Genome editing method

Abstract

The present invention provides: a single-stranded editing-promoting polynucleotide for use in combination with a single-stranded editing polynucleotide capable of modifying a target site in double-stranded genomic DNA; a genome-editing kit comprising the editing polynucleotide or an expression vector thereof, and the editing-promoting polynucleotide or an expression vector thereof; a method for modifying the target site in the double-stranded genomic DNA in a cell or non-human organism, comprising a step of processing the cell or organism with the genome-editing kit; and a method for producing a cell or an organism in which the target site of the double-stranded genomic DNA has been modified, comprising the above-described step.

Description

Method for editing genome

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

[0002] Genome editing technology is a technology for modifying the base sequence of genomic DNA using a site-specific nuclease that can recognize and cleave a specific base sequence. In particular, the genome editing technology called the CRISPR / Cas system has attracted attention as a revolutionary method that overcomes the disadvantages such as the difficulty of design, low editing efficiency, and complexity of work pointed out in the initial genome editing using ZFN (Zinc-Finger Nuclease) or TALEN (Transcription activator effector-like nuclease), and its use is being promoted not only in academic research but also in various fields such as agriculture, forestry, fisheries, livestock, and medicine.

[0003] However, in addition to the delivery of the site-specific nuclease, which is a large molecule, into the cell, the CRISPR / Cas system has been pointed out as a problem for practical use that mutations occur at positions other than the site to be edited, so-called off-target.

[0004] The research group of the present inventors has developed a new genome editing technology using a single-stranded form of polynucleotide that does not require the delivery of a site-specific nuclease (Patent Documents 1 and 2, etc.). Using this technology, genome editing can be performed simply by expressing or introducing a single-stranded form of polynucleotide into the cell, and it has the advantage that it is not necessary to introduce components other than the single-stranded form of polynucleotide, typically a site-specific nuclease, into the cell.

[0005] In addition, the research group of the present inventors has developed a single-stranded form of polynucleotide for promoting editing (referred to as editing polynucleotide) for use in combination with a single-stranded form of polynucleotide (referred to as editing polynucleotide) that can modify the target site of double-stranded genomic DNA. The polynucleotide for promoting editing can improve the efficiency of genome editing by the editing polynucleotide.

[0006] Japanese Patent Publication No. 6899564, Japanese Unexamined Patent Publication No. 2016-220639, WO2022 / 249775 A1

[0007] This invention provides a means that can be used in genome editing technology utilizing single-stranded polynucleotides to improve genome editing efficiency.

[0008] The inventors have found that the efficiency of genome editing by editing polynucleotides can be further enhanced by designing a single-stranded editing-promoting polynucleotide for use in combination with editing polynucleotides. Furthermore, the inventors have found that the efficiency of genome editing by editing polynucleotides can be further enhanced by repeatedly treating cells or organisms with editing polynucleotides at predetermined intervals.

[0009] This disclosure provides: [Item 1A] A single-stranded editing-promoting polynucleotide for use in combination with a single-stranded editing polynucleotide capable of modifying a target site of double-stranded genomic DNA, wherein 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, and the editing polynucleotide consists of, in order from the 5' end: a) a portion that can hybridize with a region adjacent to the 3' end of the primary editing site; x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site; and b) a portion that can hybridize with a region adjacent to the 5' end of the primary editing site, or portion a) and portion b). The editing-promoting polynucleotide is hybridizable with a nucleic acid sequence complementary to a predetermined region on one of the genomic DNA strands, where the predetermined region is defined as region A, where region A is located at a position corresponding to portion a) of the editing polynucleotide on the one genomic DNA strand, and region B is located at a position corresponding to portion b) of the editing polynucleotide, and the predetermined region overlaps with region A at its 5' end or with region B at its 3' end, the length of the overlap with region A at its 5' end is 16 nucleotides or more, preferably 21 nucleotides or more, and the same as or shorter than that of region A, and the length of the overlap with region B at its 3' end is 16 nucleotides or more, preferably 21 nucleotides or more, and the same as or shorter than that of region B. When the predetermined region overlaps with region A at its 5' end, the length of portion a) of the editing polynucleotide is the same as or longer than the overlap; when the predetermined region overlaps with region B at its 3' end, the length of portion b) of the editing polynucleotide is the same as or longer than the overlap; and when the editing polynucleotide consists of portions a), x), and b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a portion consisting of a base sequence complementary to portion x) can be inserted into the primary editing site, and portion x) can be inserted into the secondary editing site.The editing polynucleotide comprises portion a), portion x), and portion b), and when 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 portion consisting of a base sequence complementary to portion x), and the secondary editing site can be replaced with portion x), and when the editing polynucleotide comprises portion a) and portion b), and when the primary editing site is set to one nucleotide or multiple consecutive nucleotides on one genomic DNA strand, the target site can be deleted. [Item 1B] A single-stranded editing-promoting polynucleotide for use in combination with a single-stranded editing polynucleotide capable of modifying a target site of double-stranded genomic DNA, wherein the target site consists of a primary editing site set on one genomic DNA strand and a secondary editing site located on the other genomic DNA strand at a position corresponding to the primary editing site, and the editing polynucleotide consists of, in order from the 5' end, a) a portion consisting of a base sequence having 90% or more identity with the base sequence of the region adjacent to the 5' end of the secondary editing site, x) an arbitrary portion consisting of a base sequence not identical to the base 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 consists of portion a) and portion b). The editing-promoting polynucleotide consists of a base sequence having 90% or more identity with the base sequence of a predetermined region on one of the genomic DNA strands, where the predetermined region is defined as region A, where region A is the region on the one genomic DNA strand corresponding to portion a) of the editing polynucleotide, and region B is the region corresponding to portion b) of the editing polynucleotide, and the predetermined region overlaps with region A at its 5' end or with region B at its 3' end, the length of the overlap with region A at its 5' end is 16 nucleotides or more, preferably 21 nucleotides or more, and the same as or shorter than that of region A, and the length of the overlap with region B at its 3' end is 16 nucleotides or more, preferably 21 nucleotides or more, and the same as or shorter than that of region B.When the predetermined region overlaps with region A at its 5' end, the length of portion a) of the editing polynucleotide is the same as or longer than the overlap; when the predetermined region overlaps with region B at its 3' end, the length of portion b) of the editing polynucleotide is the same as or longer than the overlap; when the editing polynucleotide consists of portions a), x), and b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a portion consisting of a base sequence complementary to portion x) can be inserted into the primary editing site, and portion x) can be inserted into the secondary editing site; when the editing polynucleotide consists of portions a), x), and b), and the primary editing site is set between one nucleotide or more consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a portion consisting of a base sequence complementary to portion x), and the secondary editing site can be replaced with portion x); The editing-promoting polynucleotide comprising part a) and part b), which can delete a target site when the primary editing site is set on one nucleotide or multiple consecutive nucleotides on one genomic DNA strand. [Item 1C] A single-stranded editing-promoting polynucleotide for use in combination with a single-stranded editing polynucleotide capable of modifying a target site of double-stranded genomic DNA, wherein the target site comprises a primary editing site set on one genomic DNA strand and a secondary editing site located on the other genomic DNA strand at a position corresponding to the primary editing site, and the editing polynucleotide comprises, in order from the 5' end, a) a portion consisting of the same base sequence as the region adjacent to the 5' end of the secondary editing site, x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site, and b) a portion consisting of the same base sequence as the region adjacent to the 3' end of the secondary editing site, or comprising part a) and part b),The editing-promoting polynucleotide consists of the same base sequence as a predetermined region on one of the genomic DNA strands, where the predetermined region is defined as region A, where region A is the region on the one genomic DNA strand corresponding to portion a) of the editing polynucleotide, and region B is the region corresponding to portion b) of the editing polynucleotide, and the predetermined region overlaps with region A at its 5' end or with region B at its 3' end, the length of the overlap with region A at its 5' end is 16 nucleotides or more, preferably 21 nucleotides or more, and is the same as or shorter than region A, and the length of the overlap with region B at its 3' end is 16 nucleotides or more, preferably 21 nucleotides or more, and is the same as or shorter than region B. When the predetermined region overlaps with region A at its 5' end, the length of portion a) of the editing polynucleotide is the same as or longer than the overlap; when the predetermined region overlaps with region B at its 3' end, the length of portion b) of the editing polynucleotide is the same as or longer than the overlap; when the editing polynucleotide consists of portions a), x), and b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a portion consisting of a base sequence complementary to portion x) can be inserted into the primary editing site, and portion x) can be inserted into the secondary editing site; when the editing polynucleotide consists of portions a), x), and b), and the primary editing site is set between one nucleotide or more consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a portion consisting of a base sequence complementary to portion x), and the secondary editing site can be replaced with portion x); The editing-promoting polynucleotide comprising part a) and part b), wherein the primary editing site is set to one nucleotide or multiple consecutive nucleotides on one genomic DNA strand, and the target site can be deleted. [Clause 2A] The editing-promoting polynucleotide according to Clause 1A, Clause 1B, or Clause 1C, wherein the length of the editing-promoting polynucleotide is 50 nucleotides or more. [Clause 2B]The editing-promoting polynucleotide according to item 1A, item 1B, item 1C, or item 2A, wherein the overlap length is 16 to 200 nucleotides, preferably 21 to 100 nucleotides. [Item 3A] A genome editing kit comprising a polynucleotide for editing promotion described in Item 1A, Item 2A, or Item 2B, an expression vector thereof, or a conjugate of the polynucleotide for editing promotion or its expression vector with another substance, and a single-stranded polynucleotide for editing, an expression vector thereof, or a conjugate of the polynucleotide for editing or its expression vector with another substance, wherein the polynucleotide for editing comprises, in order from the 5' end, a) a portion that can hybridize with a region adjacent to the 3' end of the primary editing site, x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site, and b) a portion that can hybridize with a region adjacent to the 5' end of the primary editing site, or portion a) and portion b), wherein if the predetermined region overlaps with region A at its 5' end, the length of portion a) is the same as or longer than the overlap, and if the predetermined region overlaps with region B at its 3' end, the length of portion b) is the same as or longer than the overlap, the genome editing kit. [Item 3B] A genome editing kit comprising a polynucleotide for editing promotion described in Item 1B, Item 2A, or Item 2B, its expression vector, or a conjugate of the polynucleotide for editing promotion or its expression vector with another substance, and a single-stranded polynucleotide for editing, its expression vector, or a conjugate of the polynucleotide for editing or its expression vector with another substance, wherein the polynucleotide for editing consists of, in order from the 5' end, a) 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, x) an arbitrary 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 having 90% or more identity with the nucleotide sequence of the region adjacent to the 3' end of the secondary editing site, or portion a) and portion b)The genome editing kit comprises, wherein if the predetermined region overlaps with region A at its 5' end, the length of portion a) is the same as or longer than the overlap, and if the predetermined region overlaps with region B at its 3' end, the length of portion b) is the same as or longer than the overlap. [Item 3C] A genome editing kit comprising a polynucleotide for editing promotion described in Item 1C, Item 2A, or Item 2B, an expression vector thereof, or a conjugate of the polynucleotide for editing promotion or its expression vector with another substance, and a single-stranded polynucleotide for editing, an expression vector thereof, or a conjugate of the polynucleotide for editing or its expression vector with another substance, wherein the polynucleotide for editing comprises, in order from the 5' end, a) a portion consisting of the same base sequence as the base sequence of the region adjacent to the 5' end of the secondary editing site, x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site, and b) a portion consisting of the same base sequence as the region adjacent to the 3' end of the secondary editing site, or portion a) and portion b), wherein if the predetermined region overlaps with region A at its 5' end, the length of portion a) is the same as or longer than the overlap, and if the predetermined region overlaps with region B at its 3' end, the length of portion b) is the same as or longer than the overlap, the genome editing kit. [Clause 4] A genome editing kit according to Clause 3A, Clause 3B, or Clause 3C, comprising: an editing-promoting polynucleotide according to Clause 1A, Clause 1B, Clause 1C, Clause 2A, or Clause 2B, an expression vector thereof, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance, configured such that the predetermined region overlaps with region A at its 5' end; and an editing-promoting polynucleotide according to Clause 1A, Clause 1B, Clause 1C, Clause 2A, or Clause 2B, an expression vector thereof, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance, configured such that the predetermined region overlaps with region B at its 3' end. [Clause 5]A genome editing kit according to claim 3A, claim 3B, claim 3C, or claim 4, wherein the length of the overlap with respect to the editing-promoting polynucleotide is 16 to 200 nucleotides, preferably 21 to 100 nucleotides. [Claim 6] A method for modifying a target site of double-stranded genomic DNA in a cell or organism, comprising the step of treating the cell or organism with a genome editing kit according to claim 3A, claim 3B, claim 3C, claim 4, or claim 5. [Claim 7] A method for producing a cell or organism in which the target site of double-stranded genomic DNA has been modified, comprising the step of treating the cell or organism with a genome editing kit according to claim 3A, claim 3B, claim 3C, claim 4, or claim 5.

