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WO2022010286A1 - Donor nucleic acid used for gene correction through microhomology-mediated end joining and uses thereof - Google Patents

Donor nucleic acid used for gene correction through microhomology-mediated end joining and uses thereof Download PDF

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WO2022010286A1
WO2022010286A1 PCT/KR2021/008727 KR2021008727W WO2022010286A1 WO 2022010286 A1 WO2022010286 A1 WO 2022010286A1 KR 2021008727 W KR2021008727 W KR 2021008727W WO 2022010286 A1 WO2022010286 A1 WO 2022010286A1
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nucleic acid
sequence
donor
gene
donor nucleic
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Korean (ko)
Inventor
김재연
부티엔반
송영종
트란밀티
김지해
성연우
다스스와티
정재철
김차영
임가현
티 하이 드엉 도안
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Korea Research Institute of Bioscience and Biotechnology KRIBB
Gyeongsang National University GNU
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Korea Research Institute of Bioscience and Biotechnology KRIBB
Gyeongsang National University GNU
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/102—Mutagenizing nucleic acids
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]

Definitions

  • the present invention relates to a donor nucleic acid used for gene correction through microhomology-based end joining, and more specifically, a donor nucleic acid comprising a microhomology sequence, an altered PAM sequence, and a target gene segment; It relates to a gene editing system including the same, a gene editing method using the donor nucleic acid and the gene editing system, and a composition therefor.
  • cNHEJ Canonical non-homologous end-joining
  • MMEJ microhomology-mediated end joining pathway
  • cNHEJ does not use a homologous sequence as a template, but rather a pathway in which the cleaved ends of DNA are connected as it is by a complex of DNA-PKcs (DNA dependent protein kinase) and Ku protein, whereas the MMEJ pathway has microhomology. It is a pathway in which the DNA ends cut using the sequence as a template are linked independently of the Ku protein.
  • HR homologous recombination
  • the cNHEJ pathway method has the disadvantage that error-prone repair (mainly indel mutation addition) occurs at both ends of DNA, and the probability of precise gene insertion and replacement is very low due to such indels, This method has the disadvantage that it works with very low probability in higher plants. Therefore, the present invention was to induce precise DNA fragment insertion or replacement by the MMEJ pathway in plant cells, and to establish optimal conditions for high editing efficiency.
  • Korea Patent No. 2207728 discloses 'a method and composition for increasing the efficiency of targeted genetic modification using oligonucleotide-mediated gene repair'
  • Korea Patent No. 2002443 discloses 'a homologous recombination-based method in plants. Although a method of increasing gene editing efficiency has been disclosed, there is no description of the 'donor nucleic acid used for gene correction through microhomology-based end joining and use thereof' of the present invention.
  • the present invention was derived from the above needs, and the present inventors designed a donor nucleic acid comprising a microhomologous sequence and an altered PAM sequence in order to increase precise gene editing efficiency in plants, and using this, gene editing efficiency As a result of the analysis, it was confirmed that the editing efficiency increased compared to when using the donor nucleic acid not containing the microhomology sequence, and depending on the treatment concentration, length, or NHEJ inhibitor treatment of the donor nucleic acid containing the microhomology sequence By confirming that the editing efficiency can be different, the present invention was completed.
  • the present invention provides a 5'-end target site microhomology sequence as a donor nucleic acid used for gene correction through microhomology-mediated end joining (MMEJ). , a target gene sequence to be replaced and a 3'-terminal target site microhomology sequence, wherein the 5'-terminal and/or 3'-terminal target site microhomology sequence includes an altered PAM (Protospacer adjacent motif) sequence. It provides, characterized in that it comprises a donor (donor) nucleic acid.
  • MMEJ microhomology-mediated end joining
  • the present invention also provides a vector to which the donor nucleic acid is operably linked.
  • the present invention provides a gene scissors (CRISPR / Cas) system comprising the donor nucleic acid, CRISPR / Cas protein and guide RNA.
  • CRISPR / Cas gene scissors
  • the present invention provides a gene editing method comprising the step of replacing a target gene with a target gene segment in the donor nucleic acid using the donor nucleic acid.
  • the present invention provides a composition for gene editing comprising the donor nucleic acid.
  • the use of the donor nucleic acid used for gene correction through microhomology-based end binding according to the present invention enables precise gene editing without limiting the location and number of bases of the target gene, so that not only new breeding development of crops but also introduction or removal of SNPs are possible. And it is expected to be usefully used in the field of protein engineering through short amino acid substitution.
  • cNJ.HPAT3-1 is a donor nucleic acid (cNJ.HPAT3-1) and MMEJ-based genome editing for cNHEJ-based genome editing containing six (A1, A2, B, C, D1, D2) SNPs in the SIHPAT3 gene derived from tomato.
  • 3 is a result of analyzing the editing efficiency according to the length of the donor nucleic acid.
  • FIG. 4 is a diagram showing the sequences of donor nucleic acids (MJ.BoTT1, MJ.BoOr, and MJ.BoALS1) for MMEJ-based genome editing in cabbage-derived BoTT1 , BoOr and BoALS1 genes.
  • the underlined sequence means the guide RNA
  • the bolded sequence means the PAM sequence
  • the dotted box means the nucleotide sequence for single amino acid substitution.
  • the present invention provides a 5'-end target site microphase as a donor nucleic acid used for gene correction through microhomology-mediated end joining (MMEJ). It contains a homologous sequence, a target gene sequence to be replaced, and a 3'-terminal target site microhomology sequence, and the 5'-terminal and/or 3'-terminal target site microhomology sequence has an altered PAM (Protospacer adjacent motif) It provides a donor nucleic acid, characterized in that it comprises a sequence.
  • MMEJ microhomology-mediated end joining
  • the term “genome/gene editing” refers to a technology capable of introducing a target-directed mutation into the genome sequence of animal and plant cells, including human cells, and one or more nucleic acid molecules by DNA cleavage. Knock-out or knock-in of a specific gene by deletion, insertion, substitution, replacement, etc. of -Technology that can introduce mutations into non-coding DNA sequences.
  • the genome editing may be to introduce a mutation into a plant using an endonuclease, such as a CRISPR associated protein 9 (Cas9) protein and guide RNA.
  • an endonuclease such as a CRISPR associated protein 9 (Cas9) protein and guide RNA.
  • 'gene editing' may be used interchangeably with 'gene editing'.
  • target gene refers to some DNA in the genome of a plant to be corrected through the present invention, is not limited to the type of the gene, and may include both a coding region and a non-coding region. A person skilled in the art can select the target gene according to the target and the desired mutation for the genome editing plant to be prepared.
  • the 5'-end or 3'-end target site microhomology sequence may include one or more modified bases, but is not limited thereto.
  • Modification of the base is methylation (methylation), halogenation (halogenation), acetylation (acetylation), phosphorylation (phosphorylation), phosphorothioate linkage (phosphorothioate linkage), LNA (locked nucleic acid), MS (2' -O-methyl phosphorothioate) or MSP (2'-O-methyl plus 3'thioPACE), but is not limited thereto.
  • the 5'-terminal or 3'-terminal target site microhomology sequence may be preferably 5 or more and 24 or less oligonucleotides, and more preferably 5 or more, respectively. It may be more than 20 oligonucleotides, more preferably 11 or more and 20 or less oligonucleotides, and most preferably 20 oligonucleotides each, but is not limited thereto.
  • the modified PAM sequence is 5'-nHG-3', 5'-nGH-3, 5'-nH-3', 5'-nY-3', 5'-nHRR -3', 5'-DCn-3', 5'-CDn-3', 5'-Dn-3', 5'-Rn-3' or 5'-YYDn-3', wherein n is base A, G, T or C, H is base A, C or T, D is base A, G or T, Y is base C or T, and R is base A or G.
  • the donor nucleic acid of the present invention may be single-stranded or double-stranded.
  • the present invention provides a vector or ribonucleoprotein (RNP) to which the donor nucleic acid is operably linked.
  • RNP vector or ribonucleoprotein
  • the vector may be a plasmid vector, a viral vector, or a PCR amplicon, but is not limited thereto.
  • the present invention provides a gene scissors (CRISPR / Cas) system comprising the donor nucleic acid, CRISPR / Cas protein and guide RNA.
  • CRISPR / Cas gene scissors
  • guide RNA is a short single-stranded RNA, including RNA specific for a target DNA among nucleotide sequences encoding a target gene, and all or part of the target DNA nucleotide sequence is complementary It refers to a ribonucleic acid that binds and leads the endonuclease protein to the corresponding target DNA sequence.
  • the guide RNA may include two RNAs, that is, a dual RNA including crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as components; or a single chain comprising a first site comprising a sequence that is all or partly complementary to a nucleotide sequence in a target gene and a second site comprising a sequence that interacts with an endonuclease (especially an RNA-guided nuclease)
  • CRISPR RNA crRNA
  • tracrRNA trans-activating crRNA
  • a single chain comprising a first site comprising a sequence that is all or partly complementary to a nucleotide sequence in a target gene and a second site comprising a sequence that interacts with an endonuclease (especially an RNA-guided nuclease)
  • sgRNA single guide RNA
  • the endonuclease has activity in the target nucleotide sequence, it may be included in the scope of the present invention
  • the guide RNA may be transcribed from a plasmid template, transcribed in vitro (eg, oligonucleotide double-stranded), or synthesized guide RNA, but is not limited thereto.
  • the Cas protein may preferably be a Cas9 protein, but is not limited thereto.
  • the Cas9 protein is Streptococcus pyogenes ( Streptococcus pyogenes )-derived Cas9 protein, Campylobacter jejuni ( Campylobacter jejuni )-derived Cas9 protein, Streptococcus thermophilus ) or Streptococcus aureus ( Streptococcus aureus ) Cas9 protein derived from, Neisseria meningitidis ( Neisseria meningitidis ) Cas9 protein derived from, Pasteurella multocida ( Pasteurella multocida ) Cas9 protein derived from, Francisella novicida ( Francisella novicida ) consisting of Cas9 protein derived from It may be one or more selected from the group, but is not limited thereto. Cas9 protein or its genetic information can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI).
  • NCBI National Center for Biotechnology Information
  • Cas9 protein is an RNA-guided DNA endonuclease enzyme that induces double-stranded DNA breaks.
  • PAM Protospacer Adjacent Motif
  • the Cas protein may be SpCas9, xCas9, SpG Cas9, NG Cas9, SpRY SpCas9 or SaCas9.
  • donor nucleic acids (5'-xxxxxxxxnnnnnHGnnnnn*...*nnnnnnnnDCnnnnnyyyyyyyyyyyy-3', 5'-xxxxxxxxxxnnnnGHnnnnn*...*nnnnnnnCDnnnnnnnyyyyyyyyyyyyyyy-3' or 5'-xxxxxxxxxnnny...
  • nHG, nGH, nHH, DCn, CDn or DDn may include If the change of the PAM sequence is not desired, one or more SNPs can be added in the gRNA seed sequence within 13 nucleotides from the PAM sequence to prevent cleavage of the donor nucleic acid by the Cas protein.
  • H refers to a base A, C or T
  • D refers to a base A, G or T
  • n refers to a base A, G, C or T.
  • the donor nucleic acid (5'-xxxxxxxxxxnnnnHnnnnnn*...*nnnnnnnnDnnnnnnyyyyyyy-3') is a target site microhomology sequence (xxxxxxxx or yyyyyyyy) at both ends. ), the target gene segment sequence to be replaced (*...*) of the target gene and the altered PAM sequence (nH or Dn).
  • one or more SNPs can be added in the gRNA seed sequence within 13 nucleotides from the PAM sequence to prevent cleavage of the donor nucleic acid by the Cas protein.
  • H refers to a base A, C or T
  • D refers to a base A, G or T
  • n refers to a base A, G, C or T.
  • SpRY SpCas9 When SpRY SpCas9 is used in the gene editing system according to the present invention, SpRY SpCas9 recognizes not only the nRn PAM sequence but also nYn to a lesser extent, thereby maintaining the PAM as nYn and 1 in the gRNA seed sequence within 13 nucleotides from the PAM. More than one SNP can be added.
  • the donor nucleic acid (5'-xxxxxxxx###nYnnnnnn*...*nnnnnnnnRn###yyyyyyy-3') is a target site microhomology sequence (xxxxxxxx or yyyyyyyy) at both ends, the target gene segment sequence to be replaced ( *...*) and an altered PAM sequence (nY or Rn) and 1-3 altered gRNA seed sequences (###).
  • Y means a base C or T
  • R means a base A or G
  • n means a base A, G, C or T.
  • the donor nucleic acid (5'-xxxxxxxxxxnnnnHRRnnnn*...*nnnnnnYYDnnnnnyyyyyyy-3') is a target site microhomology sequence (xxxxxxxx or yyyyyyyy) at both ends, the target gene to be replaced It may include a target gene segment sequence (*...*) and an altered PAM sequence (nHRR or YYDn). If the change of the PAM sequence is not desired, one or more SNPs can be added in the gRNA seed sequence within 13 nucleotides of the PAM to prevent cleavage of the donor by the Cas protein.
  • H refers to a base A, C or T
  • R refers to a base A or G
  • Y refers to a base C or T
  • D refers to a base A, G or T
  • n is base A, G, C or T.
  • the present invention provides a gene editing method comprising the step of replacing a target gene with a target gene segment in the donor nucleic acid using the donor nucleic acid.
  • the gene editing method used in the present invention is a microhomology-mediated end joining (MMEJ) mediated gene editing method using microhomology having the same sequence as the specific sequence of a specific gene to be corrected.
  • MMEJ microhomology-mediated end joining
  • the editing efficiency is increased compared to when the canonical non-homologous end-joining (cNHEJ)-mediated gene editing method is used. Editing efficiency may vary depending on the treatment concentration, length, and NHEJ inhibitor treatment of the nucleic acid.
  • the gene editing method according to the present invention may be performed in bacteria, yeast, plant cells or animal cells, preferably in plant cells, but is not limited thereto.
  • the introduction of the donor nucleic acid, guide RNA and Cas protein into plant cells comprises DNA encoding the donor nucleic acid, DNA encoding the guide RNA specific to the target nucleotide sequence, and the Cas protein encoding a recombinant vector comprising a DNA sequence;
  • a donor nucleic acid or a complex of a guide RNA specific to a target nucleotide sequence and a Cas protein may be used, but is not limited thereto.
  • the method for transducing the complex of guide RNA and Cas protein into plant cells is a calcium/polyethylene glycol chamber for protoplasts, electroporation of protoplasts, micro-injection method with plant elements, various plants Particle bombardment of urea (DNA or RNA-coated), Agrobacterium tumefaciens or Agrobacterium rhizogens mediated (incomplete) bacterial infection in gene transfer, etc. may be appropriately selected from
  • introducing a recombinant vector comprising a DNA encoding a guide RNA specific for the target nucleotide sequence and a nucleic acid sequence encoding a Cas protein into a plant cell refers to a transformation method. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the recombinant vector according to the invention into suitable progenitor cells.
  • a "plant cell” into which a guide RNA specific for a target nucleotide sequence and a Cas protein are introduced may be any plant cell.
  • a plant cell is a cultured cell, cultured tissue, cultured organ or whole plant.
  • Plant tissue refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, cotyledons, hypocotyls, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, i.e. Includes single cell, protoplast, shoot and callus tissue.
  • the plant tissue may be in planta or in an organ culture, tissue culture or cell culture state.
  • any method known in the art may be used as a method of redifferentiating a plant having a corrected genome from a plant cell having a corrected genome.
  • Plant cells whose genome has been corrected must be redifferentiated into whole plants.
  • Techniques for the redifferentiation of mature plants from callus or protoplast cultures are well known in the art for a number of different species.
  • the present invention provides a composition for gene editing comprising a donor nucleic acid.
  • composition for gene editing may further include a CRISPR/Cas protein and a guide RNA, wherein the Cas protein and the guide RNA form a ribonucleoprotein complex to form RNA-guided engineered nuclease (RNA-Guided Engineered Nuclease). , RGEN).
  • RNA-Guided Engineered Nuclease RNA-Guided Engineered Nuclease
  • the CRISPR/Cas protein and the guide RNA are as described above.
  • protoplasts were isolated using a 21% sucrose density gradient method and W5 (2 mM MES pH 5.8, 154 mM NaCl, 125 mM CaCl) 2 , 5 mM KCl) solution.
  • W5 2 mM MES pH 5.8, 154 mM NaCl, 125 mM CaCl) 2 , 5 mM KCl
  • the collected protoplasts were washed three times with W5 solution, resuspended in MMG (4 mM MES pH 5.7, 0.4 M mannitol, 15 mM MgCl 2 ) solution, and the concentration was measured using a hemocytometer.
  • SlHPAT3 Solanum lycopersicum Hydroxyproline O-arabinosyltransferase 3
  • SlHPAT3 Solyc07g021170.1
  • guide RNAs were designed using the CRISPR-P 2.0 program (Table 1), and guide RNAs were synthesized using the GeneArt Precision gRNA Synthesis Kit (Invitrogen).
  • the donor nucleic acids for the cNHEJ pathway (cNJ.HPAT3-1) and the donor nucleic acids for the MMEJ pathway (MJ.HPAT3-1, MJ.HPAT3-2 and MJ.HPAT3-3) were subjected to PCR using the primers in Table 1 below. prepared.
  • the 5'-end of cNJ.HPAT3-1 was prepared to have a phosphodiester bond.
  • the PCR amplification product was modified with phosphorothioate phosphorylated at the 5' end to increase stability.
  • thermo-tolerance 1 (TT1), orange (Or) and acetolactate synthase 1 (ALS1) genes of cabbage plants
  • guide RNAs were designed using the CRISPR-P 2.0 program (Table 1), and guide RNAs were synthesized using the GeneArt Precision gRNA Synthesis Kit (Invitrogen).
  • Donor nucleic acids for the MMEJ pathway (MJ.BoTT1, MJ.BoOr and MJ.BoALS1) were prepared through PCR using the primers in Table 1 below. The concentration of donor nucleic acid was measured using a Nanodrop2000 spectrophotometer (Thermofisher, USA).
  • PBS buffer containing 20 ⁇ g SpCas9 protein (ToolGen, Inc. South Korea), 10 ⁇ g guide RNA, and 300 pmol donor nucleic acid was added to 2 ⁇ 10 5 ml of tomato or cabbage protoplasts and reacted at 25° C. for 10 minutes. Then, add RNP (ribonucleoprotein) complex, mix well, add PEG (polyethylene glycol) solution (40% (w/v) PEG4000, 0.2 M mannitol, 0.1 M CaCl 2 ), mix well, and then at room temperature for 10 minutes reacted while After washing with W5 solution and centrifuging at 100 g for 5 minutes, the supernatant was removed, and W5 solution was added thereto, followed by reaction at 25° C. and dark conditions for 48 hours.
  • RNP ribonucleoprotein
  • Example 1 Analysis of editing efficiency of cNHEJ-based and MMEJ-based genome editing methods
  • Canonical non-homologous end-joining (cNHEJ)-based genome editing is a method using donor nucleic acids that do not have microhomologous ends, and microhomology-mediated end joining (MMEJ)-based gene editing uses donor nucleic acids having microhomologous ends. way.
  • Donor nucleic acids (MJ.HPAT3-1, MJ.HPAT3-2 and MJ.HPAT3-3) for MMEJ-based genome editing were prepared.
  • MJ.HPAT3-1, MJ.HPAT3-2 and MJ.HPAT3-3 are 5, 10, and 20 microhomologous sequences from the 3rd nucleotide sequence upstream of both PAM sites of the target SlHPAT3 gene, respectively.
  • the DNA fragment at a specific site in the target gene was replaced with the donor nucleic acid sequence (FIG. 1).
  • the editing frequency for each SNP (A1, A2, B, C, D1, D2) position of the product corrected by the cNJ.HPAT3-1 donor nucleic acid was analyzed. As a result, it was confirmed that 94.74% of the total edited reads were reads in which the correction occurred at the SNP at the B-C position. In addition, reads in which correction occurred in both ends of the SNPs (A1-A2, D1-D2) were not identified, and it is predicted that the ends of the donor nucleic acid were damaged by an unknown pathway before being used for genome editing (Table). 4).
  • Editing frequency according to donor nucleic acid edited SNPs DNA Donor cNJ.HPAT3-1 MJ.HPAT3-1 Number of edited reads editing frequency(%) Number of edited reads editing frequency(%) A1-A2-B-C-D1-D2 0 0 237 9.72 A1-A2 0 0 872 35.77 A1-A2-B-C 0 0 150 6.15 B-C 72 94.74 169 6.93 B-C-D1-D2 4 5.26 249 10.21 D1-D2 0 0 761 31.21 edited reads 76 100 2438 100 total reads 93054 - 99885 -
  • the frequency of gene editing was analyzed.
  • the editing frequency appeared at a similar level as the treatment concentration of the cNJ.HPAT3-1 donor nucleic acid increased, whereas as the treatment concentration of the MJ.HPAT3-1 donor nucleic acid increased, all SNPs (A1-A2-BC-D1-D2) ), it was confirmed that the frequency of precise editing in which editing occurred also increased (FIG. 2).
  • MJ.HPAT3-1 donor nucleic acid with a microhomology sequence of 20 bp in length MJ.HPAT3-2 donor nucleic acid with a microhomology sequence of 10 bp in length
  • MJ.HPAT3- with a microhomology sequence of 5 bp in length After each of the 3 donor nucleic acids was treated, the gene editing efficiency was analyzed. As a result, as the length of the microhomologous sequence increased, the target total editing efficiency, the precision editing efficiency in all SNPs (A1-A2-BC-D1-D2), and the editing efficiency in the SNPs at positions B and C And it was confirmed that both ends (A1-A2, D1-D2) the editing efficiency that occurred in both SNPs increased (FIG. 3).
  • Nu7441 a chemical that inhibits DNA dependent protein kinase (DNA-PKcs) activity acting in the NHEJ pathway, was treated at concentrations of 0.5, 1, and 2 ⁇ M, respectively, and then gene editing efficiency was analyzed. As a result, it was confirmed that the gene editing efficiency was increased by treatment with Nu7441. In particular, it was confirmed that the editing efficiency using the cNJ.HPAT3-1 donor nucleic acid not having a microhomologous sequence was increased by about 1.88 times when Nu7441 was treated at 2 ⁇ M compared to when Nu7441 was not treated (Table 5).
  • DNA-PKcs DNA dependent protein kinase
  • thermo-tolerance 1 (TT1), orange (Or) and acetolactate synthase 1 (ALS1) genes of cabbage plants using the MMEJ-based genome editing method according to the present invention, the traits of heat resistance, carotenoid accumulation and herbicide tolerance are obtained, respectively. It was intended to manufacture a plant.
  • Donor nucleic acids for MMEJ-based genome editing (MJ.BoTT1, MJ.BoOr and MJ.BoALS1) were prepared to induce single amino acid substitutions, and upstream of both PAM sites of the targeted BoTT1 , BoOr and BoALS1 genes 3
  • a DNA fragment at a specific site in the target gene was replaced with the donor nucleic acid sequence.
  • CGC (Arg) base sequence of BoTT1 gene CAT (His) were to be replaced to CGT (Arg) base sequence of BoOr gene to CAC (His), TCT to CCT (Pro) base sequence of BoALS1 gene ( Ser) was replaced with (Fig. 4).

