US20200208166A1 - Oleic Acid-Enriched Plant Body Having Genetically Modified FAD2 And Production Method Thereof - Google Patents

Oleic Acid-Enriched Plant Body Having Genetically Modified FAD2 And Production Method Thereof Download PDF

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US20200208166A1
US20200208166A1 US16/471,443 US201716471443A US2020208166A1 US 20200208166 A1 US20200208166 A1 US 20200208166A1 US 201716471443 A US201716471443 A US 201716471443A US 2020208166 A1 US2020208166 A1 US 2020208166A1
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sequence
domain
gene
nucleic acid
unsaturated fatty
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Seok Joong Kim
Ok Jae Koo
Min Hee JUNG
Ye Seul KIM
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Toolgen Inc
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Toolgen Inc
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    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
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    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8247—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified lipid metabolism, e.g. seed oil composition
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    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases RNAses, DNAses
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPRs]
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    • C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
    • C12Y114/19—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14) with oxidation of a pair of donors resulting in the reduction of molecular oxygen to two molecules of water (1.14.19)

Definitions

  • the present invention relates to the manipulation or modification of a FAD2 gene using a CRISPR-Cas system to increase the content of oleic acid in a plant body, and more particularly, to a plant body increased in oleic acid content by modifying a FAD2 gene using a CRISPR-Cas system capable of targeting a corresponding gene, a manipulation composition capable of manipulating a FAD2 gene, and a method using the same.
  • Soybean oil is the second most consumed edible oil in the world due to being rich in essential fatty acids and the high utilization of soybean oil meal as a by-product, and 45 million tons thereof are produced annually.
  • About 62% of the fatty acids constituting soybean oil are polyunsaturated fatty acids (PUFAs), and 54% of the fatty acids are linoleic acid, and 8% of the fatty acids are linolenic acid. Since the fatty acids have two or more double bonds, oxidation easily occurs and the oil easily becomes rancid, such that it is difficult to be stored and distributed. (It has a low storage and difficulty in distributions.) Therefore, to manufacture soybean oil with a stable quality to be used in food processing or cooking, pretreatment and purification processes are required. Most soybean oil manufacturers maintain a certain level of quality by preventing rancidity by treating partial hydrogenation for adding a hydrogen to an unsaturated double bond where oxidation easily occurs during a manufacturing process.
  • partial hydrogenation has a disadvantage of producing trans-fatty acids having a risk in a process of saturating the double bond of unsaturated fatty acids with hydrogens. While, in the natural state, the production of cis-fatty acids is dominant in oxidation, however, since trans-forms are thermodynamically stable, trans-fatty acids, which are geometric isomers that do not naturally occur, are produced in hydrogenation or processing.
  • the present invention relates to an artificially manipulated unsaturated fatty acid controlling system, which has an effect of increasing the content of a specific unsaturated fatty acid. More particularly, the present invention relates to an artificially manipulated unsaturated fatty acid biosynthesis-associated factor, and a system for controlling an unsaturated fatty acid, which artificially modifies the content of a specific unsaturated fatty acid.
  • the present invention is directed to providing a plant body increased in the content of a specific unsaturated fatty acid due to an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.
  • the present invention is directed to providing a plant body decreased in the content of a specific unsaturated fatty acid by an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.
  • the present invention provides an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.
  • the present invention provides an artificially manipulated unsaturated fatty acid controlling system.
  • the present invention provides an artificially manipulated unsaturated fatty acid biosynthesis-associated factor and an expression product thereof.
  • the present invention provides a composition for manipulating a gene to manipulate an unsaturated fatty acid biosynthesis-associated factor and a method using the same.
  • the present invention provides a method of controlling the biosynthesis of an unsaturated fatty acid.
  • the present invention provides a method of controlling the type of an unsaturated fatty acid and the content thereof.
  • the present invention provides a composition for controlling an unsaturated fatty acid to control the biosynthesis of an unsaturated fatty acid and/or the content of the unsaturated fatty acid, and various uses thereof.
  • the present invention provides an artificially manipulated unsaturated fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4, FAD6, FAD7 or FAD8 and/or an expression product thereof.
  • an artificially manipulated unsaturated fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4, FAD6, FAD7 or FAD8 and/or an expression product thereof.
  • the present invention provides a composition for manipulating a gene to artificially manipulate an unsaturated fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4, FAD6, FAD7 or FAD8.
  • an unsaturated fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4, FAD6, FAD7 or FAD8.
  • the present invention provides an artificially manipulated unsaturated fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4, FADE, FAD7 or FAD8 and/or various uses of the composition for manipulating a gene for artificial manipulation.
  • an artificially manipulated unsaturated fatty acid biosynthesis-associated factor such as FAD2, FAD3, FAD4, FADE, FAD7 or FAD8 and/or various uses of the composition for manipulating a gene for artificial manipulation.
  • the present invention provides a plant body increased or decreased in the content of a specific unsaturated fatty acid and a processed product using the same.
  • the present invention provides a system capable of artificially controlling the biosynthesis of an unsaturated fatty acid and/or the content of the fatty acids, which includes an artificially manipulated unsaturated fatty acid biosynthesis-associated factor and/or a composition capable of artificially manipulating the unsaturated fatty acid biosynthesis-associated factor, for controlling the content of a specific unsaturated fatty acid.
  • the present invention provides a plant body increased in the content of a specific unsaturated fatty acid by an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.
  • the present invention provides a specific unsaturated fatty acid obtained from a plant body by using an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.
  • specific unsaturated fatty acid refers to one or more unsaturated fatty acids selected from various types of known unsaturated fatty acids, it may be one or more unsaturated fatty acids selected from the classification system represented by the number of carbons (C) and the number of double bonds (D), which are included in an unsaturated fatty acid among various types of unsaturated fatty acids.
  • C carbons
  • D double bonds
  • CN:DM unsaturated fatty acid used herein refers to an unsaturated fatty acid consisting of N number of carbons (C) and including M number of double bonds (D).
  • N may be an integer of 4 to 36
  • M may be an integer of 1 to 35.
  • the specific unsaturated fatty acid may be a C8 ⁇ 24:D1 unsaturated fatty acid.
  • the specific unsaturated fatty acid may be a C16 ⁇ 22:D1 unsaturated fatty acid.
  • the specific unsaturated fatty acid may be a C18:D1 unsaturated fatty acid.
  • the specific unsaturated fatty acid may be oleic acid, elaidic acid or vaccenic acid.
  • the specific unsaturated fatty acid may be a C8 ⁇ 24:D2 unsaturated fatty acid.
  • the specific unsaturated fatty acid may be a C16 ⁇ 22:D2 unsaturated fatty acid.
  • the specific unsaturated fatty acid may be a C18:D2 unsaturated fatty acid.
  • the specific unsaturated fatty acid may be linoleic acid or linoelaidic acid.
  • the present invention provides an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.
  • the term “unsaturated fatty acid biosynthesis-associated factor” used herein refers to all factors directly participating in or indirectly affecting the biosynthesis of an unsaturated fatty acid.
  • the factor may be DNA, RNA, a gene, a peptide, a polypeptide or a protein.
  • the factor includes various materials capable of controlling the biosynthesis of an unsaturated fatty acid, which are non-natural, that is, artificially manipulated.
  • the factor may be a genetically manipulated or modified gene or protein, which is expressed in a plant.
  • the unsaturated fatty acid biosynthesis-associated factor may increase the content of a specific unsaturated fatty acid included in a plant.
  • the unsaturated fatty acid biosynthesis-associated factor may decrease the content of a specific unsaturated fatty acid included in a plant.
  • the unsaturated fatty acid biosynthesis-associated factor may affect a direct/indirect mechanism for controlling the content of a specific unsaturated fatty acid included in a plant.
  • the unsaturated fatty acid biosynthesis-associated factor may be, for example, an artificially manipulated a FAD2 gene, a FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene or a FAD8 gene, preferably a FAD2 gene or a FAD3 gene.
  • the unsaturated fatty acid biosynthesis-associated factor may include two or more artificially manipulated genes.
  • two or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene may be artificially manipulated.
  • one or more artificially manipulated unsaturated fatty acid biosynthesis-associated factors selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene, which have undergone modification in a nucleic acid sequence, are provided.
  • the modification in a nucleic acid sequence may be non-limitedly, artificially manipulated by a guide nucleic acid-editor protein complex.
  • guide nucleic acid-editor protein complex refers to a complex formed through the interaction between a guide nucleic acid and an editor protein, and the nucleic acid-protein complex includes the guide nucleic acid and the editor protein.
  • the guide nucleic acid-editor protein complex may serve to modify a subject.
  • the subject may be a target nucleic acid, a gene, a chromosome or a protein.
  • the gene may be an unsaturated fatty acid biosynthesis-associated factor, artificially manipulated by a guide nucleic acid-editor protein complex,
  • the unsaturated fatty acid biosynthesis-associated factor artificially manipulated includes one or more modifications of nucleic acids which is
  • a deletion or insertion of one or more nucleotides at least one of a deletion or insertion of one or more nucleotides, a substitution with one or more nucleotides different from a wild-type gene, and an insertion of one or more foreign nucleotide, in a proto-spacer-adjacent motif (PAM) sequence in a nucleic acid sequence constituting the unsaturated fatty acid biosynthesis-associated factor or in a continuous 1 bp to 50 bp the base sequence region adjacent to the 5′ end and/or 3′ end thereof, or
  • PAM proto-spacer-adjacent motif
  • the modification of nucleic acids may occur in a promoter region of the gene.
  • the modification of nucleic acids may occur in an exon region of the gene. In one exemplary embodiment, 50% of the modifications may occur in the upstream section of the coding regions of the gene.
  • the modification of nucleic acids may occur in an intron region of the gene.
  • the modification of nucleic acids may occur in an enhancer region of the gene.
  • the PAM sequence may be, for example, one or more of the following sequences (described in the 5′ to 3′ direction):
  • N is A, T, C or G
  • NNNNRYAC (each of N is independently A, T, C or G, R is A or G, and Y is C or T);
  • NNAGAAW (each of N is independently A, T, C or G, and W is A or T);
  • NNNNGATT (each of N is independently A, T, C or G);
  • NNGRR(T) (each of N is independently A, T, C or G, and R is A or G);
  • TTN (N is A, T, C or G).
  • the editor protein may be derived from Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cyanothece sp., Microcystis aeruginos
  • the editor protein may be one or more selected from the group consisting of a Streptococcus pyogenes -derived Cas9 protein, a Campylobacter jejuni -derived Cas9 protein, a Streptococcus thermophilus -derived Cas9 protein, a Staphylococcus aureus -derived Cas9 protein, a Neisseria meningitidis -derived Cas9 protein, and a Cpf1 protein.
  • the editor protein may be a Streptococcus pyogenes -derived Cas9 protein or a Campylobacter jejuni -derived Cas9 protein.
  • the present invention provides a guide nucleic acid, which is capable of forming a complementary bond with respect to target sequences of SEQ ID NOs: 1 to 30, for example, SEQ ID NOs:7 or 30.
  • the guide nucleic acid may form a complementary bond with a part of nucleic acid sequences of a FAD2 gene. It may create 0 to 5, 0 to 4, 0 to 3, or 0 to 2 mismatches.
  • the guide nucleic acid may be nucleotides forming a complementary bond with one or more of the target sequences of SEQ ID NOs: 1 to 30, for example, SEQ ID NOs: 7 or 30, respectively.
  • the guide nucleic acid may be non-limitedly 18 to 25 bp, 18 to 24 bp, 18 to 23 bp, 19 to 23 bp, or 20 to 23 bp nucleotides.
  • the present invention provides a composition for gene manipulation, which may be employed in artificial manipulation of an unsaturated fatty acid biosynthesis-associated factor for a specific purpose.
  • composition for gene manipulation may include a guide nucleic acid-editor protein complex or a nucleic acid sequence encoding the same.
  • composition for gene manipulation may include:
  • a guide nucleic acid capable of forming a complementary bond with respect to each of target sequences of one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene, respectively or a nucleic acid sequence encoding the guide nucleic acid;
  • an editor protein including one or more proteins selected from the group consisting of a Streptococcus pyogenes -derived Cas9 protein, a Campylobacter jejuni -derived Cas9 protein, a Streptococcus thermophilus -derived Cas9 protein, a Staphylococcus aureus -derived Cas9 protein, a Neisseria meningitidis -derived Cas9 protein, and a Cpf1 protein or a nucleic acid sequence encoding the same.
  • the guide nucleic acid may be a nucleic acid sequence which forms a complementary bond with respect to one or more of the target sequences of SEQ ID NOs: 1 to 30, respectively.
  • the guide nucleic acid may be a nucleic acid sequence which forms a complementary bond with the target sequence of SEQ ID NOs: 7 or 30.
  • the composition for gene manipulation may be a viral vector system.
  • the viral vector may be an agrobacterium vector system using an agrobacteria.
  • the composition for gene manipulation may be a viral vector system.
  • the viral vector may include one or more selected from the group consisting of a mosaic virus, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus and a herpes simplex virus.
  • the present invention provides a method for artificially manipulating cells, which includes: introducing (a) a guide nucleic acid which is capable of forming a complementary bond with respect to the target sequences of one or more genes selected from the group consisting of a FAD2 gene, a FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene, respectively, or a nucleic acid sequence encoding the same; and
  • an editor protein including one or more proteins selected from the group consisting of a Streptococcus pyogenes -derived Cas9 protein, a Campylobacter jejuni -derived Cas9 protein, a Streptococcus thermophilus -derived Cas9 protein, a Staphylococcus aureus -derived Cas9 protein, a Neisseria meningitidis -derived Cas9 protein, and a Cpf1 protein, respectively, or a nucleic acid sequence encoding the same to cells.
  • the guide nucleic acid and the editor protein may be present in one or more vectors in the form of a nucleic acid sequence, or may be present in a complex formed by coupling the guide nucleic acid with the editor protein.
  • the introduction may be performed in vivo or ex vivo of a plant.
  • the introduction may be performed by one or more methods selected from a gene gun, an electroporation, liposomes, plasmids, agrobacterium vector system, viral vectors, nanoparticles and a protein translocation domain (PTD) fusion protein method.
  • a gene gun an electroporation, liposomes, plasmids, agrobacterium vector system, viral vectors, nanoparticles and a protein translocation domain (PTD) fusion protein method.
  • PTD protein translocation domain
  • the viral vector may include one or more selected from the group consisting of a mosaic virus, a retrovirus, a lentivirus, an adenovirus, an adeno-associated virus (AAV), a vaccinia virus, a poxvirus and a herpes simplex virus.
  • the present invention provides a composition for controlling an unsaturated fatty acid to control the biosynthesis of an unsaturated fatty acid and/or the content of the unsaturated fatty acid of a plant.
  • the composition for controlling an unsaturated fatty acid may include a composition for gene manipulation, which may be employed in artificial manipulation of an unsaturated fatty acid biosynthesis-associated factor.
  • composition for gene manipulation is the same as described above.
  • the present invention provides a processed product using a plant body increased or decreased in the content of a specific unsaturated fatty acid.
  • the plant body may include an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.
  • the processed product may be a food which can be ingested by humans and/or animals.
  • the present invention provides a kit for gene manipulation to control the content of a specific unsaturated fatty acid.
  • the kit may include a composition for gene manipulation, which may be employed in artificial manipulation of an unsaturated fatty acid biosynthesis-associated factor.
  • the gene of interest may be artificially manipulated using such a kit.
  • a plant body increased in the content of a specific unsaturated fatty acid which is good for human health or decreased in the content of a specific unsaturated fatty acid which is harmful for human health, and/or a processed product using the same can be manufactured by using an artificially manipulated unsaturated fatty acid biosynthesis-associated factor and a system for controlling an unsaturated fatty acid, which is artificially modified thereby.
  • one or more genes selected from a FAD2 gene, a FAD3 gene, a FAD4 gene, a FAD6 gene, a FAD7 gene and a FAD8 gene can be used.
  • FIG. 1 is a schematic diagram of CRISPR-Cas9 vectors, pPZP-FAD2-7 and pPZP-FAD2-30, for modifying a FAD2 gene of soybeans.
  • FIGS. 2A and 2B illustrates the growth processes of soybean transgenic plant bodies prepared by the knockout of a FAD2 gene using pPZP-FAD2-7(a) and pPZP-FAD2-30(b).
  • FIG. 3 shows T0 transformants of pPZP-FAD2-7 and pPZP-FAD2-30.
  • FIG. 4 shows the PCR results for confirming insert genes of the T0 transformants of pPZP-FAD2-7 and pPZP-FAD2-30.
  • NT is Glycine max L. Kwangan (wild type), and #1, #2, #3, #5, #8, #9, #19 and #21 are T0 transformants.
  • FIG. 5 shows the contents of oleic acid in T 1 seeds of pPZP-FAD2-7 and pPZP-FAD2-30.
  • FIG. 6 shows the indel frequency of an FAD2 gene targeted by CRISPR-Cas9 (the target sequence of FAD2A gene is SEQ ID NO: 7, and the target sequence of FAD2B is SEQ ID NO: 7).
  • FIG. 7 shows the sequencing results for an FAD2 gene of soybeans transformed using CRISPR-Cas9 (a target sequence including PAM shown with an underline).
  • the target site (Type WT) located in the chromosome #10 is SEQ ID NO: 71
  • the target site (Type WT) located in the chromosome #20 is SEQ ID NO: 72.
  • the target site includes the target sequence.
  • the sequencing results for the FAD2 gene located in the chromosome #10 are shown in SEQ ID NO: 75 to 83 (Type ⁇ 3 to +1 order).
  • the sequencing results for the FAD2 gene located in the chromosome #20 are shown in SEQ ID NO: 84 to 92 (Type ⁇ 4 to ⁇ 1 order).
  • FIGS. 8A and 8B shows the results of target site screening and indel frequency of an FAD2 gene manipulated in T 1 transformants, shows the results of target site screening and indel frequency for of an FAD2 gene in (a) Chromosome #10 (chr10), and (b) Chromosome #20 (chr20).
  • FIGS. 9A and 9B shows the target site sequencing results of an FAD2 gene manipulated in T 1 transformants, shows the target site sequencing results of an FAD2 gene in (a) Chromosome #10 (chr10), and (b) Chromosome #20 (chr20).
  • the target site located in the chromosome #10 is SEQ ID NO: 73
  • the target site located in the chromosome #20 is SEQ ID NO: 74.
  • the target site sequencing results of the FAD2 located in the chromosome #10 are shown in the following SEQ ID NOs according to samples: SEQ ID NO: 73 (FAD2-7#1-1 having no indel); SEQ ID NO:93, 94 (FAD2-30#2-4); SEQ ID NO: 73 (FAD2-30#3-1 having no indel); SEQ ID NO: 95, 96 (FAD2-30#3-2); SEQ ID NO: 97 (FAD2-30#8-1); SEQ ID NO: 98 to 100 (FAD2-30#8-2); SEQ ID NO: 101 (FAD2-30#9-1), SEQ ID NO: 73 (FAD2-30#9-1 having no indel); SEQ ID NO: 102, 103 (FAD2-30#19-1); SEQ ID NO: 104 (FAD2-30#21-2); SEQ ID NO: 105 (FAD2-30#21-5); SEQ ID NO: 106 (FAD2-30#22-5); SEQ ID NO: 107 (
  • the target site sequencing results of the FAD2 located in the chromosome #20 are shown in the following SEQ ID NOs according to samples: SEQ ID NO: 108, 109 (FAD2-7#1-1); SEQ ID NO: 74 (FAD2-30#2-4 having no indel), SEQ ID NO: 110 (FAD2-30#2-4); SEQ ID NO: 74 (FAD2-30#3-1 having no indel); SEQ ID NO: 111 (FAD2-30#3-2); SEQ ID NO: 112 (FAD2-30#8-1); SEQ ID NO: 113 (FAD2-30#8-2); SEQ ID NO: 114 (FAD2-30#9-1); SEQ ID NO: 115 (FAD2-30#19-1); SEQ ID NO: 116, 117 (FAD2-30#21-2); SEQ ID NO: 118 (FAD2-30#21-5); SEQ ID NO: 119 (FAD2-30#22-5); SEQ ID NO: 120 (FAD2-30#22-6).
  • FIG. 10 shows T 1 transformants of pPZP-FAD2-7 and pPZP-FAD2-30.
  • FIG. 11 shows the analysis results of the removal of a gene from T 1 transformants of pPZP-FAD2-7 and pPZP-FAD2-30 using PCR.
  • One aspect of the present invention relates to a transgenic plant body increased in the content of a C8 ⁇ 24:D1 unsaturated fatty acid.
  • the present invention relates to a transgenic plant body increased in the content of a specific unsaturated fatty acid by artificially manipulating an unsaturated fatty acid biosynthesis-associated factor
  • the present invention includes an unsaturated fatty acid biosynthesis-associated factor in which a function is artificially changed, an artificial manipulation composition therefor, a method of preparing the same, and a plant body including the same.
  • Another aspect of the present invention relates to a transgenic plant body decreased in the content of a C8 ⁇ 24:D2 unsaturated fatty acid.
