Composition for nucleotide sequence editing, and nucleotide sequence editing method using same

Abstract

Provided are a composition and others, the composition containing a first single-stranded polynucleotide and a third single-stranded polynucleotide, wherein: the first single-stranded polynucleotide has a length of 50-200 nucleotides; the nucleotide sequence for the first single-stranded polynucleotide has 80% or higher sequence identity with the nucleotide sequence for a portion of an editing target strand in a target region present in double-stranded DNA in a cell, and includes at least one mutation for editing purpose selected from the group consisting of deletion, substitution and insertion of one nucleotide or a plurality of nucleotides compared with the nucleotide sequence for the editing target strand; the third single-stranded polynucleotide has a length of 15-200 nucleotides; the nucleotide sequence for the third single-stranded polynucleotide has 90% or higher sequence identity with a nucleotide sequence that is included in the editing target strand and is located closer to the 5'-terminus or the 3'-terminus than a position corresponding to the position of the mutation for editing purpose in the first single-stranded polynucleotide; and, in the editing target strand, the nucleotide sequence having the sequence identity with the first single-stranded polynucleotide and the nucleotide sequence having the sequence identity with the third single-stranded polynucleotide overlap with each other, or are located adjacent to each other, or are separated from each other with 1 to 9 nucleotides interposed therebetween.

Description

Composition for editing base sequences and method for editing base sequences using the same

[0001] The present invention relates to a composition for editing a base sequence, a method for editing a base sequence using the composition, and the like.

[0002] With the report of CRISPR-Cas9 in 2013, many researchers began to experiment with genome editing (Non-Patent Documents 1 and 2). Genome editing technology itself had existed for some time, and methods using zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) were known. However, designing and constructing systems for editing desired sites in the genome was difficult, and only a few researchers used these techniques. CRISPR-Cas9 editing relies on the sequence of a nucleic acid called guide RNA to determine the cleavage site. Therefore, editing with CRISPR-Cas9 can be achieved simply by changing the guide RNA to match the target site, making it a widely used experimental technique. It is also beginning to be applied to crop and livestock improvement.

[0003] When CRISPR-Cas9 or other artificial nucleases cleave DNA at a target site in the genome, errors frequently occur during repair within the cell, allowing the target gene to be knocked out (deactivated). Meanwhile, if donor DNA containing a sequence homologous to the cleavage site is present during cleavage, that DNA may be incorporated, albeit at a low frequency, allowing for the insertion of desired sequences or the introduction of base substitution mutations. However, genome editing using these artificial nucleases involves DNA cleavage, which can lead to mutations at off-target sites and unexpected mutations at on-target sites (Non-Patent Documents 3 and 4). Therefore, its application in medicine must be approached with extreme caution. Furthermore, because the basic intellectual property rights are held by overseas organizations, this technology is difficult for Japanese companies to use.

[0004] On the other hand, a base sequence editing method using single-stranded DNA (ss DNA) or 5'-tailed duplex (TD) developed by Kamiya et al. is known as a base sequence editing method that does not use artificial nucleases (Patent Documents 1 and 2) (Figure 1). Regarding base sequence editing using TD, Kawai et al. investigated the effect of the length of the TD editor strand (E strand, a long strand containing mutations in the target sequence) on the editing efficiency and found that editing efficiency was higher when using TD with an E strand length of 100 bases or less than when using TD with an E strand length of several hundred bases (Non-Patent Documents 5 and 6).

[0005] Patent Document 3 describes a genome editing technology using a single-stranded polynucleotide. The editing polynucleotide used in this technology has a primary editing site, and also describes the use of an editing-promoting polynucleotide having a region overlapping with complementary sequences at the 5' and 3' ends of the editing polynucleotide. It also describes that the editing polynucleotide contains a nucleotide analog with high DNA affinity as a building block, and describes that improved editing activity was observed only when the editing polynucleotide contained a nucleotide analog with high DNA affinity at specific positions at the 5' and 3' ends.

[0006] International Publication No. 2005 / 075657 International Publication No. 2006 / 064813 International Publication No. 2023 / 190848

[0007] L. Cong, F. A. Ran, D. Cox, S. Lin, R. Barretto, N. Habib, P. D. Hsu, X. Wu, W. Jiang, L. A. Marraffini, F. Zhang. Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013)P. Mali, L. Yang, K. M. Esvelt, J. Aach, M. Guell, J. E. DiCarlo, J. E. Norville, G. M. Church. RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013)Y. Fu, J. A. Foden, C. Khayter, M. L. Maeder, D. Reyon, J. K. Joung, J. D. Sander. High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat. Biotechnol. 31, 822-826 (2013)M. Kosicki, K. Tomberg, A. Bradley. Repair of double-strand breaks induced by CRISPR-Cas9 leads to large deletions and complex rearrangements. Nat. Biotechnol. 36, 765-771 (2018)H. Kawai, K. Yazama, Y. Yanai, R. Kamitsubo, H. Kamiya. Gene correction by 5'-tailed duplexes with short editor oligodeoxyribonucleotides. J. Biosci. Bioengng. 132, 552-559 (2021)H. Kawai, R. Kamitsubo, H. Kamiya.Correction of monomeric enhanced green fluorescent protein (mEGFP) gene by short 5'-tailed duplexes. J. Biosci. Bioengng. 134, 175-181 (2022).

[0008] In base sequence editing techniques that do not use nucleases, such as the base sequence editing methods using ss DNA or TD disclosed in Patent Documents 1 and 2, further improvements in editing efficiency have been desired.

[0009] The present inventors have found that adding another nucleic acid (A' strand) that shares sequence identity with the editing target strand to the E strand or TD (E strand + A strand) significantly improves editing efficiency. They have also found that introducing specific chemical modifications into specific positions of the E strand or A strand significantly improves editing efficiency. The present inventors have conducted further intensive research and have completed the present invention.

[0010] That is, the present invention encompasses the following inventions: [1] A composition comprising a first single-stranded polynucleotide and a third single-stranded polynucleotide, wherein the first single-stranded polynucleotide is 50 to 200 bases long, its base sequence has 80% or more sequence identity with the base sequence of a portion of the editing target strand of a target region present in double-stranded DNA in a cell, and contains at least one editing mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides compared to the base sequence of the editing target strand, and the third single-stranded polynucleotide is 15 to 200 bases long, and its base sequence has 90% or more sequence identity with the base sequence in the editing target strand that is located 5'-terminally or 3'-terminally closer to the position in the first single-stranded polynucleotide corresponding to the editing mutation, and wherein, in the editing target strand, the base sequence that has sequence identity with the first single-stranded polynucleotide and the base sequence that has sequence identity with the third single-stranded polynucleotide overlap, are adjacent, or are separated by 1 to 9 bases. Preferably, the composition does not contain an exogenous nuclease or a polynucleotide capable of expressing the same, and is not used in combination therewith. [2] The composition according to [1], wherein the position of the editing mutation in the first single-stranded polynucleotide is within 25-75% from the 5' end, with the 5' end to 3' end being 100%. [3] The composition according to [2], wherein the position of the editing mutation in the first single-stranded polynucleotide is within 49-51% from the 5' end, with the 5' end to 3' end being 100%. [4] The composition according to any of [1] to [3], wherein the first single-stranded polynucleotide is 70-100 bases long. [5] The composition according to any of [1] to [4], wherein the first single-stranded polynucleotide consists of a base sequence 100% identical to the corresponding base sequence in the editing target strand, excluding the position of the editing mutation and a region 10 bases from the 5' end and a region 10 bases from the 3' end. [6] A composition described in any of [1] to [5], wherein the base sequence of the third single-stranded polynucleotide has 90% or more sequence identity with the base sequence of the first single-stranded polynucleotide in the editing target strand, which is located 5'-terminally closer to the position corresponding to the editing mutation.[7] The composition according to any one of [1] to [6], wherein in the editing target strand, a base sequence having sequence identity with a first single-stranded polynucleotide and a base sequence having sequence identity with a third single-stranded polynucleotide are adjacent to each other. [8] The composition according to any one of [1] to [7], wherein the third single-stranded polynucleotide is 55 to 75 bases in length. [9] The composition according to any one of [1] to [8], wherein the third single-stranded polynucleotide consists of a base sequence that is 100% identical to the corresponding base sequence in the editing target strand.

[10] The composition of any of [1] to [9], having at least one of the following characteristics: (a) at least one nucleotide is a sugar-modified nucleotide in the position of the editing mutation in the first single-stranded polynucleotide and in a region consisting of 10 bases on the 5'-end and 10 bases on the 3'-end of the position of the editing mutation, and (b) the first single-stranded polynucleotide contains at least one modified internucleotide bond, and the modified internucleotide bond is not present in the range from 3 bases on the 5'-end to 3 bases on the 3'-end of the position of the editing mutation.

[11] The composition of

[10] , wherein the sugar-modified nucleotide is a nucleotide containing a 2'-O,4'-C-methylene bridge or a 2'-O,4'-C-ethylene bridge in the ribose ring.

[12] The composition of

[10] or

[11] , wherein the first single-stranded polynucleotide contains 1 to 4 modified internucleotide bonds within 13 bases from the 5'-end and / or 3'-end. Preferably, the modified internucleotide bond is a phosphorothioate bond, an alkylphosphonate bond, or a phosphotriester bond. More preferably, the first single-stranded polynucleotide contains 1 to 4 modified internucleotide bond(s) within 5 bases from the 3'-end (more preferably, 2 within 3 bases, or 1 within 2 bases) and the modified internucleotide bond(s) is / are a phosphorothioate bond.

[13] The composition according to any of [1] to

[12] , further comprising a second single-stranded polynucleotide, wherein the second single-stranded polynucleotide is 20 to 200 bases long and contains a base sequence complementary to a base sequence of a portion of the region of the first single-stranded polynucleotide that does not contain the editing mutation, and is capable of forming a duplex with the first single-stranded polynucleotide.

[14] The composition according to

[13] , wherein the second single-stranded polynucleotide comprises a base sequence complementary to a portion of the base sequence of the first single-stranded polynucleotide that is 3'-terminal closer to the position of the editing mutation.

[15] The composition according to

[13] or

[14] , wherein the molar concentration of the third single-stranded polynucleotide is the same as or higher than the molar concentration of the second single-stranded polynucleotide.

[16] The composition according to any of

[13] to

[15] , wherein the second single-stranded polynucleotide comprises at least one modified internucleotide bond.

[17] The composition according to

[16] , wherein the second single-stranded polynucleotide comprises one or two modified internucleotide bonds within three bases from the 5'-end and / or 3'-end, or one modified internucleotide bond within two bases. Preferably, the second single-stranded polynucleotide comprises one or two modified internucleotide bonds within three bases from the 3'-end, or one modified internucleotide bond within two bases.

[18] The composition according to any one of

[10] to

[17] , wherein the modified internucleotide bond is a phosphorothioate bond, an alkylphosphonate bond, or a phosphotriester bond.

[19] The composition according to any one of [1] to

[19] , wherein the target region contains an aberrant base sequence that causes a disease, and the base sequence of the first single-stranded polynucleotide contains the editing mutation so that the aberrant base sequence can be repaired, thereby treating or preventing the disease.

[20] A pharmaceutical composition for treating or preventing a disease caused by an aberrant base sequence contained in a target region present in intracellular double-stranded DNA, comprising the composition according to

[19] .

[21] A method for treating or preventing a disease caused by an aberrant base sequence contained in a target region present in intracellular double-stranded DNA, comprising administering a therapeutically or prophylactically effective amount of the composition according to

[19] to a subject.

[22] A method for producing an isolated cell into which a mutation has been introduced or an organism having a cell into which a mutation has been introduced, the method comprising the step of introducing the composition according to any one of [1] to

[19] into the isolated cell or into a cell constituting the living body of the organism, thereby introducing a mutation into the base sequence of a target region present in double-stranded DNA in the cell. The method may be performed in vitro.

[23] Use of the composition according to any one of [1] to

[19] in the manufacture of a pharmaceutical for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell.

[0011] The present invention also encompasses the following inventions: [a1] A composition comprising a first single-stranded polynucleotide, wherein the first single-stranded polynucleotide is 50 to 200 bases in length, the base sequence of which has 80% or more sequence identity with the base sequence of a portion of an editing target strand in a target region present in double-stranded DNA in a cell, and which, compared to the base sequence of the editing target strand, comprises at least one editing mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides, and the composition has at least one of the following characteristics: (a) at least one nucleotide is a sugar-modified nucleotide in the position of the editing mutation in the first single-stranded polynucleotide and in a region consisting of 10 bases on the 5'-end and 10 bases on the 3'-end of the position of the editing mutation, and (b) the first single-stranded polynucleotide comprises at least one modified internucleotide bond, wherein the modified internucleotide bond is not present in a range from 3 bases on the 5'-end to 3 bases on the 3'-end of the position of the editing mutation. Preferably, the composition does not contain an exogenous nuclease or a polynucleotide capable of expressing the same, and is not used in combination therewith. [a2] The composition according to [a1], wherein the sugar-modified nucleotide is a nucleotide containing a 2'-O,4'-C-methylene bridge or a 2'-O,4'-C-ethylene bridge in the ribose ring. [a3] The composition according to [a1] or [a2], wherein the first single-stranded polynucleotide contains one to four modified internucleotide linkages within 13 bases from the 5'-end and / or 3'-end. Preferably, the modified internucleotide linkages are phosphorothioate linkages, alkylphosphonate linkages, or phosphotriester linkages. More preferably, the first single-stranded polynucleotide contains one to four modified internucleotide linkages within five bases from the 3'-end (more preferably, two within three bases, or one within two bases), and the modified internucleotide linkage is a phosphorothioate linkage. [a4] The composition described in any of [a1] to [a3], wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 25 to 75% from the 5' end, with the 5' end to the 3' end being 100%.[a5] The composition according to [a4], wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 49 to 51% from the 5' end, with the 5' end to the 3' end being 100%. [a6] The composition according to any of [a1] to [a5], wherein the first single-stranded polynucleotide is 70 to 100 bases in length. [a7] The composition according to any of [a1] to [a6], further comprising a second single-stranded polynucleotide, wherein the second single-stranded polynucleotide is 20 to 200 bases in length and comprises a base sequence complementary to a base sequence of a portion of the region of the first single-stranded polynucleotide that does not contain the editing mutation, and is capable of forming a duplex with the first single-stranded polynucleotide. [a8] The composition according to [a7], wherein the second single-stranded polynucleotide comprises at least one modified internucleotide bond. [a9] The composition according to [a8], wherein the second single-stranded polynucleotide comprises one or two modified internucleotide linkages within three bases from the 5'-end and / or 3'-end, or one modified internucleotide linkage within two bases from the 5'-end and / or 3'-end. Preferably, the second single-stranded polynucleotide comprises one or two modified internucleotide linkages within three bases from the 3'-end, or one modified internucleotide linkage within two bases from the 3'-end. [a10] The composition according to any of [a7] to [a9], wherein the second single-stranded polynucleotide comprises a base sequence complementary to a portion of the base sequence of the first single-stranded polynucleotide that is 3'-terminally closer to the position of the editing mutation. [a11] The composition according to any of [a1], [a3], [a8], or [a9], wherein the modified internucleotide linkage is a phosphorothioate linkage, an alkylphosphonate linkage, or a phosphotriester linkage. [a12] A composition described in any of [a1] to [a11], further comprising a third single-stranded polynucleotide, wherein the third single-stranded polynucleotide is 15 to 200 bases in length and its base sequence has 90% or more sequence identity with a base sequence in the editing target strand that is located 5' or 3' closer to the position corresponding to the editing mutation in the first single-stranded polynucleotide, and wherein, in the editing target strand, the base sequence that has sequence identity with the first single-stranded polynucleotide and the base sequence that has sequence identity with the third single-stranded polynucleotide are overlapping, adjacent, or separated by 1 to 9 bases.[a13] The composition described in [a12], wherein the base sequence of the third single-stranded polynucleotide has 90% or more sequence identity with the base sequence of the first single-stranded polynucleotide in the editing target strand, located 5'-terminally closer to the position corresponding to the editing mutation in the first single-stranded polynucleotide. [a14] The composition described in [a12] or [a13], wherein, in the editing target strand, a base sequence having sequence identity with the first single-stranded polynucleotide and a base sequence having sequence identity with the third single-stranded polynucleotide are adjacent to each other. [a15] The composition described in any of [a12] to [a14], wherein the third single-stranded polynucleotide is 55 to 75 bases long. [a16] The composition described in any of [a12] to [a15], wherein the third single-stranded polynucleotide consists of a base sequence 100% identical to the corresponding base sequence in the editing target strand. [a17] The composition according to any one of [a12] to [a16], wherein the molar concentration of the third single-stranded polynucleotide is the same as or higher than the molar concentration of the second single-stranded polynucleotide. [a18] The composition according to any one of [a1] to [a17], wherein the target region contains an aberrant base sequence that causes a disease, and the base sequence of the first single-stranded polynucleotide contains the editing mutation so that the aberrant base sequence can be repaired, thereby treating or preventing the disease. [a19] A pharmaceutical composition for treating or preventing a disease caused by an aberrant base sequence contained in a target region present in intracellular double-stranded DNA, comprising the composition according to [a18]. [a20] A method for treating or preventing a disease caused by an aberrant base sequence contained in a target region present in intracellular double-stranded DNA, comprising administering to a subject a therapeutically or prophylactically effective amount of the composition according to [a18]. [a21] A method for producing an isolated cell into which a mutation has been introduced or an organism having a cell into which a mutation has been introduced, comprising the step of introducing the composition according to any one of [a1] to [a18] into the isolated cell or into a cell constituting the living body of the organism, thereby introducing a mutation into the base sequence of a target region present in double-stranded DNA in the cell. The method may be performed in vitro.[a22] Use of a composition described in any of [a1] to [a18] in the manufacture of a pharmaceutical for the treatment or prevention of a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell.

[0012] The present invention also encompasses the following inventions: [b1] A pharmaceutical composition comprising a third single-stranded polynucleotide for use in combination with a composition comprising a first single-stranded polynucleotide, wherein the first single-stranded polynucleotide is 50 to 200 bases long, its base sequence has 80% or more sequence identity with the base sequence of a portion of the editing target strand of a target region present in double-stranded DNA in a cell, and compared to the base sequence of the editing target strand, it contains at least one editing mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides, and the third single-stranded polynucleotide is 15 to 200 bases long, its base sequence has 90% or more sequence identity with the base sequence in the editing target strand located 5'-terminally or 3'-terminally closer to the position in the first single-stranded polynucleotide corresponding to the editing mutation, and wherein, in the editing target strand, the base sequence having sequence identity with the first single-stranded polynucleotide and the base sequence having sequence identity with the third single-stranded polynucleotide overlap, are adjacent, or are separated by 1 to 9 bases, A pharmaceutical composition, wherein the target region contains an abnormal base sequence that causes a disease, and the base sequence of the first single-stranded polynucleotide contains the editing mutation so that the abnormal base sequence can be repaired, thereby treating or preventing the disease. Preferably, the composition does not contain an exogenous nuclease or a polynucleotide capable of expressing it, and is not used in combination with them. [b2] The pharmaceutical composition according to [b1], wherein the base sequence of the third single-stranded polynucleotide has 90% or more sequence identity with a base sequence in the editing target strand that is 5'-terminal of the first single-stranded polynucleotide relative to the position corresponding to the editing mutation. [b3] The pharmaceutical composition according to [b1] or [b2], wherein, in the editing target strand, a base sequence sharing sequence identity with the first single-stranded polynucleotide and a base sequence sharing sequence identity with the third single-stranded polynucleotide are adjacent. [b4] The pharmaceutical composition according to any of [b1] to [b3], wherein the third single-stranded polynucleotide is 55 to 75 bases in length. [b5] A composition described in any of [b1] to [b4], characterized in that the third single-stranded polynucleotide consists of a base sequence that is 100% identical to the corresponding base sequence of the editing target strand.[b6] The pharmaceutical composition according to any one of [b1] to [b5], wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 25 to 75% from the 5' end, with the 5' end to the 3' end being 100%. [b7] The pharmaceutical composition according to [b6], wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 49 to 51% from the 5' end, with the 5' end to the 3' end being 100%. [b8] The pharmaceutical composition according to any one of [b1] to [b7], wherein the first single-stranded polynucleotide is 70 to 100 bases long. [b9] The composition according to any one of [b1] to [b8], wherein the first single-stranded polynucleotide consists of a base sequence 100% identical to the corresponding base sequence in the editing target strand, excluding the position of the editing mutation and the regions 10 bases from the 5' end and 10 bases from the 3' end. [b10] The pharmaceutical composition according to any one of [b1] to [b9], wherein the composition comprising a first single-stranded polynucleotide further comprises a second single-stranded polynucleotide, wherein the second single-stranded polynucleotide is 20 to 200 bases long and comprises a base sequence complementary to a base sequence of a portion of the region of the first single-stranded polynucleotide that does not contain the editing mutation, and is capable of forming a double-stranded chain with the first single-stranded polynucleotide. [b11] The pharmaceutical composition according to [b10], wherein the second single-stranded polynucleotide comprises a base sequence complementary to a portion of the base sequence of the first single-stranded polynucleotide that is 3'-terminally closer to the position of the editing mutation. [b12] The pharmaceutical composition according to [b10] or [b11], wherein the molar concentration of the third single-stranded polynucleotide is used so as to be the same as or higher than the molar concentration of the second single-stranded polynucleotide. [b13] The pharmaceutical composition according to any one of [b1] to [b12], for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell. [b14] A method for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell, the method comprising administering to a subject a therapeutically or prophylactically effective amount of the pharmaceutical composition described in [b13] together with a composition containing a first single-stranded polynucleotide simultaneously, separately or consecutively.[b15] A method for producing an isolated cell into which a mutation has been introduced or an organism having a cell into which a mutation has been introduced, comprising the step of introducing the pharmaceutical composition according to any one of [b1] to [b13] into the isolated cell or a cell constituting the living body of the organism simultaneously, separately, or sequentially with a composition containing a first single-stranded polynucleotide, thereby introducing a mutation into the base sequence of a target region present in double-stranded DNA in the cell. This method may be performed in vitro. [b16] Use of the pharmaceutical composition according to any one of [b1] to [b12] in the production of a pharmaceutical for the treatment or prevention of a disease caused by an aberrant base sequence in a target region present in double-stranded DNA in a cell.

[0013] According to the present invention, desired base editing can be performed on intracellular DNA using an editing method that utilizes ss DNA or TD.

[0014]

[0023] Figure 1 is a schematic diagram of polynucleotides used in conventional base sequence editing methods using single-stranded DNA (ss DNA) or 5'-tailed duplex (TD), as well as first to third single-stranded polynucleotides in specific embodiments of the present invention.

[0024] Figure 1 shows the experimental results of Example 1, in which the change in editing efficiency due to the addition of an A' strand in TD-based base sequence editing was investigated using the copGFP gene as the target gene. The arrowhead indicates an enlarged view of a fluorescent microscope image when the A' strand was added.

[0025] Figure 1 is a graph showing the experimental results of Example 1, expressed as editing efficiency (%). Bars indicate SEM (n=3).

[0026] Figure 1 is a graph showing the results of Example 2, in which the change in editing efficiency due to the addition of an A' strand in TD-based base sequence editing was investigated using the mEGFP gene as the target gene. The arrowhead indicates an enlarged view of a fluorescent microscope image when the A' strand was added.

[0027] Figure 1 is a graph showing the experimental results of Example 2, expressed as editing efficiency (%). Bars indicate SEM (n=3).

[0028] Figure 1 is a graph showing the results of Example 3 (3-1). The modified chain, the position, and the number of PS modifications are shown in parentheses next to each sample name on the horizontal axis. Bars indicate SEM (n=3). This graph shows the results of Example 3 (3-2). The modified chain, the position, and the number of PS modifications are shown in parentheses next to each sample name on the horizontal axis. Bars indicate SEM (n=3). This graph shows the results of Example 3 (3-3). The position of PS modifications on the E chain is shown in parentheses next to each sample name on the horizontal axis. Bars indicate SEM. **: P<0.01; ***: P<0.001 (n=3); n-TD' vs others. This graph shows the results of Example 3 (3-4). The modified chain, the position of P-Me or P-OEt modifications are shown in parentheses next to each sample name on the horizontal axis. n=3. This graph shows the results of Example 3 (3-5). The position and the number of P-OEt or PS modifications on the A chain are shown in parentheses next to each sample name on the horizontal axis. Bars indicate SEM. *: P<0.05 (n=3). Graph showing the results of Example 3 (3-6). The position of LNA modification in the E chain is shown in parentheses next to each sample name on the horizontal axis.Bars indicate SEM. *: P<0.05; *: *P<0.01; ***: P<0.001 (n=3); n-TD' vs others. Graphs showing the results of Example 3 (3-7). The position of the LNA modification in the E strand is indicated in parentheses next to each sample name on the horizontal axis. In the six samples on the right, one P-OEt modification was performed on the A strand. Bars indicate SEM. *: P<0.05; **: P<0.01 (n=3). Graphs showing the results of Example 4. Graphs showing the results of Example 5. The horizontal axis indicates the molar ratio of E strand, A strand, and A' strand (E strand:A strand:A' strand) in each sample. A shows the arrangement of the components of the editing nucleic acid used in Example 6 relative to the target region. B shows the results of Example 6. The number of gaps is indicated in parentheses next to each sample name on the horizontal axis. Bars indicate SEM. *: P<0.05; ***: P<0.001 (n=3). A is a diagram showing the arrangement of the components of the editing nucleic acid used in Example 7 relative to the target region. B is a graph showing the results of Example 7. The number of overlapping bases between strand E and strand A' is shown in parentheses next to each sample name on the horizontal axis. Bars indicate SEM. *: P<0.05; **: P<0.01 (n=3). A is a diagram showing the arrangement of the components of the editing nucleic acid used in Example 8 relative to the target region. B is a graph showing the results of Example 8. The parentheses next to each sample name on the horizontal axis indicate the direction in which strand A extended (5' or 3'), the length of strand A, and whether the base sequence in strand A is wild-type (WT) or mutant (Y / H) when it contains a base sequence complementary to the portion of strand E containing a mutation. Bars indicate SEM. *: P<0.05; **: P<0.01; ***: P<0.001 (n=3); n-TD' (35A) vs others. A is a diagram showing the arrangement of the components of the editing nucleic acid used in Example 9 relative to the target region. B is a graph showing the results of Example 9.The parentheses next to each sample name on the horizontal axis indicate the position of the mutation in strand E, which places the base complementary to the base at the 3' end of strand A at position -1. When strand A contains a base sequence complementary to the portion of strand E containing the mutation, the base sequence is distinguished as wild-type (WT) or mutant (Y / H). Bars indicate SEM. *: P<0.05; **: P<0.01 (n=3); n-TD' (A 40-WT) vs others. A shows the arrangement of the components of the editing nucleic acid used in Example 10 relative to the target region. B is a graph showing the results of Example 10. The parentheses next to each sample name on the horizontal axis indicate the molar ratio of pDNA, strand E, strand A, and strand A' (pDNA: strand E: strand A: strand A') in each sample. Bars indicate SEM. *: P<0.05; ***: P<0.001. The results of Example 11 are shown. This shows the results of genome editing using TD with the addition of the A' strand. This is a graph showing the results of Example 12. Bars indicate SEM. * P<0.05 (n=3). Graph showing the results of Example 13. Bars indicate SEM. * P < 0.05, **P<0.01 (n=3). Graph showing the results of Example 14. Graph showing the results of Example 15. Diagram of the plasmid used in Example 16. Graph showing the results of Example 16. The relative editing efficiency is shown, with the efficiency when pcDNA4_mPlum_T2A_copGFP Y / H incorporating fluorescence-quenched copGFP was edited using AS n-TD' set to 1. Bars indicate SEM. *P<0.05 **P<0.01 ***P<0.001 (n=3). A is a diagram showing the arrangement of the components of the editing nucleic acid used in Example 17-1 relative to the target region. The arrangement and name of the 79-base E strand and each 35-base ODN are shown. B is a graph showing the experimental results of Example 17-1 as editing efficiency (%). Bars indicate SEM. *: P<0.05; **: P<0.01 (n=3); nE strand only vs. others. This graph shows the experimental results of Example 17-2, expressed as editing efficiency (%). The values ​​on the horizontal axis indicate the dosages of E strand, A strand, and 35-base A' strand in molar ratios, assuming that the amount of pDNA introduced for editing efficiency analysis was 1. This graph shows the experimental results of Example 17-3, expressed as editing efficiency (%). The values ​​on the horizontal axis indicate the dosages of E strand, A strand, and 35-base A' strand in molar ratios, assuming that the amount of pDNA introduced for editing efficiency analysis was 1. This graph shows the experimental results of Example 17-4, expressed as editing efficiency (%). The values ​​on the horizontal axis indicate the dosages of E strand, A strand, and 35-base A' strand in molar ratios, assuming that the amount of pDNA introduced for editing efficiency analysis was 1. FIG. 1A is a diagram showing the arrangement of the components of the editing nucleic acid used in Example 17-5 relative to the target region. The arrangement and names of the 79-base E strand, A strand, and A' strands of different lengths are shown. FIG. 1B is a graph showing the experimental results of Example 17-5 as editing efficiency (%). FIG. 1C is a graph showing the experimental results of Example 17-6 as editing efficiency (%).This graph shows the analysis values ​​from the fluorescence intensity data for Example 17-7, plotted on the vertical axis against the proportion (%) of mPlum-T2A-copGFP (normal) expression vector plasmid among the introduced plasmids (mPlum-T2A-copGFP (normal) expression vector plasmid and mPlum-T2A-copGFP(Y / H) expression vector plasmid). The straight line is a regression line determined from the analysis values ​​up to the proportion of copGFP (normal) expression vector of 50%. y is the slope of the line, and R is the linear regression line. 2indicates the coefficient of determination of the linear relationship. This graph shows the experimental results of Example 17-8 (the editing efficiency of Example 17-6 was determined by the method of Example 17-7), expressed as editing efficiency (%). This graph shows the results of Example 18-1. The position of LNA modification in the E chain is indicated in parentheses next to each sample name on the horizontal axis. This graph shows the results of Example 18-2. The position of LNA modification in the E chain is indicated in parentheses next to each sample name on the horizontal axis. This graph shows the results of Example 18-3. The position of LNA modification in the E chain is indicated in parentheses next to each sample name on the horizontal axis. This graph shows the results of Example 18-4. The position of LNA modification in the E chain is indicated in parentheses next to each sample name on the horizontal axis. Bars indicate SEM. *: P<0.05; **: P<0.01 (n=3); n-TD' vs others. This graph shows the results of Example 18-5. The position of LNA modification in the E chain is indicated in parentheses next to each sample name on the horizontal axis. Bars indicate SEM. *: P<0.05; **: P<0.01; ***: P<0.05 (n=3); n-TD' vs others. The position of the LNA modification in the E strand is shown. This graph shows the results of Example 18-6. The position of the LNA modification in the E strand is shown in parentheses next to each sample name on the horizontal axis. This graph shows the results of Example 18-7. The position of the LNA modification in the E strand is shown in parentheses next to each sample name on the horizontal axis. Figure A shows the arrangement of the components of the editing nucleic acid used in Example 18-8 relative to the target region. Figures B to E are graphs showing the results of Example 18-8. The chain length of the A strand and / or the position of the LNA modification in the E strand is shown in parentheses next to each sample name on the horizontal axis. This graph shows the results of Example 18-9. The position of the LNA modification in the E strand is shown in parentheses next to each sample name on the horizontal axis. This graph shows the results of Example 18-10. The chain length of strand A is shown in parentheses next to the name of each sample on the horizontal axis. Graphs showing the results of Example 18-11. A is a graph showing the editing efficiency for the copGFP gene. B is a graph showing the editing efficiency for the DMD gene. C is a diagram showing the editing efficiency for the WRN gene. A diagram showing the structure of pNGS2-K3. Graphs showing the results of Example 19.ns: no significant difference.