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Abstract

The present invention relates to a donor nucleic acid used for gene correction through microhomology-mediated end joining and uses thereof. The use of the donor nucleic acid according to the present invention enables precise gene editing without limiting the location of a target gene and the number of bases. Therefore, the present invention is expected to be usefully used in the field of protein engineering through the introduction or removal of SNPs and the substitution of short amino acids, as well as in the new breeding development of crops.

Description

미세상동성 기반 말단 결합을 통한 유전자 교정에 이용되는 공여자 핵산 및 이의 용도Donor nucleic acid used for gene correction through microhomology-based end joining and use thereof

본 발명은 미세상동성 기반 말단 결합을 통한 유전자 교정에 이용되는 공여자(Donor) 핵산에 관한 것으로, 보다 구체적으로는 미세상동성 서열, 변경된 PAM 서열 및 목적 유전자 절편을 포함하는 공여자(Donor) 핵산 및 이를 포함하는 유전자가위 시스템, 상기 공여자 핵산 및 유전자가위 시스템을 이용한 유전자 편집 방법과 이를 위한 조성물에 관한 것이다. The present invention relates to a donor nucleic acid used for gene correction through microhomology-based end joining, and more specifically, a donor nucleic acid comprising a microhomology sequence, an altered PAM sequence, and a target gene segment; It relates to a gene editing system including the same, a gene editing method using the donor nucleic acid and the gene editing system, and a composition therefor.

