WO2022010286A1 - Acide nucléique donneur utilisé pour la correction génique par assemblage d'extrémités médié par microhomologie et utilisations de celui-ci - Google Patents

Acide nucléique donneur utilisé pour la correction génique par assemblage d'extrémités médié par microhomologie et utilisations de celui-ci 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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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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Gyeongsang National University GNU
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    • 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
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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
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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
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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)
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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]
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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

La présente invention concerne un acide nucléique donneur utilisé pour la correction génique par l'intermédiaire d'une jonction d'extrémités médiée par microhomologie et ses utilisations. L'utilisation de l'acide nucléique donneur selon la présente invention permet une édition de gène précise sans limiter l'emplacement d'un gène cible et le nombre de bases. Par conséquent, la présente invention est prévue pour être utilement utilisée dans le domaine de l'ingénierie des protéines par l'introduction ou l'élimination de SNP et la substitution d'acides aminés courts, ainsi que dans la nouvelle amélioration génétique de cultures.
PCT/KR2021/008727 2020-07-08 2021-07-08 Acide nucléique donneur utilisé pour la correction génique par assemblage d'extrémités médié par microhomologie et utilisations de celui-ci Ceased WO2022010286A1 (fr)

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CN117904069A (zh) * 2024-01-04 2024-04-19 华中农业大学 VirEN蛋白介导的DNA拼接和基因编辑方法

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