Plant genome editing technology that does not rely on genetic modification, using cell membrane-permeable peptides

Abstract

The present invention provides: a complex comprising a genome editing enzyme and a cell membrane-permeable peptide (CPP), wherein the CPP is fused to the genome editing enzyme; a complex comprising a genome editing enzyme, a target gene-specific nucleic acid , and a CPP, wherein the CPP is fused to the genome editing enzyme and / or the a target gene-specific nucleic acid; the complex comprising a polycationic moiety fused to the CPP, wherein the polycation moiety is statically bound to the target gene-specific nucleic acid; a genome editing method using the complex; and a kit for genome-editing, the kit including these complexes.

Description

Technical Field

[0001] The present invention relates to a genome editing technique using a novel complex. Specifically, the present invention relates to a technique for directly introducing a genome editing enzyme and a nucleic acid into a plant cell without relying on genetic recombination. This application claims priority to Japanese Patent Application No. 2020-211301, and the entire contents of Japanese Patent Application No. 2020-211301 are incorporated herein by reference.

Background Art

[0002] "Genome editing technology" is an innovative technology for modifying biological functions and is being used in a wide range of fields from basic research to applied research (such as medicine and crop breeding). However, conventional genome editing technologies for plants have a fatal problem of "requiring a great deal of time and complexity in operation." The biggest reason for this is that conventional plant genome editing technologies rely on genetic recombination technology. In conventional plant genome editing technologies, a genome editing cassette gene (a gene that synthesizes a nucleic acid specific to a genome editing enzyme and a target gene) is introduced into a plant cell by genetic recombination to induce genome editing, and then cells lacking the genome editing enzyme gene are selected to obtain genome editing cells. However, since a genome editing cassette gene cannot be introduced into plant species to which genetic recombination technology cannot be applied, currently, the plant species for which genome editing is possible are limited to a part. In addition, since the genome editing enzyme gene, which is a foreign gene, is introduced into a plant cell once, it is treated as a genetically modified organism unless this foreign gene is removed, and thus commercial use is extremely difficult as it is. The removal of foreign genes is possible only by repeating mating, and thus there is a problem that it takes a great deal of time to remove the genome editing cassette gene as described above.

[0003] To overcome the problems of conventional plant genome editing technologies as described above, there is a great deal of effort being put into developing genome editing technologies that do not rely on genetic recombination. Specifically, a method has been proposed to induce genome editing without genetic recombination by directly introducing genome editing enzymes in protein form into plant cells (Non-Patent Literature 1). However, because plant cells have cell walls that carry a strong negative charge, when attempting to introduce proteins into plant cells, basic proteins are trapped by the cell wall, while acidic proteins are repelled by the cell wall. It is possible to forcibly introduce proteins into plant cells using particle guns or electroporation, but this requires specialized and expensive dedicated equipment. To date, there is no general method for easily introducing proteins into plant cells that have cell walls. Therefore, there is also no method for easily introducing genome editing enzymes into plant cells, and this situation is a major factor that is significantly delaying the modification of plant functions (especially crop breeding) using genome editing technology. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Svitashev S. et al., Nat. Commun. 2016; 7: 13274. [Overview of the project] [Problems that the invention aims to solve]

[0005] There is a need for technology that enables simple plant genome editing across a wide range of plant species without the need for genetic modification. [Means for solving the problem]

[0006] The inventors of this invention have diligently conducted research to solve the above problems and have discovered that by using a complex containing a genome editing enzyme and a cell membrane permeable peptide (hereinafter sometimes referred to as "CPP"), and a complex containing a genome editing cassette (genome editing enzyme and nucleic acid specific to the target gene) and CPP, a genome editing cassette can be directly introduced into plant cells without requiring genetic recombination, thus completing the present invention.

[0007] In other words, the present invention provides the following: (1) A complex comprising a genome editing enzyme and CPP, wherein the CPP is fused to the genome editing enzyme. (2) A complex comprising a genome editing enzyme, a nucleic acid specific to a target gene, and a CPP, wherein the CPP is fused to the genome editing enzyme and / or the nucleic acid specific to the target gene. (3) The complex according to (1) or (2), wherein the CPP is covalently bound to a genome editing enzyme and / or nucleic acid specific to the target gene. (4) The complex described in (3), wherein CPP is covalently bound to a genome editing enzyme. (5) The complex described in (2), wherein the polycationic portion is fused to the CPP, and the polycationic portion is electrostatically bound to a nucleic acid specific to the target gene. (6) The composite described in (5), wherein the polycation portion is covalently bonded to the CPP. (7) The complex described in (6), wherein the polycation portion is a polycation peptide. (8) The complex according to (7), wherein the polycationic peptide comprises 10 or more lysine residues or 10 or more arginine residues. (9) A complex described in any of (1) to (8), wherein more than 80% of the amino acid residues of the CPP are histidine residues, and the length of the CPP is 8 amino acids to several tens of amino acids. (10) The complex described in (9), wherein all amino acid residues of CPP are histidine residues. (11) The complex according to any of (1) to (10), further comprising a signal sequence. (12) A complex described in any of (1) to (11), further including subdomains. (13) A genome editing method comprising introducing any of the complexes described in (1) to (12) into cells. (14) The method according to (13), wherein the cells are plant cells, algal cells, filamentous fungal cells, or yeast cells. (15) A genome editing kit comprising any of the complexes or components described in (1) to (12). (16) The kit described in (15) for genome editing of plants, algae, filamentous fungi, or yeast. [Effects of the Invention]

[0008] According to the present invention, genome editing enzymes and genome editing cassettes can be directly introduced into plant cells without requiring genetic modification. Therefore, genome editing can be performed even in plant species to which genetic modification technology cannot be applied. Genome editing enzymes and genome editing cassettes can be introduced into plant cells simply by incubating the complex of the present invention and plant cells in a culture medium. Furthermore, genome editing according to the present invention is highly efficient. In short, by using the complex of the present invention, genome editing can be performed simply and efficiently on a wide range of plant species without requiring genetic modification. Genome-edited plants obtained by the present invention do not possess foreign genes and do not fall under the category of genetically modified organisms, so they can be immediately put into commercial use and have extremely high commercial value.