비상동 말단 결합(canonical non-homologous end-joining, cNHEJ) 및 미세상동성 기반 말단 결합(microhomology-mediated end joining, MMEJ) 경로는 CRISPR/Cas9 기반의 정밀 유전체 편집에 관여한다고 알려져 있다. cNHEJ는 상동성을 가지는 서열을 주형으로 이용하는 것이 아니라 절단된 DNA 말단이 DNA-PKcs(DNA dependent protein kinase)와 Ku 단백질의 복합체에 의하여 그대로 연결되는 경로인 반면, MMEJ 경로는 미세상동성(microhomology) 서열을 주형으로 이용하여 절단된 DNA 말단이 Ku 단백질 비의존적으로 연결되는 경로이다. 최근, 유전자의 삽입 또는 대체를 위하여 cNHEJ 경로 및 상동 재조합(homologous recombination, HR)을 이용하는 방법에 관한 연구가 보고된 바 있다. 그러나, cNHEJ 경로에 의한 방법은 DNA의 양 말단에서 error-prone repair(주로 indel 변이 추가)가 일어나는 것이 주된 경로로 이러한 indel로 인해 정밀한 유전자 삽입 및 대체의 확률이 매우 낮다는 단점이 있으며, 상동 재조합에 의한 방법은 고등식물에서 매우 낮은 확률로 작동한다는 단점이 있다. 따라서, 본 발명은 식물세포에서 MMEJ 경로에 의한 방법으로 정밀한 DNA의 절편 삽입 또는 대체를 유도하고, 높은 편집 효율을 위한 최적의 조건을 확립하고자 하였다. Canonical non-homologous end-joining (cNHEJ) and microhomology-mediated end joining (MMEJ) pathways are known to be involved in CRISPR/Cas9-based precision genome editing. cNHEJ does not use a homologous sequence as a template, but rather a pathway in which the cleaved ends of DNA are connected as it is by a complex of DNA-PKcs (DNA dependent protein kinase) and Ku protein, whereas the MMEJ pathway has microhomology. It is a pathway in which the DNA ends cut using the sequence as a template are linked independently of the Ku protein. Recently, studies on a method using the cNHEJ pathway and homologous recombination (HR) for gene insertion or replacement have been reported. However, the cNHEJ pathway method has the disadvantage that error-prone repair (mainly indel mutation addition) occurs at both ends of DNA, and the probability of precise gene insertion and replacement is very low due to such indels, This method has the disadvantage that it works with very low probability in higher plants. Therefore, the present invention was to induce precise DNA fragment insertion or replacement by the MMEJ pathway in plant cells, and to establish optimal conditions for high editing efficiency.

또한, 두 개의 가이드 RNA 및 Cas9 단백질을 사용하여 원하는 표적 부위를 절단하고, 교정하고자 하는 부위의 양 말단과 미세상동성 서열을 가지는 공여자 핵산을 주입함으로써, 원하는 서열을 가지는 DNA 절편으로 대체하는 정밀 편집 방법을 개발하고자 하였다. In addition, by cutting the desired target site using two guide RNAs and Cas9 protein, and injecting a donor nucleic acid having a microhomologous sequence with both ends of the site to be corrected, precise editing that replaces a DNA fragment with a desired sequence I tried to develop a method.

한편, 한국등록특허 제2207728호에는 '올리고뉴클레오타이드 매개 유전자 보수를 사용한 표적화된 유전자 변형의 효율을 증가시키기 위한 방법 및 조성물'이 개시되어 있고, 한국등록특허 제2002443호에는 '식물체에서 상동재조합 기반의 유전자 편집 효율을 증가시키는 방법'이 개시되어 있으나, 본 발명의 '미세상동성 기반 말단 결합을 통한 유전자 교정에 이용되는 공여자 핵산 및 이의 용도'에 대해서는 기재된 바가 없다.Meanwhile, Korea Patent No. 2207728 discloses 'a method and composition for increasing the efficiency of targeted genetic modification using oligonucleotide-mediated gene repair', and Korea Patent No. 2002443 discloses 'a homologous recombination-based method in plants. Although a method of increasing gene editing efficiency has been disclosed, there is no description of the 'donor nucleic acid used for gene correction through microhomology-based end joining and use thereof' of the present invention.

본 발명은 상기와 같은 요구에 의해 도출된 것으로서, 본 발명자들은 식물체에서 정밀한 유전자 편집 효율을 증가시키기 위해서, 미세상동성 서열 및 변경된 PAM 서열을 포함하는 공여자 핵산을 디자인하였고, 이를 이용하여 유전자 편집 효율을 분석한 결과, 미세상동성 서열을 포함하지 않는 공여자 핵산을 이용했을 때보다 편집 효율이 증가하는 것을 확인하였으며, 상기 미세상동성 서열을 포함하는 공여자 핵산의 처리 농도, 길이 또는 NHEJ 저해제 처리에 따라 편집 효율이 달라질 수 있음을 확인함으로써, 본 발명을 완성하였다. The present invention was derived from the above needs, and the present inventors designed a donor nucleic acid comprising a microhomologous sequence and an altered PAM sequence in order to increase precise gene editing efficiency in plants, and using this, gene editing efficiency As a result of the analysis, it was confirmed that the editing efficiency increased compared to when using the donor nucleic acid not containing the microhomology sequence, and depending on the treatment concentration, length, or NHEJ inhibitor treatment of the donor nucleic acid containing the microhomology sequence By confirming that the editing efficiency can be different, the present invention was completed.

상기 과제를 해결하기 위해, 본 발명은 미세상동성-기반 말단 결합(microhomology-mediated end joining, MMEJ)을 통한 유전자 교정에 이용되는 공여자(donor) 핵산으로서, 5'-말단 표적 부위 미세상동성 서열, 교체하고자 하는 목적 유전자 서열 및 3'-말단 표적 부위 미세상동성 서열을 포함하며, 상기 5'-말단 및/또는 3'-말단 표적 부위 미세상동성 서열에는 변경된 PAM(Protospacer adjacent motif) 서열을 포함하는 것을 특징으로 하는, 공여자(donor) 핵산을 제공한다. In order to solve the above problems, the present invention provides a 5'-end target site microhomology sequence as a donor nucleic acid used for gene correction through microhomology-mediated end joining (MMEJ). , a target gene sequence to be replaced and a 3'-terminal target site microhomology sequence, wherein the 5'-terminal and/or 3'-terminal target site microhomology sequence includes an altered PAM (Protospacer adjacent motif) sequence. It provides, characterized in that it comprises a donor (donor) nucleic acid.

또한, 본 발명은 상기 공여자(donor) 핵산이 작동가능하게 연결된, 벡터를 제공한다.The present invention also provides a vector to which the donor nucleic acid is operably linked.

또한, 본 발명은 상기 공여자(donor) 핵산, CRISPR/Cas 단백질 및 가이드 RNA를 포함하는, 유전자가위(CRISPR/Cas) 시스템을 제공한다.In addition, the present invention provides a gene scissors (CRISPR / Cas) system comprising the donor nucleic acid, CRISPR / Cas protein and guide RNA.

또한, 본 발명은 상기 공여자(donor) 핵산을 이용하여 표적 유전자를 상기 공여자 핵산 내의 목적 유전자 절편으로 교체하는 단계를 포함하는, 유전자 편집 방법을 제공한다.In addition, the present invention provides a gene editing method comprising the step of replacing a target gene with a target gene segment in the donor nucleic acid using the donor nucleic acid.

또한, 본 발명은 상기 공여자(donor) 핵산을 포함하는, 유전자 편집용 조성물을 제공한다. In addition, the present invention provides a composition for gene editing comprising the donor nucleic acid.

본 발명에 따른 미세상동성 기반 말단 결합을 통한 유전자 교정에 이용되는 공여자 핵산을 이용하면 표적 유전자의 위치 및 염기 수에 제한 없이 정밀한 유전자 편집이 가능하므로, 작물의 신육종 개발 뿐만 아니라 SNP의 도입이나 제거 및 짧은 아미노산 치환을 통한 단백질 엔지니어링 분야에 유용하게 사용할 수 있을 것으로 기대된다. The use of the donor nucleic acid used for gene correction through microhomology-based end binding according to the present invention enables precise gene editing without limiting the location and number of bases of the target gene, so that not only new breeding development of crops but also introduction or removal of SNPs are possible. And it is expected to be usefully used in the field of protein engineering through short amino acid substitution.

도 1은 토마토 유래의 SIHPAT3 유전자에서 6개(A1, A2, B, C, D1, D2)의 SNP를 포함하는 cNHEJ 기반 유전체 교정을 위한 공여자 핵산(cNJ.HPAT3-1) 및 MMEJ 기반 유전체 교정을 위한 공여자 핵산(MJ.HPAT3-1, MJ.HPAT3-2 및 MJ.HPAT3-3)의 서열을 보여주는 그림이다. SIHPAT3 유전자 서열에서 밑줄친 서열은 가이드 RNA를 의미하고, 진하게 표시된 서열은 PAM 서열을 의미한다. 1 is a donor nucleic acid (cNJ.HPAT3-1) and MMEJ-based genome editing for cNHEJ-based genome editing containing six (A1, A2, B, C, D1, D2) SNPs in the SIHPAT3 gene derived from tomato. Figures showing the sequences of donor nucleic acids (MJ.HPAT3-1, MJ.HPAT3-2 and MJ.HPAT3-3) for In the SIHPAT3 gene sequence, the underlined sequence means the guide RNA, and the bold sequence means the PAM sequence.

도 2는 공여자 핵산의 처리 농도에 따른 편집 빈도를 분석한 결과이다. 2 is a result of analyzing the editing frequency according to the treatment concentration of the donor nucleic acid.

도 3은 공여자 핵산의 길이에 따른 편집 효율을 분석한 결과이다. 3 is a result of analyzing the editing efficiency according to the length of the donor nucleic acid.

도 4는 양배추 유래의 BoTT1, BoOr 및 BoALS1 유전자에서 MMEJ 기반 유전체 교정을 위한 공여자 핵산(MJ.BoTT1, MJ.BoOr 및 MJ.BoALS1)의 서열을 보여주는 그림이다. BoTT1, BoOr 및 BoALS1 유전자 서열에서 밑줄친 서열은 가이드 RNA를 의미하고, 진하게 표시된 서열은 PAM 서열을 의미하며, 점선 박스는 단일 아미노산 치환을 위한 염기 서열을 의미한다.4 is a diagram showing the sequences of donor nucleic acids (MJ.BoTT1, MJ.BoOr, and MJ.BoALS1) for MMEJ-based genome editing in cabbage-derived BoTT1 , BoOr and BoALS1 genes. In the BoTT1 , BoOr and BoALS1 gene sequences, the underlined sequence means the guide RNA, the bolded sequence means the PAM sequence, and the dotted box means the nucleotide sequence for single amino acid substitution.

본 발명의 목적을 달성하기 위하여, 본 발명은 미세상동성-기반 말단 결합(microhomology-mediated end joining, MMEJ)을 통한 유전자 교정에 이용되는 공여자(donor) 핵산으로서, 5'-말단 표적 부위 미세상동성 서열, 교체하고자 하는 목적 유전자 서열 및 3'-말단 표적 부위 미세상동성 서열을 포함하며, 상기 5'-말단 및/또는 3'-말단 표적 부위 미세상동성 서열에는 변경된 PAM(Protospacer adjacent motif) 서열을 포함하는 것을 특징으로 하는, 공여자(donor) 핵산을 제공한다.In order to achieve the object of the present invention, the present invention provides a 5'-end target site microphase as a donor nucleic acid used for gene correction through microhomology-mediated end joining (MMEJ). It contains a homologous sequence, a target gene sequence to be replaced, and a 3'-terminal target site microhomology sequence, and the 5'-terminal and/or 3'-terminal target site microhomology sequence has an altered PAM (Protospacer adjacent motif) It provides a donor nucleic acid, characterized in that it comprises a sequence.

본 명세서에서 용어 "유전체/유전자 교정(genome/gene editing)"은, 인간 세포를 비롯한 동·식물세포의 유전체 염기서열에 표적지향형 변이를 도입할 수 있는 기술로서, DNA 절단에 의한 하나 이상의 핵산 분자의 결실(deletion), 삽입(insertion), 치환(substitution), 교체(replacement) 등에 의하여 특정 유전자를 녹-아웃(knock-out) 또는 녹-인(knock-in)하거나, 단백질을 생성하지 않는 비-코딩(non-coding) DNA 서열에도 변이를 도입할 수 있는 기술을 말한다. As used herein, the term “genome/gene editing” refers to a technology capable of introducing a target-directed mutation into the genome sequence of animal and plant cells, including human cells, and one or more nucleic acid molecules by DNA cleavage. Knock-out or knock-in of a specific gene by deletion, insertion, substitution, replacement, etc. of -Technology that can introduce mutations into non-coding DNA sequences.

본 발명의 표적상 상기 유전체 교정은 특히 엔도뉴클레아제(endonuclease) 예컨대, Cas9(CRISPR associated protein 9) 단백질 및 가이드 RNA를 이용하여 식물체에 변이를 도입하는 것일 수 있다. 또한, 용어 '유전자 교정'은 '유전자 편집'과 혼용되어 사용될 수 있다.In the target of the present invention, the genome editing may be to introduce a mutation into a plant using an endonuclease, such as a CRISPR associated protein 9 (Cas9) protein and guide RNA. Also, the term 'gene editing' may be used interchangeably with 'gene editing'.

