How to edit a genome

Abstract

The present invention provides: a single-strand form polynucleotide for promoting editing and to be combined with a single-strand form polynucleotide for editing and capable of modifying a target site in double-strand genomic DNA; a genome editing kit that includes said polynucleotide for editing or an expression vector therefor, and said polynucleotide for promoting editing or an expression vector therefor; a method for modifying a target site in double-strand genomic DNA of a cell or a non-human organism, the method comprising a step for treating the cell or the organism using the genome editing kit; and a method for producing a cell or an organism in which a target site in double-strand genomic DNA is modified, the method comprising said step.

Description

[Technical field]

[0001] The present invention relates to a method for editing a genome. [Background technology]

[0002] In recent years, genome editing tools such as zinc finger nucleases (ZFN), TALEN, and CRISPR / Cas systems have been developed, and genome editing using these is being actively carried out around the world. While ZFN and TALEN have the disadvantage of being time-consuming to design sequence-specific nucleases, the CRISPR / Cas system is particularly adopted as a genome editing tool around the world because targeting is extremely easy. However, the difficulty of the CRISPR / Cas system is that it can be problematic in off-target situations and it is not easy to deliver large nucleases to cells. These genome editing techniques use endonucleases, which are enzymes that cut DNA, to edit the genome, so the size and design of the nuclease is necessary.

[0003] In response to this, a method for genome editing using only single-stranded polynucleotides has been developed (Patent Documents 1 and 2). In the methods of Patent Documents 1 and 2, genome editing can be performed using only single-stranded polynucleotides without introducing components other than single-stranded polynucleotides into cells. In the methods of Patent Documents 1 and 2, genome editing can be performed not only in prokaryotic cells but also in eukaryotic cells. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 6899564 [Patent Document 2] JP 2016-220639 A Summary of the Invention

[0005] The present invention provides methods for editing a genome.The present invention provides methods for editing a genome in a cell, particularly a eukaryotic cell.

[0006] While conducting various studies on formulations for increasing the editing efficiency of single-stranded polynucleotides, the inventors surprisingly found that the editing efficiency is improved in the presence of a specific single-stranded antisense polynucleotide, thereby arriving at the present invention. That is, the present inventors synthesized a single-stranded antisense polynucleotide (also referred to as an "editing-promoting antisense polynucleotide") and tested the genome editing efficiency of a single-stranded polynucleotide for editing (also referred to as an "editing sense polynucleotide") in the presence of the editing-promoting antisense polynucleotide. When the editing-promoting antisense polynucleotide was designed to have a sequence complementary to or complementary to the end portion of the editing sense polynucleotide (i.e., when designed to bind to the end of the editing sense polynucleotide), the efficiency of genome editing was found to be increased. Without being bound by theory, when the genome region to be transcribed into RNA is the subject of editing, competition may occur between the transcribed RNA present in the vicinity of the genome DNA and the editing sense polynucleotide for binding to the genome DNA, and therefore the improvement in genome editing efficiency is believed to be due to the suppression of the competition as a result of the editing-promoting antisense polynucleotide binding to the RNA. In genome editing technology, there has never been a technical idea of ​​increasing editing efficiency by suppressing such competition for binding to genome DNA, and the present invention was made based on this technical idea.

[0007] The present disclosure provides the following: [Section 1A] An editing-facilitating polynucleotide in single-stranded form for use in combination with an editing polynucleotide in single-stranded form capable of modifying a target site in double-stranded genomic DNA, The target site comprises a primary editing site set on one genomic DNA strand and a secondary editing site on the other genomic DNA strand at a position corresponding to the primary editing site; The polynucleotide for editing is, in order from the 5' end, a) A portion capable of hybridizing to the region adjacent to the 3' end of the primary editing site x) any portion consisting of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site; and b) A portion capable of hybridizing to the region adjacent to the 5' end of the primary editing site or Part a) and part b) It consists of: The editing-facilitating polynucleotide is capable of hybridizing to a nucleic acid sequence that is complementary to a predetermined region on the one genomic DNA strand, wherein the predetermined region overlaps with region A at its 5' end or overlaps with region B at its 3' end, where region A is a region on the one genomic DNA strand that is located at a position corresponding to portion a) of the editing polynucleotide and region B is a region on the one genomic DNA strand that is located at a position corresponding to portion b) of the editing polynucleotide; the length of the overlap with region A at the 5' end of the predetermined region is the same as or shorter than region A, and the length of the overlap with region B at the 3' end of the predetermined region is the same as or shorter than region B; When the editing polynucleotide is composed of part a), part x) and part b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a part consisting of a base sequence complementary to part x) can be inserted into the primary editing site, and part x) can be inserted into the secondary editing site; When the editing polynucleotide is composed of part a), part x) and part b), and the primary editing site is set to one nucleotide or multiple consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a part consisting of a base sequence complementary to part x), and the secondary editing site can be replaced with part x), The editing-promoting polynucleotide, which is capable of deleting a target site when the editing polynucleotide consists of part a) and part b) and the primary editing site is set at one nucleotide or multiple consecutive nucleotides on one genomic DNA strand. [Section 1B] An editing-facilitating polynucleotide in single-stranded form for use in combination with an editing polynucleotide in single-stranded form capable of modifying a target site in double-stranded genomic DNA, The target site comprises a primary editing site set on one genomic DNA strand and a secondary editing site on the other genomic DNA strand at a position corresponding to the primary editing site; The polynucleotide for editing is, in order from the 5' end, a) A portion consisting of a nucleotide sequence that has 90% or more identity with the nucleotide sequence of the region adjacent to the 5' end of the secondary editing site x) any portion consisting of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site; and b) A portion consisting of a base sequence having 90% or more identity with the base sequence of the region adjacent to the 3' end of the secondary editing site or Part a) and part b) It consists of: the editing-facilitating polynucleotide is composed of a base sequence having 90% or more identity with the base sequence of a predetermined region on said one genomic DNA strand, wherein said predetermined region overlaps with region A at its 5'-end or overlaps with region B at its 3'-end, when a region on said one genomic DNA strand located at a position corresponding to portion a) of the editing polynucleotide is designated as region A and a region on said one genomic DNA strand located at a position corresponding to portion b) of the editing polynucleotide is designated as region B; the length of the overlap with region A at the 5' end of the predetermined region is the same as or shorter than region A, and the length of the overlap with region B at the 3' end of the predetermined region is the same as or shorter than region B; When the editing polynucleotide is composed of part a), part x) and part b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a part consisting of a base sequence complementary to part x) can be inserted into the primary editing site, and part x) can be inserted into the secondary editing site; When the editing polynucleotide is composed of part a), part x) and part b), and the primary editing site is set to one nucleotide or multiple consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a part consisting of a base sequence complementary to part x), and the secondary editing site can be replaced with part x), The editing-promoting polynucleotide, which is capable of deleting a target site when the editing polynucleotide consists of part a) and part b) and the primary editing site is set at one nucleotide or multiple consecutive nucleotides on one genomic DNA strand. [Section 1C] An editing-facilitating polynucleotide in single-stranded form for use in combination with an editing polynucleotide in single-stranded form capable of modifying a target site in double-stranded genomic DNA, The target site comprises a primary editing site set on one genomic DNA strand and a secondary editing site on the other genomic DNA strand at a position corresponding to the primary editing site; The polynucleotide for editing is, in order from the 5' end, a) A portion consisting of a nucleotide sequence identical to the nucleotide sequence of the region adjacent to the 5' end of the secondary editing site x) any portion consisting of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site; and b) A portion consisting of a nucleotide sequence identical to the nucleotide sequence of the region adjacent to the 3' end of the secondary editing site or Part a) and part b) It consists of: the editing-facilitating polynucleotide has a base sequence identical to that of a predetermined region on said one genomic DNA strand, wherein said predetermined region overlaps with region A at its 5'-end or overlaps with region B at its 3'-end, when a region on said one genomic DNA strand located at a position corresponding to portion a) of the editing polynucleotide is designated as region A and a region on said one genomic DNA strand located at a position corresponding to portion b) of the editing polynucleotide is designated as region B; the length of the overlap with region A at the 5' end of the predetermined region is the same as or shorter than region A, and the length of the overlap with region B at the 3' end of the predetermined region is the same as or shorter than region B; When the editing polynucleotide is composed of part a), part x) and part b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a part consisting of a base sequence complementary to part x) can be inserted into the primary editing site, and part x) can be inserted into the secondary editing site; When the editing polynucleotide is composed of part a), part x) and part b), and the primary editing site is set to one nucleotide or multiple consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a part consisting of a base sequence complementary to part x), and the secondary editing site can be replaced with part x), The editing-promoting polynucleotide, which is capable of deleting a target site when the editing polynucleotide consists of part a) and part b) and the primary editing site is set at one nucleotide or multiple consecutive nucleotides on one genomic DNA strand. [Section 2A] The editing-promoting polynucleotide according to Item 1A, Item 1B, or Item 1C, wherein the length of the editing-promoting polynucleotide is 50 nucleotides or more. [Section 2B] The editing-promoting polynucleotide according to Item 1A, Item 1B, Item 1C, or Item 2A, wherein the length of the overlap is 5 nucleotides or more. [Section 3A] The editing-promoting polynucleotide according to Item 1A, Item 2A, or Item 2B, its expression vector, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance; and Editing polynucleotide, its expression vector, or conjugate of said editing polynucleotide or its expression vector with other substance A genome editing kit comprising: The polynucleotide for editing is, in order from the 5' end, a) A portion capable of hybridizing to the region adjacent to the 3' end of the primary editing site x) any portion consisting of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site; and b) A portion capable of hybridizing to the region adjacent to the 5' end of the primary editing site or Part a) and part b) The genome editing kit comprises: [Section 3B] The editing-promoting polynucleotide according to Item 1B, Item 2A, or Item 2B, its expression vector, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance; and Editing polynucleotide, its expression vector, or conjugate of said editing polynucleotide or its expression vector with other substance A genome editing kit comprising: The polynucleotide for editing is, in order from the 5' end, a) A portion consisting of a nucleotide sequence that has 90% or more identity with the nucleotide sequence of the region adjacent to the 5' end of the secondary editing site x) any portion consisting of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site; and b) A portion consisting of a base sequence having 90% or more identity with the base sequence of the region adjacent to the 3' end of the secondary editing site or Part a) and part b) The genome editing kit comprises: [Section 3C] The editing-promoting polynucleotide according to Item 1C, Item 2A, or Item 2B, its expression vector, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance; and Editing polynucleotide, its expression vector, or conjugate of said editing polynucleotide or its expression vector with other substance A genome editing kit comprising: The polynucleotide for editing is, in order from the 5' end, a) A portion consisting of a nucleotide sequence identical to the nucleotide sequence of the region adjacent to the 5' end of the secondary editing site x) any portion consisting of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site; and b) A portion consisting of a nucleotide sequence identical to the nucleotide sequence of the region adjacent to the 3' end of the secondary editing site or Part a) and part b) The genome editing kit comprises: [Section 4] An editing-promoting polynucleotide configured so that the predetermined region overlaps with region A at its 5' end, an expression vector thereof, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance; and An editing-promoting polynucleotide configured so that the predetermined region overlaps with region B at its 3' end, an expression vector thereof, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance. The genome editing kit according to Item 3A, Item 3B or Item 3C, comprising: [Section 5] The genome editing kit according to Item 3A, Item 3B, Item 3C, or Item 4, wherein the length of the overlap for the editing-promoting polynucleotide is 5 nucleotides or more. [Section 6] A method for modifying a target site in double-stranded genomic DNA of a cell or organism, comprising treating the cell or organism with a genome editing kit according to paragraph 3A, paragraph 3B, paragraph 3C, paragraph 4 or paragraph 5. [Section 7] A method for producing a cell or organism in which a target site in double-stranded genomic DNA has been modified, comprising treating the cell or organism with a genome editing kit according to any one of claims 3A, 3B, 3C, 4, and 5. Effect of the Invention

[0008] According to the present invention, genome editing can be performed efficiently. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the designs of polynucleotides (1) and (2), which are editing polynucleotides (sense polynucleotides), and polynucleotides (3) to (16) and (22) to (27), which are editing-promoting polynucleotides (antisense polynucleotides), used in the experiments of this embodiment. [Diagram 2] Figure 2 shows the change in the efficiency of genome editing targeting introns using an editing polynucleotide (sense polynucleotide) in the presence of some of the editing-promoting polynucleotides (antisense polynucleotides) (3) to (16). [Diagram 3] Figure 3 shows the change in the efficiency of intron-targeted genome editing by an editing polynucleotide (sense polynucleotide) in the presence of some of the editing-promoting polynucleotides (antisense polynucleotides) (11)-(16) and (22)-(27). [Figure 4] Figure 4 shows the designs of polynucleotides (18), (17), and (1), which are editing polynucleotides (sense polynucleotides), and polynucleotides (3) and (4), which are editing-promoting polynucleotides (antisense polynucleotides), used in the experiments of this example. [Diagram 5]Figure 5 shows that in both cases where an editing polynucleotide (18) having a base deletion in its sequence (sense polynucleotide) is used, and where an editing polynucleotide (17) having a base insertion in its sequence (sense polynucleotide) is used, the genome editing efficiency is improved in the presence of an editing-promoting polynucleotide (antisense polynucleotide). [Figure 6] Figure 6 shows the design of polynucleotide (19), which is an exon-targeting editing polynucleotide (sense polynucleotide), and polynucleotides (20) and (21), which are editing-promoting polynucleotides (antisense polynucleotides), used in the experiments of this example. [Figure 7] Figure 7 shows that the efficiency of genome editing using a sense polynucleotide for editing is significantly improved even in the presence of one type of editing-promoting polynucleotide (antisense polynucleotide). [Figure 8A] FIG. 8A shows the mechanism of conventional genome editing (replacement) using an editing polynucleotide. [Figure 8B] Figure 8B shows the mechanism of genome editing (replacement) using an editing polynucleotide and two types of editing-promoting polynucleotides. [Figure 9A] FIG. 9A shows the mechanism of conventional genome editing using an editing sense polynucleotide. [Figure 9B] Figure 9B shows the mechanism of genome editing by an editing sense polynucleotide in the presence of an editing-promoting antisense polynucleotide. Detailed Description of the Invention

[0010] The following description may be based on a representative embodiment or specific example, but the present invention is not limited to such an embodiment or specific example. The upper and lower limit values ​​of each numerical range exemplified in this specification can be arbitrarily combined. In addition, a numerical range expressed using "~" or "-" in this specification means a range including the numerical values ​​at both ends as the upper and lower limits, unless otherwise specified.