[0009] "Directly introducing genome editing enzymes into plant cells" means introducing genome editing enzymes into plant cells as active proteins. "Directly introducing genome editing cassettes into plant cells" means introducing a complex of genome editing enzymes and nucleic acids specific to the target gene into plant cells, and this means introducing the genome editing enzyme and nucleic acid complex into plant cells as an active protein complex. [Brief explanation of the drawing]

[0010] [Figure 1]Figure 1 is a diagram illustrating a comparison between the genome editing method using the complex of the present invention and conventional methods. [Figure 2] Figure 2 shows the nucleotide sequence of the cedar magnesium chilatase gene (CjCHLI gene) used as the target gene in the example. In the figure, "gRNA1" and "gRNA2" indicate the recognition sites of gRNA1 and gRNA2, respectively. [Figure 3] Figure 3 shows the nucleotide sequence of the region recognized by gRNA1 in which genome editing (gene deletion) was confirmed in the example. The site where genome editing (gene deletion) occurred is indicated by "-". Samples shows the conditions under which the plant cells were treated, Reads shows the number of sequences for which nucleotide sequence analysis was performed and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency shows the genome editing efficiency. Underlined values ​​indicate conditions under which genome editing (gene deletion) was significantly confirmed. [Figure 4] Figure 4 shows the nucleotide sequences of regions recognized by gRNA2 in which genome editing (gene deletion) was confirmed in the examples. The sites where genome editing (gene deletion) occurred are indicated by "-". Samples shows the conditions under which the plant cells were treated, Reads shows the number of sequences for which nucleotide sequence analysis was performed and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency shows the genome editing efficiency. Underlined areas indicate conditions under which genome editing (gene deletion) was significantly confirmed. [Figure 5] Figure 5 shows the nucleotide sequences of regions recognized by gRNA2 in which genome editing (gene deletion) was confirmed in the examples. The sites where genome editing (gene deletion) occurred are indicated by "-". Samples shows the conditions under which the plant cells were treated, Reads shows the number of sequences for which nucleotide sequence analysis was performed and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency shows the genome editing efficiency. Underlined values ​​indicate conditions under which genome editing (gene deletion) was significantly confirmed. [Figure 6]Figure 6 shows the nucleotide sequences of regions recognized by gRNA2 in which genome editing (gene deletion) was confirmed in the examples. The sites where genome editing (gene deletion) occurred are indicated by "-". Samples shows the conditions under which the plant cells were treated, Reads shows the number of sequences for which nucleotide sequence analysis was performed and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency shows the genome editing efficiency. Underlined areas indicate conditions under which genome editing (gene deletion) was significantly confirmed. [Figure 7] Figure 7 shows the nucleotide sequences of regions recognized by gRNA2 in which genome editing (gene deletion) was confirmed in the examples. The sites where genome editing (gene deletion) occurred are indicated by "-". Samples shows the conditions under which the plant cells were treated, Reads shows the number of sequences for which nucleotide sequence analysis was performed and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency shows the genome editing efficiency. Underlined values ​​indicate conditions under which genome editing (gene deletion) was significantly confirmed. [Figure 8A] Figure 8A shows the nucleotide sequence of the rice E3 ubiquitin-protein ligase GW2 gene (OsGW2 gene), which was used as the target gene in the example. In the figure, "gRNA3" indicates the recognition site of gRNA3. [Figure 8B] Figure 8B is a continuation of Figure 8A and shows the base sequence of the rice E3 ubiquitin-protein ligase GW2 gene (OsGW2 gene) that was used as the target gene in the example. [Figure 9] Figure 9 shows the nucleotide sequences of regions recognized by gRNA3 in which genome editing (gene deletion) was confirmed in the examples. The sites where genome editing (gene deletion) occurred are indicated by "-". Samples shows the conditions under which the plant cells were treated, Reads shows the number of sequences for which nucleotide sequence analysis was performed and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency shows the genome editing efficiency. Underlined values ​​indicate conditions under which genome editing (gene deletion) was significantly confirmed. [Figure 10]Figure 10 shows the nucleotide sequence of the region recognized by gRNA3 in which genome editing (gene deletion) was confirmed in the examples. The site where genome editing (gene deletion) occurred is indicated by "-". Also, Samples indicates the conditions when plant cells were treated, Reads indicates the number of sequences subjected to nucleotide sequence analysis and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency indicates the genome editing efficiency. The underline indicates the conditions in which genome editing (gene deletion) was significantly confirmed. [Figure 11] Figure 11 shows the nucleotide sequence of the region recognized by gRNA3 in which genome editing (gene deletion) was confirmed in the examples. The site where genome editing (gene deletion) occurred is indicated by "-". Also, Samples indicates the conditions when plant cells were treated, Reads indicates the number of sequences subjected to nucleotide sequence analysis and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency indicates the genome editing efficiency. The underline indicates the conditions in which genome editing (gene deletion) was significantly confirmed. [Figure 12] Figure 12 shows the nucleotide sequence of the region recognized by gRNA3 in which genome editing (gene deletion) was confirmed in the examples. The site where genome editing (gene deletion) occurred is indicated by "-". Also, Samples indicates the conditions when plant cells were treated, Reads indicates the number of sequences subjected to nucleotide sequence analysis and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency indicates the genome editing efficiency. The underline indicates the conditions in which genome editing (gene deletion) was significantly confirmed. [Figure 13] Figure 13 shows the nucleotide sequence of the region recognized by gRNA3 in which genome editing (gene deletion) was confirmed in the examples. The site where genome editing (gene deletion) occurred is indicated by "-". Also, Samples indicates the conditions when plant cells were treated, Reads indicates the number of sequences subjected to nucleotide sequence analysis and the number of sequences in which genome editing (gene deletion) was confirmed, and Efficiency indicates the genome editing efficiency. The underline indicates the conditions in which genome editing (gene deletion) was significantly confirmed.

Mode for Carrying Out the Invention

[0011] In one embodiment, the present invention provides a complex comprising a genome editing enzyme and a CPP, wherein the CPP is fused to the genome editing enzyme. The fusion of CPP will be described later. Typical examples of genome editing enzymes in the complex of this embodiment are TALENs and ZFNs.