[0010] Furthermore, the Disclosure provides: [Section 8A] A method for modifying a target site in double-stranded genomic DNA of a cell or organism, or a method for producing a cell or organism in which a target site in double-stranded genomic DNA has been modified, comprising the step of treating the cell or organism with a single-stranded editing polynucleotide capable of modifying a target site in double-stranded genomic DNA, an expression vector thereof, or a conjugate of the editing polynucleotide or its expression vector with another substance, wherein 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, the editing polynucleotide consists of, in order from the 5' end: a) a portion that can hybridize with a region adjacent to the 3' end of the primary editing site; x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site; and b) a portion that can hybridize with a region adjacent to the 5' end of the primary editing site, or consists of portion a) and portion b), The method wherein, when the editing polynucleotide consists of parts a), x), and b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a portion 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 consists of parts a), x), and b), and the primary editing site is set on one nucleotide or more consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a portion consisting of a base sequence complementary to part x), and the secondary editing site can be replaced with part x); when the editing polynucleotide consists of parts a) and b), and the primary editing site is set on one nucleotide or more consecutive nucleotides on one genomic DNA strand, the target site can be deleted, and the process is performed multiple times at time intervals of 12 to 36 hours.[Item 8B] A method for modifying a target site in double-stranded genomic DNA of a cell or organism, or a method for producing a cell or organism in which a target site in double-stranded genomic DNA has been modified, comprising the step of treating the cell or organism with a single-stranded editing polynucleotide capable of modifying a target site in double-stranded genomic DNA, an expression vector thereof, or a conjugate of the editing polynucleotide or its expression vector with another substance, wherein 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, and the editing polynucleotide consists of, in order from the 5' end, a) a portion consisting of a base sequence having 90% or more identity with the base sequence of the region adjacent to the 5' end of the secondary editing site, x) an arbitrary portion consisting of a base sequence that is not identical to the base 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 consisting of portion a) and portion b), The method wherein, when the editing polynucleotide consists of parts a), x), and b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a portion 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 consists of parts a), x), and b), and the primary editing site is set on one nucleotide or more consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a portion consisting of a base sequence complementary to part x), and the secondary editing site can be replaced with part x); when the editing polynucleotide consists of parts a) and b), and the primary editing site is set on one nucleotide or more consecutive nucleotides on one genomic DNA strand, the target site can be deleted, and the process is performed multiple times at time intervals of 12 to 36 hours.[Item 8C] A method for modifying a target site in double-stranded genomic DNA of a cell or organism, or a method for producing a cell or organism in which a target site in double-stranded genomic DNA has been modified, comprising the step of treating the cell or organism with a single-stranded editing polynucleotide capable of modifying a target site in double-stranded genomic DNA, an expression vector thereof, or a conjugate of the editing polynucleotide or its expression vector with another substance, wherein 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, and the editing polynucleotide consists of, in order from the 5' end, a) a portion consisting of the same base sequence as the region adjacent to the 5' end of the secondary editing site, x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site, and b) a portion consisting of the same base sequence as the region adjacent to the 3' end of the secondary editing site, or portion a) and portion b), The method wherein the editing polynucleotide consists of parts a), x), and b), and the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a portion 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; the method wherein the editing polynucleotide consists of parts a), x), and b), and the primary editing site is set on one nucleotide or more consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a portion consisting of a base sequence complementary to part x), and the secondary editing site can be replaced with part x); the method wherein the editing polynucleotide consists of parts a) and b), and the primary editing site is set on one nucleotide or more consecutive nucleotides on one genomic DNA strand, and the target site can be deleted, and the process is performed multiple times at time intervals of 12 to 36 hours. [Clause 9] The method according to Clause 8A, Clause 8B, or Clause 8C, wherein the editing polynucleotide comprises a DNA high affinity nucleotide analog as a constituent unit.[Clause 10A] The method according to Clause 8A, Clause 8B, Clause 8C, or Clause 9, wherein an editing polynucleotide is used together with an editing-promoting polynucleotide, its expression vector, or its conjugate, the editing-promoting polynucleotide being hybridizable to a nucleic acid sequence complementary to a predetermined region on a genomic DNA strand where a primary editing site is set, wherein the predetermined region is such that, when region A is the region on the genomic DNA strand where a primary editing site is set, and region B is the region at the position corresponding to portion a) of the editing polynucleotide, the predetermined region overlaps with region A at its 5' end or with region B at its 3' end, the length of the overlap with region A at its 5' end is the same as or shorter than that of region A, and the length of the overlap with region B at its 3' end is the same as or shorter than that of region B. The method wherein, when the predetermined region overlaps with region A at its 5' end, the length of portion a) of the editing polynucleotide is the same as or longer than the overlap, and when the predetermined region overlaps with region B at its 3' end, the length of portion b) of the editing polynucleotide is the same as or longer than the overlap.[Clause 10B] The method according to Claim 8A, Claim 8B, Claim 8C, or Claim 9, wherein an editing polynucleotide is used together with an editing-promoting polynucleotide, its expression vector, or its conjugate, the editing-promoting polynucleotide consists of a base sequence having 90% or more identity with the base sequence of a predetermined region on a genomic DNA strand where a primary editing site is set, wherein the predetermined region overlaps with region A at its 5' end or with region B at its 3' end, when region A is the region on the genomic DNA strand where a primary editing site is set and the region B is the region at the position corresponding to portion b) of the editing polynucleotide, the predetermined region overlaps with region A at its 5' end or with region B at its 3' end, the length of the overlap with region A at its 5' end is the same as or shorter than that of region A, and the length of the overlap with region B at its 3' end is the same as or shorter than that of region B. The method wherein, when the predetermined region overlaps with region A at its 5' end, the length of portion a) of the editing polynucleotide is the same as or longer than the overlap, and when the predetermined region overlaps with region B at its 3' end, the length of portion b) of the editing polynucleotide is the same as or longer than the overlap.[Clause 10C] The method according to Claim 8A, Claim 8B, Claim 8C, or Claim 9, wherein an editing polynucleotide is used together with an editing-promoting polynucleotide, its expression vector, or its conjugate, the editing-promoting polynucleotide having the same base sequence as a predetermined region on a genomic DNA strand where a primary editing site is set, wherein the predetermined region overlaps with region A at its 5' end or with region B at its 3' end, when region A is the region on the genomic DNA strand where a primary editing site is set and region B is the region at the position corresponding to portion a) of the editing polynucleotide, and the length of the overlap with region A at its 5' end is the same as or shorter than that of region A, and the length of the overlap with region B at its 3' end is the same as or shorter than that of region B. The method wherein, if the predetermined region overlaps with region A at its 5' end, the length of portion a) of the editing polynucleotide is the same as or longer than the overlap, and if the predetermined region overlaps with region B at its 3' end, the length of portion b) of the editing polynucleotide is the same as or longer than the overlap. [Clause 11] The method according to Clause 8A, Clause 8B, Clause 8C, Clause 9, Clause 10A, Clause 10B, or Clause 10C, wherein the process is carried out 2 to 30 times.