또한, 용어 "표적 유전자"는 본 발명을 통해 교정하고자 하는 식물체의 유전체 내에 있는 일부 DNA를 의미하며, 그 유전자의 종류에 제한되지 않으며, 코딩 영역 및 비-코딩 영역을 모두 포함할 수 있다. 당업자는 그 표적에 따라, 제조하고자 하는 유전체 교정 식물체에 대하여 원하는 변이에 따라 상기 표적 유전자를 선별할 수 있다.In addition, the term "target gene" refers to some DNA in the genome of a plant to be corrected through the present invention, is not limited to the type of the gene, and may include both a coding region and a non-coding region. A person skilled in the art can select the target gene according to the target and the desired mutation for the genome editing plant to be prepared.

본 발명의 공여자(donor) 핵산에 있어서, 상기 5'-말단 또는 3'-말단 표적 부위 미세상동성 서열은 하나 이상의 변형된 염기(Base)를 포함하는 것일 수 있으나, 이에 제한되지 않는다. In the donor nucleic acid of the present invention, the 5'-end or 3'-end target site microhomology sequence may include one or more modified bases, but is not limited thereto.

상기 염기(Base)의 변형은 메틸화(methylation), 할로겐화(halogenation), 아세틸화(acetylation), 인산화(phosphorylation), 포스포로티오에이트 연결(phosphorothioate linkage), LNA(locked nucleic acid), MS(2'-O-methyl phosphorothioate) 또는 MSP(2'-O-methyl plus 3'thioPACE)일 수 있으나, 이에 제한되지 않는다. Modification of the base is methylation (methylation), halogenation (halogenation), acetylation (acetylation), phosphorylation (phosphorylation), phosphorothioate linkage (phosphorothioate linkage), LNA (locked nucleic acid), MS (2' -O-methyl phosphorothioate) or MSP (2'-O-methyl plus 3'thioPACE), but is not limited thereto.

또한, 본 발명의 공여자 핵산에 있어서, 상기 5'-말단 또는 3'-말단 표적 부위 미세상동성 서열은 바람직하게는 각각 5개 이상 24개 이하의 올리고뉴클레오티드일 수 있고, 더욱 바람직하게는 각각 5개 이상 20개 이하의 올리고뉴클레오티드일 수 있고, 더더욱 바람직하게는 각각 11개 이상 20개 이하의 올리고뉴클레오티드일 수 있으며, 가장 바람직하게는 각각 20개의 올리고뉴클레오티드일 수 있으나, 이에 제한되지 않는다. In addition, in the donor nucleic acid of the present invention, the 5'-terminal or 3'-terminal target site microhomology sequence may be preferably 5 or more and 24 or less oligonucleotides, and more preferably 5 or more, respectively. It may be more than 20 oligonucleotides, more preferably 11 or more and 20 or less oligonucleotides, and most preferably 20 oligonucleotides each, but is not limited thereto.

또한, 본 발명의 공여자 핵산에 있어서, 상기 변경된 PAM 서열은 5'-nHG-3', 5'-nGH-3, 5'-nH-3', 5'-nY-3', 5'-nHRR-3', 5'-DCn-3', 5'-CDn-3', 5'-Dn-3', 5'-Rn-3' 또는 5'-YYDn-3'을 포함하며, 여기서 n은 염기 A, G, T 또는 C이고, H는 염기 A, C 또는 T이고, D는 염기 A, G 또는 T이고, Y는 염기 C 또는 T이고, R은 염기 A 또는 G인 것일 수 있다. In addition, in the donor nucleic acid of the present invention, the modified PAM sequence is 5'-nHG-3', 5'-nGH-3, 5'-nH-3', 5'-nY-3', 5'-nHRR -3', 5'-DCn-3', 5'-CDn-3', 5'-Dn-3', 5'-Rn-3' or 5'-YYDn-3', wherein n is base A, G, T or C, H is base A, C or T, D is base A, G or T, Y is base C or T, and R is base A or G.

또한, 본 발명의 공여자 핵산은 단일 가닥 또는 이중 가닥인 것일 수 있다. In addition, the donor nucleic acid of the present invention may be single-stranded or double-stranded.

또한, 본 발명은 상기 공여자(donor) 핵산이 작동가능하게 연결된, 벡터 또는 리보뉴클레오단백질(Ribonucleoprotein, RNP)을 제공한다. In addition, the present invention provides a vector or ribonucleoprotein (RNP) to which the donor nucleic acid is operably linked.

상기 벡터는 플라스미드 벡터, 바이러스 벡터 또는 PCR 엠플리콘일 수 있으나, 이에 제한되지 않는다. The vector may be a plasmid vector, a viral vector, or a PCR amplicon, but is not limited thereto.

또한, 본 발명은 상기 공여자(donor) 핵산, CRISPR/Cas 단백질 및 가이드 RNA를 포함하는, 유전자가위(CRISPR/Cas) 시스템을 제공한다. In addition, the present invention provides a gene scissors (CRISPR / Cas) system comprising the donor nucleic acid, CRISPR / Cas protein and guide RNA.

본 명세서에서 용어 "가이드 RNA(guide RNA)"는 짧은 단일 가닥의 RNA로, 표적 유전자를 암호화하는 염기서열 중 표적 DNA에 특이적인 RNA를 포함하며, 표적 DNA 염기서열과 전부 또는 일부가 상보적으로 결합하여 해당 표적 DNA 염기서열로 엔도뉴클레아제 단백질을 이끄는 역할을 하는 리보핵산을 의미한다. 상기 가이드 RNA는 두 개의 RNA, 즉, crRNA(CRISPR RNA) 및 tracrRNA(trans-activating crRNA)를 구성 요소로 포함하는 이중 RNA(dual RNA); 또는 표적 유전자 내 염기서열과 전부 또는 일부 상보적인 서열을 포함하는 제1 부위 및 엔도뉴클레아제(특히, RNA-가이드 뉴클레아제)와 상호작용하는 서열을 포함하는 제2 부위를 포함하는 단일 사슬 가이드 RNA(single guide RNA, sgRNA) 형태를 말하나, 엔도뉴클레아제가 표적 염기서열에서 활성을 가질 수 있는 형태라면 제한없이 본 발명의 범위에 포함될 수 있으며, 함께 사용된 엔도뉴클레아제의 종류 또는 엔도뉴클레아제의 유래 미생물 등을 고려하여 당업계의 공지된 기술에 따라 제조하여 사용할 수 있다.As used herein, the term “guide RNA” is a short single-stranded RNA, including RNA specific for a target DNA among nucleotide sequences encoding a target gene, and all or part of the target DNA nucleotide sequence is complementary It refers to a ribonucleic acid that binds and leads the endonuclease protein to the corresponding target DNA sequence. The guide RNA may include two RNAs, that is, a dual RNA including crRNA (CRISPR RNA) and tracrRNA (trans-activating crRNA) as components; or a single chain comprising a first site comprising a sequence that is all or partly complementary to a nucleotide sequence in a target gene and a second site comprising a sequence that interacts with an endonuclease (especially an RNA-guided nuclease) Refers to the form of guide RNA (single guide RNA, sgRNA), but as long as the endonuclease has activity in the target nucleotide sequence, it may be included in the scope of the present invention without limitation, and the type or endonuclease used together It can be prepared and used according to a technique known in the art in consideration of the microorganisms derived from the nuclease and the like.

또한, 상기 가이드 RNA는 플라스미드 주형으로부터 전사된(transcribed) 것, 생체외(in vitro)에서 전사된 것(예컨대, 올리고뉴클레오티드 이중가닥) 또는 합성한 가이드 RNA 등일 수 있으나, 이에 제한되지 않는다.In addition, the guide RNA may be transcribed from a plasmid template, transcribed in vitro (eg, oligonucleotide double-stranded), or synthesized guide RNA, but is not limited thereto.

또한, 본 발명에 따른 유전자가위(CRISPR/Cas) 시스템에 있어서, 상기 Cas 단백질은 바람직하게는 Cas9 단백질일 수 있으나, 이에 제한되지 않는다.In addition, in the gene scissors (CRISPR/Cas) system according to the present invention, the Cas protein may preferably be a Cas9 protein, but is not limited thereto.

또한, 상기 Cas9 단백질은 스트렙토코커스 피요제네스(Streptococcus pyogenes) 유래의 Cas9 단백질, 캠필로박터 제주니(Campylobacter jejuni) 유래의 Cas9 단백질, 스트렙토코커스 써모필러스(Streptococcus thermophilus) 또는 스트렙토코커스 아우레우스(Streptocuccus aureus) 유래의 Cas9 단백질, 네이쎄리아 메닝기티디스(Neisseria meningitidis) 유래의 Cas9 단백질, 파스투렐라 물토시다(Pasteurella multocida) 유래의 Cas9 단백질, 프란시셀라 노비시다(Francisella novicida) 유래의 Cas9 단백질 등으로 이루어진 군에서 선택된 하나 이상일 수 있으나, 이에 제한되지 않는다. Cas9 단백질 또는 이의 유전자 정보는 NCBI (National Center for Biotechnology Information)의 GenBank와 같은 공지의 데이터베이스에서 얻을 수 있다. In addition, the Cas9 protein is Streptococcus pyogenes ( Streptococcus pyogenes )-derived Cas9 protein, Campylobacter jejuni ( Campylobacter jejuni )-derived Cas9 protein, Streptococcus thermophilus ) or Streptococcus aureus ( Streptococcus aureus ) Cas9 protein derived from, Neisseria meningitidis ( Neisseria meningitidis ) Cas9 protein derived from, Pasteurella multocida ( Pasteurella multocida ) Cas9 protein derived from, Francisella novicida ( Francisella novicida ) consisting of Cas9 protein derived from It may be one or more selected from the group, but is not limited thereto. Cas9 protein or its genetic information can be obtained from a known database such as GenBank of the National Center for Biotechnology Information (NCBI).

Cas9 단백질은 RNA-guided DNA 엔도뉴클레아제 효소로, 이중 가닥 DNA 절단(double stranded DNA break)을 유도한다. Cas9 단백질이 정확하게 표적 DNA의 염기서열에 결합하여 DNA 가닥을 잘라내기 위해서는 PAM(Protospacer Adjacent Motif)이라 알려진 3개의 염기로 이루어진 짧은 염기서열이 표적 DNA의 염기서열 옆에 존재해야 하며, Cas9 단백질은 PAM 서열(NGG)로부터 3번째와 4번째 염기쌍 사이를 추정하여 절단한다.Cas9 protein is an RNA-guided DNA endonuclease enzyme that induces double-stranded DNA breaks. In order for the Cas9 protein to accurately bind to the base sequence of the target DNA and cut the DNA strand, a short sequence of three bases known as PAM (Protospacer Adjacent Motif) must exist next to the base sequence of the target DNA. Cut by inferring between the 3rd and 4th base pairs from the sequence (NGG).

본 발명의 일 구현 예에 따른 유전자가위(CRISPR/Cas) 시스템에 있어서, 상기 Cas 단백질은 SpCas9, xCas9, SpG Cas9, NG Cas9, SpRY SpCas9 또는 SaCas9일 수 있다. In the gene scissors (CRISPR/Cas) system according to an embodiment of the present invention, the Cas protein may be SpCas9, xCas9, SpG Cas9, NG Cas9, SpRY SpCas9 or SaCas9.

본 발명에 따른 유전자 가위 시스템에서 SpCas9을 이용할 경우, 공여자 핵산(5'-xxxxxxxxnnnnHGnnnnnn*…*nnnnnnnDCnnnnyyyyyyyy-3', 5'-xxxxxxxxnnnnGHnnnnnn*…*nnnnnnnCDnnnnyyyyyyyy-3' 또는 5'-xxxxxxxxnnnnHHnnnnnn*…*nnnnnnnDDnnnnyyyyyyyy-3')은 양 말단에 표적 부위 미세상동성 서열(xxxxxxxx 또는 yyyyyyyy), 표적 유전자의 교체하고자 하는 목적 유전자 절편 서열(*…*) 및 변경된 PAM 서열(nHG, nGH, nHH, DCn, CDn 또는 DDn)을 포함할 수 있다. PAM 서열의 변경을 원하지 않는 경우 Cas 단백질에 의한 공여자 핵산의 절단을 방지하기 위해 PAM 서열로부터 13 뉴클레오티드 이내의 gRNA 씨드 서열(seed sequence)에서 1개 이상의 SNP를 추가할 수 있다. 상기 H는 염기 A, C 또는 T를 의미하고, 상기 D는 염기 A, G 또는 T를 의미하며, 상기 n은 염기 A, G, C 또는 T를 의미한다. When SpCas9 is used in the gene editing system according to the present invention, donor nucleic acids (5'-xxxxxxxxnnnnHGnnnnnn*...*nnnnnnnDCnnnnyyyyyyyy-3', 5'-xxxxxxxxnnnnGHnnnnnn*...*nnnnnnCDnnnnnyyyyyyyyy-3' or 5'-xxxxxxxxxnnny... ') are the target site microhomology sequence (xxxxxxxx or yyyyyyyy) at both ends, the target gene segment sequence to be replaced (*...*) of the target gene, and the altered PAM sequence (nHG, nGH, nHH, DCn, CDn or DDn) may include If the change of the PAM sequence is not desired, one or more SNPs can be added in the gRNA seed sequence within 13 nucleotides from the PAM sequence to prevent cleavage of the donor nucleic acid by the Cas protein. Wherein H refers to a base A, C or T, D refers to a base A, G or T, and n refers to a base A, G, C or T.