[0011] In this disclosure, "genome editing" or similar terms refers to a type of genome modification technique. Genome editing requires targeting a specific region on the genome and selectively modifying that region. In recent years, genome editing tools such as zinc finger nucleases (ZFNs), TALENs, and CRISPR / Cas systems have been developed, and genome editing using these tools is being actively carried out around the world. While ZFNs and TALENs have the disadvantage that it is time-consuming to design sequence-specific nucleases, the CRISPR / Cas system is particularly adopted as a genome editing tool around the world because targeting is extremely easy. However, the drawbacks of the CRISPR / Cas system include off-target problems and the difficulty of delivering large nucleases to cells.

[0012] In this disclosure, a "cell" is the structural and functional basic unit of an organism. A cell refers to a basic unit of life that has at least genomic DNA, cytoplasm, and a membrane structure that encases them. A cell can be a eukaryotic cell or a prokaryotic cell. A eukaryotic cell has a nucleus surrounded by a nuclear membrane and a cytoplasm. A prokaryotic cell does not have a nucleus. A genomic DNA includes the endogenous DNA of the cell, but is not necessarily composed only of endogenous factors of the cell. A eukaryotic cell can be a human cell. A cell can be a purified cell or an isolated cell. A cell can be a cultured cell. A cell can be an established cell line. A cell can be, for example, a somatic cell. A cell can be, for example, a pluripotent cell or a pluripotent stem cell. A cell can be, for example, a tissue stem cell. A cell can be, for example, a tissue progenitor cell. A cell can be, for example, a germ cell.

[0013] Organisms composed of eukaryotic cells are called eukaryotes, and organisms composed of prokaryotic cells are called prokaryotes. Eukaryotes include animals, plants, and fungi. Animals include mammals (humans and non-human mammals), especially farm animals such as livestock, for example, cows, pigs, goats, sheep, horses, llamas, and camels, birds such as chickens, and seafood such as fish, pets such as dogs, cats, rabbits, guinea pigs, hamsters, and mice, vertebrates such as birds, fish, amphibians, and reptiles, insects, crustaceans such as crabs and shrimp, mollusks such as shellfish, octopus, and squid. Plants include agricultural crops, such as food crops, e.g., rice, corn, potatoes, beans, barley, wheat, and other cereals, horticultural crops (vegetables, fruit trees, and flowers), e.g., leafy vegetables (cabbage, asparagus, etc.), fruit vegetables (eggplant, tomato, cucumber, etc.), root vegetables (radish, carrot, etc.), other vegetables, fruits (e.g., pome fruits such as apples and pears, stone fruits such as plums, apricots, plums, peaches, cherries, shell fruits such as almonds, walnuts, and chestnuts, citrus fruits such as mandarins and lemons, tropical fruits such as tropical fruits), and ornamental plants, as well as seeds, seedlings, or bulbs of any of these. Further examples of eukaryotic organisms are fungi such as yeasts, molds and basidiomycetes, examples of which include yeasts, Neurospora crassa, Matsutake, Shiitake, Enokitake, Shimeji, Nameko, King Oyster Mushroom, etc. Eukaryotic organisms may also be microalgae. Eukaryotic organisms may be human or non-human.

[0014] Examples of prokaryotes include gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Sphingomonas, Brucella, Neisseria gonorrhoeae, Burkholderia, Shigella, Salmonella, Acinetobacter, Vibrio cholerae, Klebsiella pneumoniae, Legionella, Helicobacter pylori, Campylobacter, etc.), gram-positive bacteria (Mycobacterium tuberculosis, Mycoplasma, Staphylococcus aureus, Actinomycetes, Bacillus subtilis, Bacillus anthracis, etc.), etc. Prokaryotes may be preferably gram-negative bacteria, more preferably Pseudomonas aeruginosa, Escherichia coli, Serratia marcescens, Sphingomonas, and even more preferably Pseudomonas aeruginosa and Escherichia coli.

[0015] A cell includes the genomic DNA of the cell, but may further include the genomic DNA of a foreign invader (e.g., a pathogen). In this disclosure, genomic DNA refers to the genomic DNA of the cell itself (i.e., host genomic DNA) unless otherwise specified. The genomic DNA of the invader may exist in the cell separately from the genomic DNA of the host, but may also be integrated into the genomic DNA of the host. The host genomic DNA may contain an exogenous factor (e.g., insertion of all or part of the genomic DNA of a virus, etc.). The host genomic DNA into which the genomic DNA of the invader has been integrated, and the host genomic DNA containing an exogenous factor are also encompassed by the genomic DNA as used herein.

[0016] In the present disclosure, "isolation" means to separate a cell of interest from at least one other component. Isolation can be performed, for example, by separating and removing a cell in its natural state from other components that exist together in its natural state. Isolation can be performed, for example, by separating and removing a part of a cell from a multicellular organism. In the present disclosure, the technique of handling isolated cells is referred to as an in vitro technique.

[0017] In the present disclosure, "purification" refers to further separating the isolated cells of interest from other components with which they are present. Purification can be performed, for example, by separating the cells of interest from other components based on morphology or surface markers. Purification can be performed by limiting dilution and / or cloning of the cells. Purification can be performed by establishing a cell line of interest. Purification can be performed based on the expression of a marker gene, such as a drug resistance gene or a gene encoding a fluorescent protein, when the cells of interest have the marker gene.

[0018] In the present disclosure, the terms "polynucleotide" and "nucleic acid" are used interchangeably and refer to a nucleotide polymer in which nucleotides are linked by phosphodiester bonds. A "polynucleotide" and a "nucleic acid" may be DNA, RNA, or a combination of DNA and RNA (e.g., a gapmer). A "polynucleotide" and a "nucleic acid" may be a polymer of natural nucleotides, a polymer of natural nucleotides and non-natural nucleotides (e.g., analogs of natural nucleotides, nucleotides in which at least one of the base moiety, sugar moiety, and phosphate moiety is modified (e.g., phosphorothioate backbone), etc.), or a polymer of non-natural nucleotides. A polynucleotide may be a peptide nucleic acid. A polynucleotide may be a morpholino oligo.

[0019] In this disclosure, the base sequence of a "polynucleotide" or "nucleic acid" is described in the generally accepted one-letter code unless otherwise specified. The base sequence is described from the 5' to the 3' end unless otherwise specified. The nucleotide residues constituting a "polynucleotide" or a "nucleic acid" may be described simply as adenine, thymine, cytosine, guanine, or uracil, etc., or as their one-letter codes.

[0020] In this disclosure, the term "gene" refers to a polynucleotide that includes at least one open reading frame that encodes a specific protein. A gene may include both exons and introns. "A region where RNA is transcribed" includes a region where RNA is transcribed, such as a region where a gene is present and a region where non-coding RNA is transcribed. A region where RNA is transcribed may be a region that exhibits RNA transcription activity. From a region where RNA is transcribed, RNA is transcribed using the minus strand as a template. The minus strand is called the antisense strand, and the complementary strand of the minus strand is called the sense strand.

[0021] The term "operably linked" as used with respect to a polynucleotide means that a regulatory sequence, such as a promoter, is positioned sufficiently close to a gene sequence that it can affect expression of the gene sequence. For example, a polynucleotide is operably linked to a promoter means that the polynucleotide is linked such that its expression is under the control of the promoter.

[0022] The term "expressible state" refers to a state in which a polynucleotide can be transcribed in a cell into which it has been introduced.

[0023] The term "expression vector" refers to a vector that contains a polynucleotide of interest and is equipped with a mechanism for expressing the polynucleotide of interest in a cell into which the vector is introduced. For example, a "polynucleotide expression vector" refers to a vector that is capable of expressing a polynucleotide in a cell into which the vector is introduced. In a polynucleotide expression vector, the polynucleotide is, for example, operably linked to a control sequence.

[0024] In the present disclosure, sequence identity (or homology) between base sequences is determined by juxtaposing two base sequences with gaps at the insertion and deletion sites so that the corresponding bases are most identical, and calculating the ratio of the matched bases to the entire base sequence excluding the gaps in the resulting alignment. Sequence identity between base sequences can be determined using various homology search software known in the art. For example, the value of sequence identity between base sequences can be obtained by calculation based on the alignment obtained by the known homology search software BLASTN. Here, when the base sequence to be aligned contains uracil, the identity is calculated using the base sequence after replacing uracil with thymine. In addition, when the base sequence to be aligned contains a modified nucleotide or nucleotide analog, the identity is calculated using the base sequence after replacing the modified nucleotide or nucleotide analog with the original natural deoxyribonucleotide, i.e., adenine, thymine, guanine, or cytosine.

[0025] The terms "portion" and "region" with respect to a polynucleotide are used interchangeably and each refers to one nucleotide or multiple contiguous nucleotides contained in a polynucleotide. The terms "portion" and "region" with respect to double-stranded genomic DNA are used interchangeably and each refers to one nucleotide pair or multiple contiguous nucleotide pairs contained in double-stranded genomic DNA.

[0026] In this disclosure, a portion or region that is "located corresponding to" a portion or region of a polynucleotide refers to a portion or region on another polynucleotide that is complementary to the portion or region of the polynucleotide.

[0027] In the present disclosure, a polynucleotide or a portion or region thereof is "hybridizable" with another polynucleotide or a portion or region thereof means that the polynucleotide or a portion or region thereof has the ability to bind to another polynucleotide or a portion or region thereof through hydrogen bonds between complementary bases. Hybridization can be confirmed by maintaining the hybridization state under stringent conditions. Stringent conditions can be determined based on the melting temperature (Tm) of the nucleic acid to which the probe is bound, as taught by Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). If the hybridization state between two polynucleotides or portions or regions thereof is maintained even after washing under stringent conditions, such as 1×SSC, 0.1% SDS, and 37° C., they are hybridizable to each other. Stringent conditions may be washing at 0.5xSSC, 0.1% SDS at 42°C, or washing at 0.1xSSC, 0.1% SDS at 65°C.

[0028] Polynucleotides for editing In the present disclosure, the editing polynucleotide is a single-stranded polynucleotide capable of modifying a target site in double-stranded genomic DNA. The editing polynucleotide is, in order from the 5' end, a) A portion capable of hybridizing to the region adjacent to the 3' end of the primary editing site x) any portion consisting of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site; and b) A portion capable of hybridizing to the region adjacent to the 5' end of the primary editing site or Part a) and part b) The details of the editing polynucleotide are described below (see also FIG. 8A).

[0029] In the present disclosure, the target site refers to a nucleotide pair or multiple consecutive nucleotide pairs, or a position between two consecutive nucleotide pairs, present in a double-stranded genomic DNA, where modification is desired. The target site consists of a primary editing site set on one genomic DNA strand and a secondary editing site on the other genomic DNA strand at a position corresponding to the primary editing site. For example, when the desired modification is to replace or delete a nucleotide pair or multiple consecutive nucleotide pairs in a double-stranded genomic DNA, the primary editing site is set on one genomic DNA strand at a nucleotide pair or multiple consecutive nucleotide pairs that are desired to be replaced or deleted. Also, when the desired modification is to insert a nucleotide pair or multiple consecutive nucleotide pairs into a double-stranded genomic DNA, the primary editing site is set between two consecutive nucleotides on one genomic DNA strand where insertion of a nucleotide pair or multiple consecutive nucleotides is desired.

