Crispr-cas nickase system-based precise genome editing method

Abstract

The present invention relates to a CRISPR-Cas nickase system-based precise genome editing method. The genome editing method of the present invention can improve the precision of genome editing by reducing off-target effects, and thus can be effectively used in various fields such as new drug development, genetic disease treatment, genetically modified organism (GMO) development, and production of model animals used for organ transplantation.

Description

Sophisticated genome editing method based on the CRISPR-CAS NICKASE system

[0001] The present invention relates to a sophisticated genome editing method based on a CRISPR-Cas nickase system.

[0002] This invention claims the benefit of priority from Korean Patent Application No. 10-2024-0007510, filed January 17, 2024, and Korean Patent Application No. 10-2025-0006995, filed January 16, 2025, the entire contents of which are incorporated herein by reference.

[0003] The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system was discovered as the immune system of prokaryotes. It consists of guide RNA (gRNA) that accurately recognizes and binds to target DNA and Cas9 nuclease that cleaves the target DNA. It can specifically recognize and cleave the target nucleic acid sequence. When gRNA forms a complex with Cas9 nuclease and specifically binds to the target sequence, Cas9 nuclease recognizes the protospacer adjacent motif (PAM) sequence and induces a double-strand break (DSB) 3 bp upstream of the PAM. To repair DNA damaged by DSB, cells generally undergo non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. In the absence of a donor DNA template, the DSB is reassembled via the NHEJ pathway, which often results in insertions and deletions (indels) of several base pairs, leading to mutations and loss of gene function, resulting in a knockout. Conversely, in the presence of a donor DNA template, the HDR pathway allows the genome to be edited to a desired base sequence on the target DNA.

[0004] As genome editing using the CRISPR-Cas system has been actively studied, cases of DNA sequences similar to the designed target sequence being cleaved have been observed in eukaryotic organisms with high genome complexity. These off-target effects are attributed to the inherent mismatch tolerance of CRISPR-Cas, and this phenomenon has been recognized as an obstacle to precise editing. In fact, the success rate of gene editing and correction using the third-generation CRISPR-Cas9 is less than 10%, making it difficult to use for gene correction therapy. Therefore, the development of CRISPR-Cas nickase-based genome editing technologies is urgently needed to minimize genome damage caused by undesirable off-target effects.

[0005] The CRISPR-Cas nickase system has been utilized not only to edit the genome with high accuracy using a pair of sgRNAs, but also to develop single-base editing and prime editing that can edit the genome without DSBs or HDR. Single-base editing, created by fusing a deaminase to nCas9(D10A), a nickase with the D10A mutation in the RuvC domain of Cas9, can introduce point mutations by substituting adenine for guanine or cytosine for thymine. Prime editing, consisting of nCas9(H840A) fused with reverse transcriptase and pegRNA (prime editing guide RNA), can edit not only substitutions but also indels. However, CRISPR-Cas9 itself is a large protein, and single-base editing requires a deaminase fused to the CRISPR-Cas9 nickase, while prime editing requires a reverse transcriptase fused to it, making efficient delivery into cells even more challenging. To overcome these limitations, the development of alternative genome-editing tools remains essential.

[0006] Accordingly, the present inventors developed a method capable of precisely editing a genome at the single base level by 1) using a truncated sgRNA in which the 5'-terminal sequence of the Target Recognition Sequence (TRS) in the guide RNA is deleted, and 2) designing the target recognition site and the mutation-inducing site to overlap, thereby confirming that the genome target can be precisely and efficiently edited at the single base level, thereby completing the present invention.

[0007] The purpose of the present invention is a genome editing method based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase.

[0008] A genome editing method based on a CRISPR-Cas nickase system is provided, comprising steps designed to have the following characteristics:

[0009] Truncation of the 5´ terminal sequence of the above gRNA;

[0010] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0011] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0012] Another object of the present invention is to provide a composition for genome editing based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system, comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase.

[0013] Provided is a composition for genome editing based on a CRISPR-Cas nickase system, characterized in that it is designed to have the following characteristics:

[0014] Truncation of the 5´ terminal sequence of the above gRNA;

[0015] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0016] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0017] Another object of the present invention is a method for increasing genome editing efficiency based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase,

[0018] A method for increasing genome editing efficiency based on a CRISPR-Cas nickase system is provided, comprising steps designed to have the following characteristics:

[0019] Truncation of the 5´ terminal sequence of the above gRNA;

[0020] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0021] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0022] Another object of the present invention is a method for producing a prokaryotic cell, an isolated eukaryotic cell or a non-human eukaryotic organism in which target DNA is edited based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to the target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase,

[0023] A method for producing a prokaryotic cell, an isolated eukaryotic cell, or a non-human eukaryotic organism having a target DNA edited based on a CRISPR-Cas nickase system is provided, comprising: a step of designing the cell to have the following characteristics; and a step of introducing the donor nucleic acid molecule, the dual gRNA, and the Cas nickase into the subject;

[0024] Truncation of the 5´ terminal sequence of the above gRNA;

[0025] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0026] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0027] In order to achieve the above purpose, the present invention provides a genome editing method based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase.

[0028] A genome editing method based on a CRISPR-Cas nickase system is provided, comprising steps designed to have the following characteristics:

[0029] Truncation of the 5´ terminal sequence of the above gRNA;

[0030] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0031] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0032] In one embodiment of the present invention, the 5' terminal sequence truncation of the gRNA may be truncated by 1 or 2 nt from the 5' terminal, but is not limited thereto.

[0033] In another embodiment of the present invention, the Cas nickase may be, but is not limited to, Cas9-D10A nickase or Cas9-H840A nickase.

[0034] In another embodiment of the present invention, the distance between the double nicks may be, but is not limited to, 40 to 68 bp.

[0035] In another embodiment of the present invention, the target recognition sequence of the gRNA may be 18 to 20 nt in length, but is not limited thereto.

[0036] In another embodiment of the present invention, the CRISPR-Cas nickase system can edit the genome through negative selection, but is not limited thereto.

[0037] In another embodiment of the present invention, the method may include, but is not limited to, the step of introducing the donor nucleic acid molecule, the dual gRNA, and the Cas nickase into a subject.

[0038] In another embodiment of the present invention, the introducing step may be performed by, but is not limited to, local injection, microinjection, electroporation, or lipofection.

[0039] The present invention provides a composition for genome editing based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system, comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase.

[0040] Provided is a composition for genome editing based on a CRISPR-Cas nickase system, characterized in that it is designed to have the following characteristics:

[0041] Truncation of the 5´ terminal sequence of the above gRNA;

[0042] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0043] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0044] In one embodiment of the present invention, the genome editing composition may be applied to gene editing of a subject, but is not limited thereto.

[0045] The present invention relates to a method for increasing genome editing efficiency based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase,

[0046] A method for increasing genome editing efficiency based on a CRISPR-Cas nickase system is provided, comprising steps designed to have the following characteristics:

[0047] Truncation of the 5´ terminal sequence of the above gRNA;

[0048] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0049] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0050] The present invention relates to a method for producing a prokaryotic cell, an isolated eukaryotic cell or a non-human eukaryotic organism in which target DNA is edited based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase,

[0051] A method for producing a prokaryotic cell, an isolated eukaryotic cell, or a non-human eukaryotic organism having a target DNA edited based on a CRISPR-Cas nickase system is provided, comprising: a step of designing the cell to have the following characteristics; and a step of introducing the donor nucleic acid molecule, the dual gRNA, and the Cas nickase into the subject;

[0052] Truncation of the 5´ terminal sequence of the above gRNA;

[0053] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0054] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0055] In addition, the present invention provides a genome editing application based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system, comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual gRNA), and a Cas nickase, which are designed to have the following characteristics:

[0056] Truncation of the 5´ terminal sequence of the above gRNA;

[0057] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0058] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0059] In addition, the present invention provides a use for preparing a genome editing agent based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system, comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual gRNA), and a Cas nickase, wherein the composition is designed to have the following characteristics:

[0060] Truncation of the 5´ terminal sequence of the above gRNA;

[0061] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0062] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0063] The present invention relates to a precise genome editing method based on a CRISPR-Cas nickase system. The genome editing method of the present invention can improve the precision of genome editing by reducing off-target effects, and thus can be usefully used in various fields such as new drug development, genetic disease treatment, GMO (genetically modified organism) development, and production of model animals used for organ transplantation.

[0064] Figure 1a shows the bacterial strain and sgRNA plasmid used in the present invention.

[0065] Figure 1b shows the mutagenic oligonucleotide used in the present invention.

[0066] Figure 1c shows the primers used in the present invention when constructing a plasmid.