  • the present invention relates to a transgenic plant body decreased in the content of a specific unsaturated fatty acid by artificially manipulating an unsaturated fatty acid biosynthesis-associated factor
  • the present invention includes an unsaturated fatty acid biosynthesis-associated factor in which a function is artificially changed, an artificial manipulation composition therefor, a method of preparing the same, and a plant body including the same.
  • One aspect of the present invention is a system for changing the content of fatty acids.
  • a system for changing the content of a specific saturated fatty acid in a plant body may be provided.
  • a system for changing the content of a specific unsaturated fatty acid in a plant body may be provided.
  • fatty acid used herein refers to a carboxylic acid having an aliphatic chain, and most fatty acids produced in a natural state have an even number of carbons ranging from about 4 to 36, which forms a carbon chain. Fatty acids are largely classified into saturated fatty acids and unsaturated fatty acids according to the type of a carbon bond.
  • saturated fatty acid used herein refers to fatty acids formed with a single bond.
  • Fatty acids include propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid, capric acid, lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid, behenic acid, lignoceric acid, cerotic acid and the like.
  • the term “unsaturated fatty acids” used herein refers to fatty acids having one or more carbon-carbon double bonds.
  • the unsaturated fatty acids include all of cis-unsaturated fatty acids and trans-unsaturated fatty acids.
  • the cis-unsaturated fatty acids refer to unsaturated fatty acids in which two hydrogens respectively binding to two carbons participating in a double bond are structurally placed in the same direction.
  • the trans-unsaturated fatty acids refer to unsaturated fatty acids in which two hydrogens respectively binding to two carbons participating in a double bond are structurally placed in different directions.
  • the unsaturated fatty acids may be classified into Omega-3, 6, 7 and 9 according to the position of a carbon participating in a double bond.
  • the unsaturated fatty acids include Omega-3 ( ⁇ -3) fatty acids.
  • omega-3 ( ⁇ -3) fatty acids refers to an unsaturated fatty acid in which a double bond starts from the third carbon at the end of the carbon chain, and includes alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA).
  • ALA alpha-linolenic acid
  • EPA eicosapentaenoic acid
  • DHA docosahexaenoic acid
  • the unsaturated fatty acids include omega-6 ( ⁇ -6) fatty acids.
  • omega-6 ( ⁇ -6) fatty acids refers to unsaturated fatty acids in which a double bond starts from the sixth carbon at the end of a carbon chain, and includes linoleic acid (LA), gamma-linolenic acid (GLA), dihomo-gamma-linolenic acid (DGLA) and arachidonic acid (AA).
  • LA linoleic acid
  • GLA gamma-linolenic acid
  • DGLA dihomo-gamma-linolenic acid
  • AA arachidonic acid
  • the unsaturated fatty acids include omega-7 ( ⁇ -7) fatty acids.
  • omega-7 ( ⁇ -7) fatty acids refers to unsaturated fatty acids in which a double bond starts from the seventh carbon at the end of a carbon chain, and includes paullinic acid, palmitoleic acid or vaccenic acid.
  • the unsaturated fatty acids include omega-9 ( ⁇ -9) fatty acids.
  • omega-9 ( ⁇ -9) fatty acids refers to unsaturated fatty acids in which a double bond starts from the ninth carbon at the end of a carbon chain, and includes oleic acid, elaidic acid, eicosenoic acid, erucic acid and nervonic acid.
  • the unsaturated fatty acid may be an omega-6 ( ⁇ -6) fatty acid.
  • the unsaturated fatty acid may be an omega-9 ( ⁇ -9) fatty acid.
  • the unsaturated fatty acid may be classified as a CN:DN unsaturated fatty acid by representing the number of carbons and the number of double bonds.
  • CN:DM unsaturated fatty acid refers to an unsaturated fatty acid consisting of N number of carbons (C) and including M number of double bonds (D).
  • N may be an integer of 4 to 36
  • M may be an integer of 1 to 35.
  • the unsaturated fatty acid consisting of 18 carbons and including 2 double bonds may be classified by being represented as a C18:D2 unsaturated fatty acid.
  • the unsaturated fatty acid includes a CN:D1 unsaturated fatty acid.
  • N may be an integer of 4 to 36.
  • the CN:D1 unsaturated fatty acid may be a C8:D1 unsaturated fatty acid, a C10:D1 unsaturated fatty acid, a C12:D1 unsaturated fatty acid, a C14:D1 unsaturated fatty acid, a C16:D1 unsaturated fatty acid, a C18:D1 unsaturated fatty acid, a C20:D1 unsaturated fatty acid, a C22:D1 unsaturated fatty acid or a C24:D1 unsaturated fatty acid.
  • the unsaturated fatty acid includes a CN:D2 unsaturated fatty acid.
  • N may be an integer of 4 to 36.
  • the CN:D2 unsaturated fatty acid may be a C8:D2 unsaturated fatty acid, a C10:D2 unsaturated fatty acid, a C12:D2 unsaturated fatty acid, a C14:D2 unsaturated fatty acid, a C16:D2 unsaturated fatty acid, a C18:D2 unsaturated fatty acid, a C20:D2 unsaturated fatty acid, a C22:D2 unsaturated fatty acid or a C24:D2 unsaturated fatty acid.
  • the unsaturated fatty acid includes a CN:D3 unsaturated fatty acid.
  • N may be an integer of 4 to 36.
  • the CN:D3 unsaturated fatty acid may be a C8:D3 unsaturated fatty acid, a C10:D3 unsaturated fatty acid, a C12:D3 unsaturated fatty acid, a C14:D3 unsaturated fatty acid, a C16:D3 unsaturated fatty acid, a C18:D3 unsaturated fatty acid, a C20:D3 unsaturated fatty acid, a C22:D3 unsaturated fatty acid or a C24:D3 unsaturated fatty acid.
  • the unsaturated fatty acid includes a CN:D4 unsaturated fatty acid.
  • N may be an integer of 4 to 36.
  • the CN:D4 unsaturated fatty acid may be a C8:D4 unsaturated fatty acid, a C10:D4 unsaturated fatty acid, a C12:D4 unsaturated fatty acid, a C14:D4 unsaturated fatty acid, a C16:D4 unsaturated fatty acid, a C18:D4 unsaturated fatty acid, a C20:D4 unsaturated fatty acid, a C22:D4 unsaturated fatty acid or a C24:D4 unsaturated fatty acid.
  • the unsaturated fatty acid includes a CN:D5 unsaturated fatty acid.
  • N may be an integer of 4 to 36.
  • the CN:D5 unsaturated fatty acid may be a C8:D5 unsaturated fatty acid, a C10:D5 unsaturated fatty acid, a C12:D5 unsaturated fatty acid, a C14:D5 unsaturated fatty acid, a C16:D5 unsaturated fatty acid, a C18:D5 unsaturated fatty acid, a C20:D5 unsaturated fatty acid, a C22:D5 unsaturated fatty acid or a C24:D5 unsaturated fatty acid.
  • the unsaturated fatty acid includes a CN:D6 unsaturated fatty acid.
  • N may be an integer of 4 to 36.
  • the CN:D6 unsaturated fatty acid may be a C8:D6 unsaturated fatty acid, a C10:D6 unsaturated fatty acid, a C12:D6 unsaturated fatty acid, a C14:D6 unsaturated fatty acid, a C16:D6 unsaturated fatty acid, a C18:D6 unsaturated fatty acid, a C20:D6 unsaturated fatty acid, a C22:D6 unsaturated fatty acid or a C24:D6 unsaturated fatty acid.
  • the unsaturated fatty acid includes a CN:DK unsaturated fatty acid.
  • N may be an integer of 4 to 36
  • K may be an integer of 7 to 35.
  • the unsaturated fatty acid may be a C8 to 24:D1 unsaturated fatty acid.
  • the unsaturated fatty acid may be selected from the group consisting of a C16:D1 unsaturated fatty acid, a C18:D1 unsaturated fatty acid, a C20:D1 unsaturated fatty acid and a C22:D1 unsaturated fatty acid.
  • the unsaturated fatty acid may be a C18:D1 unsaturated fatty acid or a C20:D1 unsaturated fatty acid.
  • the unsaturated fatty acid may be a C8 to 24:D2 unsaturated fatty acid.
  • the unsaturated fatty acid may be selected from the group consisting of a C16:D2 unsaturated fatty acid, a C18:D2 unsaturated fatty acid, a C20:D2 unsaturated fatty acid and a C22:D2 unsaturated fatty acid.
  • the unsaturated fatty acid may be a C18:D2 unsaturated fatty acid or a C20:D2 unsaturated fatty acid.
  • Another aspect of the present invention is an artificially manipulated or modified unsaturated fatty acid biosynthesis-associated factor.
  • saturated fatty acid biosynthesis-associated factor refers to all factors directly participating in or indirectly affecting the biosynthesis of an unsaturated fatty acid.
  • the factor may be DNA, RNA, a gene, a peptide, a polypeptide or a protein.
  • the unsaturated fatty acid biosynthesis-associated factor includes various materials capable of controlling the biosynthesis of an unsaturated fatty acid, which are non-natural, that is, artificially manipulated.
  • the unsaturated fatty acid biosynthesis-associated factor may be a genetically manipulated or modified gene or protein, which is expressed in a plant.
  • artificially manipulated means an artificially modified state, which is not a naturally occurring state.
  • genetically manipulated means that a genetic modification is artificially introduced to plant-derived substances cited in the present invention, and may be, for example, genes and gene products (polypeptides, proteins, etc.) in which their genomes are artificially modified for a specific purpose.
  • the present invention provides a unsaturated fatty acid biosynthesis-associated factor which is genetically manipulated or modified for a specific purpose.
  • Genes or proteins having the functions listed below may have multiple types of functions, not only one type of unsaturated fatty acid biosynthesis-associated function. In addition, as needed, two or more unsaturated fatty acid biosynthesis-associated functions and factors may be provided.
  • An unsaturated fatty acid biosynthesis-associated factor may produce an unsaturated fatty acid by forming one or more double bonds in a saturated fatty acid.
  • the unsaturated fatty acid biosynthesis-associated factor may form new one or more double bonds in an unsaturated fatty acid.
  • the unsaturated fatty acid biosynthesis-associated factor may change a position of one or more double bonds included in an unsaturated fatty acid.
  • the unsaturated fatty acid biosynthesis-associated factor may remove one or more double bonds of an unsaturated fatty acid having two or more double bonds.
  • the unsaturated fatty acid biosynthesis-associated factor may change a cis-unsaturated fatty acid into a trans-unsaturated fatty acid.
  • the unsaturated fatty acid biosynthesis-associated factor may change a trans-unsaturated fatty acid into a cis-unsaturated fatty acid.
  • the unsaturated fatty acid biosynthesis-associated factor may control the content of an unsaturated fatty acid included in a plant.
  • the unsaturated fatty acid biosynthesis-associated factor may increase the content of a specific unsaturated fatty acid included in a plant.
  • the unsaturated fatty acid biosynthesis-associated factor may decrease the content of a specific unsaturated fatty acid included in a plant.
  • the Unsaturated Fatty Acid Biosynthesis-Associated Factor May be an Unsaturated Fatty Acid Biosynthesis-Associated Factor of a Plant.
  • the unsaturated fatty acid biosynthesis-associated factor may be a FAD gene or FAD protein.
  • the unsaturated fatty acid biosynthesis-associated factor may be one or more selected from the group consisting of FAD2, FAD3, FADE, FAD7 and FAD8.
  • the unsaturated fatty acid biosynthesis-associated factor may be FAD2.
  • a FAD2 (omega-6 fatty acid desaturase) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding the FAD2 protein also referred to as FAD2-1, FAD2-1B or GMFAD2-1B.
  • the FAD2 gene may be one or more genes selected from the group consisting of the following genes, but the present invention is not limited thereto: genes encoding plant, for example, soybean ( Glycine max ) FAD2 (e.g., NCBI Accession No.
  • NP_001341865.1 for example, FAD2 genes represented by NCBI Accession No. NM_001354936.1, XM_006605820.2, XM_006605819.2, XM_006605822.2, XM_006605821.2, or XM_014772279.1.
  • the unsaturated fatty acid biosynthesis-associated factor may be FAD3.
  • a FAD3 (microsomal omega-3 fatty acid desaturase) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding a FAD3 protein also referred to as Fanx.
  • the FAD3 gene may be one or more genes selected from the group consisting of the following genes, but the present invention is not limited thereto: a gene encoding plant, for example, soybean ( Glycine max ) FAD3 (e.g., NCBI Accession No. NP_001237507.1), for example, an FAD3 gene represented by NCBI Accession No. NM_001250578.1.
  • the unsaturated fatty acid biosynthesis-associated factor may be FAD6.
  • a FAD6 (fatty acid desaturase 6) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding a FAD6 protein also referred to as FADC or SFD4.
  • the FAD6 gene may be one or more genes selected from the group consisting of the following genes, but the present invention is not limited thereto: a gene encoding a plant, for example, Arabidopsis thaliana FAD6 (e.g., NCBI Accession No. NP_194824.1), for example, a FAD6 gene represented by NCBI Accession No. NM_119243.4.
  • the unsaturated fatty acid biosynthesis-associated factor may be FAD7.
  • a FAD7 (chloroplast omega 3 fatty acid desaturase isoform 2) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding a FAD7 protein.
  • the FAD7 gene may be one or more genes selected from the group consisting of the following genes, but the present invention is not limited thereto: a gene encoding a plant, for example, soybean ( Glycine max ) FAD7 (e.g., NCBI Accession No. NP_001237361.1), for example, a FAD7 gene represented by NCBI Accession No. NM_001250432.1.
  • the unsaturated fatty acid biosynthesis-associated factor may be FAD8.
  • a FAD8 (omega-3 fatty acid desaturase, chloroplastic-like) gene refers to a gene (full-length DNA, cDNA or mRNA) encoding a FAD8 protein.
  • the FAD8 gene may be one or more genes selected from the group consisting of the following genes, but the present invention is not limited thereto: a gene encoding a plant, for example, soybean ( Glycine max ) FAD8 (e.g., NCBI Accession No. NP_001239777.1), for example, a FAD8 gene represented by NCBI Accession No. NM_001252848.1.
  • the unsaturated fatty acid biosynthesis-associated factor may be derived from a plant such as soybean, Arabidopsis thaliana , sesame, corn and the like, etc.
  • NCBI National Center for Biotechnology Information
  • the unsaturated fatty acid biosynthesis-associated factor for example, FAD2, FAD3, FADE, FAD7 or FAD8, may be artificially manipulated unsaturated fatty acid biosynthesis-associated factor.
  • the artificially manipulated unsaturated fatty acid biosynthesis-associated factor may be genetically manipulated.
  • the gene manipulation or modification may be achieved by artificial insertion, deletion, substitution or inversion occurring in a partial or entire region of the genomic sequence of a wild type gene.
  • the gene manipulation or modification may be achieved by fusion of manipulation or modification of two or more genes.
  • the gene may be further activated by such gene manipulation or modification, such that a protein encoded from the gene is to be expressed in the form of a protein having an improved function, compared to the innate function.
  • a function of the protein encoded by a specific gene is A
  • a function of a protein expressed by a manipulated gene may be totally different from A or may have an additional function (A+B) including A.
  • A+B additional function
  • a fusion of two or more proteins may be expressed using two or more genes having different or complementary functions due to such gene manipulation or modification.
  • two or more proteins may be expressed separately or independently in cells by using two or more genes having different or complementary functions due to such gene manipulation or modification.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may produce an unsaturated fatty acid by forming one or more double bonds in a saturated fatty acid.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may form new one or more double bonds in an unsaturated fatty acid.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may change positions of one or more double bonds included in an unsaturated fatty acid.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may remove one or more double bonds of an unsaturated fatty acid having two or more double bonds.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may change a cis-unsaturated fatty acid into a trans-unsaturated fatty acid.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may change a trans-unsaturated fatty acid into a cis-unsaturated fatty acid.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may control the content of an unsaturated fatty acid included in a plant.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may increase the content of a specific unsaturated fatty acid included in a plant.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may decrease the content of a specific unsaturated fatty acid included in a plant.
  • the manipulation includes all types of structural or functional modifications of the unsaturated fatty acid biosynthesis-associated factor.
  • the structural modification of the unsaturated fatty acid biosynthesis-associated factor includes all types of modifications, which are not the same as those of a wild type existing in a natural state.
  • the structural modification may be the loss of one or more nucleotides.
  • the structural modification may be the insertion of one or more nucleotides.
  • the inserted nucleotides include all of a subject including an unsaturated fatty acid biosynthesis-associated factor and nucleotides entering from the outside of the subject.
  • the structural modification may be the substitution of one or more nucleotides.
  • the structural modification may include the chemical modification of one or more nucleotides.
  • the chemical modification includes all of the addition, removal and substitution of chemical functional groups.
  • the structural modification may be the loss of one or more amino acids.
  • the structural modification may be the insertion of one or more amino acids.
  • the inserted amino acids include all of a subject including an unsaturated fatty acid biosynthesis-associated factor and amino acids entering from the outside of the subject.
  • the structural modification may be the substitution of one or more amino acids.
  • the structural modification may include the chemical modification of one or more amino acids.
  • the chemical modification includes all of the addition, removal and substitution of chemical functional groups.
  • the structural modification may be the partial or entire attachment of a different peptide, polypeptide or protein.
  • the different peptide, polypeptide or protein may be an unsaturated fatty acid biosynthesis-associated factor, or a peptide, polypeptide or protein having a different function.
  • the functional modification of the unsaturated fatty acid biosynthesis-associated factor may include all types having an improved or reduced function, compared to that of a wild type existing in a natural state, and having a third different function.
  • the functional modification may be a mutation of the unsaturated fatty acid biosynthesis-associated factor.
  • the mutation may be a mutation that enhances or suppresses a function of the unsaturated fatty acid biosynthesis-associated factor.
  • the functional modification may have an additional function of the unsaturated fatty acid biosynthesis-associated factor.
  • the additional function may be the same or a different function.
  • the unsaturated fatty acid biosynthesis-associated factor having the additional function may be fused with a different peptide, polypeptide or protein.
  • the functional modification may be the enhancement in functionality due to increased expression of the unsaturated fatty acid biosynthesis-associated factor.
  • the functional modification may be the degradation in functionality due to decreased expression of the unsaturated fatty acid biosynthesis-associated factor.
  • the manipulated unsaturated fatty acid biosynthesis-associated factor may be induced by one or more of the following mutations:
  • a target gene for example, deletion of 1 bp or longer nucleotides, for example, 1 to 30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide of the target gene,
  • substitution of 1 bp or longer nucleotides for example, 1 to 30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide of the target gene with a nucleotide different from a wild type, and
  • nucleotides for example, 1 to 30, 1 to 27, 1 to 25, 1 to 23, 1 to 20, 1 to 15, 1 to 10, 1 to 5, 1 to 3, or 1 nucleotide (each independently selected from A, T, C and G) into a certain position of the target gene.
  • a part of the modified target gene (“target region”) may be a continuous 1 bp or more, 3 bp or more, 5 bp or more, 7 bp or more, 10 bp or more, 12 bp or more, 15 bp or more, 17 bp or more, or 20 bp or more, for example, 1 bp to 30 bp, 3 bp to 30 bp, 5 bp to 30 bp, 7 bp to 30 bp, 10 bp to 30 bp, 12 bp to 30 bp, 15 bp to 30 bp, 17 bp to 30 bp, 20 bp to 30 bp, 1 bp to 27 bp, 3 bp to 27 bp, 5 bp to 27 bp, 7 bp to 27 bp, 10 bp to 27 bp, 12 bp to 27 bp, 15 bp to 27 bp, 17 bp to 27 bp, 20
  • One aspect of the present invention relates to a system for controlling an unsaturated fatty acid, which controls the biosynthesis of an unsaturated fatty acid by artificially manipulating an unsaturated fatty acid biosynthesis-associated factor.
  • system for controlling an unsaturated fatty acid includes all phenomena affecting the promotion or inhibition of the biosynthesis of an unsaturated fatty acid, and/or the increase or inhibition of the production of unsaturated fatty acids by changing functions of the artificially manipulated unsaturated fatty acid biosynthesis-associated factor, and includes all materials, compositions, methods and uses directly or indirectly involved in the system of controlling the biosynthesis of an unsaturated fatty acid.
  • Each factor constituting the system for controlling the biosynthesis of an unsaturated fatty acid is also referred to as an “unsaturated fatty acid controlling factor.”
  • the system of the present invention includes a modified mechanism in a plant body, which is associated with an artificially manipulated unsaturated fatty acid biosynthesis-associated factor.