[0030] The primary editing site can be set at any one nucleotide or multiple consecutive nucleotides, or between any two consecutive nucleotides, present in one genomic DNA strand, and is not limited to a specific one nucleotide or multiple consecutive nucleotides, or between a specific two consecutive nucleotides.

[0031] When the primary editing site is set at one nucleotide or multiple consecutive nucleotides, the region adjacent to the 5' end of the primary editing site and the region adjacent to the 3' end of the primary editing site are separated on the genomic DNA by the length of the primary editing site. When the primary editing site is set at a position between two consecutive nucleotides, the region adjacent to the 5' end of the primary editing site and the region adjacent to the 3' end of the primary editing site are adjacent to each other on the genomic DNA.

[0032] When the primary editing site is set at multiple consecutive nucleotides on one genomic DNA strand, the number of consecutive nucleotides is not particularly limited, and may be, for example, 2 nucleotides or more, 3 nucleotides or more, 4 nucleotides or more, 5 nucleotides or more, 6 nucleotides or more, 7 nucleotides or more, or 8 nucleotides or more, and may be, for example, 2000 nucleotides or less, 1000 nucleotides or less, 900 nucleotides or less, 800 nucleotides or less, 700 nucleotides or less, 600 nucleotides or less, 500 nucleotides or less, 450 nucleotides or less, 400 nucleotides or less, 350 nucleotides or less, 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 150 nucleotides or less, 100 nucleotides or less, 90 nucleotides or less, 80 nucleotides or less, 70 nucleotides or less, 60 nucleotides or less, 50 nucleotides or less, 40 nucleotides or less, 30 nucleotides or less, 20 nucleotides or less, 15 nucleotides or less, 12 nucleotides or less, 10 nucleotides or less, or 9 nucleotides or less.

[0033] The primary editing site may be set on either strand of the double strand constituting the genomic DNA. The secondary editing site will be set on the genomic DNA strand on which the primary editing site is not set. For example, when it is desired to modify the region of the double-stranded genomic DNA that is transcribed into RNA, the primary editing site may be set on the sense strand or on the antisense strand.

[0034] Part a) of the editing polynucleotide can hybridize to a region adjacent to the 3' end of the primary editing site.

[0035] Portion a) may also be a portion consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence of the region adjacent to the 5' end of the secondary editing site, where the identity is preferably 95% or more, more preferably 97% or more, even more preferably 99% or more, still more preferably 99.5% or more, and particularly preferably 99.9% or more.

[0036] In a preferred embodiment, portion a) consists of a base sequence complementary to the base sequence of the region adjacent to the 3' end of the primary editing site, i.e., a base sequence identical to the base sequence of the region adjacent to the 5' end of the secondary editing site.

[0037] Part b) of the editing polynucleotide can hybridize to a region adjacent to the 5' end of the primary editing site.

[0038] Part b) may also be a part consisting of a nucleotide sequence having 90% or more identity with the nucleotide sequence of the region adjacent to the 3' end of the secondary editing site, where the identity is preferably 95% or more, more preferably 97% or more, even more preferably 99% or more, still more preferably 99.5% or more, and particularly preferably 99.9% or more.

[0039] In a preferred embodiment, portion b) consists of a base sequence complementary to the base sequence of the region adjacent to the 5' end of the primary editing site, i.e., a base sequence identical to the base sequence of the region adjacent to the 3' end of the secondary editing site.

[0040] The length of the portion a) and the portion b) is not particularly limited and can be set independently. The length of the portion a) and the portion b) can be, for example, 10 nucleotides or more, 20 nucleotides or more, 30 nucleotides or more, 35 nucleotides or more, 40 nucleotides or more, 45 nucleotides or more, 50 nucleotides or more, 60 nucleotides or more, 70 nucleotides or more, 80 nucleotides or more, 90 nucleotides or more, or 100 nucleotides or more, or 1000 nucleotides or less, 500 nucleotides or less, 300 nucleotides or less, 200 nucleotides or less, 190 nucleotides or less, 180 nucleotides or less, 170 nucleotides or less, 160 nucleotides or less, 150 nucleotides or less, 140 nucleotides or less, 130 nucleotides or less, 120 nucleotides or less, 110 nucleotides or less, 100 nucleotides or less, 90 nucleotides or less, 80 nucleotides or less, 70 nucleotides or less, 60 nucleotides or less, or 50 nucleotides or less.

[0041] Furthermore, the length of each of portion a) and portion b) can be, for example, 10 to 1000 nucleotides, 10 to 500 nucleotides, 10 to 300 nucleotides, 10 to 200 nucleotides, 10 to 100 nucleotides, 32 to 100 nucleotides, 35 to 70 nucleotides, or 35 to 50 nucleotides.

[0042] Part x) is any part consisting of a base sequence that is not identical to the base sequence of the secondary editing site, and is determined by how the target site is to be modified. For example, when the desired modification is to replace one nucleotide pair or multiple consecutive nucleotide pairs in the double-stranded genomic DNA, part x) is one nucleotide or multiple consecutive nucleotides that are desired to be replaced with the secondary editing site. Also, when the desired modification is to delete one nucleotide pair or multiple consecutive nucleotide pairs in the double-stranded genomic DNA, part x) is not included in the editing polynucleotide. Furthermore, when the desired modification is to insert one nucleotide pair or multiple consecutive nucleotide pairs into the double-stranded genomic DNA, part x) is one nucleotide or multiple consecutive nucleotides that are desired to be inserted into the secondary editing site.

[0043] Part x) is composed of a base sequence that is not identical to the base sequence of the secondary editing site. Part x) may be composed of any base sequence as long as it does not completely match the base sequence of the secondary editing site, and may be, for example, a base sequence that has an identity of less than 90% with the base sequence of the secondary editing site. The identity is, for example, 50% or less, preferably 40% or less, more preferably 30% or less, even more preferably 20% or less, even more preferably 10% or less, and particularly preferably 5% or less.

[0044] The length of portion x) is not particularly limited, but can be, for example, 2000 nucleotides or less, 1000 nucleotides or less, 900 nucleotides or less, 800 nucleotides or less, 700 nucleotides or less, 600 nucleotides or less, 500 nucleotides or less, 400 nucleotides or less, 300 nucleotides or less, 200 nucleotides or less, 100 nucleotides or less, 90 nucleotides or less, 80 nucleotides or less, 70 nucleotides or less, 60 nucleotides or less, 50 nucleotides or less, 40 nucleotides or less, 30 nucleotides or less, 20 nucleotides or less, 10 nucleotides or less, 9 nucleotides or less, 8 nucleotides or less, 7 nucleotides or less, 6 nucleotides or less, 5 nucleotides or less, 4 nucleotides or less, 3 nucleotides or less, 2 nucleotides or less, or 1 nucleotide.

[0045] The editing polynucleotide consists of part a), part x) and part b), or part a) and part b), in that order from the 5'-end. Therefore, the length of the editing polynucleotide can be determined by adding up the lengths of the parts constituting the editing polynucleotide, i.e., part a), part x) and part b), or by adding up the lengths of part a) and part b).

[0046] The editing polynucleotide may contain one or more DNA high affinity nucleotide analogs in one or both of the part a) and the part b). The DNA high affinity nucleotide analog is replaced with a DNA high affinity nucleotide analog containing adenine or an adenine analog so as to be the same base as the original base, for example, when the original base is adenine, it is replaced with a DNA high affinity nucleotide analog containing adenine or an adenine analog. The DNA high affinity nucleotide analog may be included adjacently in plural, or may be included intermittently so as not to be adjacent to each other. In addition, when included in both the part a) and the part b), the DNA high affinity nucleotide analog may be included at a position selected independently of each other, and the number of included nucleotides does not have to be the same.

[0047] High-affinity nucleotide analogs for DNA are artificial nucleic acid building blocks that have high binding affinity to DNA and can form polymer structures similar to those of natural nucleic acids. Examples include nucleotides bridged between the 2' oxygen atom and the 4' carbon atom of the ribose ring, peptide nucleic acids (PNAs) having N-(2-aminoethyl)glycine instead of deoxyribose or ribose, and morpholino nucleic acids having a morpholine ring instead of deoxyribose or ribose.

[0048] In one embodiment, the DNA high affinity nucleotide analog is a nucleotide bridged between the 2' oxygen atom and the 4' carbon atom of the ribose ring, examples of which include LNA (Locked Nucleic Acid) in which the 2' oxygen atom and the 4' carbon atom are bridged via methylene, ENA bridged via ethylene, and BNA (Bridged Nucleic Acid) bridged via -CH2OCH2-. COC , BNA bridged via -NR-CH2- (R is methyl or hydrogen atom) NC Examples include cMOE cross-linked via -CH2(OCH3)-, cEt cross-linked via -CH2(CH3)-, AmNA cross-linked via an amide, and scpBNA cross-linked via a methylene to form a cyclopropane at the 6' position.

[0049] The polynucleotide for editing may further contain chemically modified nucleotides as its constituent units in addition to the DNA high affinity nucleotide analogs. Examples of chemically modified nucleotides include nucleotides in which the phosphate group is replaced with a chemically modified phosphate group such as phosphorothioate (PS), methylphosphonate, or phosphorodithioate, nucleotides in which the hydroxyl group at the 2' position of the sugar (ribose) portion is replaced with -OR (R represents, for example, CH3(2'-O-Me), CH2CHOCH3(2'-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, or CH2CH2CN), nucleotides in which a methyl group or a cationic functional group is introduced at the 5th position of the pyrimidine base, and nucleotides in which the carbonyl group at the 2nd position of the pyrimidine base is replaced with a thiocarbonyl group. Furthermore, nucleotides in which the phosphate portion or hydroxyl portion is modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group can be mentioned, but are not limited thereto.

[0050] Editing polynucleotides containing DNA high affinity nucleotide analogs and optionally further chemically modified nucleotides can be produced by known synthetic methods.

[0051] The editing polynucleotide in the present disclosure, like the single-stranded polynucleotide disclosed in Patent Document 1 (Patent No. 6899564), can modify genomic DNA within a cell as desired, and the modification does not require the introduction of site-specific nucleases (ZFN proteins, TALEN proteins, Cas proteins, etc.), guide RNAs, or their expression vectors into the cell, as is the case with conventional genome editing techniques.

[0052] Without being bound by theory, the editing polynucleotide hybridizes with a region adjacent to the 3'-end of the primary editing site set on one genomic DNA strand in part a) and with a region adjacent to the 5'-end of the primary editing site in part b). As a result, when the editing polynucleotide consists of part a), part x), and part b), part x) protrudes like a boil without hybridizing with other sequences, and when the editing polynucleotide consists of part a) and part b), part x) protrudes like a boil without hybridizing with other sequences, and when the editing polynucleotide consists of part a) and part b), part b) protrudes like a boil without hybridizing with other sequences. Then, this protruding part is recognized by the repair system, and the target site of the double-stranded genomic DNA can be modified.

[0053] Specifically, when it is desired to insert one nucleotide pair or multiple consecutive nucleotide pairs into a double-stranded genomic DNA, the primary editing site is set between two consecutive nucleotides that are desired to be inserted on one genomic DNA strand; part a) and part b) are set as described above; and part x) is set at one nucleotide or multiple consecutive nucleotides that are desired to be inserted at the secondary editing site. By performing genome editing using an editing polynucleotide consisting of parts a), x) and b) designed in this way, a part consisting of a base sequence complementary to part x) can be inserted at the primary editing site, and part x) can be inserted at the secondary editing site.

[0054] Also, when it is desired to replace one nucleotide pair or multiple consecutive nucleotide pairs in a double-stranded genomic DNA, the primary editing site is set to one nucleotide or multiple consecutive nucleotides on one genomic DNA strand that are desired to be replaced; part a) and part b) are set as described above; and part x) is set to one nucleotide or multiple consecutive nucleotides that are desired to be replaced with the secondary editing site. By performing genome editing using an editing polynucleotide consisting of part a), part x) and part b) designed in this way, a part consisting of a base sequence complementary to part x) can be inserted into the primary editing site, and part x) can be inserted into the secondary editing site (shown in FIG. 8A).

[0055] In addition, when it is desired to delete one nucleotide pair or multiple consecutive nucleotide pairs in the double-stranded genomic DNA, the primary editing site is set to one nucleotide or multiple consecutive nucleotides on one genomic DNA strand that is desired to be deleted; part a) and part b) are set as described above. By performing genome editing using an editing polynucleotide consisting of part a) and part b) designed in this way, the primary editing site can be deleted from one genomic DNA strand and the secondary editing site can be deleted from the other genomic DNA strand, that is, the target site can be deleted from the double-stranded genomic DNA.