[0011] According to the present invention, genome editing can be performed efficiently.

[0012] This graph shows the efficiency of genome editing using editing polynucleotides and editing-promoting polynucleotides for modifying the human HPRT1 gene. The top of the figure shows the positional relationship between the target genomic DNA, editing polynucleotides, and editing-promoting polynucleotides. In the following figure, "Sense strand" refers to the editing polynucleotide, "Antisense strand (upstream only)" and "Antisense strand (upstream)" refer to the editing-promoting polynucleotide whose 5' end is complementary to the 5' end of the editing polynucleotide, "Antisense strand (downstream only)" and "Antisense strand (downstream)" refer to the editing-promoting polynucleotide whose 3' end is complementary to the 3' end of the editing polynucleotide, and "Antisense strand (Pair)" refers to the combination of the two types of editing-promoting polynucleotides mentioned above. This graph shows the efficiency of genome editing using editing polynucleotides and editing-promoting polynucleotides for modifying the human HPRT1 gene. The top of the figure shows the positional relationship between the target genomic DNA, editing polynucleotides, and editing-promoting polynucleotides. This graph shows the efficiency of genome editing using editing polynucleotides and editing-promoting polynucleotides for modifying the human HPRT1 gene. The top of the figure shows the positional relationship between the target genomic DNA, editing polynucleotides, and editing-promoting polynucleotides. This graph shows the efficiency of genome editing using editing polynucleotides and editing-promoting polynucleotides for modifying the human FKTN gene. The top of the figure shows the positional relationship between the target genomic DNA, editing polynucleotides, and editing-promoting polynucleotides. This graph shows the efficiency of genome editing using editing polynucleotides and editing-promoting polynucleotides for modifying the human HPRT1 gene. The top of the figure shows the positional relationship between the target genomic DNA, editing polynucleotides, and editing-promoting polynucleotides. This figure shows the mechanism of genome editing (substitution) using editing polynucleotides. This figure shows the mechanism of genome editing (substitution) using editing polynucleotides and editing-promoting polynucleotides.

[0013] The following description may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments or specific examples. Furthermore, the upper and lower limits of each numerical range exemplified herein can be combined in any way. Also, unless otherwise specified, numerical ranges represented using "~" or "-" in this specification mean ranges that include the numbers at both ends as the upper and lower limits.

[0014] [Definition] In this disclosure, genomic DNA is the DNA contained as a genome in the cells of any organism. Genomic DNA may be the genomic DNA of a prokaryote or the genomic DNA of a eukaryote, and these may include foreign DNA such as viruses as part of their composition.

[0015] Examples of prokaryotes include Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Serratia, Sphingomonas, Brucella, Neisseria gonorrhoeae, Burkholderia, Shigella, Salmonella, Acinetobacter, Vibrio cholerae, Klebsiella pneumoniae, Legionella, Helicobacter pylori, Campylobacter, etc.) and Gram-positive bacteria (Mycoplasma tuberculosis, Staphylococcus aureus, Actinomycetes, Bacillus subtilis, Bacillus anthracis, etc.). The prokaryotes are preferably Gram-negative bacteria, more preferably Pseudomonas aeruginosa, Escherichia coli, Serratia, Sphingomonas, and even more preferably Pseudomonas aeruginosa and Escherichia coli.

[0016] Examples of eukaryotes include animals, such as mammals including humans and non-human mammals (such as monkeys, dogs, cats, rabbits, mice, rats, guinea pigs, hamsters, cows, pigs, goats, sheep, horses, llamas, and camels); birds such as chickens; reptiles such as turtles and crocodiles; amphibians such as African clawed frogs; fish such as zebrafish, medaka, and pufferfish; chordates such as sea squirts; and arthropods such as fruit flies and silkworms.

[0017] Another example of eukaryotes is plants, which include food crops such as rice, corn, potatoes, beans, barley and wheat; horticultural crops (vegetables, fruit trees and flowers), such as leafy vegetables (cabbage, asparagus, etc.), fruit vegetables (eggplant, tomato, cucumber, etc.), root vegetables (radish, carrot, etc.), other vegetables and fruits (e.g., pome fruits such as apples and pears, drupes such as plums, apricots, peaches, cherries, nut fruits such as almonds, walnuts and chestnuts, citrus fruits such as oranges and lemons, tropical fruit trees such as tropical fruits); and ornamental plants.

[0018] Another example of eukaryotes is fungi, such as yeasts, molds, and basidiomycetes, including yeasts, Neurospora crassa, Matsutake, Shiitake, Enokitake, Shimeji, Nameko, and Eryngii. Eukaryotes can also be microalgae.

[0019] The sense strand of genomic DNA refers to the DNA strand within a double helix of DNA that is transcribed into RNA, such as the coding region, that is not used as a transcription template. The antisense strand is the complementary strand to the sense strand that serves as the transcription template. Furthermore, the coding region is not limited to regions that code for proteins, but also includes regions that code for non-protein-coding RNAs (e.g., miRNA, piRNA, tRNA, rRNA, snRNA, gRNA, SRP RNA, pRNA, tncRNA, sbRNA, snlRNA, SLRNA, etc.).

[0020] The terms "genome editing," "editing of genomic DNA," and "modification of genomic DNA" are interchangeable.

[0021] The term "polynucleotide" refers to a polymer or oligomer formed by the linkage of multiple deoxyribonucleotides or ribonucleotides via phosphodiester bonds, and can be used interchangeably with the term "nucleic acid." Examples of polynucleotides include deoxyribonucleic acid (DNA), ribonucleic acid (RNA), and chimeric nucleic acids containing both deoxyribonucleotides and ribonucleotides as constituent units. Polynucleotides may contain nucleotide analogs as constituent units.

[0022] The terms "part" and "region" in relation to polynucleotides are interchangeable and both refer to a single nucleotide or a sequence of nucleotides contained within a polynucleotide. The terms "part" and "region" in relation to double-stranded genomic DNA are interchangeable and both refer to a single pair of nucleotides or a sequence of nucleotides contained within double-stranded genomic DNA.

[0023] In this disclosure, a portion or region of a polynucleotide that is "in a corresponding position" to a portion or region of that polynucleotide means a portion or region on another polynucleotide that is complementary to the portion or region of that polynucleotide.

[0024] Sequence identity refers to the percentage of identical nucleotides relative to all overlapping nucleotides in the optimal alignment calculated using an algorithm known in the art (preferably, the algorithm may consider the introduction of gaps in one or both sequences for optimal alignment). Identity can be calculated, for example, by aligning two sequences using NCBI BLAST (National Center for Biotechnology Information Basic Local Alignment Search Tool) with general settings (e.g., E-value=10; gaps allowed; filtering=ON; match score=1; mismatch score=-3, etc.). If the sequences to be aligned contain uracil, the identity calculation is performed using the sequence after replacing uracil with thymine. If the sequences to be aligned contain modified nucleotides or nucleotide analogs, the identity calculation is performed using the sequence after replacing the modified nucleotides or nucleotide analogs with the original natural deoxyribonucleotides, i.e., adenine, thymine, guanine, or cytosine.

[0025] In this disclosure, a polynucleotide or a portion or region thereof is “hybridizable” to another polynucleotide or a portion or region thereof, meaning that the polynucleotide or a portion or region thereof has the ability to bind to another polynucleotide or a portion or region thereof via complementary hydrogen bonds between bases. Hybridizability can be confirmed by the maintenance of the hybridized 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 in Berger and Kimmel (1987, Guide to Molecular Cloning Techniques Methods in Enzymology, Vol. 152, Academic Press, San Diego CA). If the hybridized state between two polynucleotides or portions or regions is maintained even after washing under stringent conditions, for example, 1×SSC, 0.1%SDS, at 37°C, then they are hybridizable to each other. Stringent conditions may include washing with 0.5×SSC, 0.1%SDS, at 42°C, or washing with 0.1×SSC, 0.1%SDS, at 65°C.