[0015] The present invention will be described in detail below. Note that the embodiment described below is an example of a typical embodiment of the present invention, and the scope of the present invention should not be construed as being narrow.

[0016] 1. Definitions As used herein, "intracellular double-stranded DNA" refers to any double-stranded DNA present in cells that constitute a living organism or in isolated cells (e.g., cultured cells). Double-stranded DNA may include regions that encode proteins or RNA products and regions for regulating their expression. Intracellular double-stranded DNA may be endogenous double-stranded DNA (genomic DNA and mitochondrial DNA) or exogenous double-stranded DNA (foreign DNA introduced into cells, viral genomes, etc.).

[0017] As used herein, the term "target region" refers to a region of intracellular double-stranded DNA that contains a base sequence into which a mutation is to be introduced. The target region may be any region of intracellular double-stranded DNA, and may include, for example, a region that contains part or all of a structural gene region, a regulatory gene region, etc. A structural gene region is a region that determines the amino acid sequence of a protein, and structural gene regions in eukaryotic cells can include both exons and introns. Examples of regulatory gene regions include operator regions, promoter regions, and attenuator regions. In relation to the gene encoded by the double-stranded DNA, one strand constituting the target region is sometimes referred to as the sense strand, and the other strand as the antisense strand.

[0018] As used herein, "nucleotide sequence editing" refers to the introduction of a targeted mutation into a nucleotide sequence. As used herein, "mutation" refers to the deletion, substitution, or insertion of one or more bases in a reference nucleotide sequence, or a combination of two or more of these. A reference nucleotide sequence typically refers to, but is not limited to, a natural or normal nucleotide sequence.

[0019] As used herein, unless otherwise specified, "editing efficiency" refers to the proportion (percentage) of cells into which an editing nucleic acid has been introduced that have been successfully edited in base sequence editing of double-stranded DNA in cells. When calculating the editing efficiency in an in vitro experiment as in the Examples, either the calculation method described in Example 1, which takes into account the copy number of the target region in the cell, or the calculation method described in Example 17-1, which does not take into account the copy number of the target region in the cell, can be used.

[0020] As used herein, the term "exogenous nuclease" refers to a nuclease that is not endogenously contained in the target biological species to which the base sequence editing of the present invention is to be performed. The exogenous nuclease may vary depending on the target biological species to which the base sequence editing of the present invention is to be performed, but may be, for example, a nuclease derived from a non-eukaryotic biological species, a nuclease derived from a non-mammalian biological species, or a nuclease derived from a biological species other than humans, primates, mice, rats, dogs, cats, rabbits, horses, or cattle. Exogenous nucleases also include completely artificial nucleases such as zinc finger nucleases (ZFNs) and TALENs, and bacterial nucleases such as Cas nucleases.

[0021] As used herein, "Tailed Duplex" or "TD" refers to a double-stranded polynucleotide having a single-stranded portion at the 5'-end or 3'-end. TD can be prepared by any method known to those skilled in the art, for example, by mixing a single-stranded polynucleotide with another single-stranded polynucleotide having a base sequence complementary to a portion of the single-stranded polynucleotide, or by hybridizing the two.

[0022] As used herein, the term "single-stranded polynucleotide" refers to a molecule in which nucleotide monomers are covalently linked in a chain, and may be any of DNA, RNA, and DNA / RNA chimeras. A single-stranded polynucleotide may contain modified nucleotides (nucleotides with modified bases and / or sugar-modified nucleotides) or modified internucleotide linkages.

[0023] As used herein, the term "modified base" refers to a nucleotide base that has been modified by the substitution or addition of one or more atoms or groups. Modified bases are structurally different from natural or synthetic unmodified bases, but are functionally interchangeable. Modified bases include all types of modification known in the art to which the present invention pertains. Such modifications include, for example, alkylation, halogenation, thiolation, amination, amidation, or acetylation, or various combinations thereof. Modified bases include, for example, 5-methylcytosine.

[0024] As used herein, the term "sugar-modified nucleotide" refers to a nucleotide in which the sugar moiety of the nucleotide has been modified. Sugar-modified nucleotides include all types of sugar modifications known in the technical field to which the present invention belongs. For example, in the case of a nucleotide having a ribose ring, sugar-modified nucleotides include 2'-modified nucleotides, 4'-thio-modified nucleotides, 4'-thio-2'-modified nucleotides, and bicyclic sugar-modified nucleotides.

[0025] Examples of 2'-modified nucleotides include halo, allyl, amino, azido, O-allyl, O-C1-C10 alkyl, OCF ３ , O-(CH ２ ) ２ -O-CH ３ , 2'-O(CH ２ ) ２ SCH ３ , O-(CH ２ ) ２ —O—N(Rm)(Rn), or O—CH ２Examples include —C(═O)—N(Rm)(Rn), where each Rm and Rn is independently H, an amino-protecting group, or a substituted or unsubstituted C1-C10 alkyl. Commercially available amidite reagents can be used to synthesize 2′-O-methylguanosine, 2′-O-methyladenosine, 2′-O-methylcytidine, and 2′-O-methyluridine. 2′-O-Aminoethylguanosine, 2′-O-aminoethyladenosine, 2′-O-aminoethylcytidine, and 2′-O-aminoethyluridine can be synthesized using amidite reagents described in the literature (Blommers et al. Biochemistry (1998), 37, 17714-17725). 2'-O-Propylguanosine, 2'-O-propyladenosine, 2'-O-propylcytidine, and 2'-O-propyluridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838). Commercially available amidite reagents can be used to synthesize 2'-O-allylguanosine, 2'-O-allyladenosine, 2'-O-allylcytidine, and 2'-O-allyluridine. 2'-O-Methoxyethylguanosine, 2'-O-methoxyethyladenosine, 2'-O-methoxyethylcytidine, and 2'-O-methoxyethyluridine can be synthesized according to the patent (US6261840) or the literature (Martin, P. Helv. Chim. Acta. (1995) 78, 486-504). 2'-O-Butylguanosine, 2'-O-butyladenosine, 2'-O-butylcytidine, and 2'-O-butyluridine can be synthesized using amidite reagents described in the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838). 2'-O-Pentylguanosine, 2'-O-pentyladenosine, 2'-O-pentylcytidine, and 2'-O-pentyluridine can be synthesized according to the literature (Lesnik, EA et al. Biochemistry (1993), 32, 7832-7838.Commercially available amidite reagents can be used for 2'-O-propargylguanosine, 2'-O-propargyladenosine, 2'-O-propargylcytidine, and 2'-O-propargyluridine.

[0026] An example of a 4'-thio modified nucleotide is a β-D-ribonucleotide in which the 4'-oxygen atom is replaced with a sulfur atom (Hoshika, S. et al. FEBS Lett. 579, pp. 3115-3118, (2005); Dande, P. et al. J. Med. Chem. 49, pp. 1624-1634 (2006); Hoshika, S. et al. ChemBioChem. 8, pp. 2133-2138, (2007)).

[0027] Examples of 4'-thio-2'-modified nucleotides include 4'-thio-2'-modified nucleotides that retain 2'-H or 2'-O-methyl (Matsugami, et al. Nucleic Acids Res. 36, 1805 (2008)).

[0028] Examples of bicyclic sugar modified nucleotides include nucleotides that contain a second ring formed by bridging two atoms of the ribose ring. Examples of bicyclic sugar modifications include 2'-O,4'-C-bridged modifications, such as 2',4'-BNA / LNA (bridged nucleic acids / locked nucleic acids) in which the 2'-oxygen atom and the 4'-carbon atom are bridged with a methylene chain (Obika, S. et al. Tetrahedron Lett., 38, pp. 8735-(1997); Obika, S. et al., Tetrahedron Lett., 39, pp. 5401-(1998); A. A. Koshkin, A. A. et al. Tetrahedron, 54, p. 3607 (1998); Obika, S. Bioorg. Med. Chem., 9, p. 1001 (2001); and ENA (2'-O,4'-C-ethylene-bridged nucleic acids), in which the methylene chain of 2',4'-BNA / LNA is bridged with an ethylene chain extending by one carbon atom (Morita, K. et al. Bioorg. Med. Chem. Lett., 12, p. 73 (2002); Morita, K. et al. Bioorg. Med. Chem., 11, p. 2211 (2003)). Other examples include AmNA described in WO2014 / 109384 and S-cEt (2',4'-constrained ethyl) described in the literature (Seth, PP et al. J. Org. Chem (2010), 75, 1569-1581.). In addition, in the modification of the ribose ring, "2'-O,4'-C-methylene bridge" and "4'-CH ２ The term "-O-2' bridge" has the same meaning.

[0029] As used herein, the term "modified internucleotide linkage" refers to a substitution or alteration of the naturally occurring phosphate bond (i.e., a phosphodiester bond) between two nucleosides. In other words, a polynucleotide containing a modified internucleotide linkage contains a modification of the phosphate group of at least one nucleotide. Modified internucleotide linkages include all types of modifications known in the art to which the present invention pertains. Modified internucleotide linkages include, for example, phosphorothioate, phosphorodithioate, alkylphosphonate, boranophosphate, phosphoramidate, and phosphotriester linkages. Examples of alkylphosphonate linkages include a phosphodiester linkage modified by substituting a non-bridging oxygen atom with a methyl group (P-CH; methylphosphonate linkage). Examples of phosphotriester linkages include a phosphodiester linkage modified by substituting a non-bridging oxygen atom with an ethoxy group (P-OCH; ethylphosphotriester linkage).

[0030] 2. Base sequence editing using strand E and strand A' or strand E + strand A (Tailed Duplex (TD)) and strand A' In one aspect, the present invention provides a composition comprising a first single-stranded polynucleotide and a third single-stranded polynucleotide, wherein the first single-stranded polynucleotide is 50 to 200 bases long, and its base sequence has 80% or more sequence identity with the base sequence of a portion of the editing target strand of a target region present in double-stranded DNA in a cell, and compared to the base sequence of the editing target strand, contains at least one mutation for editing selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides; The third single-stranded polynucleotide is 15 to 200 bases in length, and its base sequence has 90% or more sequence identity with a base sequence in the editing target strand that is located 5' or 3' of the position in the first single-stranded polynucleotide corresponding to the editing mutation, and wherein, in the editing target strand, the base sequence that has sequence identity with the first single-stranded polynucleotide and the base sequence that has sequence identity with the third single-stranded polynucleotide overlap, are adjacent, or are separated by 1 to 9 bases (hereinafter also referred to as Composition I of the present invention).

[0031] By introducing Composition I of the present invention into a cell, base sequence editing can be performed on a target region present in double-stranded DNA in the cell according to an editing mutation contained in a first single-stranded polynucleotide. That is, Composition I of the present invention can be used to introduce a mutation into a target region present in double-stranded DNA in a cell. Furthermore, base sequence editing using Composition I of the present invention does not require an exogenous nuclease. Therefore, in a preferred embodiment, Composition I of the present invention does not contain an exogenous nuclease or a polynucleotide capable of expressing the exogenous nuclease, and is not used in combination with them.

[0032] In the present invention, the editing target strand refers to either strand of the double strand in the target region, i.e., the sense strand or the antisense strand. In a preferred embodiment, the editing target strand may be the antisense strand.

[0033] The base sequence of the first single-stranded polynucleotide (herein also referred to as the E strand (editor strand)) contains at least one mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides compared to the base sequence of a portion of the editing target strand of the target region present in the double-stranded DNA in the cell. Herein, the above mutation contained in the first single-stranded polynucleotide is referred to as an editing mutation. The editing mutation may be selected from the group consisting of deletion, substitution, and insertion of 1 to 10 nucleotides (10, 9, 8, 7, 6, 5, 4, 3, 2, or 1), for example. The editing mutation may be a combination of multiple different editing mutations. When multiple editing mutations are contained, they are preferably located adjacent to each other within a range of several bases (e.g., within 10, 9, 8, 7, 6, 5, 4, 3, or 2 bases). In a more preferred embodiment, the base sequence of the first single-stranded polynucleotide may contain only one editing mutation selected from the group consisting of a deletion, substitution, and insertion of one nucleotide compared to the base sequence of a portion of the editing target strand of the target region present in the double-stranded DNA in the cell.

[0034] The editing efficiency of the editing mutation in the first single-stranded polynucleotide tends to be higher when it is located near the center rather than near the 5' or 3' end. Therefore, in a preferred embodiment, the position at which the editing mutation is introduced in the first single-stranded polynucleotide may be within a range of 25 to 75% from the 5' end, for example, within a range of 30 to 70%, within a range of 35 to 65%, within a range of 40 to 60%, or within a range of 45 to 55%, more preferably within a range of 49 to 51%, with the range from the 5' end to the 3' end being 100%.

[0035] The length of the first single-stranded polynucleotide is not particularly limited as long as it is within the range of 50 to 200 bases and allows base sequence editing, and examples thereof include 55 to 200 bases, 60 to 200 bases, 65 to 200 bases, 70 to 200 bases, 50 to 195 bases, 55 to 195 bases, 60 to 195 bases, 65 to 195 bases, 70 to 195 bases, 50 to 190 bases, 55 to 190 bases, 60 to 190 bases, 65 to 190 bases, 70 to 190 bases, 50 to 185 bases, 55 to 185 bases, 60 to 185 bases, 65 to 185 bases, 70 to 185 bases, Base length, 50-180 base length, 55-180 base length, 60-180 base length, 65-180 base length, 70-180 base length, 50-175 base length, 55-175 base length, 60-175 base length, 65-175 base length, 70-175 base length, 50-170 base length, 55-170 base length, 60-170 base length, 65-170 base length, 70-170 base length, 50-165 base length, 55-165 base length, 60-165 base length, 65-165 base length, 70-165 base length, 50-160 base length, 55-160 base length, 60-160 base length, 65-160 base length, 70-165 base length 60 base length, 50-155 base length, 55-155 base length, 60-155 base length, 65-155 base length, 70-155 base length, 50-150 base length, 55-150 base length, 60-150 base length, 65-150 base length, 70-150 base length, 50-145 base length, 55-145 base length, 60-145 base length, 65-145 base length, 70-145 base length, 50-140 base length, 55-140 base length, 60-140 base length, 65-140 base length, 70-140 base length, 50-135 base length, 55-135 base length, 60-135 base length, 65-135 base length, 70 up to 135 base length, 50 to 130 base length, 55 to 130 base length, 60 to 130 base length, 65 to 130 base length, 70 to 130 base length, 50 to 125 base length, 55 to 125 base length, 60 to 125 base length, 65 to 125 base length, 70 to 125 base length, 50 to 120 base length, 55 to 120 base length, 60 to 120 base length, 65 to 120 base length, 70 to 120 base length, 50 to 115 base length, 55 to 115 base length, 60 to 115 base length, 65 to 115 base length, 70 to 115 base length, 50 to 110 base length, 55 to 110 base length, 60 to 110 base length, 65 to 110 base length,The length may be 70 to 110 bases, 50 to 105 bases, 55 to 105 bases, 60 to 105 bases, 65 to 105 bases, 70 to 105 bases, 50 to 100 bases, 55 to 100 bases, 60 to 100 bases, 65 to 100 bases, 70 to 100 bases, 50 to 95 bases, 55 to 95 bases, 60 to 95 bases, 65 to 95 bases, 70 to 95 bases, 50 to 90 bases, 55 to 90 bases, 60 to 90 bases, 65 to 90 bases, or 70 to 90 bases, preferably 50 to 100 bases, 60 to 100 bases, 70 to 100 bases, or 60 to 90 bases. The length of the first single-stranded polynucleotide may be, for example, 50 bases or more, 55 bases or more, 60 bases or more, 65 bases or more, or 70 bases or more, and 200 bases or less, 195 bases or less, 190 bases or less, 185 bases or less, 180 bases or less, 175 bases or less, 170 bases or less, 165 bases or less, 160 bases or less, 155 bases or less, 150 bases or less, 145 bases or less, 140 bases or less, 135 bases or less, 130 bases or less, 125 bases or less, 120 bases or less, 115 bases or less, 110 bases or less, 105 bases or less, 100 bases or less, 95 bases or less, or 90 bases or less.

[0036] The base sequence of the first single-stranded polynucleotide has 80% or more sequence identity with the base sequence of a portion of the editing target strand in the target region present in double-stranded DNA in a cell. Herein, a sequence in the editing target strand that shows sequence identity to the first single-stranded polynucleotide is also referred to as an "E-strand corresponding sequence." In one embodiment, a site other than the editing mutation (e.g., near the 5'-end (a region of 10 bases, 7 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base from the end) or near the 3'-end (a region of 10 bases, 7 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base from the end)) may contain a base or sequence that differs from the E-strand corresponding sequence by several bases (e.g., 10 bases, 9 bases, 8 bases, 7 bases, 6 bases, 5 bases, 4 bases, 3 bases, 2 bases, or 1 base) as long as it can improve the editing efficiency of the editing mutation and does not introduce unintended mutations into the double-stranded DNA in the cell. The sequence identity between the base sequence of the first single-stranded polynucleotide and the E-chain corresponding sequence may vary depending on the length of the first single-stranded polynucleotide and the mutations contained therein, but may be, for example, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% or more, 88% or more, 89% or more, 90% or more, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In certain embodiments, the first single-stranded polynucleotide may consist of a base sequence that is 100% identical to the corresponding base sequence in the editing target strand, excluding the position of the editing mutation and the 10-base region from the 5' end and the 10-base region from the 3' end. In certain embodiments, the base sequence of the first single-stranded polynucleotide may differ from the E-chain corresponding sequence only in the editing mutations introduced for base sequence editing. That is, the first single-stranded polynucleotide can consist of a base sequence that is 100% identical to the corresponding base sequence in the editing target strand, excluding the editing mutation.

[0037] The sequence on the editing target strand to be selected as the E-chain corresponding sequence can be determined appropriately, taking into consideration the location of the editing site. In one embodiment of the E-chain corresponding sequence of the present invention, the E-chain corresponding sequence may be selected so as to have a higher GC content. In this case, the GC content of the first single-stranded polynucleotide may be, for example, 50%, 55% or more, 60% or more, or 65% or more.

[0038] In one embodiment, the first single-stranded polynucleotide may be a polynucleotide consisting of a nucleotide sequence arranged in the following order from the 5' end to the 3' end: a "5' terminal sequence" of 0 to 10 bases, a "5' identical sequence" of 25 to 100 bases, a "mutant sequence" of 0 to 10 bases, a "3' identical sequence" of 25 to 100 bases, and a "3' terminal sequence" of 0 to 10 bases. Here, the 5' terminal sequence and the 3' terminal sequence are nucleotide sequences that may contain a mismatch of 10 bases or less with the corresponding nucleotide sequence in the E-chain corresponding sequence. The 5' identical sequence and the 3' identical sequence are nucleotide sequences that are identical to the corresponding nucleotide sequence in the E-chain corresponding sequence. In the 5' terminal sequence, the base adjacent to the 5' identical sequence is a mismatch with the E-chain corresponding sequence. In the 3' terminal sequence, the base adjacent to the 3' identical sequence is a mismatch with the E-chain corresponding sequence. The mutant sequence is a nucleotide sequence that includes an editing mutation, and may have a portion of a sequence or base identical to the corresponding sequence or base in the E-chain corresponding sequence. If the editing mutation is a substitution or insertion, the mutant sequence is adjacent to the 5'-identical sequence or the 3'-identical sequence at the base of the editing mutation, and if the editing mutation is a deletion, the mutant sequence is adjacent to the 5'-identical sequence or the 3'-identical sequence at the deletion site. Preferably, the mismatch contained in the 5'-terminal sequence and the 3'-terminal sequence each contains one base or less (if there is no mismatch within 10 bases at the 5'-terminal of the first single-stranded polynucleotide, there is no 5'-terminal sequence, and if there is no mismatch within 10 bases at the 3'-terminal, there is no 3'-terminal sequence), and more preferably there is no 5'-terminal sequence or 3'-terminal sequence. In a preferred embodiment, the ratio of the total length of the 5'-terminal sequence and the 5'-identical sequence to the total length of the 3'-identical sequence and the 3'-terminal sequence may be within a range of 25:75 to 75:25, for example, within a range of 30:70 to 70:30, within a range of 35:65 to 65:35, within a range of 40:60 to 60:40, or within a range of 45:55 to 55:45, more preferably within a range of 49:51 to 51:49, and most preferably 50:50.

[0039] Combining a third single-stranded polynucleotide (also referred to herein as strand A') with a first single-stranded polynucleotide can improve editing efficiency compared to base sequence editing using only the first single-stranded polynucleotide. Here, the term "third single-stranded polynucleotide" is a convenient designation and does not necessarily mean that the second single-stranded polynucleotide (strand A) is a component of Composition I of the present invention. Because TDs containing strand E and strand A have long been known, strand E and strand A were initially identified as the first single-stranded polynucleotide and the second single-stranded polynucleotide, respectively. The term "third" was used to distinguish them from strand A'. Therefore, the term "third single-stranded polynucleotide" itself does not imply that Composition I of the present invention contains a second single-stranded polynucleotide. Furthermore, the first single-stranded polynucleotide (strand E) and the third single-stranded polynucleotide (strand A') do not necessarily need to be used in combination with the second single-stranded polynucleotide (strand A).

[0040] The base sequence of the third single-stranded polynucleotide has 90% or more sequence identity with the base sequence in the editing target strand that is located 5'-terminal or 3'-terminal of the position of the editing mutation in the first single-stranded polynucleotide, and in the editing target strand, the base sequence that has sequence identity with the first single-stranded polynucleotide and the base sequence that has sequence identity with the third single-stranded polynucleotide overlap, are adjacent, or are separated by 1 to 9 bases. Herein, a sequence in the editing target strand that shows sequence identity with the third single-stranded polynucleotide is also referred to as an "A' strand corresponding sequence."

[0041] In the present invention, "adjacent" means that the A'-strand corresponding sequence is located contiguous with the E-strand corresponding sequence on the 5'-end or 3'-end side of the E-strand corresponding sequence in the editing target strand, without any gap between them. Figure 1 shows an arrangement in which the A'-strand corresponding sequence is located on the 5'-end side of the E-strand corresponding sequence in the editing target strand, and the E-strand corresponding sequence and the A'-strand corresponding sequence are adjacent to each other or separated by 1 to 9 bases.

[0042] Even if the E-strand corresponding sequence and the A'-strand corresponding sequence overlap or are separated by 1 to 9 bases in the editing target strand, the editing efficiency can be improved by combining such a third single-stranded polynucleotide with the first single-stranded polynucleotide, but it is preferable that the E-strand corresponding sequence and the A'-strand corresponding sequence are adjacent in the editing target strand.

[0043] In one embodiment, the third single-stranded polynucleotide may have a sequence identity of 90% or more with the base sequence of the first single-stranded polynucleotide on the editing target strand, which is located 5'-terminally closer to the position corresponding to the editing mutation in the first single-stranded polynucleotide.

[0044] The base sequence of the third single-stranded polynucleotide does not have to be completely identical to the base sequence of the editing target strand, as long as the effect of improving editing efficiency is not lost. The sequence identity may be 90% or more, for example, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more, but most preferably 100% sequence identity.

[0045] The length of the third single-stranded polynucleotide is not particularly limited as long as it is 15 to 200 bases long and the effect of improving editing efficiency is not lost. Depending on the length of the first single-stranded polynucleotide, the length may be, for example, 20 to 200 bases long, 25 to 200 bases long, 30 to 200 bases long, 35 to 200 bases long, 40 to 200 bases long, 45 to 200 bases long, 50 to 200 bases long, 55 to 200 bases long, 15 to 195 bases long, 20 to 195 bases long, 25 to 195 bases long, 30 to 195 bases long, 35 to 195 bases long, 40 to 195 bases long, 45 to 195 bases long, 50 to 195 bases long, 55 Up to 195 bases long, 15 to 190 bases long, 20 to 190 bases long, 25 to 190 bases long, 30 to 190 bases long, 35 to 190 bases long, 40 to 190 bases long, 45 to 190 bases long, 50 to 190 bases long, 55 to 190 bases long, 15 to 185 bases long, 20 to 185 bases long, 25 to 185 bases long, 30 to 185 bases long, 35 to 185 bases long, 40 to 185 bases long, 45 to 185 bases long, 50 to 185 bases long, 55 to 185 bases long, 15 to 180 bases long, 20 to 180 bases long, 25 to 180 bases long, 30 to 180 bases long, 35 to 180 bases long, 40 to 180 bases long, 5 to 180 base length, 50 to 180 base length, 55 to 180 base length, 15 to 175 base length, 20 to 175 base length, 25 to 175 base length, 30 to 175 base length, 35 to 175 base length, 40 to 175 base length, 45 to 175 base length, 50 to 175 base length, 55 to 175 base length, 15 to 170 base length, 20 to 170 base length, 25 to 170 base length, 30 to 170 base length, 35 to 170 base length, 40 to 170 base length, 45 to 170 base length, 50 to 170 base length, 55 to 170 base length, 15 to 165 base length, 20 to 165 base length, 25 to 165 base length, 30 to 165 base length, 35 to 165 base length, 40 to 165 base length, 45 to 165 base length, 50 to 165 base length, 55 to 165 base length, 15 to 160 base length, 20 to 160 base length, 25 to 160 base length, 30 to 160 base length, 35 to 160 base length, 40 to 160 base length, 45 to 160 base length, 50 to 160 base length, 55 to 160 base length, 15 to 155 base length, 20 to 155 base length, 25 to 155 base length, 30 to 155 base length, 35 to 155 base length, 40 to 155 base length, 45 to 155 base length, 50 to 155 base length, 55 to 155 base length, 15 to 150 base length, 20 to 150 base length,25-150 base length, 30-150 base length, 35-150 base length, 40-150 base length, 45-150 base length, 50-150 base length, 55-150 base length, 15-145 base length, 20-145 base length, 25-145 base length, 30-145 base length, 35-145 base length, 40-145 base length, 45-145 base length, 50-145 base length, 55-145 base length, 15-140 base length, 20-140 base length, 25-140 base length, 30-140 base length, 35-140 base length, 40-140 base length, 45-140 base length, 50-140 base length, 55-140 base length , 15 to 135 bases long, 20 to 135 bases long, 25 to 135 bases long, 30 to 135 bases long, 35 to 135 bases long, 40 to 135 bases long, 45 to 135 bases long, 50 to 135 bases long, 55 to 135 bases long, 15 to 130 bases long, 20 to 130 bases long, 25 to 130 bases long, 30 to 130 bases long, 35 to 130 bases long, 40 to 130 bases long, 45 to 130 bases long, 50 to 130 bases long, 55 to 130 bases long, 15 to 125 bases long, 20 to 125 bases long, 25 to 125 bases long, 30 to 125 bases long, 35 to 125 bases long, 40 to 125 bases long, 45 to 125 bases length, 50-125 bases, 55-125 bases, 15-120 bases, 20-120 bases, 25-120 bases, 30-120 bases, 35-120 bases, 40-120 bases, 45-120 bases, 50-120 bases, 55-120 bases, 15-115 bases, 20-115 bases, 25-115 bases, 30-115 bases, 35-115 bases, 40-115 bases, 45-115 bases, 50-115 bases, 55-115 bases, 15-110 bases, 20-110 bases, 25-110 bases, 30-110 bases, 35-110 bases Base length, 40 to 110 base length, 45 to 110 base length, 50 to 110 base length, 55 to 110 base length, 15 to 105 base length, 20 to 105 base length, 25 to 105 base length, 30 to 105 base length, 35 to 105 base length, 40 to 105 base length, 45 to 105 base length, 50 to 105 base length, 55 to 105 base length, 15 to 100 base length, 20 to 100 base length, 25 to 100 base length, 30 to 100 base length, 35 to 100 base length, 40 to 100 base length, 45 to 100 base length, 50 to 100 base length, 55 to 100 base length, 15 to 95 base length, 20 to 95 base length, 25 to 95 base length,30-95 base length, 35-95 base length, 40-95 base length, 45-95 base length, 50-95 base length, 55-95 base length, 15-90 base length, 20-90 base length, 25-90 base length, 30-90 base length, 35-90 base length, 40-90 base length, 45-90 base length, 50-90 base length, 55-90 base length, 15-85 base length, 20-85 base length, 25-85 base length, 30-85 base length, 35-85 base length, 40-85 base length, 45-85 base length, 50-85 base length , 55 to 85 bases in length, 15 to 80 bases in length, 20 to 80 bases in length, 25 to 80 bases in length, 30 to 80 bases in length, 35 to 80 bases in length, 40 to 80 bases in length, 45 to 80 bases in length, 50 to 80 bases in length, 55 to 80 bases in length, 15 to 75 bases in length, 20 to 75 bases in length, 25 to 75 bases in length, 30 to 75 bases in length, 35 to 75 bases in length, 40 to 75 bases in length, 45 to 75 bases in length, 50 to 75 bases in length, or 55 to 75 bases in length, preferably 15 to 75 bases in length or 55 to 75 bases in length. The length of the third single-stranded polynucleotide is, for example, 15 bases or more, 20 bases or more, 25 bases or more, 30 bases or more, 35 bases or more, 40 bases or more, 45 bases or more, 50 bases or more, or 55 bases or more, and 200 bases or less, 195 bases or less, 190 bases or less, 185 bases or less, 180 bases or less, 175 bases or less, 170 bases or less, 165 bases or less. The length may be 160 bases or less, 155 bases or less, 150 bases or less, 145 bases or less, 140 bases or less, 135 bases or less, 130 bases or less, 125 bases or less, 120 bases or less, 115 bases or less, 110 bases or less, 105 bases or less, 100 bases or less, 95 bases or less, 90 bases or less, 85 bases or less, 80 bases or less, or 75 bases or less. In one embodiment, the third single-stranded polynucleotide may have a base length of, for example, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, or 70% or more, and 200% or less, 150% or less, 100% or less, or 95% or less of the base length of the first single-stranded polynucleotide, preferably 70 to 95%.

[0046] In a preferred embodiment, the position of the editing mutation in the first single-stranded polynucleotide is within a range of 49 to 51%, with the 5' end to the 3' end being 100%, the sequence identity between the base sequence of the first single-stranded polynucleotide and the E-strand corresponding sequence is 98% or more, and the length of the first single-stranded polynucleotide is 70 to 100 bases long; the third single-stranded polynucleotide has a sequence identity of 90% or more with the base sequence of the first single-stranded polynucleotide 5'-endward of the position of the editing mutation in the editing target strand, the E-strand corresponding sequence and the A'-strand corresponding sequence are adjacent, and the length of the third single-stranded polynucleotide is 55 to 75 bases long.