[0056] Without being bound by theory, considering that there is a lot of transcribed RNA around the genomic DNA, for example, when the target site is in the genomic DNA region transcribed into RNA and the primary editing site is set on the genomic DNA sense strand, the editing polynucleotide preferentially binds to a region having the same base sequence as the two regions contained in the RNA present in the vicinity, rather than the region adjacent to the 3' end side of the primary editing site of the genomic DNA sense strand and the region adjacent to the 5' end side of the primary editing site, and therefore the genome editing efficiency is considered to be poor. If genome editing efficiency is important, it is preferable to set the primary editing site on the antisense strand rather than on the genomic DNA sense strand.

[0057] As described above, the editing polynucleotide can modify the target site of the double-stranded genomic DNA by hybridizing the part a) with the region adjacent to the 3'-end of the primary editing site set on one genomic DNA strand, and the part b) with the region adjacent to the 5'-end of the primary editing site. Therefore, as long as it does not affect the hybridization of the part a) of the editing polynucleotide with the region adjacent to the 3'-end of the primary editing site and the hybridization of the part b) of the editing polynucleotide with the region adjacent to the 5'-end of the primary editing site, any base sequence may be added to one or both ends of the editing polynucleotide, i.e., to the 5'-end of the part a) and / or the 3'-end of the part b). The editing polynucleotide in the present disclosure includes those to which such any base sequence has been added.

[0058] For such editing polynucleotides having any base sequence added to their ends, the calculation of identity between the region adjacent to the 5' end and the region adjacent to the 3' end of the secondary editing site shall be performed between the base sequence of part a) and the base sequence of the region adjacent to the 5' end of the secondary editing site, or between the base sequence of part b) and the base sequence of the region adjacent to the 3' end of the secondary editing site, and the any added base sequence shall be deemed not to be present in the calculation of identity.

[0059] Editing-promoting polynucleotides The editing polynucleotide can be used in combination with an editing-promoting polynucleotide to improve genome editing efficiency. The editing-promoting polynucleotide is a polynucleotide that can hybridize with a nucleic acid sequence that is complementary to a predetermined region on a genomic DNA strand where a primary editing site is set (also called a complementary sequence of the predetermined region). The editing-promoting polynucleotide is described in detail below (see also FIG. 8B).

[0060] The specified region is set so that it overlaps with region A at its 5' end or overlaps with region B at its 3' end, when the region located at a position corresponding to portion a) of the editing polynucleotide on the genomic DNA strand in which the primary editing site is set is defined as region A, and the region located at a position corresponding to portion b) of the editing polynucleotide is defined as region B.

[0061] The length of the overlap with region A at the 5' end of the predetermined region is not limited as long as it is the same as or shorter than region A. The length of the overlap with region B at the 3' end of the predetermined region is not limited as long as it is the same as or shorter than region B. The overlap lengths can be set independently, and may be, for example, 1 nucleotide or more, 2 nucleotides or more, 3 nucleotides or more, 4 nucleotides or more, 5 nucleotides or more, 6 nucleotides or more, 7 nucleotides or more, 8 nucleotides or more, 9 nucleotides or more, or 10 nucleotides or more, or may be 20 nucleotides or less, 19 nucleotides or less, 18 nucleotides or less, 17 nucleotides or less, 16 nucleotides or less, 15 nucleotides or less, 14 nucleotides or less, 13 nucleotides or less, 12 nucleotides or less, or 11 nucleotides. The overlap lengths can be, for example, 1 to 20 nucleotides, 5 to 20 nucleotides, 5 to 15 nucleotides, 8 to 12 nucleotides, or 9 to 11 nucleotides, independently.

[0062] The complementary sequence of a given region is a region on another polynucleotide that is complementary to the given region on a genomic DNA strand. In nature, the complementary sequence of a given region is typically contained in the RNA transcribed from the genomic DNA strand in which the given region was established.

[0063] The editing-promoting polynucleotide can hybridize with a complementary sequence of a predetermined region on the genomic DNA strand in which the primary editing site is established.

[0064] The editing-promoting polynucleotide may be a polynucleotide having a base sequence that has 90% or more identity with the base sequence of the predetermined region, where the identity is preferably 95% or more, more preferably 97% or more, even more preferably 99% or more, still more preferably 99.5% or more, and particularly preferably 99.9% or more.

[0065] In a preferred embodiment, the editing-promoting polynucleotide has a base sequence identical to the base sequence of the predetermined region.

[0066] There are two types of editing-promoting polynucleotides: an editing-promoting polynucleotide in which a predetermined region is set to overlap with region A at its 5'-end, and an editing-promoting polynucleotide in which a predetermined region is set to overlap with region B at its 3'-end. Genome editing efficiency can be improved by using one of these two types of editing-promoting polynucleotides in combination with an editing polynucleotide. Genome editing efficiency can be further improved by using both of these two types of editing-promoting polynucleotides in combination with an editing polynucleotide.

[0067] In the embodiment in which the above-mentioned two types of editing-promoting polynucleotides are used, there is no limitation on the ratio of the two types of editing-promoting polynucleotides used, and the molar concentrations of the two may be approximately the same, or one of them may be significantly higher in molar concentration than the other.

[0068] The length of the editing-promoting polynucleotide is, when the predetermined region is set to overlap with region A at its 5' end, the same as or longer than the length of the overlap with region A, and, when the predetermined region is set to overlap with region B at its 3' end, the same as or longer than the length of the overlap with region B. When two types of editing-promoting polynucleotides are used, the lengths of the editing-promoting polynucleotides do not need to be the same and can be set independently.

[0069] The length of the editing-facilitating polynucleotide can be, for example, 20 nucleotides or more, 30 nucleotides or more, 40 nucleotides or more, 50 nucleotides or more, 60 nucleotides or more, 70 nucleotides or more, 80 nucleotides or more, 90 nucleotides or more, 100 nucleotides or more, 110 nucleotides or more, 120 nucleotides or more, or 130 nucleotides or more, and can be 1000 nucleotides or less, 500 nucleotides or less, 300 nucleotides or less, 250 nucleotides or less, 200 nucleotides or less, 190 nucleotides or less, 180 nucleotides or less, 170 nucleotides or less, 160 nucleotides or less, 150 nucleotides or less, 140 nucleotides or less, or 130 nucleotides or less, respectively.

[0070] Furthermore, the length of the editing-promoting polynucleotide can be, for example, 10 to 1000 nucleotides, 20 to 500 nucleotides, 30 to 300 nucleotides, 40 to 200 nucleotides, 50 to 200 nucleotides, 60 to 200 nucleotides, 70 to 150 nucleotides, or 70 to 130 nucleotides.

[0071] Without being bound by theory, it is believed that in an environment where there are many other polynucleotides having regions complementary to the primary editing site or the regions nearby it (region A and region B), the binding between the editing polynucleotide and the genomic DNA strand in which the primary editing site is set is competitively inhibited by the other polynucleotides, resulting in a decrease in the efficiency of genome editing by the editing polynucleotide.

[0072] For example, when the target site is within a genomic DNA region that is transcribed into RNA and the primary editing site is set on the antisense strand of the genomic DNA, the editing polynucleotide competes with RNA present in the vicinity for binding to the antisense strand of the genomic DNA, suppressing binding between the editing polynucleotide and the antisense strand of the genomic DNA, and it is believed that the genome editing efficiency will be lower than when there is no RNA present in the vicinity.

[0073] Under such an environment, when an editing polynucleotide is coexisted with an editing-promoting polynucleotide, the other polynucleotide (e.g., RNA) binds to the editing-promoting polynucleotide, and as a result, inhibition of binding between the editing polynucleotide and the genomic DNA strand (e.g., the antisense strand) in which the primary editing site is set is eliminated, and genome editing efficiency is thought to be improved (Figure 8B).

[0074] In an embodiment in which the target site is in a genomic DNA region to be transcribed into RNA and the primary editing site is set on the genomic DNA antisense strand, the editing-promoting polynucleotide preferably has a high binding affinity to RNA. The editing-promoting polynucleotide may contain one or more RNA high-affinity nucleotide analogs. The RNA high-affinity nucleotide analog is replaced with an RNA high-affinity nucleotide analog containing adenine or an adenine analog so as to be the same base as the original base, for example, when the original base is adenine, the original base is replaced with an RNA high-affinity nucleotide analog containing adenine or an adenine analog. The RNA high-affinity nucleotide analog may be included adjacent to each other, or may be included intermittently so as not to be adjacent to each other. In addition, when two types of editing-promoting polynucleotides are used, each editing-promoting polynucleotide may contain an RNA high-affinity nucleotide analog at a position selected independently of each other, and the number of the nucleotides does not have to be the same.

[0075] Without being bound by theory, in the embodiment in which the target site is in the genomic DNA region transcribed into RNA and the primary editing site is set on the genomic DNA antisense strand, when the target site is set in the region transcribed into the intron portion of the mRNA precursor, the competition between the editing polynucleotide and the RNA for binding with the genomic DNA antisense strand is more intense than when the target site is set in the region transcribed into the exon portion of the mRNA precursor, and the genome editing efficiency is lowered. This is because the intron portion excised from the mRNA precursor by splicing is present in large quantities around the genomic DNA.

[0076] Therefore, the editing-promoting polynucleotide can improve genome editing efficiency in both cases where the target site is within a genomic DNA region transcribed into the intron portion of an mRNA precursor and the primary editing site is set on the genomic DNA antisense strand, and where the target site is within a genomic DNA region transcribed into the exon portion of an mRNA precursor and the primary editing site is set on the genomic DNA antisense strand; however, it is believed that the effect of improving genome editing efficiency can be particularly strong when the target site is within a genomic DNA region transcribed into the intron portion of an mRNA precursor and the primary editing site is set on the genomic DNA antisense strand.

[0077] In embodiments in which the target site is set in a double-stranded genomic DNA region to be transcribed into RNA and the primary editing site is set on the antisense strand of the genomic DNA, the editing polynucleotide is also referred to as an editing sense polynucleotide and the editing-promoting polynucleotide is also referred to as an editing-promoting antisense polynucleotide.

[0078] Genome editing kits In one aspect, the present disclosure provides a genome editing kit including the above-mentioned editing polynucleotide and the above-mentioned editing promoting polynucleotide. The genome editing kit preferably includes two types of editing promoting polynucleotides: an editing promoting polynucleotide in which the predetermined region is configured to overlap with region A at its 5' end, and an editing promoting polynucleotide in which the predetermined region is configured to overlap with region B at its 3' end.

[0079] The genome editing kit may include, instead of the editing polynucleotide, an expression vector for the editing polynucleotide, or a conjugate of the editing polynucleotide or the expression vector for the editing polynucleotide and another substance. The genome editing kit may include, instead of the editing promoting polynucleotide, an expression vector for the editing promoting polynucleotide, or a conjugate of the editing promoting polynucleotide or the expression vector for the editing promoting polynucleotide and another substance.

[0080] There is no limitation on other substances conjugated to the editing polynucleotide, so long as they can form a conjugate with the editing polynucleotide and do not interfere with the function of the editing polynucleotide, i.e., the function of modifying the target site of the genome DNA. There is no limitation on other substances conjugated to the editing-promoting polynucleotide, so long as they can form a conjugate with the editing-promoting polynucleotide and do not interfere with the function of the editing-promoting polynucleotide, i.e., the function of improving the genome editing efficiency by the editing polynucleotide.

[0081] Examples of other substances that can be conjugated to the editing polynucleotide and the editing-promoting polynucleotide include functional peptides such as nuclear transport signals and membrane-permeable peptides; hydrophilic polymers such as polyethylene glycol and dextran; and labeling substances such as radioactive isotopes, fluorescein and its derivatives, rhodamine and its derivatives, fluorescent proteins, phycobiliproteins, biotin, and avidin.

[0082] The genome editing kit may further include reagents such as a nucleic acid introduction reagent such as a transfection reagent or a buffer solution.

[0083] The components contained in the genome editing kit, such as the above-mentioned editing polynucleotide, its expression vector or conjugate thereof, the above-mentioned editing-promoting polynucleotide, its expression vector or conjugate thereof, and each of the reagents, may be included in the kit in a separate form, or may be included in the kit in the form of a composition in which multiple components are combined.

[0084] The genome editing kit may further include, for example, tools used during genome editing, instructions on how to use them, etc.

[0085] In one embodiment, the genome editing kit is a kit for treating or preventing a disease, and may contain a therapeutically or prophylactically effective amount of the above-mentioned editing polynucleotide, its expression vector, or a conjugate of the editing polynucleotide or its expression vector with another substance, as well as other pharma- ceutically acceptable components, such as pharma-ceutically acceptable additives (buffers, stabilizers, preservatives, excipients, etc.), and pharma-ceutically acceptable media (water, saline, phosphate-buffered saline (PBS), etc.). The kit for treating or preventing a disease may be used to treat or prevent a disease caused by some abnormality in genomic DNA.