[0026] The term "expression vector," used in relation to polynucleotides, refers to a vector containing a polynucleotide that has a mechanism for expressing that polynucleotide within the cell into which it is introduced. A polynucleotide expression vector can also be described as a vector that has the ability to express that polynucleotide within the cell into which it is introduced. An example of a polynucleotide expression vector is a vector containing the polynucleotide operably ligated to a regulatory sequence such as a promoter.

[0027] Furthermore, the term "operably ligated" used in relation to polynucleotides means that a regulatory sequence, such as a promoter, is positioned close enough to the polynucleotide to influence its expression. For example, when a polynucleotide is operably ligated to a promoter, it means that the polynucleotide is ligated in such a way that it can be expressed under the control of the promoter.

[0028] [Editing Polynucleotides] In this disclosure, editing polynucleotides are single-stranded polynucleotides capable of modifying target sites in double-stranded genomic DNA. Editing polynucleotides consist of, in order from the 5' end: a) a portion that can hybridize with a region adjacent to the 3' end of the primary editing site; x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site; and b) a portion that can hybridize with a region adjacent to the 5' end of the primary editing site, or portions a) and b). Details of editing polynucleotides are described below (see also Figure 6).

[0029] In this disclosure, a target site means a location in double-stranded genomic DNA where modification is desired for one pair of nucleotides or multiple consecutive pairs of nucleotides, or between two consecutive pairs of nucleotides. A 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 location corresponding to the primary editing site. For example, if the desired modification is to replace or delete one pair of nucleotides or multiple consecutive pairs of nucleotides in double-stranded genomic DNA, the primary editing site is set on one genomic DNA strand at the one nucleotide or multiple consecutive nucleotides to be replaced or deleted. If the desired modification is to insert one pair of nucleotides or multiple consecutive pairs of nucleotides into double-stranded genomic DNA, the primary editing site is set on one genomic DNA strand between two consecutive nucleotides to which the insertion of one nucleotide or multiple consecutive nucleotides is desired.

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

[0031] When the primary editing site is located on 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 will be separated on the genomic DNA by the length of the primary editing site. Furthermore, when the primary editing site is located 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 will be adjacent to each other on the genomic DNA.

[0032] When the primary editing site is set on multiple consecutive nucleotides on one genomic DNA strand, there is no particular restriction on the number of such consecutive nucleotides, but it could be, for example, 2 or more nucleotides, 3 or more nucleotides, 4 or more nucleotides, 5 or more nucleotides, 6 or more nucleotides, 7 or more nucleotides, or 8 or more nucleotides, and could also be, for example, 2000 or less nucleotides, 1000 or less nucleotides, 900 or less nucleotides, 800 or less nucleotides, 700 or less nucleotides, 600 or less nucleotides, 500 or less nucleotides, 450 or less nucleotides, 400 or less nucleotides, 350 or less nucleotides, 300 or less nucleotides, 250 or less nucleotides, 200 or less nucleotides, 150 or less nucleotides, 100 or less nucleotides, 90 or less nucleotides, 80 or less nucleotides, 70 or less nucleotides, 60 or less nucleotides, 50 or less nucleotides, 40 or less nucleotides, 30 or less nucleotides, 20 or less nucleotides, 15 or less nucleotides, 12 or less nucleotides, 10 or less nucleotides, or 9 or less nucleotides.

[0033] The primary editing site may be set on either of the two strands that make up the genomic DNA. The secondary editing site will be set on the genomic DNA strand that does not have a primary editing site. For example, when modification of a region of double-stranded genomic DNA that is transcribed into RNA is desired, the primary editing site may be set on the sense strand or the antisense strand.

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

[0035] Furthermore, portion a) may consist 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. Here, the identity is preferably 95% or more, more preferably 97% or more, even more preferably 99% or more, even more preferably 99.5% or more, and particularly preferably 99.9% or more.

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

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

[0038] Furthermore, portion b) may consist 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. Here, the identity is preferably 95% or more, more preferably 97% or more, even more preferably 99% or more, even more preferably 99.5% or more, and particularly preferably 99.9% or more.

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

[0040] The lengths of parts a) and b) can be set independently. The lengths of parts a) and 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, and can also be 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 lengths of parts a) and b) may 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, respectively.

[0042] The length of part a) in the polynucleotide for editing used in combination with the polynucleotide for promoting editing is set to be the same as or longer than the overlap when the 5'-end of the predetermined region existing on the genomic DNA strand where the primary editing site is set overlaps with the 3'-end of region A. The length of part b) in the polynucleotide for editing used in combination with the polynucleotide for promoting editing is set to be the same as or longer than the overlap when the 3'-end of the predetermined region existing on the genomic DNA strand where the primary editing site is set overlaps with the 5'-end of region B. Here, the predetermined region is a region defined in relation to the polynucleotide for promoting editing on the genomic DNA strand where the primary editing site is set. Region A is a region at the position corresponding to part a) of the polynucleotide for editing on the genomic DNA strand. Region B is a region at the position corresponding to part b) of the polynucleotide for editing on the genomic DNA strand. The descriptions of the predetermined region, region A, and region B are described in detail in the item of the polynucleotide for promoting editing.

[0043] For example, when the length of the overlap between the 5'-end of the predetermined region existing on the genomic DNA strand where the primary editing site is set and the 3'-end of region A is 21 nucleotides, the length of part a) in the polynucleotide for editing used in combination with the polynucleotide for promoting editing is set to be 21 nucleotides or more. Also, for example, when the length of the overlap between the 3'-end of the predetermined region existing on the genomic DNA strand where the primary editing site is set and the 5'-end of region B is 21 nucleotides, the length of part b) in the polynucleotide for editing used in combination with the polynucleotide for promoting editing is set to be 21 nucleotides or more.

[0044] Part x) is any part consisting of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site, and is determined by how the target site is to be modified. For example, if the desired modification is to replace one pair of nucleotides or multiple consecutive pairs of nucleotides in the double-stranded genomic DNA, part x) is the one pair of nucleotides or multiple consecutive nucleotides that are to be replaced with the secondary editing site. Also, if the desired modification is to delete one pair of nucleotides or multiple consecutive pairs of nucleotides in the double-stranded genomic DNA, part x) is not included in the editing polynucleotide. Furthermore, if the desired modification is to insert one pair of nucleotides or multiple consecutive pairs of nucleotides into the double-stranded genomic DNA, part x) is the one pair of nucleotides or multiple consecutive nucleotides that are to be inserted into the secondary editing site.

[0045] Part x) consists of a nucleotide sequence that is not identical to the nucleotide sequence of the secondary editing site. Part x) can consist of any nucleotide sequence as long as it is not an exact match to the nucleotide sequence of the secondary editing site, and for example, it may be a nucleotide sequence having less than 90% identity with the nucleotide 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.

[0046] The length of part x) is not particularly limited, but 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, 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.

[0047] The polynucleotide for editing consists of, in order from the 5'-end side, part a), part x), and part b), or consists of part a) and part b). Therefore, the length of the polynucleotide for editing can be determined by summing the lengths of each part constituting the polynucleotide for editing, that is, the lengths of part a), part x), and part b), or by summing the lengths of part a) and part b).

[0048] The polynucleotide for editing may contain one or more DNA high-affinity nucleotide analogs in one or both of part a) and part b). The DNA high-affinity nucleotide analog is replaced with, for example, an adenine or an adenine analog-containing DNA high-affinity nucleotide analog so as to be the same base as the original base when the original nucleotide is a nucleotide containing adenine. A plurality of DNA high-affinity nucleotide analogs may be contained adjacently, or may be intermittently contained so as not to be adjacent to each other. Also, when contained in both part a) and part b), the DNA high-affinity nucleotide analogs may be contained at positions independently selected from each other, and the number of contained ones does not have to be the same.

[0049] The DNA high-affinity nucleotide analog is an artificial nucleic acid constituent unit having a high binding affinity for DNA that can form a polymer structure similar to that of natural nucleic acids. Examples include nucleotides in which the oxygen atom at the 2'-position and the carbon atom at the 4'-position of the ribose ring are cross-linked, peptide nucleic acids (PNA) having N-(2-aminoethyl)glycine instead of deoxyribose or ribose, and morpholino nucleic acids having a morpholine ring instead of deoxyribose or ribose, etc.

[0050] In one embodiment, the DNA high affinity nucleotide analog is a nucleotide in which the oxygen atom at the 2' position and the carbon atom at the 4' position of the ribose ring are cross-linked. Examples include LNA (Locked Nucleic Acid), where the oxygen atom at the 2' position and the carbon atom at the 4' position are cross-linked via methylene; ENA, where they are cross-linked via ethylene; and BNA (Bridged Nucleic Acid), where they are cross-linked via -CH2OCH2-. COC BNA cross-linked via -NR-CH2- (where R is a methyl or hydrogen atom) NC Examples include cMOE crosslinked via -CH2(OCH3)-, cEt crosslinked via -CH2(CH3)-, AmNA crosslinked via amide, and scpBNA crosslinked via methylene with cyclopropane formed at the 6' position.

