Regulating editing of sequences of allergen and fatty acid genes in peanut

Abstract

A method for producing a modified peanut plant having at least one improved phenotypic trait is disclosed. The method comprises the step of genetically modifying at least one regulatory gene involving allergen synthesis and / or at least one regulatory gene involving fatty acid metabolism, said improved phenotypic traits being relative to a corresponding peanut plant lacking a genetic modification.

Description

Field of the Invention

[0001] The present disclosure relates to conferring desirable traits in peanut plants. More specifically, the present invention relates to producing peanut plants with reduced allergen content or with differential expression of allergen-related genes and / or with improved fatty acid content by manipulating nucleotide sequences that regulate the expression of regulatory genes controlling allergen synthesis and fatty acid metabolism in peanut. Background of the Invention

[0003] Peanut (Arachis hypogaea L.), also known as groundnut, is a plant from the Fabaceae family. Peanut is of great economic importance, especially in the food industry, and is the second most harvested legume in the world after soybean.

[0004] The peanut plant is an oilseed crop, and its oil content is about 50%. The peanut kernel contains 45% to 55% oil by weight, which is rich in monounsaturated fatty acids (MUFA) and polyunsaturated fatty acids (PUFA). Peanut oil is mainly composed of triglycerides of eight fatty acids. About 80% of these fatty acids are oleic acid (monounsaturated, C18:1) or linoleic acid (polyunsaturated, C18:2). Generally, these two fatty acids are inversely proportional.

[0005] However, harvested peanuts suffer from relatively short shelf-life storage due to their fatty acid content which is sensitive to rapid oxidation.

[0006] Additionally, peanut allergy is one of the most common allergies in the general population and is most common in children under 18 years of age.

[0007] Allergic individuals exposed to peanuts can experience severe, life-threatening reactions. Exposure can occur through ingestion or skin contact, and even small amounts can trigger an allergic reaction.

[0008] Symptoms range from oral allergy syndrome (primarily associated with pollen-related peanut allergies) to severe breathing difficulties, anaphylaxis, and sometimes death.

[0009] Allergies are most likely due to the presence of certain proteins in peanuts, including glycoproteins, arachidyl globulin, conarachidyl globulin, peanut agglutinin, and peanut phospholipase.

[0010] Therefore, the development of allergen-free peanuts or peanuts imparting reduced allergy symptoms is highly desirable to minimize the risk to endangered population segments and to improve the fatty acid content to extend the shelf life of harvested peanuts. SUMMARY OF THE INVENTION

[0012] It is an object of the present invention to disclose a method for producing a modified peanut plant having at least one improved phenotypic trait relative to a corresponding peanut plant lacking the genetic modification, wherein the method comprises the step of genetically modifying at least one regulatory gene involved in allergen synthesis and / or at least one regulatory gene involved in fatty acid metabolism.

[0013] It is another object of the present invention to disclose the method as defined above, wherein said genetic modification is introduced by targeted genome editing.

[0014] It is another object of the present invention to disclose the method as defined in any above, wherein at least one regulatory gene involved in allergen synthesis is chosen from Ara h (Peanut (Arachis hypogaea)) regulatory genes, and at least one regulatory gene involved in fatty acid metabolism is chosen from FAD (Fatty acid desaturase) regulatory genes.