[0047] In one embodiment, Composition I of the present invention may further comprise a second single-stranded polynucleotide (also referred to herein as Strand A (Assistant Strand)). The second single-stranded polynucleotide is 20 to 200 bases long and comprises a base sequence complementary to a portion of the base sequence of the region of the first single-stranded polynucleotide that does not contain the editing mutation, and can form a double-stranded chain with the first single-stranded polynucleotide. If the second single-stranded polynucleotide comprises a base sequence complementary to the region of the first single-stranded polynucleotide that contains the editing mutation (mutated portion or mutated sequence), editing efficiency may be reduced. Therefore, the second single-stranded polynucleotide does not comprise a base sequence complementary to the region of the first single-stranded polynucleotide that contains the editing mutation. The second single-stranded polynucleotide may comprise a base sequence complementary to the base sequence of either the 5'-end or 3'-end of the editing mutation of the first single-stranded polynucleotide, as long as it can form a double-stranded chain with the first single-stranded polynucleotide. In a preferred embodiment, the second single-stranded polynucleotide may comprise a base sequence complementary to a portion of the base sequence of the first single-stranded polynucleotide on the 3'-terminal side of the position of the editing mutation (see Figure 1).

[0048] In the second single-stranded polynucleotide, the length of the base sequence complementary to the first single-stranded polynucleotide is not particularly limited as long as base sequence editing is possible, and may be, for example, 20 to 100 bases long (e.g., 25 to 100 bases long, 30 to 100 bases long, 20 to 95 bases long, 25 to 95 bases long, 30 to 95 bases long, 20 to 90 bases long, 25 to 90 bases long, 30 to 90 bases long, 20 to 85 bases long, 25 to 85 bases long, 30 to 85 bases long, 20 to 80 bases long, 25 to 80 bases long, 30 to 80 bases long, 20 to 75 bases long, 25 to 75 bases long, 30 to 75 bases long, 20 to 70 bases long, 25 to 70 bases long, 30 to The length may be 70 bases, 20 to 65 bases, 25 to 65 bases, 30 to 65 bases, 20 to 60 bases, 25 to 60 bases, 30 to 60 bases, 20 to 55 bases, 25 to 55 bases, 30 to 55 bases, 20 to 50 bases, 25 to 50 bases, 30 to 50 bases, 20 to 45 bases, 25 to 45 bases, 30 to 45 bases, 20 to 40 bases, 25 to 40 bases, 30 to 40 bases, 20 to 35 bases, 25 to 35 bases, or 30 to 35 bases), preferably 25 to 50 bases, 30 to 50 bases, 35 to 50 bases, or 30 to 45 bases, more preferably 30 to 35 bases.

[0049] The length of the second single-stranded polynucleotide is not particularly limited as long as it is 20 to 200 bases long and allows base sequence editing. Depending on the length and position of the mutation of the first single-stranded polynucleotide, the length may be, for example, 25 to 200 bases long, 30 to 200 bases long, 20 to 195 bases long, 25 to 195 bases long, 30 to 195 bases long, 20 to 190 bases long, 25 to 190 bases long, 30 to 190 bases long, 20 to 185 bases long, 25 to 185 bases long, 30 to 185 bases long, 20 to 180 bases long, 25 to 180 bases long, 30 to 180 bases long, 20 to 175 bases long, 25 to 175 bases long, 30-175 base length, 20-170 base length, 25-170 base length, 30-170 base length, 20-165 base length, 25-165 base length, 30-165 base length, 20-160 base length, 25-160 base length, 30-160 base length, 20-155 base length, 25-155 base length, 30-155 base length, 20-150 base length, 25-150 base length, 30-150 base length, 20-145 base length, 25-145 base length, 30-145 base length, 20-140 base length, 25-140 base length, 30-140 base length, 20-135 base length, 25-135 base length, 30-135 base length , 20-130 base length, 25-130 base length, 30-130 base length, 20-125 base length, 25-125 base length, 30-125 base length, 20-120 base length, 25-120 base length, 30-120 base length, 20-115 base length, 25-115 base length, 30-115 base length, 20-110 base length, 25-110 base length, 30-110 base length, 20-105 base length, 25-105 base length, 30-105 base length, 20-100 base length, 25-100 base length, 30-100 base length, 20-95 base length, 25-95 base length, 30-95 base length, 20-90 base length, 25 Up to 90 bases long, 30 to 90 bases long, 20 to 85 bases long, 25 to 85 bases long, 30 to 85 bases long, 20 to 80 bases long, 25 to 80 bases long, 30 to 80 bases long, 20 to 75 bases long, 25 to 75 bases long, 30 to 75 bases long, 20 to 70 bases long, 25 to 70 bases long, 30 to 70 bases long, 20 to 65 bases long, 25 to 65 bases long, 30 to 65 bases long, 20 to 60 bases long, 25 to 60 bases long, 30 to 60 bases long, 20 to 55 bases long, 25 to 55 bases long, 30 to 55 bases long, 20 to 50 bases long, 25 to 50 bases long, 30 to 50 bases long, 20 to 45 bases long, 25 to 45 bases long,The length may be 30 to 45 bases, 20 to 40 bases, 25 to 40 bases, 30 to 40 bases, 20 to 35 bases, 25 to 35 bases, or 30 to 35 bases, preferably 25 to 100 bases, 30 to 100 bases, 35 to 100 bases, 30 to 90 bases, 25 to 50 bases, 30 to 50 bases, 35 to 50 bases, or 30 to 45 bases, more preferably 30 to 35 bases (e.g., 33 bases, 34 bases, or 35 bases), and even more preferably 34 bases or 35 bases.

[0050] In one embodiment, the second single-stranded polynucleotide may be a polynucleotide consisting of a base sequence complementary to a base sequence on the 5' or 3' side of the editing site in the editing target strand (referred to as the "A-strand corresponding sequence"). The A-strand corresponding sequence may be within the range of the E-strand corresponding sequence or may be a sequence extending beyond the end of the E-strand corresponding sequence. If the A-strand corresponding sequence extends beyond the range of the E-strand corresponding sequence, the length of the excess bases may be the same or shorter than the length from the editing site to the end of the E-strand corresponding sequence, and preferably may be 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, or 10% or less of the length from the editing site to the end of the E-strand corresponding sequence.

[0051] The second single-stranded polynucleotide may have any nucleotide sequence inserted at its 5'-end or 3'-end, as long as it does not reduce editing efficiency. Examples of such nucleotide sequences include those that form a hairpin structure similar to that of the single-stranded DNA in the TD disclosed in WO 2006 / 064813.

[0052] When Composition I of the present invention comprises three types of single-stranded polynucleotides, first to third, the molar ratio of the first, second, and third single-stranded polynucleotides in the composition is not particularly limited as long as base sequence editing is possible. However, the lower the molar ratio of the second single-stranded polynucleotide, or the lower the ratio of the molar concentration or dose of the second single-stranded polynucleotide to the sum of the molar concentrations or doses (molar) of the first and third single-stranded polynucleotides, the higher the editing efficiency may be. Thus, in one embodiment, the molar concentration or dose of the second single-stranded polynucleotide may be 0.2 to 2, preferably 0.2 to 1, and more preferably 0.2 to 0.5, where the sum of the molar concentrations or doses of the first and third single-stranded polynucleotides is 1. Alternatively, the molar concentration or dose of the second single-stranded polynucleotide may preferably be the same as or lower than the sum of the molar concentrations or doses of the first and third single-stranded polynucleotides. Note that, in this specification, "dosage" refers to the molar concentration at the time of administration to a cell or subject to be subjected to base sequence editing.

[0053] Furthermore, when Composition I of the present invention comprises three types of single-stranded polynucleotides, first to third, or two types of single-stranded polynucleotides, first and third, the editing efficiency may be higher as the ratio of the molar concentration or dose (mol) of the third single-stranded polynucleotide to the molar concentration or dose (mol) of the first single-stranded polynucleotide increases. Thus, in one embodiment, the molar concentration or dose of the third single-stranded polynucleotide may be 0.25 to 4, preferably 1 to 4, where the molar concentration or dose of the first single-stranded polynucleotide is 1. Alternatively, it may be preferable that the molar concentration or dose of the third single-stranded polynucleotide is the same as or higher than the molar concentration or dose of the first single-stranded polynucleotide.

[0054] Furthermore, the higher the ratio of the molar concentration or dose (molar) of the third single-stranded polynucleotide to the molar concentration or dose (molar) of the second single-stranded polynucleotide, the higher the editing efficiency may be. Thus, in one embodiment, when Composition I of the present invention comprises three types of single-stranded polynucleotides, first to third, the molar concentration or dose of the third single-stranded polynucleotide may be 0.25 to 4, preferably 1 to 4, where the molar concentration or dose of the second single-stranded polynucleotide is 1. Alternatively, it may be preferable that the molar concentration or dose of the third single-stranded polynucleotide is the same as or higher than the molar concentration or dose of the second single-stranded polynucleotide.

[0055] In a preferred embodiment, the position of the editing mutation in the first single-stranded polynucleotide is within a range of 49 to 51%, with the 5' end to the 3' end being 100%, the sequence identity between the base sequence of the first single-stranded polynucleotide and the sequence corresponding to the E chain is 98% or more, and the length of the first single-stranded polynucleotide is 70 to 100 bases; the second single-stranded polynucleotide comprises a base sequence complementary to a portion of the base sequence of the first single-stranded polynucleotide on the 3' end side of the position of the editing mutation, and the length of the second single-stranded polynucleotide is 30 to 100 bases; and the third single-stranded polynucleotide has a sequence identity of 90% or more with the base sequence of the first single-stranded polynucleotide on the 5' end side of the position of the editing mutation in the editing target strand, the sequence corresponding to the E chain and the sequence corresponding to the A' chain are adjacent, and the length of the third single-stranded polynucleotide is 55 to 75 bases.

[0056] The first to third single-stranded polynucleotides may be DNA, RNA, or a DNA / RNA chimera, respectively, as long as their base sequences can be edited, but are preferably DNA. The first to third single-stranded polynucleotides may contain modified nucleotides or modified internucleotide bonds, as long as their base sequences can be edited.

[0057] In one embodiment, the first single-stranded polynucleotide may have at least one of the following characteristics: (a) at least one nucleotide is a sugar-modified nucleotide in the position of the editing mutation in the first single-stranded polynucleotide and in a region consisting of 10 bases on the 5'-end and 10 bases on the 3'-end of the position of the editing mutation; and (b) the first single-stranded polynucleotide includes at least one modified internucleotide bond, and the modified internucleotide bond is not present in a range from 3 bases on the 5'-end to 3 bases on the 3'-end of the position of the editing mutation.

[0058] In feature (a), when the position of the sugar-modified nucleotide is specified, among the nucleotides identical to the editing target strand on the 5'-terminal and 3'-terminal sides of the position of the editing mutation in the first single-stranded polynucleotide, the nucleotide closest to the position of the editing mutation is designated as position 1. For example, "the N-position on the 5'-terminal side of the position of the editing mutation" refers to the nucleotide N-th closest to the editing mutation among the nucleotides identical to the editing target strand on the 5'-terminal side of the position of the editing mutation in the first single-stranded polynucleotide. In this specification, "the N-position on the 5'-terminal side of the position of the editing mutation" may be referred to as "-N-position," and "the N-position on the 3'-terminal side of the position of the editing mutation" may be referred to as "+N-position." "The N-position on the 3'-terminal side of the position of the editing mutation" refers to the nucleotide N-th closest to the editing mutation among the nucleotides identical to the editing target strand on the 3'-terminal side of the position of the editing mutation in the first single-stranded polynucleotide. In addition, when the first single-stranded polynucleotide contains multiple editing mutations, the position of the sugar-modified nucleotide on the 5'-end is determined based on the position of the editing mutation located most 5' among the editing mutations, and the position of the sugar-modified nucleotide on the 3'-end is determined based on the position of the editing mutation located most 3' among the editing mutations. "10 bases on the 5'-end side of the position of the editing mutation" refers to the bases 1 to 10 on the 5'-end side of the position of the editing mutation. "10 bases on the 3'-end side of the position of the editing mutation" refers to the bases 1 to 10 on the 3'-end side of the position of the editing mutation.

[0059] The number and positions of sugar-modified nucleotides to be introduced into the first single-stranded polynucleotide can be appropriately selected from the nucleotides contained in the region consisting of the position of the mutation for editing and the 10 bases on the 5'-end and 10 bases on the 3'-end of the position of the mutation for editing, as long as base sequence editing is possible. The number of sugar-modified nucleotides is not particularly limited, but may be, for example, 15 or less, and preferably 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0060] In one embodiment, in the first single-stranded polynucleotide, the position of the editing mutation, at least one selected from the nucleotides at positions 1, 3, 5, 7, and 9 on the 5'-end side of the position of the editing mutation, and at least one selected from the nucleotides at positions 1, 3, 5, 7, and 9 on the 3'-end side of the position of the editing mutation may be a sugar-modified nucleotide, for example, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 1 and 3 on the 5'-end side of the position of the editing mutation, and the nucleotide at position 3 on the 3'-end side of the position of the editing mutation may be a sugar-modified nucleotide. The nucleotides at positions 1 and 3 on the terminal side, the nucleotides at positions 1, 3, and 5 on the 5'-terminal side and the nucleotides at positions 1, 3, and 5 on the 3'-terminal side of the position of the editing mutation, the nucleotides at positions 1, 3, 5, and 7 on the 5'-terminal side and the nucleotides at positions 1, 3, 5, and 7 on the 3'-terminal side of the position of the editing mutation, or the nucleotides at positions 1, 3, 5, 7, and 9 on the 5'-terminal side and the nucleotides at positions 1, 3, 5, 7, and 9 on the 3'-terminal side of the position of the editing mutation may be sugar-modified nucleotides. In a particular embodiment, the first single-stranded polynucleotide comprises: the nucleotide at the position of the editing mutation; the nucleotide at position 1 on the 5'-end side of the position of the editing mutation; the nucleotide at position 1 and the 3'-end side of the position of the editing mutation; the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the 1st nucleotide on the 3'-end side of the position of the editing mutation; the nucleotide at position 1 on the 5'-end side of the position of the editing mutation; the nucleotide at position 1 and the 3'-end side of the position of the editing mutation; the nucleotide at position 3 and the 1st nucleotide on the 5'-end side of the position of the editing mutation and the 1st and 3rd nucleotide on the 3'-end side of the position of the editing mutation; the nucleotides at positions 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the 1st, 3rd and 5th nucleotides on the 3'-end side of the position of the editing mutation; the 2nd nucleotide on the 5'-end side of the position of the editing mutation and the 2nd nucleotide on the 3'-end side of the position of the editing mutation;the nucleotides at positions 2 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 2 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3 and 2 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 2 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3, 2 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 2 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 7, 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3, 5 and 7 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 9, 7, 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3, 5, 7 and 9 on the 3'-end side of the position of the editing mutation, any one of the nucleotides at positions 1 to 6 on the 5'-end of the position of the editing mutation, any one of the nucleotides at positions 1 to 6 on the 3'-end of the position of the editing mutation, the nucleotide at position 2 on the 5'-end of the position of the editing mutation and the nucleotide at position 2 on the 3'-end, the nucleotide at position 3 on the 5'-end of the position of the editing mutation and the nucleotide at position 3 on the 3'-end, the nucleotide at position 4 on the 5'-end of the position of the editing mutation and the nucleotide at position 4 on the 3'-end, the nucleotide at position 5 on the 5'-end of the position of the editing mutation and the nucleotide at position 5 on the 3'-end, the nucleotide at position 6 on the 5'-end of the position of the editing mutation and the nucleotide at position 6 on the 3'-end, the nucleotides at positions 5 and 3 on the 5'-end of the position of the editing mutation, the nucleotides at positions 4 and 2 on the 5'-end of the position of the editing mutation, the nucleotides at positions 3 and 1 on the 5'-end of the position of the editing mutation, the nucleotides at positions 1 and 3 on the 3'-end of the position of the editing mutation, the nucleotides at positions 2 and 4 on the 3'-end of the position of the editing mutation, the nucleotides at positions 3 and 5 on the 3'-terminal side of the position of the editing mutation, the nucleotide at position 7 on the 5'-terminal side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 6 on the 5'-terminal side of the position of the editing mutation and the nucleotide at the position of the editing mutation,the nucleotide at position 5 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 4 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 5 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 6 on the 3'-end of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 7 on the 3'-end of the position of the editing mutation, the nucleotide at position 1 on the 5'-end of the position of the editing mutation, the nucleotide at position 1 on the 3'-end of the position of the editing mutation, the nucleotide at position 2 on the 5'-end of the position of the editing mutation, the nucleotide at position 2 on the 3'-end of the position of the editing mutation, the nucleotide at position 3 on the 5'-end of the position of the editing mutation, the nucleotide at position 3 on the 3'-end of the position of the editing mutation, the nucleotide at position 4 on the 5'-end of the position of the editing mutation, the nucleotide at position 4 on the 3'-end of the position of the editing mutation, the nucleotide at position 5 on the 5'-end of the position of the editing mutation, and the nucleotide at position 5 on the 3'-end of the position of the editing mutation,the nucleotide at position 6 on the 5'-end side of the position of the editing mutation, the nucleotide at position 6 on the 3'-end side of the position of the editing mutation, the nucleotide at position 7 on the 5'-end side of the position of the editing mutation, the nucleotide at position 7 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation and the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 2 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 2 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3 and 2 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 2 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3, 2 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 2 and 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end side of the position of the editing mutation, The following may be sugar-modified nucleotides: the nucleotides at positions 3 and 1 on the 5'-end of the position of the editing mutation and the nucleotides at positions 1 and 3 on the 3'-end of the position of the editing mutation; the nucleotides at positions 3 and 1 on the 5'-end of the position of the editing mutation and the nucleotides at positions 1, 3 and 4 on the 3'-end of the position of the editing mutation; the nucleotide at the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end of the position of the editing mutation; the nucleotide at position 1 on the 5'-end of the position of the editing mutation, the nucleotide at position 4 on the 3'-end of the position of the editing mutation; the nucleotide at position 2 and 4 on the 3'-end of the position of the editing mutation; or the nucleotide at position 2 on the 5'-end of the position of the editing mutation, the nucleotide at position 2 and the nucleotide at position 4 on the 3'-end of the position of the editing mutation, and the nucleotide at position 4 on the 3'-end of the position of the editing mutation. In a preferred embodiment, the first single-stranded polynucleotide comprises: a nucleotide at the position of the editing mutation; a nucleotide at the first position on the 5'-terminal side of the position of the editing mutation; a nucleotide at the position of the editing mutation; and a nucleotide at the first position on the 3'-terminal side of the position of the editing mutation.the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation, the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotide at position 1 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3 and 5 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation and the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 and 3 on the 3'-end of the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation and the nucleotide at position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotide at position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 2 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 4 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 5 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 6 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 7 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation, the nucleotide at position 2 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation, the nucleotide at position 3 on the 3'-end side of the position of the editing mutation,the nucleotide at position 4 on the 5'-end side of the position of the editing mutation, the nucleotide at position 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 5 on the 5'-end side of the position of the editing mutation, the nucleotide at position 5 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3 and 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation, the nucleotide at position 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 and 4 on the 3'-end side of the position of the editing mutation, or The second nucleotide on the 5'-terminal side of the position of the editing mutation, the nucleotide at the position of the editing mutation, and the fourth nucleotide on the 3'-terminal side of the position of the editing mutation can be sugar-modified nucleotides.

[0061] In a preferred embodiment, the sugar-modified nucleotide may be a nucleotide containing a 4'-lower linear alkylene-O-2' bridge in the ribose ring, more preferably a nucleotide containing a 2'-O,4'-C-methylene bridge or a 2'-O,4'-C-ethylene bridge in the ribose ring, and even more preferably an LNA or ENA.

[0062] In feature (b), the position of the modified internucleotide bond is represented by the position of the base having a modification at the 3'-side phosphate moiety. Regarding the position near the editing mutation, the base closest to the editing mutation among the bases identical to the editing target strand on the 5'-end and 3'-end sides of the editing mutation in the first single-stranded polynucleotide is used as the reference (position 1). Note that, when the first single-stranded polynucleotide contains multiple editing mutations, the position of the modified internucleotide bond on the 5'-end side is determined based on the position of the editing mutation located closest to the 5'-end of the editing mutations, and the position of the modified internucleotide bond on the 3'-end side is determined based on the editing mutation located closest to the 3'-end of the editing mutations.

[0063] When a modified internucleotide bond is present in the first single-stranded polynucleotide within a range from 3 bases on the 5'-end side to 3 bases on the 3'-end side of the position of the editing mutation (i.e., within a range from the third base on the 5'-end side to the third base on the 3'-end side of the editing mutation), the editing efficiency may decrease. Therefore, a modified internucleotide bond is not positioned within this range.

[0064] In one embodiment, the first single-stranded polynucleotide may contain 1 to 4 (e.g., 1, 2, 3, or 4) modified internucleotide linkages near the 5'-end (e.g., within 20 bases, 15 bases, 13 bases, 10 bases, 8 bases, 6 bases, 5 bases, 4 bases, 3 bases, or 2 bases from the 5'-end) and / or near the 3'-end (e.g., within 20 bases, 15 bases, 13 bases, 10 bases, 8 bases, 6 bases, 5 bases, 4 bases, 3 bases, or 2 bases from the 3'-end). The modified internucleotide linkages in this case are not particularly limited, but are preferably phosphorothioate linkages (also referred to as PS modification), alkylphosphonate linkages, or phosphotriester linkages, more preferably phosphorothioate linkages. In a preferred embodiment, the first single-stranded polynucleotide may contain 1 to 4 modified internucleotide linkages within 13 bases (more preferably within 5 bases) from the 3'-end. In a more preferred embodiment, the first single-stranded polynucleotide may contain two modified internucleotide linkages within three bases or one modified internucleotide linkage within two bases from the 3'-end. In an even more preferred embodiment, the first single-stranded polynucleotide may contain one to four (preferably two modified internucleotide linkages within three bases or one modified internucleotide linkage within two bases) within 13 bases from the 3'-end, and the modified internucleotide linkage may be a phosphorothioate linkage.

[0065] In a preferred embodiment, the position of the editing mutation in the first single-stranded polynucleotide is within a range of 49 to 51%, assuming that the sequence from the 5' end to the 3' end is 100%, the sequence identity between the base sequence of the first single-stranded polynucleotide and the E-chain corresponding sequence is 98% or more, the length of the first single-stranded polynucleotide is 70 to 100 bases long, and the first single-stranded polynucleotide comprises: a nucleotide at the position of the editing mutation, a nucleotide at position 1 on the 5' end side of the position of the editing mutation, a nucleotide at position 1 and a nucleotide at position 1 on the 3' end side of the position of the editing mutation, a nucleotide at position 1 on the 5' end side of the position of the editing mutation and a nucleotide at position 1 on the 3' end side of the position of the editing mutation, a nucleotide at position 1 on the 5' end side of the position of the editing mutation, a nucleotide at position 1 on the 5' end side of the position of the editing mutation, and a nucleotide at position 1 on the 3' end side of the position of the editing mutation, the nucleotides at positions 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3 and 5 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation and the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 and 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position of the editing mutation and the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at position of the editing mutation and the nucleotide at position 2 on the 3'-end side of the position of the editing mutation, the nucleotide at position of the editing mutation and the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the 4th nucleotide on the 3'-terminal side of the position of the editing mutation; the nucleotide at the position of the editing mutation and the 5th nucleotide on the 3'-terminal side of the position of the editing mutation;the nucleotide at the position of the editing mutation and the nucleotide at position 6 on the 3'-end of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 7 on the 3'-end of the position of the editing mutation, the nucleotide at position 2 on the 5'-end of the position of the editing mutation, the nucleotide at position 2 on the 3'-end of the position of the editing mutation, the nucleotide at position 3 on the 5'-end of the position of the editing mutation, the nucleotide at position 3 on the 3'-end of the position of the editing mutation, the nucleotide at position 4 on the 5'-end of the position of the editing mutation, the nucleotide at position 4 on the 3'-end of the position of the editing mutation, the nucleotide at position 5 on the 5'-end of the position of the editing mutation, the nucleotide at position 5 on the 3'-end of the position of the editing mutation, the nucleotide at position 1 on the 5'-end of the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end of the position of the editing mutation, the nucleotides at positions 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3 and 4 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation, the nucleotide at position 2 and 4 on the 3'-end side of the position of the editing mutation, or the nucleotide at position 2 on the 5'-end side of the position of the editing mutation, the nucleotide at position 3 and the nucleotide at position 4 on the 3'-end side of the position of the editing mutation are sugar-modified nucleotides, the sugar-modified nucleotide is a nucleotide containing a 4'-lower linear alkylene-O-2' bridge in the ribose ring, more preferably a nucleotide containing a 2'-O,4'-C-methylene bridge or a 2'-O,4'-C-ethylene bridge in the ribose ring, even more preferably an LNA or an ENA;the first single-stranded polynucleotide contains one to four modified internucleotide bonds within five bases from the 3' end, more preferably two modified internucleotide bonds within three bases or one modified internucleotide bond within two bases from the 3' end, the modified internucleotide bond is a phosphorothioate bond, the second single-stranded polynucleotide contains a base sequence complementary to a portion of the base sequence of the first single-stranded polynucleotide on the 3' end side relative to the position of the editing mutation, the length of the second single-stranded polynucleotide is 30 to 100 bases, and the third single-stranded polynucleotide has 90% or more sequence identity with the base sequence of the first single-stranded polynucleotide on the 5' end side relative to the position of the editing mutation in the editing target strand, the E-strand corresponding sequence and the A'-strand corresponding sequence are adjacent, and the length of the third single-stranded polynucleotide is 55 to 75 bases.

[0066] In one embodiment, the second single-stranded polynucleotide may contain at least one modified internucleotide bond. The number and position of the modified internucleotide bond may be selected appropriately as long as base sequence editing is possible. In a preferred embodiment, the second single-stranded polynucleotide may contain one or two modified internucleotide bonds within three bases from the 5'-end and / or 3'-end, or one modified internucleotide bond within two bases, more preferably one or two modified internucleotide bonds within three bases from the 3'-end. In a preferred embodiment, the modified internucleotide bond may be a phosphorothioate bond, an alkylphosphonate bond, or a phosphotriester bond, more preferably an alkylphosphonate bond or a phosphotriester bond, and even more preferably a methylphosphonate bond or an ethylphosphotriester bond.

[0067] In one embodiment, the first to third single-stranded polynucleotides may have a chemical modification at the 5'-end or 3'-end. Examples of the chemical modification include a linker such as an amino linker. The amino linker is a linker represented by the following formula (AL): (wherein the dashed line represents a bond, and the methylene group represents a phosphodiester bond with the 5'-phosphate or 3'-phosphate group of the 5'-terminal nucleotide of one single-stranded polynucleotide) (hereinafter also referred to as an AL linker). When the AL linker is attached to the 5'-terminal of a single-stranded polynucleotide, 5'-Amino-Modifier C6 (product number 0-1906-02, manufactured by Glen Research) or the like can be used, and when it is attached to the 3'-terminal, Phthalamido Amino C6 lcaa CPG (product number N-8217-10, manufactured by ChemGenes) or the like can be used.

[0068] Other linkers having an amino group at the terminal include the aforementioned AL linker having six methylene groups, as well as linkers having an amino acid group at the terminal of 3 to 12 methylene groups. When this linker is attached to the 5'-end of a single-stranded polynucleotide, linkers having an amino acid group at the terminal of 4, 5, or 12 methylene groups can be obtained using, for example, TFA-Amino C-4 CED phosphoramidite (Product No. CLP-1453, ChemGenes), TFA amino C-5 CED phosphoramidite (Product No. CLP-1357, ChemGenes), or MMT-Amino C-12 CED phosphoramidite (Product No. CLP-1453, ChemGenes), respectively. When this linker is attached to the 3'-end of a single-stranded polynucleotide, a linker having an amino acid group at the end of three or six methylene groups can be obtained, for example, by using 3'-Amino Modifier C-3 lcaa CPG (product number N-9750-10, manufactured by ChemGenes) or 3'-Amino Modifier TFA Amino C-6 lcaa CPG (product number N-1004-10, manufactured by ChemGenes).

[0069] Other chemical modifications include linkers with terminal hydroxyl groups. When this linker is attached to the 5' end of a single-stranded polynucleotide, linkers with hydroxyl groups at the terminals of 2, 3, 4, 6, 9, or 12 methylene groups can be obtained using, for example, DMT-ethane-Diol phosphoramidite (product number CLP-2250, manufactured by ChemGenes), DMT-propane-Diol phosphoramidite (product number CLP-1368, manufactured by ChemGenes), DMT-butane-Diol phosphoramidite (product number CLP-9775, manufactured by ChemGenes), DMT-hexane-Diol phosphoramidite (product number CLP-9765, manufactured by ChemGenes), DMT-nonane-Diol phosphoramidite (product number CLP-9009, manufactured by ChemGenes), or DMT-dodecane-Diol phosphoramidite (product number CLP-1114, manufactured by ChemGenes). Further examples include linkers in which an ethyloxy group having a hydroxyl group at the terminal is linked. Linkers in which three, four, or six ethyloxy groups are linked and a hydroxyl group at the terminal can be obtained, for example, using DMT-triethyloxy-Glycol phosphoramidite (product number CLP-1113, ChemGenes), DMT-tetraethyloxy-Glycol CED phosphoramidite (product number CLP-1368, ChemGenes), or DMT Hexaethylene Glycol phosphoramidite (product number CLP-9765, ChemGenes), respectively. When linking this linker to the 3' end of a single-stranded polynucleotide, it can be obtained by coupling the above phosphoramidite reagent to a Universal UnyLinker Support (product numbers N-4000-20, N-4000-10, N-4000-05, N-4000-03, ChemGenes) or the like.

[0070] In one embodiment, any two or three of the first to third single-stranded polynucleotides may be linked by a linker. The linker may link the end of one single-stranded polynucleotide to the end of the other single-stranded polynucleotide. For example, the 3' end of the first single-stranded polynucleotide may be linked to the 5' end of the second or third single-stranded polynucleotide, the 3' end of the second single-stranded polynucleotide to the 5' end of the first or third single-stranded polynucleotide, or the 3' end of the third single-stranded polynucleotide to the 5' end of the first or second single-stranded polynucleotide. In the present invention, pharmaceutically acceptable linkers known in the art can be used, such as those described in WO2012 / 074038. In a specific embodiment, the linker is represented by the following formula (Z): (wherein the dashed line represents a bond, the oxygen atom bonded to the phenyl group represents a phosphodiester bond with the 5'-phosphate group of the 5'-terminal nucleotide of one single-stranded polynucleotide, and the methylene group at the other end represents a phosphodiester bond with the 3'-phosphate group of the 3'-terminal nucleotide of another single-stranded polynucleotide) (hereinafter also referred to as a Z linker). Z linkers can be appropriately prepared according to the description in WO2012 / 074038. Other linkers include linkers having a disulfide bond that is cleaved under reductive conditions in cells (SS linkers). SS linkers can be obtained, for example, using 5'-Thiol C-6 Disulfide Modifier CED phosphoramidite (product number CLP-8506, manufactured by ChemGenes).

[0071] The first, second, and third single-stranded polynucleotides can be prepared using known chemical synthesis methods. A single-stranded polynucleotide of the desired base length can also be prepared by synthesizing multiple short polynucleotides (e.g., about 100 bases long) and ligating them using known ligation methods. Alternatively, the first single-stranded polynucleotide can be prepared by restriction enzyme treatment of phage or phagemid DNA prepared by mutagenesis PCR using double-stranded DNA containing the target region as a template, or by a commercially available mutagenesis kit.