[0067] Figure 1d is a schematic diagram of a dual sgRNA plasmid expressing two sgRNAs that bind to different DNA strands within the same target gene. Specifically, the dual sgRNA plasmid was constructed by linking two PCR fragments carrying the ori or spectinomycin resistance gene, each end of which is ligated with the corresponding N 20 Same as TRS. Black arrows indicate primers used in PCR, and TRS represents the target recognition sequence.

[0068] Figure 2a is a schematic diagram of the PAM-in and PAM-out designs distinguished by the relative positions of the PAM and the dual nicks, with triangles indicating the nick sites formed by the dual sgRNA / nickase complexes.

[0069] Figures 2b to 2d show various dual sgRNA plasmids with different distances between the double nicks. Specifically, four base substitutions ( 504 TAAC → ATCA) is indicated in bold red letters, the underlined sequence indicates the BclI restriction enzyme site, and the filled triangle indicates the double nick formed by the Cas9-NG nickase and the dual sgRNA complex.

[0070] Figure 2e schematically illustrates genome editing using dual sgRNA plasmids in Cas9-NG nickase. Specifically, dual sgRNA plasmids and quadruple base mutations ( 504 After electroporation of mutant oligonucleotides containing TAAC → ATCA) into E. coli SH169 cells overexpressing Cas9-NG nickase and Bet protein, the recovered cells were plated on MacConkey agar plates supplemented with D-galactose, and Gal + Wow Gal - Cells appear as red and white colonies, respectively, on MacConkey agar. Editing efficiency was calculated by counting the number of white colonies, which is the phenotype of cells with edited target sites.

[0071] Figure 2f graphically compares the quadruple-base editing efficiency using the PAM-in and PAM-out designs with various double-nick distances (DBN). Specifically, the editing efficiency (%) was calculated as the proportion of white colonies among the total colonies (red colonies + white colonies) formed on MacConkey agar containing D-galactose. The P value was calculated by comparing the proportion of white colonies of DBN 44 and DBN 53 with those of DBN 0, 10, 20, and 26 (*P < 0.05), and the negative control (NC) was expressed using dual sgRNA plasmids targeting the galK and xylB genes.

[0072] Figure 2g is a graph showing the number of surviving colonies according to DBN. Specifically, it indirectly indicates whether the Cas9-NG nickase complex can induce cell death by creating double nicks in target DNA. Each bar represents the average of three independent experiments, and DBN represents the distance between double nicks.

[0073] Figure 2h schematically illustrates the target DNA sequence recognized by dual sgRNAs with different distances between the double nicks. Specifically, blue and green boxes represent PAM positions, filled triangles represent the formation sites of the double nicks, and red cones represent the four-base substitutions introduced at the target site ( 504 TAAC → ATCA). In the colony PCR product treated with BclI restriction enzyme, the edited target is cleaved into DNA fragments of 155 and 733 bp in size.

[0074] Figure 2i shows the results of electrophoresis on a 2% agarose gel of PCR products treated with BclI restriction enzyme. Specifically, "U" and "C" represent uncut and cleaved PCR products, respectively. The numbers in parentheses indicate the number of correctly edited colonies confirmed through restriction enzyme digestion among randomly selected white colonies, and PAM denotes the protospacer adjacent motif.

[0075] Figure 2j shows the sequence analysis results of the edited galK target using dual sgRNAs with different double nick distances. Specifically, the underlined bold letters indicate the target sequence for genome editing, the blue and green shaded chromatograms indicate the complementary region to the sgRNA, and the red shaded chromatogram indicates the correctly edited base ( 504TAAC → ATCA). Gray boxes and gray-shaded chromatograms indicate unwanted mutations, and the numbers in parentheses indicate the number of correctly edited colonies among the white colonies selected for sequencing. del indicates base deletion.

[0076] Figures 3a and 3b illustrate mutagenic oligonucleotides with homology arms of varying lengths that generate four-nucleotide mutations in Target1 (Figure 3a) and Between Targets (Figure 3b), respectively. Specifically, blue and green shaded letters indicate complementary regions to the sgRNA, red shaded sequences indicate target nucleotides, and filled triangles indicate double nicking sites. Red letters indicate the designed mutations.

[0077] Figures 3c to 3e show the results comparing editing efficiency. Specifically, the blue and green shaded areas represent the target DNA sequence recognized by each sgRNA, and the filled triangles represent the nick sites formed by the dual sgRNA / nickase complex. Mutagenic oligonucleotides with homology arms of various lengths were used, with L44 having a length equal to the nick distance (DBN) of 44 bp. L67-5′E contains a DBN of 44 nt and a homology arm (23 nt) extending 5′ from the nick site (67 nt = 44 nt + 23 nt). L90 contains a DBN of 44 nt and a homology arm (23 nt) extending in both directions from the nick site (90 nt = 44 nt + 46 nt). L120 contains a 44-nt double-stranded nucleotide nucleotide (DBN) and 38-nt homology arms extending in both directions from the nick site (120 nt = 44 nt + 76 nt). Editing efficiency was calculated as the number of white colonies out of all colonies (red + white colonies), and editing accuracy was calculated based on the number of correctly edited colonies (in parentheses) out of the white colonies confirmed by sequencing. Each bar represents the average of three independent experiments.

[0078] Figure 3c shows the results comparing the editing efficiency when the target sequence recognized by sgRNA overlaps with the editing site (Target1).

[0079] Figure 3d shows the results comparing the editing efficiency when the editing site does not overlap with the target sequence recognized by the sgRNA.

[0080] Figure 3e shows the results comparing the editing efficiency when the target sequence recognized by sgRNA overlaps with the editing site (Target2).

[0081] Figures 3f and 3g show the results of base sequence analysis of the quadruple-base edited galK target using various mutant oligonucleotides. Specifically, the underlined bold letters indicate the target sequence for genome editing, and the blue and green shaded sequences represent the target DNA recognized by each sgRNA. The red-shaded chromatogram indicates the correctly edited base, the gray boxes and gray-shaded chromatograms indicate unwanted mutations, and the bold red sequence indicates the designed mutation. The numbers in parentheses indicate the number of correctly edited colonies among the white colonies selected on MacConkey agar plates supplemented with D-galactose, and del and ins indicate deletion and insertion, respectively.

[0082] Figures 3h and 3i compare the genome editing efficiency according to the electroporation order of the dual sgRNA plasmid and the mutant oligonucleotide. Specifically, the genome editing efficiency of the galK target was evaluated when the mutant oligonucleotide was introduced first, followed by the dual sgRNA plasmid (Figure 3h) and when the dual sgRNA plasmid was introduced first, followed by the mutant oligonucleotide (Figure 3i). Each bar represents the average of three independent experiments.

[0083] Figure 4a schematically illustrates PAM-in and PAM-out designs with similar nick-to-nick distances. Specifically, the blue and green shaded areas represent the target DNA sequences recognized by each sgRNA, and the filled triangles indicate the locations where nicks are created.

[0084] Figure 4b illustrates the target sequences recognized by the PAM-in and PAM-out dual sgRNA plasmids, which have similar distances between their dual nicks. Specifically, the sequences shaded in blue and green represent the target DNA recognized by each sgRNA, and the filled triangles represent the nick sites formed by the dual sgRNA / Cas9-NG nickase complex.

[0085] Figure 4c is a graphical representation of the results comparing the quadruple-base editing efficiency and the number of viable colonies at the galK target using sgRNAs of PAM-in and PAM-out designs. Specifically, the editing efficiency was calculated as the number of white colonies on MacConkey agar plates supplemented with D-galactose, and the P value was calculated by comparing the white colony ratio between PAM-in (48) and PAM-out (46) using the same mutant oligonucleotide (*P < 0.05). Each bar represents the mean of three independent experiments, and the negative control (NC) was represented using a dual sgRNA plasmid targeting the galK and xylB genes.

[0086] Figure 4d shows the sequencing results of the galK target of Figure 4c. Specifically, the underlined bold letters indicate the target nucleotides for genome editing, and the blue and green shaded chromatograms indicate the target sequences recognized by each sgRNA. The red shaded chromatograms indicate correctly edited bases ( 528 AATT → GTAG), gray boxes and gray-shaded chromatograms indicate unwanted mutations. Numbers in parentheses indicate correctly edited colonies among the white colonies selected for sequencing.

[0087] Figure 5a graphically represents the results of Cas9-NG nickase-mediated single-nucleotide editing at various sites of the galK target. Specifically, editing efficiency was calculated as the proportion of white colonies among all red and white colonies, and the numbers in parentheses represent the number of correctly edited colonies among the white colonies selected for sequencing. The negative control (NC) was represented using a dual sgRNA plasmid targeting the galK and xylB genes.