[0086] Genome editing method and method for producing genome-edited cells or organisms In one aspect, the present disclosure provides a method for modifying a target site of a double-stranded genomic DNA of a cell or organism (also referred to as a "genome editing method" in this disclosure), comprising treating the cell or organism with the above-mentioned genome editing kit. In another aspect, the present disclosure provides a method for producing a cell or organism in which a target site of a double-stranded genomic DNA has been modified (also referred to as a "genome-edited cell or organism production method" in this disclosure), comprising treating the cell or organism with the above-mentioned genome editing kit.

[0087] The cells treated with the genome editing kit are cells in which modification of genomic DNA is desired, and may originate from various organisms including the prokaryotes and eukaryotes mentioned above. When the cells are cells of a multicellular organism, the cells may be derived from various tissues of the organism. The cells may be somatic cells or germ cells. The cells may also be differentiated or undifferentiated cells, and may be tissue stem cells, embryonic stem cells, or induced pluripotent stem cells (iPS cells).

[0088] The cells treated with the genome editing kit may be in the form of a single cell or a cell population containing one or more types of cells.

[0089] The organism to be treated with the genome editing kit is an organism in which modification of genomic DNA is desired, and may be various organisms including the prokaryotes and eukaryotes described above. The organism may be a mature individual or an embryo or fetus in a developmental stage, or in the case of a plant, it may be a seed, seedling, or bulb.

[0090] In certain embodiments, the cells do not include human gametes and zygotes, and may be referred to as cells (but excluding human gametes and zygotes).

[0091] In some embodiments, the organism does not include humans, and may be referred to as a non-human organism. In other embodiments, the organism does not include humans and animals, and may be referred to as a non-human non-animal organism.

[0092] Moreover, in certain embodiments, the cells do not include human gametes and zygotes and the organism does not include humans, hi yet another preferred embodiment, the cells do not include human gametes and zygotes and the organism does not include humans or animals.

[0093] Treatment of cells or organisms using a genome editing kit means that the editing polynucleotide, its expression vector, or a conjugate of the editing polynucleotide or its expression vector with other substances and the editing promotion polynucleotide, its expression vector, or a conjugate of the editing promotion polynucleotide or its expression vector with other substances contained in the genome editing kit are coexisted with cells or administered to an organism. By coexistence with cells or administration to an organism, the editing polynucleotide, its expression vector, or a conjugate of the editing polynucleotide or its expression vector with other substances and the editing promotion polynucleotide, its expression vector, or a conjugate of the editing promotion polynucleotide or its expression vector with other substances are introduced into the cell or organism, and the genomic DNA of the cell or organism can be edited.

[0094] Treatment of cells or organisms using a genome editing kit can be carried out by various known methods appropriately selected by a person skilled in the art taking into consideration factors such as the type or properties of the cells or organisms. Examples of treatment of cells or organisms include microinjection, electroporation, DEAE-dextran treatment, transfection using a transfection reagent (jetPEI (PolyPlus-transfection), lipofectamine, etc.), transfection using lipid nanoparticles, and hydrodynamics method.

[0095] In certain embodiments, the treatment of human cells is performed in vitro or ex vivo.

[0096] In general, genome editing increases or decreases the characteristics of a cell or organism, such as nutritional requirements, sensitivity or resistance to chemicals, or imparts characteristics not present in the cell or organism before editing, or loses the characteristics of the cell or organism before editing. Using such changes in characteristics as an indicator, a cell or organism with the desired genome editing can be selected. For example, when it is expected that drug resistance will be increased or imparted by genome editing, a cell or organism with genome editing can be selected by culturing a cell treated with a genome editing kit in an appropriate medium containing the drug, or by raising an organism treated with a genome editing kit in an appropriate feed containing the drug. The selection and evaluation method of the characteristics of a cell or organism, and the culture or rearing conditions that enable the selection of a cell or organism can be appropriately determined within the scope of the ordinary working ability of a person skilled in the art.

[0097] The above-mentioned genome editing method and method for producing a genome-edited cell or organism may include, in addition to a step of treating the cell or organism using a genome editing kit, a step of selecting a cell or organism whose genomic DNA has been edited using as an indicator the characteristics of the cell or organism that are changed by the editing polynucleotide.

[0098] Further inventions provided by this disclosure According to the present disclosure, the following inventions are also provided. Table 1 shows examples of the correspondence between the terms used in the following inventions and the terms used in the above inventions. [Table 1]

[0099] [1] A method for editing a genome in a eukaryotic cell, comprising: (A) allowing, in the cell, a single-stranded form of an editing polynucleotide capable of hybridizing to adjacent sequences upstream and downstream of a target region of the genome (e.g., a target region corresponding to an antisense strand of a genomic region to which RNA is transcribed) to interact in a sequence-specific manner with the genome; The single-stranded form of the editing polynucleotide (e.g., editing sense polynucleotide) has an upstream homology arm and a downstream homology arm, the upstream homology arm has a sequence capable of hybridizing to a sequence adjacent to the upstream of the target region, and the downstream homology arm has a sequence capable of hybridizing to a sequence adjacent to the downstream of the target region, the upstream homology arm and the downstream homology arm can hybridize to at least the upstream and downstream of the target region, and there may be any sequence or no sequence between the upstream homology arm and the downstream homology arm; the step (A) is carried out in the presence of another single-stranded polynucleotide (e.g., a single-stranded antisense polynucleotide), and the other polynucleotide (e.g., a single-stranded antisense polynucleotide) has a sequence that can hybridize to a region having a sequence corresponding to a terminal portion of the editing polynucleotide (e.g., a partial region of RNA transcribed from a genomic region, the partial region having a nucleotide sequence corresponding to a terminal portion of the editing sense polynucleotide) (e.g., a sequence that has complementarity to the partial region); The other polynucleotide (e.g., a single-stranded antisense polynucleotide) has a length of 40 bases or more, By the above process, the target region is replaced with the base sequence between the upstream homology arm and the downstream homology arm of the single-stranded editing polynucleotide (e.g., the single-stranded editing sense polynucleotide), provided that if there is no base sequence between the upstream homology arm and the downstream homology arm, the target region is deleted. method. [2] The method according to [1] above, wherein the single-stranded editing polynucleotide is a polynucleotide selected from the group consisting of DNA, RNA, and combinations thereof. [3] The method according to [1] or [2] above, wherein the other single-stranded polynucleotide is DNA. [4] The method according to any one of [1] to [3] above, wherein the region having a sequence corresponding to the terminal portion has a length of 5 bases or more. [5] The method according to any one of [1] to [4] above, wherein the region having a sequence corresponding to the terminal portion has a length of 20 bases or less. [6] The method according to any one of [1] to [5] above, wherein the single-stranded editing polynucleotide has a length of 60 to 200 bases. [7] The method according to any one of [1] to [6] above, wherein the other single-stranded polynucleotide has a length of 50 to 200 bases. [8] The method according to any one of [1] to [7] above, wherein the target region is present in an intron. [9] The method according to any of [1] to [8] above, wherein the step (A) is carried out in the presence of at least two types of hybridizable other single-stranded polynucleotides, and at least one type of other single-stranded polynucleotide (first other polynucleotide) has a sequence (e.g., a sequence having complementarity) that can hybridize to a region having a sequence corresponding to the 5'-terminal portion of the editing polynucleotide, and at least one type of other single-stranded polynucleotide (second other polynucleotide) has a sequence (e.g., a sequence having complementarity) that can hybridize to a region having a sequence corresponding to the 3'-terminal portion of the editing polynucleotide.

[10] A genome editing kit for use in the method according to any one of [1] to [8] above, comprising a combination of the single-stranded editing polynucleotide and the other single-stranded polynucleotide.

[11] A genome editing kit for use in the method described in any one of [1] to [8] above, comprising a DNA encoding the editing polynucleotide in single-stranded form and a DNA encoding the other polynucleotide in single-stranded form, the DNAs being expressibly contained in one or more expression vectors.

[12] A genome editing kit for use in the method described in any one of [1] to [8] above, comprising a DNA encoding the single-stranded editing polynucleotide and the other single-stranded polynucleotide, wherein the DNA encoding the single-stranded editing polynucleotide is expressibly contained in an expression vector, and the other single-stranded polynucleotide is DNA.

[13] A genome editing kit for use in the method described in any one of [1] to [8] above, comprising a polynucleotide encoding the editing polynucleotide in single-stranded form and DNA encoding the other single-stranded polynucleotide, the DNAs being expressibly contained in one or more expression vectors.

[14] A genome editing kit for use in the method described in [9] above, comprising a combination of the single-stranded editing polynucleotide and at least two other single-stranded polynucleotides.

[15] A genome editing kit for use in the method described in [9] above, comprising a DNA encoding the editing polynucleotide in single-stranded form and a DNA encoding at least two other types of single-stranded polynucleotides, the DNAs being expressibly contained in one or more expression vectors.

[16] A genome editing kit for use in the method described in [9] above, comprising a DNA encoding the single-stranded editing polynucleotide and at least two other single-stranded polynucleotides, wherein the DNA encoding the single-stranded editing polynucleotide is expressibly contained in an expression vector, and the other single-stranded polynucleotides are DNA.

[17] A genome editing kit for use in the method described in [9] above, comprising a polynucleotide encoding the editing polynucleotide in single-stranded form and DNA encoding the at least two other types of single-stranded polynucleotides, the DNAs being expressibly contained in one or more expression vectors.

[0100] [1A] A method for editing a genome in a eukaryotic cell, comprising: (A) allowing a single-stranded sense polynucleotide for editing capable of hybridizing to a sequence adjacent to the upstream and downstream of a target region corresponding to an antisense strand of a genomic region into which RNA is transcribed to interact in a sequence-specific manner with the genome in the cell; The single-stranded sense polynucleotide for editing has an upstream homology arm and a downstream homology arm, the upstream homology arm has a sequence capable of hybridizing to a sequence adjacent to the upstream of the target region, and the downstream homology arm has a sequence capable of hybridizing to a sequence adjacent to the downstream of the target region, the upstream homology arm and the downstream homology arm can hybridize to at least the upstream and downstream of the target region, and there may be any sequence or no sequence between the upstream homology arm and the downstream homology arm; the step (A) is carried out in the presence of a single-stranded antisense polynucleotide, the antisense polynucleotide having a sequence capable of hybridizing to a partial region of RNA transcribed from a genomic region and having a sequence corresponding to a terminal portion of the editing sense strand polynucleotide (e.g., a sequence having complementarity to the partial region); The antisense polynucleotide has a length of 40 bases or more, By the above process, the target region is replaced with a base sequence between the upstream homology arm and the downstream homology arm of the single-stranded form of the editing sense polynucleotide, provided that if there is no base sequence between the upstream homology arm and the downstream homology arm, the target region is deleted. method. [2A] The method according to [1A] above, wherein the single-stranded sense polynucleotide for editing is a polynucleotide selected from the group consisting of DNA, RNA, and combinations thereof. [3A] The method according to [1A] or [2A] above, wherein the single-stranded antisense polynucleotide is DNA. [4A] The method according to any one of [1A] to [3A] above, wherein the terminal portion has a length of 5 bases or more. [5A] The method according to any one of [1A] to [4A] above, wherein the terminal portion has a length of 20 bases or less. [6A] The method described in any one of [1A] to [5A] above, wherein the single-stranded sense polynucleotide for editing has a length of 60 to 200 bases. [7A] The method according to any one of [1A] to [6A] above, wherein the single-stranded antisense polynucleotide has a length of 50 to 200 bases. [8A] The method according to any one of [1A] to [7A] above, wherein the target region is present in an intron. [9A] The method according to any one of [1A] to [8A] above, wherein the step (A) is carried out in the presence of at least two types of hybridizable single-stranded antisense polynucleotides, and at least one type of antisense polynucleotide has a sequence (e.g., a sequence having complementarity) that can hybridize to a partial region of RNA transcribed from a genomic region and having a sequence corresponding to the 5'-terminal portion of the sense strand polynucleotide for editing, and at least one type of antisense polynucleotide has a sequence (e.g., a sequence having complementarity) that can hybridize to a partial region of RNA transcribed from a genomic region and having a sequence corresponding to the 3'-terminal portion of the sense strand polynucleotide for editing. [10A] A genome editing kit for use in the method described in any one of [1A] to [8A] above, comprising a combination of the single-stranded sense polynucleotide for editing and the single-stranded antisense polynucleotide. [11A] A genome editing kit for use in the method described in any one of [1A] to [8A] above, comprising a DNA encoding the single-stranded sense polynucleotide for editing and a DNA encoding the single-stranded antisense polynucleotide, the DNAs being expressibly contained in one or more expression vectors. [12A] A genome editing kit for use in the method described in any one of [1A] to [8A] above, comprising a DNA encoding the single-stranded sense polynucleotide for editing and the single-stranded antisense polynucleotide, wherein the DNA encoding the single-stranded sense polynucleotide for editing is expressibly contained in an expression vector, and the single-stranded antisense polynucleotide is DNA. [13A] A genome editing kit for use in the method described in any one of [1A] to [8A] above, comprising a polynucleotide encoding the single-stranded sense polynucleotide for editing and DNA encoding the single-stranded antisense polynucleotide, the DNAs being expressibly contained in one or more expression vectors. [14A] A genome editing kit for use in the method described in [9A] above, comprising a combination of the single-stranded sense polynucleotide for editing and at least two types of single-stranded antisense polynucleotides. [15A] A genome editing kit for use in the method described in [9A] above, comprising a DNA encoding the single-stranded sense polynucleotide for editing and a DNA encoding the at least two types of single-stranded antisense polynucleotides, the DNAs being expressibly contained in one or more expression vectors. [16A] A genome editing kit for use in the method described in [9A] above, comprising a DNA encoding the single-stranded sense polynucleotide for editing and at least two types of single-stranded antisense polynucleotides, wherein the DNA encoding the single-stranded sense polynucleotide for editing is expressibly contained in an expression vector, and the single-stranded antisense polynucleotide is DNA. [17A] A genome editing kit for use in the method described in [9A] above, comprising a polynucleotide encoding the single-stranded sense polynucleotide for editing and DNA encoding the at least two types of single-stranded antisense polynucleotides, the DNAs being expressibly contained in one or more expression vectors.