  • an artificially manipulated unsaturated fatty acid biosynthesis-associated factor By the artificially manipulated unsaturated fatty acid biosynthesis-associated factor,
  • the biosynthesis of a C8 to 24:D1 unsaturated fatty acid may be controlled,
  • the biosynthesis of a C8 to 24:D2 unsaturated fatty acid may be controlled,
  • the production amount of a C8 to 24:D1 unsaturated fatty acid may be controlled
  • the production amount of a C8 to 24:D2 unsaturated fatty acid may be controlled
  • the content of a C8 to 24:D1 unsaturated fatty acid in a plant body may be controlled
  • the content of a C8 to 24:D2 unsaturated fatty acid in a plant body may be controlled
  • the content ratio of the C8 to 24:D1 unsaturated fatty acid and the C8 to 24:D2 unsaturated fatty acid in a plant body may be controlled
  • a double bond of the C8 to 24:D1 unsaturated fatty acid may be added or removed, and
  • a double bond of the C8 to 24:D2 unsaturated fatty acid may be added or removed.
  • the system for controlling an unsaturated fatty acid of the present invention includes a composition for manipulating an unsaturated fatty acid biosynthesis-associated factor.
  • the composition for manipulation may be a composition capable of artificially manipulating an unsaturated fatty acid biosynthesis-associated factor, and preferably, a composition for gene manipulation.
  • Manipulation or modification of substances involved in the unsaturated fatty acid biosynthesis-associated factor and the system for controlling an unsaturated fatty acid of the present invention is preferably accomplished by genetic manipulation.
  • composition and method for manipulating a gene by targeting a partial or entire non-coding or coding region of the unsaturated fatty acid biosynthesis-associated factor may be provided.
  • the composition and method may be used in manipulation or modification of one or more unsaturated fatty acid biosynthesis-associated genes involved in the formation of a desired system for controlling an unsaturated fatty acid.
  • the manipulation or modification may be performed by modification of nucleic acids constituting a gene. As a result of the manipulation, all of knock down, knock out, and knock in are included.
  • the manipulation may be performed by targeting a promoter region, or a transcription sequence, for example, an intron or exon sequence.
  • a coding sequence for example, a coding region, an initial coding region may be targeted for the modification of expression and knockout.
  • the modification of nucleic acids may be substitution, deletion, and/or insertion of one or more nucleotides, for example, 1 to 30 bp, 1 to 27 bp, 1 to 25 bp, 1 to 23 bp, 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, 1 to 5 bp, 1 to 3 bp, or 1 bp nucleotides.
  • the above-mentioned region may be targeted such that one or more unsaturated fatty acid biosynthesis-associated genes contain a deletion or mutation.
  • the knockdown of a gene may be used to decrease the expression of undesired alleles or transcriptomes.
  • non-coding sequences of a promoter, an enhancer, an intron, a 3′UTR, and/or a polyadenylation signal may be targeted to be used in modifying an unsaturated fatty acid biosynthesis-associated gene affecting an unsaturated fatty acid biosynthesis function.
  • the activity of an unsaturated fatty acid biosynthesis-associated gene may be regulated, for example, activated or inactivated by the modification of nucleic acids of the gene.
  • the modification of nucleic acids of the gene may catalyze cleavage of a single strand or double strands, that is, breaks of nucleic acid strands in a specific region of the target gene by a guide nucleic acid-editor protein complex, resulting in inactivation of the target gene.
  • the nucleic acid strand breaks may be repaired through a mechanism such as homologous recombination or non-homologous end joining (NHEJ).
  • a mechanism such as homologous recombination or non-homologous end joining (NHEJ).
  • NHEJ when the NHEJ mechanism takes place, a change in DNA sequence is induced at the cleavage site, resulting in inactivation of the gene.
  • the repair by NHEJ may induce substitution, insertion or deletion of a short gene fragment, and may be used in the induction of a corresponding gene knockout.
  • the present invention provides a composition for manipulating an unsaturated fatty acid biosynthesis-associated factor.
  • the composition for manipulation is a composition that is able to artificially manipulate an unsaturated fatty acid biosynthesis-associated factor, and preferably, a composition for gene manipulation.
  • composition may be employed in gene manipulation for one or more unsaturated fatty acid biosynthesis-associated factors involved in formation of a desired system for controlling an unsaturated fatty acid.
  • the gene manipulation may be performed in consideration of a gene expression regulating process.
  • it may be performed by selecting a suitable manipulation means for each stage of transcription, RNA processing, RNA transporting, RNA degradation, translation, and protein modification regulating stages.
  • small RNA interferes with mRNA or reduces stability thereof using RNA interference (RNAi) or RNA silencing, and in some cases, breaks up mRNA to interrupt the delivery of protein synthesis information, resulting in regulation of the expression of genetic information.
  • RNAi RNA interference
  • RNA silencing RNA silencing
  • the gene manipulation may be performed by modification of nucleic acids constituting an unsaturated fatty acid biosynthesis-associated factor. As manipulation results, all of knockdown, knockout, and knockin are included.
  • the modification of nucleic acids may be substitution, deletion, and/or insertion of one or more nucleotides, for example, 1 to 30 bp, 1 to 27 bp, 1 to 25 bp, 1 to 23 bp, 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, 1 to 5 bp, 1 to 3 bp, or 1 bp nucleotides.
  • the gene for knockout of one or more unsaturated fatty acid biosynthesis-associated factors, elimination of the expression of one or more factors, or one or more knockouts of one or two alleles, the gene may be manipulated such that one or more unsaturated fatty acid biosynthesis-associated factors contain a deletion or mutation.
  • knockdown of the unsaturated fatty acid biosynthesis-associated factor may be used to decrease expression of undesired alleles or transcriptomes.
  • the modification of nucleic acids may be insertion of one or more nucleic acid fragments or genes.
  • the nucleic acid fragment may be a nucleic acid sequence consisting of one or more nucleotides, and a length of the nucleic acid fragment may be 1 to 40 bp, 1 to 50 bp, 1 to 60 bp, 1 to 70 bp, 1 to 80 bp, 1 to 90 bp, 1 to 100 bp, 1 to 500 bp or 1 to 1000 bp.
  • the inserted gene may be one of the unsaturated fatty acid biosynthesis-associated factors, or a gene having a different function.
  • the modification of nucleic acids may employ a wild type or variant enzyme which is capable of catalyzing hydrolysis (cleavage) of bonds between nucleic acids in a DNA or RNA molecule, preferably, a DNA molecule. It may also employ a guide nucleic acid-editor protein complex.
  • the gene may be manipulated using one or more nucleases selected from the group consisting of a meganuclease, a zinc finger nuclease, CRISPR/Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein) and a TALE-nuclease, thereby regulating the expression of genetic information.
  • nucleases selected from the group consisting of a meganuclease, a zinc finger nuclease, CRISPR/Cas9 (Cas9 protein), CRISPR-Cpf1 (Cpf1 protein) and a TALE-nuclease, thereby regulating the expression of genetic information.
  • the gene manipulation may be mediated by NHEJ or homology-directed repair (HDR) using a guide nucleic acid-editor protein complex, for example, a CRISPR/Cas system.
  • NHEJ homology-directed repair
  • HDR homology-directed repair
  • NHEJ when the NHEJ mechanism takes place, a change in DNA sequence may be induced at a cleavage site, thereby inactivating the gene. Repair by NHEJ may induce substitution, insertion or deletion of a short gene fragment, and may be used in the induction of the knockout of a corresponding gene.
  • the present invention may provide the gene manipulation site.
  • the gene manipulation site may be a site in the gene, triggering the decrease or elimination of expression of an unsaturated fatty acid biosynthesis-associated gene product.
  • the site may be in an initial coding region
  • composition for manipulating an unsaturated fatty acid biosynthesis-associated factor may target
  • an unsaturated fatty acid biosynthesis-associated factor affecting the regulation of biosynthesis of unsaturated fatty acid such as an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, or an FAD8 gene, as a manipulation subject.
  • an FAD gene preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, or an FAD8 gene
  • the composition for manipulating an unsaturated fatty acid biosynthesis-associated factor may target an FAD2 gene as a manipulation subject.
  • target regions of the FAD2 gene that is, target sequences for regions in which gene manipulation occurs or which are recognized for gene manipulation are summarized in Table 1.
  • the target sequence may target one or more genes.
  • the target sequence may simultaneously target two or more genes.
  • the two or more genes may be homologous genes or heterologous genes.
  • the gene may contain one or more target sequences.
  • the gene may be simultaneously targeted at two or more target sequences.
  • the gene may be changed in the site and number of gene manipulations according to the number of target sequences.
  • the gene manipulation may be designed in various forms depending on the number and positions of the target sequences.
  • the gene manipulation may simultaneously occur in two or more target sequences.
  • the two or more target sequences may be present in the homologous gene or heterologous gene.
  • the gene manipulation may be simultaneously performed with respect to the two or more genes.
  • the two or more genes may be homologous genes or heterologous genes.
  • Target sequences of FAD2 gene No. Target sequence(including PAM) 1 ATAGATTGGCCATGCAATGAGGG (SEQ ID NO: 1) 2 AATAGATTGGCCATGCAATGAGG (SEQ ID NO: 2) 3 CCTTGGAGAACCCAATAGATTGG (SEQ ID NO: 3) 4 TGGGTGATTGCTCACGAGTGTGG (SEQ ID NO: 4) 5 TTTTAGTCCCTTATTTCTCATGG (SEQ ID NO: 5) 6 AAACACTTCATCACGGTCAAGGG (SEQ ID NO: 6) 7 GTGTTTGGAACCCTTGAGAGAGG (SEQ ID NO: 7) 8 GTGAATGGTGGCTTTGTGTTTGG (SEQ ID NO: 8) 9 ACAAAGCCACCATTCACTGTTGG (SEQ ID NO: 9) 10 AGTTGGCCAACAGTGAATGGTGG (SEQ ID NO: 10) 11 TTGAGTTGGCCAACAGTGAATGG (SEQ ID NO: 11) 12 TGAAAGGTCATAAACAACAACAACA
  • the system for controlling an unsaturated fatty acid of the present invention may include a guide nucleic acid-editor protein complex as a composition for manipulating an unsaturated fatty acid biosynthesis-associated factor.
  • guide nucleic acid-editor protein complex refers to a complex formed through the interaction between a guide nucleic acid and an editor protein, and the nucleic acid-protein complex includes a guide nucleic acid and an editor protein.
  • guide nucleic acid refers to a nucleic acid capable of recognizing a target nucleic acid, gene, chromosome or protein.
  • the guide nucleic acid may be present in the form of DNA, RNA or a DNA/RNA hybrid, and may have a nucleic acid sequence of 5 to 150 bases.
  • the guide nucleic acid may include one or more domains.
  • the domains may be, but are not limited to, a guide domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain, or a tail domain.
  • the guide nucleic acid may include two or more domains, which may be the same domain repeats, or different domains.
  • the guide nucleic acid may have one continuous nucleic acid sequence.
  • the one continuous nucleic acid sequence may be (N)m, where N represents A, T, C or G, or A, U, C or G, and m is an integer of 1 to 150.
  • the guide nucleic acid may have two or more continuous nucleic acid sequences.
  • the two or more continuous nucleic acid sequences may be (N)m and (N)o, where N represents A, T, C or G, or A, U, C or G, m and o are an integer of 1 to 150, and m and o may be the same as or different from each other.
  • editing protein refers to a peptide, polypeptide or protein which is able to directly bind to or interact with, without direct binding to, a nucleic acid.
  • the editor protein may be an enzyme.
  • the editor protein may be a fusion protein.
  • fusion protein refers to a protein that is produced by fusing an enzyme with an additional domain, peptide, polypeptide or protein.
  • enzyme refers to a protein that contains a domain capable of cleaving a nucleic acid, gene, chromosome or protein.
  • the additional domain, peptide, polypeptide or protein may be a functional domain, peptide, polypeptide or protein, which has a function the same as or different from the enzyme.
  • the fusion protein may include an additional domain, peptide, polypeptide or protein at one or more regions of the amino terminus (N-terminus) of the enzyme or the vicinity thereof; the carboxyl terminus (C-terminus) or the vicinity thereof; the middle part of the enzyme; and a combination thereof.
  • the fusion protein may include a functional domain, peptide, polypeptide or protein at one or more regions of the N-terminus of the enzyme or the vicinity thereof; the C-terminus or the vicinity thereof; the middle part of the enzyme; and a combination thereof.
  • the guide nucleic acid-editor protein complex may serve to modify a subject.
  • the subject may be a target nucleic acid, gene, chromosome or protein.
  • the guide nucleic acid-editor protein complex may result in final regulation (e.g., inhibition, suppression, reduction, increase or promotion) of the expression of a protein of interest, removal of the protein, or expression of a new protein.
  • final regulation e.g., inhibition, suppression, reduction, increase or promotion
  • the guide nucleic acid-editor protein complex may act at a DNA, RNA, gene or chromosome level.
  • the guide nucleic acid-editor protein complex may act in gene transcription and translation stages.
  • the guide nucleic acid-editor protein complex may act at a protein level.
  • the guide nucleic acid is a nucleic acid that is capable of recognizing a target nucleic acid, gene, chromosome or protein, and forms a guide nucleic acid-protein complex.
  • the guide nucleic acid is configured to recognize or target a nucleic acid, gene, chromosome or protein targeted by the guide nucleic acid-protein complex.
  • the guide nucleic acid may be present in the form of DNA, RNA or a DNA/RNA mixture, and have a 5 to 150-nucleic acid sequence.
  • the guide nucleic acid may be present in a linear or circular shape.
  • the guide nucleic acid may be one continuous nucleic acid sequence.
  • the one continuous nucleic acid sequence may be (N)m, where N is A, T, C or G, or A, U, C or G, and m is an integer of 1 to 150.
  • the guide nucleic acid may be two or more continuous nucleic acid sequences.
  • the two or more continuous nucleic acid sequences may be (N)m and (N)o, where N represents A, T, C or G, or A, U, C or G, m and o are an integer of 1 to 150, and may be the same as or different from each other.
  • the guide nucleic acid may include one or more domains.
  • the domains may be, but are not limited to, a guide domain, a first complementary domain, a linker domain, a second complementary domain, a proximal domain, or a tail domain.
  • the guide nucleic acid may include two or more domains, which may be the same domain repeats, or different domains.
  • guide domain is a domain having a complementary guide sequence which is able to form a complementary bond with a target sequence on a target gene or nucleic acid, and serves to specifically interact with the target gene or nucleic acid.
  • the guide sequence is a nucleic acid sequence complementary to the target sequence on a target gene or nucleic acid, which has, for example, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more complementarity or complete complementarity.
  • the guide domain may be a sequence of 5 to 50 bases.
  • the guide domain may be a sequence of 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50 or 45 to 50 bases.
  • the guide domain may be a sequence of 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50 bases.
  • the guide domain may have a guide sequence.
  • the guide sequence may be a complementary base sequence which is able to form a complementary bond with the target sequence on the target gene or nucleic acid.
  • the guide sequence may be a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90%, 95% or more complementarity or complete complementarity.
  • the guide sequence may be a 5 to 50 bases sequence.
  • the guide domain may be a 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, or 45 to 50-base sequence.
  • the guide sequence may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50-base sequence.
  • the guide domain may include a guide sequence and an additional base sequence.
  • the additional base sequence may be utilized to improve or degrade the function of the guide domain.
  • the additional base sequence may be utilized to improve or degrade the function of the guide sequence.
  • the additional base sequence may be a 1 to 35-base sequence.
  • the additional base sequence may be a 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35 or 30 to 35-base sequence.
  • the additional base sequence may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30 or 30 to 35-base sequence.
  • the additional base sequence may be located at the 5′ end of the guide sequence.
  • the additional base sequence may be located at the 3′ end of the guide sequence.
  • first complementary domain is a nucleic acid sequence including a nucleic acid sequence complementary to a second complementary domain, and has enough complementarity so as to form a double strand with the second complementary domain.
  • the first complementary domain may be a 5 to 35-base sequence.
  • the first complementary domain may be a 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35, or 30 to 35-base sequence.
  • the first complementary domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30 or 30 to 35-base sequence.
  • linker domain is a nucleic acid sequence connecting two or more domains, which are two or more identical or different domains.
  • the linker domain may be connected with two or more domains by covalent bonding or non-covalent bonding, or may connect two or more domains by covalent bonding or non-covalent bonding.
  • the linker domain may be a 1 to 30-base sequence.
  • the linker domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30-base sequence.
  • the linker domain may be a 1 to 30, 5 to 30, 10 to 30, 15 to 30, 20 to 30, or 25 to 30-base sequence.
  • second complementary domain is a nucleic acid sequence including a nucleic acid sequence complementary to the first complementary domain, and has enough complementarity so as to form a double strand with the first complementary domain.
  • the second complementary domain may have a base sequence complementary to the first complementary domain, and a base sequence having no complementarity to the first complementary domain, for example, a base sequence not forming a double strand with the first complementary domain, and may have a longer base sequence than the first complementary domain.
  • the second complementary domain may have a 5 to 35-base sequence.
  • the second complementary domain may be a 1 to 35, 5 to 35, 10 to 35, 15 to 35, 20 to 35, 25 to 35, or 30 to 35-base sequence.
  • the second complementary domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, or 30 to 35-base sequence.
  • proximal domain is a nucleic acid sequence located adjacent to the second complementary domain.
  • the proximal domain may have a complementary base sequence therein, and may be formed in a double strand due to a complementary base sequence.
  • the proximal domain may be a 1 to 20-base sequence.
  • the proximal domain may be a 1 to 20, 5 to 20, 10 to 20 or 15 to 20-base sequence.
  • the proximal domain may be a 1 to 5, 5 to 10, 10 to 15 or 15 to 20-base sequence.
  • tail domain is a nucleic acid sequence located at one or more ends of the both ends of the guide nucleic acid.
  • the tail domain may have a complementary base sequence therein, and may be formed in a double strand due to a complementary base sequence.
  • the tail domain may be a 1 to 50-base sequence.
  • the tail domain may be a 5 to 50, 10 to 50, 15 to 50, 20 to 50, 25 to 50, 30 to 50, 35 to 50, 40 to 50, or 45 to 50-base sequence.
  • the tail domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50-base sequence.
  • a part or all of the nucleic acid sequences included in the domains may selectively or additionally include a chemical modification.
  • the chemical modification may be, but is not limited to, methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP).
  • LNA locked nucleic acid
  • MS 2′-O-methyl 3′phosphorothioate
  • MSP 2′-O-methyl 3′thioPACE
  • the guide nucleic acid includes one or more domains.
  • the guide nucleic acid may include a guide domain.
  • the guide nucleic acid may include a first complementary domain.
  • the guide nucleic acid may include a linker domain.
  • the guide nucleic acid may include a second complementary domain.
  • the guide nucleic acid may include a proximal domain.
  • the guide nucleic acid may include a tail domain.
  • the guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more guide domains.
  • the guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more first complementary domains.
  • the guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more linker domains.
  • the guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more second complementary domains.
  • the guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more proximal domains.
  • the guide nucleic acid may include 1, 2, 3, 4, 5, 6 or more tail domains.
  • one type of domain may be duplicated.
  • the guide nucleic acid may include several domains with or without duplication.
  • the guide nucleic acid may include the same type of domain.
  • the same type of domain may have the same nucleic acid sequence or different nucleic acid sequences.
  • the guide nucleic acid may include two types of domains.
  • the two different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.
  • the guide nucleic acid may include three types of domains.
  • the three different types of domains may have different nucleic acid sequences or the same nucleic acid sequence.
  • the guide nucleic acid may include four types of domains.
  • the four different types of domains may have different nucleic acid sequences, or the same nucleic acid sequence.
  • the guide nucleic acid may include five types of domains.
  • the five different types of domains may have different nucleic acid sequences, or the same nucleic acid sequence.
  • the guide nucleic acid may include six types of domains.
  • the six different types of domains may have different nucleic acid sequences, or the same nucleic acid sequence.
  • the guide nucleic acid may consist of [guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[linker domain]-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain].
  • the two guide domains may include guide sequences for different or the same targets, the two first complementary domains and the two second complementary domains may have the same or different nucleic acid sequences.
  • the guide domains include guide sequences for different targets, the guide nucleic acids may specifically bind to two different targets, and here, the specific bindings may be performed simultaneously or sequentially.
  • the linker domains may be cleaved by specific enzymes, and the guide nucleic acids may be divided into two or three parts in the presence of specific enzymes.
  • gRNA As a specific example of the guide nucleic acid of the present invention, gRNA will be described below.
  • gRNA refers to a nucleic acid capable of specifically targeting a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, with respect to a target gene or nucleic acid.
  • the gRNA is a nucleic acid-specific RNA which may bind to a CRISPR enzyme and guide the CRISPR enzyme to the target gene or nucleic acid.
  • the gRNA may include multiple domains. Due to each domain, interactions may occur in a three-dimensional structure or active form of a gRNA strand, or between these strands.
  • the gRNA may be called single-stranded gRNA (single RNA molecule); or double-stranded gRNA (including more than one, generally, two discrete RNA molecules).
  • the single-stranded gRNA may include a guide domain, that is, a domain including a guide sequence capable of forming a complementary bond with a target gene or nucleic acid; a first complementary domain; a linker domain; a second complementary domain, a domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain; a proximal domain; and optionally a tail domain in the 5′ to 3′ direction.