[0072] In one embodiment, the target region contains an abnormal base sequence that causes a disease, and the base sequence of the first single-stranded polynucleotide may include the editing mutation so that the abnormal base sequence can be repaired and thereby the disease can be treated or prevented (e.g., to a normal base sequence, or to a base sequence that differs from the normal base sequence but eliminates the cause of the disease). The abnormal base sequence is a base sequence that has at least one mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides in the normal base sequence. In this specification, repair of the abnormal base sequence is not limited to restoring the normal base sequence (or wild-type base sequence), and may also include, for example, introducing a mutation that results in a synonymous codon encoding the same amino acid as the codon in the normal base sequence, or introducing a mutation that results in a codon encoding an amino acid different from the codon in the normal base sequence.Examples of disease-causing mutations include the factor 9 gene in hemophilia, the ASS1 gene in type I citrullinemia, the SLC25A13 gene in type II citrullinemia, and the Factor V gene in hemophilia. Leiden gene, SERPINA1 gene for α1-antitrypsin deficiency, CBS gene for homocystinuria, PAH gene for phenylketonuria, HFE gene for hemochromatosis, TTR gene for transthyretin amyloidosis, LDLR gene, apo(a) gene, and ANGPTL3 gene for PCSK9 gene for familial hypercholesterolemia, LIPA gene for cholesterol ester storage disease, ATP7B gene for Wilson disease, ALAS1 gene for acute intermittent porphyria, GHR gene for acromegaly, UGT1A1 gene for Gilbert syndrome, AGXT gene for primary hyperoxaluria, G6PC gene for glycogen storage disease type Ia, SLC26A1 gene for Hurler syndrome: mucopolysaccharidosis (IDUA), GFAP gene for Alexander disease, GRIA2 gene for amyotrophic lateral sclerosis gene, the MECP2 gene of Rett syndrome, the SCN1A gene of Dravet syndrome, the SNCA gene and LRRK2 gene of Parkinson's disease, the APP gene and MAPT gene of Alzheimer's disease, the UBE3A gene of Angelman syndrome, the PANK2 gene of pantothenic acid-related neurodegenerative disease, the SMN1 gene of spinal muscular atrophy, the EYS gene and RP1 gene of retinitis pigmentosa, the ABCA4 gene of Stargardt disease, the USH2A gene of Usher syndrome, the CEP290 gene of Leber congenital amaurosis, the GNE gene of distal myopathy, the GAA gene of Pompe disease, the CFTR gene and ENAC gene of cystic fibrosis, the NPHS2 gene of monosegmental glomerulosclerosis, the COL4A5 gene of Alport syndrome, and the SLCO2A1 gene of hypertrophic dermatoperiostosis.

[0073] In a preferred embodiment, the base sequence of the first single-stranded polynucleotide may include the editing mutation so that the abnormal base sequence becomes a normal base sequence.

[0074] In one aspect, the present invention provides a pharmaceutical composition for treating or preventing a disease caused by an aberrant nucleotide sequence contained in a target region present in intracellular double-stranded DNA, comprising composition I of the present invention (hereinafter also referred to as pharmaceutical composition I of the present invention). Here, the nucleotide sequence of the first single-stranded polynucleotide in composition I of the present invention contains the editing mutation so that the aberrant nucleotide sequence can be repaired, thereby treating or preventing the disease (e.g., to a normal nucleotide sequence, or to a nucleotide sequence that differs from the normal nucleotide sequence but from which the cause of the disease has been eliminated). As a result, when nucleotide sequence editing occurs using pharmaceutical composition I of the present invention, the aberrant nucleotide sequence is repaired (e.g., to a normal nucleotide sequence), and the disease caused by the aberrant nucleotide sequence can be treated or prevented.

[0075] Pharmaceutical composition I of the present invention preferably contains the single-stranded polynucleotide in a form that can be introduced into cells, which may be any form known in the art, such as a dispersion in an appropriate solvent (e.g., water), an encapsulation in hollow nanoparticles, liposomes, lipid nanoparticles (LNPs), or the like, or a complex with a cationic polymer.

[0076] The subject to which the pharmaceutical composition I of the present invention is administered is, for example, a mammal, preferably a human.

[0077] The administration route of the pharmaceutical composition I of the present invention may be either oral or parenteral, and a suitable administration route may be selected depending on the target symptoms, etc. The administration route may be either systemic or local. Examples of parenteral administration include intravenous administration, intraarterial administration, intrathecal administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, and intraarticular administration.

[0078] The pharmaceutical composition I of the present invention is administered to a subject in a therapeutically or prophylactically effective amount. The term "therapeutically or prophylactically effective amount" refers to an amount that exhibits a therapeutic or prophylactic effect for a specific disease, administration form, and administration route, and is determined appropriately depending on the subject's species, type of disease, symptoms, sex, age, chronic illnesses, and other factors.

[0079] The dosage of the pharmaceutical composition I of the present invention can be appropriately determined depending on the species of the subject, the type of disease, symptoms, sex, age, chronic illnesses, and other factors.

[0080] The pharmaceutical composition I of the present invention can be used to treat or prevent any disease caused by a mutation in a base sequence. Examples of such diseases include type I citrullinemia (ASS1), type II citrullinemia (SLC25A13), hemophilia (Factor V), and the like. Leiden), alpha 1-antitrypsin deficiency (SERPINA1), homocystinuria (CBS), phenylketonuria (PAH), hemochromatosis (HFE), transthyretin amyloidosis (TTR), familial hypercholesterolemia (PCSK9, LDLR, apo(a), ANGPTL3), cholesterol ester storage disease (LIPA), Wilson's disease (ATP7B), acute intermittent porphyria (ALAS1), acromegaly (GHR), Gilbert's syndrome (UGT1A1), primary hyperoxaluria (AGXT), glycogen storage disease type Ia (G6PC), Hurler's syndrome: mucopolysaccharidosis (IDUA(SLC26A1)), Alexander disease (GFAP), amyotrophic lateral cord blood disorder These include sclerosis (GRIA2), Rett syndrome (MECP2), Dravet syndrome (SCN1A), Parkinson's disease (SNCA, LRRK2), Alzheimer's disease (APP, MAPT), Angelman syndrome (UBE3A), pantothenic acid-related neurodegenerative disease (PANK2), spinal muscular atrophy (SMN1), retinitis pigmentosa (EYS, RP1), Stargardt disease (ABCA4), Usher syndrome (USH2A), Leber congenital amaurosis (CEP290), distal myopathy (GNE), Pompe disease (GAA), cystic fibrosis (CFTR, ENAC), monosegmental glomerulosclerosis (NPHS2), Alport syndrome (COL4A5), and hypertrophic dermatoperiostosis (SLCO2A1). The target gene name is in parentheses.

[0081] In one aspect, the present invention provides a method for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell, the method comprising administering a therapeutically or prophylactically effective amount of Composition I of the present invention to a subject. Here, the base sequence of the first single-stranded polynucleotide in Composition I of the present invention contains the editing mutation so that the abnormal base sequence is repaired, thereby treating or preventing the disease (e.g., so that the abnormal base sequence becomes a normal base sequence, or so that the base sequence differs from the normal base sequence but eliminates the cause of the disease). Specific embodiments of the present invention are the same as those for administering Pharmaceutical Composition I of the present invention.

[0082] In one aspect, the present invention provides a method for producing an isolated cell into which a mutation has been introduced or an organism having a cell into which a mutation has been introduced, the method comprising the step of introducing Composition I of the present invention into the isolated cell or into a cell constituting the living body of the organism, thereby introducing a mutation into the base sequence of a target region present in double-stranded DNA in the cell (hereinafter also referred to as Production Method I of the present invention).

[0083] The cells used in Production Method I of the present invention may be either cells that do not constitute a living organism (i.e., isolated cells) or cells that exist within a living organism and constitute a living organism. In one embodiment, human cells may be excluded from cells that exist within a living organism. In another embodiment, Production Method I of the present invention may involve introducing Composition I of the present invention into cells by administering Composition I of the present invention to a human for the purpose of treating a human disease. When isolated cells are the target, Production Method I of the present invention may be performed in vitro. The cells may be either eukaryotic or prokaryotic, and the biological species from which the cells originate is not particularly limited. The biological species from which the cells originate may be, for example, animals such as mammals, birds, and insects, plants, microorganisms, etc., but animals are preferred, and mammals are more preferred. Examples of mammals include humans, monkeys, cows, sheep, goats, horses, pigs, rabbits, dogs, cats, rats, mice, and guinea pigs. The microorganism may be either a prokaryotic or eukaryotic microorganism. Examples of prokaryotic microorganisms include bacteria such as gram-positive bacteria and gram-negative bacteria, and examples of eukaryotic microorganisms include fungi such as filamentous fungi (molds) and yeasts.

[0084] The type of cell is not particularly limited, and examples include somatic cells, germ cells, stem cells, and cultured cells thereof. Specific examples of somatic cells include nervous system tissues such as the brain and spinal cord; sensory organs such as retinal cells and olfactory cells; digestive organs such as the esophagus, stomach, small intestine, and large intestine; respiratory organs such as the lungs and bronchi; reproductive organs such as the testes, ovaries, uterus, and placenta; urinary organs such as the kidneys and bladder; hematopoietic organs such as bone marrow cells and blood cells; muscle tissues such as skeletal muscle, smooth muscle, and cardiac muscle; bone tissues such as osteoblasts and osteoclasts; and skin tissues such as skin and hair root cells, as well as live cells or cultured cells thereof isolated from various tissues and organs. Specific examples of germ cells include eggs, sperm, and cultured cells thereof. Specific examples of stem cells include embryonic stem cells (ES cells), embryonic germ cells (EG cells), trophoblast stem cells (TS cells), bone marrow stem cells, and neural stem cells.

[0085] Composition I of the present invention can be introduced into cells using methods known in the art, such as electroporation, calcium phosphate injection, lipofection, microinjection, etc. As described with respect to pharmaceutical composition I of the present invention, the first, second, and third single-stranded polynucleotides may be made into a form that can be introduced into cells and administered to a human or non-human organism.

[0086] When Composition I of the present invention is introduced into a cell, a mutation similar to that observed when the editing target strand of the target region present in the double-stranded DNA in the cell is homologously replaced with the first single-stranded polynucleotide contained in Composition I of the present invention is introduced into the editing target strand, and the other strand is also modified to become complementary to it, resulting in mutations being introduced into both strands that make up the double-stranded DNA in the cell.

[0087] If the target region contains an abnormal base sequence that causes a disease, the disease caused by the abnormal base sequence can be treated or prevented by introducing a mutation into the abnormal base sequence to repair it (for example, by converting it to a normal base sequence, or by converting it to a base sequence that differs from the normal base sequence but from which the cause of the disease has been eliminated).

[0088] Cells in which a mutation has been introduced into the target region and individual organisms harboring such cells are useful as screening systems, disease models, etc., in which sensitivity to specific compounds, etc. differs from that of normal cells.

[0089] 3. Base Sequence Editing Using a Composition Comprising a Chemically Modified Single-Stranded Polynucleotide In one aspect, the present invention provides a composition comprising a first single-stranded polynucleotide, wherein the first single-stranded polynucleotide is 50 to 200 bases in length, the base sequence of which has 80% or more sequence identity to the base sequence of a portion of the editing target strand of a target region present in double-stranded DNA in a cell, and which, compared to the base sequence of the editing target strand, comprises at least one editing mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides, and the composition has at least one of the following characteristics: (a) at least one nucleotide is a sugar-modified nucleotide in the position of the editing mutation in the first single-stranded polynucleotide and in a region consisting of 10 bases on the 5' side and 10 bases on the 3' side of the position of the editing mutation; and (b) the first single-stranded polynucleotide comprises at least one modified internucleotide bond, wherein the modified internucleotide bond is not present in a range from 3 bases on the 5' side to 3 bases on the 3' side of the position of the editing mutation (hereinafter also referred to as composition II of the present invention).

[0090] Introducing Composition II of the present invention into a cell enables base sequence editing of a target region in double-stranded DNA in the cell according to an editing mutation contained in a first single-stranded polynucleotide. That is, Composition II of the present invention can be used to introduce mutations into a target region in double-stranded DNA in the cell. Furthermore, base sequence editing using Composition II of the present invention does not require an exogenous nuclease. Therefore, in a preferred embodiment, Composition II of the present invention does not contain an exogenous nuclease or a polynucleotide capable of expressing such an exogenous nuclease, and is not used in combination with such an exogenous nuclease. Here, the term "first single-stranded polynucleotide" is a convenient designation and does not necessarily mean that the second single-stranded polynucleotide (strand A) or the third single-stranded polynucleotide (strand A') are components of Composition II of the present invention. The term "first single-stranded polynucleotide" itself does not imply that Composition II of the present invention contains the second single-stranded polynucleotide (strand A) or the third single-stranded polynucleotide (strand A'). When using Composition II of the present invention, base sequence editing may be performed using the first single-stranded polynucleotide alone, or may be performed in combination with at least one of the second single-stranded polynucleotide (Strand A) and the third single-stranded polynucleotide (Strand A'). As described below, Composition II of the present invention may contain at least one of the second single-stranded polynucleotide (Strand A) and the third single-stranded polynucleotide (Strand A').

[0091] The first single-stranded polynucleotide in Composition II of the present invention may have the same characteristics as the first single-stranded polynucleotide in Composition I of the present invention, except that it must have at least one of the above characteristics (a) and (b). Characteristics (a) and (b) are the same as characteristics (a) and (b) in Composition I of the present invention.

[0092] In one embodiment, in the first single-stranded polynucleotide, the position of the editing mutation, at least one selected from the nucleotides at positions 1, 3, 5, 7, and 9 on the 5'-end side of the editing mutation, and at least one selected from the nucleotides at positions 1, 3, 5, 7, and 9 on the 3'-end side of the editing mutation may be a sugar-modified nucleotide, for example, the nucleotide at positions 1 on the 5'-end side and 1 on the 3'-end side of the editing mutation, and the nucleotides at positions 1 and 3 on the 5'-end side of the editing mutation. and the nucleotides at positions 1 and 3 on the 3'-terminus, the nucleotides at positions 1, 3, and 5 on the 5'-terminus and the nucleotides at positions 1, 3, and 5 on the 3'-terminus of the editing mutation, the nucleotides at positions 1, 3, 5, and 7 on the 5'-terminus and the nucleotides at positions 1, 3, 5, and 7 on the 3'-terminus of the editing mutation, or the nucleotides at positions 1, 3, 5, 7, and 9 on the 5'-terminus and the nucleotides at positions 1, 3, 5, 7, and 9 on the 3'-terminus of the editing mutation may be sugar-modified nucleotides. In a particular embodiment, the first single-stranded polynucleotide comprises: the nucleotide at the position of the editing mutation; the nucleotide at position 1 on the 5'-end side of the position of the editing mutation; the nucleotide at position 1 and the 3'-end side of the position of the editing mutation; the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the 1st nucleotide on the 3'-end side of the position of the editing mutation; the nucleotide at position 1 on the 5'-end side of the position of the editing mutation; the nucleotide at position 1 and the 3'-end side of the position of the editing mutation; the nucleotide at position 3 and the 1st nucleotide on the 5'-end side of the position of the editing mutation and the 1st and 3rd nucleotide on the 3'-end side of the position of the editing mutation; the nucleotides at positions 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the 1st, 3rd and 5th nucleotides on the 3'-end side of the position of the editing mutation; the 2nd nucleotide on the 5'-end side of the position of the editing mutation and the 2nd nucleotide on the 3'-end side of the position of the editing mutation;the nucleotides at positions 2 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 2 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3 and 2 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 2 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3, 2 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 2 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 7, 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3, 5 and 7 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 9, 7, 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3, 5, 7 and 9 on the 3'-end side of the position of the editing mutation, any one of the nucleotides at positions 1 to 6 on the 5'-end of the position of the editing mutation, any one of the nucleotides at positions 1 to 6 on the 3'-end of the position of the editing mutation, the nucleotide at position 2 on the 5'-end of the position of the editing mutation and the nucleotide at position 2 on the 3'-end, the nucleotide at position 3 on the 5'-end of the position of the editing mutation and the nucleotide at position 3 on the 3'-end, the nucleotide at position 4 on the 5'-end of the position of the editing mutation and the nucleotide at position 4 on the 3'-end, the nucleotide at position 5 on the 5'-end of the position of the editing mutation and the nucleotide at position 5 on the 3'-end, the nucleotide at position 6 on the 5'-end of the position of the editing mutation and the nucleotide at position 6 on the 3'-end, the nucleotides at positions 5 and 3 on the 5'-end of the position of the editing mutation, the nucleotides at positions 4 and 2 on the 5'-end of the position of the editing mutation, the nucleotides at positions 3 and 1 on the 5'-end of the position of the editing mutation, the nucleotides at positions 1 and 3 on the 3'-end of the position of the editing mutation, the nucleotides at positions 2 and 4 on the 3'-end of the position of the editing mutation, the nucleotides at positions 3 and 5 on the 3'-terminal side of the position of the editing mutation, the nucleotide at position 7 on the 5'-terminal side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 6 on the 5'-terminal side of the position of the editing mutation and the nucleotide at the position of the editing mutation,the nucleotide at position 5 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 4 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 5 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 6 on the 3'-end of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 7 on the 3'-end of the position of the editing mutation, the nucleotide at position 1 on the 5'-end of the position of the editing mutation, the nucleotide at position 1 on the 3'-end of the position of the editing mutation, the nucleotide at position 2 on the 5'-end of the position of the editing mutation, the nucleotide at position 2 on the 3'-end of the position of the editing mutation, the nucleotide at position 3 on the 5'-end of the position of the editing mutation, the nucleotide at position 3 on the 3'-end of the position of the editing mutation, the nucleotide at position 4 on the 5'-end of the position of the editing mutation, the nucleotide at position 4 on the 3'-end of the position of the editing mutation, the nucleotide at position 5 on the 5'-end of the position of the editing mutation, and the nucleotide at position 5 on the 3'-end of the position of the editing mutation,the nucleotide at position 6 on the 5'-end side of the position of the editing mutation, the nucleotide at position 6 on the 3'-end side of the position of the editing mutation, the nucleotide at position 7 on the 5'-end side of the position of the editing mutation, the nucleotide at position 7 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation and the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 2 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 2 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3 and 2 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 2 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3, 2 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 2 and 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end side of the position of the editing mutation, The following may be sugar-modified nucleotides: the nucleotides at positions 3 and 1 on the 5'-end of the position of the editing mutation and the nucleotides at positions 1 and 3 on the 3'-end of the position of the editing mutation; the nucleotides at positions 3 and 1 on the 5'-end of the position of the editing mutation and the nucleotides at positions 1, 3 and 4 on the 3'-end of the position of the editing mutation; the nucleotide at the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end of the position of the editing mutation; the nucleotide at position 1 on the 5'-end of the position of the editing mutation, the nucleotide at position 4 on the 3'-end of the position of the editing mutation; the nucleotide at position 2 and 4 on the 3'-end of the position of the editing mutation; or the nucleotide at position 2 on the 5'-end of the position of the editing mutation, the nucleotide at position 2 and the nucleotide at position 4 on the 3'-end of the position of the editing mutation, and the nucleotide at position 4 on the 3'-end of the position of the editing mutation. In a preferred embodiment, the first single-stranded polynucleotide comprises: a nucleotide at the position of the editing mutation; a nucleotide at the first position on the 5'-terminal side of the position of the editing mutation; a nucleotide at the position of the editing mutation; and a nucleotide at the first position on the 3'-terminal side of the position of the editing mutation.the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation, the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotide at position 1 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3 and 5 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation and the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 and 3 on the 3'-end of the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation and the nucleotide at position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotide at position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 2 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 4 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 5 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 6 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 7 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation, the nucleotide at position 2 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation, the nucleotide at position 3 on the 3'-end side of the position of the editing mutation,the nucleotide at position 4 on the 5'-end side of the position of the editing mutation, the nucleotide at position 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 5 on the 5'-end side of the position of the editing mutation, the nucleotide at position 5 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3 and 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation, the nucleotide at position 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 and 4 on the 3'-end side of the position of the editing mutation, or The nucleotide at position 2 on the 5'-terminal side of the position of the editing mutation, the nucleotide at position 3' on the 5'-terminal side of the position of the editing mutation, and the nucleotide at position 4 on the 3'-terminal side of the position of the editing mutation can be sugar-modified nucleotides. In a preferred embodiment, the sugar-modified nucleotide can be a nucleotide comprising a 4'-lower linear alkylene-O-2' bridge in the ribose ring, more preferably a nucleotide comprising a 2'-O,4'-C-methylene bridge or a 2'-O,4'-C-ethylene bridge in the ribose ring, and even more preferably an LNA or ENA.

[0093] When feature (b) is present, in one embodiment, the first single-stranded polynucleotide may comprise one to four (e.g., one, two, three, or four) modified internucleotide linkages near the 5'-end (e.g., within 20 bases, 15 bases, 13 bases, 10 bases, 8 bases, 6 bases, 5 bases, 4 bases, 3 bases, or 2 bases from the 5'-end) and / or near the 3'-end (e.g., within 20 bases, 15 bases, 13 bases, 10 bases, 8 bases, 6 bases, 5 bases, 4 bases, 3 bases, or 2 bases from the 3'-end). In a preferred embodiment, the first single-stranded polynucleotide may comprise one to four modified internucleotide linkages within 13 bases (more preferably within 5 bases) from the 3'-end. In a more preferred embodiment, the first single-stranded polynucleotide may comprise two modified internucleotide linkages within 3 bases or one modified internucleotide linkage within 2 bases from the 3'-end. In a preferred embodiment, the modified internucleotide bond may be a phosphorothioate bond, an alkylphosphonate bond, or a phosphotriester bond. In a more preferred embodiment, the first single-stranded polynucleotide contains 1 to 4 (preferably 2 within 3 bases, or 1 within 2 bases) modified internucleotide bond(s) within 13 bases from the 3'-end, and the modified internucleotide bond(s) may be a phosphorothioate bond.

[0094] In a preferred embodiment, the position of the editing mutation in the first single-stranded polynucleotide is within a range of 49 to 51%, assuming that the sequence from the 5' end to the 3' end is 100%, the sequence identity between the base sequence of the first single-stranded polynucleotide and the E-chain corresponding sequence is 98% or more, the length of the first single-stranded polynucleotide is 70 to 100 bases long, and the first single-stranded polynucleotide comprises: a nucleotide at the position of the editing mutation, a nucleotide at position 1 on the 5' end side of the position of the editing mutation, a nucleotide at position 1 and a nucleotide at position 1 on the 3' end side of the position of the editing mutation, a nucleotide at position 1 on the 5' end side of the position of the editing mutation and a nucleotide at position 1 on the 3' end side of the position of the editing mutation, a nucleotide at position 1 on the 5' end side of the position of the editing mutation, a nucleotide at position 1 on the 5' end side of the position of the editing mutation, and a nucleotide at position 1 on the 3' end side of the position of the editing mutation, the nucleotides at positions 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1 and 3 on the 3'-end side of the position of the editing mutation, the nucleotides at positions 5, 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3 and 5 on the 3'-end side of the position of the editing mutation, the nucleotide at position 3 on the 5'-end side of the position of the editing mutation and the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 and 3 on the 3'-end side of the position of the editing mutation, the nucleotide at position 2 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation and the nucleotide at the position of the editing mutation, the nucleotide at position of the editing mutation and the nucleotide at position 1 on the 3'-end side of the position of the editing mutation, the nucleotide at position of the editing mutation and the nucleotide at position 2 on the 3'-end side of the position of the editing mutation, the nucleotide at position of the editing mutation and the nucleotide at position 3 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the 4th nucleotide on the 3'-terminal side of the position of the editing mutation; the nucleotide at the position of the editing mutation and the 5th nucleotide on the 3'-terminal side of the position of the editing mutation;the nucleotide at the position of the editing mutation and the nucleotide at position 6 on the 3'-end of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotide at position 7 on the 3'-end of the position of the editing mutation, the nucleotide at position 2 on the 5'-end of the position of the editing mutation, the nucleotide at position 2 on the 3'-end of the position of the editing mutation, the nucleotide at position 3 on the 5'-end of the position of the editing mutation, the nucleotide at position 3 on the 3'-end of the position of the editing mutation, the nucleotide at position 4 on the 5'-end of the position of the editing mutation, the nucleotide at position 4 on the 3'-end of the position of the editing mutation, the nucleotide at position 5 on the 5'-end of the position of the editing mutation, the nucleotide at position 5 on the 3'-end of the position of the editing mutation, the nucleotide at position 1 on the 5'-end of the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end of the position of the editing mutation, the nucleotides at positions 3 and 1 on the 5'-end side of the position of the editing mutation and the nucleotides at positions 1, 3 and 4 on the 3'-end side of the position of the editing mutation, the nucleotide at the position of the editing mutation and the nucleotides at positions 1 and 4 on the 3'-end side of the position of the editing mutation, the nucleotide at position 1 on the 5'-end side of the position of the editing mutation, the nucleotide at position 2 and 4 on the 3'-end side of the position of the editing mutation, or the nucleotide at position 2 on the 5'-end side of the position of the editing mutation, the nucleotide at position 3 and the nucleotide at position 4 on the 3'-end side of the position of the editing mutation are sugar-modified nucleotides, the sugar-modified nucleotide is a nucleotide containing a 4'-lower linear alkylene-O-2' bridge in the ribose ring, more preferably a nucleotide containing a 2'-O,4'-C-methylene bridge or a 2'-O,4'-C-ethylene bridge in the ribose ring, even more preferably an LNA or an ENA;The first single-stranded polynucleotide contains 1 to 4 modified internucleotide linkages within 5 bases from the 3' end, more preferably 2 modified internucleotide linkages within 3 bases or 1 modified internucleotide linkage within 2 bases from the 3' end, and the modified internucleotide linkage is a phosphorothioate linkage, an alkylphosphonate linkage, or a phosphotriester linkage.

[0095] In one embodiment, Composition II of the present invention may further comprise a second single-stranded polynucleotide. The second single-stranded polynucleotide in Composition II of the present invention may have the same characteristics as the second single-stranded polynucleotide in Composition I of the present invention.

[0096] When the second single-stranded polynucleotide comprises at least one modified internucleotide bond, in a preferred embodiment, the second single-stranded polynucleotide may comprise one or two or one modified internucleotide bond within three bases from the 5'-end and / or 3'-end, more preferably one or two or one modified internucleotide bond within three bases from the 3'-end. In a preferred embodiment, the modified internucleotide bond may be a phosphorothioate bond, an alkylphosphonate bond, or a phosphotriester bond, more preferably an alkylphosphonate bond or a phosphotriester bond, and even more preferably a methylphosphonate bond or an ethylphosphotriester bond.

[0097] In one embodiment, Composition II of the present invention may further comprise a third single-stranded polynucleotide. The third single-stranded polynucleotide in Composition II of the present invention may have the same characteristics as the third single-stranded polynucleotide in Composition I of the present invention.

[0098] In one embodiment, the target region contains an aberrant base sequence that causes a disease, and the base sequence of the first single-stranded polynucleotide may contain the editing mutation so that the aberrant base sequence can be repaired, thereby treating or preventing the disease (for example, so that the aberrant base sequence becomes a normal base sequence, or so that the aberrant base sequence becomes a base sequence that differs from the normal base sequence but eliminates the cause of the disease).

[0099] In one aspect, the present invention provides a pharmaceutical composition for treating or preventing a disease caused by an aberrant base sequence contained in a target region of double-stranded DNA in a cell, comprising Composition II of the present invention (hereinafter also referred to as Pharmaceutical Composition II of the present invention). Here, the base sequence of the first single-stranded polynucleotide in Composition II of the present invention contains the editing mutation so that the aberrant base sequence can be repaired, thereby treating or preventing the disease (e.g., to a normal base sequence, or to a base sequence that differs from the normal base sequence but eliminates the cause of the disease). Thus, when base sequence editing occurs using Pharmaceutical Composition II of the present invention, the aberrant base sequence is repaired (e.g., to a normal base sequence, or to a base sequence that differs from the normal base sequence but eliminates the cause of the disease), thereby treating or preventing the disease caused by the aberrant base sequence. Specific embodiments of Pharmaceutical Composition II of the present invention are the same as those of Pharmaceutical Composition I of the present invention.

[0100] In one aspect, the present invention provides a method for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in intracellular double-stranded DNA, comprising administering a therapeutically or prophylactically effective amount of Composition II of the present invention to a subject. Here, the base sequence of the first single-stranded polynucleotide in Composition II of the present invention contains the editing mutation so that the abnormal base sequence is repaired, thereby treating or preventing the disease (e.g., to a normal base sequence, or to a base sequence that differs from the normal base sequence but eliminates the cause of the disease). Specific embodiments of the present invention are similar to those for administering Pharmaceutical Composition II of the present invention.

[0101] In one aspect, the present invention provides a method for producing an isolated cell into which a mutation has been introduced or an organism having a cell into which a mutation has been introduced, the method comprising the step of introducing composition II of the present invention into the isolated cell or into a cell constituting the living body of the organism, thereby introducing a mutation into a base sequence of a target region present in double-stranded DNA in the cell (hereinafter also referred to as production method II of the present invention). A specific embodiment of production method II of the present invention is the same as production method I of the present invention, except that composition II of the present invention is used instead of composition I of the present invention.

[0102] 4. Pharmaceutical composition comprising A' chain to be used in combination with a composition comprising E chain or TD In one aspect, the present invention provides a pharmaceutical composition comprising a third single-stranded polynucleotide to be used in combination with a composition comprising a first single-stranded polynucleotide, wherein the first single-stranded polynucleotide is 50 to 200 bases long, and its base sequence has 80% or more sequence identity with the base sequence of a portion of the editing target strand of a target region present in double-stranded DNA in a cell, and compared to the base sequence of the editing target strand, it contains at least one editing mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides; the third single-stranded polynucleotide is 15 to 200 bases long, and its base sequence has 90% or more sequence identity with the base sequence in the editing target strand that is located 5'-terminally or 3'-terminally closer to the position in the first single-stranded polynucleotide that corresponds to the editing mutation, and wherein, in the editing target strand, the base sequence that has sequence identity with the first single-stranded polynucleotide and the base sequence that has sequence identity with the third single-stranded polynucleotide overlap, are adjacent, or are separated by 1 to 9 bases; The target region contains an abnormal base sequence that causes a disease, and the base sequence of the first single-stranded polynucleotide contains the editing mutation so that the abnormal base sequence can be repaired, thereby treating or preventing the disease (for example, so that the abnormal base sequence becomes a normal base sequence, or so that the base sequence differs from the normal base sequence but eliminates the cause of the disease) (hereinafter also referred to as pharmaceutical composition III of the present invention).

[0103] By introducing pharmaceutical composition III of the present invention into a cell in combination with a composition containing a first single-stranded polynucleotide, base sequence editing occurs, and the abnormal base sequence can be repaired (e.g., converted to a normal base sequence, or converted to a base sequence that differs from the normal base sequence but eliminates the cause of the disease). Therefore, pharmaceutical composition III of the present invention can be a pharmaceutical composition for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell. Examples of such diseases include those listed for pharmaceutical composition I of the present invention.

[0104] Base sequence editing using pharmaceutical composition III of the present invention in combination with a composition comprising a first single-stranded polynucleotide does not require an exogenous nuclease. Thus, in a preferred embodiment, pharmaceutical composition III of the present invention does not contain an exogenous nuclease or a polynucleotide capable of expressing such an exogenous nuclease, and is not used in combination with them.

[0105] The third single-stranded polynucleotide in pharmaceutical composition III of the present invention may have the same characteristics as the third single-stranded polynucleotide in composition I of the present invention. Here, the term "third single-stranded polynucleotide" is a convenient name and does not mean that the first single-stranded polynucleotide (strand E) or the second single-stranded polynucleotide (strand A) is a component of pharmaceutical composition III of the present invention.