또한, 본 발명에 따른 유전자 가위 시스템에서 xCas9, SpG Cas9 또는 NG Cas9을 이용할 경우, 공여자 핵산(5'-xxxxxxxxnnnnHnnnnnnn*…*nnnnnnnDnnnnyyyyyyyy-3')은 양 말단에 표적 부위 미세상동성 서열(xxxxxxxx 또는 yyyyyyyy), 표적 유전자의 교체하고자 하는 목적 유전자 절편 서열(*…*) 및 변경된 PAM 서열(nH 또는 Dn)을 포함할 수 있다. PAM 서열의 변경을 원하지 않는 경우 Cas 단백질에 의한 공여자 핵산의 절단을 방지하기 위해 PAM 서열로부터 13 뉴클레오티드 이내의 gRNA 씨드 서열(seed sequence)에서 1개 이상의 SNP를 추가할 수 있다. 상기 H는 염기 A, C 또는 T를 의미하고, 상기 D는 염기 A, G 또는 T를 의미하며, 상기 n은 염기 A, G, C 또는 T를 의미한다. In addition, when xCas9, SpG Cas9 or NG Cas9 is used in the gene editing system according to the present invention, the donor nucleic acid (5'-xxxxxxxxnnnnHnnnnnnn*...*nnnnnnnDnnnnyyyyyyyy-3') is a target site microhomology sequence (xxxxxxxx or yyyyyyyy) at both ends. ), the target gene segment sequence to be replaced (*...*) of the target gene and the altered PAM sequence (nH or Dn). If the change of the PAM sequence is not desired, one or more SNPs can be added in the gRNA seed sequence within 13 nucleotides from the PAM sequence to prevent cleavage of the donor nucleic acid by the Cas protein. Wherein H refers to a base A, C or T, D refers to a base A, G or T, and n refers to a base A, G, C or T.

본 발명에 따른 유전자 가위 시스템에서 SpRY SpCas9을 이용할 경우, SpRY SpCas9는 nRn PAM 서열뿐만 아니라 더 약한 정도로 nYn을 인식함으로 PAM을 nYn으로 유지하고 PAM으로부터 13 뉴클레오티드 이내의 gRNA 씨드 서열(seed sequence)에서 1개 이상의 SNP를 추가할 수 있다. 공여자 핵산(5'-xxxxxxxx###nYnnnnnnn*…*nnnnnnnRn###yyyyyyyy-3')은 양 말단에 표적 부위 미세상동성 서열(xxxxxxxx 또는 yyyyyyyy), 표적 유전자의 교체하고자 하는 목적 유전자 절편 서열(*…*) 및 변경된 PAM 서열(nY 또는 Rn) 및 1-3개의 변경된 gRNA 씨드 서열(###)을 포함할 수 있다. 상기 Y는 염기 C 또는 T를 의미하고, 상기 R은 염기 A 또는 G를 의미하며, 상기 n은 염기 A, G, C 또는 T를 의미한다. When SpRY SpCas9 is used in the gene editing system according to the present invention, SpRY SpCas9 recognizes not only the nRn PAM sequence but also nYn to a lesser extent, thereby maintaining the PAM as nYn and 1 in the gRNA seed sequence within 13 nucleotides from the PAM. More than one SNP can be added. The donor nucleic acid (5'-xxxxxxxx###nYnnnnnnn*…*nnnnnnnRn###yyyyyyyy-3') is a target site microhomology sequence (xxxxxxxx or yyyyyyyy) at both ends, the target gene segment sequence to be replaced ( *...*) and an altered PAM sequence (nY or Rn) and 1-3 altered gRNA seed sequences (###). Wherein Y means a base C or T, R means a base A or G, wherein n means a base A, G, C or T.

본 발명에 따른 유전자 가위 시스템에서 SaCas9을 이용할 경우, 공여자 핵산(5'-xxxxxxxxnnnnHRRnnnnn*…*nnnnnYYDnnnnyyyyyyyy-3')은 양 말단에 표적 부위 미세상동성 서열(xxxxxxxx 또는 yyyyyyyy), 표적 유전자의 교체하고자 하는 목적 유전자 절편 서열(*…*) 및 변경된 PAM 서열(nHRR 또는 YYDn)을 포함할 수 있다. PAM 서열의 변경을 원하지 않는 경우 Cas 단백질에의한 공여자의 절단을 방지하기 위해 PAM으로부터 13 뉴클레오티드 이내의 gRNA 씨드 서열(seed sequence)에서 1개 이상의 SNP를 추가할 수 있다. 상기 H는 염기 A, C 또는 T를 의미하고, 상기 R은 염기 A 또는 G를 의미하고, Y는 염기 C 또는 T를 의미하고, 상기 D는 염기 A, G 또는 T를 의미하며, 상기 n은 염기 A, G, C 또는 T를 의미한다. When SaCas9 is used in the gene editing system according to the present invention, the donor nucleic acid (5'-xxxxxxxxnnnnHRRnnnnn*…*nnnnnYYDnnnnyyyyyyyy-3') is a target site microhomology sequence (xxxxxxxx or yyyyyyyy) at both ends, the target gene to be replaced It may include a target gene segment sequence (*...*) and an altered PAM sequence (nHRR or YYDn). If the change of the PAM sequence is not desired, one or more SNPs can be added in the gRNA seed sequence within 13 nucleotides of the PAM to prevent cleavage of the donor by the Cas protein. wherein H refers to a base A, C or T, R refers to a base A or G, Y refers to a base C or T, and D refers to a base A, G or T, wherein n is base A, G, C or T.

또한, 본 발명은 상기 공여자(donor) 핵산을 이용하여 표적 유전자를 상기 공여자 핵산 내의 목적 유전자 절편으로 교체하는 단계를 포함하는, 유전자 편집 방법을 제공한다. In addition, the present invention provides a gene editing method comprising the step of replacing a target gene with a target gene segment in the donor nucleic acid using the donor nucleic acid.

본 발명에서 사용된 유전자 편집 방법은 교정하고자 하는 특정 유전자의 특정 서열과 동일한 서열을 가지는 미세상동성(microhomology)을 이용한 MMEJ(microhomology-mediated end joining) 매개 유전자 편집 방법이다. The gene editing method used in the present invention is a microhomology-mediated end joining (MMEJ) mediated gene editing method using microhomology having the same sequence as the specific sequence of a specific gene to be corrected.

본 발명에 따른 공여자 핵산을 이용한 MMEJ 매개 유전자 편집 방법을 이용하면 비상동 말단 결합(canonical non-homologous end-joining, cNHEJ) 매개 유전자 편집 방법을 이용했을 때보다 편집 효율이 증가하는 것이 특징이며, 공여자 핵산의 처리 농도, 길이 및 NHEJ 저해제 처리에 따라 편집 효율이 달라질 수 있다. When the MMEJ-mediated gene editing method using the donor nucleic acid according to the present invention is used, the editing efficiency is increased compared to when the canonical non-homologous end-joining (cNHEJ)-mediated gene editing method is used. Editing efficiency may vary depending on the treatment concentration, length, and NHEJ inhibitor treatment of the nucleic acid.

또한, 본 발명에 따른 유전자 편집 방법은 박테리아, 효모, 식물 세포 또는 동물 세포에서 이루어질 수 있으며, 바람직하게는 식물 세포에서 이루어질 수 있으나, 이에 제한되지 않는다. In addition, the gene editing method according to the present invention may be performed in bacteria, yeast, plant cells or animal cells, preferably in plant cells, but is not limited thereto.

본 발명에 따른 유전자 편집 방법에 있어서, 공여자 핵산, 가이드 RNA 및 Cas 단백질을 식물 세포로 도입하는 것은, 공여자 핵산을 암호화하는 DNA, 표적 염기서열에 특이적인 가이드 RNA를 암호화하는 DNA 및 Cas 단백질을 암호화하는 DNA 서열을 포함하는 재조합 벡터; 또는 공여자 핵산, 표적 염기서열에 특이적인 가이드 RNA와 Cas 단백질의 복합체(ribonucleoprotein);를 이용하는 것일 수 있으나, 이에 제한되는 것은 아니다. In the gene editing method according to the present invention, the introduction of the donor nucleic acid, guide RNA and Cas protein into plant cells comprises DNA encoding the donor nucleic acid, DNA encoding the guide RNA specific to the target nucleotide sequence, and the Cas protein encoding a recombinant vector comprising a DNA sequence; Alternatively, a donor nucleic acid or a complex of a guide RNA specific to a target nucleotide sequence and a Cas protein (ribonucleoprotein) may be used, but is not limited thereto.

본 발명에 따른 유전자 편집 방법에 있어서, 가이드 RNA와 Cas 단백질의 복합체를 식물 세포에 형질도입하는 방법은 원형질체에 대한 칼슘/폴리에틸렌 글리콜 방, 원형질체의 전기천공법, 식물 요소로의 현미주사법, 각종 식물 요소의(DNA 또는 RNA-코팅된) 입자 충격법, 아그로박테리움 튜메파시엔스(Agrobacterium tumefaciens) 또는 아그로박테리움 리조젠(Agrobacterium rhizogens)에 의해 매개된 유전자 전이에서(비완전성) 박테리아에 의한 감염 등으로부터 적당하게 선택될 수 있다.In the gene editing method according to the present invention, the method for transducing the complex of guide RNA and Cas protein into plant cells is a calcium/polyethylene glycol chamber for protoplasts, electroporation of protoplasts, micro-injection method with plant elements, various plants Particle bombardment of urea (DNA or RNA-coated), Agrobacterium tumefaciens or Agrobacterium rhizogens mediated (incomplete) bacterial infection in gene transfer, etc. may be appropriately selected from

또한, 상기 표적 염기서열에 특이적인 가이드 RNA를 암호화하는 DNA 및 Cas 단백질을 암호화하는 핵산 서열을 포함하는 재조합 벡터를 식물 세포에 도입하는 것은 형질전환 방법을 의미한다. 식물 종의 형질전환은 이제는 쌍자엽 식물뿐만 아니라 단자엽 식물 양자를 포함한 식물 종에 대해 일반적이다. 원칙적으로, 임의의 형질전환 방법은 본 발명에 따른 재조합 벡터를 적당한 선조 세포로 도입시키는데 이용될 수 있다. In addition, introducing a recombinant vector comprising a DNA encoding a guide RNA specific for the target nucleotide sequence and a nucleic acid sequence encoding a Cas protein into a plant cell refers to a transformation method. Transformation of plant species is now common for plant species including both monocots as well as dicots. In principle, any transformation method can be used to introduce the recombinant vector according to the invention into suitable progenitor cells.

본 발명에 따른 유전자 편집 방법에 있어서, 표적 염기서열에 특이적인 가이드 RNA 및 Cas 단백질이 도입되는 "식물 세포"는 어떤 식물 세포도 된다. 식물 세포는 배양 세포, 배양 조직, 배양 기관 또는 전체 식물이다. "식물 조직"은 분화된 또는 미분화된 식물의 조직, 예를 들면 이에 한정되진 않으나, 떡잎, 하배축, 뿌리, 줄기, 잎, 꽃가루, 종자, 암 조직 및 배양에 이용되는 다양한 형태의 세포들, 즉 단일 세포, 원형질체(protoplast), 싹 및 캘러스 조직을 포함한다. 식물 조직은 인 플란타(in planta)이거나 기관 배양, 조직 배양 또는 세포 배양 상태일 수 있다. In the gene editing method according to the present invention, a "plant cell" into which a guide RNA specific for a target nucleotide sequence and a Cas protein are introduced may be any plant cell. A plant cell is a cultured cell, cultured tissue, cultured organ or whole plant. "Plant tissue" refers to tissues of differentiated or undifferentiated plants, such as, but not limited to, cotyledons, hypocotyls, roots, stems, leaves, pollen, seeds, cancer tissues and various types of cells used in culture, i.e. Includes single cell, protoplast, shoot and callus tissue. The plant tissue may be in planta or in an organ culture, tissue culture or cell culture state.