  • a guide domain that is, a domain including a guide sequence capable of forming a complementary bond with a target gene or nucleic acid
  • a first complementary domain a linker domain
  • a second complementary domain a domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain
  • a proximal domain and optionally a tail domain in the 5′ to 3′ direction.
  • the double-stranded gRNA may include a first strand which includes a guide domain, that is, a domain including a guide sequence capable of forming a complementary bond with a target gene or nucleic acid and a first complementary domain; and a second strand which includes a second complementary domain, a domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain, a proximal domain; and optionally a tail domain in the 5′ to 3′ direction.
  • a guide domain that is, a domain including a guide sequence capable of forming a complementary bond with a target gene or nucleic acid and a first complementary domain
  • a second strand which includes a second complementary domain, a domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain, a proximal domain
  • optionally a tail domain in the 5′ to 3′ direction optionally a tail domain
  • the first strand may be referred to as crRNA
  • the second strand may be referred to as tracrRNA.
  • the crRNA may include a guide domain and a first complementary domain
  • the tracrRNA may include a second complementary domain, a proximal domain and optionally a tail domain.
  • the single-stranded gRNA may include a guide domain, that is, a domain including a guide sequence capable of forming a complementary bond with a target gene or nucleic acid; a first complementary domain; a second complementary domain, and a domain having a sequence complementary to the first complementary domain sequence, thereby forming a double-stranded nucleic acid with the first complementary domain in the 5′ to 3′ direction.
  • a guide domain that is, a domain including a guide sequence capable of forming a complementary bond with a target gene or nucleic acid
  • a first complementary domain a second complementary domain
  • a domain having a sequence complementary to the first complementary domain sequence thereby forming a double-stranded nucleic acid with the first complementary domain in the 5′ to 3′ direction.
  • the guide domain includes a complementary guide sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid.
  • the guide sequence may be a nucleic acid sequence having complementarity to the target sequence on the target gene or nucleic acid, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • the guide domain is considered to allow a gRNA-Cas complex, that is, a CRISPR complex to specifically interact with the target gene or nucleic acid.
  • the guide domain may be a 5 to 50-base sequence.
  • the guide domain may be a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide domain may include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide domain may include a guide sequence.
  • the guide sequence may be a complementary base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid.
  • the guide sequence may be a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • the guide sequence may be a nucleic acid sequence complementary to a target gene, that is, a target sequence of a unsaturated fatty acid biosynthesis-associated factor such as an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 or an FAD8 gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • a target gene that is, a target sequence of a unsaturated fatty acid biosynthesis-associated factor such as an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 or an FAD8 gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • the guide sequence may be a 5 to 50-base sequence.
  • the guide sequence may be a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD2 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD3 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD6 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD7 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD8 gene, which is a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • target sequences of the target genes that is, the unsaturated fatty acid biosynthesis-associated factors such as the FAD2 gene for the guide sequence are listed above in Table 1, but the present invention is not limited thereto.
  • the guide domain may include a guide sequence and an additional base sequence.
  • the additional base sequence may be a 1 to 35-base sequence.
  • the additional base sequence may be a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-base sequence.
  • the additional base sequence may be a single base sequence, guanine (G), or a sequence of two bases, GG.
  • the additional base sequence may be located at the 5′ end of the guide sequence.
  • the additional base sequence may be located at the 3′ end of the guide sequence.
  • a part or all of the base sequence of the guide domain may include a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′ phosphorothioate (MS) or 2′-O-methyl 3′ thioPACE (MSP), but the present invention is not limited thereto.
  • the first complementary domain includes a nucleic acid sequence complementary to a second complementary domain, and has enough complementarity such that it is able to form a double strand with the second complementary domain.
  • the first complementary domain may be a 5 to 35-base sequence.
  • the first complementary domain may include a 5 to 35-base sequence.
  • the first complementary domain may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-base sequence.
  • the first complementary domain may include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25-base sequence.
  • the first complementary domain may have homology with a natural first complementary domain, or may be derived from a natural first complementary domain.
  • the first complementary domain may have a difference in the base sequence of a first complementary domain depending on the species existing in nature, may be derived from a first complementary domain contained in the species existing in nature, or may have partial or complete homology with the first complementary domain contained in the species existing in nature.
  • the first complementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides , or a first complementary domain derived therefrom.
  • the first complementary domain when the first complementary domain is the first complementary domain of Streptococcus pyogenes or a first complementary domain derived therefrom, the first complementary domain may be 5′-GUUUUAGAGCUA-3′(SEQ ID NO: 42) or a base sequence having partial, that is, at least 50% or more, or complete homology with 5′-GUUUUAGAGCUA-3′(SEQ ID NO: 42).
  • the first complementary domain may further include (X)n, resulting in 5′-GUUUUAGAGCUA(X)n-3′(SEQ ID NO: 42).
  • the X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 5 to 15.
  • the (X)n may be n repeats of the same base, or a mixture of n bases of A, T, U and G.
  • the first complementary domain when the first complementary domain is the first complementary domain of Campylobacter jejuni or a first complementary domain derived therefrom, the first complementary domain may be 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′(SEQ ID NO: 43), or a base sequence having partial, that is, at least 50% or more, or complete homology with 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′(SEQ ID NO: 43).
  • the first complementary domain may further include (X)n, resulting in 5′-GUUUUAGUCCCUUUUUAAAUUUCUU(X)n-3′(SEQ ID NO: 43).
  • the X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 5 to 15.
  • the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.
  • the first complementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Parcubacteria bacterium (GWC2011 GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp.
  • GWC2011 GWC2_44_17 Lachnospiraceae bacterium
  • Butyrivibrio proteoclasiicus Peregrinibacteria bacterium (GW2011_GWA_33_10)
  • Acidaminococcus sp. BV3L6
  • SC_KO8D17 Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum or Eubacterium eligens , or a first complementary domain derived therefrom.
  • the first complementary domain when the first complementary domain is the first complementary domain of Parcubacteria bacterium or a first complementary domain derived therefrom, the first complementary domain may be 5′-UUUGUAGAU-3′, or a base sequence having partial, that is, at least 50% or more homology with 5′-UUUGUAGAU-3′.
  • the first complementary domain may further include (X)n, resulting in 5′-(X)nUUUGUAGAU-3′.
  • the X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 5.
  • the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.
  • a part or all of the base sequence of the first complementary domain may have a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′ phosphorothioate (MS) or 2′-O-methyl 3′ thioPACE (MSP), but the present invention is not limited thereto.
  • the linker domain is a nucleic acid sequence connecting two or more domains, and connects two or more identical or different domains.
  • the linker domain may be connected with two or more domains by covalent bonding or non-covalent bonding, or may connect two or more domains by covalent or non-covalent bonding.
  • the linker domain may be a nucleic acid sequence connecting a first complementary domain with a second complementary domain to produce single-stranded gRNA.
  • the linker domain may be connected with the first complementary domain and the second complementary domain by covalent or non-covalent bonding.
  • the linker domain may connect the first complementary domain with the second complementary domain by covalent or non-covalent bonding
  • the linker domain may be a 1 to 30-base sequence.
  • the linker domain may include a 1 to 30-base sequence.
  • the linker domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25 or 25 to 30-base sequence.
  • the linker domain may include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, or 25 to 30-base sequence.
  • the linker domain is suitable to be used in a single-stranded gRNA molecule, and may be used to produce single-stranded gRNA by being connected with a first strand and a second strand of double-stranded gRNA or connecting the first strand with the second strand by covalent or non-covalent bonding.
  • the linker domain may be used to produce single-stranded gRNA by being connected with crRNA and tracrRNA of double-stranded gRNA or connecting the crRNA with the tracrRNA by covalent or non-covalent bonding.
  • the linker domain may have homology with a natural sequence, for example, a partial sequence of tracrRNA, or may be derived therefrom.
  • a part or all of the base sequence of the linker domain may have a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′ phosphorothioate (MS) or 2′-O-methyl 3′ thioPACE (MSP), but the present invention is not limited thereto.
  • the second complementary domain includes a nucleic acid sequence complementary to the first complementary domain, and has enough complementarity so as to form a double strand with the first complementary domain.
  • the second complementary domain may include a base sequence complementary to the first complementary domain, and a base sequence having no complementarity with the first complementary domain, for example, a base sequence not forming a double strand with the first complementary domain, and may have a longer base sequence than the first complementary domain.
  • the second complementary domain may be a 5 to 35-base sequence.
  • the first complementary domain may include a 5 to 35-base sequence.
  • the second complementary domain may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the second complementary domain may include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the second complementary domain may have homology with a natural second complementary domain, or may be derived from the natural second complementary domain.
  • the second complementary domain may have a difference in base sequence of a second complementary domain according to a species existing in nature, and may be derived from a second complementary domain contained in the species existing in nature, or may have partial or complete homology with the second complementary domain contained in the species existing in nature.
  • the second complementary domain may have partial, that is, at least 50% or more, or complete homology with a second complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides , or a second complementary domain derived therefrom.
  • the second complementary domain when the second complementary domain is a second complementary domain of Streptococcus pyogenes or a second complementary domain derived therefrom, the second complementary domain may be 5′-UAGCAAGUUAAAAU-3′(SEQ ID NO: 44), or a base sequence having partial, that is, at least 50% or more homology with 5′-UAGCAAGUUAAAAU-3′(SEQ ID NO: 44) (a base sequence forming a double strand with the first complementary domain is underlined).
  • the second complementary domain may further include (X)n and/or (X)m, resulting in 5′-(X)n UAGCAAGUUAAAAU(X)m-3′(SEQ ID NO: 44).
  • the X may be selected from the group consisting of bases A, T, U and G, and each of the n and m may represent the number of bases, in which the n may be an integer of 1 to 15, and the m may be an integer of 1 to 6.
  • the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.
  • (X)m may represent m repeats of the same base, or a mixture of m bases of A, T, U and G.
  • the second complementary domain when the second complementary domain is the second complementary domain of Campylobacter jejuni or a second complementary domain derived therefrom, the second complementary domain may be 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′(SEQ ID NO: 45), or a base sequence having partial, that is, at least 50% or more homology with 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′(SEQ ID NO: 45) (a base sequence forming a double strand with the first complementary domain is underlined).
  • the second complementary domain may further include (X)n and/or (X)m, resulting in 5′-(X)nAAGAAAUUUAAAAAGGGACUAAAAU(X)m-3′(SEQ ID NO: 45).
  • the X may be selected from the group consisting of bases A, T, U and G, and each of the n and m may represent the number of bases, in which the n may be an integer of 1 to 15, and the m may be an integer of 1 to 6.
  • (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.
  • (X)m may represent m repeats of the same base, or a mixture of m bases of A, T, U and G.
  • the first complementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp.
  • SC_KO8D17 Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum or Eubacterium eligens , or a first complementary domain derived therefrom.
  • the second complementary domain when the second complementary domain is a second complementary domain of Parcubacteria bacterium or a second complementary domain derived therefrom, the second complementary domain may be 5′-AAAUUUCUACU(SEQ ID NO: 46)-3′, or a base sequence having partial, that is, at least 50% or more homology with 5′-AAAUUUCUACU-3′(SEQ ID NO: 46) (a base sequence forming a double strand with the first complementary domain is underlined).
  • the second complementary domain may further include (X)n and/or (X)m, resulting in 5′-(X)nAAAUUUCUACU(X)m-3′(SEQ ID NO: 46).
  • the X may be selected from the group consisting of bases A, T, U and G, and each of the n and m may represent the number of bases, in which the n may be an integer of 1 to 10, and the m may be an integer of 1 to 6.
  • the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.
  • the (X)m may represent m repeats of the same base, or a mixture of m bases of A, T, U and G.
  • a part or all of the base sequence of the second complementary domain may have a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.
  • the proximal domain is a sequence of 1 to 20 bases located adjacent to the second complementary domain, and a domain located at the 3′end direction of the second complementary domain.
  • the proximal domain may be used to form a double strand between complementary base sequences therein.
  • the proximal domain may be a 5, 6, 7, 8, 8, 9, 10, 11, 12, 13, 14 or 15-base sequence.
  • the proximal domain may include a 5, 6, 7, 8, 8, 9, 10, 11, 12, 13, 14 or 15-base sequence.
  • proximal domain may have homology with a natural proximal domain, or may be derived from the natural proximal domain.
  • proximal domain may have a difference in base sequence according to a species existing in nature, may be derived from a proximal domain contained in the species existing in nature, or may have partial or complete homology with the proximal domain contained in the species existing in nature.
  • the proximal domain may have partial, that is, at least 50% or more, or complete homology with a proximal domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides , or a proximal domain derived therefrom.
  • the proximal domain when the proximal domain is a proximal domain of Streptococcus pyogenes or a proximal domain derived therefrom, the proximal domain may be 5′-AAGGCUAGUCCG-3′(SEQ ID NO: 47), or a base sequence having partial, that is, at least 50% or more homology with 5′-AAGGCUAGUCCG-3′(SEQ ID NO: 47).
  • the proximal domain may further include (X)n, resulting in 5′-AAGGCUAGUCCG(X)n-3′(SEQ ID NO: 47).
  • the X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 15.
  • the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.
  • the proximal domain when the proximal domain is a proximal domain of Campylobacter jejuni or a proximal domain derived therefrom, the proximal domain may be 5′-AAAGAGUUUGC-3′(SEQ ID NO: 48), or a base sequence having at least 50% or more homology with 5′-AAAGAGUUUGC-3′(SEQ ID NO: 48).
  • the proximal domain may further include (X)n, resulting in 5′-AAAGAGUUUGC(X)n-3′(SEQ ID NO: 48).
  • the X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 40.
  • the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.
  • a part or all of the base sequence of the proximal domain may have a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.
  • the tail domain is a domain which is able to be selectively added to the 3′ end of single-stranded gRNA or double-stranded gRNA.
  • the tail domain may be a 1 to 50-base sequence, or include a 1 to 50-base sequence.
  • the tail domain may be used to form a double strand between complementary base sequences therein.
  • the tail domain may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50-base sequence.
  • the tail domain may include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45, or 45 to 50-base sequence.
  • the tail domain may have homology with a natural tail domain, or may be derived from the natural tail domain.
  • the tail domain may have a difference in base sequence according to a species existing in nature, may be derived from a tail domain contained in a species existing in nature, or may have partial or complete homology with a tail domain contained in a species existing in nature.
  • the tail domain may have partial, that is, at least 50% or more, or complete homology with a tail domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides or a tail domain derived therefrom.
  • the tail domain when the tail domain is a tail domain of Streptococcus pyogenes or a tail domain derived therefrom, the tail domain may be 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′(SEQ ID NO: 49), or a base sequence having partial, that is, at least 50% or more homology with 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′(SEQ ID NO: 49).
  • the tail domain may further include (X)n, resulting in 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC(X)n-3′(SEQ ID NO: 49).
  • the X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 15.
  • the (X)n may represent n repeats of the same base, or a mixture of n bases such as A, T, U and G.
  • the tail domain when the tail domain is a tail domain of Campylobacter jejuni or a tail domain derived therefrom, the tail domain may be 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′(SEQ ID NO: 50), or a base sequence having partial, that is, at least 50% or more homology with 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′(SEQ ID NO: 50).
  • the tail′ domain may further include (X)n, resulting in 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU(X)n-3′(SEQ ID NO: 50).
  • the X may be selected from the group consisting of bases A, T, U and G, and the n may represent the number of bases, which is an integer of 1 to 15.
  • the (X)n may represent n repeats of the same base, or a mixture of n bases of A, T, U and G.
  • the tail domain may include a 1 to 10-base sequence at the 3′ end involved in an in vitro or in vivo transcription method.
  • the tail domain when a T7 promoter is used in in vitro transcription of gRNA, the tail domain may be an arbitrary base sequence present at the 3′ end of a DNA template.
  • the tail domain when a U6 promoter is used in in vivo transcription, the tail domain may be UUUUUU, when an H1 promoter is used in transcription, the tail domain may be UUUU, and when a pol-III promoter is used, the tail domain may include several uracil bases or alternative bases.
  • a part or all of the base sequence of the tail domain may have a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.
  • the gRNA may include a plurality of domains as described above, and therefore, the length of the nucleic acid sequence may be regulated according to a domain contained in the gRNA, and interactions may occur in strands in a three-dimensional structure or active form of gRNA or between theses strands due to each domain.
  • the gRNA may be referred to as single-stranded gRNA (single RNA molecule); or double-stranded gRNA (including more than one, generally two discrete RNA molecules).
  • the double-stranded gRNA consists of a first strand and a second strand.
  • the first strand may consist of
  • the second strand may consist of
  • the first strand may be referred to as crRNA
  • the second strand may be referred to as tracrRNA.
  • the guide domain includes a complementary guide sequence which is able to form a complementary bond with a target sequence on a target gene or nucleic acid.
  • the guide sequence is a nucleic acid sequence complementary to the target sequence on the target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • the guide domain is considered to allow a gRNA-Cas complex, that is, a CRISPR complex to specifically interact with the target gene or nucleic acid.
  • the guide domain may be a 5 to 50-base sequence, or includes a 5 to 50-base sequence.
  • the guide domain may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide domain may include a guide sequence.
  • the guide sequence may be a complementary base sequence which is able to form a complementary bond with a target sequence on a target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • the guide sequence may be a nucleic acid sequence complementary to a target gene, that is, a target sequence of a unsaturated fatty acid biosynthesis-associated factor such as an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8 gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • a target gene that is, a target sequence of a unsaturated fatty acid biosynthesis-associated factor
  • an FAD gene preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8 gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • the guide sequence may be a 5 to 50-base sequence or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence is a nucleic acid sequence complementary to a target sequence of the FAD2 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence is a nucleic acid sequence complementary to a target sequence of the FAD3 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence is a nucleic acid sequence complementary to a target sequence of the FAD6 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence is a nucleic acid sequence complementary to a target sequence of the FAD7 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence is a nucleic acid sequence complementary to a target sequence of the FAD8 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • target sequences of a target gene that is, unsaturated fatty acid biosynthesis-associated factors such as an FAD2 gene are listed above in Table 1, but the present invention is not limited thereto.
  • the guide domain may include a guide sequence and an additional base sequence.
  • the additional base sequence may be a 1 to 35-base sequence.
  • the additional base sequence may be a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-base sequence.
  • the additional base sequence may include one base, guanine (G), or two bases, GG.
  • the additional base sequence may be located at the 5′ end of the guide domain, or at the 5′ end of the guide sequence.
  • the additional base sequence may be located at the 3′ end of the guide domain, or at the 3′ end of the guide sequence.
  • the first complementary domain includes a nucleic acid sequence complementary to a second complementary domain of the second strand, and is a domain having enough complementarity so as to form a double strand with the second complementary domain.
  • the first complementary domain may be or include a 5 to 35-base sequence.
  • the first complementary domain may be or include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the first complementary domain may have homology with a natural first complementary domain, or may be derived from a natural first complementary domain.
  • the first complementary domain may have a difference in base sequence according to a species existing in nature, may be derived from the first complementary domain contained in the species existing in nature, or may have partial or complete homology with the first complementary domain contained in the species existing in nature.
  • the first complementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides , or a first complementary domain derived therefrom.
  • the first complementary domain may include an additional base sequence which does not undergo complementary bonding with the second complementary domain of the second strand.
  • the additional base sequence may be a sequence of 1 to 15 bases.
  • the additional base sequence may be a sequence of 1 to 5, 5 to 10, or 10 to 15 bases.
  • a part or all of the base sequence of the guide domain and/or first complementary domain may have a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′ phosphorothioate (MS) or 2′-O-methyl 3′ thioPACE (MSP), but the present invention is not limited thereto.
  • the first strand may consist of 5′-[guide domain]-[first complementary domain]-3′ as described above.
  • first strand may optionally include an additional base sequence.
  • the first strand may be
  • the Ntarget is a base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid, and a base sequence region which may be changed according to a target sequence on a target gene or nucleic acid.
  • Ntarget may be a base sequence capable of forming a complementary bond with a target gene, that is, a target sequence of an unsaturated fatty acid biosynthesis-associated factor such as an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8 gene.
  • an FAD gene preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8 gene.
  • the (Q)m is a base sequence including the first complementary domain, which is able to form a complementary bond with the second complementary domain of the second strand.
  • the (Q)m may be a sequence having partial or complete homology with the first complementary domain of a species existing in nature, and the base sequence of the first complementary domain may be changed according to the species of origin.
  • the Q may be each independently selected from the group consisting of A, U, C and G, and the m may be the number of bases, which is an integer of 5 to 35.
  • the (Q)m may be 5′-GUUUUAGAGCUA-3′(SEQ ID NO: 42), or a base sequence having at least 50% or more homology with 5′-GUUUUAGAGCUA-3′(SEQ ID NO: 42).
  • the (Q)m may be 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′(SEQ ID NO: 43), or a base sequence having at least 50% or more homology with 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′(SEQ ID NO: 43).