[0051] Editing polynucleotides may further contain chemically modified nucleotides in addition to DNA high-affinity nucleotide analogs as their constituent units. 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 phosphorodithionate; nucleotides in which the hydroxyl group at the 2' position of the sugar (ribose) moiety is replaced with -OR (where R represents, for example, CH3 (2'-O-Me), CH2CH2OCH3 (2'-MOE), CH2CH2NHC(NH)NH2, CH2CONHCH3, CH2CH2CN, etc.); nucleotides in which a methyl group or 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 or hydroxyl moiety is modified with, for example, biotin, an amino group, a lower alkylamine group, or an acetyl group can be included, but are not limited to these.

[0052] Editable polynucleotides, including DNA high-affinity nucleotide analogs and optionally further chemically modified nucleotides, can be produced by known synthetic methods.

[0053] The editing polynucleotides in this disclosure, like the single-stranded polynucleotides disclosed in Patent Document 1 (Japanese Patent No. 6899564), can modify genomic DNA in cells as desired, and do not require the intracellular introduction of site-specific nucleases (ZFN proteins, TALEN proteins, Cas proteins, etc.), guide RNA, or expression vectors thereof, as is required in conventional genome editing technologies.

[0054] Although not bound by theory, the editing polynucleotide hybridizes its portion a) with the region adjacent to the 3' end of the primary editing site on one of the genomic DNA strands, and its portion b) with the region adjacent to the 5' end of the primary editing site. As a result, if the editing polynucleotide consists of portions a), x), and b), portion x) protrudes like a bump without hybridizing with other sequences. If the editing polynucleotide consists of portions a) and b), the primary editing site on the genomic DNA protrudes like a bump without hybridizing with other sequences. This protruding portion is then recognized by the repair system, and the target site on the double-stranded genomic DNA can be modified.

[0055] Specifically, when it is desired to insert one pair of nucleotides or multiple consecutive pairs of nucleotides into a double-stranded genomic DNA, the primary editing site is set between two consecutive nucleotides on one of the genomic DNA strands that are to be inserted; portions a) and b) are set as described above; and portion x) is set to one or more consecutive nucleotides that are to be inserted into the secondary editing site. By performing genome editing using the editing polynucleotide consisting of portions a), x), and b) designed in this way, a portion consisting of a base sequence complementary to portion x) can be inserted into the primary editing site, and portion x) can be inserted into the secondary editing site.

[0056] Furthermore, if it is desired to replace one pair of nucleotides or multiple consecutive pairs of nucleotides within a double-stranded genomic DNA, the primary editing site is set to one or more consecutive nucleotides on one of the genomic DNA strands that are to be replaced; portions a) and b) are set as described above; and portion x) is set to one or more consecutive nucleotides that are to be replaced with the secondary editing site. By performing genome editing using the editing polynucleotide consisting of portions a), x), and b) designed in this way, a portion consisting of a base sequence complementary to portion x) can be inserted into the primary editing site, and portion x) can be inserted into the secondary editing site (as shown in Figure 6).

[0057] In addition, if it is desired to delete one pair of nucleotides or multiple consecutive pairs of nucleotides within a double-stranded genomic DNA, the primary editing site is set to one nucleotide or multiple consecutive nucleotides on one of the genomic DNA strands that are to be deleted; and parts a) and b) are set as described above. By performing genome editing using the editing polynucleotide consisting of parts a) and b) designed in this way, it is possible to delete the primary editing site from one genomic DNA strand and the secondary editing site from the other genomic DNA strand, that is, to delete the target site from the double-stranded genomic DNA.

[0058] While not bound by theory, considering the presence of a large amount of transcribed RNA surrounding genomic DNA, if, for example, the target site is located within a genomic DNA region transcribed into RNA, and the primary editing site is set on the sense strand of genomic DNA, the editing polynucleotide will preferentially bind to regions in the surrounding RNA that have the same base sequence as the two regions adjacent to the 3' end and the 5' end of the primary editing site on the sense strand of genomic DNA. Therefore, the genome editing efficiency is likely to be poor. If genome editing efficiency is a priority, it is preferable to set the primary editing site on the antisense strand of genomic DNA rather than on the sense strand.

[0059] Furthermore, as described above, the editing polynucleotide can modify the target site of double-stranded genomic DNA by having portion a) hybridize with a region adjacent to the 3' end of the primary editing site set on one genomic DNA strand, and portion b) hybridize with a region adjacent to the 5' end of the primary editing site. Therefore, the editing polynucleotide may have any nucleotide sequence added to one or both ends, i.e., the 5' end of portion a) and / or the 3' end of portion b), as long as it does not affect the hybridization of portion a) of the editing polynucleotide with the region adjacent to the 3' end of the primary editing site, and the hybridization of portion b) of the editing polynucleotide with the region adjacent to the 5' end of the primary editing site. The editing polynucleotide in this disclosure includes those with such arbitrary nucleotide sequences added.

[0060] For such editing polynucleotides to which an arbitrary base sequence has been added to the terminal, the identity calculation 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 added arbitrary base sequence shall be considered not to exist in the identity calculation.

[0061] [Editing-Enhancing Polynucleotides] Editing polynucleotides can be used in combination with single-stranded editing-enhancing polynucleotides to improve genome editing efficiency. Editing-enhancing polynucleotides are polynucleotides that can hybridize with nucleic acid sequences (also called complementary sequences of the specified region) that are complementary to a specified region on the genomic DNA strand where the primary editing site is set. Details of editing-enhancing polynucleotides are described below (see also Figure 7).

[0062] The predetermined region is set such that, when region A is defined as the region on the genomic DNA strand where the primary editing site is set, corresponding to portion a) of the editing polynucleotide, and region B is defined as the region at the position corresponding to portion b) of the editing polynucleotide, its 5' end overlaps with region A, or its 3' end overlaps with region B.

[0063] The length of the overlap between the 5' end of the predetermined region and region A is the same as or shorter than that of region A. Similarly, the length of the overlap between the 3' end of the predetermined region and region B is the same as or shorter than that of region B. These 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, 10 nucleotides or more, 15 nucleotides or more, 16 nucleotides or more, 20 nucleotides or more, 21 nucleotides or more, 25 nucleotides or more, 30 nucleotides or more, 35 nucleotides or more, 40 nucleotides or more, 45 nucleotides or more, or 50 nucleotides or more, and also 500 nucleotides or less, 300 nucleotides or less, 200 nucleotides or less, 190 nucleotides or less. The following may be 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, 50 nucleotides or less, 40 nucleotides or less, 30 nucleotides or less, 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. Furthermore, the lengths of these overlaps can be independently, for example, 1–20 nucleotides, 5–20 nucleotides, 5–15 nucleotides, 8–12 nucleotides, 9–11 nucleotides, 15–200 nucleotides, 16–150 nucleotides, 20–100 nucleotides, 21–100 nucleotides, 25–100 nucleotides, 30–100 nucleotides, 35–90 nucleotides, 40–90 nucleotides, 45–80 nucleotides, or 50–80 nucleotides.

[0064] The inventors have found that when editing polynucleotides are designed to hybridize across both exons and introns on a genomic DNA strand, editing-enhancing polynucleotides can improve the genome editing efficiency of the editing polynucleotides, but the degree of improvement may not be significant. Such editing polynucleotides can be used when the target site is located in a region near the boundary between exons and introns on the genomic DNA. One example is an editing polynucleotide that hybridizes with exons on a genomic DNA strand to a length of 10 nucleotides or more and with introns to a length of 11 nucleotides or more.

[0065] Edit-promoting polynucleotides designed to hybridize with complementary sequences of a predetermined region can have their genome editing efficiency significantly improved, even for editing polynucleotides designed to hybridize across both exons and introns, by setting the length of the overlap between the predetermined region and region A at its 5' end, or the overlap between the predetermined region and region B at its 3' end, to 16 nucleotides or more, for example, 20 nucleotides or more, 21 nucleotides or more, 25 nucleotides or more, 30 nucleotides or more, 35 nucleotides or more, 40 nucleotides or more, 45 nucleotides or more, or 50 nucleotides or more. The length of the overlap between region A at the 5' end of a given region, or the length of the overlap between region B at the 3' end of a given region, is, for example, 16-200 nucleotides, 20-150 nucleotides, 21-100 nucleotides, 26-100 nucleotides, 31-100 nucleotides, 36-100 nucleotides, 41-100 nucleotides, 46-100 nucleotides, 21-90 nucleotides, 26-90 nucleotides, 31-90 nucleotides, 36-90 nucleotides, 41-90 nucleotides, 46-90 nucleotides, 21-80 nucleotides, 26-80 nucleotides, 31- It may be 80 nucleotides, 36-80 nucleotides, 41-80 nucleotides, 46-80 nucleotides, 21-70 nucleotides, 26-70 nucleotides, 31-70 nucleotides, 36-70 nucleotides, 41-70 nucleotides, 46-70 nucleotides, 21-60 nucleotides, 26-60 nucleotides, 31-60 nucleotides, 36-60 nucleotides, 41-60 nucleotides, 46-60 nucleotides, 21-50 nucleotides, 26-50 nucleotides, 31-50 nucleotides, 36-50 nucleotides, 41-50 nucleotides, or 46-50 nucleotides.