[0088] Figure 5b shows the sequence analysis of the galK target in cells that appeared as white colonies on MacConkey agar plates supplemented with D-galactose. Specifically, the bolded sequence indicates the target nucleotide for single-base editing, and the blue and green shaded sequences indicate the complementary region to the dual sgRNA. The gray boxes and gray-shaded chromatograms indicate unwanted mutations. "del" indicates deletion.

[0089] Figure 6a is a graph showing the results of confirming the minimum length of the target recognition sequence (TRS) within the dual sgRNA required for double nick formation. Specifically, △0 to △3 represent the number of nucleotides truncated from the 5'-end of the dual sgRNA, and the negative control (NC) was represented using a dual sgRNA plasmid targeting the galK and xylB genes.

[0090] Figure 6b demonstrates overcoming single-mismatch tolerance of Cas9-NG nickase using 5'-terminally truncated sgRNA. Specifically, the sgRNA(Δ2) / Cas9-NG nickase complex with up to 5'-terminally truncated sgRNA fails to nick DNA edited by a single nucleotide (blue shading), but nicks DNA perfectly matching the sgRNA's target recognition sequence (green shading).

[0091] Figure 6c schematically illustrates the transcriptional repression of the xylose operon by the sgRNA / Cas9-NG nickase complex. Specifically, it was shown that the sgRNA / Cas9-NG nickase complex could bind to the xylA promoter and repress transcription of the xylAB gene when L-arabinose was present in the medium.

[0092] Figure 6d shows the various lengths (N 20 , N 17 , N 14 , N 11 , N 10 , N9 and N8) showed 5'-terminal truncated sgRNAs.

[0093] Figure 6e shows the results of suppression of xylAB gene expression by 5'-terminal truncated sgRNA / Cas9-NG nickase complex on MacConkey agar plates supplemented with D-xylose. Specifically, white colonies indicate inability to metabolize xylose due to suppression of xylAB gene expression, and TRS represents the target recognition sequence.

[0094] Figure 7a schematically illustrates single nucleotide editing at various sites of the galK target using a cleaved dual sgRNA / Cas9-NG nickase complex. Specifically, filled triangles represent nick sites formed by the sgRNA / Cas9-NG nickase complex, where the target recognition sequence is maximally cleaved.

[0095] Figure 7b is a graph showing the percentage of colonies with phenotypic changes due to single nucleotide editing in the galK gene and the number of surviving colonies. Specifically, editing accuracy was calculated as the number of correctly edited colonies among randomly selected white colonies for sequencing. The negative control (NC) was expressed using a dual sgRNA plasmid targeting the galK and xylB genes.

[0096] Figure 7c shows the sequence analysis of the galK target edited using the cleaved dual sgRNA / Cas9-NG nickase complex of Figure 7b. Specifically, the target nucleotides to be edited are indicated in bold, and the sequences shaded in blue and green represent the target DNA sequences recognized by each sgRNA. The chromatogram shaded in red indicates correctly edited bases, and the altered bases are indicated in bold red. The gray boxes and gray-shaded chromatograms indicate unwanted mutations, and each bar represents the average of three independent experiments.

[0097] Figure 7d is a schematic representation of genome editing using a mutagenic PCR product and a duplex sgRNA truncated by 2 nt at the 5'-end.

[0098] Figure 7e shows the results comparing the efficiency of single-nucleotide editing at various sites in the galK gene. Specifically, the number of single-nt edited colonies (%) was calculated as the percentage of correctly edited cells among randomly selected white colonies. The negative control (NC) was expressed using a dual sgRNA plasmid targeting the galK and xylB genes.

[0099] Figure 7f shows the sequence analysis results of the single-nucleotide edited galK target. Specifically, the bolded sequence represents the target nucleotide to be edited, and the blue and green shaded sequences represent the target DNA hybridized with the dual sgRNA. The red-shaded chromatogram represents the correctly edited base, the bold red sequence represents the intended mutation, and the numbers in parentheses indicate the number of correctly edited colonies among the white colonies selected for sequencing.

[0100] Figure 7g is a schematic diagram illustrating the process of obtaining edited cells by electroporating E. coli cells overexpressing Cas9-NG nickase and Bet protein with a double sgRNA plasmid and a mutant oligonucleotide having a C490T mutation in the galK gene, and then plating the recovered cells on an LB agar plate.

[0101] Figure 7h shows the results of base sequence analysis of the galK target from randomly selected colonies grown on LB agar containing spectinomycin. Specifically, the bold blue sequence represents the target nucleotide for single-nucleotide editing, while the shaded blue and green sequences indicate the target DNA recognized by the dual sgRNA. The shaded red chromatogram indicates the correctly edited base, and the altered base is indicated in bold red.

[0102] The terminology used herein is for descriptive purposes only and should not be construed as limiting.

[0103] Singular expressions include plural expressions unless the context clearly dictates otherwise. In this specification, terms such as "comprise" or "have" are intended to indicate the presence of a feature, number, step, operation, component, part, or combination thereof described in the specification, but should be understood to not preclude the presence or addition of one or more other features, numbers, steps, operations, components, parts, or combinations thereof.

[0104] Additionally, unless otherwise defined, all terms used herein, including technical or scientific terms, have the same meaning as commonly understood by a person of ordinary skill in the art to which the embodiments pertain. Terms defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the relevant technology, and shall not be interpreted in an idealized or overly formal sense unless explicitly defined herein.

[0105]

[0106] Hereinafter, the present invention will be described in detail.

[0107]

[0108] The present invention relates to a genome editing method based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase,

[0109] A genome editing method based on a CRISPR-Cas nickase system is provided, comprising steps designed to have the following characteristics:

[0110] Truncation of the 5´ terminal sequence of the above gRNA;

[0111] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0112] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0113] In the present invention, "target DNA" refers to DNA that is the target of editing by the CRISPR-Cas system. In the present invention, the target DNA refers to a region of the genome of a microbial strain that is completely complementary or substantially complementary to a crRNA or sgRNA. In some embodiments, the target DNA is located completely adjacent to a PAM sequence (a PAM sequence located completely adjacent to the 5' or 3' side of the target region) in the genome of the organism, and may be 10 to 40 nt in length, but is not limited thereto.

[0114] In the present invention, the term "donor nucleic acid molecule" may be used interchangeably with "donor nucleic acid sequence" or "mutagenic oligonucleotide", and the term "donor nucleic acid molecule" refers to a natural or modified polynucleotide, RNA-DNA chimera, or DNA fragment, or PCR-amplified ssDNA or dsDNA fragment, or an analog thereof, containing the desired nucleotide sequence to be inserted into the target DNA. Such donor nucleic acid molecules may include any form, such as single-stranded and double-stranded forms, but are not limited thereto, as long as they can induce a modification on the target DNA to achieve the purpose of the present invention.

[0115] The modification on the target DNA may include substitution of one or more nucleotides at any desired position, insertion of one or more nucleotides, deletion of one or more nucleotides, knockout, knockin, homology to an endogenous nucleic acid sequence, orthology, substitution with an endogenous or heterologous nucleic acid sequence, or a combination thereof.

[0116] In the present invention, preferably, the modification on the target DNA is a point mutation introduced (induced) by substitution of one or more nucleotides in a wild-type DNA sequence, and the introduction of such point mutation is, for example, by an oligonucleotide.

[0117] In the present invention, “mutation” means a mutation introduced into a genome by an oligonucleotide composed of single-stranded or double-stranded synthetic DNA.

[0118] The term "oligonucleotide" as used herein in reference to mutagenesis refers to a nucleic acid sequence of 10 to 150 nucleotides in length, preferably 25 to 145 nucleotides (mer), more preferably 40 to 130 nucleotides (which may be used as a probe or amplifier).

[0119] In an embodiment of the present invention, the effect of mutation sites in a mutagenic oligo on genome editing efficiency was investigated. When the mutation site was within the target sequence recognized by the sgRNA, a high percentage of white colonies were observed when using a mutagenic oligo with extended homology arms at the nick site (Figures 3c and 3e). On the other hand, when the mutation site was between targets, more than 35% of white colonies were obtained when using the M67-5′ and M67-3′ oligos (Figure 3d). This may be because the quadruple mutagenic oligo has sufficient homology on both sides of the mutagenic site. These results indicate that editing can be performed efficiently when the homologous regions of the donor DNA nucleic acid are equally balanced on both sides of the mutagenic sequence.

[0120] In the present invention, based on the mutagenic site of the donor nucleic acid molecule, the 5'-end and 3'-end lengths of the donor nucleic acid molecule may be 7 nt, 8 nt, 9 nt, 10 nt, 11 nt, 12 nt, 13 nt, 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt, 22 nt, 23 nt, 24 nt, 25 nt, 26 nt, 27 nt, 28 nt, 29 nt, 30 nt, 31 nt, 32 nt, 33 nt, 34 nt, or 35 nt or more, but are not limited thereto.