[0101] The term "corresponding sequence" in the section "further inventions provided by the present disclosure" refers to a sequence (particularly a base sequence) at a portion corresponding to the sequence. For example, DNA and RNA transcribed from the DNA have corresponding sequences. It is known that the correspondence between DNA and RNA sequences is such that A, T, G, and C in DNA are A, U, G, and C, respectively. In addition, the term "having complementarity" means that two polynucleotides can hybridize by Watson-Crick type binding in an intracellular environment or an equivalent in vitro environment. In general, A and T (or U) have complementarity, and G and C have complementarity. The "complementarity" in the term "having complementarity" can be 90% or more, 95% or more, or complete complementarity (i.e., 100% complementarity).

[0102] The term "target region" refers to the region that is targeted by a genome modification system.

[0103] <Method of editing genome and method of producing cells having edited genome> According to the present invention, there is provided a method for editing a genome. According to the present invention, there is provided a method for targeting a target region of a genome. According to the present invention, there is provided a method for obtaining a cell having an edited genome.

[0104] In all embodiments, the cell may be a eukaryotic cell. In all embodiments, the cell may be preferably a cloned cell. In all embodiments, the method of the present invention may also be preferably an in vitro method. Thus, in the method of the present invention, an isolated cell may be obtained. The isolated cell obtained in the present invention may be a cell in which the base sequence to be inserted into the target sequence is inserted.

[0105] The cells obtained by the present invention may or may not be cloned. In a preferred embodiment, the cells obtained by the present invention may be cloned and obtained as cloned cells. By making the genome uniform, there is an advantage that subsequent industrial application or genetic manipulation, etc., can be facilitated.

[0106] In the following, the method for editing a genome (or a method for producing a cell having an edited genome) provided by the present invention will be described with reference to Figures 9A and 9B. The method described in Figure 9A is disclosed in detail in WO2018 / 030536 and JP2016-220639A. The region (100) that transcribes RNA of the genome is composed of a sense strand (110) and an antisense strand (120).

[0107] According to the present invention, there is provided a method for editing a genome (or for producing a cell having an edited genome), comprising the steps of: (A) The method includes a step of allowing a single-stranded sense polynucleotide for editing (10) capable of hybridizing to a sequence (122) adjacent to the upstream side and a sequence (123) adjacent to the downstream side of a target region (121) corresponding to the antisense strand of a genomic region into which RNA is transcribed to interact in a sequence-specific manner with the genome (100) in the cell. Here, "adjacent" means adjacent to the target region (i.e., consecutive in sequence).

[0108] According to the present invention, there is provided a method for editing a genome in a cell, comprising the steps of: (A) A method is provided, comprising a step of allowing a single-stranded form of an editing sense polynucleotide (10) capable of hybridizing to a sequence (122) adjacent to the upstream side and a sequence (123) adjacent to the downstream side of a target region (121) corresponding to the antisense strand of a genomic region from which RNA is transcribed to interact in a sequence-specific manner with the genome in the cell.

[0109] According to the present invention there is provided a method for targeting a genome in a cell, comprising the steps of: (A) A method is provided, comprising a step of allowing a single-stranded sense polynucleotide for editing (10) capable of hybridizing to a sequence (122) adjacent to the upstream side and a sequence (123) adjacent to the downstream side of a target region (121) corresponding to the antisense strand of a genomic region into which RNA is transcribed to interact in a sequence-specific manner with the genome (100) in the cell.

[0110] In one embodiment of the present invention, the single-stranded sense polynucleotide for editing (10) has an upstream homology arm (11) and a downstream homology arm (12). The upstream homology arm (11) has a sequence capable of hybridizing to a sequence (122) adjacent to the upstream of the target region, and the downstream homology arm (12) has a sequence capable of hybridizing to a sequence (123) adjacent to the downstream of the target region (121), and the upstream homology arm (11) and the downstream homology arm (12) can hybridize to at least the sequence (122) adjacent to the upstream of the target region and the sequence (123) adjacent to the downstream of the target region, respectively.

[0111] The single-stranded form of the editing sense polynucleotide (10) can have an arbitrary sequence (13) between the upstream homology arm (11) and the downstream homology arm (12). The arbitrary sequence (13) is, for example, replaced with the sequence (111) of the corresponding region of the complementary strand of the target region (121). Specifically, the target region (121) is replaced with a sequence (23) complementary to the arbitrary sequence (13), and the region (111) corresponding to the target sequence on the complementary strand is replaced with the sequence of the arbitrary sequence (13). Thus, the arbitrary sequence (13) is determined by how one wishes to modify the region (111) corresponding to the target sequence on the complementary strand. That is, the region (111) can be replaced with the arbitrary sequence (13). For example, a part or all of the region (111) corresponding to the target sequence on the complementary strand can be replaced with a new sequence (i.e., the sequence of the arbitrary sequence (13)). When the sequence of the arbitrary sequence (13) has an insertion of a sequence relative to the region (111) corresponding to the target sequence on the complementary strand, the new sequence can be inserted into the region (111). When the sequence of the arbitrary sequence (13) has a deletion of a part or the whole of a sequence relative to the region (111) corresponding to the target sequence on the complementary strand, the sequence can be deleted from the region (111). The single-stranded sense polynucleotide (10) for editing may not have a base in the arbitrary sequence (13) {i.e., the upstream homology arm (11) and the downstream homology arm (12) may be seamlessly connected}. When the arbitrary sequence (13) has no base, the target region (121) and the region (111) corresponding to the target sequence on the complementary strand are deleted in their entirety. This is a method for editing a genome using a single-stranded polynucleotide that applies the mechanism of PODiR. A cell with an edited genome can be obtained, for example, by cloning and analyzing the sequence of a region including the target region of the genome.

[0112] In a preferred embodiment, as shown in Figure 9B, the step (A) may be performed in the presence of either or both of a single-stranded antisense polynucleotide (20) and a single-stranded antisense polynucleotide (30). The antisense polynucleotide (20) is a partial region of RNA (50) transcribed from a genomic region (120), and has a sequence capable of hybridizing to a region (51) having a sequence corresponding to the 5'-end portion of the editing sense strand polynucleotide and a sequence (53) upstream of the region. The antisense polynucleotide (30) is a partial region of RNA (50) transcribed from a genomic region (120), and has a sequence capable of hybridizing to a region (52) having a sequence corresponding to the 3'-end portion of the editing sense strand polynucleotide and a sequence (54) downstream of the region.

[0113] In a preferred embodiment, the length of region (51) and region (52) may be 1 to 20 bases, for example, 2 to 19 bases, 3 to 18 bases, 4 to 17 bases, 5 to 16 bases, 6 to 15 bases, 7 to 14 bases, 8 to 13 bases, 9 to 12 bases, 10 bases, or 11 bases. Accordingly, the length of region (21) of single-stranded antisense polynucleotide (20) and region (31) of single-stranded antisense polynucleotide (30) that bind to this region may also be 1 to 20 bases, for example, 2 to 19 bases, 3 to 18 bases, 4 to 17 bases, 5 to 16 bases, 6 to 15 bases, 7 to 14 bases, 8 to 13 bases, 9 to 12 bases, 10 bases, or 11 bases, respectively.

[0114] In certain preferred embodiments, the upstream and downstream homology arms can each be 10 or more, 20 or more, 30 or more, 40 or more, 50 or more, 60 or more, 70 or more, 80 or more, 90 or more, or 100 or more nucleotides in length. In certain embodiments, the upstream and downstream homology arms can each be 200 or less, 190 or less, 180 or less, 170 or less, 160 or less, 150 or less, 140 or less, 130 or less, 120 or less, 110 or less, 100 or less, 90 or less, 80 or less, 70 or less, 60 or less, 50 or less, or 40 or less. In certain embodiments, the upstream homology arm and the downstream homology arm can each be 10-200 nucleotides, 20-150 nucleotides, 30-100 nucleotides, 35-70 nucleotides, or 40-50 nucleotides in length. The upstream homology arm and the downstream homology arm can be of different lengths or the same length. With respect to the upstream homology arm and the downstream homology arm, the shorter arm can be 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more in length of the longer arm.

[0115] In a preferred embodiment, the antisense polynucleotide (20) and the antisense polynucleotide (30) can independently have a length of more than 10 bases, more than 15 bases, more than 20 bases, more than 25 bases, more than 30 bases, more than 35 bases, or more than 40 bases. In a more preferred embodiment, the antisense polynucleotide (20) and the antisense polynucleotide (30) can independently have a length of more than 50 bases, more than 60 bases, more than 70 bases, more than 80 bases, more than 90 bases, more than 100 bases, more than 110 bases, more than 120 bases, or more than 130 bases. The antisense polynucleotide (20) and the antisense polynucleotide (30) may independently have a length of, for example, 200 bases or less, 190 bases or less, 180 bases or less, 170 bases or less, 160 bases or less, 150 bases or less, 140 bases or less, or 130 bases or less. The antisense polynucleotide (20) and the antisense polynucleotide (30) may have the same length. The antisense polynucleotide (20) may be within ±50%, ±40%, ±30%, ±25%, ±20%, ±15%, ±10%, or ±5% of the length of the antisense polynucleotide (30). In addition, the length of the antisense polynucleotide (30) may be within ±50%, ±40%, ±30%, ±25%, ±20%, ±15%, ±10%, or ±5% of the length of the antisense polynucleotide (20).

[0116] Without being bound by any theory, this reduces the effect of RNA (50) hybridizing to the antisense strand (120) of the genome in the presence of RNA (50), preventing the sense polynucleotide for editing (10) from binding to the antisense strand (120), thereby making it easier for the sense polynucleotide for editing (10) to bind to the antisense strand (120).

[0117] In one embodiment of the present invention, the step (A) is carried out in the presence of at least two types of hybridizable single-stranded antisense polynucleotides, at least one type of antisense polynucleotide having a sequence capable of hybridizing to a partial region of RNA transcribed from a genomic region and having a sequence corresponding to the 5'-end portion of the editing sense strand polynucleotide, and at least one type of antisense polynucleotide having a sequence capable of hybridizing to a partial region of RNA transcribed from a genomic region and having a sequence corresponding to the 3'-end portion of the editing sense strand polynucleotide. This makes it easier for the editing sense polynucleotide (10) to bind to the antisense strand (120) compared to the case where one type of single-stranded antisense polynucleotide is used.

[0118] The sense polynucleotide for editing contains, as a building block, a nucleotide selected from the group consisting of DNA, RNA, and derivatives thereof. The derivative may be a modified nucleotide or modified nucleic acid. Examples of modified nucleic acids include nucleic acids modified with fluorescent dyes, biotinylated nucleic acids, and nucleic acids with cholesteryl groups introduced therein. In order to enhance the stability of RNA, the bases may be modified with 2'-O-methyl, 2'-fluoro, or 2'-methoxyethyl (MOE), and the phosphodiester bond in the nucleic acid backbone may be replaced with a phosphorothioate bond. Examples of artificial nucleic acids include nucleic acids in which the oxygen atom at the 2' position and the carbon atom at the 4' position are crosslinked. Examples of such artificial nucleic acids include locked nucleic acid (LNA), which is a crosslinked DNA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are crosslinked via methylene, ENA, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are crosslinked via ethylene, and BNA, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are crosslinked via -CHOCH-. COC BNA in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -NR-CH2- {where R is a methyl or hydrogen atom} NCExamples of such bridged nucleic acids (BNAs) include cMOE, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH2(OCH3)-, cEt, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via -CH2(CH3)-, AmNA, in which the carbon atoms at the 2' position and the 4' position are bridged via an amide, scpBNA, in which the oxygen atom at the 2' position and the carbon atom at the 4' position are bridged via a methylene and a cyclopropane is formed at the 6' position, and peptide nucleic acids (PNAs) in which a polymer in which N-(2-aminoethyl)glycine is amide-bonded instead of deoxyribose or ribose is the main chain. In addition, the sense polynucleotide for editing may be a hybrid molecule of DNA and RNA.