본 발명의 제조방법에 있어서, 유전체가 교정된 식물세포로부터 유전체가 교정된 식물을 재분화하는 방법은 당업계에 공지된 임의의 방법을 이용할 수 있다. 유전체가 교정된 식물세포는 전식물로 재분화되어야 한다. 캘러스 또는 원형질체 배양으로부터 성숙한 식물의 재분화를 위한 기술은 수많은 여러 가지 종에 대해서 당업계에 주지되어 있다.In the production method of the present invention, any method known in the art may be used as a method of redifferentiating a plant having a corrected genome from a plant cell having a corrected genome. Plant cells whose genome has been corrected must be redifferentiated into whole plants. Techniques for the redifferentiation of mature plants from callus or protoplast cultures are well known in the art for a number of different species.

또한, 본 발명은 공여자(donor) 핵산을 포함하는, 유전자 편집용 조성물을 제공한다. In addition, the present invention provides a composition for gene editing comprising a donor nucleic acid.

본 발명에 따른 유전자 편집용 조성물은 CRISPR/Cas 단백질 및 가이드 RNA를 더 포함할 수 있으며, 상기 Cas 단백질 및 가이드 RNA는 리보핵산-단백질(ribonucleoprotein) 복합체를 형성하여 RNA 유전자 가위(RNA-Guided Engineered Nuclease, RGEN)로 작동할 수 있다. The composition for gene editing according to the present invention may further include a CRISPR/Cas protein and a guide RNA, wherein the Cas protein and the guide RNA form a ribonucleoprotein complex to form RNA-guided engineered nuclease (RNA-Guided Engineered Nuclease). , RGEN).

본 발명에 따른 유전자 편집용 조성물에서, 상기 CRISPR/Cas 단백질 및 가이드 RNA는 전술한 바와 같다. In the composition for gene editing according to the present invention, the CRISPR/Cas protein and the guide RNA are as described above.

이하, 본 발명을 실시예에 의해 상세히 설명한다. 단, 하기 실시예는 본 발명을 예시하는 것일 뿐, 본 발명의 내용이 하기 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in detail by way of Examples. However, the following examples only illustrate the present invention, and the content of the present invention is not limited to the following examples.

재료 및 방법Materials and Methods

1. 식물 재료1. Plant material 및 원형질체 분리and protoplast isolation

토마토(Solanum lycopersicum cv. Micro-Tom) 또는 양배추(Brassica oleracea)의 종자를 70%(v/v) 에탄올로 멸균한 후, 멸균수로 5회 세척하고 0.4 mg/ℓ 티아민 HCl, 100 mg/ℓ 미오-이노시톨, 30 g/ℓ 수크로스 및 8 g/ℓ 젤라이트, pH 5.7가 첨가된 1/2 MS(Murashige and Skoog) 배지에 치상하여 16시간 명/8시간 암의 광주기, 25℃(토마토) 또는 23℃(양배추)의 온도 조건으로 배양하였다. After sterilizing the seeds of tomato ( Solanum lycopersicum cv. Micro-Tom ) or cabbage ( Brassica oleracea ) with 70% (v/v) ethanol, washed 5 times with sterile water and 0.4 mg/ℓ thiamine HCl, 100 mg/ℓ Myo-inositol, 30 g/L sucrose, 8 g/L gelite, and 1/2 MS (Murashige and Skoog) medium supplemented with pH 5.7 added to a photoperiod of 16 hours light/8 hours dark, 25°C ( Tomato) or 23 ℃ (cabbage) incubated at a temperature condition.

원형질체(protoplast) 분리를 위해, 4일령의 토마토 또는 7일령의 양배추 자엽(cotyledon)을 0.5%(v/v) 셀룰라아제, 0.5%(v/v) 펙티나아제, 1%(v/v) 비스코자임, 3 mM MES(2-(N-morpholino)ethanesulfonic acid, pH 5.8) 버퍼 및 9%(w/v) 만니톨이 첨가된 CPW(cell and protoplast washing) 용액에 15분 동안 진공 침투(vacuum infiltration)를 유도한 후, 50 rpm, 25℃ 조건으로 2~4시간 동안 배양하였다. 이후, 8겹 거즈를 이용하여 여과하고 100 g에서 5분 동안 원심분리한 후, 21% 수크로스 밀도 구배 방법을 이용하여 원형질체를 분리하고 W5(2 mM MES pH 5.8, 154 mM NaCl, 125 mM CaCl2, 5 mM KCl) 용액에 수거하였다. 수거된 원형질체는 W5 용액으로 3회 세척하고 MMG(4 mM MES pH 5.7, 0.4 M 만니톨, 15 mM MgCl2) 용액에 재현탁하고 헤모사이토미터(hemocytometer)를 이용하여 농도를 측정하였다. For protoplast isolation, 4-day-old tomato or 7-day-old cabbage cotyledon was treated with 0.5% (v/v) cellulase, 0.5% (v/v) pectinase, and 1% (v/v) biscotti. Vacuum infiltration for 15 min in cell and protoplast washing (CPW) solution supplemented with zyme, 3 mM MES (2-(N-morpholino)ethanesulfonic acid, pH 5.8) buffer and 9% (w/v) mannitol After induction, incubated for 2 to 4 hours at 50 rpm, 25 ℃ conditions. Then, after filtration using 8-ply gauze and centrifugation at 100 g for 5 minutes, protoplasts were isolated using a 21% sucrose density gradient method and W5 (2 mM MES pH 5.8, 154 mM NaCl, 125 mM CaCl) 2 , 5 mM KCl) solution. The collected protoplasts were washed three times with W5 solution, resuspended in MMG (4 mM MES pH 5.7, 0.4 M mannitol, 15 mM MgCl 2 ) solution, and the concentration was measured using a hemocytometer.

2. 공여자(donor) 핵산 및 가이드 RNA(gRNA) 준비2. Donor nucleic acid and guide RNA (gRNA) preparation

토마토 식물체에서 신초 분열조직의 형성에 관여하는 당전이효소 유전자인 SlHPAT3(Solanum lycopersicum Hydroxyproline O-arabinosyltransferase 3)를 표적으로 하는 유전자 교정을 실시하기 위해서 Sol Genomics network database로부터 SlHPAT3(Solyc07g021170.1) 유전자 서열을 얻었다. 이후, CRISPR-P 2.0 프로그램을 이용하여 가이드 RNA(guide RNA, gRNA)를 디자인하고(표 1), GeneArt Precision gRNA Synthesis Kit(Invitrogen)를 이용하여 가이드 RNA를 합성하였다. cNHEJ 경로를 위한 공여자 핵산(cNJ.HPAT3-1) 및 MMEJ 경로를 위한 공여자 핵산(MJ.HPAT3-1, MJ.HPAT3-2 및 MJ.HPAT3-3)을 하기 표 1의 프라이머를 이용한 PCR을 통해 준비하였다. cNJ.HPAT3-1의 5'-말단은 포스포다이에스터(phosphodiester) 결합을 가지도록 제조하였다. PCR 증폭산물은 안정성을 증가시키기 위하여 5' 말단에 인산화된 포스포로티오에이트(phosphorothioate)로 변형시켰다.In order to perform gene editing targeting SlHPAT3 ( Solanum lycopersicum Hydroxyproline O-arabinosyltransferase 3), a glycotransferase gene involved in the formation of shoot meristems in tomato plants, the SlHPAT3 (Solyc07g021170.1) gene sequence was extracted from the Sol Genomics network database. got it Then, guide RNAs (gRNAs) were designed using the CRISPR-P 2.0 program (Table 1), and guide RNAs were synthesized using the GeneArt Precision gRNA Synthesis Kit (Invitrogen). The donor nucleic acids for the cNHEJ pathway (cNJ.HPAT3-1) and the donor nucleic acids for the MMEJ pathway (MJ.HPAT3-1, MJ.HPAT3-2 and MJ.HPAT3-3) were subjected to PCR using the primers in Table 1 below. prepared. The 5'-end of cNJ.HPAT3-1 was prepared to have a phosphodiester bond. The PCR amplification product was modified with phosphorothioate phosphorylated at the 5' end to increase stability.

또한, 양배추 식물체의 thermo-tolerance 1(TT1), orange(Or) 및 acetolactate synthase 1(ALS1) 유전자를 표적으로 하는 유전자 교정을 실시하기 위해서 NCBI로부터 BoTT1(Bo2g095110), BoOr(Bo9g014710) 및 BoALS1(Bo1g076910) 유전자 서열을 얻었다. 이후, CRISPR-P 2.0 프로그램을 이용하여 가이드 RNA를 디자인하고(표 1), GeneArt Precision gRNA Synthesis Kit(Invitrogen)를 이용하여 가이드 RNA를 합성하였다. MMEJ 경로를 위한 공여자 핵산(MJ.BoTT1, MJ.BoOr 및 MJ.BoALS1)을 하기 표 1의 프라이머를 이용한 PCR을 통해 준비하였다. 공여자 핵산의 농도는 Nanodrop2000 분광광도계(Thermofisher, USA)를 이용하여 측정하였다. In addition, in order to perform gene editing targeting the thermo-tolerance 1 (TT1), orange (Or) and acetolactate synthase 1 (ALS1) genes of cabbage plants, BoTT1 (Bo2g095110), BoOr (Bo9g014710) and BoALS1 (Bo1g076910) were obtained from NCBI. ) the gene sequence was obtained. Then, guide RNAs were designed using the CRISPR-P 2.0 program (Table 1), and guide RNAs were synthesized using the GeneArt Precision gRNA Synthesis Kit (Invitrogen). Donor nucleic acids for the MMEJ pathway (MJ.BoTT1, MJ.BoOr and MJ.BoALS1) were prepared through PCR using the primers in Table 1 below. The concentration of donor nucleic acid was measured using a Nanodrop2000 spectrophotometer (Thermofisher, USA).

Figure PCTKR2021008727-appb-img-000001
Figure PCTKR2021008727-appb-img-000001

3. PEG-매개 형질전환3. PEG-Mediated Transformation

2×105 ㎖의 토마토 또는 양배추 원형질체에 20 μg SpCas9 단백질(ToolGen, Inc. South Korea), 10 μg 가이드 RNA 및 300 pmol 공여자 핵산을 포함한 PBS 버퍼를 넣고 25℃에서 10분 동안 반응시켰다. 이후, RNP(ribonucleoprotein) 복합체를 첨가하여 잘 섞어주고 PEG(polyethylene glycol) 용액(40%(w/v) PEG4000, 0.2 M mannitol, 0.1 M CaCl2)을 첨가하고 잘 섞어준 후, 실온에서 10분 동안 반응시켰다. W5 용액으로 세척하고, 100 g에서 5분 동안 원심분리한 후, 상층액을 제거하고, W5 용액을 첨가하여 25℃, 암조건으로 48시간 동안 반응시켰다. PBS buffer containing 20 μg SpCas9 protein (ToolGen, Inc. South Korea), 10 μg guide RNA, and 300 pmol donor nucleic acid was added to 2×10 5 ml of tomato or cabbage protoplasts and reacted at 25° C. for 10 minutes. Then, add RNP (ribonucleoprotein) complex, mix well, add PEG (polyethylene glycol) solution (40% (w/v) PEG4000, 0.2 M mannitol, 0.1 M CaCl 2 ), mix well, and then at room temperature for 10 minutes reacted while After washing with W5 solution and centrifuging at 100 g for 5 minutes, the supernatant was removed, and W5 solution was added thereto, followed by reaction at 25° C. and dark conditions for 48 hours.

4. 염기서열 분석4. Sequence analysis

형질전환된 원형질체로부터 CTAB(Cetyl trimethylammonium bromide) 방법을 이용하여 게노믹 DNA를 추출하고 3번의 PCR을 수행하여 miniseq sequencing service(MiniSeqTM System, USA) 분석을 위한 샘플을 준비한 후, 표적 부위에 대한 딥 시퀀싱(deep sequencing) 분석을 수행하였다. miniseq 샘플 준비를 위한 첫 번째 및 두 번째 PCR은 하기 표 2와 같은 프라이머를 이용하여 수행하였고, 세 번째 PCR은 제조사에서 제공하는 프라이머를 이용하여 수행하였으며, 시퀀싱 결과는 Cas-Analyzer tool을 이용하여 편집 효율을 분석하였다. After extracting genomic DNA from the transformed protoplast using the CTAB (Cetyl trimethylammonium bromide) method, performing PCR 3 times to prepare a sample for analysis by miniseq sequencing service (MiniSeq TM System, USA), dip into the target site A deep sequencing analysis was performed. The first and second PCRs for miniseq sample preparation were performed using the primers shown in Table 2 below, the third PCR was performed using the primers provided by the manufacturer, and the sequencing results were edited using the Cas-Analyzer tool. Efficiency was analyzed.