[0012] In a further embodiment, the present invention provides a complex comprising a genome editing enzyme, a nucleic acid specific to a target gene, and a CPP, wherein the CPP is fused to the genome editing enzyme and / or the nucleic acid specific to the target gene.

[0013] The fusion of CPPs may take any form, as long as it does not interfere with the introduction of the complex into plant cells and genome editing. The fusion may be by covalent bonds, such as peptide bonds, or by non-covalent bonds, such as electrostatic bonds or van der Waals forces. When CPPs covalently bond to genome editing enzymes, the covalent bond may take any form, but is typically a peptide bond.

[0014] The genome editing enzyme and CPP may be in any positional relationship. The CPP may be fused to the N-terminus of the genome editing enzyme, to the C-terminus of the genome editing enzyme, to both the N-terminus and C-terminus of the genome editing enzyme, or to amino acid residues other than the N-terminus and C-terminus of the genome editing enzyme. Preferably, the CPP is fused to the N-terminus or C-terminus of the genome editing enzyme. The fusion of the genome editing enzyme and CPP may be mediated by a linker. Various linkers are known and can be used. A preferred linker is one that does not hinder the introduction of the complex of the present invention into plant cells and genome editing. In the case of fusion via a peptide bond, examples of linkers include, but are not limited to, peptides consisting of one to several glycine residues. One CPP may be fused to one genome editing enzyme, or two or more CPPs may be fused.

[0015] CPP may be fused with a nucleic acid specific to the target gene. The fusion may be performed at the 3' end, the 5' end, or other parts, such as the sugar and / or base portion of the nucleic acid. Preferably, the fusion is performed at the 3' end of the nucleic acid. Known methods such as organic synthesis may be used for the fusion. One CPP may be fused to one nucleic acid, or two or more CPPs may be fused.

[0016] Various CPPs are known. The CPP used in the present invention may be any peptide, as long as it allows the complex of the present invention to be directly introduced into plant cells and does not interfere with genome editing. Examples of CPPs that can be used in the present invention include, but are not limited to, peptides rich in basic amino acids (e.g., arginine, lysine, histidine) or polyhistidines.

[0017] Further examples of CPPs that can be used in the present invention include peptides with a length of several amino acids or more, in which more than half of the constituent amino acids are histidine. Such peptides have excellent cell membrane permeability.

[0018] The length of the CPP that can be used in the present invention is not particularly limited, but is typically several amino acids or more, for example, several to several tens of amino acids. For example, it may be 6 to 40 amino acids, 7 to 30 amino acids, 8 to 20 amino acids, for example, 6 amino acids, 7 amino acids, 8 amino acids, 9 amino acids, 10 amino acids, 11 amino acids, 12 amino acids, 13 amino acids, 14 amino acids, 15 amino acids, 16 amino acids, 17 amino acids, 18 amino acids, 19 amino acids, 20 amino acids, 21 amino acids, 22 amino acids, 23 amino acids, 24 amino acids, 25 amino acids, 26 amino acids, 27 amino acids, 28 amino acids, 29 amino acids, 30 amino acids, or 30 amino acids or more. In this specification, several amino acids means 2, 3, 4, 5, 6, 7, 8 or 9 amino acids. In this specification, several tens of amino acids means any number of amino acids in the range of 10 to 100.

[0019] A preferred example of a CPP that can be used in the present invention is a peptide (polyhistidine) in which approximately 80% or more of the constituent amino acid residues are histidine residues, more preferably approximately 90% or more of the constituent amino acid residues are histidine residues, and even more preferably all of the constituent amino acid residues are histidine residues. The length of the polyhistidine is the same as the length of the CPP described above.

[0020] In the present invention, any amino acid residue other than histidine can constitute the CPP. Preferably, the amino acid residue other than histidine that can constitute the CPP in the present invention is a basic amino acid residue such as arginine or lysine, or an amino acid residue having properties similar to histidine. The amino acid residue that can constitute the CPP in the present invention may be a natural amino acid residue, a non-natural amino acid residue, a modified amino acid residue, or a synthetic amino acid residue. The synthesis and modification of amino acids can be carried out as appropriate by those skilled in the art.

[0021] The CPPs that can be used in the present invention can be prepared by known methods such as peptide synthesis methods including Fmoc solid-phase synthesis and genetic recombination.

[0022] By using peptides with high cell membrane permeability (such as those described above), the efficiency of introducing the complex of the present invention into plant cells can be increased, resulting in higher genome editing efficiency.

[0023] The genome editing enzyme used in the present invention may be any genome editing enzyme and is not particularly limited. Various genome editing enzymes are known. Examples of genome editing enzymes that can be used in the present invention include, but are not limited to, Cas family nucleases such as Cas9, Cas12, Cas13, Casφ, and TiD, nucleases such as TALEN and ZFN, and deaminases such as activated-induced cytidine deaminase (AID) and Target-G. In this specification, genome editing enzymes include wild-type and mutant types. Mutant genome editing enzymes include both naturally occurring and artificially induced mutants. Mutant genome editing enzymes may have increased, decreased, or absent editing efficiency compared to the original enzyme. Methods for producing mutant genome editing enzymes are known and include, but are not limited to, genetic recombination, peptide chemical synthesis, and chemical modification. Furthermore, genome editing enzymes may cleave single-stranded DNA or double-stranded DNA.

[0024] In a particular example of the present invention, the genome editing enzyme is Cas9. In a further particular example of the present invention, the genome editing enzyme is an AID bound to Cas9 whose cleavage activity has been regulated by functional modification.

[0025] If the genome editing enzyme is a TALEN or ZFN, these proteins are designed to match the target gene. Such design can be carried out by methods known to those skilled in the art.

[0026] A target gene-specific nucleic acid is a nucleic acid that can position a genome editing enzyme at the site in the target gene where mutation is desired. A typical example of a target gene-specific nucleic acid is guide RNA (gRNA), but it is not limited to this. Target gene-specific nucleic acids can be designed and created using known methods, taking into account the base sequence of the target gene. gRNA can be designed using known software such as CRISPRdirect, CRISPR-P2.0, Geneious, and ApE. Preferably, the target gene-specific nucleic acid is specific to the genome editing enzyme and forms a complex with the genome editing enzyme when incubated with it. Examples of such nucleic acids and genome editing enzymes include, but are not limited to, gRNA and Cas9.