[0015] Another object of the present invention is to disclose a method as defined in any of the above, wherein the at least one regulatory gene involved in allergen synthesis is selected from Arah1 having a genomic nucleotide sequence as shown in SEQ ID NO: 1 or a functional variant thereof, Ara h1.1 having a genomic nucleotide sequence as shown in SEQ ID NO: 341 or a functional variant thereof, Ara h2 having a genomic nucleotide sequence as shown in SEQ ID NO: 686 or a functional variant thereof, Ara h3 having a genomic nucleotide sequence as shown in SEQ ID NO: 908 or a functional variant thereof, Ara h5 having a genomic nucleotide sequence as shown in SEQ ID NO: 1161 or a functional variant thereof, Ara h6 having a genomic nucleotide sequence as shown in SEQ ID NO: 1348 or a functional variant thereof, Ara h7 having a genomic nucleotide sequence as shown in SEQ ID NO: 1514 or a functional variant thereof, Ara h8 having a genomic nucleotide sequence as shown in SEQ ID NO: 1690 or a functional variant thereof, Ara h9 having a genomic nucleotide sequence as shown in SEQ ID NO: 1960 or a functional variant thereof, Ara h10 having a genomic nucleotide sequence as shown in SEQ ID NO: 2208 or a functional variant thereof, Ara h11 having a genomic nucleotide sequence as shown in SEQ ID NO: 2476 or a functional variant thereof, Ara h14 having a genomic nucleotide sequence as shown in SEQ ID NO: 2665 or a functional variant thereof, Ara h15 having a genomic nucleotide sequence as shown in SEQ ID NO: 2854 or a functional variant thereof, or any combination thereof.

[0016] Another object of the present invention is to disclose a method as defined in any of the above, wherein at least one regulatory gene involved in allergen synthesis encodes a polypeptide sequence selected from the group consisting of: Ara h1 having a polypeptide sequence as shown in SEQ ID NO: 2 or a functional variant thereof, Ara h1.1 having a polypeptide sequence as shown in SEQ ID NO: 342 or a functional variant thereof, Ara h2 having a polypeptide sequence as shown in SEQ ID NO: 687 or a functional variant thereof, Ara h3 having a polypeptide sequence as shown in SEQ ID NO: 909 or a functional variant thereof, Ara h5 having a polypeptide sequence as shown in SEQ ID NO: 1162 or a functional variant thereof, Ara h6 having a polypeptide sequence as shown in SEQ ID NO: 1349 or a functional variant thereof, Ara h7 having a polypeptide sequence as shown in SEQ ID NO: 1515 or a functional variant thereof, Ara h8 having a polypeptide sequence as shown in SEQ ID NO: 1691 or a functional variant thereof, Ara h9 having a polypeptide sequence as shown in SEQ ID NO: 1961 or a functional variant thereof, Ara h10 having a polypeptide sequence as shown in SEQ ID NO: 2209 or a functional variant thereof, Ara h11 having a polypeptide sequence as shown in SEQ ID NO: 2477 or a functional variant thereof, Ara h14 having a polypeptide sequence as shown in SEQ ID NO: 2666 or a functional variant thereof, Ara h15 having a polypeptide sequence as shown in SEQ ID NO: 2855 or a functional variant thereof, or any combination thereof.

[0017] It is a further object of the present invention to disclose a method as defined in any of the above, wherein the functional variant has at least 75% sequence identity with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665, SEQ ID NO: 2854, and / or with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 342, SEQ ID NO: 687, SEQ ID NO: 909, SEQ ID NO: 1162, SEQ ID NO: 1349, SEQ ID NO: 1514, SEQ ID NO: 1691, SEQ ID NO: 1961, SEQ ID NO: 2209, SEQ ID NO: 2476, SEQ ID NO: 2666, SEQ ID NO: The Ara h polypeptide sequence of ID NO: 2855 has at least 75% sequence identity, or any combination thereof.

[0018] Another object of the present invention is to disclose a method as defined in any of the above, wherein at least one regulatory gene involved in fatty acid metabolism is selected from FAD2-1 having a genomic nucleotide sequence as shown in SEQ ID NO:3065 or a functional variant thereof, FAD2-2 having a genomic nucleotide sequence as shown in SEQ ID NO:3410 or a functional variant thereof, FAD2-3 having a genomic nucleotide sequence as shown in SEQ ID NO:3809 or a functional variant thereof, FAD2-4 having a genomic nucleotide sequence as shown in SEQ ID NO:4105 or a functional variant thereof, or any combination thereof.