[0066] The complementary sequence of a given region is a region on another polynucleotide that is complementary to the given region on the genomic DNA strand. In nature, the complementary sequence of a given region is typically contained in RNA transcribed from the genomic DNA strand in which the given region is located.

[0067] Edit-promoting polynucleotides can hybridize with the complementary sequence of a predetermined region on the genomic DNA strand where the primary editing site is set.

[0068] Furthermore, the editing-promoting polynucleotide may be a polynucleotide consisting of a base sequence having 90% or more identity with the base sequence of the predetermined region. Here, the identity is preferably 95% or more, more preferably 97% or more, even more preferably 99% or more, even more preferably 99.5% or more, and particularly preferably 99.9% or more.

[0069] In a preferred embodiment, the editing-promoting polynucleotide has the same base sequence as the base sequence of the predetermined region.

[0070] There are two types of editing-promoting polynucleotides: one in which a predetermined region overlaps with region A at its 5' end, and another in which a predetermined region overlaps with region B at its 3' end. By using one of these two types of editing-promoting polynucleotides in combination with the editing polynucleotide, genome editing efficiency can be improved. By using both of these two types of editing-promoting polynucleotides in combination with the editing polynucleotide, genome editing efficiency can be further improved.

[0071] In the embodiment using the two types of editing-promoting polynucleotides described above, there is no restriction on the ratio of the two types of editing-promoting polynucleotides used. The molar concentrations of both may be approximately the same, or the molar concentration of one may be significantly higher than the other.

[0072] The length of the editing-promoting polynucleotide is equal to or longer than the overlap length with region A when the predetermined region is set to overlap with region A at its 5' end, and equal to or longer than the overlap length with region B when the predetermined region is set to overlap with region B at its 3' end. When two types of editing-promoting polynucleotides are used, the lengths of each editing-promoting polynucleotide do not need to be the same and can be set independently. Furthermore, the overlap lengths in relation to each editing-promoting polynucleotide do not need to be the same and can be set independently.

[0073] The length of the editing-promoting polynucleotides may 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 may also 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.

[0074] Furthermore, the lengths of the editing-promoting polynucleotides can be, for example, 20-1000 nucleotides, 20-500 nucleotides, 30-300 nucleotides, 40-200 nucleotides, 50-200 nucleotides, 60-200 nucleotides, 70-150 nucleotides, or 70-130 nucleotides, respectively.

[0075] Furthermore, the distance between two predetermined regions on the genomic DNA strand (the distance between the ends closer to the other predetermined region) in relation to each editing-promoting polynucleotide is preferably 0 to 70 nucleotides, for example, 1 to 70 nucleotides, 2 to 65 nucleotides, 3 to 60 nucleotides, 4 to 55 nucleotides, or 5 to 50 nucleotides.

[0076] Although not bound by theory, in an environment where many other polynucleotides have regions complementary to the primary editing site or its vicinity (regions A and B), the binding of the editing polynucleotide to the genomic DNA strand in which the primary editing site is set is competitively inhibited by these other polynucleotides, and as a result, the genome editing efficiency by the editing polynucleotide is thought to decrease.

[0077] For example, if the target site is located within a genomic DNA region transcribed into RNA, and the primary editing site is set on the genomic DNA antisense strand, the editing polynucleotide will compete with surrounding RNA for binding to the genomic DNA antisense strand. This suppresses the binding of the editing polynucleotide to the genomic DNA antisense strand, and therefore, the genome editing efficiency is thought to be lower than when there is no surrounding RNA.

[0078] Under these conditions, when editing polynucleotides are coexisted with editing polynucleotides, surrounding RNA binds to the editing-promoting polynucleotides. As a result, the inhibition of binding between the editing polynucleotides and the genomic DNA antisense strand is resolved, and genome editing efficiency is expected to improve (Figure 7). Furthermore, with respect to the editing-promoting polynucleotides, when the overlap with region A at the 5' end of a predetermined region or with region B at the 3' end of a predetermined region is relatively long, for example, 21 nucleotides or more, the recognition of the hybrid of surrounding RNA and the editing-promoting polynucleotide by RNase H and RNA degradation are enhanced. As a result, the inhibition of binding between the editing polynucleotides and the genomic DNA antisense strand is resolved, and genome editing efficiency is expected to improve.

[0079] In embodiments where the target site is located within a genomic DNA region transcribed into RNA, and the primary editing site is set on the genomic DNA antisense strand, it is preferable that the editing-promoting polynucleotide 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 if the original nucleotide is an adenine-containing nucleotide, for example. Multiple RNA high-affinity nucleotide analogs may be included adjacent to each other, or they may be included intermittently so as not to be adjacent to each other. Furthermore, when two types of editing-promoting polynucleotides are used, each editing-promoting polynucleotide may contain an RNA high-affinity nucleotide analog at independently selected positions, and the number of analogs does not need to be the same.

[0080] While not bound by theory, in embodiments where the target site is located within a genomic DNA region transcribed into RNA and the primary editing site is set on the genomic DNA antisense strand, if the target site is set within a region transcribed into an intron portion of the mRNA precursor, the competition for binding between the editing polynucleotide and RNA and the genomic DNA antisense strand is considered to be more intense, resulting in a lower genome editing efficiency, compared to cases where the target site is set within a region transcribed into an exon portion of the mRNA precursor. This is because a large amount of intron portions, excised from the mRNA precursor by splicing, are present around the genomic DNA.

[0081] Therefore, the editing-promoting polynucleotide can improve genome editing efficiency in both cases: when the target site is located within a genomic DNA region transcribed to the intron portion of the mRNA precursor and the primary editing site is set on the genomic DNA antisense strand; and when the target site is located within a genomic DNA region transcribed to the exon portion of the mRNA precursor and the primary editing site is set on the genomic DNA antisense strand. However, it is believed that the genome editing efficiency-improving effect is particularly strong when the target site is located within a genomic DNA region transcribed to the intron portion of the mRNA precursor and the primary editing site is set on the genomic DNA antisense strand.

[0082] In an embodiment where the target site is set to a double-stranded genomic DNA region transcribed into RNA, and the primary editing site is set on the antisense strand of genomic DNA, the editing polynucleotide is also called the editing sense polynucleotide, and the editing-promoting polynucleotide is also called the editing-promoting antisense polynucleotide.

[0083] [Genome Editing Kit] This disclosure provides a genome editing kit comprising the editing polynucleotide described above and the editing-promoting polynucleotide described above. Preferably, the genome editing kit includes two types of editing-promoting polynucleotides: one configured such that the predetermined region overlaps with region A at its 5' end, and another configured such that the predetermined region overlaps with region B at its 3' end.

[0084] Genome editing kits may include, instead of editing polynucleotides, an expression vector for editing polynucleotides, or a conjugate of editing polynucleotides or an expression vector for editing polynucleotides with other substances. Furthermore, genome editing kits may include, instead of editing-promoting polynucleotides, an expression vector for editing-promoting polynucleotides, or a conjugate of editing-promoting polynucleotides or an expression vector for editing-promoting polynucleotides with other substances.

[0085] Other substances that can be conjugated to editing polynucleotides are not limited as long as they can form conjugates with the editing polynucleotides and do not interfere with the function of the editing polynucleotides, i.e., their function of modifying target sites in genomic DNA. Similarly, other substances that can be conjugated to editing-promoting polynucleotides are not limited as long as they can form conjugates with the editing-promoting polynucleotides and do not interfere with the function of the editing-promoting polynucleotides, i.e., their function of improving the genome editing efficiency of the editing polynucleotides.

[0086] Examples of other substances that can be conjugated to editing polynucleotides and editing-promoting polynucleotides include functional peptides such as nuclear localization signals and membrane-permeable peptides; hydrophilic polymers such as polyethylene glycol and dextran; and labeling substances such as radioisotopes, fluorescein and its derivatives, rhodamine and its derivatives, fluorescent proteins, phycobiliproteins, biotin, and avidin.

[0087] Genome editing kits may further include reagents such as nucleic acid delivery reagents or buffers, including transfection reagents.

[0088] The components included in a genome editing kit, such as the editing polynucleotides, their expression vectors or conjugates, the editing-promoting polynucleotides, their expression vectors or conjugates, and each of the reagents, may be included in the kit in separate forms, or in the form of a composition in which multiple components are combined.

[0089] The genome editing kit may further include, for example, the instruments used during genome editing, instructions on how to use them, etc.

[0090] In one embodiment, the genome editing kit is a disease treatment or prevention kit and may include, in addition to a therapeutically or prophylactically effective amount of the aforementioned editing polynucleotide, its expression vector, or a conjugate of the editing polynucleotide or its expression vector with other substances, other pharmaceutically acceptable components, such as pharmaceutically acceptable additives (buffers, stabilizers, preservatives, excipients, etc.) and pharmaceutically acceptable media (water, saline, phosphate-buffered saline (PBS), etc.). The disease treatment or prevention kit can be used to treat or prevent diseases caused by any abnormality in genomic DNA.

[0091] [Genome editing method and method for producing genome-edited cells or organisms] This disclosure provides a method for modifying a target site in the double-stranded genomic DNA of a cell or organism (also referred to in this disclosure as the "genome editing method"), which includes the step of treating the cell or organism with the genome editing kit described above. This disclosure also provides a method for producing a cell or organism in which the target site in the double-stranded genomic DNA has been modified (also referred to in this disclosure as the "method for producing genome-edited cells or organisms"), which includes the step of treating the cell or organism with the genome editing kit described above.