[0027] The plants whose genomes are edited according to this invention include all types of plants. These plants include seed plants, ferns, and mosses. Seed plants include angiosperms and gymnosperms. Angiosperms include dicotyledons and monocotyledons. Dicotyledons include sympetalous and polypetalous flowers. Examples of sympetalous flowers include, but are not limited to, plants of the Asteraceae, Ericaceae, Lamiaceae, Solanaceae, Convolvulaceae, Pedagoaceae, Primulaceae, and Campanulaceae families. Examples of polypetalous flowers include, but are not limited to, plants of the Brassicaceae, Rosaceae, Theaceae, Caryophyllaceae, Portulacaceae, Myricaceae, Cucurbitaceae, Rutaceae, Apiaceae, Fabaceae, and Lauraceae families. Examples of monocots include, but are not limited to, plants of the Iridaceae, Poaceae, Juncaceae, Araceae, Zingiberaceae, Commelinaceae, Hypochnoraceae, Musaceae, Liliaceae, and Orchidaceae families. Examples of gymnosperms include, but are not limited to, plants of the Cupressaceae, Pinaceae, Cupressaceae, Taxaceae, Ginkgoaceae, and Cycadaceae families. Examples of ferns include, but are not limited to, Osmunda japonica, Dwarf Fern, Dryopteris, Horsetail, and Equisetum hyemale. Examples of bryophytes include, but are not limited to, Marchantia polymorpha, Polytrichum commune, luminescent moss, and Sphagnum moss.

[0028] The present invention may be used to introduce genome editing enzymes into various edible plants, horticultural plants, ornamental plants, trees for building materials, street trees, and windbreak trees, and perform genome editing. Examples of genome editing applications include, but are not limited to, plant breeding and genetic research.

[0029] The complex of the present invention may be used not only for plants, but also for genome editing of animals, microorganisms such as filamentous fungi, yeasts, bacteria and actinomycetes, and algae.

[0030] The above complex can be produced using known methods such as chemical synthesis or genetic recombination. For example, a fusion of the genome editing enzyme and CPP may be obtained by genetic recombination using a fusion of DNA encoding the genome editing enzyme and DNA encoding CPP, and the complex may be obtained by incubation of this fusion with nucleic acid specific to the target gene. Incubation is usually carried out in an aqueous solution at room temperature or about 37°C. The aqueous solution may be a buffer solution. If necessary, the complex may be purified using known means such as column chromatography.

[0031] An example of the complex of the present invention in which CPP is fused non-covalently is a complex in which a polycationic portion is fused to the CPP, and the polycationic portion is electrostatically bound to a nucleic acid specific to the target gene. In the above complex, the genome editing cassette and the CPP are fused via the polycationic portion.

[0032] The polycationic moiety is a portion having two or more positively charged groups under physiological conditions, which can electrostatically bind to nucleic acids specific to the target gene. Physiological conditions may include, for example, pH conditions under which plant cells can survive or proliferate, or pH conditions within plant cells.

[0033] Electrostatic bonding refers to the binding of negatively charged nucleic acids and positively charged polycationic moieties under physiological conditions due to electrostatic attraction.

[0034] The fusion of the polycation moiety and the CPP may be carried out in any manner, as long as it does not interfere with the introduction of the complex into plant cells and genome editing of the present invention. The fusion may be by means of covalent bonds, electrostatic bonds, or van der Waals forces, for example. Typically, the fusion of the polycation moiety and the CPP is by covalent bonds. A typical example of a covalent bond is a peptide bond. The polycation moiety and the CPP may be in any positional relationship. The polycation moiety may be bound to the N-terminus of the CPP, to the C-terminus of the CPP, to both the N-terminus and the C-terminus of the CPP, or to amino acid residues other than the N-terminus and C-terminus of the CPP. The binding of the polycation moiety and the CPP may be via a linker. Various linkers are known and can be used. A preferred linker is one that does not interfere with the introduction of the complex into plant cells and genome editing. In the case of fusion via a peptide bond, examples of linkers include, but are not limited to, peptides consisting of one to several glycine residues. A single CPP may have one polycation moiety fused to it, or it may have two or more polycation moieties fused to it. Furthermore, a single polycation moiety may have one CPP fused to it, or it may have two or more CPPs fused to it.

[0035] The polycationic moiety may be of any type, as long as it does not interfere with the introduction of the complex of the present invention into plant cells and genome editing. Examples of polycationic moieties include, but are not limited to, peptides that are positively charged under physiological conditions (preferably polycationic peptides), oligosaccharides, and cationic polymers. The peptides and oligosaccharides may be wild-type, mutant, or modified. Mutant peptides and mutant oligosaccharides, as well as their modifications, have electrostatic binding ability to target genes that is equivalent to or greater than that of the original peptides and oligosaccharides. The cationic polymer may be naturally derived or chemically synthesized.

[0036] Polycationic peptides are peptides having two or more amino acid residues that have a positive charge under physiological conditions, and such peptides are well known. Examples of polycationic peptides include, but are not limited to, peptides rich in basic amino acids (e.g., lysine, arginine, histidine). The length of a polycationic peptide is not particularly limited as long as it does not hinder the introduction of the complex into plant cells and genome editing, but is typically several to several tens of amino acids, for example, 6 to 40 amino acids, and may be 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more than 20 amino acids. Examples of polycationic peptides include peptides consisting of lysine and / or arginine residues. Specific examples of peptides consisting of lysine and / or arginine residues include K8, K9, K10, K11, K12, R8, R9, R10, R11, and R12. Further specific examples of polycationic peptides include peptides consisting of several KH repeat sequences. Polycationic peptides are not limited to the above examples. The amino acid residues constituting the polycationic peptides that can be used in the present invention may be natural amino acid residues, unnatural amino acid residues, modified amino acid residues, or synthetic amino acid residues. The synthesis and modification of amino acids can be carried out as appropriate by those skilled in the art.

[0037] Examples of positively charged oligosaccharides include, but are not limited to, polymers of hexosamines such as glucosamine, fructosamine, galactosamine, and mannosamine, as well as chitosan. The number of sugar residues in the positively charged oligosaccharide is not particularly limited, as long as it does not hinder the introduction of the complex of the present invention into plant cells and genome editing.