[0121] In addition, in the present invention, based on the mutagenic site of the donor nucleic acid molecule, the difference in length between the 5' end and the 3' end of the donor nucleic acid molecule may be 0 to 26 nt, 0 to 25 nt, 0 to 24 nt, 0 to 23 nt, 0 to 22 nt, 0 to 21 nt, 0 to 20 nt, 0 to 19 nt, 0 to 18 nt, 0 to 17 nt, 0 to 16 nt, 0 to 15 nt, 0 to 14 nt, 0 to 13 nt, 0 to 12 nt, 0 to 11 nt, 0 to 10 nt, 0 to 9 nt, 0 to 8 nt, 0 to 7 nt, 0 to 6 nt, 0 to 5 nt, 0 to 4 nt, 0 to 3 nt, 0 to 2 nt, or 0 to 1 nt, but is not limited thereto. no.

[0122] In the present invention, nucleic acids or oligonucleotides may be chemically modified, such as, but not limited to, base methylation and 5' or 3' modifications. Modified nucleic acids or oligonucleotides can prevent cleavage by nucleases and improve in vivo stability, thereby enhancing editing efficiency. In particular, modified nucleic acids may be recognized as parent strands during nucleic acid repair processes, thereby improving in vivo stability, but are not limited thereto.

[0123] In the present invention, "gRNA (guide RNA)" refers to an RNA molecule that binds to the Cas protein in the CRISPR-Cas system, guides the Cas protein to a target DNA sequence, and enables genome editing by accurately recognizing and cleaving the target DNA sequence. The gRNA may be a dual guide RNA (dual gRNA) comprising a crRNA (CRISPR RNA) and a tracrRNA (trans-activating crRNA) that hybridize with the target DNA, a dual single-stranded guide RNA (dual sgRNA), or a single-stranded guide RNA (sgRNA) that comprises portions of the crRNA and tracrRNA and hybridizes with the target DNA. Any gRNA may be used in the present invention as long as it comprises essential portions of the crRNA and tracrRNA and a portion complementary to the target.

[0124] The gRNA can be delivered to a cell or organism in the form of RNA or DNA encoding the gRNA. Additionally, the gRNA may be in the form of isolated RNA, RNA contained in a viral vector, or encoded within a vector. Preferably, the vector may be, but is not limited to, a viral vector, a plasmid vector, or an Agrobacterium vector.

[0125] In the present invention, the gRNA may be one or more gRNAs forming a dual gRNA, and may have a portion of the nucleotides at the 5' end truncated. Preferably, the number of truncated nucleotides may be 1 or 2, but is not limited thereto. In addition, the gRNA may be a dual gRNA or a dual sgRNA, but is not limited thereto.

[0126] In one embodiment of the present invention, the 5' terminal sequence truncation of the gRNA may be truncated by 1 or 2 nt from the 5' terminal, but is not limited thereto. More specifically, the 5' terminal sequence truncation of the gRNA may be truncated by 2 nt from the 5' terminal, but is not limited thereto.

[0127] In an embodiment of the present invention, in order to determine the minimum length of the target recognition sequence of sgRNA that can maintain nickase activity, plasmids carrying dual sgRNAs with 1 to 3 nt truncations were constructed and the number of surviving colonies was compared. It was confirmed that the target DNA was effectively cleaved when 1 to 2 nt were cleaved from the 5′ end of the sgRNA, but the target was not cleaved when 3 nt were cleaved from the 5′ end (Fig. 6a). In addition, the single-nucleotide editing results using dual sgRNA / nickase complexes with maximal cleavage at various positions of the galK target showed that when donor DNA that causes mutations in the target sequence to which the sgRNA hybridizes was used, only the target nucleotide was precisely edited (Fig. 7c).

[0128] Specifically, in an embodiment of the present invention, successful editing at the single nucleotide level was confirmed using a maximally truncated dual sgRNA by examining the target sequence of randomly selected colonies. When an intact sgRNA was used, unwanted mutations were observed in the DNA sequence to which the sgRNA binds, independent of editing of the target nucleotide. On the other hand, when a maximally truncated dual sgRNA was used, it was confirmed that the target nucleotide remained unedited without unwanted mutations in all unedited colonies. This suggests that the in vivo activity of the truncated sgRNA / nickase is relatively weaker than that of the intact sgRNA / nickase, enabling donor DNA-dependent, precise genome editing without unwanted mutations in the surrounding sequence.

[0129] In the present invention, "Cas protein" means an essential protein element in the CRISPR-Cas system, and forms an active endonuclease or nickase when forming a complex with two RNAs called crRNA and tracrRNA. Information on Cas genes and proteins can be obtained from GenBank of the National Center for Biotechnology Information (NCBI), but is not limited thereto. Depending on whether there are multiple types of Cas proteins (class 1) or a single type (class 2), they are divided into two classes, and the two classes are further subdivided into six types (I-VI) depending on the CRISPR-Cas loci on the genome and the type of Cas protein in action. Any Cas protein can be used as long as it can achieve the purpose of the present invention, but preferably Cas9, Cpf1, or Cas13a, and preferably Cas9. Any nucleic acid encoding the Cas9 protein or the Cas9 protein may be used as long as it can achieve the purpose of the present invention, but may be, for example, SpCas9 or SaCas9.

[0130] In one embodiment of the present invention, the Cas nickase may be, but is not limited to, Cas9, Cpf1, or Cas13a. Furthermore, in one embodiment of the present invention, the Cas nickase may be, but is not limited to, Cas9 nickase or Cas9-NG nickase.

[0131] In the present invention, the Cas9-NG nickase uses 5'-NG as a PAM sequence, and may be capable of recognizing a shorter PAM sequence than the wild-type Cas9, thereby reducing PAM sequence constraints and enabling recognition across the entire genome, but is not limited thereto. In addition, the Cas9-NG nickase and truncated sgRNA system requires only the SpCas9-NG nickase portion (∼160 kDa) and a smaller truncated sgRNA (∼100 nt) for genome editing, thereby overcoming difficulties in integrating it into a vector and delivering it to cells.

[0132] In the present invention, the "Cas nickase" may be a Cas9 variant having a mutation in D10, for example, a D10A mutant; or a Cas9 variant having a mutation in H840, for example, an H840A mutant. D10A creates a nick in the target strand, whereas H840A creates a nick in the non-target strand DNA. Specifically, a Cas9 nickase that forms a single-strand break (SSB) or nick in the target DNA can be created through a D10A mutant in which aspartic acid (D), the 10th amino acid in the RuvC domain of Cas9 nuclease, is substituted with alanine (A); or a H840A mutant in which histidine (H), the 840th amino acid in the HNH domain, is mutated to alanine (A). The Cas9 nickase system utilizes a pair of sgRNAs to create nicks in complementary strands of two adjacent DNA targets, resulting in a DSB in the target DNA. Furthermore, the length of the target-recognizing sequence can be doubled, preventing cleavage of sequences similar to the target, thereby reducing genomic damage caused by off-target effects.

[0133] In one embodiment of the present invention, the Cas nickase may be, but is not limited to, Cas9-D10A nickase or Cas9-H840A nickase. More specifically, the Cas9 nickase may be, but is not limited to, Cas9-D10A nickase. Since the Cas9-D10A nickase or Cas9-H840A nickase can be identified by a substance known in the art, it is clear that a person skilled in the art can determine which nickase is meant even if it is not at position 10 or 840 from a specific sequence number.

[0134] In the present invention, "hybridization" means the formation of a double-stranded nucleic acid by complementary single-stranded nucleic acids. Hybridization may occur when the complementarity between two nucleic acid strands is perfect (perfect match), or may occur even when some mismatched bases exist.

[0135] In the present invention, the distance (DBN) between the double nicks generated by the dual gRNA and Cas nickase may be 40 to 68 bp, and specifically, 44 to 53 bp. If the distance between the double nicks decreases, steric hindrance may affect the simultaneous recognition and complete cleavage of the target DNA by two different Cas9 nickases, and this mutual interference may prevent the formation of DSBs, thereby increasing the proportion of viable cells. When the DBN is 44 bp, the Cas9-NG (D10A) nickase-sgRNA complex acts independently without interfering with each other to form DSBs, thereby reducing the number of viable colonies and effectively inducing genome editing.

[0136] In an embodiment of the present invention, when the distance between the double nicks was 10 to 26 bp, the percentage of surviving cells was high, but the editing efficiency of the target sequence, confirmed by the phenotypic change of the colonies, was low at approximately 9%. In contrast, when the distance between the two nicks was approximately 44 to 53 bp, unedited cells were fatally affected, and edited cells exhibiting a phenotype of white colonies could be efficiently obtained on MacConkey agar (Figs. 2f and 2g).