Figure PCTKR2021008727-appb-img-000002
Figure PCTKR2021008727-appb-img-000002

실시예 1. cNHEJ 기반 및 MMEJ 기반 유전체 교정 방법의 편집 효율 분석Example 1. Analysis of editing efficiency of cNHEJ-based and MMEJ-based genome editing methods

cNHEJ(canonical non-homologous end-joining) 기반 유전체 교정은 미세상동성 말단을 가지지 않은 공여자 핵산을 이용하는 방법이고, MMEJ(microhomology-mediated end joining) 기반 유전자 교정은 미세상동성 말단을 가지는 공여자 핵산을 이용하는 방법이다. 본 발명에서는 토마토 유래의 SlHPAT3 유전자에서 6개(A1, A2, B, C, D1, D2)의 SNP(single nucleotide polymorphisms)를 포함하는 cNHEJ 기반 유전체 교정을 위한 공여자 핵산(cNJ.HPAT3-1) 및 MMEJ 기반 유전체 교정을 위한 공여자 핵산(MJ.HPAT3-1, MJ.HPAT3-2 및 MJ.HPAT3-3)을 제조하였다. MJ.HPAT3-1, MJ.HPAT3-2 및 MJ.HPAT3-3는 표적으로 하는 SlHPAT3 유전자의 양쪽 PAM 부위의 상류(upstream) 3번째 염기 서열부터 각각 5개, 10개 및 20개의 미세상동성 서열을 가지도록 디자인함으로써, 표적으로 하는 유전자에서 특정 부위의 DNA 절편이 공여자 핵산 서열로 대체되도록 하였다(도 1).Canonical non-homologous end-joining (cNHEJ)-based genome editing is a method using donor nucleic acids that do not have microhomologous ends, and microhomology-mediated end joining (MMEJ)-based gene editing uses donor nucleic acids having microhomologous ends. way. In the present invention, the donor nucleic acid (cNJ.HPAT3-1) and in SlHPAT3 gene of tomato-derived 6 for cNHEJ based dielectric correction containing SNP (single nucleotide polymorphisms) of (A1, A2, B, C , D1, D2) Donor nucleic acids (MJ.HPAT3-1, MJ.HPAT3-2 and MJ.HPAT3-3) for MMEJ-based genome editing were prepared. MJ.HPAT3-1, MJ.HPAT3-2 and MJ.HPAT3-3 are 5, 10, and 20 microhomologous sequences from the 3rd nucleotide sequence upstream of both PAM sites of the target SlHPAT3 gene, respectively. By designing to have a , the DNA fragment at a specific site in the target gene was replaced with the donor nucleic acid sequence (FIG. 1).

또한, 토마토 원형질체에 SpCas9 단백질, 가이드 RNA(gR1.HPAT3, gR2.HPAT3) 및 공여자 핵산을 주입한 후, 편집 효율을 분석한 결과, SpCas9 단백질 및 가이드 RNA만 주입한 경우, 표적하는 유전체 교정이 일어나지 않은 반면, SpCas9 단백질, 가이드 RNA 및 공여자 핵산을 모두 주입한 경우, 표적하는 유전체 교정이 일어나는 것을 확인하였다. 특히, cNJ.HPAT3-1를 공여자 핵산으로 주입했을 때보다, MJ.HPAT3-1를 공여자 핵산으로 주입했을 때의 편집 효율이 높은 것을 확인하였다(표 3).In addition, as a result of analyzing the editing efficiency after injection of SpCas9 protein, guide RNA (gR1.HPAT3, gR2.HPAT3) and donor nucleic acid into tomato protoplasts, when only SpCas9 protein and guide RNA are injected, targeted genome editing does not occur On the other hand, when all of the SpCas9 protein, guide RNA and donor nucleic acid were injected, it was confirmed that targeted genome editing occurred. In particular, it was confirmed that the editing efficiency was higher when MJ.HPAT3-1 was injected as a donor nucleic acid than when cNJ.HPAT3-1 was injected as a donor nucleic acid (Table 3).

공여자 핵산에 따른 편집 효율Editing efficiency according to donor nucleic acid 그룹group SpCas9SpCas9 gR1.HPAT3gR1.HPAT3 gR2.HPAT3gR2.HPAT3 DNA donorDNA donor total readstotal reads edited readsedited reads editing efficiency
(%)editing efficiency
(%) 1One -- -- -- -- 121368121368 00 00 22 ++ ++ -- -- 110285110285 00 00 33 ++ -- ++ -- 105799105799 00 00 44 ++ ++ ++ -- 117136117136 00 00 55 ++ ++ ++ cNJ.HPAT3-1cNJ.HPAT3-1 9305493054 7676 0.080.08 66 ++ ++ ++ MJ.HPAT3-1MJ.HPAT3-1 9988599885 24382438 2.442.44

상기 결과를 통해, cNHEJ 기반보다 MMEJ 기반 유전체 교정 방법의 편집 효율이 약 30배 이상 높은 것을 알 수 있었다. Through the above results, it was found that the editing efficiency of the MMEJ-based genome editing method was about 30 times higher than that of the cNHEJ-based method.

실시예 2. cNHEJ 기반 및 MMEJ 기반 유전체 교정 방법의 편집 빈도 분석Example 2. Editing frequency analysis of cNHEJ-based and MMEJ-based genome editing methods

cNJ.HPAT3-1 공여자 핵산에 의해 교정된 산물의 각 SNP(A1, A2, B, C, D1, D2) 위치별 편집 빈도를 분석하였다. 그 결과, 총 edited reads 중 94.74%가 B-C 위치의 SNP에서 교정이 일어난 reads인 것을 확인하였다. 또한, 양쪽 말단의 SNP(A1-A2, D1-D2)에서 교정이 일어난 reads는 확인되지 않았으며, 이는 공여자 핵산의 말단이 유전체 교정에 이용되기 전에 알 수 없는 경로에 의해 손상된 것으로 예측된다(표 4). The editing frequency for each SNP (A1, A2, B, C, D1, D2) position of the product corrected by the cNJ.HPAT3-1 donor nucleic acid was analyzed. As a result, it was confirmed that 94.74% of the total edited reads were reads in which the correction occurred at the SNP at the B-C position. In addition, reads in which correction occurred in both ends of the SNPs (A1-A2, D1-D2) were not identified, and it is predicted that the ends of the donor nucleic acid were damaged by an unknown pathway before being used for genome editing (Table). 4).

반면, MJ.HPAT3-1 공여자 핵산에 의해 교정된 산물의 각 SNP(A1, A2, B, C, D1, D2) 위치별 편집 빈도를 분석한 결과, 총 edited reads 중에서 9.72% 만이 표적으로 하는 모든 SNP(A1-A2-B-C-D1-D2)에서 교정이 일어난 것을 확인하였다. 또한, B 및 C 위치의 SNP를 포함하는 reads는 총 edited reads 중에서 33.01%로 나타난 것을 확인하였다. 양쪽 말단의 SNP(A1-A2, D1-D2)에서 교정이 일어난 reads는 66.98%로 나타나는 것을 확인함으로써, CRISPR/Cas9 시스템에 의한 recurrent cleavage가 발생하지 않은 것을 알 수 있었다(표 4). On the other hand, as a result of analyzing the editing frequency for each SNP (A1, A2, B, C, D1, D2) position of the product corrected by the MJ.HPAT3-1 donor nucleic acid, only 9.72% of the total edited reads targeted all It was confirmed that the correction occurred in the SNP (A1-A2-BC-D1-D2). In addition, it was confirmed that reads containing SNPs at positions B and C appeared in 33.01% of the total edited reads. By confirming that 66.98% of reads in which the correction occurred in the SNPs (A1-A2, D1-D2) at both ends, it was confirmed that recurrent cleavage by the CRISPR/Cas9 system did not occur (Table 4).

또한, cNJ.HPAT3-1 공여자 핵산에 의한 B 및 C 위치의 SNP를 포함하는 reads는 94.74%인 반면, MJ.HPAT3-1 공여자 핵산에 의한 B 및 C 위치의 SNP를 포함하는 reads는 6.93%인 것으로 보아, cNHEJ 기반 유전체 교정 방법은 ODM(oligonucleotide directed mutagenesis)이 관여하는 것으로 예측된다.Also, reads containing SNPs at positions B and C by the cNJ.HPAT3-1 donor nucleic acid were 94.74%, whereas reads containing SNPs at positions B and C by the MJ.HPAT3-1 donor nucleic acid were 6.93%. As it can be seen, the cNHEJ-based genome editing method is predicted to involve oligonucleotide directed mutagenesis (ODM).

공여자 핵산에 따른 편집 빈도Editing frequency according to donor nucleic acid
edited
SNPs
edited
SNPs DNA DonorDNA Donor cNJ.HPAT3-1cNJ.HPAT3-1 MJ.HPAT3-1MJ.HPAT3-1 Number of
edited readsNumber of
edited reads editing frequency(%)editing frequency(%) Number of
edited readsNumber of
edited reads editing frequency(%)editing frequency(%) A1-A2-B-C-D1-D2A1-A2-B-C-D1-D2 00 00 237237 9.729.72 A1-A2A1-A2 00 00 872872 35.7735.77 A1-A2-B-CA1-A2-B-C 00 00 150150 6.156.15 B-CB-C 7272 94.7494.74 169169 6.936.93 B-C-D1-D2B-C-D1-D2 44 5.265.26 249249 10.2110.21 D1-D2D1-D2 00 00 761761 31.2131.21 edited readsedited reads 7676 100100 24382438 100100 total readstotal reads 9305493054 -- 9988599885 --

상기 결과를 통해, cNHEJ 기반보다 MMEJ 기반 유전체 교정 방법의 각 SNP 위치별 편집 빈도가 보다 다양하게 나타나는 것을 알 수 있었다. Through the above results, it was found that the editing frequency for each SNP position of the MMEJ-based genome editing method was more varied than that of the cNHEJ-based method.

실시예 3. 공여자 핵산의 처리 농도에 따른 편집 빈도 분석Example 3. Analysis of editing frequency according to treatment concentration of donor nucleic acid

유전체 교정의 주형(template)인 공여자 핵산을 50, 100, 200 및 300 pmol의 농도로 각각 처리한 후, 유전자 편집 빈도를 분석하였다. 그 결과, cNJ.HPAT3-1 공여자 핵산의 처리 농도가 증가할수록 편집 빈도는 유사한 수준으로 나타난 반면, MJ.HPAT3-1 공여자 핵산의 처리 농도가 증가할수록 모든 SNP(A1-A2-B-C-D1-D2)에서 편집이 일어난 정밀 편집 빈도도 증가하는 것을 확인하였다(도 2). After the donor nucleic acid, which is a template for genome editing, was treated at concentrations of 50, 100, 200 and 300 pmol, respectively, the frequency of gene editing was analyzed. As a result, the editing frequency appeared at a similar level as the treatment concentration of the cNJ.HPAT3-1 donor nucleic acid increased, whereas as the treatment concentration of the MJ.HPAT3-1 donor nucleic acid increased, all SNPs (A1-A2-BC-D1-D2) ), it was confirmed that the frequency of precise editing in which editing occurred also increased (FIG. 2).

실시예 4. 공여자 핵산의 길이에 따른 편집 효율 분석Example 4. Analysis of editing efficiency according to the length of the donor nucleic acid

미세상동성 서열의 길이가 20 bp인 MJ.HPAT3-1 공여자 핵산, 미세상동성 서열의 길이가 10 bp인 MJ.HPAT3-2 공여자 핵산 및 미세상동성 서열의 길이가 5 bp인 MJ.HPAT3-3 공여자 핵산을 각각 처리한 후, 유전자 편집 효율을 분석하였다. 그 결과, 미세상동성 서열의 길이가 증가할수록 표적으로 하는 총 편집 효율, 모든 SNP(A1-A2-B-C-D1-D2)에서 편집이 일어난 정밀 편집 효율, B, C 위치의 SNP에서 일어난 편집 효율 및 양쪽 말단(A1-A2, D1-D2) SNP에서 일어난 편집 효율이 모두 증가하는 것을 확인하였다(도 3). MJ.HPAT3-1 donor nucleic acid with a microhomology sequence of 20 bp in length, MJ.HPAT3-2 donor nucleic acid with a microhomology sequence of 10 bp in length, and MJ.HPAT3- with a microhomology sequence of 5 bp in length. After each of the 3 donor nucleic acids was treated, the gene editing efficiency was analyzed. As a result, as the length of the microhomologous sequence increased, the target total editing efficiency, the precision editing efficiency in all SNPs (A1-A2-BC-D1-D2), and the editing efficiency in the SNPs at positions B and C And it was confirmed that both ends (A1-A2, D1-D2) the editing efficiency that occurred in both SNPs increased (FIG. 3).

상기 결과를 바탕으로, 미세상동서열의 길이가 20 bp인 공여자 핵산을 이용하였을 때, 편집 효율이 가장 높은 것을 알 수 있었다. Based on the above results, it was found that the editing efficiency was the highest when a donor nucleic acid having a length of 20 bp of the microhomologous sequence was used.