[0038] Examples of cationic polymers include, but are not limited to, polyethyleneimine, polypropyleneimine, poly(β-aminoester), polylactic acid / polyglycolic acid, and 2-hydroxypropyl methacrylamide. The length of the cationic polymer is not particularly limited, as long as it does not hinder the introduction of the complex of the present invention into plant cells and genome editing.

[0039] Polycationic peptides, positively charged oligosaccharides, and cationic polymers can be manufactured or extracted from nature by methods known to those skilled in the art.

[0040] The above complex can be produced using known methods. For example, the complex may be produced by (i) incubating a genome editing enzyme with nucleic acid specific to the target gene to obtain a complex (genome editing cassette), and (ii) incubating a fusion of a polycation moiety and CPP, thereby electrostatically bonding the negative charge of the nucleic acid with the positive charge of the polycation moiety. Incubation is usually carried out in an aqueous solution at room temperature or about 37°C. The aqueous solution may be a buffer solution. The fusion of the polycation moiety and CPP can be produced by known methods, such as peptide synthesis methods such as the Fmoc method or genetic recombination methods. If necessary, the complex may be purified using known means such as column chromatography.

[0041] Specific examples of polycationic peptide and CPP fusions include, but are not limited to, K10(G)H8, K10(G)H12, K10(G)H16, K10(G)H20, and R10(G)H20. The parentheses indicate that glycine residues may or may not be present.

[0042] The complex of the present invention may further include a signal sequence. The signal sequence is also called a signal peptide. By including a signal sequence in the complex of the present invention, the complex of the present invention can be localized to a desired intracellular compartment. Various types of signal sequences are known, and examples of signal sequences include, but are not limited to, nuclear localization signal sequences (NLS), mitochondrial localization signal sequences (MLS), and chloroplast localization signal sequences (CLS). A signal sequence can be selected according to the desired localization site within the cell and fused to the complex of the present invention. Using the complex of the present invention, which further includes a signal sequence, genome editing can be performed at a desired location within the cell. For example, using the complex of the present invention, which includes a nuclear localization signal, genome editing can be performed in the nucleus without using genetic recombination. Using the complex of the present invention, which includes a mitochondrial localization signal sequence, genome editing can be performed in the mitochondria without using genetic recombination. Using the complex of the present invention, which includes a chloroplast localization signal sequence, genome editing can be performed in the mitochondria without using genetic recombination.

[0043] The signal sequence may be fused to any part of the complex of the present invention, such as a genome editing enzyme, a nucleic acid specific to the target gene, or a subdomain (described later). The mode of fusion of the signal sequence may be any mode, as long as it localizes the complex to a desired location within the cell and does not hinder the introduction of the complex into plant cells and genome editing. The fusion may be by covalent bonds, such as peptide bonds, or by non-covalent bonds, such as electrostatic bonds or van der Waals forces. Typically, the signal sequence is fused to the complex by covalent bonds. When the signal sequence is covalently fused to a genome editing enzyme, the covalent bond may take any form, but is typically a peptide bond.

[0044] The genome editing enzyme and the signal sequence may be in any positional relationship. The signal sequence may be fused to the N-terminus of the genome editing enzyme, to the C-terminus, to both the N-terminus and C-terminus, or to an amino acid residue other than the N-terminus and C-terminus. Alternatively, the signal sequence may be inserted into the amino acid sequence of the genome editing enzyme. Preferably, the signal sequence is fused to the N-terminus or C-terminus of the genome editing enzyme. More preferably, the signal sequence is fused to the N-terminus of the genome editing enzyme. The fusion of the genome editing enzyme and the signal sequence may be mediated by a linker. Various linkers are known and can be used. A preferred linker is one that localizes the complex to a desired location within the cell and does not hinder the introduction of the complex into plant cells and genome editing. In the case of fusion via a peptide bond, examples of linkers include, but are not limited to, peptides consisting of one to several glycine residues. One signal sequence may be fused to one genome editing enzyme, or two or more signal sequences may be fused.

[0045] The signal sequence may be fused with a subdomain. The fusion may be performed at the N-terminus, the C-terminus, or at an amino acid residue other than the N-terminus or C-terminus. Known methods such as genetic recombination or organic synthesis may be used for the fusion. The fusion of the signal sequence and the subdomain may be mediated by a linker. One signal sequence may be fused to one subdomain, or two or more signal sequences may be fused to one subdomain.

[0046] The signal sequence may be fused with a nucleic acid specific to the target gene. The fusion may occur at the 3' end, the 5' end, or at other parts, such as the sugar and / or base portions of the nucleic acid. Preferably, the fusion occurs at the 3' end of the nucleic acid. Known methods, such as organic synthesis, may be used for the fusion. The fusion of the signal sequence and the nucleic acid may be mediated by a linker. One signal sequence may be fused to one nucleic acid, or two or more signal sequences may be fused to one nucleic acid.

[0047] The complex of the present invention may further include subdomains. In this specification, a subdomain means a functional protein. By using such a complex of the present invention, various desired types of genome editing can be performed. The type of subdomain is not particularly limited, but examples include base substitution enzymes, DNA methyltransferases, DNA demethyltransferases, transcription activators, transcription repressors, etc. Those skilled in the art can appropriately select subdomains and use them in the complex of the present invention. For example, by using the complex of the present invention which includes a base substitution enzyme as a subdomain, genome base substitution can be performed without using genetic recombination. By using the complex of the present invention which includes a DNA methyltransferase as a subdomain, genome methylation can be performed without using genetic recombination. By using the complex of the present invention which includes a DNA demethyltransferase as a subdomain, genome demethylation can be performed without using genetic recombination. By using the complex of the present invention which includes a transcription activator as a subdomain, genome transcriptional activation can be performed without using genetic recombination. By using the complex of the present invention which includes a transcription repressor as a subdomain, genome transcriptional repression can be performed without using genetic recombination.