[0137] In one embodiment of the present invention, the distance between the double nicks is 40 to 68 bp, 40 to 67 bp, 40 to 66 bp, 40 to 65 bp, 40 to 64 bp, 40 to 63 bp, 40 to 62 bp, 40 to 61 bp, 40 to 60 bp, 40 to 59 bp, 40 to 58 bp, 40 to 57 bp, 40 to 56 bp, 40 to 55 bp, 40 to 54 bp, 40 to 53 bp, 41 to 68 bp, 41 to 67 bp, 41 to 66 bp, 41 to 65 bp, 41 to 64 bp, 41 to 63 bp, 41 to 62 bp, 41 to 61bp, 41 to 60bp, 41 to 59bp, 41 to 58bp, 41 to 57bp, 41 to 56bp, 41 to 55bp, 41 to 54bp, 41 to 53bp, 42 to 68bp, 42 to 67bp, 42 to 66bp, 42 to 65bp, 42 to 64bp, 42 to 63bp, 42 to 62bp, 42 to 61bp, 42 to 60bp, 42 to 59bp, 42 to 58bp, 42 to 57bp, 42 to 56bp, 42 to 55bp, 42 to 54bp, 42 to 53bp, 43 to 68bp, 43 to 67bp, 43 to 66bp, 43 to 65bp, 43 to 64bp, 43 to 63bp, 43 to 62bp, 43 to 61bp, 43 to 60bp, 43 to 59bp, 43 to 58bp, 43 to 57bp, 43 to 56bp, 43 to 55bp, 43 to 54bp, 43 to 53bp, 44 to 68bp, 44 to 67bp, 44 to 66bp, 44 to 65bp, 44 to 64bp, 44 to 63bp, 44 to 62bp, 44 to 61bp, It may be 44 to 60 bp, 44 to 59 bp, 44 to 58 bp, 44 to 57 bp, 44 to 56 bp, 44 to 55 bp, 44 to 54 bp, or 44 to 53 bp,More specifically, the distance between the double nicks may be, but is not limited to, 44 ​​to 53 bp.

[0138] In the present invention, the “target recognition portion (TRS)” may be used interchangeably with a target recognition sequence, but is not limited thereto.

[0139] In the present invention, if the target recognition sequence of the gRNA does not overlap with the mutagenesis site of the donor nucleic acid molecule, the cleaved sgRNA / Cas9 nickase complex may continuously cleave the target DNA, potentially leading to indel formation during the repair process. Conversely, if the target recognition sequence of the gRNA does overlap with the mutagenesis site of the donor nucleic acid molecule, the binding and cleavage efficiency of the cleaved sgRNA / Cas9 nickase may be reduced, preventing additional cleavage of the target DNA, thereby enabling the effective acquisition of cells in which a single nucleotide has been edited.

[0140] In the present invention, “overlap between the target recognition site of the gRNA and the mutagenic site of the donor nucleic acid molecule” may mean, but is not limited to, that at least one of the two target recognition sites of the double guide RNA overlaps with the mutagenic site of the donor nucleic acid molecule.

[0141] In one embodiment of the present invention, the target recognition sequence of the gRNA may be 18 nt or more, 19 nt or more, 20 nt or more, or 18 to 20 nt in length, but is not limited thereto.

[0142] In the present invention, "PAM (protospacer-adjacent motif)" refers to a short sequence essential for the Cas protein in the CRISPR-Cas system to recognize and cleave target DNA. The PAM sequence helps the Cas protein accurately recognize and cleave DNA, and may generally be composed of a specific base sequence, but is not limited thereto.

[0143] In the present invention, we confirmed that the PAM position relative to the nick formation site is an important factor affecting genome editing. In the PAM-in design, no white colonies were observed even when the distance between the double nicks was 48 bp, and the number of surviving colonies was high, showing low editing efficiency. On the other hand, when PAM-out (46) was used, the percentage of white colonies increased significantly from 40 to 94% compared to PAM-in (48), and the number of surviving colonies was also significantly reduced. These results demonstrate that the use of DBN 44 in the PAM-out design reduces the number of surviving colonies and achieves high genome editing efficiency. In other words, when using the sgRNA in the PAM-out design, two different Cas9 nickases form nicks simultaneously, inducing DSBs, reducing the number of surviving cells and enabling high-efficiency genome editing. In the case of the PAM-in design, since double nicks are not formed simultaneously, cells do not die, and consequently, mutations are not introduced into the target site.

[0144] In one embodiment of the present invention, the CRISPR-Cas nickase system can edit the genome through negative selection, but is not limited thereto.

[0145] In the present invention, we confirmed that oligonucleotides with homology arms were ineffective in promoting the repair of truncated chromosomes, and that most cells died when the target was truncated, preventing the generation of edited cells. Consequently, these results confirmed that edited cells can be successfully obtained through negative selection using a dual sgRNA / Cas9-NG nickase complex after mutagenesis.

[0146] In the present invention, “negative selection” may mean a process in which cells in which a desired mutation is induced survive and cells in which no mutation is introduced are eliminated using a CRISPR-Cas nickase system that operates by targeting a specific gene or sequence after mutagenesis, and this can be confirmed using a dual sgRNA / Cas9-NG nickase complex after mutagenesis, but is not limited thereto.

[0147] In one embodiment of the present invention, the method may include, but is not limited to, introducing the donor nucleic acid molecule, the dual gRNA, and the Cas nickase into a subject.

[0148] In the present invention, the subject may be, but is not limited to, a cell or an organism. Furthermore, the cell may be, but is not limited to, a prokaryotic cell, an isolated cell, an isolated eukaryotic cell, a cell isolated from a eukaryotic animal, or a cell isolated from a eukaryotic plant. The organism may be, but is not limited to, a eukaryotic organism, a eukaryotic animal, a eukaryotic plant, or an organism other than a human.

[0149] In one embodiment of the present invention, the introducing step may be performed by, but is not limited to, local injection, microinjection, electroporation, or lipofection.

[0150] The present invention provides a composition for genome editing based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system, comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase.

[0151] Provided is a composition for genome editing based on a CRISPR-Cas nickase system, characterized in that it is designed to have the following characteristics:

[0152] Truncation of the 5´ terminal sequence of the above gRNA;

[0153] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0154] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0155] In one embodiment of the present invention, the entire editing composition may be for application to gene editing of a subject, but is not limited thereto.

[0156] In the present invention, the subject may be, but is not limited to, a cell or an organism. Furthermore, the cell may be, but is not limited to, a prokaryotic cell, an isolated cell, an isolated eukaryotic cell, a cell isolated from a eukaryotic animal, or a eukaryotic plant. The organism may be, but is not limited to, a eukaryotic organism, a eukaryotic animal, a eukaryotic plant, or an organism other than a human.

[0157] In addition, in the present invention, the subject is not limited to a plasmid, a virus, a prokaryotic cell, an isolated eukaryotic cell, or a eukaryotic organism other than a human, as long as the method of the present invention can be applied.

[0158] The eukaryotic cell may be a cell of yeast, fungus, plant, insect, amphibian, mammal, etc., and may be, but is not limited to, cells cultured in vitro, transplanted cells, primary cell cultures, in vivo cells, and cells of mammals including humans, which are commonly used in the art.

[0159] The terms "introducing," "inserting," "delivering," and "administering" (and grammatical variations thereof) as used herein mean presenting a polynucleotide of interest to a host organism or a cell of said organism (e.g., a host cell, such as a bacterial cell) in such a manner that said polynucleotide gains access to the interior of the cell, and such methods include "transformation," "transfection," and / or "transduction."

[0160] The present invention relates to a method for increasing genome editing efficiency based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase,

[0161] A method for increasing genome editing efficiency based on a CRISPR-Cas nickase system is provided, comprising steps designed to have the following characteristics:

[0162] Truncation of the 5´ terminal sequence of the above gRNA;

[0163] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0164] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0165] The present invention relates to a method for producing a prokaryotic cell, an isolated eukaryotic cell or a non-human eukaryotic organism in which target DNA is edited based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to a target DNA, a dual guide RNA (dual guide RNA, dual gRNA), and a Cas nickase,

[0166] A method for producing a prokaryotic cell, an isolated eukaryotic cell, or a non-human eukaryotic organism having a target DNA edited based on a CRISPR-Cas nickase system is provided, comprising: a step of designing the cell to have the following characteristics; and a step of introducing the donor nucleic acid molecule, the dual gRNA, and the Cas nickase into the subject;

[0167] Truncation of the 5´ terminal sequence of the above gRNA;

[0168] Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and

[0169] Position of the protospacer adjacent motif (PAM) outside the double nick by the above double gRNA and Cas nickase.

[0170]

[0171] The above-described matters of the present invention may have the same meaning when utilized in the method and composition of the present invention, but are not limited thereto.

[0172] The following examples are provided solely to illustrate the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention.