[0119] The binding between DNA and RNA is stronger than that between DNA and DNA. In consideration of this, the antisense polynucleotide (20) and the antisense polynucleotide (30) may be a polynucleotide having a strong binding affinity to RNA in order to improve the affinity to RNA, and for example, the polynucleotide may be DNA, LNA, BNA, morpholino oligo, or a combination thereof. In addition, the sense polynucleotide for editing may be a polynucleotide having a strong binding affinity to DNA in order to improve the binding to DNA, and may include, for example, a building block selected from the group consisting of RNA, LNA, BNA, and modified RNA, or a combination thereof.

[0120] As a result, the method of the present invention allows genome editing in eukaryotic cells, specifically, replacing a target region (121) of the genome with the sequence of the complementary strand of an arbitrary sequence (13) between the upstream homology arm (11) and the downstream homology arm (12), and replacing the sequence of a region (111) corresponding to the said region of the complementary strand of the target region (121) with the arbitrary sequence (13). In the absence of the arbitrary sequence (13), the target region (121) of the genome and the region (111) corresponding to the said region of the complementary strand will be deleted.

[0121] In some embodiments, the target region is present in the region where the RNA is transcribed. The region where the RNA is transcribed can be a region where transcriptional activity is activated. In some embodiments, the target region can be an exon portion. In some embodiments, the target region is an intron portion. The size of the target region is not particularly limited, and may be, for example, 10 kbp or less, 9 kbp or less, 8 kbp or less, 7 kbp or less, 6 kbp or less, 5 kbp or less, 4 kbp or less, 3 kbp or less, 2 kbp or less, 1 kbp or less, 900 bp or less, 800 bp or less, 700 bp or less, 600 bp or less, 500 bp or less, 400 bp or less, 300 bp or less, 200 bp or less, 100 bp or less, 90 bp or less, 80 bp or less, 70 bp or less, 60 bp or less, 50 bp or less, 40 bp or less, 30 bp or less, 20 bp or less, 10 bp or less, 9 bp or less, 8 bp or less, 7 bp or less, 6 bp or less, 5 bp or less, 4 bp or less, 3 bp or less, 2 bp or less, 1 bp or less, or 0 bp {0 bp may be set, for example, when inserting a sequence into the target region}.

[0122] In some embodiments, the method of the present invention does not include the cleavage of the target region by the endonuclease of exogenous DNA.In some embodiments, the method of the present invention does not use the endonuclease of exogenous DNA in the editing and targeting of the target region.In some embodiments, the method of the present invention does not use ZFN, TALEN and CRISPR / Cas system in the editing and targeting of the target region.In some embodiments, other genome editing tools can be used in combination to edit regions other than the target region of the method of the present invention.

[0123] According to the present invention, there is provided a method for targeting a target region of a genome, comprising: (A) allowing a single-stranded sense polynucleotide for editing capable of hybridizing to a sequence adjacent to the upstream and downstream of the target region corresponding to the antisense strand of the genome region into which RNA is transcribed to interact in a sequence-specific manner with the genome in the cell; The single-stranded sense polynucleotide for editing has an upstream homology arm and a downstream homology arm, the upstream homology arm has a sequence capable of hybridizing to a sequence adjacent to the upstream of the target region, and the downstream homology arm has a sequence capable of hybridizing to a sequence adjacent to the downstream of the target region, the upstream homology arm and the downstream homology arm can hybridize to at least the upstream and downstream of the target region, and there may be any sequence or no sequence between the upstream homology arm and the downstream homology arm; the step (A) is carried out in the presence of a single-stranded antisense polynucleotide, the antisense polynucleotide having a sequence capable of hybridizing to a partial region of an RNA transcribed from a genomic region, the region having a sequence corresponding to a terminal portion of the editing sense strand polynucleotide; The antisense polynucleotide has a length of 40 bases or more. A method is provided.

[0124] A substance (e.g., a label or a protein) can be conjugated to the single-stranded form of the editing sense polynucleotide, thereby allowing the substance (e.g., the label or protein) to be recruited (or targeted) to a target region of the genome. The label can be a label such as a fluorescent label, a dye, an RI label, and a tag. In this embodiment, the single-stranded form of the editing sense polynucleotide (10) may have, as an arbitrary sequence (13) between the upstream homology arm (11) and the downstream homology arm (12), a sequence identical to the sequence (111) of a region corresponding to the target region in the complementary strand of the target region.

[0125] According to the present invention, there is provided a genome targeting kit or genome editing kit, comprising a single-stranded sense polynucleotide for editing (10), and at least one, preferably both, of a single-stranded antisense polynucleotide (20) and a single-stranded antisense polynucleotide (30). The single-stranded sense polynucleotide for editing (10), the single-stranded antisense polynucleotide (20), and the single-stranded antisense polynucleotide (30) are as described above.

[0126] According to the present invention, there is provided a genome targeting kit or genome editing kit, comprising a polynucleotide encoding a single-stranded form of a sense polynucleotide for editing (10) and at least one, preferably both, of a single-stranded form of an antisense polynucleotide (20) and a single-stranded form of an antisense polynucleotide (30). The polynucleotide encoding the single-stranded form of the sense polynucleotide for editing (10) is operably linked to a control sequence, thereby enabling these polynucleotides to be expressed.

[0127] According to the present invention, there is provided a genome targeting kit or genome editing kit, comprising a single-stranded sense polynucleotide for editing (10) and at least one, preferably both, of a polynucleotide encoding a single-stranded antisense polynucleotide (20) and a polynucleotide encoding a single-stranded antisense polynucleotide (30). The polynucleotide encoding the single-stranded antisense polynucleotide (20) and the polynucleotide encoding the single-stranded antisense polynucleotide (30) are each operably linked to a control sequence, thereby enabling these polynucleotides to be expressed.

[0128] According to the present invention, there is provided a genome targeting kit or genome editing kit, comprising a polynucleotide encoding a single-stranded form of an editing sense polynucleotide (10), and at least one of, and preferably both of, a single-stranded form of an antisense polynucleotide (20) and a single-stranded form of an antisense polynucleotide (30). The polynucleotide encoding the single-stranded form of an editing sense polynucleotide (10), the polynucleotide encoding the single-stranded form of an antisense polynucleotide (20), and the polynucleotide encoding the single-stranded form of an antisense polynucleotide (30) are each operably linked to a control sequence. This allows these polynucleotides to be expressed.

[0129] In the above embodiment, the polynucleotide encoding the single-stranded form of the editing sense polynucleotide (10), the polynucleotide encoding the single-stranded form of the antisense polynucleotide (20), and the polynucleotide encoding the single-stranded form of the antisense polynucleotide (30) are each operably linked to a control sequence and may be contained in one or more expression vectors.

[0130] In the above embodiment, the genome targeting kit is preferably used in the genome targeting method of the present invention. In the genome targeting kit, the single-stranded sense polynucleotide for editing (10) is RNA, and can target the target region of the genome in the form of RNA.

[0131] In the above aspect, the genome editing kit is preferably used in the genome editing method of the present invention. The genome editing kit may further include an introduction reagent for introducing a polynucleotide into a cell.

[0132] In all of the above embodiments, the single-stranded sense polynucleotide for editing (10), the single-stranded antisense polynucleotide (20) and the single-stranded antisense polynucleotide (30) may be provided in a state of being incorporated into a single strand. Thus, according to the present invention, there is provided a single-stranded chimeric polynucleotide comprising the single-stranded antisense polynucleotide (20), the single-stranded sense polynucleotide for editing (10) and the single-stranded antisense polynucleotide (30) in this order. The present invention also provides an expression vector comprising DNA encoding the single-stranded chimeric polynucleotide in an expressible manner.

[0133] According to the present invention, there are provided a cell having a genome modified by the genome editing method of the present invention, and an organism containing said cell. EXAMPLES

[0134] Example 1: Genome editing using sense polynucleotides for editing and antisense polynucleotides for promoting editing

[0135] A single-stranded sense strand polynucleotide (editing sense polynucleotide) that directly contributes to genome editing binds to genomic DNA and modifies the genomic DNA to the sequence of the editing sense polynucleotide. The first step of genome editing is for the editing sense polynucleotide to hybridize with the antisense DNA of the genomic DNA. However, when the transcription region of the genomic DNA is targeted, newly synthesized RNA such as mRNA or a fragment thereof (e.g., a fragment removed by splicing) is hybridized on the DNA, which may inhibit the editing sense polynucleotide from hybridizing to the antisense side of the DNA. In particular, the inventors' experiments have revealed that genome editing targeting introns is less efficient than genome editing targeting exons. In introns, RNA fragments from the processed and excised mRNA may bind to the intron portion of the genomic DNA and inhibit genome editing.

[0136] In order to dissociate the newly synthesized RNA hybridized to the antisense strand of the genomic DNA from the genomic DNA, we designed an antisense polynucleotide for detachment. The antisense polynucleotide for detachment was designed to bind to a DNA region that partially overlaps with the DNA region to which the sense polynucleotide for editing hybridizes. The synthesized antisense polynucleotide for detachment was introduced into the system, and genome editing by the sense polynucleotide for editing was tested in the presence of the antisense polynucleotide for detachment.

[0137] Specifically, as shown in Figure 1, an editing sense polynucleotide (101 bases long) represented by (1) was prepared. Also, as shown in Figure 1, peeling antisense polynucleotides represented by (3), (4), (7), (8), (11) to (16), and (22) to (27) were prepared. The peeling antisense polynucleotides represented by (3), (4), (7), (8), (11) to (16), and (22) to (27) are designed to have a sequence that can hybridize to the terminal 10 bases (sometimes called "glue") of the editing sense polynucleotide represented by (1). (3) and (4) are 130 bases long, (7) and (8) are 10 bases long, (11) and (12) are 100 bases long, (13) and (14) are 70 bases long, (15) and (16) are 40 bases long, (22) and (23) are 80 bases long, (24) and (25) are 60 bases long, and (26) and (27) are 50 bases long.

[0138] Also, an editing sense polynucleotide (81 bases long) represented by (2) was prepared as shown in Figure 1. Next, antisense polynucleotides for peeling represented by (5) and (6) each having a length of 120 bases and designed to hybridize to a position not overlapping with (1), and antisense polynucleotides for peeling represented by (9) and (10) each having a length of 130 bases and designed to have a sequence capable of hybridizing to the terminal 10 bases of (2) were prepared.

[0139] Further, as shown in Figure 4, sense polynucleotides for editing represented by (17) and (18) were prepared. In addition, as shown in Figure 6, a sense polynucleotide for editing represented by (19) and antisense polynucleotides for exfoliation represented by (20) and (21) were prepared.

[0140] The polynucleotides (1) to (18) and (22) to (27) are set in the first intron of the region encoding ARFGEF3 (BIG3) of human genomic DNA. The sense polynucleotides (1) and (2) for editing have a base sequence in which one nucleotide has been replaced in the partial sequence on the sense strand of the intron, the sense polynucleotide (17) for editing has a base sequence in which three nucleotides have been inserted in the partial sequence on the sense strand of the intron, and the sense polynucleotide (18) for editing has a base sequence in which two nucleotides have been deleted in the partial sequence on the sense strand of the intron. The antisense polynucleotides (3) to (18) for peeling have a partial sequence on the antisense strand of the partial sequence on the antisense strand of the intron.

[0141] The 10 nucleotides on the 5'-terminus of the editing sense polynucleotides (1), (17) and (18) are complementary to the 10 nucleotides on the 5'-terminus of the exfoliation antisense polynucleotides (3), (11), (13), (15), (22), (24) and (26) and to the exfoliation antisense polynucleotide (7). The 10 nucleotides on the 3'-terminus of the editing sense polynucleotides (1), (17) and (18) are complementary to the 10 nucleotides on the 3'-terminus of the exfoliation antisense polynucleotides (4), (12), (14), (16), (23), (25) and (27) and to the exfoliation antisense polynucleotide (8).

[0142] The 10 nucleotides on the 5' end of the editing sense polynucleotide (2) are complementary to the 10 nucleotides on the 5' end of the exfoliation antisense polynucleotide (9), and the 10 nucleotides on the 3' end are complementary to the 10 nucleotides on the 3' end of the exfoliation antisense polynucleotide (10).

[0143] The editing sense polynucleotide (1) and the exfoliation antisense polynucleotides (5) and (6) do not have complementary sequences, and the editing sense polynucleotide (2) and the exfoliation antisense polynucleotides (3) and (4) do not have complementary sequences.