[0092] Cells processed using genome editing kits are cells whose genomic DNA modification is desired, and can originate from a variety of organisms, including the prokaryotes and eukaryotes mentioned earlier. If the cells are from a multicellular organism, they can originate from various tissues of that organism. The cells may be somatic cells or germ cells. Furthermore, the cells may be differentiated or undifferentiated, and may be tissue stem cells, embryonic stem cells, or induced pluripotent stem cells (iPS cells).

[0093] Cells processed using a genome editing kit may be single cells or in the form of a cell population containing one or more types of cells.

[0094] Organisms processed using genome editing kits are those whose genomic DNA modification is desired, and can be a variety of organisms, including the prokaryotes and eukaryotes mentioned earlier. The organisms may be mature individuals, embryos or fetuses in the developmental stage, or, in the case of plants, seeds, seedlings, or bulbs.

[0095] In one embodiment, cells may be expressed as cells (excluding human gametes and fertilized eggs), excluding human gametes and fertilized eggs.

[0096] In one embodiment, organisms do not include humans and can be described as non-human organisms. In another embodiment, organisms do not include humans or animals and can be described as non-human, non-animal organisms.

[0097] Furthermore, in one embodiment, the cells do not contain human gametes and fertilized eggs, and the organism does not contain humans. In another preferred embodiment, the cells do not contain human gametes and fertilized eggs, and the organism does not contain humans or animals.

[0098] Processing cells or organisms using a genome editing kit means co-existing the editing polynucleotides, their expression vectors, or conjugates of the editing polynucleotides or their expression vectors with other substances, and the editing-promoting polynucleotides, their expression vectors, or conjugates of the editing-promoting polynucleotides or their expression vectors with other substances, contained in the genome editing kit, with cells, or administering these to an organism. By co-existing with cells or administering them to an organism, the editing polynucleotides, their expression vectors, or conjugates of the editing polynucleotides or their expression vectors with other substances, and the editing-promoting polynucleotides, their expression vectors, or conjugates of the editing-promoting polynucleotides or their expression vectors with other substances are introduced into the cells or organism, allowing for the editing of the genomic DNA of the cells or organism.

[0099] The processing of cells or organisms using genome editing kits can be carried out by various known methods appropriately selected by those skilled in the art, taking into consideration factors such as the type or properties of the cells or organisms. Examples of cell or organism processing methods include microinjection, electroporation, DEAE-dextran treatment, transfection using transfection reagents (jetPEI (PolyPlus-transfection), lipofectamine, etc.), transfection using lipid nanoparticles, and hydrodynamic methods.

[0100] In one embodiment, the treatment of human cells is performed in vitro or ex vivo.

[0101] Generally, genome editing can increase or decrease the characteristics of cells or organisms, such as nutritional requirements, sensitivity to or tolerance to chemicals, or confer characteristics not present in the cells or organisms before editing, or cause them to lose characteristics present in the cells or organisms before editing. These changes in characteristics can be used as indicators to select cells or organisms that have undergone the desired genome editing. For example, when it is expected that genome editing will increase or confer drug resistance, genome-edited cells or organisms can be selected by culturing cells treated with a genome editing kit in a suitable medium containing the drug, or by feeding organisms treated with a genome editing kit in a suitable feed containing the drug. The methods for selecting and evaluating the characteristics of cells or organisms, and the culture or rearing conditions that enable the selection of cells or organisms, can be appropriately determined within the scope of the ordinary capabilities of those skilled in the art.

[0102] The genome editing method and the method for producing genome-edited cells or organisms described above may include, in addition to the step of processing cells or organisms using a genome editing kit, a step of selecting cells or organisms in which genomic DNA has been edited, using the characteristics of the cells or organisms altered by the editing polynucleotides as indicators.

[0103] [Further Methods] The Disclosure further provides a method for modifying a target site in the double-stranded genomic DNA of a cell or organism, comprising the step of treating the cell or organism multiple times at time intervals of 12 to 36 hours using the editing polynucleotide described above, its expression vector, or a conjugate of the editing polynucleotide or its expression vector with other substances, and a method for producing a cell or organism in which the target site of the double-stranded genomic DNA has been modified (in the Disclosure, these methods are collectively referred to as the "multiple-treatment method").

[0104] In the multiple-processing method, the processing of cells or organisms using the above-mentioned editing polynucleotide, its expression vector, or a conjugate of the editing polynucleotide or its expression vector with another substance is performed multiple times at time intervals of 12 to 36 hours. The time interval may be within the range of 12 to 36 hours, for example, 16 to 32 hours, 18 to 30 hours, 20 to 28 hours, or 22 to 26 hours. The number of processing steps may be two or more, for example, 2 to 30 times, 3 to 25 times, or 5 to 20 times. Details of the cells or organisms to be processed and the processing method in the multiple-processing method are as described in the genome editing method and the method for producing genome-edited cells or organisms described above.

[0105] In the multi-pass processing method, the editing polynucleotide may include a DNA high-affinity nucleotide analog as a constituent unit. Furthermore, in the multi-pass processing method, the editing polynucleotide may be used together with the editing-promoting polynucleotide, its expression vector, or its conjugate. Additionally, in the multi-pass processing method, the editing polynucleotide may be used together with a substance that enhances genome editing efficiency, such as Hfq or its homolog, DnaK or its homolog, their variants, or nucleic acids containing an encoding base sequence.

[0106] The present invention will be described in more detail by the following examples, but the present invention is not limited thereto.

[0107] [Example 1. Genome editing of the human HPRT1 gene] Polynucleotides To modify the human hypoxanthine phosphoribosyltransferase 1 (HPRT1) gene, three single-stranded DNAs (polynucleotides 1, 10, and 20) consisting of the nucleotide sequences shown in Table 1 were used as editing polynucleotides, and fourteen single-stranded DNAs (polynucleotides 2-9 and 11-16) consisting of the nucleotide sequences shown in Table 2 were used as editing-promoting polynucleotides.

[0108] Polynucleotides 1, 10, and 20 consist of sequences obtained by substituting one nucleotide from the sequences shown in SEQ ID NOs: 21-23, which are partial sequences of the sense strand of the human HPRT1 gene (NCBI Reference Sequence: NG_012329.2, Location: 5,021..45,524). The nucleotide sequence shown in SEQ ID NO: 21 corresponds to exon 7 of the human HPRT1 gene and its adjacent introns on both sides (27 nucleotides on the 5' side and 25 nucleotides on the 3' side). The nucleotide sequence shown in SEQ ID NO: 22 corresponds to exon 7 of the human HPRT1 gene and its adjacent introns on both sides (26 nucleotides on the 5' side and 26 nucleotides on the 3' side). The nucleotide sequence shown in SEQ ID NO: 23 corresponds to exon 7 of the human HPRT1 gene and its adjacent introns on both sides (28 nucleotides on the 5' side and 26 nucleotides on the 3' side). Polynucleotides 1, 10, and 20 can replace the cytosine at position 50 of the nucleotide sequence shown in SEQ ID NO: 21 (corresponding to position 49 in SEQ ID NO: 22 and position 51 in SEQ ID NO: 23) with thymine.

[0109] Polynucleotides 2-9 and 11-16 all consist of partial sequences of the human HPRT1 gene antisense strand. Polynucleotides 2 and 3, 4 and 5, 6 and 7, 8 and 9, 11 and 12, 13 and 14, and 15 and 16 can each be used as editing-promoting polynucleotide pairs. The 5' ends of polynucleotides 4, 6, 8, 11, 13, and 15 are designed to overlap with the genomic DNA antisense strand region to which the 5' ends of polynucleotides 1, 10, and 20 bind, and are complementary to the 5' ends of polynucleotides 1, 10, and 20. The 3' ends of polynucleotides 5, 7, 9, 12, 14, and 16 are designed to overlap with the genomic DNA antisense strand region to which the 3' ends of polynucleotides 1, 10, and 20 bind, and are complementary to the 3' ends of polynucleotides 1, 10, and 20.

[0110] Genome editing 2.5x10 5 HEK293T cells were seeded in 2 ml of Dulbecco's Modified Eagle Medium (containing 10% (v / v) Fetal Bovine Serum (Sigma, F7524) and 1% (v / v) Antibiotic-Antimycotic (Gibco, 15240-062)) into a 6-well plate (Corning, 3516) and cultured at 37°C under 5% CO2 conditions for 48 hours until approximately 70-80% confluence was achieved. Next, 200 μl of a DNA mixture (Opti-MEM (Gibco, 11058-021) + Viafect Transfection Reagent (Promega)) containing 5 μg of one of the editing polynucleotides shown in Table 1 (10 μg if "double dose" is indicated, 15 μg if "triple dose" is indicated) and 5 μg of one of the editing polynucleotides shown in Table 2, or 5 μg of both, per well, was added to each well, and the cells were cultured for another day. After removing the medium, the cells were detached and collected using 0.025% Trypsin-EDTA (Gibco, 25300-062), seeded in a 10 cm dish, and cultured for 4 days in a medium containing 1% (v / v) Antibiotic-Antimycotic until approximately 90-100% confluence was reached. After removing the medium, the cells were detached and collected using 0.025% Trypsin-EDTA.