[0106] The first single-stranded polynucleotide in the composition used in combination with pharmaceutical composition III of the present invention may have the same characteristics as the first single-stranded polynucleotide in composition I of the present invention. Furthermore, a composition containing the single-stranded polynucleotide of the first single-stranded polynucleotide may correspond to an embodiment of pharmaceutical composition II of the present invention that contains the first single-stranded polynucleotide but does not contain the second and third single-stranded polynucleotides.

[0107] In one embodiment, a composition comprising a first single-stranded polynucleotide used in combination with pharmaceutical composition III of the present invention may further comprise a second single-stranded polynucleotide, wherein the second single-stranded polynucleotide may have the same characteristics as the second single-stranded polynucleotide in composition I of the present invention. Furthermore, a composition comprising the first and second single-stranded polynucleotides may correspond to an embodiment of pharmaceutical composition II of the present invention that comprises the first and second single-stranded polynucleotides but does not comprise a third single-stranded polynucleotide.

[0108] As used herein, the terms "coadministration" and "use in combination" refer to the administration of two or more drugs or pharmaceutical compositions to the same individual. These drugs or pharmaceutical compositions may be administered simultaneously or nearly simultaneously (e.g., within one hour), or may be administered separately or sequentially with an interval (e.g., several hours) between them. Pharmaceutical composition III of the present invention may be administered simultaneously or nearly simultaneously with a composition comprising a first single-stranded polynucleotide, or may be administered with an interval between them. Furthermore, pharmaceutical composition III of the present invention and a composition comprising a first single-stranded polynucleotide may be administered in any order. In a preferred embodiment, pharmaceutical composition III of the present invention and a composition comprising a first single-stranded polynucleotide may be administered simultaneously.

[0109] Pharmaceutical composition III of the present invention preferably contains the third single-stranded polynucleotide in a form that can be introduced into cells, which may be any form known in the art, such as a dispersion in an appropriate solvent (e.g., water), an encapsulation in hollow nanoparticles, liposomes, lipid nanoparticles (LNPs), or the like, or a complex with a cationic polymer.

[0110] The subject to which the pharmaceutical composition III of the present invention is administered is, for example, a mammal, preferably a human.

[0111] The administration route of the pharmaceutical composition III of the present invention may be either oral or parenteral, and a suitable administration route may be selected depending on the target symptoms, etc. The administration route may be either systemic or local. Examples of parenteral administration include intravenous administration, intraarterial administration, intrathecal administration, intramuscular administration, intradermal administration, subcutaneous administration, intraperitoneal administration, transdermal administration, intraosseous administration, and intraarticular administration.

[0112] The pharmaceutical composition III of the present invention is administered to a subject in a therapeutically or prophylactically effective amount. The term "therapeutically or prophylactically effective amount" refers to an amount that exhibits a therapeutic or prophylactic effect for a specific disease, administration form, and administration route, and is determined appropriately depending on the subject's species, type of disease, symptoms, sex, age, chronic illnesses, and other factors.

[0113] The dosage of the pharmaceutical composition III of the present invention can be appropriately determined depending on the species of the subject, the type of disease, symptoms, sex, age, chronic illnesses, and other factors.

[0114] The ratio of the dosage of the pharmaceutical composition III of the present invention to the dosage of the composition comprising a first single-stranded polynucleotide is not particularly limited, as long as it can improve the editing efficiency of the composition comprising the first single-stranded polynucleotide.

[0115] In one embodiment, pharmaceutical composition III of the present invention can be used so that the dose (molar) of the second single-stranded polynucleotide is 0.2 to 2, preferably 0.2 to 1, and more preferably 0.2 to 0.5, where the sum of the doses of the first and third single-stranded polynucleotides is 1. Alternatively, pharmaceutical composition III of the present invention is preferably used so that the dose of the second single-stranded polynucleotide is the same as or lower than the sum of the doses of the first and third single-stranded polynucleotides.

[0116] In one embodiment, pharmaceutical composition III of the present invention can be used such that the dose (molar) of the third single-stranded polynucleotide is 0.25 to 4, preferably 1 to 4, relative to the dose of the first single-stranded polynucleotide, which is 1. Alternatively, pharmaceutical composition III of the present invention is preferably used such that the dose of the third single-stranded polynucleotide is the same as or higher than the dose of the first single-stranded polynucleotide.

[0117] In one embodiment, pharmaceutical composition III of the present invention can be used such that the dose (molar) of the third single-stranded polynucleotide is 0.25 to 4, preferably 1 to 4, relative to the dose of the second single-stranded polynucleotide, which is 1. Alternatively, pharmaceutical composition III of the present invention is preferably used such that the dose of the third single-stranded polynucleotide is the same as or higher than the dose of the second single-stranded polynucleotide.

[0118] In one aspect, the present invention provides a method for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in intracellular double-stranded DNA, comprising administering to a subject a therapeutically or prophylactically effective amount of composition III of the present invention simultaneously, separately, or sequentially with a composition containing a first single-stranded polynucleotide. A specific embodiment of the present invention is the same as the case of administering pharmaceutical composition III of the present invention.

[0119] In one aspect, the present invention provides a method for producing an isolated cell into which a mutation has been introduced or an organism having a cell into which a mutation has been introduced, comprising the step of introducing pharmaceutical composition III of the present invention into the isolated cell or into a cell constituting the living body of the organism simultaneously, separately, or sequentially with a composition containing a first single-stranded polynucleotide, thereby introducing a mutation into the base sequence of a target region present in double-stranded DNA in the cell (hereinafter also referred to as production method III of the present invention). A specific embodiment of production method III of the present invention is the same as production method I of the present invention, except that the third single-stranded polynucleotide and the first polynucleotide are contained in different compositions and are introduced into the cell simultaneously, separately, or sequentially.

[0120] The pharmaceutical composition III of the present invention may be introduced into cells separately or sequentially from the composition containing the first single-stranded polynucleotide, but is preferably introduced simultaneously.

[0121] The present invention will be described in more detail below with reference to examples and test examples, but the scope of the present invention is not limited thereto. In the examples, "n-" is used to indicate that heat treatment and annealing treatment were not performed during the preparation of the editing nucleic acid. In the examples, "nE" indicates that an E chain was introduced into a cell, "n-TD" indicates that a TD (an E chain and an A chain) was introduced into a cell, and "n-TD'" indicates that a TD (an E chain and an A chain) and an A' chain were introduced into a cell.

[0122] Example 1: Improving Base Sequence Editing Efficiency by Adding an A' Strand. Using green fluorescent protein, we investigated the effect of adding an A' strand on the editing efficiency of TD-mediated base sequence editing. The target gene was the copGFP gene, encoding green fluorescent protein derived from copepods. Plasmid DNA containing a mutant copGFP gene (SEQ ID NO: 1), in which the base sequence TAC corresponding to the Tyr in the fluorophore Gly-Tyr-Gly was changed to CAC (encoding His), was co-transfected with TD into human U2OS cells. TD consists of a 79-nt E strand (SEQ ID NO: 5), which has a sequence identical to a portion of the antisense strand of the target gene and a wild-type fluorophore-encoding sequence, and an A strand (SEQ ID NO: 6), which hybridizes to a region 35 nt from the 3' end of the E strand. Upon TD-mediated base sequence editing, the mutant copGFP gene on the plasmid DNA becomes the wild-type copGFP gene, resulting in the observation of green fluorescence. A' strands of various lengths were introduced together with the target plasmid and TD, and the percentage of cells exhibiting green fluorescence was examined using a fluorescence microscope. E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' A strand copGFP-153-79b-AS-35TD3'-ODN (A strand for AS_E strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' A' strand copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3'

[0123] The specific experimental method is as follows. 1 μM aqueous solutions of E chain, A chain, and A' chain were mixed at the desired ratio and left to stand at room temperature for 1 hour without heat treatment to prepare nucleic acid for editing. U2OS cells (8 × 10 3Cells (obtained from the American Type Culture Collection (Manassas, VA, USA)) were seeded and cultured overnight. For analysis of editing efficiency, the mPlum-T2A-copGFP(Y / H) expression vector plasmid (30.0 ng; H. Kawai, R. Kamitsubo, H. Kamiya. Correction of monomeric enhanced green fluorescent protein (mEGFP) gene by short 5'-tailed duplexes. J. Biosci. Bioengng. 134, 175-181 (2022)) and editing nucleic acids (strand E only, strand E + strand A, or strand E + strand A + strand A') were co-transfected using Lipofectamine 3000. After 4 hours of culture, the medium was replaced. After 48 hours of culture, the nuclei were stained with Hoechst 33342, fixed in 4% paraformaldehyde (100 μL / well), and observed under a fluorescence microscope.

[0124] Editing efficiency was calculated using the following method. Fluorescence microscopy images were acquired using the Opera Phenix® High-Throughput High-Content Imaging System (PerkinElmer). Fluorescence intensity data for Hoechst33342, mPlum, and copGFP were acquired for each cell type using image analysis software Harmony (PerkinElmer). For each 384-well plate in each experiment, only cell nuclei with average fluorescence intensity and morphology were extracted from the Hoechst33342 fluorescence intensity data and analyzed for editing efficiency. The median fluorescence intensity / pixel integrated values ​​for mPlum and copGFP at the fluorescence wavelengths in each cell nucleus were calculated. A calibration curve was created based on the abundance of vectors and the fluorescence intensity of copGFP in samples prepared by mixing mPlum-T2A-copGFP (normal) expression vector plasmid and mPlum-T2A-copGFP (Y / H) expression vector plasmid at a fixed ratio. Using this calibration curve, editing efficiency was calculated from the integrated fluorescence intensity values ​​calculated for each well.

[0125] As a result, when the E chain, A chain, and A' chain were introduced, more cells with copGFP fluorescence were observed, demonstrating improved editing efficiency compared to when only the E chain and A chain were introduced. In particular, when the E chain and A chain used in this example were combined with the 65-base A' chain (SEQ ID NO: 7; in the antisense strand of the target gene, the base sequence sharing sequence identity with the E chain and the base sequence sharing sequence identity with the A' chain are adjacent (i.e., the distance between the two is 0 bases)), the editing efficiency was significantly improved (Figures 2 and 3). Furthermore, when only the E chain was used instead of the TD, the effect of adding the A' chain on improving editing efficiency was also confirmed.

[0126] Example 2: Enhancement of Base Sequence Editing Efficiency by Addition of A' Strand The target gene was changed to the mEGFP gene, and the enhancement of editing efficiency by addition of the A' strand was confirmed as in Example 1. mEGFP is a green fluorescent protein, but it shares only 27% amino acid identity with copGFP and almost no homology at the DNA sequence level encoding it. U2OS cells were co-transfected with TD (E strand + A strand) or TD + A' strand together with a plasmid (mPlum-T2A-mEGFP(Y / H) expression vector plasmid; H. Kawai, R. Kamitsubo, H. Kamiya. Correction of monomeric enhanced green fluorescent protein (mEGFP) gene by short 5'-tailed duplexes. J. Biosci. Bioengng. 134, 175-181 (2022)) containing a mutant gene in which the base sequence TAC corresponding to the Tyr in the fluorophore Thr-Tyr-Gly of mEGFP was changed to CAC (SEQ ID NO: 3). Fluorescence microscopy observation was carried out in the same manner as in Example 1. The editing nucleic acids used are shown below.

[0127] E strand mEGFP-79_AS_E: SEQ ID NO: 8 5'-TCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAG -3' A strand mEGFP-35_S_A: SEQ ID NO: 9 5'-CTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCT -3' A' strand mEGFP-65_AS_Ap: SEQ ID NO: 10 5'-GAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCT -3'

[0128] As a result, even in experiments using the mEGFP gene, when the A' strand was added, more cells with green fluorescence were observed than when only the E strand and A strand were added, demonstrating significantly improved editing efficiency (Figures 4 and 5).

[0129] Example 3: Improving base sequence editing efficiency by chemical modification of E strand or A strand Using the analytical method (Example 1) targeting the copGFP gene, the effect of chemical modification of E strand and A strand on editing efficiency was analyzed. Unless otherwise specified in the examples, the A' strand in n-TD' is the same as in Example 1, copGFP_A_prime65.

[0130] (3-1) Effect of phosphorothioate (PS) modification on base sequence editing efficiency 1 For each of the E, A, and A' strands, PS modifications were introduced at one or two positions from the 5'-end or 3'-end, respectively, and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used (only those containing modifications) are shown below (PS modification is present at the phosphate moiety 3'-side of the underlined position):

[0131] E chain copGFP-79_AS_E 5'3'S: SEQ ID NO: 11 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 5'S: SEQ ID NO: 12 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 3'S: SEQ ID NO: 13 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 5'3'SS: SEQ ID NO: 14 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 5'SS: SEQ ID NO: 15 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP -79_AS_E 3'SS: SEQ ID NO: 16 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0132] Chain A copGFP-35_S_A 5'3'S: SEQ ID NO: 17 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-35_S_A 5'S: SEQ ID NO: 18 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-35_S_A 3'S: SEQ ID NO: 19 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-35_S_A 5'3'SS: SEQ ID NO: 20 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-35_S_A 5'SS: SEQ ID NO: 21 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-35_S_A 3'SS: SEQ ID NO: 22 5'- CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3'

[0133] A' strand copGFP-79_AS_Ap 5'3'S: SEQ ID NO: 23 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_Ap 5'S: SEQ ID NO: 24 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_Ap 3'S: SEQ ID NO: 25 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_Ap 5'3'SS: SEQ ID NO: 26 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_Ap 5'SS: SEQ ID NO: 27 5'- CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_Ap 3'SS: SEQ ID NO: 28 5'- CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3'

[0134] The results are shown in Figure 6. For both strand E and strand A, PS modification at the 3' end of each strand improved editing efficiency. Although the A' strand, which had PS modifications at the 5' and 3' ends, did not show any improvement in editing efficiency in this experimental system, the internucleotide modifications in strand A may be useful for conferring nuclease resistance during cell transfection.

[0135] (3-2) Effect of PS modification on base sequence editing efficiency 2 PS modifications were introduced into one to four positions from the 3' end of each of the E and A strands, and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used (only those containing modifications) are shown below (PS modification is present at the phosphate moiety 3'-side of the underlined position).

[0136] E chain copGFP-79_AS_E 3'S: SEQ ID NO: 13 copGFP -79_AS_E 3'SS: SEQ ID NO: 16 copGFP-79_AS_E 3'SSS: SEQ ID NO: 29 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 3'SSSS: SEQ ID NO: 30 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0137] Chain A copGFP-35_S_A 3'S: SEQ ID NO: 19 copGFP-35_S_A 3'SS: SEQ ID NO: 22 copGFP-35_S_A 3'SSS: SEQ ID NO: 31 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-35_S_A 3'SSSS: SEQ ID NO: 32 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3'

[0138] The results are shown in Figure 7. PS modification at up to two sites at the 3' end of the E strand and at up to three sites on the A strand tended to improve editing efficiency.

[0139] (3-3) Effect of PS modification on base sequence editing efficiency 3 Using E chains in which the positions of the two PS modifications were changed, the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used (only those containing modifications) are shown below (PS modification is present at the phosphate moiety 3' on the underlined side).

[0140] E strand copGFP-79_AS_E 3'SSp11: SEQ ID NO: 33 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 3'SSp21: SEQ ID NO: 34 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 3'SSp31: SEQ ID NO: 35 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 3'SSp41: SEQ ID NO: 36 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 3'SSp51: SEQ ID NO: 37 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 3'SSp61: SEQ ID NO: 38 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E 3'SSp71: SEQ ID NO: 39 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0141] The results are shown in Figure 8. Two PS modifications in the E strand improved editing efficiency when they were located near the 3' end of the E strand. On the other hand, when the two PS modifications were located within 3 bases from the 5' end to 1 base from the 3' end of the mutation introduced for base sequence editing, editing efficiency decreased.

[0142] (3-4) Effect of PCH3(P-Me) and POCH2CH3(P-OEt) Modifications of Strand A on Base Sequence Editing Efficiency A PCH3(P-Me) modification (substitution of a phosphodiester bond with a methylphosphonate bond) or a POCH2CH3(P-OEt) modification (substitution of a phosphodiester bond with an ethylphosphotriester bond) was introduced into strand A at two positions from the 5' or 3' end, and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used (only those containing modifications) are shown below (P-Me or P-OEt modification is present in the phosphate moiety on the 3' side of the underlined position):

[0143] Chain A copGFP_forAS_E79 A35 5' p-met x2: SEQ ID NO: 40 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP_forAS_E79 A35 3' p-met x2: SEQ ID NO: 41 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP_forAS_E79 A35 5' p-etho x2: SEQ ID NO: 42 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP_forAS_E79 A35 3' p-etho x2: SEQ ID NO: 43 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3'

[0144] The results are shown in Figure 9. P-Me or P-OEt modification at two sites from the 3' end of strand A improved the editing efficiency.

[0145] (3-5) Effect of P-OEt Modification of Strand A on Base Sequence Editing Efficiency P-OEt modifications were introduced into strand A at one or two positions from the 3' end, and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used (only those containing P-OEt modifications) are shown below (P-OEt modifications are present at the phosphate moiety on the 3' side of the underlined nucleotide). Strand A containing PS modifications at one or two positions from the 3' end (copGFP-35_S_A 3'S and copGFP-35_S_A 3'SS) were also used.

[0146] A chain copGFP-35_S_A 3'S: SEQ ID NO: 19 copGFP-35_S_A 3'SS: SEQ ID NO: 22 copGFP_forAS_E79 A35 3' p-etho x1: SEQ ID NO: 44 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP_forAS_E79 A35 3' p-etho x2: SEQ ID NO: 45 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3'

[0147] The results are shown in Figure 10. P-OEt modification of the 3'-end of strand A improved the editing efficiency.

[0148] (3-6) Effect of LNA Modification of E Strand on Base Sequence Editing Efficiency LNA modifications were introduced into the E strand near the mutations introduced for base sequence editing, and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used (only those containing modifications) are shown below (underlined nucleotides have LNA modifications). In the name of each sequence, the position of the mutation is designated as 0, and the position of the base on the 5' side is designated as minus (-), and the position of the base on the 3' side is designated as plus (+).

[0149] E strand copGFP-79_AS_E LNA0: SEQ ID NO: 46 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+0+1: SEQ ID NO: 47 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+1: SEQ ID NO: 48 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3-1+1+3: SEQ ID NO: 49 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-5-3-1+1+3+5: SEQ ID NO: 50 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2+2: SEQ ID NO: 51 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2-1+1+2: SEQ ID NO: 52 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3-2+2+3: SEQ ID NO: 53 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'copGFP-79_AS_ELNA-3-2-1+1+2+3: SEQ ID NO: 54 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0150] The results are shown in Figure 11. LNA modification at specific positions flanking the mutation improved editing efficiency. In particular, the highest editing efficiency was achieved when LNA modifications were introduced at six positions (-5, -3, -1, +1, +3, and +5, with the mutation position designated as 0). On the other hand, the editing efficiency decreased when LNA modifications were introduced at positions -2 and +2. Similar results were obtained when editing the E strand alone.

[0151] (3-7) Improving base sequence editing efficiency by combining LNA modification of E strand and P-OEt modification of A strand Regarding the introduction of LNA modification near the mutation in E strand, the modification site was further expanded and combined with P-OEt modification of A strand, and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used (only those containing modifications) are shown below (underlined nucleotides have LNA modifications). In the name of each sequence, the position of the mutation is designated as 0, and the position of the base on the 5' side is designated as minus (-), and the position of the base on the 3' side is designated as plus (+).

[0152] E chain copGFP-79_AS_E LNA-1+1: SEQ ID NO: 48 copGFP-79_AS_E LNA-3-1+1+3: SEQ ID NO: 49 copGFP-79_AS_E LNA-5-3-1+1+3+5: SEQ ID NO: 50 copGFP-79_AS_E LNA-7-5-3-1+1+3+5+7: SEQ ID NO: 55 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-9-7-5-3-1+1+3+5+7+9: SEQ ID NO: 56 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0153] Chain A copGFP_forAS_E79 A35 3' p-etho x 1: SEQ ID NO: 44

[0154] The results are shown in Figure 12. Editing efficiency was also improved when LNA modifications were introduced at eight positions (-7, -5, -3, -1, +1, +3, +5, and +7) or ten positions (-9, -7, -5, -3, -1, +1, +3, +5, +7, and +9), with the mutation position set to 0. Similar results were obtained when editing the E strand alone. Furthermore, when combined with P-OEt modification of the A strand, the editing efficiency tended to increase additively, with particularly high editing efficiency observed in the E strand with LNA modifications at 2 to 6 positions.

[0155] Example 4: Improvement of base sequence editing efficiency by LNA modification of E strand and P-OEt modification of A strand Similar to Example 2, improvement of base sequence editing efficiency by LNA modification of E strand and P-OEt modification of A strand was investigated using a mutant mEGFP gene as the target gene. The editing nucleic acids used (only those containing modifications for E strand and A strand) are shown below. The modified E strand has an LNA modification at the underlined nucleotide. The modified A strand has a P-OEt modification at the phosphate moiety 3' of the underlined nucleotide. As controls, unmodified E strand (SEQ ID NO: 8) and A strand (SEQ ID NO: 9) were used.

[0156] E strand mEGFP-79_AS_E LNA-2,0: SEQ ID NO: 57 5'-TCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAG -3' A strand mEGFP-35_S_A 3' p-etho×1: SEQ ID NO: 58 5'- CTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCT -3' A' strand mEGFP-65_AS_Ap: SEQ ID NO: 59 5'- GAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCT -3'

[0157] The results are shown in Figure 13. The introduction of LNA modifications at positions -2 and 0 in the E strand and the P-OEt modification of the A strand improved the editing efficiency. In particular, the LNA modification of the E strand significantly improved the editing efficiency. Similar results were obtained when editing the E strand alone.

[0158] Example 5: Examination of the ratio of E strand, A strand, and A' strand The molar ratio (pDNA:editing nucleic acid) of editing nucleic acid (total of E strand, A strand, and A' strand) to plasmid DNA (pDNA) containing the target gene was set to 1:60, and the molar ratio of E strand:A strand:A' strand was varied to examine the base sequence editing efficiency in the same manner as in Example 1. The editing nucleic acids used are shown below.

[0159] E chain copGFP-153-79b-AS (AS_E chain): SEQ ID NO: 5 A chain copGFP-153-79b-AS-35TD3'-ODN (A chain for AS_E chain): SEQ ID NO: 6 A' chain copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' chain for AS_E chain): SEQ ID NO: 7

[0160] The results are shown in Figure 14. When the molar concentration of the E chain was constant, the editing efficiency tended to be higher when the molar concentration of the A' chain was the same as or higher than the molar concentration of the A chain. When the molar concentration of the A chain was constant, the editing efficiency tended to be higher when the molar concentration of the A' chain was the same as or higher than the molar concentration of the E chain. When the molar concentration of the A' chain was constant, the editing efficiency tended to be higher when the molar concentration of the E chain was the same as or higher than the molar concentration of the A chain. When the molar concentration of the A chain was lower than the sum of the molar concentrations of the E chain and the A' chain, the editing efficiency tended to be higher.

[0161] Example 6: Effect of the positional relationship between A' strand and E strand on base sequence editing efficiency 1 In the antisense strand of a target gene, the A' strand was designed so that the distance (i.e., gap) between the base sequence sharing sequence identity with the E strand and the base sequence sharing sequence identity with the A' strand was 0 to 9 bases, and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used are listed below. In the name of the A' strand, the number after "Gap" indicates the number of bases in the gap. In the case of copGFP_A_prime65 (SEQ ID NO: 7), the gap is 0 bases (this positional relationship is also referred to as "nick").

[0162] E chain copGFP-153-79b-AS (AS_E chain): SEQ ID NO: 5 A chain copGFP-153-79b-AS-35TD3'-ODN (A chain for AS_E chain): SEQ ID NO: 6 A' chain copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' chain for AS_E chain): SEQ ID NO: 7 65Ap_AS_Gap1: SEQ ID NO: 60 5'- CCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTC -3' 65Ap_AS_Gap2: SEQ ID NO: 61 5'- TCCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTT -3' 65Ap_AS_Gap3: SEQ ID NO: 62 5'- GTCCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGT -3' 65Ap_AS_Gap6: SEQ ID NO: 63 5'- GCCGTCCTCGTACTTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGG -3' 65Ap_AS_Gap9: SEQ ID NO: 64 5'- GCCGCCGTCCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGA -3'

[0163] The results are shown in Figure 15. Although the editing efficiency decreased as the gap became larger, it was confirmed that the addition of the A' strand improved the editing efficiency even when the gap was 9 bases.

[0164] Example 7: Effect of the positional relationship between A' strand and E strand on base sequence editing efficiency 2 In the antisense strand of a target gene, the A' strand was designed so that the overlap between the base sequence sharing sequence identity with the E strand and the base sequence sharing sequence identity with the A' strand was 3 to 39 bases, and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used are shown below.

[0165] E chain copGFP-153-79b-AS (AS_E chain): SEQ ID NO: 5 A chain copGFP-153-79b-AS-35TD3'-ODN (A chain for AS_E chain): SEQ ID NO: 6 A' chain copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' chain for AS_E chain): SEQ ID NO: 7 copGFP-79_AS_Ap 3-overlap: SEQ ID NO: 65 5'- GTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGT -3' copGFP-79_AS_Ap 6-overlap: SEQ ID NO: 66 5'- CTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGC -3' copGFP-79_AS_Ap 9-overlap: SEQ ID NO: 67 5'- CTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGC -3' copGFP-79_AS_Ap 12-overlap: SEQ ID NO: 68 5'- GATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGG -3' copGFP-79_AS_Ap 15-overlap: SEQ ID NO: 69 5'- GCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGT -3' copGFP-79_AS_Ap 19-overlap: SEQ ID NO: 70 5'- GTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGT -3' copGFP-79_AS_Ap 29-overlap: SEQ ID NO: 71 5'- AGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGG -3' copGFP-79_AS_Ap 39-overlap: SEQ ID NO: 72 5'-GTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGT -3'

[0166] The results are shown in Figure 16. Although there was a tendency for the editing efficiency to be lower when there was overlap than when there was no overlap (n-TD'(nick)), it was confirmed that the addition of the A' strand improved the editing efficiency even when the overlap was 3 to 39 bases.

[0167] Example 8: Effect of A strand length on base sequence editing efficiency A strands were designed with different lengths and positions at which they form a duplex with the E strand, and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used are shown below. Using a 35-base A strand (SEQ ID NO: 7) as a reference, the chain length was extended on the 5' or 3' side. When the A strand length is extended on the 3' side, it contains a base sequence complementary to the portion of the E strand containing a mutation. Therefore, both an A strand having the sequence of the wild-type (WT) copGFP gene (i.e., completely complementary to the E strand; SEQ ID NOs: 71 to 75) and an A strand having the sequence of the mutant (Y / H) copGFP gene (i.e., not complementary to the E strand at the mutation site; SEQ ID NOs: 76 to 80) were designed.

[0168] E chain copGFP-153-79b-AS (AS_E chain): SEQ ID NO: 5 A chain copGFP-153-79b-AS-35TD 3'-ODN (A chain for AS_E chain): SEQ ID NO: 6 copGFP-153-79b-AS-45TD 3'-ODN 5': SEQ ID NO: 73 5'-CAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-55TD 3'-ODN 5': SEQ ID NO: 74 5'-TGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TD 3'-ODN 5': SEQ ID NO: 75 5'-ACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-75TD3'-ODN5': SEQ ID NO: 76 5'-GGGCCGCATGACCAACAAGATGAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-45TD3'-ODN3': SEQ ID NO: 77 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGC -3' copGFP-153-79b-AS-55TD3'-ODN3': SEQ ID NO: 78 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACT -3' copGFP-153-79b-AS-65TD3'-ODN3': SEQ ID NO: 79 5'- CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTA -3' copGFP-153-79b-AS-75TD3'-ODN3': SEQ ID NO: 80 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGC -3' copGFP-153-79b-AS-79TD3'-ODN3': SEQ ID NO: 815'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCTACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACG -3' 79E_AS_45TD3'-ODN3': SEQ ID NO: 82 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCCACGGC -3' 79E_AS_55TD3'-ODN3': SEQ ID NO: 83 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCCACGGCTTCTACCACT -3' 79E_AS_65TD3'-ODN3': SEQ ID NO: 84 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCCACGGCTTCTACCACTTCGGCACCTA -3' 79E_AS_75TD3'-ODN3': SEQ ID NO: 85 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCCACGGCTTCTACCACTTCGGCACCTACCCCAGCGGC -3' 79E_AS_79TD3'-ODN3': SEQ ID NO: 86 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGCCACGGCTTCTACCACTTCGGCACCTACCCCAGCGGCTACG -3' A' strand copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7

[0169] The results are shown in Figure 17. When the 5' end of strand A extended beyond the 3' end of strand E, the editing efficiency tended to decrease with increasing base length, but the high editing efficiency achieved by adding strand A was maintained. On the other hand, when the 3' end of strand A extended to include a base sequence complementary to the mutation-containing portion of strand E, the editing efficiency decreased.

[0170] Example 9: Effect of distance between double-stranded portion of n-TD' and mutation on base sequence editing efficiency The E and A strands were designed so that the mutation on the E strand was located at +44 (5' end of E strand), +34, +24, +14, +5, +4, +1, -6, -16, -26, or -34 (+ indicates that the double-stranded portion of TD does not overlap with the mutation site, and - indicates that the double-stranded portion of TD overlaps with the mutation site), with the base on the E strand complementary to the base at the 3' end of the A strand being considered as position -1, and the base sequence editing efficiency was examined as in Example 1. The editing nucleic acids used are shown below.

