2 expression vector (reference example 1): ipt expression vector (reference example 1): AtCUC1 expression vector: AtARR1ΔDDK expression vector: AtCycD2;1 expression vector: NtMYBA2Δ631-1042 expression vector: AtE2F3 expression vector: Dpa expression vector = 1:1:1:1:1:1:1:1:1:1:1 • Test group 10: Cas expression vector: tdTomato expression vector: PP2AC1 expression vector: PP2AC2 expression vector: PP2AC3 expression vector: PP2AC4 expression vector: PP2AC5 expression vector = 1:1:1:1:1:1:1 • Test group 11: Cas expression vector: tdTomato expression vector: TON2 expression suppression vector = 1:1:1 <Installation Confirmation> The procedure was carried out in the same manner as in Test Example 2-1.

[0197] <Transplant> The procedure was carried out in the same manner as in Test Example 2-1.

[0198] <Detection of genome editing by Sanger sequencing> A newly developed fifth leaf was sampled, and the leaf was crushed using a pestle in extraction buffer (1M KCl, 10mM EDTA, 100mM Tris-HCl with HCl) until the buffer turned green. The extract was thoroughly mixed in a vortex and incubated at 95°C for 10 minutes. After incubation, the mixture was thoroughly mixed again in a vortex, centrifuged, and the supernatant was used as a PCR template.

[0199] Fragments containing the target sequence were amplified by PCR using KOD Fx Neo (TOYOBO). After processing the PCR products with the Exo-CIP Rapid PCR Cleanup Kit (NEB), the presence or absence of mutations was analyzed by Sanger sequencing. The results for test group 1, test group 2 (ZmWUS2+ipt), test group 3 (AtCUC1), test group 5 (AtCyc2;1), test group 7 (AtE2F3+Dpa), and test group 11 (TON2) are shown in Table 3.

[0200] <result>

[0201] [Table 3]

[0202] Comparing the mutation rate with and without the introduction of cell-dividing activators (5.7%), the mutation rates increased when ZmWUS2+ipt, AtCUC1, AtCyc2;1, AtE2F3+Dpa, and TON2 were used (13.9%, 7.4%, 10.3%, 10.0%, and 8.8%, respectively). These rates were significantly higher than when other cell-dividing activators were introduced.

[0203] <Detection of genome editing in next-generation technologies> Individuals in which mutations were confirmed in the fifth leaf were grown, and the resulting next-generation seeds were sown. The germinated cotyledons were then detected using the same method as described above for detecting the fifth leaf. As a result, mutations were also detected in the next-generation individuals.

[0204] Example 9. Preparation of LbCpf1 expression vector An LbCpf1 expression vector was obtained by inserting an LbCpf1 protein expression cassette and a guide RNA expression cassette into a pTTK352 binary vector containing the sequence of a geminivirus vector (bean yellow dwarf virus (BeYDV)).

[0205] Regarding LbCpf1 expression vectors, (3) The LbCpf1 protein coding sequence (SEQ ID NO: 67) in which an epitope tag and nuclear localization signal have been added, (9) Direct repeat sequence for LbCpf1 (sequence number 68), An expression vector was prepared in the same manner as in Example 8, except for the use of [specific component].

[0206] Example Test 6. Genome Editing Test 6 Genome editing experiments were conducted in plants using the effector expression vector and silencing suppressor expression vector (pDGB3alpha2_35S:P19:Tnos, reference Sarrion-Perdigones A, Vazquez-Vilar M, Palaci J, Castelijns B, Forment J, Ziarsolo P, Blanca J, Granell A, Orzaez D. (2013) GoldenBraid 2.0: a comprehensive DNA assembly framework for plant synthetic biology. Plant Physiol. 162(3):1618-1631) from Example 9, and the mutation rate was analyzed. Specifically, the procedure was carried out in the same manner as in Example 4.

[0207] <result> CAPS analysis revealed that mutations were introduced using the genome editing tool LbCpf1, similar to the MAD7 method. Therefore, in addition to Cas9 and MAD7, LbCpf1 can also induce genome editing, making this vector applicable to a variety of genome editing tools.

Claims

1. A polynucleotide comprising a site-specific nuclease expression cassette and an insulator and / or scaffold / matrix binding region, wherein the site-specific nuclease expression cassette is (a) promoter, (b) Site-specific nuclease coding sequences, and (c) Terminator and other 3'UTR Includes, The insulator and / or the scaffold / matrix coupling region is located within the 3'UTR and downstream of the terminator. The promoter is located on the 5' side of the site-specific nuclease coding sequence. Polynucleotide.

2. The polynucleotide according to claim 1, wherein the insulator is at least one selected from the group consisting of Ars insulator, Gypsy insulator, Bead1 insulator, TEF insulator, and TEF2 insulator.

3. The polynucleotide according to claim 2, wherein the insulator is an Ars insulator.

4. The polynucleotide according to claim 1, wherein the terminator is a linkage of at least two terminators.

5. The polynucleotide according to claim 1, wherein the terminator is at least one selected from the group consisting of a 35S terminator, an ACT3 terminator, and an extensin terminator.

6. The polynucleotide according to claim 1, wherein the promoter is a shoot apical expression promoter.

7. The polynucleotide according to claim 1, wherein the promoter is a CmYLCV promoter, a UBQ promoter, an EF-1α promoter, or an RPS5A promoter.

8. The polynucleotide according to claim 1, comprising a viral vector sequence.

9. The polynucleotide according to claim 8, wherein the viral vector sequence is a geminivirus vector sequence.

10. The polynucleotide according to claim 8, wherein the viral vector sequence is a BeYDV sequence.

11. The insulator is an Ars insulator, The Terminator is an Extensin Terminator, The promoter is the CmYLCV promoter, The polynucleotide comprises a viral vector sequence, and The aforementioned viral vector sequence is a geminivirus vector sequence. The polynucleotide according to claim 1.

12. Furthermore, the polynucleotide according to claim 1, comprising a guide RNA expression cassette.

13. A plant genome editing vector comprising a polynucleotide according to any one of claims 1 to 12.

14. A plant genome editing kit comprising a polynucleotide according to any one of claims 1 to 12.

15. A method for editing a plant genome, comprising introducing a polynucleotide according to any one of claims 1 to 12 into a plant.

16. Furthermore, the plant genome editing method according to claim 15, further comprising introducing a cell-dividing activator to the polynucleotide introduction site.

17. A method for producing a genome-edited plant, comprising introducing a polynucleotide according to any one of claims 1 to 12 into the plant.

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