[0173]

[0174] [Example]

[0175]

[0176] Bacterial strains and culture conditions

[0177]

[0178] The bacterial strains used in the present invention are shown in Fig. 1a. Escherichia coli was cultured in LB broth (LPS solution, Cat. LB-05) at 30°C or 37°C depending on the replication origin (ori) sequence of the plasmid. E. coli DH5α was used as a cloning host for constructing a dual sgRNA plasmid. To introduce the D10A mutation into the cas9-NG gene, E. coli HK1159 strain carrying the cas9-NG gene in the chromosome of E. coli MG1655 was used, and pHK463 expressing the λBet protein was used to introduce the D10A mutation by single-stranded oligonucleotide. E. coli HK1159 carrying the pHK463 plasmid was cultured in LB broth (30°C) containing ampicillin. OD 600nmWhen the concentration reached 0.4, L-arabinose (TCI, Cat. A0515) was added to a final concentration of 1 mM to overexpress Cas9-NG nuclease and λ red Bet protein. After 3 h, cultured cells were harvested, washed twice with 10% glycerol, resuspended, and stored at -80°C. pHL143 sgRNA plasmid (200 ng) and D10A mutagenic oligonucleotide (100 pmol) were electroporated into pHK463 / HK1159 cells using a 0.1 cm electroporation cuvette (Bio-Rad, Cat. 1652089) at 25 μF, 200 Ω, and 1.8 kV. Cells were then immediately transferred to 950 μL of SOC medium and allowed to recover at 30°C and 180 rpm for 1 h. Afterwards, the cells were plated on LB agar containing spectinomycin and cultured at 37°C for 16 hours. Sanger sequencing confirmed that the D10A mutation was successfully introduced into the cas9-NG gene. After curing the pHK463 plasmid at 42°C, the strain was named Escherichia coli SH169. The strain contained L-arabinose-inducible P in the chromosome. BAD The cas9-NG (D10A) gene is located downstream of the promoter. Kanamycin (25 μg / mL), ampicillin (50 μg / mL), and spectinomycin (75 μg / mL) were added to the medium as needed. Figure 1b summarizes the mutagenic oligonucleotides used in the present invention.

[0179]

[0180] Plasmid production

[0181]

[0182] The sgRNA plasmid used in the present invention is shown in Figure 1a. The primers used for plasmid construction are shown in Figure 1c. To create nicks on different strands of the double-stranded target DNA, a plasmid was constructed that simultaneously expresses two sgRNAs targeting different strands within the galK gene (Figure 1d). The inventors designed the plasmid to enable stable replication by placing the spectinomycin resistance gene and the plasmid's replication origin between the two sgRNAs. This arrangement prevents recombination between identical nucleotide sequences in the sgRNA scaffold. A DNA fragment containing the first sgRNA gene and the spectinomycin resistance gene, and another DNA fragment containing the second sgRNA gene and the ori sequence of the plasmid, were amplified by PCR. PCR was performed using a KOD FX (ToYoBo, Cat. KFX-101). The two purified DNA fragments were ligated using Gibson Assembly Master Mix (NEB, Cat. E2611) and transformed into DH5α competent cells. The sequence of the double sgRNA plasmid was confirmed by Sanger sequencing.

[0183]

[0184] Mutagenicity

[0185]

[0186] Mutagenic oligonucleotides (Fig. 1b) were synthesized to produce four base substitutions ( 494 TTGT → GATC, 504 TAAC → ATCA or 528A premature termination codon was generated in the target galK gene by introducing a mutagenic oligonucleotide (AATT → GTAG) or a single nucleotide substitution (C490T, T504A, or C523T). The mutagenic oligonucleotide (500 pmol) and the dual sgRNA plasmid (200 ng) were electroporated into Escherichia coli SH169 cells carrying the pHK463 plasmid, resulting in overexpression of both Cas9-NG(D10A) nickase and λred Bet proteins in response to the addition of L-arabinose. Electroporation was performed as described above. The recovered cells were cultured on MacConkey agar (BD Difco, Cat. 281810) containing D-galactose (0.5%) (Samcheon Chemical, Cat. G0476) and spectinomycin or LB agar containing spectinomycin, and then cultured at 37°C for 16 hours.

[0187] For single-nucleotide editing using PCR products as donor DNA, double-stranded donor DNA was amplified using PCR from strains harboring the C490T, T504A, or C523T mutations in the galK gene. The 1-kb mutagenic PCR product (500 ng) and the 5′-terminal 2-nucleotide (nt)-truncated duplex sgRNA plasmid (200 ng) were co-electroporated into SH169 cells harboring the pKD46 plasmid. Transformed cells were plated on MacConkey agar containing D-galactose (0.5%) and spectinomycin.

[0188]

[0189] Two-Step Electroporation

[0190]

[0191] A mutagenic oligonucleotide was synthesized to produce a quadruple base substitution in the galK gene ( 494TTGT → GATC) was induced, and the lengths of the oligonucleotides were 44 mer, 67 mer, 90 mer, and 120 mer. The mutagenic oligonucleotides (500 pmol) or pSH356 sgRNA plasmid (200 ng) were individually electroporated into L-arabinose-induced SH169 cells carrying the pHK463 plasmid and then incubated at 37°C for 30 min. The recovered cells were harvested, washed twice with 10% glycerol solution, and resuspended. The pSH356 sgRNA plasmid or mutagenic oligonucleotides were then electroporated into each electrocompetent cell. After 30 min of recovery at 37°C, the cells were plated on MacConkey agar supplemented with D-galactose (0.5%) and spectinomycin.

[0192]

[0193] Calculating editing efficiency

[0194]

[0195] Genome editing efficiency for the target gene galK was calculated by counting the number of red and white colonies on MacConkey agar [white colonies / (white colonies + red colonies)]. To confirm whether the target gene was cleaved by the CRISPR-Cas nickase system, the number of viable colonies was counted on MacConkey agar supplemented with D-galactose (0.5%) and spectinomycin or LB agar supplemented with spectinomycin. White colonies were randomly selected, and the edited DNA target was analyzed by restriction enzyme digestion and Sanger sequencing to assess the accuracy of Cas9-NG(D10A) nickase-mediated genome editing.

[0196] For restriction enzyme digestion analysis, a 1 kb DNA fragment containing the target region was amplified from randomly selected white colonies grown on MacConkey agar supplemented with D-galactose using the galK-F and galK-R primers. The PCR product was then treated with BclI restriction enzyme (Enzynomics, Cat. R048S) for 90 min (37°C). The size of the digested DNA fragment was confirmed by 2% agarose gel electrophoresis. The primers used for PCR amplification and Sanger sequencing are shown in Figure 1c.

[0197]

[0198] In vivo nickase binding assay

[0199]

[0200] We constructed an sgRNA plasmid targeting the promoter of the xylA gene in the xylose operon. Various sgRNA plasmids with target recognition sequences (TRS) of various lengths were transformed into SH169 cells. Each single colony on LB agar supplemented with spectinomycin was streaked onto MacConkey agar supplemented with D-galactose (0.3%) and spectinomycin. Transformed cells were also streaked onto MacConkey agar supplemented with L-arabinose (0.3%) and incubated at 37°C for 12 h. When the Cas9-NG(D10A) nickase-sgRNA complex represses the xylA promoter, preventing the cells from metabolizing D-xylose, the colonies remain white. On the other hand, if the xylA promoter is not repressed and the cell can metabolize D-xylose, it turns red.

[0201]

[0202] Statistical analysis

[0203]

[0204] The collected data were analyzed in Microsoft Excel 2016 (Microsoft Corporation, USA) using a two-tailed unpaired t-test to assess significance.

[0205]

[0206] Example 1. The effect of double-nick distance (DBN) on genome editing.

[0207]

[0208] In this example, we investigated the effect of various nicks occurring around the genome editing site on gene editing efficiency.

[0209] Specifically, dual sgRNA plasmids were designed to vary the distances between nicks (DBN), and mutagenic oligonucleotides (120mers) were synthesized to introduce four base substitutions in the galK gene ( 504 TAAC → ATCA) was introduced. The mutagenic oligonucleotide can generate a quadruple mutation including a stop codon in the galK target. This construct was electroporated into E. coli SH169 cells overexpressing the λ Bet protein and the Cas9 nickase mutant (5′-NG) Cas9-NG nickase with an extended PAM (Fig. 2a to 2d). When the target sequence of galK is appropriately mutated by the mutagenic oligonucleotide through the introduction of a stop codon, the edited cells form white colonies on MacConkey agar containing D-galactose. Conversely, when the target is not edited, D-galactose can be metabolized normally, resulting in the formation of red colonies (Fig. 2e).