[0171] 79GFP_AS_5'-1-mut(+44-WT): SEQ ID NO: 87 5'-AGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAGGGCGCCTTTGGTGCTCTTCATCTTGTTGGTCATGCGGCC -3' 35mer_for_79GFP_AS_5'-1-mut(+44-WT): SEQ ID NO: 88 5'- GGCCGCATGACCAACAAGATGAAGAGCACCAAAGG -3' 65Ap_for_79E_AS_5'-1-mut(+44-WT): SEQ ID NO: 89 5'- GTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGT -3' 79GFP_AS_5'-11-mut(+34-WT): SEQ ID NO: 90 5'-TAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAGGGCGCCTTTGGTGCTCTTCATCTTGTTGG -3' 35mer_for_79GFP_AS_5'-11-mut(+34-WT): SEQ ID NO:91 5'-CCAACAAGATGAAGAGCACCAAAGGCGCCCTGACC -3' 65Ap_for_79E_AS_5'-11-mut(+34-WT): SEQ ID NO: 92 5'-AGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGG -3' 79GFP_AS_5'-21-mut(+24-WT): SEQ ID NO: 93 5'-GCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAGGGCGCCTTTGGTGCTCTTC -3' 35mer_for_79GFP_AS_5'-21-mut(+24-WT): SEQ ID NO: 94 5'-GAAGAGCACCAAAGGCGCCCTGACCTTCAGCCCCT -3' 65Ap_for_79E_AS_5'-21-mut(+24-WT): SEQ ID NO: 95 5'-GTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGT -3'79GFP_AS_5'-31-mut(+14-WT): SEQ ID NO: 96 5'-TGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAGGGCGCCTTT -3' 35mer_for_79GFP_AS_5'-31-mut(+14-WT): SEQ ID NO: 97 5'-AAAGGCGCCCTGACCTTCAGCCCCTACCTGCTGAG -3' 65Ap_for_79E_AS_5'-31-mut(+14-WT): SEQ ID NO: 98 5'-CTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGC -3' 79GFP_AS_5'-41-mut(+4-WT): SEQ ID NO: 99 5'-TCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCA -3' 35mer_for_79GFP_AS_5'-41-mut(+4-WT): SEQ ID NO: 100 5'-TGACCTTCAGCCCCTACCTGCTGAGCCACGTGATG -3' 65Ap_for_79E_AS_5'-41-mut(+4-WT): SEQ ID NO: 101 5'-CCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTC -3' 79GFP_AS_5'-44-mut(+1-WT): SEQ ID NO: 102 5'-TTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGG -3' 35mer_for_79GFP_AS_5'-44-mut(+1-WT): SEQ ID NO: 103 5'-CCTTCAGCCCCTACCTGCTGAGCCACGTGATGGGC -3' 65Ap_for_79E_AS_5'-44-mut(+1-WT): SEQ ID NO: 104 5'-CGTCCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGG -3'79GFP_AS_5'-51-mut(-6): SEQ ID NO: 105 5'-GAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGG -3' 35mer_for_79GFP_AS_5'-51-mut(-6-WT): SEQ ID NO: 106 5'-CCCCTACCTGCTGAGCCACGTGATGGGCTACGGCT -3' 35A_AS_5'-51-mut(-6-YH): SEQ ID NO: 107 5'-CCCCTACCTGCTGAGCCACGTGATGGGCCACGGCT -3' 65Ap_for_79E_AS_5'-51-mut(-6): SEQ ID NO: 108 5'-ACGCCGCCGTCCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAG -3' 79GFP_AS_5'-61-mut(-16): SEQ ID NO: 109 5'-TGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAG -3' 35mer_for_79GFP_AS_5'-61-mut(-16-WT): SEQ ID NO: 110 5'-CTGAGCCACGTGATGGGCTACGGCTTCTACCACTT -3' 35A_AS_5'-61-mut(-16-YH): SEQ ID NO: 111 5'-CTGAGCCACGTGATGGGCCACGGCTTCTACCACTT -3' 65Ap_for_79E_AS_5'-61-mut(-16): SEQ ID NO: 112 5'-CACGTGCAGCACGCCGCCGTCCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGA -3' 79GFP_AS_5'-71-mut(-26): SEQ ID NO: 113 5'-CCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCA -3' 35mer_for_79GFP_AS_5'-71-mut(-26-WT): SEQ ID NO: 114 5'-TGATGGGCTACGGCTTCTACCACTTCGGCACCTAC-3' 35A_AS_5'-71-mut(-26-YH): SEQ ID NO: 115 5'-TGATGGGCCACGGCTTCTACCACTTCGGCACCTAC -3' 65Ap_for_79E_AS_5'-71-mut(-26): SEQ ID NO: 116 5'-AGCTGAAGCTCACGTGCAGCACGCCGCCGTCCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCG -3' 79GFP_AS_5'-79-mut(-34): SEQ ID NO: 117 5'-TGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTA -3' 35mer_for_79GFP_AS_5'-79-mut(-34-WT): SEQ ID NO: 118 5'-TACGGCTTCTACCACTTCGGCACCTACCCCACGGG -3' 35A_AS_5'-79-mut(-34-YH): SEQ ID NO: 119 5'-CACGGCTTCTACCACTTCGGCACCTACCCCACGGG -3' 65Ap_for_79E_AS_5'-79-mut(-34): SEQ ID NO: 120 5'-GGTTGTGGGCGTAGCTCTTCATGCTCCTGCCGCCGCACGACGGTGCACTCGAAGTCGATGGCGATG -3'

[0172] The results are shown in Figure 18. The highest editing efficiency was achieved when mutations were introduced into the central base 35 from both ends of the 79-base-long E strand ("+5-WT"). When the mutation position was shifted by changing the base sequence while maintaining the length and relative positions of the three strands, whether it was moved to the 5' or 3' side, editing efficiency decreased. Editing efficiency was particularly significant when the mutation was moved further away than +14 or closer than +4.

[0173] Example 10: Examination of the Nick Position in n-TD' When a sequence with sequence identity to the E strand and a sequence with sequence identity to the A' strand are adjacent to each other on the editing target strand, the E strand and the A' strand can be considered to be two single-stranded polynucleotides obtained by introducing a single non-linked portion (nick) into a single-stranded polynucleotide of a certain length. Therefore, E strands and A' strands with shifted nick positions were designed, and the base sequence editing efficiency was examined in the same manner as in Example 1. Specifically, antisense E strands of 109, 79, 65, and 35 bases in length were created, as well as A' strands of 35, 65, 79, and 109 bases in length adjacent to the 5' side of the E strand. A 144-nucleotide E strand was also created without combining with the A' strand. The A strand was a single-stranded polynucleotide consisting of a base sequence complementary to the 35 nucleotides at the 3' end of each E strand. The editing nucleic acids used are listed below.

[0174] 144E_AS_5'-105-mut: SEQ ID NO: 121 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' 109E_AS_5'-70-mut_for_35Ap: SEQ ID NO: 122 5'-CGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' 35Ap_for_109E_AS_5'-70-mut: SEQ ID NO: 123 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGC -3' 65E_AS_5'-26-mut_for_79Ap: SEQ ID NO: 124 5'-TAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' 79Ap_for_65E_AS_5'-26-mut: SEQ ID NO: 125 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGG -3' 35E_AS_notmut_for_109Ap: SEQ ID NO: 126 5'-ATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' 109Ap_5'-105-mut_for_35E_AS_notmut: SEQ ID NO: 127 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCTCGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCC -3'

[0175] The results are shown in Figure 19. High editing efficiency was obtained when a combination of a 79-mer E strand and a 65-mer A' strand, or a combination of a 65-mer E strand and a 79-mer A' strand, was used.

[0176] Example 11: Genome editing by TD with added A' strand The efficiency of base sequence editing was examined using U2OS cells with a mutant copGFP gene inserted into the genome as the target gene, as in Example 1. The cells used in this example were prepared by lipofecting U2OS cells with the mPlum-T2A-copGFP(Y / H) expression vector plasmid (H. Kawai, R. Kamitsubo, H. Kamiya. Correction of monomeric enhanced green fluorescent protein (mEGFP) gene by short 5'-tailed duplexes. J. Biosci. Bioengng. 134, 175-181 (2022)), followed by cloning using drug (Zeocin) resistance and mPlum fluorescence as indicators.

[0177] For each of the E, A, and A' strands, in which LNA was introduced at two positions (-1 and +1), PS modification was introduced at one position from the 5' end. The editing nucleic acids used were (the modified positions are underlined. For PS modifications, the phosphate moiety is modified on the 3' side of the underlined position). E strand copGFP-79_AS_E LNA-1+1_5'PS: SEQ ID NO: 128 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' A strand copGFP-35_S_A_5'PS (alias: copGFP-35_S_A 5'S): SEQ ID NO: 18 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' A strand copGFP_A_prime65_5'PS: SEQ ID NO: 129 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3'

[0178] Fluorescence microscopy was performed using the Opera Phenix® high-throughput, high-content imaging system (PerkinElmer).

[0179] The results are shown in Figure 20. As a result of introducing the TD and A' strands, cells emitting copGFP fluorescence were observed. Therefore, it was confirmed that genome editing is possible using the method of the present invention.

[0180] Example 12: Improvement of base sequence editing efficiency by LNA modification of E strand and P-OEt modification of A strand Similar to Example 2, improvement of base sequence editing efficiency by LNA modification of E strand and P-OEt modification of A strand was investigated using a mutant mEGFP gene as the target gene. The editing nucleic acids used (only those containing modifications for E strand and A strand) are shown below. The modified E strand has an LNA modification at the underlined nucleotide. The modified A strand has a P-OEt modification at the phosphate moiety 3' of the underlined nucleotide. Unmodified E strand (SEQ ID NO: 8) and A strand (SEQ ID NO: 9) were used as controls.

[0181] E strand mEGFP-79_AS_E LNA-1+1: SEQ ID NO: 130 5'-TCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAG -3' A strand mEGFP-35_S_A 3' p-etho×1: SEQ ID NO: 58 5'- CTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCT -3' A' strand mEGFP-65_AS_Ap: SEQ ID NO: 59 5'- GAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCT -3'

[0182] The results are shown in Figure 21. As with the copGFP gene as the target gene, the introduction of LNA modifications near the mutation in the E strand and the P-OEt modification of the A strand improved the editing efficiency. In particular, the LNA modification of the E strand significantly improved the editing efficiency. Similar results were obtained when editing the E strand alone. In Figure 21, "AS_TD'" indicates the results for TD' without any modifications, "AS_E(LNAx2)" indicates the results for E strands with modifications, "AS_TD'(LNAx2)" indicates the results for TD' with E strands with modifications, and "AS_TD'(LNAx2,3'-Etho)" indicates the results for TD' with E strands with modifications and A strands with modifications.

[0183] Example 13: Effect of ENA modification of E strand on base sequence editing efficiency For the E strand, an ENA modification was introduced near the mutation introduced for base sequence editing (the position where the LNA or ENA modification was introduced is underlined in the sequence), and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used (only those containing modifications) are shown below (the underlined nucleotides have an LNA or ENA modification). In the name of each sequence, the position of the mutation is designated as 0, the position of the base on the 5' side is designated as minus (-), and the position of the base on the 3' side is designated as plus (+).

[0184] copGFP-79_AS_E LNA-1+1: SEQ ID NO: 48 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E ENA-1+1: SEQ ID NO: 131 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0185] The results are shown in Figure 22. In editing with nE (E strand only) and n-TD' (E strand + A strand + A strand'), LNA modification and ENA modification showed almost the same effect.

[0186] Example 14: Effect of introducing an amino linker into the 5' or 3' end of each oligodeoxynucleotide (ODN) on base sequence editing efficiency. An amino linker (AL linker) was introduced into the 5' or 3' end of one of the ODNs contained in n-TD' (Strand E + Strand A + Strand A'), and the base sequence editing efficiency was examined in the same manner as in Example 1. The editing nucleic acids used (only those containing AL linkers) are listed below. 5'-Amino-Modifier C6 (Product No. 0-1906-02, Glen Research) and Phthalamido Amino C6 lcaa CPG (Product No. N-8217-10, ChemGenes) were used to introduce the AL linker into the 5' and 3' ends, respectively.

[0187] E strand copGFP-153-79b-AS(E)_5'NH2: AL linker introduced at the 5' end of SEQ ID NO: 5 5'-AL-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG-3' copGFP-153-79b-AS(E)_3'NH2: AL linker introduced at the 3' end of SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG-AL-3' A strand copGFP-153-79b-AS-35TD3'-ODN(A)_5'NH2: AL linker introduced at the 5' end of SEQ ID NO: 6 5'-AL-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT-3' copGFP-153-79b-AS-35TD3'-ODN(A)_3'NH2: AL linker introduced at the 3' end of SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT-AL-3' A' strand copGFP-153-79b-AS-65TDdown-ODN(Aprime)_5'NH2: AL linker introduced at the 5' end of SEQ ID NO: 7 5'-AL-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT-3' copGFP-153-79b-AS-65TDdown-ODN(Aprime)_3'NH2: AL linker introduced at the 3' end of SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT-AL-3'

[0188] The results are shown in Figure 23. When an AL linker was introduced into the 5' or 3' end of any of the E, A, and A' strands (5'-Linker or 3'-Linker in the figure), there was no effect on editing efficiency compared to when an AL linker was not introduced (EAA'(n-TD') in the figure). This result demonstrates that it is possible to modify each end of each ODN and to link ODNs with a cleavable linker.

[0189] Example 15: Comparison of base sequence editing efficiency when using ODNs linked by linkers The E and A strands, and the E and A' strands were each linked by a Z linker, and the base sequence editing efficiency was examined in the same manner as in Example 1. The 79-nt antisense strand E strand (SEQ ID NO: 5) was used as the editing nucleic acid and linked to the A strand (SEQ ID NO: 6) or A' strand (SEQ ID NO: 7) by a Z linker. Linking using a Z linker was performed as described in WO2012 / 074038. Regardless of whether or not the ODNs were linked by a Z linker, they were mixed so that the molar concentrations of each ODN were the same. That is, the molar ratio of E strand:A strand was 1:1 in n-TD and Linker_(E+A) (the 3' end of the E strand linked to the 5' end of the A strand), the molar ratio of E strand:A strand was 1:1 in Linker_(E+A') (the 3' end of the E strand linked to the 5' end of the A' strand), and the molar ratio of E strand:A strand:A' strand was 1:1:1 in n-TD', Linker_(E+A)+A' (Linker_(E+A) plus A' strand), and Linker_(E+A')+A (Linker_(E+A') plus A strand). The editing nucleic acids used (only those linked with Z linkers) are shown below.

[0190] Construct in which E chain and A chain are linked copGFP-79_AS_5'E3'_C8Ph_5'A3': the 3' end of SEQ ID NO: 5 and the 5' end of SEQ ID NO: 6 are linked via a Z linker 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG-Z-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT-3' Construct in which E chain and A' chain are linked copGFP-79_AS_5'E3'_C8Ph_5'A'3': the 3' end of SEQ ID NO: 5 and the 5' end of SEQ ID NO: 7 are linked via a Z linker 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG-Z-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT-3'

[0191] The results are shown in Figure 24. Linker_(E+A) showed the same effect as when E strand and A strand were mixed (n-TD). On the other hand, Linker_(E+A') had almost the same editing efficiency as E strand alone, and there was no effect of the A' strand connected by the Z linker.

[0192] Example 16: Editing efficiency for nonsense mutations in causative genes reported in human diseases Nonsense mutation sequences reported in eight types of human diseases were used as target sequences for gene editing (https: / / www.ncbi.nlm.nih.gov / snp / ). Table 1 shows the disease names, causative genes, and sequences near the nonsense mutations. In Table 1, underlines indicate stop codons resulting from nonsense mutations, and shaded letters indicate nonsense mutations. GC is shown in bold. The gene information for each gene is registered on the National Center for Biotechnology Information website (https: / / www.ncbi.nlm.nih.gov / snp / ) under the following numbers: DMD: rs128626235 WRN: rs121908446 HBB: rs33950507 ADA: rs780014431 LCA: rs780667159 PRKN: rs1562485799 BRAF: rs121913357 NRAS: rs1562485799

[0193] We inserted 55-nt sequences before and after these nonsense mutations between the red fluorescent protein mPlum gene and the T2A sequence of the expression vector, generating pcDNA4_mPlum_S_copGFP WT (Figure 25). The nonsense mutations were then edited by n-TD or n-TD' to allow translation of the copGFP gene. n-TD' was prepared using a 79-nt antisense (AS) E strand, a 35-nt A strand complementary to the 3' end of the E strand, and a 65-nt A' strand extending from the 5' end of the E strand. The n-TD' was prepared so that the molar ratio of E:A:A' when transfected into cells was 10:20:30, with the plasmid DNA being 1. We also prepared n-TD without the A' strand, so that the molar ratio of E:A when transfected into cells was 10:20, with the plasmid DNA being 1. As a control, similar to Example 1, editing efficiency was measured using pcDNA4_mPlum_T2A_copGFP Y / H, E strand (SEQ ID NO: 5), A strand (SEQ ID NO: 6), and A′ strand (SEQ ID NO: 7) incorporating fluorescence-quenched copGFP.

[0194] The results are shown in Figure 26 and Table 2. For all sequences, AS n-TD' showed higher gene editing efficiency than AS n-TD (Figure 26). Furthermore, gene sequences with high gene editing efficiency using AS n-TD' tended to have a high GC content in the E strand, suggesting that the GC content of the E strand affects editing efficiency.

[0195] Example 17-1: Effect of Addition of a 35-Base Oligodeoxynucleotide (ODN) with a Different Position in the Target Region from the E-Strand on the E-Strand-Based Base Sequence Editing Efficiency. The effect of adding a 35-base ODN with a different position in the target region from the E-Strand on the editing efficiency of the E-Strand-Based Base Sequence Editing Method using green fluorescent protein (Green Fluorescent Protein) was examined. The target gene was the copGFP gene encoding green fluorescent protein from copepods. Plasmid DNA containing a mutant copGFP gene (SEQ ID NO: 1), in which the base sequence TAC corresponding to Tyr in the fluorophore-constituting Gly-Tyr-Gly was changed to CAC (encoding His), E-Strand, and ODN were co-transfected into human U2OS cells, and the percentage of cells exhibiting green fluorescence was compared using a fluorescence microscope. A 79-base single-stranded polynucleotide (AS_E-Strand, SEQ ID NO: 5) with a sequence identical to a portion of the antisense strand of the target gene and a wild-type fluorophore-encoding sequence was used as the E-Strand. We used 35-base ODNs with sequence identity to the antisense or sense strand, which are in a different position from the E strand relative to the target region. The sequences of the ODNs used are shown below.

[0196] copGFP-153-79b-S-35TDup-ODN (AS_Up_O): SEQ ID NO: 140 5'-GGCGCCTTTGGTGCTCTTCATCTTGTTGGTCATGC -3' copGFP-153-79b-S-35TD5'-ODN (AS_Up_T): SEQ ID NO: 141 5'-ATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-S-35TDcn-ODN (AS_Cnt_Y): SEQ ID NO: 142 5'-GAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCA -3' copGFP-153-79b-S-35TDcnYH-ODN (AS_Cnt_H): SEQ ID NO: 143 5'-GAAGTGGTAGAAGCCGTGGCCCATCACGTGGCTCA -3' copGFP-153-79b-S-35TD 3'-ODN (AS_Dwn_T): SEQ ID NO: 144 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAG -3' copGFP-153-79b-S-35TD down-ODN (AS_Dwn_O): SEQ ID NO: 145 5'-GCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-153-79b-AS-35TD up-ODN (S_Up_O): SEQ ID NO: 146 5'-GCATGACCAACAAGATGAAGAGCACCAAAGGCGCC -3' copGFP-153-79b-AS-35TD 3'-ODN (S_Up_T, A chain for AS_E chain): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-35TDcn-ODN (S_Cnt_Y): SEQ ID NO: 147 5'-TGAGCCACGTGATGGGCTACGGCTTCTACCACTTC -3' copGFP-153-79b-AS-35TDcnYH-ODN (S_Cnt_H): SEQ ID NO: 148 5'-TGAGCCACGTGATGGGCCACGGCTTCTACCACTTC -3' copGFP-153-79b-AS-35TD5'-ODN (S_Dwn_T): SEQ ID NO: 149 5'-CTTCTACCACTTCGGCACCTACCCCAGCGGCTACG -3'copGFP-153-79b-AS-35TDdown-ODN (S_Dwn_O): SEQ ID NO: 150 5'-AGAACCCCTTCCTGCACGCCATCAACAACGGCGGC -3'

[0197] Figure 27A shows the arrangement of the E strand and each 35-base ODN in the target region. The name of each 35-base ODN includes AS if it has sequence identity with the antisense strand of the copGFP gene, and S if it has sequence identity with the sense strand. If it is located upstream of the cpoGFP gene from the editing base (editing mutation on the E strand), it includes Up (upstream), if it is located downstream, it includes Dwn (downstream), and if it is located on the editing base, it includes Cnt (center). If it does not overlap with the base sequence of the E strand but is adjacent, it includes O (outside), and if it is a base sequence that overlaps with the end of the E strand, it includes T (terminal). For the ODN located on the edited base, if it contains the sequence 5'-TAC-3' (sense strand) encoding Tyr in the chromophore of fluorescent copGFP or its complementary strand 5'-GTA-3' (antisense strand), it contains Y, and if it contains the sequence 5'-CAC-3' (sense strand) encoding His of mutant copGFP or its complementary strand 5'-GTG-3' (antisense strand), it contains H. Note that S_Up_T is the same ODN as the A strand of TD (SEQ ID NO: 6).

[0198] The specific experimental method is as follows. Solutions of the E strand and a 35-base ODN with a different configuration relative to the target region from the E strand were mixed at a ratio of 1:10 and left to stand at room temperature for 1 hour to prepare editing nucleic acids. The mPlum-T2A-copGFP (Y / H) expression vector plasmid (pDNA; 30.0 ng, 7.33 fmol) containing the mutant copGFP gene for editing efficiency analysis was mixed with each of the prepared editing nucleic acids at a molar ratio of pDNA to E strand of 1:10, and seeded (8 x 10) cells into each well of a 384-well plate. 3After incubation with 1000 cells, the cells were co-transfected into U2OS cells cultured overnight using Lipofectamine 3000. After 4 hours, the medium was replaced and the cells were cultured for 48 hours. Nuclei were stained with Hoechst 33342, and the cells were fixed with 4% paraformaldehyde. Fluorescence microscopy images were obtained.

[0199] Editing efficiency was calculated as follows. Fluorescence microscopy images of each well of a 384-well plate were taken using Opera Phoenix. Using the Hoechst33342 fluorescence intensity data from all wells, the image analysis software Harmony was used to identify each cell nucleus. The median fluorescence intensity / pixel integration values ​​for Hoechst33342, mPlum, and copGFP were calculated for each cell nucleus. From the Hoechst33342 fluorescence intensity data from all wells, cell nuclei with average fluorescence intensity and morphology were selected for analysis of editing efficiency. Based on the mPlum and copGFP fluorescence intensity data from cell nuclei in wells transfected with only the mPlum-T2A-copGFP(Y / H) expression vector plasmid, a threshold was established between cells with negative copGFP fluorescence (cells without the edited fluorescent-type copGFP gene pDNA) and cells with positive copGFP fluorescence (cells containing the fluorescent-type copGFP gene pDNA). In addition, a threshold was set for the mPlum-positive cells (cells containing pDNA) and mPlum-negative cells (cells not containing pDNA) based on the mPlum fluorescence intensity of the nuclei of all analyzed wells. The editing efficiency was calculated by dividing the number of copGFP-positive cells by the number of mPlum-positive cells in each well.

[0200] The results are shown in Figure 27B. Compared to the editing efficiency of the E strand alone (-), the editing efficiency was most improved in the 5'-tailed duplex (TD) in which the ODN S_Up_T (A strand) complementary to the 3'-end of the E strand was added. Furthermore, the editing efficiency was significantly improved when the ODN S_Dwn_T complementary to the 5'-end of the E strand was added. Furthermore, the editing efficiency was significantly improved when the ODN AS_Dwn_O adjacent to the 5'-end of the E strand was added.

[0201] Example 17-2: Effect of different amounts of E strand, A strand, and ODN adjacent to the 5'-end of E strand (AS_Dwn_O, 35-base A' strand) introduced on base sequence editing efficiency In a base sequence editing method using E strands with green fluorescent protein, the effect on editing efficiency of varying the total amount of E strand, A strand, and ODN adjacent to the 5'-end of E strand (AS_Dwn_O, 35-base A' strand) introduced into the target plasmid was examined. Different combinations and doses of E strand, A strand, and 35-base A' strand were introduced into cells with target plasmid DNA, and the percentage of cells that exhibited green fluorescence was compared using a fluorescence microscope.

[0202] The specific experimental method is as follows. Solutions of E strand (SEQ ID NO: 5), A strand (SEQ ID NO: 6), and 35-base A' strand (AS_Dwn_O, SEQ ID NO: 145) were mixed at a molar ratio of 1:0:0, 1:10:0, 1:0:10, or 1:5:5 and allowed to stand at room temperature for 1 hour to prepare editing nucleic acids. The mPlum-T2A-copGFP(Y / H) expression vector plasmid (pDNA; 30.0 ng, 7.33 fmol) containing the mutant copGFP gene for editing efficiency analysis was mixed with each of the prepared editing nucleic acids at a pDNA to E strand molar ratio of 1:10, 1:1, or 1:0.1, and seeded (8 × 10 cells) into each well of a 384-well plate. 3 After incubation with 1000 cells, the cells were co-transfected into U2OS cells cultured overnight using Lipofectamine 3000. After 4 hours, the medium was replaced and the cells were cultured for 48 hours. Nuclei were stained with Hoechst 33342, and the cells were fixed with 4% paraformaldehyde. Fluorescence microscopy images were obtained.

[0203] The editing efficiency was calculated in the same manner as in Example 17-1.

[0204] The results are shown in Figure 28. The editing efficiency increased depending on the amount of E strand. Furthermore, compared to the editing efficiency with E strand alone, the addition of A strand or the 35-base A' strand improved the editing efficiency. Furthermore, compared to the editing efficiency with the addition of either A strand or A' strand alone, the addition of both strands further improved the editing efficiency.

[0205] Example 17-3: Effect of the amount of A chain and 35-base A' chain introduced into E chain on base sequence editing efficiency In a base sequence editing method using E chains that uses green fluorescent protein, the effect on editing efficiency was examined when the amount of A chain and 35-base A' chain introduced into the target plasmid was varied while the dose of E chain was kept constant. Cells were introduced with different doses of A chain and 35-base A' chain at a molar ratio of 1:1 between target plasmid DNA and E chain, and the percentage of cells that exhibited green fluorescence was compared using a fluorescence microscope.

[0206] The specific experimental method is as follows. 1 μM solutions of E strand (SEQ ID NO: 5), A strand (SEQ ID NO: 6), and 35-base A' strand (AS_Dwn_O, SEQ ID NO: 145) were mixed with E strand, A strand, 35-base A' strand, or a mixture of A strand and 35-base A' strand (1:1 molar ratio) at 1:0.1, 1:1, 1:10, 1:100, or 1:1000 ratios and allowed to stand at room temperature for 1 hour to prepare editing nucleic acids. The mPlum-T2A-copGFP(Y / H) expression vector plasmid (pDNA; 30.0 ng, 7.33 fmol) containing the mutant copGFP gene for editing efficiency analysis was mixed with each of the prepared editing nucleic acids at a pDNA to E strand molar ratio of 1:1 and seeded (8 × 10 cells) into each well of a 384-well plate. 3 After incubation with 1000 cells, the cells were co-transfected into U2OS cells cultured overnight using Lipofectamine 3000. After 4 hours, the medium was replaced and the cells were cultured for 48 hours. Nuclei were stained with Hoechst 33342, and the cells were fixed with 4% paraformaldehyde. Fluorescence microscopy images were obtained.

[0207] The editing efficiency was calculated in the same manner as in Example 17-1.

[0208] The results are shown in Figure 29. When the molar ratio of pDNA to E strand was 1:1 and A strand or 35-base A' strand was added, the editing efficiency improved as the dosage of each strand increased. Furthermore, when the same dosage of A strand and 35-base A' strand was added, the editing efficiency was more improved with the addition of A strand. When A strand and A' strand were mixed at a 1:1 ratio and added, the editing efficiency was higher than when either strand was added alone at the same dosage.

[0209] Example 17-4: Effect of the ratio of the amount of A chain and the 35-base A' chain introduced relative to the amount of E chain introduced on base sequence editing efficiency In a base sequence editing method using an E chain that utilizes green fluorescent protein, the effect on editing efficiency was examined when the dosage of E chain relative to the target plasmid was kept constant and the dosage ratio of A chain and the 35-base A' chain was changed. The molar ratio of the target plasmid DNA, E chain, and the sum of A chain and the 35-base A' chain was set to 1:1:100, and A chain and the 35-base A' chain were introduced into cells at different molar ratios, and the percentage of cells that exhibited green fluorescence using a fluorescence microscope was compared.

[0210] The specific experimental method is as follows: 1 μM solutions of E strand (SEQ ID NO: 5), A strand (SEQ ID NO: 6), and 35-base A' strand (AS_Dwn_O, SEQ ID NO: 145) were mixed at molar ratios of 100:0, 90:10, 80:20, 70:30, 60:40, 50:50, 40:60, 30:70, 20:80, 10:90, and 0:100, with E strand being 1, and the mixture was left to stand at room temperature for 1 hour to prepare editing nucleic acids. The mPlum-T2A-copGFP (Y / H) expression vector plasmid (pDNA; 30.0 ng, 7.33 fmol) containing the mutant copGFP gene for editing efficiency analysis and each prepared editing nucleic acid were mixed at a molar ratio of pDNA to E strand of 1:1, and seeded (8 × 10 cells) into each well of a 384-well plate. 3 After incubation with 1000 cells, the cells were co-transfected into U2OS cells cultured overnight using Lipofectamine 3000. After 4 hours, the medium was replaced and the cells were cultured for 48 hours. Nuclei were stained with Hoechst 33342, and the cells were fixed with 4% paraformaldehyde. Fluorescence microscopy images were obtained.

[0211] The editing efficiency was calculated in the same manner as in Example 17-1.

[0212] The results are shown in Figure 30. The effect of changing the dosage ratio of A strand to 35-base A' strand on editing efficiency was observed. When the dosage of 35-base A' strand was higher than that of A strand, editing efficiency tended to be more improved.

[0213] Example 17-5: Effect of differences in the length of the ODN (A' strand) adjacent to the 5'-end of the E strand on base sequence editing efficiency In a base sequence editing method using an E strand using green fluorescent protein, the editing efficiency was analyzed by adding an ODN (A' strand; strand length 15-75 bases) adjacent to the 5'-end of the E strand to examine the effect of the A' strand length on editing efficiency. Target plasmid DNA and E strand (SEQ ID NO: 5) were supplemented with A' strand (SEQ ID NO: 6), or A' strands with lengths of 15 bases (A' strand 15-nt), 25 bases (A' strand 25-nt), 35 bases (A' strand 35-nt, SEQ ID NO: X11), 45 bases (A' strand 45-nt), 55 bases (A' strand 55-nt), 65 bases (A' strand 65-nt), or 75 bases (A' strand 75-nt), and these were introduced into cells, and the percentage of cells that exhibited green fluorescence was compared using a fluorescence microscope. The sequences of the ODNs used are shown below: Figure 31A shows the location of the E strand and each ODN in the target region.

[0214] copGFP-153-79b-AS-35TD3'-ODN (A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP_A_prime15 (A' strand_15-nt): SEQ ID NO: 151 5'-GCAGGAAGGGGTTCT -3' copGFP_A_prime25 (A' strand_25-nt): SEQ ID NO: 152 5'-TTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP_A_prime35 (copGFP-153-79b-S-35TDdown-ODN) (A' strand_35-nt): SEQ ID NO: 145 5'-GCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP_A_prime45 (A' strand_45-nt): SEQ ID NO: 153 copGFP_A_prime55 (A' strand_55-nt): SEQ ID NO: 154 5'-TCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP_A_prime65 (A' strand_65-nt) (A' strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP_A_prime75 (A' strand_75-nt): SEQ ID NO: 155 5'-CGCCGCCGTCCTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3'

[0215] The specific experimental method is as follows. 1 μM solutions of E strand and A strand or A' strands of different lengths were mixed at a molar ratio of 1:10 and left to stand at room temperature for 1 hour to prepare editing nucleic acids. The mPlum-T2A-copGFP(Y / H) expression vector plasmid (pDNA; 30.0 ng, 7.33 fmol) containing the mutant copGFP gene for editing efficiency analysis was mixed with each of the prepared editing nucleic acids at a molar ratio of pDNA to E strand of 1:10, and seeded (8 × 10 cells) into each well of a 384-well plate. 3 After incubation with 1000 cells, the cells were co-transfected into U2OS cells cultured overnight using Lipofectamine 3000. After 4 hours, the medium was replaced and the cells were cultured for 48 hours. Nuclei were stained with Hoechst 33342, and the cells were fixed with 4% paraformaldehyde. Fluorescence microscopy images were obtained.

[0216] The editing efficiency was calculated in the same manner as in Example 17-1.