  • the (Q)m may be 5′-GUUUUAGAGCUGUGUUGUUUCG-3′(SEQ ID NO: 51), or a base sequence having at least 50% or more homology with 5′-GUUUUAGAGCUGUGUUGUUUCG-3′(SEQ ID NO: 51).
  • each of the (X)a, (X)b and (X)c is selectively an additional base sequence, where the X may be each independently selected from the group consisting of A, U, C and G, and each of the a, b and c may be the number of bases, which is 0 or an integer of 1 to 20.
  • the second strand may consist of a second complementary domain and a proximal domain, and selectively include a tail domain.
  • the second complementary domain includes a nucleic acid sequence complementary to the first complementary domain of the first strand, and has enough complementarity so as to form a double strand with the first complementary domain.
  • the second complementary domain may include a base sequence complementary to the first complementary domain and a base sequence not complementary to the first complementary domain, for example, a base sequence not forming a double strand with the first complementary domain, and may have a longer base sequence than the first complementary domain.
  • the second complementary domain may be a 5 to 35-base sequence, or include a 5 to 35-base sequence.
  • the second complementary domain may be or include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence, but the present invention is not limited thereto.
  • the second complementary domain may have homology with a natural second complementary domain, or may be derived from a natural second complementary domain.
  • the second complementary domain may have a difference in base sequence thereof according to a species existing in nature, may be derived from a second complementary domain contained in the species existing in nature, or may have partial or complete homology with the second complementary domain contained in the species existing in nature.
  • the second complementary domain may have partial, that is, at least 50% or more, or complete homology with a second complementary domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides , or a second complementary domain derived therefrom.
  • the second complementary domain may further include an additional base sequence which does not undergo complementary bonding with the first complementary domain of the first strand.
  • the additional base sequence may be a 1 to 25-base sequence.
  • the additional base sequence may be a 1 to 5, 5 to 10, 10 to 15, 15 to 20 or 20 to 25-base sequence.
  • the proximal domain is a sequence of 1 to 20 bases, and a domain located at the 3′ end direction of the second complementary domain.
  • the proximal domain may be or include a sequence of 5, 6, 7, 8, 8, 9, 10, 11, 12, 13, 14 or 15 bases.
  • the proximal domain may have a double strand bond between complementary base sequences therein.
  • proximal domain may have homology with a natural proximal domain, or may be derived from a natural proximal domain.
  • proximal domain may have a difference in base sequence according to a species existing in nature, may be derived from a proximal domain of a species existing in nature, or may have partial or complete homology with the proximal domain of a species existing in nature.
  • the proximal domain may have partial, that is, at least 50% or more, or complete homology with a proximal domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides , or a proximal domain derived therefrom.
  • the tail domain may be a domain selectively added to the 3′ end of the second strand, and the tail domain may be or include a 1 to 50-base sequence.
  • the tail domain may be or include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25, 25 to 30, 30 to 35, 35 to 40, 40 to 45 or 45 to 50-base sequence.
  • the tail domain may have a double strand bond between complementary base sequences therein.
  • the tail domain may have homology with a natural tail domain, or may be derived from a natural tail domain.
  • the tail domain may have a difference in base sequence according to a species existing in nature, may be derived from a tail domain contained in the species existing in nature, or may have partial or complete homology with the tail domain contained in the species existing in nature.
  • the tail domain may have partial, that is, at least 50% or more, or complete homology with a tail domain of Streptococcus pyogenes, Campylobacter jejuni, Streptococcus thermophilus, Staphylococcus aureus or Neisseria meningitides , or a tail domain derived therefrom.
  • the tail domain may include a sequence of 1 to 10 bases at the 3′ end involved in an in vitro or in vivo transcription method.
  • the tail domain when a T7 promoter is used in in vitro transcription of gRNA, the tail domain may be an arbitrary base sequence present at the 3′ end of a DNA template.
  • the tail domain when a U6 promoter is used in in vivo transcription, the tail domain may be UUUUUU, when an H1 promoter is used in transcription, the tail domain may be UUUU, and when a pol-III promoter is used, the tail domain may include several uracil bases or alternative bases.
  • each of the base sequence of the second complementary domain, the proximal domain and/or the tail domain may have a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.
  • the second strand may consist of 5′-[second complementary domain]-[proximal domain]-3′ or 5′-[second complementary domain]-[proximal domain]-[tail domain]-3′ as described above.
  • the second strand may selectively include an additional base sequence.
  • the second strand may be 5′-(Z)h-(P)k-3′; or 5′-(X)d-(Z)h-(X)e-(P)k-(X)f-3′.
  • the second strand may be 5′-(Z)h-(P)k-(F)i-3′; or 5′-(X)d-(Z)h-(X)e-(P)k-(X)f-(F)i-3′.
  • the (Z)h is a base sequence including a second complementary domain, which is able to form a complementary bond with the first complementary domain of the first strand.
  • the (Z)h may be a sequence having partial or complete homology with the second complementary domain of a species existing in nature, and the base sequence of the second complementary domain may be modified according to the species of origin.
  • the Z may be each independently selected from the group consisting of A, U, C and G, and the h may be the number of bases, which is an integer of 5 to 50.
  • the (Z)h may be 5′-UAGCAAGUUAAAAU-3′(SEQ ID NO: 44), or a base sequence having at least 50% or more homology with 5′-UAGCAAGUUAAAAU-3′(SEQ ID NO: 44).
  • the (Z)h may be 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′(SEQ ID NO: 45), or a base sequence having at least 50% or more homology with 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′(SEQ ID NO: 45).
  • the (Z)h may be 5′-CGAAACAACACAGCGAGUUAAAAU-3′(SEQ ID NO: 52), or a base sequence having at least 50% or more homology with 5′-CGAAACAACACAGCGAGUUAAAAU-3′(SEQ ID NO: 52).
  • the (P)k is a base sequence including a proximal domain, which may have partial or complete homology with a proximal domain of a species existing in nature, and the base sequence of the proximal domain may be modified according to the species of origin.
  • the P may be each independently selected from the group consisting of A, U, C and G, and the k may be the number of bases, which is an integer of 1 to 20.
  • the (P)k may be 5′-AAGGCUAGUCCG-3′(SEQ ID NO: 47), or a base sequence having at least 50% or more homology with 5′-AAGGCUAGUCCG-3′(SEQ ID NO: 47).
  • the (P)k may be 5′-AAAGAGUUUGC-3′(SEQ ID NO: 48), or a base sequence having at least 50% or more homology with 5′-AAAGAGUUUGC-3′(SEQ ID NO: 48).
  • the (P)k may be 5′-AAGGCUUAGUCCG-3′(SEQ ID NO: 53), or a base sequence having at least 50% or more homology with 5′-AAGGCUUAGUCCG-3′(SEQ ID NO: 53).
  • the (F)i may be a base sequence including a tail domain, and having partial or complete homology with a tail domain of a species existing in nature, and the base sequence of the tail domain may be modified according to the species of origin.
  • the F may be each independently selected from the group consisting of A, U, C and G, and the i may be the number of bases, which is an integer of 1 to 50.
  • the (F)i may be 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′(SEQ ID NO: 49), or a base sequence having at least 50% or more homology with 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′(SEQ ID NO: 49).
  • the (F)i when the tail domain has partial or complete homology with a tail domain of Campylobacter jejuni or a tail domain derived therefrom, the (F)i may be 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′(SEQ ID NO: 50), or a base sequence having at least 50% or more homology with 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′(SEQ ID NO: 50).
  • the (F)i may be 5′-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUUUUU-3′(SEQ ID NO: 54), or a base sequence having at least 50% or more homology with 5′-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3′(SEQ ID NO: 54).
  • the (F)i may include a sequence of 1 to 10 bases at the 3′ end involved in an in vitro or in vivo transcription method.
  • the tail domain when a T7 promoter is used in in vitro transcription of gRNA, the tail domain may be an arbitrary base sequence present at the 3′ end of a DNA template.
  • the tail domain when a U6 promoter is used in in vivo transcription, the tail domain may be UUUUUU, when an H1 promoter is used in transcription, the tail domain may be UUUU, and when a pol-III promoter is used, the tail domain may include several uracil bases or alternative bases.
  • the (X)d, (X)e and (X)f may be base sequences selectively added, where the X may be each independently selected from the group consisting of A, U, C and G, and each of the d, e and f may be the number of bases, which is 0 or an integer of 1 to 20.
  • Single-stranded gRNA may be classified into two types.
  • the single-stranded gRNA in which a first strand or a second strand of the double-stranded gRNA is linked by a linker domain, and here, the single-stranded gRNA consists of 5′-[first strand]-[linker domain]-[second strand]-3′.
  • the single-stranded gRNA may consist of
  • Each domain except the linker domain is the same as the description of each domain of the first and second strands of the double-stranded gRNA.
  • the linker domain is a domain connecting a first strand and a second strand, and specifically, is a nucleic acid sequence which connects a first complementary domain with a second complementary domain to produce single-stranded gRNA.
  • the linker domain may be connected with the first complementary domain and the second complementary domain by covalent bonding or non-covalent bonding, or connect the first complementary domain with the second complementary domain by covalent or non-covalent bonding.
  • the linker domain may be or include a 1 to 30-base sequence.
  • the linker domain may be or include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25 or 25 to 30-base sequence.
  • the linker domain is suitable to be used in a single-stranded gRNA molecule, and may be connected with the first strand and the second strand of the double-stranded gRNA, or connect the first strand with the second strand by covalent or non-covalent bonding to be used in production of the single-stranded gRNA.
  • the linker domain may be connected with crRNA and tracrRNA of the double-stranded gRNA, or connect crRNA with tracrRNA by covalent or non-covalent bonding to be used in production of the single-stranded gRNA.
  • the linker domain may have homology with a natural sequence, for example, a partial sequence of tracrRNA, or may be derived therefrom.
  • a part or all of the base sequence of the linker domain may have a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.
  • the single-stranded gRNA may consist of 5′-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-3′ or 5′-[guide domain]-[first complementary domain]-[linker domain]-[second complementary domain]-[proximal domain]-[tail domain]-3′ as described above.
  • the single-stranded gRNA may selectively include an additional base sequence.
  • the single-stranded gRNA may be
  • the single-stranded gRNA may be
  • the Ntarget is a base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid, and a base sequence region capable of being changed according to a target sequence on a target gene or nucleic acid.
  • Ntarget is a base sequence capable of forming a complementary bond with a target gene, that is, a target sequence of an unsaturated fatty acid biosynthesis-associated factor such as an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, or an FAD8 gene.
  • an FAD gene preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, or an FAD8 gene.
  • the (Q)m includes a base sequence including the first complementary domain, which is able to form a complementary bond with a second complementary domain.
  • the (Q)m may be a sequence having partial or complete homology with a first complementary domain of a species existing in nature, and the base sequence of the first complementary domain may be changed according to the species of origin.
  • the Q may be each independently selected from the group consisting of A, U, C and G, and the m may be the number of bases, which is an integer of 5 to 35.
  • the (Q)m may be 5′-GUUUUAGAGCUA-3′(SEQ ID NO: 42), or a base sequence having at least 50% or more homology with 5′-GUUUUAGAGCUA-3′(SEQ ID NO: 42).
  • the (Q)m may be 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′(SEQ ID NO: 43), or a base sequence having at least 50% or more homology with 5′-GUUUUAGUCCCUUUUUAAAUUUCUU-3′(SEQ ID NO: 43).
  • the (Q)m may be 5′-GUUUUAGAGCUGUGUUGUUUCG-3′(SEQ ID NO: 51), or a base sequence having at least 50% or more homology with 5′-GUUUUAGAGCUGUGUUGUUUCG-3′(SEQ ID NO: 51).
  • the (L)j is a base sequence including the linker domain, and connecting the first complementary domain with the second complementary domain, thereby producing single-stranded gRNA.
  • the L may be each independently selected from the group consisting of A, U, C and G, and the j may be the number of bases, which is an integer of 1 to 30.
  • the (Z)h is a base sequence including the second complementary domain, which is able to have a complementary bond with the first complementary domain.
  • the (Z)h may be a sequence having partial or complete homology with the second complementary domain of a species existing in nature, and the base sequence of the second complementary domain may be changed according to the species of origin.
  • the Z may be each independently selected from the group consisting of A, U, C and G, and the h is the number of bases, which may be an integer of 5 to 50.
  • the (Z)h may be 5′-UAGCAAGUUAAAAU-3′(SEQ ID NO: 44), or a base sequence having at least 50% or more homology with 5′-UAGCAAGUUAAAAU-3′(SEQ ID NO: 44).
  • the (Z)h may be 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′(SEQ ID NO: 45), or a base sequence having at least 50% or more homology with 5′-AAGAAAUUUAAAAAGGGACUAAAAU-3′(SEQ ID NO: 45).
  • the (Z)h may be 5′-CGAAACAACACAGCGAGUUAAAAU-3′(SEQ ID NO: 52), or a base sequence having at least 50% or more homology with 5′-CGAAACAACACAGCGAGUUAAAAU-3′(SEQ ID NO: 52).
  • the (P)k is a base sequence including a proximal domain, which may have partial or complete homology with a proximal domain of a species existing in nature, and the base sequence of the proximal domain may be modified according to the species of origin.
  • the P may be each independently selected from the group consisting of A, U, C and G, and the k may be the number of bases, which is an integer of 1 to 20.
  • the (P)k may be 5′-AAGGCUAGUCCG-3′(SEQ ID NO: 47), or a base sequence having at least 50% or more homology with 5′-AAGGCUAGUCCG-3′(SEQ ID NO: 47).
  • the (P)k may be 5′-AAAGAGUUUGC-3′(SEQ ID NO: 48), or a base sequence having at least 50% or more homology with 5′-AAAGAGUUUGC-3′(SEQ ID NO: 48).
  • the (P)k may be 5′-AAGGCUUAGUCCG-3′(SEQ ID NO: 53), or a base sequence having at least 50% or more homology with 5′-AAGGCUUAGUCCG-3′(SEQ ID NO: 53).
  • the (F)i may be a base sequence including a tail domain, and having partial or complete homology with a tail domain of a species existing in nature, and the base sequence of the tail domain may be modified according to the species of origin.
  • the F may be each independently selected from the group consisting of A, U, C and G, and the i may be the number of bases, which is an integer of 1 to 50.
  • the (F)i may be 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′(SEQ ID NO: 49), or a base sequence having at least 50% or more homology with 5′-UUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC-3′(SEQ ID NO: 49).
  • the (F)i when the tail domain has partial or complete homology with a tail domain of Campylobacter jejuni or a tail domain derived therefrom, the (F)i may be 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′(SEQ ID NO: 50), or a base sequence having at least 50% or more homology with 5′-GGGACUCUGCGGGGUUACAAUCCCCUAAAACCGCUUUU-3′(SEQ ID NO: 50).
  • the (F)i may be 5′-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUUUUU-3′(SEQ ID NO: 54), or a base sequence having at least 50% or more homology with 5′-UACUCAACUUGAAAAGGUGGCACCGAUUCGGUGUUUUU-3′(SEQ ID NO: 54).
  • the (F)i may include a sequence of 1 to 10 bases at the 3′ end involved in an in vitro or in vivo transcription method.
  • the tail domain when a T7 promoter is used in in vitro transcription of gRNA, the tail domain may be an arbitrary base sequence present at the 3′ end of a DNA template.
  • the tail domain when a U6 promoter is used in in vivo transcription, the tail domain may be UUUUUU, when an H1 promoter is used in transcription, the tail domain may be UUUU, and when a pol-III promoter is used, the tail domain may include several uracil bases or alternative bases.
  • the (X)a, (X)b, (X)c, (X)d, (X)e and (X)f may be base sequences selectively added, where the X may be each independently selected from the group consisting of A, U, C and G, and each of the a, b, c, d, e and f may be the number of bases, which is 0 or an integer of 1 to 20.
  • the single-stranded gRNA may be single-stranded gRNA consisting of a guide domain, a first complementary domain and a second complementary domain, and here, the single-stranded gRNA may consist of:
  • the guide domain includes a complementary guide sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid.
  • the guide sequence may be a nucleic acid sequence having complementarity to the target sequence on the target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or more complementarity or complete complementarity.
  • the guide domain is considered to allow a gRNA-Cas complex, that is, a CRISPR complex to specifically interact with the target gene or nucleic acid.
  • the guide domain may be or include a 5 to 50-base sequence.
  • the guide domain may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide domain may include a guide sequence.
  • the guide sequence may be a complementary base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • the guide sequence may be a nucleic acid sequence complementary to a target gene, that is, a target sequence of an unsaturated fatty acid biosynthesis-associated factor such as an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8 gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • an FAD gene preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8 gene, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD2 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD3 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD6 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD7 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the guide sequence may be a nucleic acid sequence complementary to a target sequence of the FAD8 gene.
  • the guide sequence may be or include a 5 to 50-base sequence.
  • the guide sequence may be or include a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • target sequences of the target genes that is, the unsaturated fatty acid biosynthesis-associated factor such as the FAD2 gene for the guide sequence are listed above in Table 1, but the present invention is not limited thereto.
  • the guide domain may include a guide sequence and an additional base sequence.
  • the additional base sequence may be a 1 to 35-base sequence.
  • the additional base sequence may be a 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10-base sequence.
  • the additional base sequence may be a single base sequence, guanine (G), or a sequence of two bases, GG.
  • the additional base sequence may be located at the 5′ end of the guide domain, or at the 5′ end of the guide sequence.
  • the additional base sequence may be located at the 3′ end of the guide domain, or at the 3′ end of the guide sequence.
  • the first complementary domain is a domain including a nucleic acid sequence complementary to the second complementary domain, and having enough complementarity so as to form a double strand with the second complementary domain.
  • the first complementary domain may be or include a 5 to 35-base sequence.
  • the first complementary domain may be or include a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the first complementary domain may have homology with a natural first complementary domain, or may be derived from a natural first complementary domain.
  • the first complementary domain may have a difference in the base sequence of a first complementary domain depending on the species existing in nature, may be derived from a first complementary domain contained in the species existing in nature, or may have partial or complete homology with the first complementary domain contained in the species existing in nature.
  • the first complementary domain may have partial, that is, at least 50% or more, or complete homology with a first complementary domain of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp.
  • SC_KO8D17 Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum or Eubacterium eligens , or a first complementary domain derived therefrom.
  • the first complementary domain may include an additional base sequence which does not undergo complementary bonding with the second complementary domain.
  • the additional base sequence may be a 1 to 15-base sequence.
  • the additional base sequence may be a 1 to 5, 5 to 10, or 10 to 15-base sequence.
  • the second complementary domain includes a nucleic acid sequence complementary to the first complementary domain, and has enough complementarity so as to form a double strand with the first complementary domain.
  • the second complementary domain may include a base sequence complementary to the first complementary domain, and a base sequence having no complementarity with the first complementary domain, for example, a base sequence not forming a double strand with the first complementary domain, and may have a longer base sequence than the first complementary domain.
  • the second complementary domain may be or include a 5 to 35-base sequence.
  • the second complementary domain may be a 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the second complementary domain may have homology with a natural second complementary domain, or may be derived from the natural second complementary domain.
  • the second complementary domain may have a difference in base sequence of the second complementary domain according to a species existing in nature, and may be derived from second complementary domain contained in the species existing in nature, or may have partial or complete homology with the second complementary domain contained in the species existing in nature.
  • the second complementary domain may have partial, that is, at least 50% or more, or complete homology with a second complementary domain of Parcubacteria bacterium (GWC2011_GWC2_44_17), Lachnospiraceae bacterium (MC2017), Butyrivibrio proteoclasiicus, Peregrinibacteria bacterium (GW2011_GWA_33_10), Acidaminococcus sp. (BV3L6), Porphyromonas macacae, Lachnospiraceae bacterium (ND2006), Porphyromonas crevioricanis, Prevotella disiens, Moraxella bovoculi (237), Smiihella sp.
  • SC_KO8D17 Leptospira inadai, Lachnospiraceae bacterium (MA2020), Francisella novicida (U112), Candidatus Methanoplasma termitum or Eubacterium eligens , or a second complementary domain derived therefrom.
  • the second complementary domain may include an additional base sequence which does not undergo complementary bonding with the first complementary domain.
  • the additional base sequence may be a 1 to 15-base sequence.
  • the additional base sequence may be a 1 to 5, 5 to 10, or 10 to 15-base sequence.
  • the linker domain is a nucleic acid sequence connecting a first complementary domain with a second complementary domain to produce single-stranded gRNA.
  • the linker domain may be connected with the first complementary domain and the second complementary domain by covalent or non-covalent bonding, or may connect the first and second complementary domains by covalent or non-covalent bonding.
  • the linker domain may be or include a 1 to 30-base sequence.
  • the linker domain may be or include a 1 to 5, 5 to 10, 10 to 15, 15 to 20, 20 to 25 or 25 to 30-base sequence.
  • a part or all of the base sequence of the guide domain, the first complementary domain, the second complementary domain and the linker domain may have a chemical modification.