[0210] As a result, for the PAM-in design in which the PAM sequence is located inside the double nick, as shown in Figs. 2f and 2g, no white colonies were observed on MacConkey agar containing D-galactose, and the number of surviving colonies was high (≥10 7 CFU / μg DNA). On the other hand, in the PAM-out design, where the PAM sequence is located outside the double nick, white colonies were observed at rates of 9%, 11%, and 7% when the distance between double nicks (DBN) was 10, 20, and 26 bp, respectively. When the double nicks were formed at intervals of 44 or 53 bp, the number of surviving colonies was 10 5 CFU / μg DNA was significantly reduced, and the percentage of white colonies was very high at 71% or 49%, respectively.

[0211] A quadruple nucleotide substitution in the galK target of randomly selected white colonies was confirmed by BclI restriction enzyme digestion and Sanger sequencing.

[0212] As a result of the restriction enzyme digestion experiment, in the case of DBN 10, DBN 20, DBN 26, DBN 44, and DBN 53, where white colonies were observed, it was confirmed that the BclI restriction enzyme site was inserted into the target sequence in 4, 4, 4, 2, and 2 of the 4 white colonies, respectively (Fig. 2h and Fig. 2i).

[0213] Sanger sequencing results showed that all four white colonies for DBN 10, DBN 20, and DBN 26 were correctly edited. However, for DBN 44 and DBN 53, unwanted mutations were observed within the target sequence in all four colonies (Fig. 2j).

[0214] These results demonstrate that when the nucleotide sequence to be edited is located between two overlapping target sequences (DBN 10, DBN 20, DBN 26), the probability of obtaining edited cells decreases, but the editing accuracy is higher. Conversely, when the nucleotide sequence to be edited is located between the targets, the editing accuracy decreases, but the target DNA is efficiently cleaved by the CRISPR-Cas9 nickase system. Furthermore, these findings suggest that the results of nickase-mediated genome editing cannot be conclusively verified using restriction enzymes. Therefore, in subsequent experiments, the edited target sequences were analyzed by Sanger sequencing.

[0215]

[0216] Example 2. Effective genome editing by nickase-mediated negative selection.

[0217]

[0218] To improve efficient cleavage of target DNA and genome editing accuracy under PAM-out (DBN 44 bp) conditions, experiments were conducted to analyze editing efficiency according to mutation location.

[0219] Specifically, mutations were designed to be introduced into the target DNA or between targets hybridized with the sgRNA (Editing on Target1 and Editing between Targets). Mutagenic oligonucleotides of various lengths (44 mer, 67 mer, 90 mer, and 120 mer) were synthesized to induce four base substitutions at two mutagenic sites (Figures 3A and 3B). Each mutagenic oligonucleotide was electroporated together with a 44-bp DBN duplex sgRNA plasmid into cells overexpressing Cas9-NG (D10A) nickase and λ Bet protein.

[0220] As a result, as shown in Fig. 3c, when the mutation was located in the target DNA (On Target1), the percentage of white colonies was 96–97% when L67-5′E, L90, and L120 were used, indicating that almost all cells exhibited an edited phenotype. However, when L44 and L67-3′E were used, white colonies were observed in less than 5%.

[0221] Also, as shown in Fig. 3d, when the mutation was located between targets (Between Targets), the proportion of white colonies was not significantly different when M67-5′E and M67-3′E were used, at 54% and 34%, respectively. In addition, when the mutagenesis site was present in Target2, the editing efficiency was relatively low when R67-5′E was used compared to Target1, but the editing efficiency increased to approximately 77% when R67-3′E was used (Fig. 3e). In addition, under the conditions using 90mer and 120mer, genome editing was possible with high efficiency in most cases, suggesting that the length of the homologous DNA fragments on both sides from the mutagenesis site is an important factor for effective genome editing by HDR.

[0222]

[0223] White colonies were randomly selected, and the nucleotide sequence of the edited galK target was amplified by PCR and analyzed by Sanger sequencing (Fig. 3f and 3g). When mutagenic oligos (L44, L67-5′E, L67-3′E, L90, L120) were used to induce mutations (On Target1) in the target DNA, 3, 4, 2, 4, and 4 out of the 4 white colonies showed accurate sequence editing, respectively. Conversely, when M67-3′E was used to induce mutations between targets, only 1 out of the 4 white colonies was accurately edited, whereas when M44, M67-5′E, M90, and M120 were used, all 4 white colonies showed unwanted mutations in the target DNA recognized by the sgRNA.

[0224] The above results demonstrate that the accuracy of Cas9-NG nickase-mediated genome editing can be improved when the nucleotide to be edited is located at the target sequence hybridized with the sgRNA.

[0225]

[0226] We investigated the genome editing mechanism of the nickase system using a two-step electroporation method that changes the electroporation order of a dual sgRNA plasmid (pSH356) and a mutagenic oligonucleotide.

[0227] As a result, when the mutagenic oligo was electroporated first and then the pSH356 sgRNA plasmid was electroporated, the editing efficiency was lower than when the oligo and sgRNA plasmid were electroporated simultaneously, but the result pattern was similar (Fig. 3c and 3h). However, when the sgRNA plasmid was electroporated first, few white colonies were obtained (Fig. 3i).

[0228] These results indicate that oligonucleotides with homology arms are ineffective in promoting the repair of broken chromosomes. When the target is cleaved, most cells die, preventing the generation of edited cells. Consequently, these results suggest that edited cells can be successfully obtained through negative selection using a dual sgRNA / Cas9-NG nickase complex after mutagenesis.

[0229]

[0230] Example 3. Genome editing efficiency according to PAM-Out design

[0231]

[0232] When the PAM-in design sgRNA plasmid with a distance between nicks (DBN) of 48 bp was used, genome editing was not successful, and the number of surviving colonies increased even when the DBN was sufficiently spaced (Figs. 2f and 2g). Based on this result, to determine whether not only the DBN but also the PAM-in or PAM-out design affects the editing efficiency, we designed PAM-out (48) to create double nicks at similar positions to PAM-in (46) (Figs. 4a and 4b).

[0233] In the case of PAM-in(48) dual sgRNA, when R120 oligos that induce mutations in the target were used, the percentage of white colonies was low at 3%, and the number of surviving colonies was 10 6.9 / μg DNA.

[0234] For PAM-out(46) dual sgRNA, when R120 oligo was used, the percentage of white colonies was 94%, and CFU was 10 5 / μg DNA (Fig. 4c).

[0235] To verify editing accuracy, raw sequencing was performed (Fig. 4d).

[0236] When the R120 oligo was used in PAM-in (48), three out of four white colonies showed accurate sequence editing. For PAM-out (46) using the R120 oligo, only the desired mutation was introduced in all confirmed white colonies (Fig. 4d).

[0237] These results indicate that the PAM-out design effectively promotes duplex formation, and that designing base editing into the target sequence hybridized with sgRNA can achieve genome editing with significantly high efficiency.

[0238]

[0239] Example 4. Failure of precise editing using an uncleaved sgRNA / nickase complex.

[0240]

[0241] Experiments were performed to determine whether a dual sgRNA with a 20 nt long sequence as a target recognition sequence (TRS) could be used for single nucleotide editing using mutagenic oligonucleotides for each target at multiple mutagenic sites.

[0242] Mutagenic oligonucleotides introducing single nucleotide substitutions at three mutagenic sites and N 20 Double sgRNA (DBN 44 bp) was electroporated into Cas9-NG (D10A) nickase and λ Bet overexpressing cells.

[0243] The percentages of white colonies obtained using mutagenic oligonucleotides inducing mutations at Target 1, between Targets, and Target 2 were 71%, 72%, and 70%, respectively (Fig. 5a). The edited nucleotide sequences were then confirmed using Sanger sequencing. However, unwanted mutations were observed in all confirmed white colonies (Fig. 5b). These results indicate that the accuracy of single-nucleotide editing was very low when using dual sgRNAs with 20 nt target sequences.

[0244]

[0245] Example 5. Minimum TRS length required for in vivo nickase activity

[0246]

[0247] To improve the accuracy of single-nucleotide editing, we identified the Target Recognition Sequence (TRS) length required for dual sgRNAs to recognize the target and maintain nickase activity. We confirmed that dual sgRNAs with a minimal TRS length can enhance the accuracy of single-nucleotide editing by preventing mismatches between the target DNA and sgRNA.

[0248] Specifically, after introducing a duplex sgRNA with a 1-3 nt truncation at the 5′ end into Cas9-NG (D10A) nickase cells, the nickase activity was confirmed according to the 5′ end truncation length of the sgRNA.

[0249] As a result, when dual sgRNAs with 1-2 nt truncations at the 5′ end were introduced, the number of surviving colonies was almost the same as the number of transformants containing intact dual sgRNAs (10 4 CFU / μg DNA). However, when a 3 nt truncated duplex sgRNA was used, the CFU was 10 7 / μg DNA was increased (Fig. 6a).