[0144] The sense polynucleotide for editing (19) is set at a position including the entire first exon of the region encoding ARFGEF3 (BIG3) in human genomic DNA, and has a base sequence in which one nucleotide has been substituted in the sequence on the sense strand side at that position. The antisense polynucleotide for peeling (20) is set at a position including a part of the 5'-end untranslated region and a part of the first exon of the region encoding ARFGEF3, and the antisense polynucleotide for peeling (21) is set within the first intron of the region encoding ARFGEF3, and both have partial sequences on the antisense strand side.

[0145] The 10 nucleotides on the 5' end of the editing sense polynucleotide (19) are complementary to the 10 nucleotides on the 5' end of the exfoliation antisense polynucleotide (20), and the 10 nucleotides on the 3' end are complementary to the 10 nucleotides on the 3' end of the exfoliation antisense polynucleotide (21).

[0146] Specific sequences and SEQ ID NOs of the above polynucleotides are shown in the table below. [Table 2]

[0147] In the above table, capital letters indicate parts that differ from the sequence of the genome to be edited.

[0148] In this way, genome editing was induced in cells using a sense polynucleotide for editing in the presence of an antisense polynucleotide for excision.

[0149] Specifically, the test was carried out as follows. [1] First, HEK293T cells were cultured in Dulbecco's Modified Eagle's Medium-high glucose; DMEM (Sigma, D5796) medium containing 10% (v / v) Fetal Bovine Serum (Sigma, F7524) and 1% (v / v) Antibiotic-Antimycotic (Gibco, 15240-062) until confluent cells in a 10 cm dish. [2] Next, the supernatant was removed using an aspirator, and 1 mL of PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd., 166-23555) and 1 mL of 0.05% Trypsin-EDTA (Gibco, Ltd., 25300-062) were added thereto, followed by incubation at 37°C for 3 minutes. [3] Next, 3.6 mL of DMEM (Sigma, D5796), 0.36 mL of Fetal Bovine Serum (Biowest, S1760), and 0.04 mL of Antibiotic-Antimycotic (Gibco, 15240-062) were added, and the cells were detached from the plate using a pipette bag and collected in a 15 mL centrifuge tube. The tube was then centrifuged at 1000 rpm for 5 minutes to collect the cells at the bottom of the tube, and the supernatant was removed using an aspirator. [4] Then, 4.5 mL of DMEM (Sigma, D5796), 0.45 mL of Fetal Bovine Serum (Sigma, F7524), and 0.05 mL of Antibiotic-Antimycotic (Gibco, 15240-062) were added and the cells were resuspended. [5] Count the number of cells in the suspension and measure it to 2.5 x 10 5 The cells were dispensed into a 6-well multiwell plate (Corning, 3516) so as to give 10 cells / well. [6] The cells were cultured in a CO2 incubator at 37°C for 2 days until they reached 60-80% confluent cells. [7] 150 μl of DNA mixture (Opti-MEM (Gibco, 11058-021) + Fugene HD solution) containing 5 μg of each polynucleotide was added to the wells, and the wells were cultured at 37°C in a CO2 incubator for one day. [8] The supernatant from the HEK293T cells was removed using an aspirator, and 0.3 mL of PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd., 166-23555) and 0.3 mL of 0.05% Trypsin-EDTA (Gibco, Ltd., 25300-062) were added and incubated at 37°C for 3 minutes. [9] Next, 2.7 mL of DMEM (Sigma, D5796), 0.27 mL of Fetal Bovine Serum (Biowest, S1760), and 0.03 mL of Antibiotic-Antimycotic (Gibco, 15240-062) were added, and the cells were detached from the plate using a pipette bag and collected in a 15 mL centrifuge tube. The cells were then collected at the bottom of the tube at 1000 rpm for 5 minutes, and the supernatant was removed using an aspirator.

[10] Then, 9 mL of DMEM (Sigma, D5796), 0.9 mL of Fetal Bovine Serum (Sigma, F7524), and 0.1 mL of Antibiotic-Antimycotic (Gibco, 15240-062) were added, the cells were resuspended, and the cells were seeded onto a 10 cm dish.

[11] The cells were cultured at 37°C in a CO2 incubator for 4 days to allow sufficient proliferation.

[12] The supernatant from HEK293T cells was removed using an aspirator, and 1 mL of PBS(-) (Fujifilm Wako Pure Chemical Industries, Ltd., 166-23555) and 1 mL of 0.05% Trypsin-EDTA (Gibco, Ltd., 25300-062) were added and incubated at 37°C for 3 min.

[13] Next, 3.6 mL of DMEM (Sigma, D5796), 0.36 mL of Fetal Bovine Serum (Biowest, S1760), and 0.04 mL of Antibiotic-Antimycotic (Gibco, 15240-062) were added, and the cells were detached from the plate using a pipette bag and collected in a 15 mL centrifuge tube. The cells were collected at the bottom of the centrifuge tube at 1000 rpm for 5 min, and the supernatant was removed using an aspirator.

[14] Then, 4.5 mL of DMEM (Sigma, D5796), 0.45 mL of Fetal Bovine Serum (Sigma, F7524), and 0.05 mL of Antibiotic-Antimycotic (Gibco, 15240-062) were added and the cells were resuspended.

[15] Measure the number of cells in the suspension and determine a cell count of 2.0 × 10 6 The cell suspension was transferred to a 1.5 mL tube and the cells were collected at the bottom of the tube at 1000 rpm for 5 minutes, and the supernatant was removed using an aspirator.

[16] The cell pellet (2.0 × 10 6 Genome DNA was extracted from 1000 ribosomal DNA (1000 ribosomal DNA; ...

[0150] The results are shown in Figure 2. As shown in Figure 2, the genome editing efficiency using the editing sense polynucleotide represented by (1) in the absence of the exfoliation antisense polynucleotide was 0.0026%, whereas the genome editing efficiency was significantly improved in the presence of the exfoliation antisense polynucleotides of the combinations of (3) and (4), (11) and (12), and (13) and (14).

[0151] As further shown in Figure 2, the genome editing efficiency with the editing sense polynucleotide represented by (2) was 0.0056%, whereas in the presence of the exfoliation antisense polynucleotides (9) and (10), the genome editing efficiency was 12.1425%.

[0152] In addition, genome editing was performed using the sense polynucleotide for editing (1) in the presence of the antisense polynucleotides for peeling (11) to (16) and (22) to (27) using the same method as in the above test method [7], except that the amount of each polynucleotide contained in the DNA mixture solution used was changed from 5 μg to 50 pmol. As shown in Figure 3, the genome editing efficiency with the sense polynucleotide for editing (1) was improved in the presence of any combination of antisense polynucleotides for peeling. In particular, the improvement in genome editing efficiency was remarkable in the presence of the combination of (11) and (12), the combination of (13) and (14), and the combination of (22) and (23).

[0153] In the above experiment, the genome editing efficiency was confirmed using eukaryotic cells. As a result, the efficiency of genome editing by the sense polynucleotide for editing was improved in the presence of the antisense polynucleotide for exfoliation. This suggests that the antisense polynucleotide for exfoliation removed the factors that hinder genome editing by the sense polynucleotide for editing. The antisense polynucleotide for exfoliation was originally designed with the intention of inhibiting RNA transcribed from genomic DNA from binding to DNA. It was suggested that the transcribed RNA was probably linked to the genomic DNA by DNA-RNA interaction, which inhibited the sense polynucleotide for editing from hybridizing to the region. Since the DNA-RNA interaction is stronger than the DNA-DNA interaction, it is considered that the inhibitory effect is particularly large when DNA is used as the sense polynucleotide for editing.

[0154] Furthermore, as shown in Figure 4, instead of the editing sense polynucleotide (1) having a point mutation, the same experiment as above was performed using an editing sense polynucleotide (18) having a base deletion and an editing sense polynucleotide (17) having a base insertion. The results are shown in Figure 5. As shown in Figure 5, in both cases where an editing sense polynucleotide (18) having a base deletion was used and where an editing sense polynucleotide (17) having a base insertion was used, the editing efficiency was greatly improved in the presence of an antisense polynucleotide.

[0155] As shown in FIG. 6, the same experiment as above was performed using the sense polynucleotide for editing (19) in the presence of antisense polynucleotides (20) and (21) targeting exons. The results are shown in FIG. 7. As shown in FIG. 7, even when an exon was targeted, the editing efficiency by the sense polynucleotide for editing was greatly improved in the presence of antisense polynucleotides on both sides (antisense polynucleotides (20) and (21)) compared to the absence of the antisense polynucleotide. Also, as shown in FIG. 7, even in the presence of only one antisense polynucleotide (21), the editing efficiency was improved by about 7 to 8 times compared to the absence of the antisense polynucleotide. From this, it was revealed that an antisense polynucleotide designed to be partially overlapped with the sense strand significantly improves the editing efficiency even if it is only on one side.

[0156] From the above, it has been demonstrated that an antisense polynucleotide designed to bind to a DNA region that partially overlaps with the DNA region to which an editing sense polynucleotide hybridizes can be used as an editing-promoting antisense polynucleotide.

Claims

1. A genome editing promoter comprising a single-stranded form of an editing-promoting polynucleotide for use in combination with a single-stranded form of an editing polynucleotide capable of modifying a target site in a double-stranded genomic DNA, The target site comprises a primary editing site set on one genomic DNA strand and a secondary editing site on the other genomic DNA strand at a position corresponding to the primary editing site; The polynucleotide for editing is, in order from the 5' end, a) A portion capable of hybridizing to the region adjacent to the 3' end of the primary editing site x) any portion consisting of a base sequence that is not identical to the base sequence of the secondary editing site; and b) A portion capable of hybridizing to the region adjacent to the 5' end of the primary editing site. or Part a) and part b) It consists of: the length of portion a) and portion b) are each independently 10 to 100 nucleotides; The editing-facilitating polynucleotide is capable of hybridizing to a nucleic acid sequence that is complementary to a predetermined region on the one genomic DNA strand, wherein the predetermined region overlaps with region A at its 5' end or overlaps with region B at its 3' end, where region A is a region on the one genomic DNA strand that is located at a position corresponding to portion a) of the editing polynucleotide and region B is a region on the one genomic DNA strand that is located at a position corresponding to portion b) of the editing polynucleotide; the length of the overlap with region A at the 5' end of the predetermined region is the same as or shorter than region A, and the length of the overlap with region B at the 3' end of the predetermined region is the same as or shorter than region B; the length of the editing-promoting polynucleotide is 50 nucleotides or more; When the editing polynucleotide is composed of part a), part x), and part b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a part consisting of a base sequence complementary to part x) can be inserted into the primary editing site, and part x) can be inserted into the secondary editing site, When the editing polynucleotide is composed of part a), part x), and part b), and the primary editing site is set to one nucleotide or a plurality of consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a part consisting of a base sequence complementary to part x), and the secondary editing site can be replaced with part x; The genome editing promoter, which is capable of deleting a target site when the editing polynucleotide consists of part a) and part b) and the primary editing site is set at one nucleotide or multiple consecutive nucleotides on one genomic DNA strand.

2. The genome editing promoter of claim 1, wherein the length of the overlap is 5 nucleotides or more.

3. The editing-promoting polynucleotide according to claim 1 or 2, its expression vector, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance, and Editing polynucleotide, its expression vector, or conjugate of said editing polynucleotide or its expression vector with other substance A genome editing kit comprising: The polynucleotide for editing is, in order from the 5' end, a) A portion capable of hybridizing to the region adjacent to the 3' end of the primary editing site x) any portion consisting of a base sequence that is not identical to the base sequence of the secondary editing site; and b) A portion capable of hybridizing to the region adjacent to the 5' end of the primary editing site. or Part a) and part b) The genome editing kit comprises:

4. An editing-promoting polynucleotide configured so that the predetermined region overlaps with region A at its 5' end, an expression vector thereof, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance; and An editing-promoting polynucleotide configured so that the predetermined region overlaps with region B at its 3' end, an expression vector thereof, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance. The genome editing kit of claim 3 .

5. The genome editing kit of claim 3 or 4, wherein the length of the overlap for the editing-promoting polynucleotide is 5 nucleotides or more.

6. A method for modifying a target site in double-stranded genomic DNA of a cell (excluding human gametes and fertilized eggs) or a non-human organism, comprising a step of treating the cell (excluding human gametes and fertilized eggs) or a non-human organism with a genome editing kit described in any one of claims 3 to 5, with the proviso that when the cell is a human cell excluding human gametes and fertilized eggs, the step is carried out in vitro.

7. A method for producing a cell (excluding human gametes and fertilized eggs) or a non-human organism in which a target site in double-stranded genomic DNA has been modified, comprising a step of treating the cell (excluding human gametes and fertilized eggs) or non-human organism with the genome editing kit described in any one of claims 3 to 5, with the proviso that when the cell is a human cell excluding human gametes and fertilized eggs, the step is carried out in vitro.

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