[0217] The results are shown in Figure 31B. The editing efficiency of 5'-tailed duplex (TD), in which the A strand was added to the E strand, was higher when an A' strand with a length of 45 bases or more was added at the same molar dose, and the highest editing efficiency was achieved when an A' strand with a length of 65 bases was added.

[0218] Example 17-6: Effect of A' strand length on base sequence editing efficiency In base sequence editing methods using green fluorescent protein (GFP)-based E or TD (E and A strands), the editing efficiency was analyzed by adding A' strands with lengths of 15 to 75 bases to examine the effect of A' strand length on editing efficiency. A' strands with lengths of 15 bases (A' strand 15-nt), 25 bases (A' strand 25-nt), 35 bases (A' strand 35-nt, SEQ ID NO: X11), 45 bases (A' strand 45-nt), 55 bases (A' strand 55-nt), 65 bases (A' strand 65-nt), or 75 bases (A' strand 75-nt) were added to the target plasmid DNA and E strand or TD, and these were introduced into cells, and the percentage of cells that exhibited green fluorescence was compared using a fluorescence microscope. The ODNs used were the same as those used in Example 17-5 (SEQ ID NOs: 7, 145, and 151 to 155).

[0219] The specific experimental method is as follows. Solutions of E chain (SEQ ID NO: 5), A chain (SEQ ID NO: 6), and A' chains of different lengths (SEQ ID NOs: 145 and 151-155) were mixed at a molar ratio of 1:10 for E chain:A chain or E chain:A' chain, and 1:5:5 for E chain:A chain:A' chain, and the mixture was left to stand at room temperature for 1 hour to prepare editing nucleic acids. An mPlum-T2A-copGFP(Y / H) expression vector plasmid (pDNA; 30.0 ng, 7.33 fmol) containing a mutant copGFP gene for editing efficiency analysis was mixed with each of the prepared editing nucleic acids at a molar ratio of pDNA to E chain of 1:10, and the mixture was seeded (8 × 10 cells) into each well of a 384-well plate. 3 After incubation with 1000 cells, the cells were co-transfected into U2OS cells cultured overnight using Lipofectamine 3000. After 4 hours, the medium was replaced and the cells were cultured for 48 hours. Nuclei were stained with Hoechst 33342, and the cells were fixed with 4% paraformaldehyde. Fluorescence microscopy images were obtained.

[0220] The editing efficiency was calculated in the same manner as in Example 17-1.

[0221] The results are shown in Figure 32. Compared with the editing efficiency of TD, the editing efficiency was higher when an A' strand having a chain length of 35 bases or more was added to TD.

[0222] Example 17-7: Establishment of a method for calculating quantitative editing efficiency using the integrated fluorescence intensity of each cell. In Examples 17-1 and 17-2, the editing efficiency was calculated by dividing the number of copGFP-positive cells by the number of mPlum-positive cells. This calculation method does not reflect the copy number of the target plasmid DNA introduced into each cell. Furthermore, if the majority of the analyzed cell nuclei become copGFP-positive due to improved editing efficiency (e.g., Example 17-6), it becomes difficult to compare editing efficiencies. Therefore, we investigated whether editing efficiency reflecting the introduced copy number of the target plasmid DNA could be calculated from the total integrated fluorescence intensity of copGFP and mPlum in the analyzed cell nuclei of each well.

[0223] The specific experimental method is as follows: mPlum-T2A-copGFP (normal) expression vector plasmid and mPlum-T2A-copGFP(Y / H) expression vector plasmid were mixed at the following molar ratios: 1:0 (100% normal copGFP), 2:1 (66.7%), 5:5 (50%), 1:2 (33.3%), 1:3 (25%), 1:7 (12.5%), 1:15 (6.25%), 1:31 (3.13%), 1:63 (1.56%), 1:127 (0.781%), 1:255 (0.391%), 1:511 (0.195%), 1:1023 (0.0977%), 1:2047 (0.0488%), 1:4095 (0.244%), and 0:1 (0%). Seed each well of a 384-well plate (8 x 10 3 After incubation with 1000 cells, 30.0 ng of the mixed plasmids was transfected into U2OS cells cultured overnight using Lipofectamine 3000. The medium was replaced after 4 hours and the cells were cultured for 48 hours. Nuclei were stained with Hoechst 33342, and the cells were fixed with 4% paraformaldehyde. Fluorescence microscopy images were taken.

[0224] Fluorescence intensity analysis values ​​for each well were calculated from fluorescence microscopy images using the following method. Fluorescence microscopy images of each well of a 384-well plate were taken using Opera Phoenix. Using the Hoechst33342 fluorescence intensity data from all wells, each cell nucleus was identified using the image analysis software Harmony, and the median fluorescence intensity / pixel values ​​of the Hoechst33342, mPlum, and copGFP fluorescence wavelengths for each cell nucleus were calculated. Cell nuclei with average fluorescence intensity and morphology were selected for analysis from the Hoechst33342 fluorescence intensity data from all wells. A threshold line was set between copGFP fluorescence-negative cells (cells without pDNA containing the fluorescent copGFP gene) and copGFP fluorescence-positive cells (cells containing pDNA containing the fluorescent copGFP gene) based on the mPlum and copGFP fluorescence intensity data from cell nuclei in wells transfected with only the mPlum-T2A-copGFP(Y / H) expression vector plasmid. In addition, a threshold was set for mPlum-positive cells (cells containing pDNA) and mPlum-negative cells (cells without pDNA) based on the mPlum fluorescence intensity of the cell nuclei analyzed in each well. The integrated copGFP fluorescence intensity of copGFP-positive cells and the integrated mPlum fluorescence intensity of mPlum-positive cells were calculated for each well transfected with different ratios of mPlum-T2A-copGFP (normal) expression vector plasmid and mPlum-T2A-copGFP(Y / H) expression vector plasmid. The integrated copGFP value for each well was corrected by the fluorescence intensity data from a well transfected with only the mPlum-T2A-copGFP(Y / H) expression vector plasmid, and the analytical value was calculated.

[0225] The results are shown in Figure 33. The analysis values ​​from the fluorescence intensity data are plotted on the vertical axis against the percentage (%) of mPlum-T2A-copGFP (normal) expression vector plasmid in the introduced plasmid on the horizontal axis. A regression line was obtained using the analysis values ​​up to 50% copGFP (normal) expression vector, and the coefficient of determination R 2This result suggests that editing efficiencies can be compared accurately, particularly up to 50%, based on the analysis of fluorescence intensity data using this method.

[0226] Example 17-8: Effect of differences in A' strand length on base sequence editing efficiency The results of Example 17-6 were reanalyzed using the method of Example 17-7, and the effect of A' strand length on editing efficiency was compared.

[0227] The results are shown in Figure 34. Similar to the results shown in Figure 32, it was confirmed that the editing efficiency was higher when an A' strand with a chain length of 35 bases or more was added to the TD compared to the editing efficiency of the TD. Furthermore, the editing efficiency was highest when an A' strand with a chain length of 65 bases was added to the TD.

[0228] Example 18-1: Effect of LNA Modification of E Strand on Base Sequence Editing Efficiency (2) The base sequence editing efficiency when an LNA modification was introduced at one site around the edited base on the E strand was examined in the same manner as in Example 1. In Example 3 (3-6), the editing efficiency when an LNA modification was introduced at specific positions symmetrical to the edited base was examined. The editing efficiency was most improved when LNA modifications were introduced at six sites: -5, -3, -1, +1, +3, and +5 (the position of the edited base on the E strand is designated as 0, the position of the base 5' to it as minus (-), and the position of the base 3' to it as plus (+)) (Figures 11 and 12). The editing nucleic acids used are shown below (the underlined nucleotides have LNA modifications): E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD3'-ODN (A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA0: SEQ ID NO: 46 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA+1: SEQ ID NO: 156 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA+2: SEQ ID NO: 1575'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA+3: SEQ ID NO: 158 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA+4: SEQ ID NO: 159 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA+5: SEQ ID NO: 160 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA+6: SEQ ID NO: 161 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1: SEQ ID NO: 162 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2: SEQ ID NO: 163 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3: SEQ ID NO: 164 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-4: SEQ ID NO: 1655'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-5: SEQ ID NO: 166 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-6: SEQ ID NO: 167 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0229] The results are shown in Figure 35. When a single LNA modification was introduced, the editing efficiency improved only when the LAN modification was introduced at the edited base position (0). Similar results were obtained when editing the E strand alone.

[0230] Example 18-2: Effect of LNA modification of E strand on base sequence editing efficiency (3) The base sequence editing efficiency when LNA modifications were introduced at two positions symmetrically around the edited base position (0) of the E strand was examined in the same manner as in Example 1. The editing nucleic acids used are shown below (the underlined nucleotides have LNA modifications):

[0231] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD3'-ODN (A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA0: SEQ ID NO: 46 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+1: SEQ ID NO: 48 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2+2: SEQ ID NO: 168 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3+3: SEQ ID NO: 169 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-4+4: SEQ ID NO: 170 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-5+5: SEQ ID NO: 1715'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-6+6: SEQ ID NO: 172 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0232] The results are shown in Figure 36. Editing efficiency was particularly improved when LNA modifications were introduced into -1 and +1. Similar results were obtained when editing the E strand alone.

[0233] Example 18-3: Effect of LNA modification of E strand on base sequence editing efficiency (4) The base sequence editing efficiency when LNA modifications were introduced at two sites, one base apart, around the edited base of the E strand was examined in the same manner as in Example 1. The editing nucleic acids used are shown below (the underlined nucleotides have LNA modifications):

[0234] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD3'-ODN (A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA-5-3: SEQ ID NO: 173 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-4-2: SEQ ID NO: 174 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3-1: SEQ ID NO: 175 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA+1+3: SEQ ID NO: 176 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA+2+4: SEQ ID NO: 177 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA+3+5: SEQ ID NO: 1785'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0235] The results are shown in Figure 37. Editing efficiency improved when LNA modifications were introduced at both positions +1 and +3. Similar results were obtained when editing the E strand alone.

[0236] Example 18-4: Effect of LNA modification of E strand on base sequence editing efficiency (5) The base sequence editing efficiency when LNA modifications were introduced at the edited base position (0) of the E strand and at one additional position was examined in the same manner as in Example 1. The editing nucleic acids used are shown below (the underlined nucleotides have LNA modifications):

[0237] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD3'-ODN (A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA0: SEQ ID NO: 46 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+1: SEQ ID NO: 179 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+2: SEQ ID NO: 180 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+3: SEQ ID NO: 181 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+4: SEQ ID NO: 182 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+5: SEQ ID NO: 1835'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+6: SEQ ID NO: 184 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+7: SEQ ID NO: 185 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+0: SEQ ID NO: 186 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2+0: SEQ ID NO: 187 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3+0: SEQ ID NO: 188 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-4+0: SEQ ID NO: 189 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-5+0: SEQ ID NO: 190 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-6+0: SEQ ID NO: 1915'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-7+0: SEQ ID NO: 192 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0238] The results are shown in Figure 38. When LNA modifications were introduced at the editing base position (0) and one additional position, editing efficiency was improved in some combinations compared to when no LNA modifications were introduced (particularly -2+0, -1+0, 0+3, 0+4, and 0+6). Editing efficiency was particularly improved when LNA modifications were introduced at positions 0 and +4 (0+4). Similar results were obtained when editing the E strand alone.

[0239] Example 18-5: Effect of LNA modification of E strand on base sequence editing efficiency (6) The base sequence editing efficiency when LNA modifications were introduced at the edited base position (0) of the E strand and at two additional positions was examined in the same manner as in Example 1. The editing nucleic acids used are shown below (LNA modifications were made to the underlined nucleotides).

[0240] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD3'-ODN (A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA0: SEQ ID NO: 46 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+0+1: SEQ ID NO: 47 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2+0+2: SEQ ID NO: 193 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3+0+3: SEQ ID NO: 194 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-4+0+4: SEQ ID NO: 195 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-5+0+5: SEQ ID NO: 1965'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-6+0+6: SEQ ID NO: 197 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-7+0+7: SEQ ID NO: 198 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0241] The results are shown in Figure 39. In some combinations, the editing efficiency was improved compared to when no LAN modifications were introduced (particularly -2 + 0 + 2, -3 + 0 + 3, -4 + 0 + 4, and -5 + 0 + 5). The editing efficiency was particularly improved when LNA modifications were introduced at positions (0), -4, and +4 of the edited base (-4 + 0 + 4). Similar results were obtained when editing the E strand alone.

[0242] Example 18-6: Effect of LNA modification of E strand on base sequence editing efficiency (7) The base sequence editing efficiency when LNA modifications were introduced into two or three consecutive bases at two positions symmetrically centered around the edited base position (0) of the E strand was examined in the same manner as in Example 1. The editing nucleic acids used are shown below (underlined nucleotides have LNA modifications):

[0243] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD3'-ODN (A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA-2-1+1+2: SEQ ID NO: 199 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3-2+2+3: SEQ ID NO: 200 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3-2-1+1+2+3: SEQ ID NO: 201 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0244] The results are shown in Figure 40. Editing efficiency decreased with each combination. Similar results were obtained when editing the E strand alone.

[0245] Example 18-7: Effect of LNA modification of E strand on base sequence editing efficiency (8) The base sequence editing efficiency when the LNA modifications that improved the editing efficiency in Examples 3 (3-6) and 18-1 to 18-6 (i.e., the edited base positions (0), -1+1, -3-1+1+3, -2+0, -1+0, 0+1, and 0+2) were combined with an LNA modification at +4 was examined in the same manner as in Example 1. The editing nucleic acids used are shown below (the underlined nucleotides have LNA modifications):

[0246] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD3'-ODN (A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA-1+1: SEQ ID NO: 48 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+1+4: SEQ ID NO: 202 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3-1+1+3: SEQ ID NO: 49 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-3-1+1+3+4: SEQ ID NO: 203 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+1: SEQ ID NO: 179 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+1+4: SEQ ID NO: 2045'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+0: SEQ ID NO: 186 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+0+4: SEQ ID NO: 205 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+2: SEQ ID NO: 180 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+2+4: SEQ ID NO: 206 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2+0: SEQ ID NO: 187 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2+0+4: SEQ ID NO: 207 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+4: SEQ ID NO: 182 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0247] The results are shown in Figure 41. Editing efficiency was improved by combining LNA modification at +4, except for -3-1+1+3.

[0248] Example 18-8: Effect of LNA Modification of E Strand on Base Sequence Editing Efficiency (9) The base sequence editing efficiency was examined in the same manner as in Example 1 when using an unmodified E strand or an E strand with an LNA modification at edited base position 0, with additional LNA modifications at positions +4, +5, +6, or +7, and an A strand of different lengths (31-35 bases) shortened by one base from the 3' end. The experimental results up to Example 18-7 showed that combining an LNA modification at the +4 position of the E strand with LNA modifications at other positions significantly improved editing efficiency. When an ODN consisting of a sequence complementary to the sequence from the 3' end of the E strand to 35 bases is used as the A strand, the +4 position of the E strand corresponds to the base adjacent to the 3' end of the A strand, i.e., the 3' end of the sequence in the E strand that does not base pair with the A strand. Therefore, the relationship between the position of the LNA modification in the E strand and the 3' end of the A strand was examined. The editing nucleic acid used is shown below (with LNA modification at the underlined nucleotides):

[0249] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD3'-ODN (A strand, 35-nt A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA0+4: SEQ ID NO: 182 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-34TD 3'-ODN (34-nt A strand): SEQ ID NO: 208 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGA -3' copGFP-153-79b-AS-33TD 3'-ODN (33-nt A strand): SEQ ID NO: 209 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTG -3' copGFP-153-79b-AS-32TD 3'-ODN (32-nt A strand): SEQ ID NO: 210 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGT -3' copGFP-153-79b-AS-31TD 3'-ODN (31-nt A chain): SEQ ID NO: 211 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACG -3' copGFP-79_AS_E LNA0+5: SEQ ID NO: 183 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+6: SEQ ID NO: 1845'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+7: SEQ ID NO: 185 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0250] The results are shown in Figure 42. In n-TD's using unmodified E strands or E strands modified with LNA at two positions (0 and +4), the editing efficiency was highest when the A strand length was 34 bases, and significantly decreased when the A strand length was 32 bases or less. Furthermore, even when A strands of different lengths were used, higher editing efficiency was obtained with n-TD's using E strands modified with LNA at two positions (0 and +4) than with n-TD's using unmodified E strands. The improvement in editing efficiency due to LNA modification of the E strand was independent of the positional relationship between the LNA modification position of the E strand and the 3' end of the A strand, but was dependent on both the LNA modification position of the E strand and the appropriate A strand length.

[0251] Example 18-9: Effect of LNA modification of E strand on base sequence editing efficiency (10) The base sequence editing efficiency by n-TD' using an E strand with LNA modifications introduced at two sites, which showed improved editing efficiency in Example 18-4, and an A strand of 33, 34, or 35 bases in length, was investigated in the same manner as in Example 1. The editing nucleic acids used are shown below (the underlined nucleotides have LNA modifications):

[0252] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD 3'-ODN (A strand, 35-nt A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-34TD 3'-ODN (34-nt A strand): SEQ ID NO: 208 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGA -3' copGFP-153-79b-AS-33TD 3'-ODN (33-nt A strand): SEQ ID NO: 209 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTG -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA0+4: SEQ ID NO: 182 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+5: SEQ ID NO: 183 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+6: SEQ ID NO: 184 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0+7: SEQ ID NO: 1855'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0253] The results are shown in Figure 43. The editing efficiency of n-TD' containing a 35- or 34-mer A strand was improved when LNA modifications were introduced into the E strand at two positions (position 0 and +4, +5, or +6) compared to when no LNA modifications were introduced. The editing efficiency was highest when an E strand with LNA modifications introduced at positions 0 and +4 was used, and the further the modification positions were (+5, +6), the lower the editing efficiency. There was no difference in the trend toward improved editing efficiency between the 35-mer A strand and the 34-mer A strand. Furthermore, the editing efficiency did not improve when an E strand with LNA modifications introduced at positions 0 and +7 was used. When a 33-mer A strand was used, the editing efficiency was improved by introducing two LNA modifications into the E strand, but there was no difference depending on the position of the LNA modifications.

[0254] Example 18-10: Effect of LNA modification of E strand on base sequence editing efficiency (11) The base sequence editing efficiency by n-TD' using the E strand, whose editing efficiency was improved in the experimental results of Example 18-7, and an A strand of 33, 34, or 35 bases in length, was investigated in the same manner as in Example 1. The editing nucleic acids used are shown below (underlined nucleotides have LNA modifications):

[0255] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD 3'-ODN (A strand, 35-nt A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-34TD 3'-ODN (34-nt A strand): SEQ ID NO: 208 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGA -3' copGFP-153-79b-AS-33TD 3'-ODN (33-nt A strand): SEQ ID NO: 209 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTG -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 copGFP-79_AS_E LNA0: SEQ ID NO: 46 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA0+4: SEQ ID NO: 182 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+1+4: SEQ ID NO: 202 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+0+4: SEQ ID NO: 2055'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2+0+4: SEQ ID NO: 207 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3'

[0256] The results are shown in Figure 44. In n-TD' containing an LNA-modified E strand, the editing efficiency improved to the same extent when the A strand was 35 bases long and when it was 34 bases long.

[0257] Example 18-11: Effect of LNA Modification of E Strand on Base Sequence Editing Efficiency (12) The effect of the LNA modification position on the base sequence editing efficiency of the copGFP gene on the editing efficiency for nonsense mutations in the DMD and WRN genes used in the experiment of Example 16 was examined. The base sequence editing efficiency of each gene by n-TD' was examined in the same manner as in Example 1. The copGFP and DMD genes were introduced into cells at a molar ratio of 10:10 E strand, 20:20 A strand, and 30:30 A' strand relative to pDNA, and the WRN gene was introduced at a molar ratio of 0.1:0.1 E strand, 0.2:0.2 A strand, and 0.3 A' strand relative to pDNA, and the editing efficiency was analyzed. The editing nucleic acids used are shown below (underlined nucleotides have LNA modifications):

[0258] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-153-79b-AS-35TD3'-ODN (A strand, 35-nt A strand): SEQ ID NO: 6 5'-CTGACCTTCAGCCCCTACCTGCTGAGCCACGTGAT -3' copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 5'-CTCGTACTTCTCGATGCGGGTGTTGGTGTAGCCGCCGTTGTTGATGGCGTGCAGGAAGGGGTTCT -3' copGFP-79_AS_E LNA0+4: SEQ ID NO: 182 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-4+0: SEQ ID NO: 189 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-1+1: SEQ ID NO: 48 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA0: SEQ ID NO: 46 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' copGFP-79_AS_E LNA-2+2: SEQ ID NO: 51 5'-CGTAGCCGCTGGGGTAGGTGCCGAAGTGGTAGAAGCCGTAGCCCATCACGTGGCTCAGCAGGTAGGGGCTGAAGGTCAG -3' DMD-79_AS_E: SEQ ID NO: 2125'-TGATTACATTAACCTGTGGATAATTACGAGTTGATTGTCGGACCCAGCTCAGGAGAATCTTTTCACTGTTGGTTTGTTG -3' DMD-35_forAS-E_A: SEQ ID NO: 213 5'-CAACAAACCAACAGTGAAAAGATTCTCCTGAGCTG -3' DMD-65_forAS-E_Aprime: SEQ ID NO: 214 5'-GCCGGGATTCTCCTCCACGTCACCGCATGTTAGAAGACTTCCTCTGCCCTCGCTGGTGGTGAAGT -3' DMD-79_AS_E LNA0+4: SEQ ID NO: 215 5'-TGATTACATTAACCTGTGGATAATTACGAGTTGATTGTCGGACCCAGCTCAGGAGAATCTTTTCACTGTTGGTTTGTTG -3' DMD-79_AS_E LNA-4+0: SEQ ID NO: 216 5'-TGATTACATTAACCTGTGGATAATTACGAGTTGATTGTCGGACCCAGCTCAGGAGAATCTTTTCACTGTTGGTTTGTTG -3' DMD-79_AS_E LNA-1+1: SEQ ID NO: 217 5'-TGATTACATTAACCTGTGGATAATTACGAGTTGATTGTCGGACCCAGCTCAGGAGAATCTTTTCACTGTTGGTTTGTTG -3' DMD-79_AS_E LNA0: SEQ ID NO: 218 5'-TGATTACATTAACCTGTGGATAATTACGAGTTGATTGTCGGACCCAGCTCAGGAGAATCTTTTCACTGTTGGTTTGTTG -3' DMD-79_AS_E LNA-2+2: SEQ ID NO: 219 5'-TGATTACATTAACCTGTGGATAATTACGAGTTGATTGTCGGACCCAGCTCAGGAGAATCTTTTCACTGTTGGTTTGTTG -3' WRN-79_AS_E: SEQ ID NO: 220 5'-CAGCAATAATCTTCTGAACCTCTGGAGTCAGGCCTGCTCGCTCCAAATCAAGGGGGCAGCCAGCTTTCACCGCTTGGGA -3' WRN-35_forAS-E_A: SEQ ID NO: 2215'-TCCCAAGCGGTGAAAGCTGGCTGCCCCCTTGATTT -3' WRN-65_forAS-E_Aprime: SEQ ID NO: 222 GCCGGGATTCTCCTCCACGTCACCGCATGTTAGAAGACTTCCTCTGCCCTCGTTTCGGATAACAT WRN-79_AS_E LNA0+4: SEQ ID NO: 223 5'-CAGCAATAATCTTCTGAACCTCTGGAGTCAGGCCTGCTCGCTCCAAATCAAGGGGGCAGCCAGCTTTCACCGCTTGGGA -3' WRN-79_AS_E LNA-4+0: SEQ ID NO: 224 5'-CAGCAATAATCTTCTGAACCTCTGGAGTCAGGCCTGCTCGCTCCAAATCAAGGGGGCAGCCAGCTTTCACCGCTTGGGA -3' WRN-79_AS_E LNA-1+1: SEQ ID NO: 225 5'-CAGCAATAATCTTCTGAACCTCTGGAGTCAGGCCTGCTCGCTCCAAATCAAGGGGGCAGCCAGCTTTCACCGCTTGGGA -3' WRN-79_AS_E LNA0: SEQ ID NO: 226 5'-CAGCAATAATCTTCTGAACCTCTGGAGTCAGGCCTGCTCGCTCCAAATCAAGGGGGCAGCCAGCTTTCACCGCTTGGGA -3' WRN-79_AS_E LNA-2+2: SEQ ID NO: 227 5'-CAGCAATAATCTTCTGAACCTCTGGAGTCAGGCCTGCTCGCTCCAAATCAAGGGGGCAGCCAGCTTTCACCGCTTGGGA -3'

[0259] The results are shown in Figure 45. For all genes, the editing efficiency was particularly improved in n-TD's containing an E strand with LNA modifications introduced at two positions, 0 and +4 or -1 and +1, compared to n-TD's containing an unmodified E strand. For the WRN gene, the editing efficiency was improved in n-TD's containing an LNA-modified E strand compared to n-TD's containing an unmodified E strand. For n-TD(E+A), the editing efficiency was improved in n-TDs containing an LNA-modified E strand compared to n-TDs containing an unmodified E strand for the DMD gene and the WRN gene.

[0260] Example 19: Examination of mutagenesis by ethyl phosphotriester bond, phosphorothioate bond, or LNA modification In the above examples, it was shown that ODN modification improves the editing efficiency of n-TD'. The possibility of mutagenesis was examined when a similarly modified polynucleotide served as a template for DNA replication in human cells. Specific experimental methods are as follows. Mutations induced by each modified polynucleotide were analyzed using the supF-NGS method (H. Kawai, R. Iwata, S. Ebi, R. Sugihara, S. Masuda, C. Fujiwara, S. Kimura, H. Kamiya. Development of a versatile high-throughput mutagenesis assay with multiplexed short-read NGS using DNA-barcoded supF shuttle vector library amplified in E. coli. Elife. 11, e83780 (2022)). The supF-NGS method uses a shuttle vector plasmid (replicable in both mammalian cells and E. coli) called pNGS2-K3 (Figure 46) containing the supF (amber suppressor tRNA) gene as a mutation detection marker and a molecular barcode (a 12-base random base sequence used for mutation data analysis). It also uses the indicator strain E. coli RF01 (R. Fukushima, T. Suzuki, H. Kamiya. New indicator Escherichia coli strain for rapid and accurate detection of supF mutations. Genes Environ. 42, 28 (2020)). This method analyzes the frequency and frequency of mutations in the supF gene sequence using next-generation sequencing (NGS). First, five ODNs (SEQ ID NOs: 228–232) with 5′-phosphorylated (Phos-) modifications were extracted from pNGS2-K3 as single-stranded circular DNA. The modifications are underlined.) was used to construct double-stranded circular DNA by in vitro enzymatic reaction (R. Fukushima, T. Suzuki, Y. Komatsu, H. Kamiya. Biased distribution of action-at-a-distance mutations by 8-oxo-7,8-dihydroguanine. Mutation Res. (Fundam. Mol. Mech. Mutagen.), 825, 111794 (2022)). SupF top_strand: SEQ ID NO: 228 5'-Phos-GAGCAGACTCTAAATCTGCCG -3' SupF top_strand p-etho (ethyl phosphotriester bond): SEQ ID NO: 229 5'-Phos-GAGCAGACTCTAAATCTGCCG -3' SupF top_strand S (phosphorothioate bond): SEQ ID NO: 230 5'-Phos-GAGCAGACTCTAAATCTGCCG -3' SupF top_strand LNA-1+1: SEQ ID NO: 231 5'-Phos-GAGCAGACTCTAAATCTGCCG -3' SupF top_strand LNA-1+1 T>C: SEQ ID NO: 232 5'-Phos-GAGCAGACTCCAAATCTGCCG -3' A control experiment, pNGS2-K3(Ctrl), was constructed using the unmodified ODN of SEQ ID NO: 228. Plasmids (pNGS2-K3(P-OEt), pNGS2-K3(PS), and pNGS2-K3(LNA-1+1)) were constructed by introducing modifications into the supF gene sequence: an ethyl phosphotriester bond with the ODN of SEQ ID NO: 229, a phosphorothioate bond with the ODN of SEQ ID NO: 230, and two LNA modifications with the ODN of SEQ ID NO: 231. pNGS2-K3(LNA-1+1 T>C) was also constructed using the ODN of SEQ ID NO: 232, with a C:A mismatch between the two LNA modifications. Each double-stranded circular DNA (400 ng) constructed was then transfected into U2OS cells (5 x 10) in a 6-well plate. 5The plasmids were transfected into RF01 cells (cells / well, seeded the day before) using Lipofectamine 2000. After 48 hours of culture, the plasmids replicated from the double-stranded circular DNA within the cells were recovered and transfected into RF01. RF01 was plated on a Kanamycin-containing LB plate, and plasmids were extracted from the colonies that formed. Sequence information for the supF gene was obtained using the supF-NGS method, and the mutant frequency was calculated.

[0261] The results are shown in Figure 47. The average of the mutant frequencies of the supF gene from triplicate experiments was 6.73 x 10 for pNGS2-K3(Ctrl), pNGS2-K3(P-OEt), pNGS2-K3(PS), and pNGS2-K3(LNA-1+1). -3 , 6.67×10 -3 , 7.04×10 -3 and 5.41 × 10 -3 These modifications did not significantly induce mutations. In addition, the average mutant frequency of pNGS2-K3 (LNA-1+1 T>C) was 5.52 × 10 -1 Of which, 5.49 × 10 -1 The mutation was a T to C mutation resulting from a C:A mismatch between the LNA modifications. This result suggests that the DNA strand containing the two LNA modifications in pNGS2-K3 and the unmodified DNA strand were replicated with almost the same efficiency, suggesting that the LNA modifications may not inhibit DNA replication. Furthermore, the average mutant frequency excluding the T to C mutation (LNA-1+1 T>C w / oC) was 8.12 × 10 -3 This indicates that even when mismatched base pairs for editing are present, LNA modification on both sides of the mismatched base pair does not significantly induce mutations.

[0262] Example 20: Improvement of base sequence editing efficiency by combining LNA modification of E strand and P-OEt modification of A strand (2) The LNA modification of E strand, which showed an improvement in editing efficiency, was combined with P-OEt modification of A strand to examine the base sequence editing efficiency in the same manner as in Example 1. The editing nucleic acids used are shown below.

[0263] E strand copGFP-153-79b-AS (AS_E strand): SEQ ID NO: 5 copGFP-153-79b-AS-35TD3'-ODN (A strand, 35-nt A strand): SEQ ID NO: 6 copGFP_forAS_E79 A35 3' p-etho ×1: SEQ ID NO: 44 copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' strand for AS_E strand): SEQ ID NO: 7 copGFP-79_AS_E LNA-1+1: SEQ ID NO: 48 copGFP-79_AS_E LNA-1+1+4: SEQ ID NO: 202 copGFP-79_AS_E LNA-1+0: SEQ ID NO: 186 copGFP-79_AS_E LNA-1+0+4: SEQ ID NO: 205 copGFP-79_AS_E LNA-2+0: SEQ ID NO: 187 copGFP-79_AS_E LNA-2+0+4: SEQ ID NO: 207 copGFP-79_AS_E LNA0: SEQ ID NO: 46 copGFP-79_AS_E LNA0+4: SEQ ID NO: 182

[0264] The results are shown in Figure 48. In all cases where LNA modification was introduced into the E strand, a tendency for the editing efficiency to be additively improved by combining it with P-OEt modification of the A strand was observed.

[0265] According to the present invention, it is possible to efficiently edit a target base sequence in genomic DNA without using artificial nucleases. Therefore, the present invention can be used for genome editing in the fields of cell engineering, biotechnology, medicine, etc. In particular, the present invention can be used for the treatment or prevention of diseases caused by gene mutations.