[0048] Both the signal sequence and the subdomain may be included in the complex of the present invention. By using such a complex of the present invention, a desired type of genome editing can be performed in a desired intracellular compartment. For example, base substitution of the nuclear genome can be performed without genetic recombination using the complex of the present invention comprising a nuclear localization signal sequence and a base substitution enzyme. Nuclear genome methylation can be performed without genetic recombination using the complex of the present invention comprising a nuclear localization signal sequence and a DNA methyltransferase. Nuclear genome demethylation can be performed without genetic recombination using the complex of the present invention comprising a nuclear localization signal sequence and a DNA demethylase. Transcriptional activation of the nuclear genome can be performed without genetic recombination using the complex of the present invention comprising a nuclear localization signal and a transcription activator. Mitochondrial genome base substitution can be performed without genetic recombination using the complex of the present invention comprising a mitochondrial localization signal sequence and a base substitution enzyme. Transcriptional repression of the chloroplast genome can be performed without genetic recombination using the complex of the present invention comprising a chloroplast localization signal sequence and a transcription repressor.

[0049] The subdomain may be fused to any part of the complex of the present invention. Typically, the subdomain is fused to the genome editing enzyme. The subdomain may be fused to the N-terminus of the genome editing enzyme, to the C-terminus of the genome editing enzyme, to both the N-terminus and the C-terminus of the genome editing enzyme, or to an amino acid residue other than the N-terminus and C-terminus of the genome editing enzyme. Preferably, the subdomain is fused to the N-terminus or the C-terminus of the genome editing enzyme.

[0050] The mode of fusion between the genome editing enzyme and the subdomain may be any mode, as long as it does not impede the function of the subdomain and does not hinder the introduction of the complex into plant cells and genome editing. The fusion may be by covalent bonds, such as peptide bonds, or by non-covalent bonds, such as electrostatic bonds or van der Waals forces. Typically, the signal sequence is fused to the complex by covalent bonds. When the signal sequence is covalently bonded to the genome editing enzyme, the covalent bond may take any form, but it is typically a peptide bond.

[0051] Fusion of a genome editing enzyme with a subdomain may be mediated by a linker. Various linkers are known and can be used. Preferred linkers do not interfere with the function of the subdomain and do not hinder the introduction of the complex into plant cells or genome editing. In the case of fusion via peptide bonds, examples of linkers include, but are not limited to, peptides consisting of one to several glycine residues. A single genome editing enzyme may be fused with one subdomain, or with two or more subdomains.

[0052] CPP may be fused to the subdomain. The fusion mode and fusion region between the subdomain and CPP are described in the explanation regarding the fusion of genome editing enzymes and CPP.

[0053] In the complex of the present invention that includes a subdomain, if the CPP is fused to the subdomain and the subdomain is fused to a genome editing enzyme, then the CPP can be understood as being fused to the genome editing enzyme via the subdomain. Accordingly, in this specification, "CPP is fused to a genome editing enzyme" includes the above-described case.

[0054] The above description relates to introducing the complex of the present invention into plant cells. However, since the complex of the present invention has high permeability to cells of all species, it can be introduced not only into plants but also into the cells of animals, filamentous fungi, bacteria, actinomycetes, yeasts, algae, and other organisms, making it useful for genome editing in a wide range of species. In particular, since the complex of the present invention has high permeability to plant cells, algal cells, filamentous fungal cells, and yeast cells that have cell walls, it is suitable for genome editing in these species.

[0055] Accordingly, in a further embodiment, the present invention provides a genome editing method comprising introducing the complex of the present invention into cells. The complex of the present invention may be introduced into cells by incubation of the complex and cells in a culture medium. The method of introducing the complex of the present invention into cells, the type of culture medium, and the incubation conditions can be appropriately selected and modified by those skilled in the art depending on the type of cell. In this embodiment of the genome editing method, the cells are typically plant cells.

[0056] When performing genome editing of plants, the complex of the present invention can be introduced into any form of plant cell or any plant tissue. For example, the complex of the present invention can be introduced into plant leaves, stems, shoot apex, winter buds, roots, seeds, spores, pollen, cultured cells, etc. In this specification, plant leaves, stems, shoot apex, winter buds, roots, seeds, spores, pollen, cultured cells, etc., may be collectively referred to as plant cells.

[0057] In the genome editing method of the present invention, nucleic acids specific to one or more target genes may be introduced. Alternatively, one or more genome editing enzymes may be introduced. That is, one type of complex of the present invention may be used for genome editing, or two or more types of complexes of the present invention may be used.

[0058] In a further embodiment, the present invention provides a genome editing kit comprising the complex of the present invention or its components. Examples of components of the complex of the present invention include CPP, polycationic moieties, fusion products of genome editing enzymes and CPP, and fusion products of polycationic moieties and CPP. The kit may be designed so that the components of the complex of the present invention can be combined to obtain the complex of the present invention. Typically, the kit is accompanied by instructions for use. The species of organisms that can be genome edited using the kit of the present invention are not particularly limited as described above. The kit of the present invention is suitably used for genome editing of plants, algae, filamentous fungi, and yeasts that have cell walls.

[0059] In the following, genome editing methods using the complex of the present invention (recombinant protein method and peptide method) and conventional genome editing methods will be compared and explained with reference to Figure 1. The following explanation is intended to provide a clear and easy-to-understand description of the present invention and does not limit its scope.

[0060] Conventional genome editing technologies have the following problems: Because this technology relies on genetic engineering, genome editing is only possible for plant species to which genetic engineering is applicable. Since genome-edited plants will contain foreign genes, they will be treated as genetically modified organisms, making commercial use extremely difficult. Removing foreign genes is only possible through repeated crossbreeding, while removing genome editing cassettes requires a considerable amount of time.

[0061] This invention relates to a complex obtained by fusing a genome editing cassette (e.g., genome editing enzyme Cas9 and gRNA) with CPP (e.g., H8-H20 peptides), and to a method of genome editing in plants by directly introducing the complex into plant cells. CPP fusion can be performed by covalent or non-covalent bonding (e.g., electrostatic bonding). Specific examples of fusion methods include a method of preparing and using a recombinant protein obtained by genetically engineering the fusion of genome editing enzyme Cas9 and CPP (recombinant protein method), and a method of electrostatically binding CPP to a genome editing cassette (peptide method). The complex of this invention can be prepared using these methods.

[0062] According to the recombinant protein method, for example, a Cas9-CPP recombinant fusion protein (e.g., a fusion protein of Cas9 and H8-H2O CPP) can be prepared using an E. coli expression system, incubated with gRNA for cleaving the target region to form a complex, and the resulting complex can then be introduced into plant cells to perform genome editing.