실시예 5. NHEJ 저해제 처리에 따른 편집 효율 분석Example 5. Analysis of editing efficiency according to NHEJ inhibitor treatment

동물 시스템에서 NHEJ 경로에서 작동하는 DNA-PKcs(DNA dependent protein kinase) 활성을 저해하는 화학물질인 Nu7441를 0.5, 1 및 2 μM의 농도로 각각 처리한 후, 유전자 편집 효율을 분석하였다. 그 결과, Nu7441의 처리에 의해 유전자 편집 효율이 증가하는 것을 확인하였다. 특히, 미세상동서열을 가지지 않는 cNJ.HPAT3-1 공여자 핵산을 이용한 편집 효율은 Nu7441을 처리하지 않았을 때보다 2 μM의 Nu7441을 처리하였을 때 약 1.88배 증가하는 것을 확인하였다(표 5). In an animal system, Nu7441, a chemical that inhibits DNA dependent protein kinase (DNA-PKcs) activity acting in the NHEJ pathway, was treated at concentrations of 0.5, 1, and 2 μM, respectively, and then gene editing efficiency was analyzed. As a result, it was confirmed that the gene editing efficiency was increased by treatment with Nu7441. In particular, it was confirmed that the editing efficiency using the cNJ.HPAT3-1 donor nucleic acid not having a microhomologous sequence was increased by about 1.88 times when Nu7441 was treated at 2 μM compared to when Nu7441 was not treated (Table 5).

Figure PCTKR2021008727-appb-img-000003
Figure PCTKR2021008727-appb-img-000003

상기 결과를 통해, cNJ.HPAT3-1 공여자 핵산에 의한 유전자 편집은 NHEJ 경로에 의한 것이 아니라, Nu7441에 의해 활성화되는 다른 경로에 의해 이루어진다는 것을 알 수 있었다. 또한, MJ.HPAT3-1 공여자 핵산에 의한 유전자 편집은 1 μM의 Nu7441 처리에 의해 편집 효율이 증가할 수 있음을 알 수 있었다. From the above results, it was found that gene editing by the cNJ.HPAT3-1 donor nucleic acid is not by the NHEJ pathway but by another pathway activated by Nu7441. In addition, it was found that the editing efficiency of gene editing by MJ.HPAT3-1 donor nucleic acid can be increased by treatment with 1 μM Nu7441.

실시예 6. MMEJ 기반 유전체 교정 방법의 편집 효율 검정Example 6. Editing efficiency test of MMEJ-based genome editing method

본 발명에 따른 MMEJ 기반 유전체 교정 방법을 이용하여 양배추 식물체의 thermo-tolerance 1(TT1), orange(Or) 및 acetolactate synthase 1(ALS1) 유전자를 교정함으로써 각각 내열성, 카로티노이드 축적 및 제초제 내성의 형질을 얻는 식물체를 제조하고자 하였다. MMEJ 기반 유전체 교정을 위한 공여자 핵산(MJ.BoTT1, MJ.BoOr 및 MJ.BoALS1)을 제조하여 단일 아미노산 치환을 유도하였고, 표적으로 하는 BoTT1, BoOr 및 BoALS1 유전자의 양쪽 PAM 부위의 상류(upstream) 3번째 염기 서열부터 20개의 미세상동서열을 가지도록 디자인함으로써, 표적으로 하는 유전자에서 특정 부위의 DNA 절편이 공여자 핵산 서열로 대체되도록 하였다. BoTT1 유전자의 CGC(Arg) 염기 서열을 CAT(His)로 대체되도록 하였고, BoOr 유전자의 CGT(Arg) 염기 서열을 CAC(His)로 대체되도록 하였으며, BoALS1 유전자의 CCT(Pro) 염기 서열을 TCT(Ser)로 대체되도록 하였다(도 4). By correcting the thermo-tolerance 1 (TT1), orange (Or) and acetolactate synthase 1 (ALS1) genes of cabbage plants using the MMEJ-based genome editing method according to the present invention, the traits of heat resistance, carotenoid accumulation and herbicide tolerance are obtained, respectively. It was intended to manufacture a plant. Donor nucleic acids for MMEJ-based genome editing (MJ.BoTT1, MJ.BoOr and MJ.BoALS1) were prepared to induce single amino acid substitutions, and upstream of both PAM sites of the targeted BoTT1 , BoOr and BoALS1 genes 3 By designing to have 20 microhomologous sequences from the first nucleotide sequence, a DNA fragment at a specific site in the target gene was replaced with the donor nucleic acid sequence. Was to replace the CGC (Arg) base sequence of BoTT1 gene CAT (His), were to be replaced to CGT (Arg) base sequence of BoOr gene to CAC (His), TCT to CCT (Pro) base sequence of BoALS1 gene ( Ser) was replaced with (Fig. 4).

양배추 원형질체에 SpCas9 단백질, 가이드 RNA(표 1) 및 공여자 핵산을 주입한 후, 편집 효율을 분석한 결과, BoTT1, BoOr 및 BoALS1 유전자를 표적으로 하는 편집 효율이 각각 약 7.27%, 약 3.94% 및 약 2.51%로 나타나는 것을 확인함으로써(표 6), 본 발명에 따른 공여자 핵산을 이용한 유전체 교정 방법이 토마토 뿐만 아니라, 양배추 식물체에서도 정상적으로 작동하는 것을 알 수 있었다. After injection of SpCas9 protein, guide RNA (Table 1) and donor nucleic acid into cabbage protoplasts, the editing efficiency was analyzed. As a result, the editing efficiency targeting the BoTT1 , BoOr and BoALS1 genes was about 7.27%, about 3.94% and about 3.94%, respectively. By confirming that it appears as 2.51% (Table 6), it was found that the genome editing method using the donor nucleic acid according to the present invention works normally not only in tomatoes but also in cabbage plants.

Figure PCTKR2021008727-appb-img-000004
Figure PCTKR2021008727-appb-img-000004

Claims (13)

미세상동성-기반 말단 결합(microhomology-mediated end joining, MMEJ)을 통한 유전자 교정에 이용되는 공여자(donor) 핵산으로서, 5'-말단 표적 부위 미세상동성 서열, 교체하고자 하는 목적 유전자 서열 및 3'-말단 표적 부위 미세상동성 서열을 포함하며, 상기 5'-말단 및/또는 3'-말단 표적 부위 미세상동성 서열에는 변경된 PAM(Protospacer adjacent motif) 또는 변경된 gRNA 씨드 서열(seed sequence)을 포함하는 것을 특징으로 하는, 공여자(donor) 핵산.As a donor nucleic acid used for gene correction through microhomology-mediated end joining (MMEJ), the 5'-end target site microhomology sequence, the target gene sequence to be replaced, and 3' -Contains a terminal target site microhomology sequence, and the 5'-terminal and/or 3'-terminal target site microhomology sequence includes an altered PAM (Protospacer adjacent motif) or an altered gRNA seed sequence (seed sequence) Characterized in that, donor nucleic acid. 제1항에 있어서, 상기 5'-말단 또는 3'-말단 표적 부위 미세상동성 서열은 하나 이상의 변형된 염기(Base)를 포함하는 것을 특징으로 하는, 공여자(donor) 핵산.The donor nucleic acid according to claim 1, wherein the 5'-terminal or 3'-terminal target site microhomology sequence comprises one or more modified bases. 제2항에 있어서, 상기 염기(Base)의 변형은 메틸화(methylation), 할로겐화(halogenation), 아세틸화(acetylation), 인산화(phosphorylation), 포스포로티오에이트 연결(phosphorothioate linkage), LNA(locked nucleic acid), MS(2'-O-methyl phosphorothioate) 또는 MSP(2'-O-methyl plus 3'thioPACE)인 것을 특징으로 하는, 공여자(donor) 핵산.According to claim 2, wherein the modification of the base (Base) is methylation (methylation), halogenation (halogenation), acetylation (acetylation), phosphorylation (phosphorylation), phosphorothioate linkage (phosphorothioate linkage), LNA (locked nucleic acid) ), MS (2'-O-methyl phosphorothioate) or MSP (2'-O-methyl plus 3'thioPACE), characterized in that, the donor (donor) nucleic acid. 제1항에 있어서, 상기 5'-말단 또는 3'-말단 표적 부위 미세상동성 서열은 각각 5개 이상 24개 이하의 올리고뉴클레오티드인 것을 특징으로 하는, 공여자(donor) 핵산.The donor nucleic acid according to claim 1, wherein the 5'-terminal or 3'-terminal target site microhomology sequence is 5 or more and 24 or less oligonucleotides, respectively. 제1항에 있어서, 상기 변경된 PAM(Protospacer adjacent motif) 서열은 5'-nHG-3', 5'-nGH-3', 5'-nHH-3', 5'-nH-3', 5'-nY-3', 5'-nHRR-3', 5'-DCn-3', 5'-CDn-3', 5'-DDn-3', 5'-Dn-3', 5'-Rn-3' 또는 5'-YYDn-3'을 포함하며, 여기서 n은 염기 A, G, T 또는 C이고, H는 염기 A, C 또는 T이고, D는 염기 A, G 또는 T이고, Y는 염기 C 또는 T이고, R은 염기 A 또는 G인 것; 또는 PAM 서열의 변경을 원하지 않는 경우 Cas 단백질에 의한 공여자 핵산의 절단을 방지하기 위해 PAM 서열로부터 13 뉴클레오티드 이내의 gRNA 씨드 서열(seed sequence)에서 1개 이상의 SNP가 추가된 것을 특징으로 하는, 공여자(donor) 핵산.According to claim 1, wherein the modified PAM (Protospacer adjacent motif) sequence is 5'-nHG-3', 5'-nGH-3', 5'-nHH-3', 5'-nH-3', 5' -nY-3', 5'-nHRR-3', 5'-DCn-3', 5'-CDn-3', 5'-DDn-3', 5'-Dn-3', 5'-Rn -3' or 5'-YYDn-3', wherein n is a base A, G, T or C, H is a base A, C or T, D is a base A, G or T, and Y is base C or T and R is base A or G; Or, when the change of the PAM sequence is not desired, one or more SNPs are added to the gRNA seed sequence within 13 nucleotides from the PAM sequence to prevent cleavage of the donor nucleic acid by the Cas protein, the donor ( donor) nucleic acid. 제1항 내지 제5항 중 어느 한 항의 공여자(donor) 핵산이 작동 가능하게 연결된, 벡터.A vector to which the donor nucleic acid of any one of claims 1 to 5 is operably linked. 제6항에 있어서, 상기 벡터는 플라스미드 벡터, 바이러스 벡터 또는 PCR 엠플리콘인 것을 특징으로 하는, 벡터. The vector according to claim 6, wherein the vector is a plasmid vector, a viral vector or a PCR amplicon. 제1항 내지 제5항 중 어느 한 항의 공여자(donor) 핵산, CRISPR/Cas 단백질 및 가이드 RNA를 포함하는, 유전자가위(CRISPR/Cas) 시스템.Claims 1 to 5 of any one of the donor (donor) nucleic acid, CRISPR / Cas protein and comprising a guide RNA, a gene scissors (CRISPR / Cas) system. 제8항에 있어서, 상기 CRISPR/Cas 단백질은 SpCas9, xCas9, SpG Cas9, NG Cas9, SpRY SpCas9 또는 SaCas9인 것을 특징으로 하는, 유전자가위(CRISPR/Cas) 시스템.According to claim 8, wherein the CRISPR / Cas protein is SpCas9, xCas9, SpG Cas9, NG Cas9, SpRY SpCas9 or SaCas9, characterized in that the, gene scissors (CRISPR / Cas) system. 제1항 내지 제5항 중 어느 한 항의 공여자(donor) 핵산을 이용하여 표적 유전자를 상기 공여자 핵산 내의 표적 유전자 절편으로 교체하는 단계를 포함하는, 유전자 편집 방법.A gene editing method comprising the step of replacing a target gene with a target gene segment in the donor nucleic acid using the donor nucleic acid of any one of claims 1 to 5. 제10항에 있어서, 상기 방법은 박테리아, 효모, 식물 세포 또는 동물 세포에서 이루어지는 것을 특징으로 하는, 유전자 편집 방법.11. The method according to claim 10, characterized in that the method is carried out in bacteria, yeast, plant cells or animal cells. 제1항 내지 제5항 중 어느 한 항의 공여자(donor) 핵산을 포함하는, 유전자 편집용 조성물.According to any one of claims 1 to 5, comprising the donor (donor) nucleic acid of any one, gene editing composition. 제12항에 있어서, 상기 조성물은 CRISPR/Cas 단백질 및 가이드 RNA를 더 포함하는 것을 특징으로 하는, 유전자 편집용 조성물.The composition of claim 12, wherein the composition further comprises a CRISPR/Cas protein and a guide RNA.
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