[0266] SEQ ID NO: 1: nucleotide sequence of mPlum-T2A-copGFP(Y / H) (sequence containing mutant copGFP gene) SEQ ID NO: 2: amino acid sequence of mPlum-T2A-copGFP(Y / H) SEQ ID NO: 3: nucleotide sequence of mPlum-T2A-mEGFP(Y / H) (sequence containing mutant mEGFP gene) SEQ ID NO: 4: amino acid sequence of mPlum-T2A-mEGFP(Y / H) SEQ ID NO: 5: copGFP-153-79b-AS (AS_E chain) SEQ ID NO: 6: copGFP-153-79b-AS-35TD3'-ODN (A chain for AS_E chain) SEQ ID NO: 7: copGFP-153-79b-AS-65TDdown-ODN (alias: copGFP_A_prime65) (A' chain for AS_E chain) SEQ ID NO: 8: mEGFP-79_AS_E SEQ ID NO: 9: mEGFP-35_S_A SEQ ID NO: 10: mEGFP-65_AS_Ap SEQ ID NO: 11: copGFP-79_AS_E 5'3'S SEQ ID NO: 12: copGFP-79_AS_E 5'S SEQ ID NO: 13: copGFP-79_AS_E 3'S SEQ ID NO: 14: copGFP-79_AS_E 5'3'SS SEQ ID NO: 15: copGFP-79_AS_E 5'SS SEQ ID NO: 16: copGFP -79_AS_E 3'SS SEQ ID NO: 17: copGFP-35_S_A 5'3'S SEQ ID NO: 18: copGFP-35_S_A 5'S SEQ ID NO: 19: copGFP-35_S_A 3'S SEQ ID NO: 20: copGFP-35_S_A 5'3'SS SEQ ID NO: 21: copGFP-35_S_A 5'SS SEQ ID NO:22: copGFP-35_S_A 3'SS SEQ ID NO:23: copGFP-79_AS_Ap 5'3'S SEQ ID NO:24: copGFP-79_AS_Ap 5'S SEQ ID NO:25: copGFP-79_AS_Ap 3'S SEQ ID NO:26: copGFP-79_AS_Ap 5'3'SS SEQ ID NO:27: copGFP-79_AS_Ap 5'SS SEQ ID NO:28: copGFP-79_AS_Ap 3'SS SEQ ID NO:29: copGFP-79_AS_E 3'SSS SEQ ID NO:30: copGFP-79_AS_E 3'SSSS SEQ ID NO:31: copGFP-35_S_A 3'SSS SEQ ID NO:32: copGFP-35_S_A 3'SSSSSEQ ID NO: 33: copGFP-79_AS_E 3'SSp11 SEQ ID NO: 34: copGFP-79_AS_E 3'SSp21 SEQ ID NO: 35: copGFP-79_AS_E 3'SSp31 SEQ ID NO: 36: copGFP-79_AS_E 3'SSp41 SEQ ID NO: 37: copGFP-79_AS_E 3'SSp51 SEQ ID NO: 38: copGFP-79_AS_E 3'SSp61 SEQ ID NO: 39: copGFP-79_AS_E 3'SSp71 SEQ ID NO: 40: copGFP_forAS_E79 A35 5' p-met x 2 SEQ ID NO: 41: copGFP_forAS_E79 A35 3' p-met x 2 SEQ ID NO: 42: copGFP_forAS_E79 A35 5' p-etho x 2 SEQ ID NO: 43: copGFP_forAS_E79 A35 3' p-etho x 2 SEQ ID NO: 44: copGFP_forAS_E79 A35 3' p-etho x 1 SEQ ID NO: 45: copGFP_forAS_E79 A35 3' p-etho x 2 SEQ ID NO: 46: copGFP-79_AS_E LNA0 SEQ ID NO: 47: copGFP-79_AS_E LNA-1+0+1 SEQ ID NO: 48: copGFP-79_AS_E LNA-1+1 SEQ ID NO: 49: copGFP-79_AS_E LNA-3-1+1+3 SEQ ID NO: 50: copGFP-79_AS_E LNA-5-3-1+1+3+5 SEQ ID NO: 51: copGFP-79_AS_E LNA-2+2 SEQ ID NO: 52: copGFP-79_AS_E LNA-2-1+1+2 SEQ ID NO: 53: copGFP-79_AS_E LNA-3-2+2+3 SEQ ID NO: 54: copGFP-79_AS_E LNA-3-2-1+1+2+3 SEQ ID NO: 55: copGFP-79_AS_E LNA-7-5-3-1+1+3+5+7 SEQ ID NO: 56: copGFP-79_AS_E LNA-9-7-5-3-1+1+3+5+7+9 SEQ ID NO: 57: mEGFP-79_AS_E LNA-2,0 SEQ ID NO: 58: mEGFP-35_S_A 3' p-etho×1 SEQ ID NO: 59: mEGFP-65_AS_Ap SEQ ID NO: 60: 65Ap_AS_Gap1 SEQ ID NO: 61: 65Ap_AS_Gap2 SEQ ID NO: 62: 65Ap_AS_Gap3SEQ ID NO: 63: 65Ap_AS_Gap6 SEQ ID NO: 64: 65Ap_AS_Gap9 SEQ ID NO: 65: copGFP-79_AS_Ap 3-overlap SEQ ID NO: 66: copGFP-79_AS_Ap 6-overlap SEQ ID NO: 67: copGFP-79_AS_Ap 9-overlap SEQ ID NO: 68: copGFP-79_AS_Ap 12-overlap SEQ ID NO: 69: copGFP-79_AS_Ap 15-overlap SEQ ID NO: 70: copGFP-79_AS_Ap 19-overlap SEQ ID NO: 71: copGFP-79_AS_Ap 29-overlap SEQ ID NO: 72: copGFP-79_AS_Ap 39-overlap SEQ ID NO: 73: copGFP-153-79b-AS-45TD3'-ODN5' SEQ ID NO: 74: copGFP-153-79b-AS-55TD3'-ODN5' SEQ ID NO: 75: copGFP-153-79b-AS-65TD3'-ODN5' SEQ ID NO: 76: copGFP-153-79b-AS-75TD3'-ODN5' SEQ ID NO: 77: copGFP-153-79b-AS-45TD3'-ODN3' SEQ ID NO: 78: copGFP-153-79b-AS-55TD3'-ODN3' SEQ ID NO: 79: copGFP-153-79b-AS-65TD3'-ODN3' SEQ ID NO: 80: copGFP-153-79b-AS-75TD3'-ODN3' SEQ ID NO: 81: copGFP-153-79b-AS-79TD3'-ODN3' SEQ ID NO: 82: 79E_AS_45TD3'-ODN3' SEQ ID NO: 83: 79E_AS_55TD3'-ODN3' SEQ ID NO: 84: 79E_AS_65TD3'-ODN3' SEQ ID NO: 85: 79E_AS_75TD3'-ODN3' SEQ ID NO: 86: 79E_AS_79TD3'-ODN3' SEQ ID NO: 87: 79GFP_AS_5'-1-mut(+44-WT) SEQ ID NO: 88: 35mer_for_79GFP_AS_5'-1-mut(+44-WT) SEQ ID NO: 89: 65Ap_for_79E_AS_5'-1-mut(+44-WT) SEQ ID NO: 90: 79GFP_AS_5'-11-mut(+34-WT) SEQ ID NO: 91: 35mer_for_79GFP_AS_5'-11-mut(+34-WT)SEQ ID NO: 92: 65Ap_for_79E_AS_5'-11-mut(+34-WT) SEQ ID NO: 93: 79GFP_AS_5'-21-mut(+24-WT) SEQ ID NO: 94: 35mer_for_79GFP_AS_5'-21-mut(+24-WT) SEQ ID NO: 95: 65Ap_for_79E_AS_5'-21-mut(+24-WT) SEQ ID NO: 96: 79GFP_AS_5'-31-mut(+14-WT) SEQ ID NO: 97: 35mer_for_79GFP_AS_5'-31-mut(+14-WT) SEQ ID NO: 98: 65Ap_for_79E_AS_5'-31-mut(+14-WT) SEQ ID NO: 99: 79GFP_AS_5'-41-mut(+4-WT) SEQ ID NO: 100: 35mer_for_79GFP_AS_5'-41-mut(+4-WT) SEQ ID NO: 101: 65Ap_for_79E_AS_5'-41-mut(+4-WT) SEQ ID NO: 102: 79GFP_AS_5'-44-mut(+1-WT) SEQ ID NO: 103: 35mer_for_79GFP_AS_5'-44-mut(+1-WT) SEQ ID NO: 104: 65Ap_for_79E_AS_5'-44-mut(+1-WT) SEQ ID NO: 105: 79GFP_AS_5'-51-mut(-6) SEQ ID NO: 106: 35mer_for_79GFP_AS_5'-51-mut(-6-WT) SEQ ID NO: 107: 35A_AS_5'-51-mut(-6-YH) SEQ ID NO: 108: 65Ap_for_79E_AS_5'-51-mut(-6) SEQ ID NO: 109: 79GFP_AS_5'-61-mut(-16) SEQ ID NO: 110: 35mer_for_79GFP_AS_5'-61-mut(-16-WT) SEQ ID NO: 111: 35A_AS_5'-61-mut(-16-YH) SEQ ID NO: 112: 65Ap_for_79E_AS_5'-61-mut(-16) SEQ ID NO: 113: 79GFP_AS_5'-71-mut(-26) SEQ ID NO: 114: 35mer_for_79GFP_AS_5'-71-mut(-26-WT) SEQ ID NO: 115: 35A_AS_5'-71-mut(-26-YH) SEQ ID NO: 116: 65Ap_for_79E_AS_5'-71-mut(-26) SEQ ID NO: 117: 79GFP_AS_5'-79-mut(-34)SEQ ID NO:118: 35mer_for_79GFP_AS_5'-79-mut(-34-WT) SEQ ID NO:119: 35A_AS_5'-79-mut(-34-YH) SEQ ID NO:120: 65Ap_for_79E_AS_5'-79-mut(-34) SEQ ID NO:121: 144E_AS_5'-105-mut SEQ ID NO:122: 109E_AS_5'-70-mut_for_35Ap SEQ ID NO:123: 35Ap_for_109E_AS_5'-70-mut SEQ ID NO:124: 65E_AS_5'-26-mut_for_79Ap SEQ ID NO:125: 79Ap_for_65E_AS_5'-26-mut SEQ ID NO:126: 35E_AS_notmut_for_109Ap SEQ ID NO: 127: 109Ap_5'-105-mut_for_35E_AS_notmut SEQ ID NO: 128: copGFP-79_AS_E LNA-1+1_5'PS SEQ ID NO: 129: copGFP_A_prime65_5'PS SEQ ID NO: 130: mEGFP-79_AS_E LNA-1+1 SEQ ID NO: 131: copGFP-79_AS_E ENA-1+1 SEQ ID NO: 132: Nonsense mutation sequence of DMD gene SEQ ID NO: 133: Nonsense mutation sequence of WRN gene SEQ ID NO: 134: Nonsense mutation sequence of HBB gene SEQ ID NO: 135: Nonsense mutation sequence of ADA gene SEQ ID NO: 136: Nonsense mutation sequence of LCA gene SEQ ID NO: 137: Nonsense mutation sequence of PRKN gene SEQ ID NO: 138: Nonsense mutation sequence of BRAF gene SEQ ID NO: 139: Nonsense mutation sequence of NRAS gene SEQ ID NO: 140: copGFP-153-79b-S-35TDup-ODN (AS_Up_O) SEQ ID NO: 141: copGFP-153-79b-S-35TD5'-ODN (AS_Up_T) SEQ ID NO: 142: copGFP-153-79b-S-35TDcn-ODN (AS_Cnt_Y) SEQ ID NO: 143: copGFP-153-79b-S-35TDcnYH-ODN (AS_Cnt_H) SEQ ID NO: 144: copGFP-153-79b-S-35TD3'-ODN (AS_Dwn_T) SEQ ID NO: 145: copGFP-153-79b-S-35TDdown-ODN (AS_Dwn_O)SEQ ID NO: 146: copGFP-153-79b-AS-35TDup-ODN (S_Up_O) SEQ ID NO: 147: copGFP-153-79b-AS-35TDcn-ODN (S_Cnt_Y) SEQ ID NO: 148: copGFP-153-79b-AS-35TDcnYH-ODN (S_Cnt_H) SEQ ID NO: 149: copGFP-153-79b-AS-35TD5'-ODN (S_Dwn_T) SEQ ID NO: 150: copGFP-153-79b-AS-35TDdown-ODN (S_Dwn_O) SEQ ID NO: 151: copGFP_A_prime15 SEQ ID NO: 152: copGFP_A_prime25 SEQ ID NO: 153: copGFP_A_prime45 SEQ ID NO: 154: copGFP_A_prime55 SEQ ID NO: 155: copGFP_A_prime75 SEQ ID NO: 156: copGFP-79_AS_E LNA+1 SEQ ID NO: 157: copGFP-79_AS_E LNA+2 SEQ ID NO: 158: copGFP-79_AS_E LNA+3 SEQ ID NO: 159: copGFP-79_AS_E LNA+4 SEQ ID NO: 160: copGFP-79_AS_E LNA+5 SEQ ID NO: 161: copGFP-79_AS_E LNA+6 SEQ ID NO: 162: copGFP-79_AS_E LNA-1 SEQ ID NO: 163: copGFP-79_AS_E LNA-2 SEQ ID NO: 164: copGFP-79_AS_E LNA-3 SEQ ID NO: 165: copGFP-79_AS_E LNA-4 SEQ ID NO: 166: copGFP-79_AS_E LNA-5 SEQ ID NO: 167: copGFP-79_AS_E LNA-6 SEQ ID NO: 168: copGFP-79_AS_E LNA-2+2 SEQ ID NO: 169: copGFP-79_AS_E LNA-3+3 SEQ ID NO: 170: copGFP-79_AS_E LNA-4+4 SEQ ID NO: 171: copGFP-79_AS_E LNA-5+5 SEQ ID NO: 172: copGFP-79_AS_E LNA-6+6 SEQ ID NO: 173: copGFP-79_AS_E LNA-5-3 SEQ ID NO: 174: copGFP-79_AS_E LNA-4-2 SEQ ID NO: 175: copGFP-79_AS_E LNA-3-1 SEQ ID NO: 176: copGFP-79_AS_E LNA+1+3SEQ ID NO: 177: copGFP-79_AS_E LNA+2+4 SEQ ID NO: 178: copGFP-79_AS_E LNA+3+5 SEQ ID NO: 179: copGFP-79_AS_E LNA0+1 SEQ ID NO: 180: copGFP-79_AS_E LNA0+2 SEQ ID NO: 181: copGFP-79_AS_E LNA0+3 SEQ ID NO: 182: copGFP-79_AS_E LNA0+4 SEQ ID NO: 183: copGFP-79_AS_E LNA0+5 SEQ ID NO: 184: copGFP-79_AS_E LNA0+6 SEQ ID NO: 185: copGFP-79_AS_E LNA0+7 SEQ ID NO: 186: copGFP-79_AS_E LNA-1+0 SEQ ID NO: 187: copGFP-79_AS_E LNA-2+0 SEQ ID NO: 188: copGFP-79_AS_E LNA-3+0 SEQ ID NO: 189: copGFP-79_AS_E LNA-4+0 SEQ ID NO: 190: copGFP-79_AS_E LNA-5+0 SEQ ID NO: 191: copGFP-79_AS_E LNA-6+0 SEQ ID NO: 192: copGFP-79_AS_E LNA-7+0 SEQ ID NO: 193: copGFP-79_AS_E LNA-2+0+2 SEQ ID NO: 194: copGFP-79_AS_E LNA-3+0+3 SEQ ID NO: 195: copGFP-79_AS_E LNA-4+0+4 SEQ ID NO: 196: copGFP-79_AS_E LNA-5+0+5 SEQ ID NO: 197: copGFP-79_AS_E LNA-6+0+6 SEQ ID NO: 198: copGFP-79_AS_E LNA-7+0+7 SEQ ID NO: 199: copGFP-79_AS_E LNA-2-1+1+2 SEQ ID NO: 200: copGFP-79_AS_E LNA-3-2+2+3 SEQ ID NO: 201: copGFP-79_AS_E LNA-3-2-1+1+2+3 SEQ ID NO: 202: copGFP-79_AS_E LNA-1+1+4 SEQ ID NO: 203: copGFP-79_AS_E LNA-3-1+1+3+4 SEQ ID NO: 204: copGFP-79_AS_E LNA0+1+4 SEQ ID NO: 205: copGFP-79_AS_E LNA-1+0+4 SEQ ID NO: 206: copGFP-79_AS_E LNA0+2+4 SEQ ID NO: 207: copGFP-79_AS_E LNA-2+0+4SEQ ID NO: 208: copGFP-153-79b-AS-34TD3'-ODN SEQ ID NO: 209: copGFP-153-79b-AS-33TD3'-ODN SEQ ID NO: 210: copGFP-153-79b-AS-32TD3'-ODN SEQ ID NO: 211: copGFP-153-79b-AS-31TD3'-ODN SEQ ID NO: 212: DMD-79_AS_E SEQ ID NO: 213: DMD-35_forAS-E_A SEQ ID NO: 214: DMD-65_forAS-E_Aprime SEQ ID NO: 215: DMD-79_AS_E LNA0+4 SEQ ID NO: 216: DMD-79_AS_E LNA-4+0 SEQ ID NO: 217: DMD-79_AS_E LNA-1+1 SEQ ID NO: 218: DMD-79_AS_E LNA0 SEQ ID NO: 219: DMD-79_AS_E LNA-2+2 SEQ ID NO: 220: WRN-79_AS_E SEQ ID NO: 221: WRN-35_forAS-E_A SEQ ID NO: 222: WRN-65_forAS-E_Aprime SEQ ID NO: 223: WRN-79_AS_E LNA0+4 SEQ ID NO: 224: WRN-79_AS_E LNA-4+0 SEQ ID NO: 225: WRN-79_AS_E LNA-1+1 SEQ ID NO: 226: WRN-79_AS_E LNA0 SEQ ID NO: 227: WRN-79_AS_E LNA-2+2 SEQ ID NO: 228: SupF top_strand SEQ ID NO: 229: SupF top_strand p-etho SEQ ID NO: 230: SupF top_strand S SEQ ID NO: 231: SupF top_strand LNA-1+1 SEQ ID NO: 232: SupF top_strand LNA-1+1 T>C

[0267] Phosphorothioate bond (as modified internucleotide bond) PCH ３ Modification (as modified internucleotide bond) POCH ２ CH ３ Modifications (as modified internucleotide linkages) 2'-O,4'-C-methylene-bridged nucleic acids (as sugar-modified nucleotides) 2'-O,4'-C-ethylene-bridged nucleic acids (as sugar-modified nucleotides)

Claims

1. A composition comprising a first single-stranded polynucleotide and a third single-stranded polynucleotide, wherein the first single-stranded polynucleotide is 50-200 bases long, its base sequence has 80% or more sequence identity with the base sequence of a portion of an editing target strand of a target region present in double-stranded DNA in a cell, and compared to the base sequence of the editing target strand, contains at least one editing mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides; and the third single-stranded polynucleotide is 15-200 bases long, its base sequence has 90% or more sequence identity with a base sequence in the editing target strand that is located 5'-terminal or 3'-terminal of a position in the first single-stranded polynucleotide corresponding to the editing mutation, and wherein, in the editing target strand, the base sequence having sequence identity with the first single-stranded polynucleotide and the base sequence having sequence identity with the third single-stranded polynucleotide overlap, are adjacent, or are separated by 1-9 bases.

2. The composition of claim 1, wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 25 to 75% from the 5' end, with the 5' end to the 3' end being 100%.

3. The composition of claim 2, wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 49 to 51% from the 5' end, with the 5' end to the 3' end being 100%.

4. The composition according to any one of claims 1 to 3, wherein the first single-stranded polynucleotide is 70 to 100 bases in length.

5. The composition according to any one of claims 1 to 4, characterized in that the first single-stranded polynucleotide consists of a base sequence that is 100% identical to the corresponding base sequence in the editing target strand in the sequence excluding the position of the editing mutation and a region of 10 bases from the 5' end and a region of 10 bases from the 3' end.

6. The composition according to any one of claims 1 to 5, wherein the base sequence of the third single-stranded polynucleotide has 90% or more sequence identity with the base sequence of the first single-stranded polynucleotide on the 5'-terminal side of the position corresponding to the editing mutation in the editing target strand.

7. The composition of any one of claims 1 to 6, wherein in the editing target strand, a base sequence having sequence identity to a first single-stranded polynucleotide and a base sequence having sequence identity to a third single-stranded polynucleotide are adjacent to each other.

8. The composition of any one of claims 1 to 7, wherein the third single-stranded polynucleotide is 55 to 75 bases in length.

9. The composition according to any one of claims 1 to 8, characterized in that the third single-stranded polynucleotide consists of a base sequence that is 100% identical to the corresponding base sequence of the editing target strand.

10. The composition according to any one of claims 1 to 9, further comprising a second single-stranded polynucleotide, the second single-stranded polynucleotide having a length of 20 to 200 bases, comprising a base sequence complementary to a base sequence of a portion of the region of the first single-stranded polynucleotide that does not contain the editing mutation, and capable of forming a double strand with the first single-stranded polynucleotide.

11. The composition of claim 10, wherein the second single-stranded polynucleotide comprises a base sequence complementary to a portion of the base sequence of the first single-stranded polynucleotide on the 3'-terminal side relative to the position of the editing mutation.

12. The composition of claim 10 or 11, wherein the molar concentration of the third single-stranded polynucleotide is the same as or higher than the molar concentration of the second single-stranded polynucleotide.

13. The composition of any one of claims 1 to 12, wherein the target region contains an aberrant base sequence that causes a disease, and the base sequence of the first single-stranded polynucleotide contains the editing mutation such that the aberrant base sequence can be repaired, thereby treating or preventing the disease.

14. A pharmaceutical composition for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell, comprising the composition according to claim 13.

15. A method for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell, comprising administering to a subject a therapeutically or prophylactically effective amount of the composition described in claim 13.

16. A method for producing an isolated cell into which a mutation has been introduced or an organism having a cell into which a mutation has been introduced, the method comprising the step of introducing a composition according to any one of claims 1 to 13 into the isolated cell or into a cell constituting the living body of the organism, thereby introducing a mutation into the base sequence of a target region present in double-stranded DNA in the cell.

17. A composition comprising a first single-stranded polynucleotide, the first single-stranded polynucleotide being 50 to 200 bases in length, the base sequence of which has 80% or more sequence identity with the base sequence of a portion of the editing target strand of a target region present in double-stranded DNA in a cell, and which, compared to the base sequence of the editing target strand, contains at least one editing mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides, and which has at least one of the following characteristics: (a) at least one nucleotide is a sugar-modified nucleotide in the position of the editing mutation in the first single-stranded polynucleotide and in a region consisting of 10 bases on the 5' side and 10 bases on the 3' side of the position of the editing mutation, and (b) the first single-stranded polynucleotide contains at least one modified internucleotide bond, which is not present within a range from 3 bases on the 5' end to 3 bases on the 3' end of the position of the editing mutation.

18. The composition of claim 17, wherein the sugar-modified nucleotide is a nucleotide containing a 2'-O,4'-C-methylene or 2'-O,4'-C-ethylene bridge in the ribose ring.

19. The composition of claim 17 or 18, wherein the first single-stranded polynucleotide comprises 1 to 4 modified internucleotide bonds within 13 bases from the 5' end and / or the 3' end.

20. The composition of any one of claims 17 to 19, wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 25 to 75% from the 5' end, with the 5' end to the 3' end being 100%.

21. The composition of claim 20, wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 49 to 51% from the 5' end, with the 5' end to the 3' end being 100%.

22. The composition of any one of claims 17 to 21, wherein the first single-stranded polynucleotide is 70 to 100 bases in length.

23. The composition according to any one of claims 17 to 22, further comprising a second single-stranded polynucleotide, the second single-stranded polynucleotide having a length of 20 to 200 bases, comprising a base sequence complementary to a base sequence of a portion of a region of the first single-stranded polynucleotide that does not contain the editing mutation, and capable of forming a duplex with the first single-stranded polynucleotide.

24. The composition of claim 23, wherein the second single-stranded polynucleotide comprises at least one modified internucleotide bond.

25. The composition of claim 24, wherein the second single-stranded polynucleotide comprises one or two modified internucleotide linkages within 3 bases or one within 2 bases from the 5'-terminus and / or 3'-terminus.

26. The composition according to any one of claims 23 to 25, wherein the second single-stranded polynucleotide comprises a base sequence complementary to a portion of the base sequence of the first single-stranded polynucleotide on the 3'-terminal side relative to the position of the editing mutation.

27. The composition of any one of claims 17, 19, 24 or 25, wherein the modified internucleotide linkage is a phosphorothioate linkage, an alkylphosphonate linkage, or a phosphotriester linkage.

28. The composition according to any one of claims 17 to 27, further comprising a third single-stranded polynucleotide, the third single-stranded polynucleotide being 15 to 75 bases in length and having a base sequence that has 90% or more sequence identity with a base sequence in the editing target strand that is 5' or 3' terminal of a position in the first single-stranded polynucleotide corresponding to the editing mutation, and wherein, in the editing target strand, the base sequence that has sequence identity with the first single-stranded polynucleotide and the base sequence that has sequence identity with the third single-stranded polynucleotide overlap, are adjacent, or are separated by 1 to 9 bases.

29. The composition described in claim 28, wherein the base sequence of the third single-stranded polynucleotide has a sequence identity of 90% or more with the base sequence of the first single-stranded polynucleotide on the 5'-terminal side of the position corresponding to the editing mutation in the editing target strand.

30. The composition of claim 28 or 29, wherein in the editing target strand, a base sequence having sequence identity to a first single-stranded polynucleotide and a base sequence having sequence identity to a third single-stranded polynucleotide are adjacent.

31. The composition of any one of claims 28 to 30, wherein the third single-stranded polynucleotide is 55 to 75 bases in length.

32. The composition according to any one of claims 28 to 31, wherein the third single-stranded polynucleotide consists of a base sequence that is 100% identical to the corresponding base sequence of the editing target strand.

33. The composition of any one of claims 28 to 32, wherein the molar concentration of the third single-stranded polynucleotide is the same as or higher than the molar concentration of the second single-stranded polynucleotide.

34. The composition of any one of claims 17 to 33, wherein the target region contains an aberrant base sequence that causes a disease, and the base sequence of the first single-stranded polynucleotide contains the editing mutation such that the aberrant base sequence can be repaired, thereby treating or preventing the disease.

35. A pharmaceutical composition for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell, comprising the composition according to claim 34.

36. A method for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell, comprising administering to a subject a therapeutically or prophylactically effective amount of the composition described in claim 34.

37. A method for producing an isolated cell into which a mutation has been introduced, or an organism having a cell into which a mutation has been introduced, comprising the step of introducing a composition according to any one of claims 17 to 34 into the isolated cell or into a cell constituting the living body of the organism, thereby introducing a mutation into the base sequence of a target region present in double-stranded DNA in the cell.

38. A pharmaceutical composition comprising a third single-stranded polynucleotide for use in combination with a composition comprising a first single-stranded polynucleotide, wherein the first single-stranded polynucleotide is 50 to 200 bases long, and its base sequence has 80% or more sequence identity with the base sequence of a portion of the editing target strand of a target region present in double-stranded DNA in a cell, and compared to the base sequence of the editing target strand, contains at least one editing mutation selected from the group consisting of deletion, substitution, and insertion of one or more nucleotides; the third single-stranded polynucleotide is 15 to 200 bases long, and its base sequence has 90% or more sequence identity with the base sequence in the editing target strand located 5'-terminal or 3'-terminal of the position in the first single-stranded polynucleotide corresponding to the editing mutation, wherein, in the editing target strand, the base sequence having sequence identity with the first single-stranded polynucleotide and the base sequence having sequence identity with the third single-stranded polynucleotide overlap, are adjacent, or are separated by 1 to 9 bases; The target region contains an abnormal base sequence that causes a disease, and the base sequence of the first single-stranded polynucleotide contains the editing mutation such that the abnormal base sequence can be repaired, thereby treating or preventing the disease.

39. The pharmaceutical composition described in claim 38, wherein the base sequence of the third single-stranded polynucleotide has a sequence identity of 90% or more with the base sequence of the first single-stranded polynucleotide on the 5'-terminal side of the position corresponding to the editing mutation in the editing target strand.

40. The pharmaceutical composition of claim 38 or 39, wherein in the editing target strand, a base sequence having sequence identity to a first single-stranded polynucleotide and a base sequence having sequence identity to a third single-stranded polynucleotide are adjacent.

41. The pharmaceutical composition of any one of claims 38 to 40, wherein the third single-stranded polynucleotide is 55 to 75 bases in length.

42. The composition of any one of claims 38 to 41, wherein the third single-stranded polynucleotide consists of a base sequence that is 100% identical to the corresponding base sequence of the editing target strand.

43. The pharmaceutical composition of any one of claims 38 to 42, wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 25 to 75% from the 5' end, with the 5' end to the 3' end being 100%.

44. The pharmaceutical composition of claim 43, wherein the position of the editing mutation in the first single-stranded polynucleotide is within a range of 49 to 51% from the 5' end, with the 5' end to the 3' end being 100%.

45. The pharmaceutical composition of any one of claims 38 to 44, wherein the first single-stranded polynucleotide is 70 to 100 bases in length.

46. ​​The composition described in any one of claims 38 to 45, characterized in that the first single-stranded polynucleotide consists of a base sequence that is 100% identical to the corresponding base sequence in the editing target strand in a sequence excluding the position of the editing mutation and a region of 10 bases from the 5' end and a region of 10 bases from the 3' end.

47. A pharmaceutical composition according to any one of claims 38 to 46, wherein the composition comprising a first single-stranded polynucleotide further comprises a second single-stranded polynucleotide, the second single-stranded polynucleotide having a length of 20 to 200 bases, comprising a base sequence complementary to a base sequence of a portion of the region of the first single-stranded polynucleotide that does not contain the editing mutation, and capable of forming a duplex with the first single-stranded polynucleotide.

48. The pharmaceutical composition of claim 47, wherein the second single-stranded polynucleotide comprises a base sequence complementary to a portion of the base sequence of the first single-stranded polynucleotide on the 3'-terminal side relative to the position of the editing mutation.

49. The pharmaceutical composition of claim 47 or 48, wherein the molar concentration of the third single-stranded polynucleotide is used to be the same as or higher than the molar concentration of the second single-stranded polynucleotide.

50. A pharmaceutical composition according to any one of claims 38 to 49, for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell.

51. A method for treating or preventing a disease caused by an abnormal base sequence contained in a target region present in double-stranded DNA in a cell, comprising administering to a subject a therapeutically or prophylactically effective amount of the pharmaceutical composition described in claim 50 simultaneously, separately or successively with a composition containing a first single-stranded polynucleotide.

52. A method for producing an isolated cell into which a mutation has been introduced or an organism having a cell into which a mutation has been introduced, the method comprising the step of introducing into the isolated cell or into a cell constituting the living body of the organism the pharmaceutical composition according to any one of claims 38 to 50 simultaneously, separately or sequentially with a composition comprising a first single-stranded polynucleotide, thereby introducing a mutation into the base sequence of a target region present in double-stranded DNA in the cell.