[0063] According to the peptide method, for example, a complex is formed by incubation of the genome editing enzyme Cas9 and gRNA, and a fusion of the polycation moiety and CPP (for example, a fusion of K10, H8-H20, and CPP) is electrostatically bound to the nucleic acid of the complex (utilizing the negative charge of the gRNA bases and the positive charge of the lysine residues of the polycation moiety). The resulting complex can then be introduced into plant cells to perform genome editing.

[0064] Both methods directly introduce genome editing enzyme cassettes into plant cells without relying on genetic recombination, thus overcoming all the problems of conventional genome editing technologies and allowing for the acquisition of genome-edited cells simply and quickly.

[0065] Unless otherwise specified, terms used herein shall be understood in the sense commonly understood in the fields of biology, biochemistry, chemistry, pharmacology, medicine, and other related fields.

[0066] Numerical values ​​in this specification may include values ​​within a range of ±5%, ±10%, or ±20% of that value.

[0067] In this specification, amino acids are denoted using either the known one-letter or three-letter notation. In this specification, when a peptide is represented, a number is added to the right of the amino acid shown using the one-letter notation. For example, H20 means a peptide consisting of 20 histidine residues. K10 means a peptide consisting of 10 lysine residues. K10H20 means a peptide in which the N-terminus of a peptide consisting of 20 histidine residues is bonded to the C-terminus of a peptide consisting of 10 lysine residues. K10GH20 means a peptide in which a peptide consisting of 10 lysine residues, one glycine residue, and a peptide consisting of 20 histidine residues are bonded from the N-terminus to the C-terminus. In this specification, peptides may contain bonds other than peptide bonds. Unless otherwise specified, bonds between amino acid residues in a peptide are peptide bonds.

[0068] In this specification, fusions of genome editing enzymes and CPPs are represented by a hyphen (-). For example, Cas9-H20 refers to a fusion in which CPP(H20) is attached to the C-terminus of the genome editing enzyme Cas9. Unless otherwise specified, the bond between the genome editing enzyme and the CPP is a peptide bond.

[0069] The present invention will be described in more detail and specifically below with reference to examples, but these examples are not intended to limit the scope of the present invention. [Examples]

[0070] 1) Test cells BL21(DE3) strain of Escherichia coli was used to express Cas9 and CPP-fused Cas9. As plant cells, callus and callus-derived cells of the tree plant Japanese cedar (Cryptomeria japonica) and cultured cells of the herbaceous plant rice (Oryza sativa) were used. Japanese cedar callus was subcultured in 1 / 2 MD agar medium at one-week intervals. Japanese cedar callus-derived cells were suspended and tested in 1 / 2 MD liquid medium, cultured in the dark at 25°C with shaking at 120 rpm. Rice cultured cells were subcultured in MS liquid medium at one-week intervals. Rice cultured cells were suspended and tested in MS liquid medium, cultured in the dark at 27°C with shaking at 120 rpm.

[0071] 2) Expression of CPP-fused Cas9 Recombinant proteins Cas9-H8, Cas9-H12, Cas9-H16, and Cas9-H20 were prepared by fusing the cell membrane permeable peptides (CPPs) H8, H12, H16, and H20 peptides, respectively. The expression plasmid pET24b was used, and the host E. coli strain BL21(DE3) was employed. The recombinant proteins were expressed intracellularly at 20°C for 18 hours, and the cell lysates were purified using a cobalt ion-immobilized resin (GE Healthcare).

[0072] 3) Cas9 expression Instead of CPP, a recombinant Cas9 protein was prepared by fusing it with the FLAG tag (DYKDDDDK), a peptide tag that does not have cell membrane permeability. The expression plasmid pET24b was used, and the host E. coli strain BL21(DE3) was used. The recombinant protein was expressed intracellularly at 20°C for 18 hours, and the cell lysate was purified using anti-FLAG antibody immobilization resin (MBL).

[0073] 4) Preparation of gRNA+CPP fusion Cas9 complex (Preparation of complex used in recombinant protein synthesis) A 10 μM gRNA+CPP-fused Cas9 complex was prepared by mixing equal volumes of CPP-fused Cas9 (Cas9-H8, Cas9-H12, Cas9-H16, Cas9-H20) (20 μM) dissolved in SEC Buffer (20 mM HEPES-KOH, 500 mM KCl, pH 7.5) and gRNA (20 μM) dissolved in Duplex Buffer (30 mM HEPES-KOH, 100 mM potassium acetate, pH 7.5), and incubating at room temperature for 15 minutes. A 10 μM gRNA+Cas9 complex was prepared using the same method. Three types of gRNA (gRNA1, gRNA2, gRNA3) were used (the same was used in the following experiments). These gRNAs target specific regions of the cedar magnesium chilatase gene (whose nucleotide sequence is shown in SEQ ID NO: 1): CjCHLI gene (indicated as gRNA1 and gRNA2 in Figure 2) and specific regions of the rice E3 ubiquitin-protein ligase GW2 gene (whose nucleotide sequence is shown in SEQ ID NO: 2): OsGW2 gene (indicated as gRNA3 in Figure 8). The gRNA1 and gRNA2 regions are represented by nucleotides 56-78 and 1094-1116, respectively, in SEQ ID NO: 1. The gRNA3 region is represented by nucleotides 1796-1818, in SEQ ID NO: 2. gRNA1 and gRNA2 were designed using known software (ApE), and gRNA3 was designed using known software (CRISPRdirect and CRISPR-P2.0).

[0074] 5) Preparation of the gRNA+Cas9+CPP complex (Preparation of the complex used in the peptide method) A gRNA+Cas9 complex (10 μM) was prepared by mixing equal volumes of Cas9 (20 μM) dissolved in SEC Buffer (20 mM HEPES-KOH, 500 mM KCl, pH 7.5) and gRNA (20 μM) dissolved in Duplex Buffer (30 mM HEPES-KOH, 100 mM potassium acetate, pH 7.5) and incubating at room temperature for 15 minutes. Next, an equivalent volume of K10G-CPP (K10GH8, K10H12, K10H16, K10H20 peptides) (20, 200, 2000 μM) dissolved in Duplex Buffer (30 mM HEPES-KOH, 100 mM potassium acetate, pH 7.5) with the gRNA+Cas9 complex and incubating at room temperature for 60 minutes. The gRNAs used were gRNA1, gRNA2, and gRNA3 as described above.