[0111] - Evaluation of genome editing efficiency: Genomic DNA was extracted from the recovered cells using the NucleoSpin Tissue Kit (Macherey-Nagel) and 2 x 10⁻¹⁶ 5Two-step PCR was performed using primeSTAR Max DNA polymerase (Takara) with 1600 ng of genomic DNA, equivalent to one cell, as a template. Next, index-addition PCR was performed to prepare samples for sequencing, and the sequences were analyzed using a next-generation sequencer (iSeq 100 Sequencing System, Illumina) and CLC Genomic Workbench 20 (QIAGEN Digital Insights). Genome editing efficiency was calculated by determining the percentage of reads containing the nucleotide substitution sequence among reads that included the 10 nucleotides before and after the nucleotide substitution site (a total of 21 nucleotides).

[0112] Figure 1 shows the efficiency of genome editing using polynucleotide 1 as the editing polynucleotide and polynucleotides 2-9 as editing-promoting polynucleotides. In genome editing using only the editing polynucleotide, the genome editing efficiency remained low even when the amount of editing polynucleotide added was increased (2x and 3x in the figure). In contrast, when editing-promoting polynucleotides with an overlap length of 26 nucleotides or more were used in combination, the genome editing efficiency improved even when only one of the editing-promoting polynucleotide pairs was used.

[0113] Figure 2 shows the efficiency of genome editing using polynucleotide 10 as the editing polynucleotide and polynucleotides 11-16 as editing-promoting polynucleotides. In genome editing using only the editing polynucleotide, the genome editing efficiency remained low even when the amount of editing polynucleotide added was increased (three times the amount in the figure). In contrast, the genome editing efficiency improved when editing-promoting polynucleotide pairs with overlap lengths of 16 nucleotides or more, especially 26 nucleotides or more, were used in combination.

[0114] Figure 3 shows the efficiency of genome editing using polynucleotide 10 as the editing polynucleotide and polynucleotides 15 and 16 as editing-promoting polynucleotides. In genome editing using only the editing polynucleotide, the genome editing efficiency remained low even when the amount of editing polynucleotide added was increased (three times the amount in the figure). In contrast, when editing-promoting polynucleotides with an overlap length of 36 nucleotides were used in combination, the genome editing efficiency improved even when only one of the editing-promoting polynucleotide pairs was used.

[0115] [Example 2. Genome editing of the human FKTN gene] Polynucleotides: To modify the human fukutin (FKTN) gene, a single-stranded DNA (polynucleotide 17) consisting of the nucleotide sequence shown in Table 3 was used as the editing polynucleotide, and two types of single-stranded DNA (polynucleotides 18 and 19) consisting of the nucleotide sequences shown in Table 4 were used as editing-promoting polynucleotides.

[0116] Polynucleotide 17 consists of a sequence in which the sequence shown in SEQ ID NO: 24, which is a partial sequence of the sense strand of the human FKTN gene (NCBI Reference Sequence: NC_000009.12, Location: 105,558,131..105,641,118), has been replaced by one nucleotide. The nucleotide sequence shown in SEQ ID NO: 24 corresponds to a part of exon 11 of the human FKTN gene. Polynucleotide 17 can replace the 50th thymine in the nucleotide sequence shown in SEQ ID NO: 24 with cytosine.

[0117] Polynucleotides 18 and 19 both consist of partial sequences of the human FKTN gene antisense strand. Polynucleotides 18 and 19 can be used as an editing-enhancing polynucleotide pair. The 5' end of polynucleotide 18 is designed to overlap with the genomic DNA antisense strand region to which the 5' end of polynucleotide 17 binds, and is complementary to the 5' end of polynucleotide 17. The 3' end of polynucleotide 19 is designed to overlap with the genomic DNA antisense strand region to which the 3' end of polynucleotide 17 binds, and is complementary to the 3' end of polynucleotide 17.

[0118] Using these polynucleotides, genome editing was performed using the same method as in Example 1, and the efficiency was calculated. The results are shown in Figure 4. In genome editing using only editing polynucleotides, the genome editing efficiency remained low even when the amount of editing polynucleotides added was increased (3 times the amount in the figure). In contrast, when editing-promoting polynucleotides with an overlap length of 36 nucleotides were used in combination, the genome editing efficiency improved even when only one of the editing-promoting polynucleotide pairs was used.

[0119] [Example 3. Genome editing by multiple treatments] Genome editing was performed using the same method as in Example 1, except that polynucleotide 20 was used as the editing polynucleotide and polynucleotides 15 and 16 were used as editing-promoting polynucleotides, and the DNA mixture solution was used for 1, 3, or 10 treatments. The efficiency was then calculated. Multiple treatments with the DNA mixture solution were performed with a one-day interval, which extended the culture time. The genome editing efficiency is shown in Figure 5. The genome editing efficiency improved by performing multiple treatments with the DNA mixture solution.

Claims

1. A single-stranded editing-promoting polynucleotide for use in combination with a single-stranded editing polynucleotide capable of modifying target sites in double-stranded genomic DNA, wherein 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, and the editing polynucleotide consists of, in order from the 5' end, a) a portion that can hybridize with the region adjacent to the 3' end of the primary editing site, x) an arbitrary portion consisting of a base sequence not identical to the base sequence of the secondary editing site, and b) a portion that can hybridize with the region adjacent to the 5' end of the primary editing site, or consists of portion a) and portion b). The editing-promoting polynucleotide is hybridizable with a nucleic acid sequence complementary to a predetermined region on one of the genomic DNA strands, where the predetermined region is defined as region A, where region A is located at a position corresponding to portion a) of the editing polynucleotide on the one genomic DNA strand, and region B is located at a position corresponding to portion b) of the editing polynucleotide, and the predetermined region overlaps with region A at its 5' end or with region B at its 3' end, the length of the overlap with region A at its 5' end is 16 nucleotides or more and is the same as or shorter than region A, and the length of the overlap with region B at its 3' end is 16 nucleotides or more and is the same as or shorter than region B. The lengths of portions a) and b) of the editing polynucleotide are each independently 16 to 100 nucleotides, provided that if the predetermined region overlaps with region A at its 5' end, the length of portion a) is the same as or longer than the overlap, and if the predetermined region overlaps with region B at its 3' end, the length of portion b) is the same as or longer than the overlap, and the length of the editing-promoting polynucleotide is 50 nucleotides or more.The editing polynucleotide comprises portions a), x), and b), and when the primary editing site is set between two consecutive nucleotides on one genomic DNA strand, a portion consisting of a base sequence complementary to portion x) can be inserted into the primary editing site, and portion x) can be inserted into the secondary editing site. The editing polynucleotide comprises portions a), x), and b), and when the primary editing site is set on one nucleotide or more consecutive nucleotides on one genomic DNA strand, the primary editing site can be replaced with a portion consisting of a base sequence complementary to portion x), and the secondary editing site can be replaced with portion x). The editing polynucleotide comprises portions a) and b), and when the primary editing site is set on one nucleotide or more consecutive nucleotides on one genomic DNA strand, a target site can be deleted.

2. The editing-promoting polynucleotide according to claim 1, wherein the overlap length is 16 to 200 nucleotides.

3. A genome editing kit comprising the editing-promoting polynucleotide described in claim 1, its expression vector, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance, and a single-stranded editing polynucleotide, its expression vector, or a conjugate of the editing polynucleotide or its expression vector with another substance, wherein the editing polynucleotide consists of, in order from the 5' end, a) a portion that can hybridize with a region adjacent to the 3' end of the primary editing site, x) an arbitrary portion consisting of a base sequence that is not identical to the base sequence of the secondary editing site, and b) a portion that can hybridize with a region adjacent to the 5' end of the primary editing site, or consists of portion a) and portion b), wherein if the predetermined region overlaps with region A at its 5' end, the length of portion a) is the same as or longer than the overlap, and if the predetermined region overlaps with region B at its 3' end, the length of portion b) is the same as or longer than the overlap.

4. A genome editing kit according to claim 3, comprising the editing-promoting polynucleotide according to claim 1, its expression vector, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance, wherein the predetermined region is configured to overlap with region A at its 5' end; and the editing-promoting polynucleotide according to claim 1, its expression vector, or a conjugate of the editing-promoting polynucleotide or its expression vector with another substance, wherein the predetermined region is configured to overlap with region B at its 3' end.

5. The genome editing kit according to claim 3, wherein the length of the overlap with respect to the editing-promoting polynucleotide is 16 to 200 nucleotides.

6. A method for modifying a target site of double-stranded genomic DNA of a cell (excluding human gametes and fertilized eggs) or a non-human organism, comprising the step of treating the cell (excluding human gametes and fertilized eggs) or a non-human organism with a genome editing kit according to any one of claims 3 to 5, wherein the step is performed in vitro or ex vivo if the cell is a human cell excluding human gametes and fertilized eggs.

7. A method for producing cells (excluding human gametes and fertilized eggs) or non-human organisms in which the target sites of double-stranded genomic DNA have been modified, comprising the step of treating cells (excluding human gametes and fertilized eggs) or non-human organisms with a genome editing kit as described in any one of claims 3 to 5, wherein the step is performed in vitro or ex vivo if the cells are human cells excluding human gametes and fertilized eggs.

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