[0250] The above results indicate that the nickase activity of the sgRNA / Cas9-NG(D10A) nickase complex can be maintained even when the 5′ end of the duplex sgRNA is truncated by up to 2 nt. This suggests that cleaving 2 nt from the 5′ end of the duplex sgRNA is necessary to enhance the accuracy of single-nucleotide editing (Fig. 6b).

[0251]

[0252] Furthermore, we used the CRISPR interference system to determine the minimum TRS length required for sgRNA binding to the xylA promoter. We confirmed that gene expression could be repressed even when the sgRNA TRS length was shortened to 9 nt (Figures 6c to 6e). These results imply that the 5′-end-truncated sgRNA / Cas9-NG(D10A) nickase complex can bind to a relatively short target sequence (~9 bp) in vivo, but a target length of at least 18 bp is required for nick formation.

[0253]

[0254] Example 6. Precise genome editing using 5′ truncated sgRNA / Cas9-NG nickase.

[0255]

[0256] Experiments were performed to determine whether single nucleotide editing could be efficiently performed using duplex sgRNAs with 2 nt truncations at the 5′ end.

[0257] Specifically, cells were electroporated with mutagenic oligonucleotides and a double-cutting sgRNA plasmid that introduced single nucleotide substitutions at each of the three mutagenic sites (Fig. 7a).

[0258] The percentages of white colonies obtained using oligonucleotides that induce mutations on Target1, between Targets, and on Target2 using duplex sgRNAs with 2 nt truncations at the 5′ end were 17%, 15%, and 29%, respectively (Fig. 7b).

[0259]

[0260] White colonies were randomly selected, and the nucleotide sequence of the edited galK target was analyzed using Sanger sequencing.

[0261] For On Target 1 and On Target 2, three and four of the four white colonies were accurately edited, respectively. In contrast, for Between Targets, unwanted mutations were found near the target sequence in four of the four white colonies (Fig. 7c).

[0262] These results demonstrated that nickase-mediated single-nucleotide editing can be performed using up to 5′-end truncated duplex sgRNAs and oligonucleotides introducing mutations into the target.

[0263]

[0264] Additionally, genome editing was performed in cells overexpressing Cas9-NG nickase and λ Red using the PCR product (~1 kb) and a 5′-end-truncated duplex sgRNA plasmid (Fig. 7d). For On Target1 and On Target2, the proportion of white colonies among the total colonies was 20% and 30%, respectively. For Between Targets, white colonies were rarely observed on MacConkey agar containing D-galactose (less than 2%, Fig. 7e).

[0265] The accuracy of single nucleotide editing was confirmed using Sanger sequencing (Fig. 7f).

[0266] When the nucleotide to be edited was located on the target, four and four of the four white colonies, respectively, showed accurate single-nucleotide substitutions (C490T and C523T). Conversely, when the nucleotide to be edited was located between the targets, single-nucleotide editing was unsuccessful.

[0267] The above results demonstrate that Cas9-NG nickase-mediated single nucleotide editing can be efficiently performed using a duplex sgRNA truncated by 2 nt at the 5′ end and a donor DNA (oligonucleotide or PCR product) containing the nucleotide to be edited at the target sequence.

[0268] Additionally, the proportion of single nucleotide edited colonies among 10 randomly selected colonies on LB agar was calculated.

[0269] After electroporation of pHK463 / SH169 cells with a 5′ truncated sgRNA and a mutagenic oligonucleotide inducing mutations in Target1, 10 candidate colonies that underwent single nucleotide editing were randomly selected on LB agar containing spectinomycin (Fig. 7g).

[0270] Sanger sequencing confirmed that three of ten randomly selected colonies were precisely edited at the single nucleotide level (Fig. 7h). This is consistent with the editing efficiency determined through phenotypic observation (Figs. 7b and 7e).

[0271]

[0272] The foregoing description of the present invention is provided for illustrative purposes only. Those skilled in the art will readily appreciate that the present invention can be readily modified into other specific forms without altering the technical spirit or essential characteristics of the present invention. Therefore, the embodiments described above should be understood as illustrative in all respects and not restrictive.

[0273] The present invention relates to a precise genome editing method based on a CRISPR-Cas nickase system. The genome editing method of the present invention can improve the precision of genome editing by reducing off-target effects, and is expected to be usefully utilized in various fields such as new drug development, genetic disease treatment, GMO (genetically modified organism) development, and production of model animals used for organ transplantation, and thus has industrial applicability.

Claims

1. A genome editing method based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to target DNA, dual guide RNA (dual gRNA), and Cas nickase, A genome editing method based on a CRISPR-Cas nickase system, comprising steps designed to have the following features: Truncation of the 5´ terminal sequence of the above gRNA; Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and Position of the protospacer adjacent motif (PAM) outside the double nick by the dual gRNA and Cas nickase.

2. In paragraph 1, The 5' terminal sequence truncation of the above gRNA is characterized by 1 or 2 nt truncation at the 5' terminal. Genome editing method based on the CRISPR-Cas nickase system.

3. In paragraph 1, The above Cas nickase is characterized in that it is Cas9-D10A nickase or Cas9-H840A nickase. Genome editing method based on the CRISPR-Cas nickase system.

4. In paragraph 1, The distance between the above double nicks is characterized by being 40 to 68 bp. Genome editing method based on the CRISPR-Cas nickase system.

5. In paragraph 1, The target recognition sequence of the above gRNA is characterized by being 18 to 20 nt long. Genome editing method based on the CRISPR-Cas nickase system.

6. In paragraph 1, The CRISPR-Cas nickase system is characterized by editing the genome through negative selection. Genome editing method based on the CRISPR-Cas nickase system.

7. In paragraph 1, The method is characterized by comprising the step of introducing the donor nucleic acid molecule, the dual gRNA and the Cas nickase into the subject. Genome editing method based on the CRISPR-Cas nickase system.

8. In paragraph 7, The above-mentioned introducing step is characterized in that it is performed by a local injection method, microinjection method, electroporation method or lipofection method. Genome editing method based on the CRISPR-Cas nickase system.

9. A composition for genome editing based on the CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system, comprising a donor nucleic acid molecule complementarily binding to target DNA, dual guide RNA (dual guide RNA, dual gRNA), and Cas nickase, A composition for genome editing based on the CRISPR-Cas nickase system, characterized in that it is designed to have the following features: Truncation of the 5´ terminal sequence of the above gRNA; Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and Position of the protospacer adjacent motif (PAM) outside the double nick by the dual gRNA and Cas nickase.

10. In paragraph 9, The above genome editing composition is a genome editing composition based on the CRISPR-Cas nickase system, which is intended for application to genome editing of a target organism.

11. A method for increasing genome editing efficiency based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to target DNA, dual guide RNA (dual gRNA), and Cas nickase, A method for increasing genome editing efficiency based on a CRISPR-Cas nickase system, comprising steps designed to have the following features: Truncation of the 5´ terminal sequence of the above gRNA; Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and Position of the protospacer adjacent motif (PAM) outside the double nick by the dual gRNA and Cas nickase.

12. A method for producing a prokaryotic cell, an isolated eukaryotic cell or a eukaryotic organism other than a human, wherein the target DNA is edited based on a CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system comprising a donor nucleic acid molecule complementarily binding to the target DNA, dual guide RNA (dual gRNA), and Cas nickase, A method for producing a prokaryotic cell, an isolated eukaryotic cell or a non-human eukaryotic organism having edited target DNA based on a CRISPR-Cas nickase system, comprising: a step of designing to have the following characteristics; and a step of introducing the donor nucleic acid molecule, the dual gRNA and the Cas nickase into the subject; Truncation of the 5´ terminal sequence of the above gRNA; Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and Position of the protospacer adjacent motif (PAM) outside the double nick by the dual gRNA and Cas nickase.

13. A composition comprising a donor nucleic acid molecule complementarily binding to target DNA, a dual guide RNA (dual gRNA), and a Cas nickase, wherein the composition is designed to have the following characteristics: CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system-based genome editing: Truncation of the 5´ terminal sequence of the above gRNA; Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and Position of the protospacer adjacent motif (PAM) outside the double nick by the dual gRNA and Cas nickase.

14. Use for manufacturing a genome editing agent based on CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated protein) nickase system, comprising a composition comprising a donor nucleic acid molecule complementarily binding to target DNA, a dual guide RNA (dual gRNA), and a Cas nickase, wherein the composition is designed to have the following characteristics: Truncation of the 5´ terminal sequence of the above gRNA; Overlapping of the target recognition sequence of the gRNA and the mutagenesis site of the donor nucleic acid molecule; and Position of the protospacer adjacent motif (PAM) outside the double nick by the dual gRNA and Cas nickase.

Images