  • the chemical modification may be methylation, acetylation, phosphorylation, phosphorothioate linkage, a locked nucleic acid (LNA), 2′-O-methyl 3′phosphorothioate (MS) or 2′-O-methyl 3′thioPACE (MSP), but the present invention is not limited thereto.
  • the single-stranded gRNA may consist of 5′-[second complementary domain]-[first complementary domain]-[guide domain]-3′ or 5′-[second complementary domain]-[linker domain]-[first complementary domain]-[guide domain]-3′ as described above.
  • the single-stranded gRNA may selectively include an additional base sequence.
  • the single-stranded gRNA may be
  • the single-stranded gRNA may be
  • the Ntarget is a base sequence capable of forming a complementary bond with a target sequence on a target gene or nucleic acid, and a base sequence region which may be changed according to a target sequence on a target gene or nucleic acid.
  • Ntarget may be a base sequence capable of forming a complementary bond with a target gene, that is, a target sequence of an unsaturated fatty acid biosynthesis-associated factor such as an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8 gene.
  • an FAD gene preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene or an FAD8 gene.
  • the (Q)m is a base sequence including the first complementary domain, which is able to form a complementary bond with the second complementary domain of the second strand.
  • the (Q)m may be a sequence having partial or complete homology with the first complementary domain of a species existing in nature, and the base sequence of the first complementary domain may be changed according to the species of origin.
  • the Q may be each independently selected from the group consisting of A, U, C and G, and the m may be the number of bases, which is an integer of 5 to 35.
  • the (Q)m may be 5′-UUUGUAGAU-3′, or a base sequence having at least 50% or more homology with 5′-UUUGUAGAU-3′.
  • the (Z)h is a base sequence including a second complementary domain, which is able to form a complementary bond with the first complementary domain of the first strand.
  • the (Z)h may be a sequence having partial or complete homology with the second complementary domain of a species existing in nature, and the base sequence of the second complementary domain may be modified according to the species of origin.
  • the Z may be each independently selected from the group consisting of A, U, C and G, and the h may be the number of bases, which is an integer of 5 to 50.
  • the (Z)h may be 5′-AAAUUUCUACU-3′(SEQ ID NO: 46), or a base sequence having at least 50% or more homology with 5′-AAAUUUCUACU-3′(SEQ ID NO: 46).
  • the (L)j is a base sequence including the linker domain, which connects the first complementary domain with the second complementary domain.
  • the L may be each independently selected from the group consisting of A, U, C and G, and the j may be the number of bases, which is an integer of 1 to 30.
  • each of the (X)a, (X)b and (X)c is selectively an additional base sequence, where the X may be each independently selected from the group consisting of A, U, C and G, and the a, b and c may be the number of bases, which is 0 or an integer of 1 to 20.
  • An editor protein refers to a peptide, polypeptide or protein which is able to directly bind to or interact with, without direct binding to, a nucleic acid.
  • the nucleic acid may be a nucleic acid contained in a target nucleic acid, gene or chromosome.
  • the nucleic acid may be a guide nucleic acid.
  • the editor protein may be an enzyme.
  • the editor protein may be a fusion protein.
  • the fusion protein refers to a protein produced by fusing an enzyme with an additional domain, peptide, polypeptide or protein.
  • the enzyme refers to a protein including a domain which is able to cleave a nucleic acid, gene, chromosome or protein.
  • the enzyme may be a nuclease, protease or restriction enzyme.
  • the additional domain, peptide, polypeptide or protein may be a functional domain, peptide, polypeptide or protein, which has a function the same as or different from the enzyme.
  • the fusion protein may include an additional domain, peptide, polypeptide or protein at one or more of an N-terminus of an enzyme or the proximity thereof; a C-term inus of the enzyme or the proximity thereof; the middle region of an enzyme; and a combination thereof.
  • the fusion protein may include a functional domain, peptide, polypeptide or protein at one or more of an N-terminus of an enzyme or the proximity thereof; a C-term inus of the enzyme or the proximity thereof; the middle region of an enzyme; and a combination thereof.
  • the functional domain, peptide, polypeptide or protein may be a domain, peptide, polypeptide or protein having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for isolation and purification of a protein (including a peptide), but the present invention is not limited thereto.
  • the functional domain, peptide, polypeptide or protein may be a deaminase.
  • the tag includes a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag and a thioredoxin (Trx) tag
  • the reporter gene includes glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) ⁇ -galactosidase, p-glucoronidase, luciferase, autofluorescent proteins including the green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP), but the present invention is not limited thereto.
  • the functional domain, peptide, polypeptide or protein may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).
  • NLS nuclear localization sequence or signal
  • NES nuclear export sequence or signal
  • the NLS may be NLS of SV40 virus large T-antigen with an amino acid sequence PKKKRKV(SEQ ID NO: 55); NLS derived from nucleoplasmin (e.g., nucleoplasmin bipartite NLS with a sequence KRPAATKKAGQAKKKK(SEQ ID NO: 56)); c-myc NLS with an amino acid sequence PAAKRVKLD(SEQ ID NO: 57) or RQRRNELKRSP(SEQ ID NO: 58); hRNPA1 M9 NLS with a sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 59); an importin- ⁇ -derived IBB domain sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV(SEQ ID NO: 60); myoma T protein sequences VSRKRPRP(SEQ ID NO: 61) and PPKKARED(SEQ ID NO: 62
  • the editor protein may include a complete active enzyme.
  • the “complete active enzyme” refers to an enzyme having the same function as a function of a wild-type enzyme, and for example, the wild-type enzyme cleaving the double strand of DNA has complete enzyme activity of entirely cleaving the double strand of DNA.
  • the complete active enzyme includes an enzyme having an improved function compared to the function of the wild-type enzyme, and for example, a specific modification or manipulation type of the wild-type enzyme cleaving the double strand of DNA has full enzyme activity which is improved compared to the wild-type enzyme, that is, activity of cleaving the double strand of DNA.
  • the editor protein may include an incomplete or partially active enzyme.
  • the “incomplete or partially active enzyme” refers to an enzyme having some of the functions of the wild-type enzyme, and for example, a specific modification or manipulation type of the wild-type enzyme cleaving the double strand of DNA has incomplete or partial enzyme activity of cleaving a part of the double strand, that is, a single strand of DNA.
  • the editor protein may include an inactive enzyme.
  • the “inactive enzyme” refers to an enzyme in which the function of a wild-type enzyme is completely inactivated.
  • a specific modification or manipulation type of the wild-type enzyme cleaving the double strand of DNA has inactivity so as not to completely cleave the DNA double strand.
  • the editor protein may be a natural enzyme or fusion protein.
  • the editor protein may be present in the form of a partially modified natural enzyme or fusion protein.
  • the editor protein may be an artificially produced enzyme or fusion protein, which does not exist in nature.
  • the editor protein may be present in the form of a partially modified artificial enzyme or fusion protein, which does not exist in nature.
  • the modification may be substitution, removal, addition of amino acids contained in the editor protein, or a combination thereof.
  • the modification may be substitution, removal, addition of some bases in the base sequence encoding the editor protein, or a combination thereof.
  • CRISPR enzyme As one exemplary embodiment of the editor protein of the present invention, a CRISPR enzyme will be described below.
  • CRISPR enzyme is a main protein component of a CRISPR-Cas system, and forms a complex with gRNA, resulting in the CRISPR-Cas system.
  • the CRISPR enzyme is a nucleic acid or polypeptide (or a protein) having a sequence encoding the CRISPR enzyme, and representatively, a Type II CRISPR enzyme or Type V CRISPR enzyme is widely used.
  • the Type II CRISPR enzyme is Cas9, which may be derived from various microorganisms such as Streptococcus pyogenes, Streptococcus thermophilus, Streptococcus sp., Staphylococcus aureus, Nocardiopsis rougevillei, Streptomyces pristinaespiralis, Streptomyces viridochromogenes, Streptomyces viridochromogenes, Streptosporangium roseum, Streptosporangium roseum, AlicyclobacHlus acidocaldarius, Bacillus pseudomycoides, Bacillus selenitireducens, Exiguobacterium sibiricum, Lactobacillus delbrueckii, Lactobacillus salivarius, Microscilla marina, Burkholderiales bacterium, Polaromonas naphthalenivorans, Polaromonas sp., Crocosphaera watsonii, Cya
  • Cas9 is an enzyme which binds to gRNA so as to cleave or modify a target sequence or position on a target gene or nucleic acid, and may consist of an HNH domain capable of cleaving a nucleic acid strand forming a complementary bond with gRNA, an RuvC domain capable of cleaving a nucleic acid strand forming a complementary bond with gRNA, an REC domain recognizing a target and a PI domain recognizing PAM.
  • Hiroshi Nishimasu et al. (2014) Cell 156:935-949 may be referenced for specific structural characteristics of Cas9.
  • Type V CRISPR enzyme may be Cpf1, which may be derived from Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacillus, Methylobacterium or Acidaminococcus
  • the Cpf1 may consist of an RuvC domain similar and corresponding to the RuvC domain of Cas9, an Nuc domain without the HNH domain of Cas9, an REC domain recognizing a target, a WED domain and a PI domain recognizing PAM.
  • RuvC domain similar and corresponding to the RuvC domain of Cas9
  • Nuc domain without the HNH domain of Cas9 an Nuc domain without the HNH domain of Cas9
  • an REC domain recognizing a target
  • WED domain a WED domain
  • PI domain recognizing PAM recognizing PAM.
  • the CRISPR enzyme of the Cas9 or Cpf1 protein may be isolated from a microorganism existing in nature or non-naturally produced by a recombinant or synthetic method.
  • the crystal structure of the type II CRISPR enzyme was determined according to studies on two or more types of natural microbial type II CRISPR enzyme molecules (Jinek et al., Science, 343(6176):1247997, 2014) and studies on Streptococcus pyogenes Cas9 (SpCas9) complexed with gRNA (Nishimasu et al., Cell, 156:935-949, 2014; and Anders et al., Nature, 2014, doi: 10.1038/nature13579).
  • the type II CRISPR enzyme includes two lobes, that is, recognition (REC) and nuclease (NUC) lobes, and each lobe includes several domains.
  • the REC lobe includes an arginine-rich bridge helix (BH) domain, an REC1 domain and an REC2 domain.
  • BH arginine-rich bridge helix
  • the BH domain is a long ⁇ -helix and arginine-rich region
  • the REC1 and REC2 domains play an important role in recognizing a double strand formed in gRNA, for example, single-stranded gRNA, double-stranded gRNA or tracrRNA.
  • the NUC lobe includes an RuvC domain, an HNH domain and a PAM-interaction (PI) domain.
  • the RuvC domain encompasses RuvC-like domains, or the HNH domain is used to include HNH-like domains.
  • the RuvC domain shares structural similarity with members of the microorganism family existing in nature having the type II CRISPR enzyme, and cleaves a single strand, for example, a non-complementary strand of a target gene or nucleic acid, that is, a strand not forming a complementary bond with gRNA.
  • the RuvC domain is sometimes referred to as an RuvCI domain, RuvCII domain or RuvCIII domain in the art, and generally called an RuvC I, RuvCII or RuvCIII.
  • the RuvC domain is assembled from each of three divided RuvC domains (RuvC I, RuvCII and RuvCIII) located at the sequences ofamino acids 1 to 59, 718 to 769 and 909 to 1098 of SpCas9, respectively.
  • the HNH domain shares structural similarity with the HNH endonuclease, and cleaves a single strand, for example, a complementary strand of a target nucleic acid molecule, that is, a strand forming a complementary bond with gRNA.
  • the HNH domain is located between RuvC II and III motifs.
  • the HNH domain is located at amino acid sequence 775 to 908 of SpCas9.
  • the PI domain recognizes a specific base sequence in a target gene or nucleic acid, that is, a protospacer adjacent motif (PAM) or interacts with PAM.
  • PAM protospacer adjacent motif
  • the PI domain is located at the sequence of amino acids1099 to 1368 of SpCas9.
  • the PAM may vary according to the origin of the type II CRISPR enzyme.
  • Type V CRISPR enzyme includes similar RuvC domains corresponding to the RuvC domains of the type II CRISPR enzyme, and may consist of an Nuc domain, instead of the HNH domain of the type II CRISPR enzyme, REC and WED domains, which recognize a target, and a PI domain recognizing PAM.
  • RuvC domains corresponding to the RuvC domains of the type II CRISPR enzyme, and may consist of an Nuc domain, instead of the HNH domain of the type II CRISPR enzyme, REC and WED domains, which recognize a target, and a PI domain recognizing PAM.
  • the type V CRISPR enzyme may interact with gRNA, thereby forming a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, and may allow a guide sequence to approach a target sequence including a PAM sequence in cooperation with gRNA.
  • a gRNA-CRISPR enzyme complex that is, a CRISPR complex
  • the ability of the type V CRISPR enzyme for interaction with a target gene or nucleic acid is dependent on the PAM sequence.
  • the PAM sequence is a sequence present in a target gene or nucleic acid, and may be recognized by the PI domain of the type V CRISPR enzyme.
  • the PAM sequence may vary according to the origin of the type V CRISPR enzyme. That is, there are different PAM sequences which are able to be specifically recognized depending on a species.
  • the PAM sequence recognized by Cpf1 may be 5′-TTN-3′ (N is A, T, C or G).
  • a CRISPR enzyme cleaves a double or single strand of a target gene or nucleic acid, and has nuclease activity causing breakage or deletion of the double or single strand.
  • the wild-type type II CRISPR enzyme or type V CRISPR enzyme cleaves the double strand of the target gene or nucleic acid.
  • the CRISPR enzyme may be manipulated or modified, such a manipulated or modified CRISPR enzyme may be modified into an incompletely or partially active or inactive enzyme.
  • a CRISPR enzyme modified to change enzyme activity, thereby exhibiting incomplete or partial activity is called a nickase.
  • nickase refers to a CRISPR enzyme manipulated or modified to cleave only one strand of the double strand of the target gene or nucleic acid, and the nickase has nuclease activity of cleaving a single strand, for example, a strand that is not complementary or complementary to gRNA of the target gene or nucleic acid. Therefore, to cleave the double strand, nuclease activity of the two nickases is needed.
  • the nickase may have nuclease activity by the RuvC domain. That is, the nickase may include nuclease activity of the HNH domain, and to this end, the HNH domain may be manipulated or modified.
  • the CRISPR enzyme is the type II CRISPR enzyme
  • the residue 840 in the amino acid sequence of SpCas9 is mutated from histidine to alanine
  • the nuclease activity of the HNH domain is inactivated to be used as a nickase. Since the nickase produced thereby has nuclease activity of the RuvC domain, it is able to cleave a strand which does not form a complementary bond with a non-complementary strand of the target gene or nucleic acid, that is, gRNA.
  • the nuclease activity of the HNH domain is inactivated to be used as a nickase.
  • the nickase produced thereby has nuclease activity by the RuvC domain, and thus is able to cleave a non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with gRNA.
  • the nickase may have nuclease activity by the HNH domain. That is, the nickase may include the nuclease activity of the RuvC domain, and to this end, the RuvC domain may be manipulated or modified.
  • the CRISPR enzyme is the type II CRISPR enzyme
  • the nuclease activity of the RuvC domain is inactivated to be used as a nickase.
  • the nickase produced thereby has the nuclease activity of the HNH domain, and thus is able to cleave a complementary strand of the target gene or nucleic acid, that is, a strand that forms a complementary bond with gRNA.
  • the nuclease activity of the RuvC domain is inactivated to be used as a nickase.
  • the nickase produced thereby has the nuclease activity of the HNH domain, and thus is able to cleave a complementary strand of the target gene or nucleic acid, that is, a strand that forms a complementary bond with gRNA.
  • a CRISPR enzyme which is modified to make enzyme activity completely inactive is called an inactive CRISPR enzyme.
  • inactive CRISPR enzyme refers to a CRISPR enzyme which is modified not to completely cleave the double strand of the target gene or nucleic acid, and the inactive CRISPR enzyme has nuclease inactivity due to the mutation in the domain with nuclease activity of the wild-type CRISPR enzyme.
  • the inactive CRISPR enzyme may be one in which the nuclease activities of the RuvC domain and the HNH domain are inactivated.
  • the inactive CRISPR enzyme may be manipulated or modified in the RuvC domain and the HNH domain so as to inactive nuclease activity.
  • the CRISPR enzyme is the type II CRISPR enzyme
  • the residues 10 and 840 in the amino acid sequence of SpCas9 are mutated from aspartic acid and histidine to alanine, respectively, nuclease activities by the RuvC domain and the HNH domain are inactivated, such that the double strand may not cleave completely the double strand of the target gene or nucleic acid.
  • CjCas9 when the residues 8 and 559 in the amino acid sequence of CjCas9 are mutated from aspartic acid and histidine to alanine, the nuclease activities by the RuvC domain and the HNH domain are inactivated, such that the double strand may not cleave completely the double strand of the target gene or nucleic acid.
  • the CRISPR enzyme may have endonuclease activity, exonuclease activity or helicase activity, that is, an ability to anneal the helix structure of the double-stranded nucleic acid, in addition to the above-described nuclease activity.
  • the CRISPR enzyme may be modified to completely, incompletely, or partially activate the endonuclease activity, exonuclease activity or helicase activity.
  • the CRISPR enzyme may interact with gRNA, thereby forming a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, and lead a guide sequence to approach a target sequence including a PAM sequence in cooperation with gRNA.
  • a gRNA-CRISPR enzyme complex that is, a CRISPR complex
  • the ability of the CRISPR enzyme to interact with the target gene or nucleic acid is dependent on the PAM sequence.
  • the PAM sequence is a sequence present in the target gene or nucleic acid, which may be recognized by the PI domain of the CRISPR enzyme.
  • the PAM sequence may vary depending on the origin of the CRISPR enzyme. That is, there are various PAM sequences which are able to be specifically recognized according to species.
  • the CRISPR enzyme is the type II CRISPR enzyme
  • the PAM sequence may be 5′-NGG-3′, 5′-NAG-3′ and/or 5′-NGA-3′,
  • the PAM sequence may be 5′-NNNNGATT-3′ and/or 5′-NNNGCTT-3′,
  • the CRISPR enzyme is the type V CRISPR enzyme
  • the PAM sequence may be 5′-TTN-3′.
  • the N may be A, T, G or C; or A, U, G or C.
  • the CRISPR enzyme capable of recognizing a specific PAM sequence may be manipulated or modified using the PAM sequence capable of being specifically recognized according to species.
  • the PI domain of SpCas9 may be replaced with the PI domain of CjCas9 so as to have the nuclease activity of SpCas9 and recognize a CjCas9-specific PAM sequence, thereby producing SpCas9 recognizing the CjCas9-specific PAM sequence.
  • a specifically recognized PAM sequence may be changed by substitution or replacement of the PI domain.
  • the CRISPR enzyme may be modified to improve or inhibit various characteristics such as nuclease activity, helicase activity, an ability to interact with gRNA, and an ability to approach the target gene or nucleic acid, for example, PAM recognizing ability of the CRISPR enzyme.
  • the CRISPR enzyme mutant may be a CRISPR enzyme which interacts with gRNA to form a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, and is modified or manipulated to improve target specificity, when approaching or localized to the target gene or nucleic acid, such that only a double or single strand of the target gene or nucleic acid is cleaved without cleavage of a double or single strand of a non-target gene or nucleic acid which partially forms a complementary bond with gRNA and a non-target gene or nucleic acid which does not form a complementary bond therewith.
  • a CRISPR enzyme complex that is, a CRISPR complex
  • an effect of cleaving the double or single strand of the non-target gene or nucleic acid partially forming a complementary bond with gRNA and the non-target gene or nucleic acid not forming a complementary bond therewith is referred to as an off-target effect
  • a position or base sequence of the non-target gene or nucleic acid partially forming a complementary bond with gRNA and the non-target gene or nucleic acid not forming a complementary bond therewith is referred to as an off-target.
  • the cleavage effect of the double or single strand of the target gene or nucleic acid is referred to as an on-target effect, and a location or target sequence of the target gene or nucleic acid is referred to as an on-target.
  • the CRISPR enzyme mutant is modified in at least one of the amino acids of a naturally-occurring CRISPR enzyme, and may be modified, for example, improved or inhibited in one or more of the various characteristics such as nuclease activity, helicase activity, an ability to interact with gRNA, an ability to approach the target gene or nucleic acid and target specificity, compared to the unmodified CRISPR enzyme.
  • the modification may be substitution, removal, addition of an amino acid, or a mixture thereof.
  • the modification may be a modification of one or two or more amino acids located in a region consisting of amino acids having positive charges, present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more amino acids of the positively-charged amino acids such as lysine (K), arginine (R) and histidine (H), present in the naturally-occurring CRISPR enzyme.
  • the positively-charged amino acids such as lysine (K), arginine (R) and histidine (H)
  • the modification may be a modification of one or two or more amino acids located in a region composed of non-positively-charged amino acids present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more amino acids of the non-positively-charged amino acids, that is, aspartic acid (D), glutamic acid (E), serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more amino acids of non-charged amino acids, that is, serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more of the amino acids having hydrophobic residues present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more amino acids of glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.