[0075] 6) Genome editing trials 360 μL of cedar cells (20 mg / mL in 1 / 2 MD liquid medium) or rice cells (20 mg / mL in MS liquid medium) that had been passaged for one week were dispensed into 5 mL polystyrene Falcon round tubes. To this 360 μL of cedar cells or rice cells, 40 μL of the gRNA+CPP fusion Cas9 complex or gRNA+Cas9+CPP complex prepared above was mixed. Subsequently, the cells were cultured in the dark at 25°C and 120 rpm for 24 to 72 hours with shaking. Under the above experimental conditions, it was confirmed that the fluorescently modified gRNA+CPP fusion Cas9 complex or gRNA+Cas9+CPP complex was taken up into the cedar cells and rice cells.

[0076] Japanese cedar or rice cells were harvested by centrifugation (500g x 10min, 4°C) after culturing and washed multiple times with 1 / 2 MD liquid medium. Genomic DNA was extracted from the Japanese cedar or rice cells using a plant cell genome DNA extraction kit (DNA Suisui P: manufactured by Rizo Co., Ltd.), and the gene region targeted for genome editing was amplified by PCR. The presence or absence of genome editing was evaluated by amplicon sequencing analysis of the PCR product. In addition, the presence or absence of genome editing was evaluated by cloning the PCR product and subjecting it to Sanger sequencing analysis.

[0077] 7) Results Genome editing (gene deletions) that were not detected in cells treated with gRNA alone or the gRNA+Cas9 complex were confirmed in cedar cells treated with the recombinant protein method (gRNA+CPP fusion Cas9 complex) or the peptide method (gRNA+Cas9+CPP complex). On the other hand, genome editing (gene deletions) that were not detected in cells treated with the recombinant protein method (gRNA+CPP fusion Cas9 complex) were confirmed in rice cells treated with the recombinant protein method (gRNA+CPP fusion Cas9 complex) were confirmed.

[0078] Using two types of gRNA (gRNA1 or gRNA2) corresponding to the target gene (CjCHLI gene), four types of genome editing (gene deletion) were confirmed using the recombinant protein method (gRNA+CPP fusion Cas9 complex) (Figures 3, 4, and 7). On the other hand, six types of genome editing (gene deletion) were confirmed using the peptide method (gRNA+Cas9+CPP complex) (Figures 3, 4, 5, 6, and 7). Furthermore, genome editing (gene deletion) was confirmed using four types of CPP fusion Cas9 (Cas9-H8, Cas9-H12, Cas9-H16, and Cas9-H20) with the recombinant protein method (gRNA+CPP fusion Cas9 complex). In the peptide method (gRNA+Cas9+CPP complex), genome editing (gene deletion) was confirmed when using four types of K10G-CPP (K10GH8, K10GH12, K10GH16, and K10GH20 peptides).

[0079] Using gRNA3 corresponding to the target gene (OsGW2 gene), five types of genome editing (gene deletion) were confirmed using the recombinant protein method (gRNA+CPP fusion Cas9 complex) (Figures 8, 9, 10, 11, and 12). Genome editing (gene deletion) was confirmed using three types of CPP fusion Cas9 (Cas9-H8, Cas9-H16, and Cas9-H20) with the recombinant protein method (gRNA+CPP fusion Cas9 complex).

[0080] The region where genome editing (gene deletion) was confirmed coincided with the recognition region of gRNA, confirming that genome editing could be induced in the targeted region.

[0081] These results demonstrate the effectiveness of genome editing using the complex of the present invention. Specifically, the complex of the present invention demonstrated the introduction of genome editing enzymes, which are difficult to directly introduce into plant cells, and the induction of genome editing.

[0082] By utilizing this invention, crop breeding that does not rely on genetic modification becomes possible. Since the crops obtained by this invention do not fall under the category of genetically modified organisms, they have high commercial value. In particular, the Japanese cedar used in the examples of this invention is a crop with an extremely long generation time, and if conventional genome editing technology that relies on genetic modification were to be used, it would take decades to remove the foreign gene (genome editing enzyme Cas9 gene) through crossbreeding, making breeding impractical. This invention is expected to be extremely powerful even in genome editing (molecular breeding using genome editing, for example, breeding) of crops with such long generation times. [Industrial applicability]

[0083] This invention can be used in fields such as agriculture, forestry, food, pharmaceuticals, and plant research, breeding, and variety improvement.

Claims

1. A complex comprising a genome editing enzyme and a cell membrane permeable peptide (CPP), wherein the CPP is fused to the genome editing enzyme, and the CPP is a polyhistidine consisting of 12 to 30 histidine residues, for use in genome editing of plant cells, algal cells, filamentous fungal cells, or yeast cells.

2. A complex comprising a genome editing enzyme, a nucleic acid specific to a target gene, and a CPP, which is used for genome editing of plant cells, algal cells, filamentous fungal cells, or yeast cells, CPP is a polyhistidine consisting of 12 to 30 histidine residues. The N-terminus of the CPP is fused to a polycationic moiety which is a polycationic peptide consisting of lysine and / or arginine residues or a KH repeat sequence of 10 to 40 amino acids in length, and the polycationic moiety is electrostatically bound to the nucleic acid specific to the target gene, thereby fusing the CPP to the nucleic acid specific to the target gene. A complex.

3. The complex according to claim 1, wherein CPP is covalently bound to a genome editing enzyme.

4. The composite according to claim 2, wherein the polycation portion is covalently bonded to the CPP.

5. The complex according to any one of claims 1 to 4, wherein CPP is a polyhistidine consisting of 16 to 25 histidine residues.

6. The complex according to any one of claims 1 to 5, further comprising a signal sequence.

7. A genome editing method comprising introducing a complex according to any one of claims 1 to 6 into plant cells, algal cells, filamentous fungal cells, or yeast cells.

8. A genome editing kit comprising a complex according to any one of claims 1 to 6, or a fusion of the CPP and the polycation moiety which are components of the complex according to claim 2, the genome editing enzyme, and a nucleic acid specific to the target gene, the genome editing kit comprising instructions for use with the genome editing kit.

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