  • G glycine
  • A alanine
  • V valine
  • I isoleucine
  • L leucine
  • M methionine
  • F phenylalanine
  • Y tyrosine
  • W tryptophan
  • the modification may be a modification of one or two or more of the amino acids having polar residues, present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more amino acids of serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), lysine (K), arginine (R), histidine (H), aspartic acid (D) and glutamic acid (E), present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more of the amino acids including lysine (K), arginine (R) and histidine (H), present in the naturally-occurring CRISPR enzyme.
  • the modification may be a substitution of one or two or more of the amino acids including lysine (K), arginine (R) and histidine (H), present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more of the amino acids including aspartic acid (D) and glutamic acid (E), present in the naturally-occurring CRISPR enzyme.
  • the modification may be a substitution of one or two or more of the amino acids including aspartic acid (D) and glutamic acid (E), present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more of the amino acids including serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.
  • S serine
  • T threonine
  • N asparagine
  • C cysteine
  • P proline
  • G glycine
  • A alanine
  • V valine
  • I isoleucine
  • L leucine
  • M methionine
  • F phenylalanine
  • Y tyrosine
  • W tryptophan
  • the modification may be a substitution of one or two or more of the amino acid including serine (S), threonine (T), asparagine (N), glutamine (Q), cysteine (C), proline (P), glycine (G), alanine (A), valine (V), isoleucine (I), leucine (L), methionine (M), phenylalanine (F), tyrosine (Y) and tryptophan (W), present in the naturally-occurring CRISPR enzyme.
  • S serine
  • T threonine
  • N asparagine
  • C cysteine
  • P proline
  • G glycine
  • A alanine
  • V valine
  • I isoleucine
  • L leucine
  • M methionine
  • F phenylalanine
  • Y tyrosine
  • W tryptophan
  • the modification may be a modification of one, two, three, four, five, six, seven or more of the amino acids present in the naturally-occurring CRISPR enzyme.
  • the modification may be a modification of one or two or more of the amino acids present in the RuvC domain of the CRISPR enzyme.
  • the RuvC domain may be an RuvCI, RuvCII or RuvCIII domain.
  • the modification may be a modification of one or two or more of the amino acids present in the HNH domain of the CRISPR enzyme.
  • the modification may be a modification of one or two or more of the amino acids present in the REC domain of the CRISPR enzyme.
  • the modification may be one or two or more of the amino acids present in the PI domain of the CRISPR enzyme.
  • the modification may be a modification of two or more of the amino acids contained in at least two or more domains of the REC, RuvC, HNH and PI domains of the CRISPR enzyme.
  • the modification may be a modification of two or more of the amino acids contained in the REC and RuvC domains of the CRISPR enzyme.
  • the modification in the SpCas9 mutant, may be a modification of at least two or more of the A203, H277, G366, F539, 1601, M763, D965 and F1038 amino acids contained in the REC and RuvC domains of SpCas9.
  • the modification may be a modification of two or more of the amino acids contained in the REC and HNH domains of the CRISPR enzyme.
  • the modification in the SpCas9 mutant, may be a modification of at least two or more of the A203, H277, G366, F539, 1601 and K890 amino acids contained in the REC and HNH domains of SpCas9.
  • the modification may be a modification of two or more of the amino acids contained in the REC and PI domains of the CRISPR enzyme.
  • the modification in the SpCas9 mutant, may be a modification of at least two or more of the A203, H277, G366, F539, 1601, T1102 and D1127 amino acids contained in the REC and PI domains of SpCas9.
  • the modification may be a modification of three or more of the amino acids contained in the REC, RuvC and HNH domains of the CRISPR enzyme.
  • the modification in the SpCas9 mutant, may be a modification of at least three or more of the A203, H277, G366, F539, 1601, M763, K890, D965 and F1038 amino acids contained in the REC, RuvC and HNH domains of SpCas9.
  • the modification may be a modification of three or more of the amino acids contained in the REC, RuvC and PI domains contained in the CRISPR enzyme.
  • the modification in the SpCas9 mutant, may be a modification of at least three or more of the A203, H277, G366, F539, 1601, M763, D965, F1038, T1102 and D1127 amino acids contained in the REC, RuvC and PI domains of SpCas9.
  • the modification may be a modification of three or more of the amino acids contained in the REC, HNH and PI domains of the CRISPR enzyme.
  • the modification in the SpCas9 mutant, may be a modification of at least three or more of the A203, H277, G366, F539, 1601, K890, T1102 and D1127 amino acids contained in the REC, HNH and PI domains of SpCas9.
  • the modification may be a modification of three or more of the amino acids contained in the RuvC, HNH and PI domains of the CRISPR enzyme.
  • the modification in the SpCas9 mutant, may be a modification of at least three or more of the M763, K890, D965, F1038, T1102 and D1127 amino acids contained in the RuvC, HNH and PI domains of SpCas9.
  • the modification may be a modification of four or more of the amino acids contained in the REC, RuvC, HNH and PI domains of the CRISPR enzyme.
  • the modification in the SpCas9 mutant, may be a modification of at least four or more of the A203, H277, G366, F539, 1601, M763, K890, D965, F1038, T1102 and D1127 amino acids contained in the REC, RuvC, HNH and PI domains of SpCas9.
  • the modification may be a modification of one or two or more of the amino acids participating in the nuclease activity of the CRISPR enzyme.
  • the modification may be a modification of one or two or more of the group consisting of the amino acids D10, E762, H840, N854, N863 and D986, or one or two or more of the group consisting of the amino acids corresponding to other Cas9 orthologs.
  • the modification may be a modification for partially inactivating the nuclease activity of the CRISPR enzyme, and such a CRISPR enzyme mutant may be a nickase.
  • the modification may be a modification for inactivating the nuclease activity of the RuvC domain of the CRISPR enzyme, and such a CRISPR enzyme mutant may not cleave a non-complementary strand of a target gene or nucleic acid, that is, a strand which does not form a complementary bond with gRNA.
  • the SpCas9 when residue 10 of the amino acid sequence of SpCas9 is mutated from aspartic acid to alanine, that is, when mutated to D10A, the nuclease activity of the RuvC domain is inactivated, and thus the SpCas9 may be used as a nickase.
  • the nickase produced thereby may not cleave a non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with gRNA.
  • CjCas9 when residue 8 of the amino acid sequence of CjCas9 is mutated from aspartic acid to alanine, that is, when mutated to D8A, the nuclease activity of the RuvC domain is inactivated, and thus the CjCas9 may be used as a nickase.
  • the nickase produced thereby may not cleave a non-complementary strand of the target gene or nucleic acid, that is, a strand that does not form a complementary bond with gRNA.
  • the modification may be a modification for inactivating the nuclease activity of the HNH domain of the CRISPR enzyme, and such a CRISPR enzyme mutant may not cleave a complementary strand of the target gene or nucleic acid, that is, a strand forming a complementary bond with gRNA.
  • the SpCas9 when residue 840 of the amino acid sequence of SpCas9 is mutated from histidine to alanine, that is, when mutated to H840A, the nuclease activity of the HNH domain is inactivated, and thus the SpCas9 may be used as a nickase.
  • the nickase produced thereby may not cleave a complementary strand of the target gene or nucleic acid, that is, a strand that forms a complementary bond with gRNA.
  • CjCas9 when residue 559 of the amino acid sequence of CjCas9 is mutated from histidine to alanine, that is, when mutated to H559A, the nuclease activity of the HNH domain is inactivated, and thus the CjCas9 may be used as a nickase.
  • the nickase produced thereby may not cleave a complementary strand of the target gene or nucleic acid, that is, a strand that forms a complementary bond with gRNA.
  • the modification may be a modification for completely inactivating the nuclease activity of the CRISPR enzyme, and such a CRISPR enzyme mutant may be an inactive CRISPR enzyme.
  • the modification may be a modification for inactivating the nuclease activities of the RuvC and HNH domains of the CRISPR enzyme, and such a CRISPR enzyme mutant may does not cleave a double strand of the target gene or nucleic acid.
  • the residues 10 and 840 in the amino acid sequence of SpCas9 are mutated from aspartic acid and histidine to alanine, that is, mutated to D10A and H840A, respectively, the nuclease activities of the RuvC domain and the HNH domain are inactivated, the double strand of the target gene or nucleic acid may not be completely cleaved.
  • CjCas9 when residues 8 and 559 of the amino acid sequence of CjCas9 are mutated from aspartic acid and histidine to alanine, that is, mutated to D8A and H559A, respectively, the nuclease activities by the RuvC and HNH domains are inactivated, and thus the double strand of the target gene or nucleic acid may not be completely cleaved.
  • the CRISPR enzyme mutant may further include an optionally functional domain, in addition to the innate characteristics of the CRISPR enzyme, and such a CRISPR enzyme mutant may have an additional characteristic in addition to the innate characteristics.
  • the functional domain may be a domain having methylase activity, demethylase activity, transcription activation activity, transcription repression activity, transcription release factor activity, histone modification activity, RNA cleavage activity or nucleic acid binding activity, or a tag or reporter gene for isolating and purifying a protein (including a peptide), but the present invention is not limited thereto.
  • the functional domain, peptide, polypeptide or protein may be a deaminase.
  • an incomplete or partial CRISPR enzyme may additionally include a cytidine deaminase as a functional domain.
  • a cytidine deaminase for example, apolipoprotein B editing complex 1 (APOBEC1) may be added to SpCas9 nickase, thereby producing a fusion protein.
  • the [SpCas9 nickase]-[APOBEC1] formed thereby may be used in base repair or editing of C into T or U, or G into A.
  • the tag includes a histidine (His) tag, a V5 tag, a FLAG tag, an influenza hemagglutinin (HA) tag, a Myc tag, a VSV-G tag and a thioredoxin (Trx) tag
  • the reporter gene includes glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT) ⁇ -galactosidase, ⁇ -glucoronidase, luciferase, autofluorescent proteins including the green fluorescent protein (GFP), HcRed, DsRed, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP) and blue fluorescent protein (BFP), but the present invention is not limited thereto.
  • the functional domain may be a nuclear localization sequence or signal (NLS) or a nuclear export sequence or signal (NES).
  • NLS nuclear localization sequence or signal
  • NES nuclear export sequence or signal
  • the CRISPR enzyme may include one or more NLSs.
  • one or more NLSs may be included at an N-terminus of an CRISPR enzyme or the proximity thereof; a C-terminus of the enzyme or the proximity thereof; or a combination thereof.
  • the NLS may be an NLS sequence derived from the following NLSs, but the present invention is not limited thereto: NLS of a SV40 virus large T-antigen having the amino acid sequence PKKKRKV(SEQ ID NO: 55); NLS from nucleoplasmin (e.g., nucleoplasmin bipartite NLS having the sequence KRPAATKKAGQAKKKK(SEQ ID NO: 56)); c-myc NLS having the amino acid sequence PAAKRVKLD(SEQ ID NO: 57) or RQRRNELKRSP(SEQ ID NO: 58); hRNPA1 M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY(SEQ ID NO: 59); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV(SEQ ID NO: 60) of the IBB domain from importin- ⁇ ; the sequences VSRKRPRP(SEQ ID
  • the CRISPR enzyme mutant may include a split-type CRISPR enzyme prepared by dividing the CRISPR enzyme into two or more parts.
  • split refers to functional or structural division of a protein or random division of a protein into two or more parts.
  • the split-type CRISPR enzyme may be a completely, incompletely or partially active enzyme or inactive enzyme.
  • the SpCas9 may be divided into two parts between the residue 656, tyrosine, and the residue 657, threonine, thereby generating split SpCas9.
  • split-type CRISPR enzyme may selectively include an additional domain, peptide, polypeptide or protein for reconstitution.
  • substitution refers to formation of the split-type CRISPR enzyme to be structurally the same or similar to the wild-type CRISPR enzyme.
  • the additional domain, peptide, polypeptide or protein for reconstitution may be FRB and FKBP dimerization domains; intein; ERT and VPR domains; or domains which form a heterodimer under specific conditions.
  • the SpCas9 may be divided into two parts between the residue 713, serine, and the residue 714, glycine, thereby generating split SpCas9.
  • the FRB domain may be connected to one of the two parts, and the FKBP domain may be connected to the other one.
  • the FRB domain and the FKBP domain may be formed in a dimer in an environment in which rapamycine is present, thereby producing a reconstituted CRISPR enzyme.
  • the CRISPR enzyme or CRISPR enzyme mutant described in the present invention may be a polypeptide, protein or nucleic acid having a sequence encoding the same, and may be codon-optimized for a subject to introduce the CRISPR enzyme or CRISPR enzyme mutant.
  • codon optimization refers to a process of modifying a nucleic acid sequence by maintaining a native amino acid sequence while replacing at least one codon of the native sequence with a codon more frequently or the most frequently used in host cells so as to improve expression in the host cells.
  • a variety of species have a specific bias to a specific codon of a specific amino acid, and the codon bias (the difference in codon usage between organisms) is frequently correlated with efficiency of the translation of mRNA, which is considered to be dependent on the characteristic of a translated codon and availability of a specific tRNA molecule.
  • the dominance of tRNA selected in cells generally reflects codons most frequently used in peptide synthesis. Therefore, a gene may be customized by optimal gene expression in a given organism based on codon optimization.
  • target sequence is a base sequence present in a target gene or nucleic acid, and has complementarity to a guide sequence contained in a guide domain of a guide nucleic acid.
  • the target sequence is a base sequence which may vary according to a target gene or nucleic acid, that is, a subject for gene manipulation or correction, which may be designed in various forms according to the target gene or nucleic acid.
  • the target sequence may form a complementary bond with the guide sequence contained in the guide domain of the guide nucleic acid, and a length of the target sequence may be the same as that of the guide sequence.
  • the target sequence may be a 5 to 50-base sequence.
  • the target sequence may be a 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25-base sequence.
  • the target sequence may be a nucleic acid sequence complementary to the guide sequence contained in the guide domain of the guide nucleic acid, which has, for example, at least 70%, 75%, 80%, 85%, 90% or 95% or more complementarity or complete complementarity.
  • the target sequence may be or include a 1 to 8-base sequence, which is not complementary to the guide sequence contained in the guide domain of the guide nucleic acid.
  • the target sequence may be a base sequence adjacent to a nucleic acid sequence that is able to be recognized by an editor protein.
  • the target sequence may be a continuous 5 to 50-base sequence adjacent to the 5′ end and/or 3′ end of the nucleic acid sequence that is able to be recognized by the editor protein.
  • target sequences for a gRNA-CRISPR enzyme complex will be described below.
  • the target sequence When the target gene or nucleic acid is targeted by the gRNA-CRISPR enzyme complex, the target sequence has complementarity to the guide sequence contained in the guide domain of gRNA.
  • the target sequence is a base sequence which varies according to the target gene or nucleic acid, that is, a subject for gene manipulation or correction, which may be designed in various forms according to the target gene or nucleic acid.
  • the target sequence may be a base sequence adjacent to a PAM sequence which is able to be recognized by the CRISPR enzyme, that is, Cas9 or Cpf1.
  • the target sequence may be a continuous 5 to 50-base sequence adjacent to the 5′ end and/or 3′ end of the PAM sequence which is recognized by the CRISPR enzyme.
  • the target sequence may be a nucleic acid sequence contained in one or more genes selected from the group consisting of an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and an FAD8 gene.
  • the target sequence may be a nucleic acid sequence contained in the FAD2 gene.
  • the target sequence may be a nucleic acid sequence contained in the FAD3 gene.
  • the target sequence may be a nucleic acid sequence contained in the FAD6 gene.
  • the target sequence may be a nucleic acid sequence contained in the FAD7 gene.
  • the target sequence may be a nucleic acid sequence contained in the FAD8 gene.
  • the target sequence may be a partial nucleic acid sequence of one or more genes selected from the group consisting of an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and an FAD8 gene.
  • the target sequence may be a partial nucleic acid sequence of the FAD2 gene.
  • the target sequence may be a partial nucleic acid sequence of the FAD3 gene.
  • the target sequence may be a partial nucleic acid sequence of the FAD6 gene.
  • the target sequence may be a partial nucleic acid sequence of the FAD7 gene.
  • the target sequence may be a partial nucleic acid sequence of the FAD8 gene.
  • the target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of one or more genes selected from the group consisting of an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and an FAD8 gene.
  • the target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the FAD2 gene.
  • the target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the FAD3 gene.
  • the target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the FAD6 gene.
  • the target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the FAD7 gene.
  • the target sequence may be a nucleic acid sequence of the coding or non-coding region or a mixture thereof of the FAD8 gene.
  • the target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of one or more genes selected from the group consisting of an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and an FAD8 gene.
  • polyA polyadenyl
  • the target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the FAD2 gene.
  • the target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the FAD3 gene.
  • the target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the FAD6 gene.
  • the target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the FAD7 gene.
  • the target sequence may be a nucleic acid sequence of the promoter, enhancer, 3′UTR or polyadenyl (polyA) region or a mixture thereof of the FAD8 gene.
  • the target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of one or more genes selected from the group consisting of an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and an FAD8 gene.
  • the target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the FAD2 gene.
  • the target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the FAD3 gene.
  • the target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the FAD6 gene.
  • the target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the FAD7 gene.
  • the target sequence may be a nucleic acid sequence of an exon, an intron or a mixture thereof of the FAD8 gene.
  • the target sequence may be a nucleic acid sequence including or adjacent to a mutated region (e.g., a region different from a wild-type gene) of one or more genes selected from the group consisting of an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and an FAD8 gene.
  • a mutated region e.g., a region different from a wild-type gene
  • the target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the FAD2 gene.
  • the target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the FAD3 gene.
  • the target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the FAD6 gene.
  • the target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the FAD7 gene.
  • the target sequence may be a nucleic acid sequence including or adjacent to a mutated region of the FAD8 gene.
  • the target sequence may be a continuous 5 to 50-nucleic acid sequence of one or more genes selected from the group consisting of an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and an FAD8 gene.
  • the target sequence may be a continuous 5 to 50-nucleic acid sequence of the FAD2 gene.
  • the target sequence may be a continuous 5 to 50-nucleic acid sequence of the FAD3 gene.
  • the target sequence may be a continuous 5 to 50-nucleic acid sequence of the FAD6 gene.
  • the target sequence may be a continuous 5 to 50-nucleic acid sequence of the FAD7 gene.
  • the target sequence may be a continuous 5 to 50-nucleic acid sequence of the FAD8 gene.
  • the above target sequences of the FAD2 gene are summarized in Table 1.
  • a guide nucleic acid-editor protein complex may modify a target.
  • the guide nucleic acid-editor protein complex may be used to ultimately regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein of interest, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a new protein.
  • the guide nucleic acid-editor protein complex may act at a DNA, RNA, gene or chromosomal level.
  • the guide nucleic acid-editor protein complex may regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein encoded by target DNA, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a modified protein through manipulation or modification of the target DNA.
  • the guide nucleic acid-editor protein complex may regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein encoded by target DNA, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a modified protein through manipulation or modification of target RNA.
  • the guide nucleic acid-editor protein complex may regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein encoded by target DNA, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a modified protein through manipulation or modification of a target gene.
  • the guide nucleic acid-editor protein complex may regulate (e.g., inhibit, suppress, reduce, increase or promote) the expression of a protein encoded by target DNA, remove a protein, regulate (e.g., inhibit, suppress, reduce, increase or promote) protein activity, or express a modified protein through manipulation or modification of a target chromosome.
  • the guide nucleic acid-editor protein complex may act at gene transcription and translation stages.
  • the guide nucleic acid-editor protein complex may promote or suppress the transcription of a target gene, thereby regulating (e.g., inhibiting, suppressing, reducing, increasing or promoting) the expression of a protein encoded by the target gene.
  • the guide nucleic acid-editor protein complex may promote or suppress the translation of a target gene, thereby regulating (e.g., inhibiting, suppressing, reducing, increasing or promoting) the expression of a protein encoded by the target gene.
  • the guide nucleic acid-editor protein complex may act at a protein level.
  • the guide nucleic acid-editor protein complex may manipulate or modify a target protein, thereby removing the target protein or regulating (e.g., inhibiting, suppressing, reducing, increasing or promoting) protein activity.
  • the present invention provides a guide nucleic acid-editor protein complex used to manipulate a unsaturated fatty acid biosynthesis-associated factor, for example, an FAD gene, preferably an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and/or an FAD8 gene.
  • a gRNA-CRISPR enzyme complex is provided.
  • the present invention may provide gRNA including a guide domain capable of forming a complementary bond with a target sequence from a gene, for example, isolated or non-natural gRNA and DNA encoding the same.
  • the gRNA and the DNA sequence encoding the same may be designed to be able to complementarily bind to a target sequence listed in Table 1.
  • a target region of the gRNA is designed to provide a third gene, which has a nucleic acid modification, for example, double or single strand breaks; or a specific function at a target site in an FAD2 gene, an FAD3 gene, an FAD6 gene, an FAD7 gene, and/or an FAD8 gene.
  • the two or more cleaving events may occur due to the same or different Cas9 proteins.
  • the gRNA may target, for example, two or more of the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and/or the FAD8 gene, or
  • the FAD2 gene may independently induce the cleavage of a double strand and/or a single strand of the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and/or the FAD8 gene, or
  • the FAD2 gene may induce the insertion of one foreign nucleotide into a cleavage site of the FAD2 gene, the FAD3 gene, the FAD6 gene, the FAD7 gene, and/or the FAD8 gene.
  • a nucleic acid constituting the guide nucleic acid-editor protein complex may include:
  • the (b) may employ the same or two or more editor proteins.
  • the nucleic acid may be designed to target an enzymatically inactive editor protein or a fusion protein (e.g., a transcription repressor domain fusion) thereof to place it sufficiently adjacent to a knockdown target site in order to reduce, decrease or inhibit expression of the FAD2 gene.
  • a fusion protein e.g., a transcription repressor domain fusion
  • the manipulation or modification of target DNA, RNA, genes or chromosomes using the gRNA-CRISPR enzyme complex will be described below.
  • a target gene or nucleic acid may be manipulated or corrected using the above-described gRNA-CRISPR enzyme complex, that is, the CRISPR complex.
  • the manipulation or correction of the target gene or nucleic acid includes all of the stages of i) cleaving or damaging the target gene or nucleic acid and ii) repairing the damaged target gene or nucleic acid.
  • the cleavage or damage of the target gene or nucleic acid may be cleavage or damage of the target gene or nucleic acid using the CRISPR complex, and particularly, cleavage or damage of a target sequence in the target gene or nucleic acid.
  • the cleavage or damage of the target gene or nucleic acid using the CRISPR complex may be complete cleavage or damage to the double strand of a target sequence.
  • the double strand of a target sequence forming a complementary bond with gRNA may be completely cleaved.
  • a complementary single strand of a target sequence forming a complementary bond with gRNA may be cleaved by the SpCas9 nickase (D10A), and a non-complementary single strand of the target sequence forming a complementary bond with gRNA may be cleaved by the SpCas9 nickase (H840A), and the cleavages may take place sequentially or simultaneously.
  • a complementary single strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the SpCas9 nickase (D10A)
  • a non-complementary single strand of a target sequence forming a complementary bond with the second gRNS may be cleaved by the SpCas9 nickase (H840A)
  • the cleavages may take place sequentially or simultaneously.
  • the cleavage or damage of a target gene or nucleic acid using the CRISPR complex may be cleavage or damage to only the single strand of a target sequence.
  • the single strand may be a complementary single strand of a target sequence forming a complementary bond with gRNA, or a non-complementary single strand of the target sequence forming a complementary bond with gRNA.
  • a complementary single strand of a target sequence forming a complementary bond with gRNA may be cleaved by the SpCas9 nickase (D10A), but a non-complementary single strand of the target sequence forming a complementary bond with gRNA may not be cleaved.
  • a complementary single strand of a target sequence forming a complementary bond with gRNA may be cleaved by the SpCas9 nickase (H840A), but a non-complementary single strand of the target sequence forming a complementary bond with gRNA may not be cleaved.
  • the cleavage or damage of a target gene or nucleic acid using the CRISPR complex may be partial removal of a nucleic acid fragment.
  • a double strand of a target sequence forming a complementary bond with the first gRNA may be cleaved, and a double strand of a target sequence forming a complementary bond with the second gRNA may be cleaved, resulting in the removal of nucleic acid fragments by the first and second gRNAs and SpCas9.
  • a double strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the wild-type SpCas9
  • a complementary single strand of a target sequence forming a complementary bond with the second gRNA may be cleaved by the SpCas9 nickase (D10A)
  • a non-complementary single strand nay be cleaved by the SpCas9 nickase (H840A)
  • a complementary single strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the SpCas9 nickase (D10A)
  • a non-complementary single strand may be cleaved by the SpCas9 nickase (H840A)
  • a complementary double strand of a target sequence forming a complementary bond with the second gRNA may be cleaved by the SpCas9 nickase (D10A)
  • a non-complementary single strand may be cleaved by the SpCas9 nickase (H840A)
  • a double strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the wild-type SpCas9
  • a complementary single strand of a target sequence forming a complementary bond with the second gRNA may be cleaved by the SpCas9 nickase (D10A)
  • a non-complementary single strand of a target sequence forming a complementary bond with the third gRNA may be cleaved by the SpCas9 nickase (H840A), resulting in the removal of nucleic acid fragments by the first gRNA, the second gRNA, the third gRNA, the wild-type SpCas9, the SpCas9 nickase (D
  • a complementary single strand of a target sequence forming a complementary bond with the first gRNA may be cleaved by the SpCas9 nickase (D10A)
  • a non-complementary single strand of a target sequence forming a complementary bond with the second gRNA may be cleaved by the SpCas9 nickase (H840A)
  • a complementary single strand of a target sequence forming a complementary bond with the third gRNA may be cleaved by the SpCas9 nickase (D10A)
  • a non-complementary single strand of a target sequence forming a complementary bond with fourth gRNA may be cleaved by the SpCas9 nickase (H840A), resulting in the removal of
  • the target gene or nucleic acid cleaved or damaged by the CRISPR complex may be repaired or restored through non-homologous end joining (NHEJ) and homology-directed repairing (HDR).
  • NHEJ non-homologous end joining
  • HDR homology-directed repairing
  • NHEJ Non-Homologous End Joining
  • NHEJ is a method of restoration or repairing double strand breaks in DNA by joining both ends of a cleaved double or single strand together, and generally, when two compatible ends formed by breaking of the double strand (for example, cleavage) are frequently in contact with each other to completely join the two ends, the broken double strand is recovered.
  • the NHEJ is a restoration method that is able to be used in the entire cell cycle, and usually occurs when there is no homologous genome to be used as a template in cells, like the G1 phase.
  • insertions and/or deletions in the nucleic acid sequence occur in the NHEJ-repaired region, such insertions and/or deletions cause the leading frame to be shifted, resulting in frame-shifted transcriptome mRNA.
  • innate functions are lost because of nonsense-mediated decay or the failure to synthesize normal proteins.
  • mutations in which insertion or deletion of a considerable amount of sequence may be caused to destroy the functionality of the proteins.
  • the mutation is locus-dependent because mutation in a significant functional domain is probably less tolerated than mutations in a non-significant region of a protein.
  • the deletion length ranges from 1 bp to 50 bp, insertions tend to be shorter, and frequently include a short repeat sequence directly surrounding a broken region.
  • the NHEJ is a process causing a mutation, and when it is not necessary to produce a specific final sequence, may be used to delete a motif of the small sequence.
  • a specific knockout of a gene targeted by the CRISPR complex may be performed using such NHEJ.
  • a double strand or two single strands of a target gene or nucleic acid may be cleaved using the CRISPR enzyme such as Cas9 or Cpf1, and the broken double strand or two single strands of the target gene or nucleic acid may have indels through the NHEJ, thereby inducing specific knockout of the target gene or nucleic acid.
  • the site of a target gene or nucleic acid cleaved by the CRISPR enzyme may be a non-coding or coding region, and in addition, the site of the target gene or nucleic acid restored by NHEJ may be a non-coding or coding region.
  • HDR Homology Directed Repairing
  • HDR is a correction method without an error, which uses a homologous sequence as a template to repair or restoration a damaged gene or nucleic acid, and generally, to repair or restoration broken DNA, that is, to restore innate information of cells, the broken DNA is repaired using information of a complementary base sequence which is not modified or information of a sister chromatid.
  • the most common type of HDR is homologous recombination (HR).
  • HDR is a repair or restoration method usually occurring in the S or G2/M phase of actively dividing cells.
  • a DNA template artificially synthesized using information of a complementary base sequence or homologous base sequence that is, a nucleic acid template including a complementary base sequence or homologous base sequence may be provided to the cells, thereby repairing the broken DNA.
  • a nucleic acid sequence or nucleic acid fragment is further added to the nucleic acid template to repair the broken DNA, the nucleic acid sequence or nucleic acid fragment further added to the broken DNA may be subjected to knockin.
  • the further added nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment for correcting the target gene or nucleic acid modified by a mutation to a normal gene or nucleic acid, or a gene or nucleic acid to be expressed in cells, but the present invention is not limited thereto.
  • a double or single strand of a target gene or nucleic acid may be cleaved using the CRISPR complex, a nucleic acid template including a base sequence complementary to a base sequence adjacent to the cleavage site may be provided to cells, and the cleaved base sequence of the target gene or nucleic acid may be repaired or restored through HDR.
  • the nucleic acid template including the complementary base sequence may have broken DNA, that is, a cleaved double or single strand of a complementary base sequence, and further include a nucleic acid sequence or nucleic acid fragment to be inserted into the broken DNA.
  • An additional nucleic acid sequence or nucleic acid fragment may be inserted into a cleaved site of the broken DNA, that is, the target gene or nucleic acid using the nucleic acid template including a nucleic acid sequence or nucleic acid fragment to be inserted into the complementary base sequence.
  • the nucleic acid sequence or nucleic acid fragment to be inserted and the additional nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment for correcting a target gene or nucleic acid modified by a mutation to a normal gene or nucleic acid or a gene or nucleic acid to be expressed in cells.
  • the complementary base sequence may be a base sequence having complementary bonds with broken DNA, that is, right and left base sequences of the cleaved double or single strand of the target gene or nucleic acid.
  • the complementary base sequence may be a base sequence having complementary bonds with broken DNA, that is, 3′ and 5′ ends of the cleaved double or single strand of the target gene or nucleic acid.
  • the complementary base sequence may be a 15 to 3000-base sequence, a length or size of the complementary base sequence may be suitably designed according to a size of the nucleic acid template or the target gene.
  • the nucleic acid template a double- or single-stranded nucleic acid may be used, or it may be linear or circular, but the present invention is not limited thereto.
  • a double- or single-stranded target gene or nucleic acid is cleaved using the CRISPR complex, a nucleic acid template including a homologous base sequence with a base sequence adjacent to a cleavage site is provided to cells, and the cleaved base sequence of the target gene or nucleic acid may be repaired or restored by HDR.
  • the nucleic acid template including the homologous base sequence may be broken DNA, that is, a cleaved double- or single-stranded homologous base sequence, and further include a nucleic acid sequence or nucleic acid fragment to be inserted into the broken DNA.
  • An additional nucleic acid sequence or nucleic acid fragment may be inserted into broken DNA, that is, a cleaved site of a target gene or nucleic acid using the nucleic acid template including a homologous base sequence and a nucleic acid sequence or nucleic acid fragment to be inserted.
  • the nucleic acid sequence or nucleic acid fragment to be inserted and the additional nucleic acid sequence or nucleic acid fragment may be a nucleic acid sequence or nucleic acid fragment for correcting a target gene or nucleic acid modified by a mutation to a normal gene or nucleic acid or a gene or nucleic acid to be expressed in cells.
  • the homologous base sequence may be broken DNA, that is, a base sequence having homology with cleaved double-stranded base sequence or right and left single-stranded base sequences of a target gene or nucleic acid.
  • the complementary base sequence may be a base sequence having homology with broken DNA, that is, the 3′ and 5′ ends of a cleaved double or single strand of a target gene or nucleic acid.
  • the homologous base sequence may be a 15 to 3000-base sequence, and a length or size of the homologous base sequence may be suitably designed according to a size of the nucleic acid template or a target gene or nucleic acid.
  • the nucleic acid template a double- or single-stranded nucleic acid may be used and may be linear or circular, but the present invention is not limited thereto.
  • SSA is a method of repairing double strand breaks between two repeat sequences present in a target nucleic acid, and generally uses a repeat sequence of more than 30 bases.
  • the repeat sequence is cleaved (to have sticky ends) to have a single strand with respect to a double strand of the target nucleic acid at each of the broken ends, and after the cleavage, a single-strand overhang containing the repeat sequence is coated with an RPA protein such that it is prevented from inappropriately annealing the repeat sequences to each other.
  • RAD52 binds to each repeat sequence on the overhang, and a sequence capable of annealing a complementary repeat sequence is arranged.
  • a single-stranded flap of the overhang is cleaved, and synthesis of new DNA fills a certain gap to restore a DNA double strand.
  • a DNA sequence between two repeats is deleted, and a deletion length may be dependent on various factors including the locations of the two repeats used herein, and a path or degree of the progress of cleavage.
  • SSA similar to HDR, utilizes a complementary sequence, that is, a complementary repeat sequence, and in contrast, does not requires a nucleic acid template for modifying or correcting a target nucleic acid sequence.
  • Single strand breaks in a genome are repaired through a separate mechanism, SSBR, from the above-described repair mechanisms.
  • PARP1 and/or PARP2 recognizes the breaks and recruits a repair mechanism.
  • PARP1 binding and activity with respect to the DNA breaks are temporary, and SSBR is promoted by promoting the stability of an SSBR protein complex in the damaged regions.
  • the most important protein in the SSBR complex is XRCC1, which interacts with a protein promoting 3′ and 5′ end processing of DNA to stabilize the DNA. End processing is generally involved in repairing the damaged 3′ end to a hydroxylated state, and/or the damaged 5′ end to a phosphatic moiety, and after the ends are processed, DNA gap filling takes place.
  • There are two methods for the DNA gap filling that is, short patch repair and long patch repair, and the short patch repair involves insertion of a single base. After DNA gap filling, a DNA ligase promotes end joining.
  • MMR Mismatch Repair
  • MMR works on mismatched DNA bases.
  • Each of an MSH2/6 or MSH2/3 complex has ATPase activity and thus plays an important role in recognizing a mismatch and initiating a repair, and the MSH2/6 primarily recognizes base-base mismatches and identifies one or two base mismatches, but the MSH2/3 primarily recognizes a larger mismatch.
  • BER is a repair method which is active throughout the entire cell cycle, and used to remove a small non-helix-distorting base damaged region from the genome.
  • damaged bases are removed by cleaving an N-glycoside bond joining a base to the phosphate-deoxyribose backbone, and then the phosphodiester backbone is cleaved, thereby generating breaks in single-strand DNA.
  • the broken single strand ends formed thereby were removed, a gap generated due to the removed single strand is filled with a new complementary base, and then an end of the newly-filled complementary base is ligated with the backbone by a DNA ligase, resulting in repair of the damaged DNA.
  • NER is an excision mechanism important for removing large helix-distorting damage from DNA, and when the damage is recognized, a short single-strand DNA segment containing the damaged region is removed, resulting in a single strand gap of 22 to 30 bases.
  • the generated gap is filled with a new complementary base, and an end of the newly filled complementary base is ligated with the backbone by a DNA ligase, resulting in the repair of the damaged DNA.
  • Manipulation or correction of a target gene or nucleic acid may largely lead to effects of knockout, knockdown, and knockin.
  • knockout refers to inactivation of a target gene or nucleic acid
  • activation of a target gene or nucleic acid refers to a state in which transcription and/or translation of a target gene or nucleic acid does not occur. Transcription and translation of a gene causing a disease or a gene having an abnormal function may be inhibited through knockout, resulting in the prevention of protein expression.
  • the target gene or nucleic acid when edited or corrected using a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, the target gene or nucleic acid may be cleaved using the CRISPR complex.
  • the damaged target gene or nucleic acid may be repaired through NHEJ using the CRISPR complex.
  • the damaged target gene or nucleic acid may have indels due to NHEJ, and thereby, specific knockout for the target gene or nucleic acid may be induced.
  • knockdown refers to a decrease in transcription and/or translation of a target gene or nucleic acid or the expression of a target protein.
  • the onset of a disease may be prevented or a disease may be treated by regulating the overexpression of a gene or protein through the knockdown.
  • the CRISPR inactive complex may specifically bind to the target gene or nucleic acid, transcription of the target gene or nucleic acid may be inhibited by the transcription inhibitory activity domain included in the CRISPR inactive complex, thereby inducing knockdown in which expression of the corresponding gene or nucleic acid is inhibited.
  • knockin refers to insertion of a specific nucleic acid or gene into a target gene or nucleic acid
  • the “specific nucleic acid” refers to a gene or nucleic acid of interest to be inserted or expressed.
  • a mutant gene triggering a disease may be utilized in disease treatment by correction to normal or insertion of a normal gene to induce expression of the normal gene through the knockin.
  • the knockin may further need a donor.
  • the target gene or nucleic acid when edited or corrected using a gRNA-CRISPR enzyme complex, that is, a CRISPR complex, the target gene or nucleic acid may be cleaved using the CRISPR complex.
  • the target gene or nucleic acid damaged using the CRISPR complex may be repaired through HDR.
  • a specific nucleic acid may be inserted into the damaged gene or nucleic acid using a donor.
  • donor refers to a nucleic acid sequence that helps HDR-based repair of the damaged gene or nucleic acid, and here, the donor may include a specific nucleic acid.
  • the donor may be a double- or single-stranded nucleic acid.
  • the donor may be present in a linear or circular shape.
  • the donor may include a nucleic acid sequence having homology with a target gene or nucleic acid.
  • the donor may include a nucleic acid sequence having homology with each of base sequences at a location into which a specific nucleic acid is to be inserted, for example, upstream (left) and downstream (right) of a damaged nucleic acid.
  • the specific nucleic acid to be inserted may be located between a nucleic acid sequence having homology with a base sequence downstream of the damaged nucleic acid and a nucleic acid sequence having homology with a base sequence upstream of the damaged nucleic acid.
  • the homologous nucleic acid sequence may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% or more homology or complete homology.

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PCT/KR2017/010576 WO2018117377A1 (ko) 2016-12-22 2017-09-26 Fad2 유전자 조작된 올레인산 강화 식물체 및 이의 제조 방법

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CN111073904B (zh) * 2019-12-10 2023-12-22 北大荒垦丰种业股份有限公司 大豆主栽品种的遗传转化、基因编辑及分析方法
KR20230026234A (ko) 2021-08-17 2023-02-24 세종대학교산학협력단 고올레인산 함유 종자를 생성하는 식물체 및 이의 제조 방법
CN114107370A (zh) * 2021-12-03 2022-03-01 山东舜丰生物科技有限公司 利用Cas12i在大豆中进行基因编辑的方法
KR102588622B1 (ko) * 2022-01-19 2023-10-12 세종대학교산학협력단 지방산 함량이 변형된 종자를 생성하는 대두 형질전환체 제조용 재조합 벡터

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011005998A1 (en) * 2009-07-08 2011-01-13 The Curators Of The University Of Missouri Method to develop high oleic acid soybeans using conventional soybean breeding techniques
WO2015026886A1 (en) * 2013-08-22 2015-02-26 E. I. Du Pont De Nemours And Company Methods for producing genetic modifications in a plant genome without incorporating a selectable transgene marker, and compositions thereof

Family Cites Families (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7566813B2 (en) * 2002-03-21 2009-07-28 Monsanto Technology, L.L.C. Nucleic acid constructs and methods for producing altered seed oil compositions
CN101516181B (zh) * 2006-07-14 2015-09-30 联邦科学技术研究组织 改变水稻的脂肪酸组成
UA118090C2 (uk) * 2012-09-07 2018-11-26 ДАУ АГРОСАЙЄНСІЗ ЕлЕлСі Спосіб інтегрування послідовності нуклеїнової кислоти, що представляє інтерес, у ген fad2 у клітині сої та специфічний для локусу fad2 білок, що зв'язується, здатний індукувати спрямований розрив
JP6517143B2 (ja) * 2012-10-23 2019-05-22 ツールゲン インコーポレイテッド 標的ｄｎａに特異的なガイドｒｎａおよびｃａｓタンパク質コード核酸またはｃａｓタンパク質を含む、標的ｄｎａを切断するための組成物、ならびにその使用
KR101601868B1 (ko) * 2013-02-13 2016-03-09 경북대학교 산학협력단 지방산 함량이 조절된 콩 식물체 및 이의 제조방법
EP2966983B1 (en) * 2013-03-15 2021-07-28 Cellectis Modifying soybean oil composition through targeted knockout of the fad2-1a/1b genes
US20150067922A1 (en) * 2013-05-30 2015-03-05 The Penn State Research Foundation Gene targeting and genetic modification of plants via rna-guided genome editing

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011005998A1 (en) * 2009-07-08 2011-01-13 The Curators Of The University Of Missouri Method to develop high oleic acid soybeans using conventional soybean breeding techniques
WO2015026886A1 (en) * 2013-08-22 2015-02-26 E. I. Du Pont De Nemours And Company Methods for producing genetic modifications in a plant genome without incorporating a selectable transgene marker, and compositions thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Sequence Accession BBN97124, 06 November 2014, sequence alignment attached at the end of the office action. (Year: 2014) *

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BR112019012779A2 (pt) 2020-02-27
AU2017379402B2 (en) 2022-03-10
KR102117110B1 (ko) 2020-05-29
EA202091316A1 (ru) 2020-08-18
US20240102035A1 (en) 2024-03-28
CN110325643A (zh) 2019-10-11
WO2018117377A1 (ko) 2018-06-28
US20220228160A1 (en) 2022-07-21
KR20180073430A (ko) 2018-07-02

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