[0019] Another object of the present invention is to disclose a method as defined in any of the above, wherein at least one regulatory gene involved in fatty acid metabolism encodes a polypeptide selected from the group consisting of: FAD2-1 having a polypeptide sequence as shown in SEQ ID NO: 3066 or a functional variant thereof, FAD2-2 having a polypeptide sequence as shown in SEQ ID NO: 3411 or a functional variant thereof, FAD2-3 having a polypeptide sequence as shown in SEQ ID NO: 3810 or a functional variant thereof, FAD2-4 having a polypeptide sequence as shown in SEQ ID NO: 4106 or a functional variant thereof, or any combination thereof.

[0020] It is a further object of the present invention to disclose a method as defined in any of the above, wherein the functional variant has at least 75% sequence identity with a FAD nucleotide sequence selected from the group consisting of SEQ ID NO: 3065, SEQ ID NO: 3410, SEQ ID NO: 3809, SEQ ID NO: 4105, and / or has at least 75% sequence identity with a FAD polypeptide sequence selected from the group consisting of SEQ ID NO: 3066, SEQ ID NO: 3411, SEQ ID NO: 3810, SEQ ID NO: 4106, or any combination thereof.

[0021] It is another object of the present invention to disclose the method as defined in any above, wherein said genetic modification in Ara h (peanut) regulatory genes confers altered allergen levels, different allergen expression, modified or mutated allergen molecule expression and / or lower, reduced, decreased and / or eliminated allergic response.

[0022] It is another object of the present invention to disclose the method as defined in any above, wherein said altered allergen level is lowered, reduced, decreased and / or eliminated.

[0023] It is another object of the present invention to disclose the method as defined in any above, wherein said altered allergen levels, different allergen expression, modified or mutated allergen molecule expression confers lower, reduced, decreased and / or eliminated allergic response.

[0024] It is a further object of the present invention to disclose the method as defined in any above, wherein the genetic modification in the FAD (fatty acid desaturase) regulatory gene confers an altered fatty acid content level or ratio.

[0025] It is another object of the present invention to disclose the method as defined in any above, wherein said altered fatty acid content levels or ratios are characterized by altered fatty acid transport and / or altered fatty acid conversion.

[0026] It is another object of the present invention to disclose the method as defined in any above, wherein said altered fatty acid content level or ratio is selected from oleic acid:linoleic acid ratio of high oleic acid / linoleic acid ratio.

[0027] It is another object of the present invention to disclose the method as defined in any of the above, wherein said at least one phenotypic trait is selected from the group consisting of lower allergic response, reduced allergic response, decreased allergic response, eliminated allergic response, longer shelf life, high oleic acid / linoleic acid ratio, healthier fatty acid profile, and any combination thereof.

[0028] It is another object of the present invention to disclose the method as defined in any above, wherein said peanut plant is an Arah / FAD double mutant.

[0029] It is another object of the present invention to disclose the method as defined in any above, wherein the step of introducing the genetic modification is using mutagenesis, small interfering RNA (siRNA), micro RNA (miRNA), artificial miRNA (amiRNA), DNA gene introgression, endonuclease or any combination thereof.

[0030] It is another object of the present invention to disclose the method as defined in any of the above, wherein the targeted genome editing is selected from the group consisting of meganucleases, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes (CRISPR / Cas), transcription activator-like effector nucleases (TALENs) or zinc finger nucleases (ZFNs) or any combination thereof.

[0031] It is another object of the present invention to disclose the method as defined in any above, wherein the Cas protein is selected from the group including but not limited to Cpf1, Cas9, Cas12, Cas13, Cas14, CasX or CasY.

[0032] It is another object of the present invention to disclose the method as defined in any above, wherein said mutated regulatory gene is a CRISPR / Cas9 induced heritable mutant allele.

[0033] It is another object of the present invention to disclose the method as defined in any above, wherein at least one regulatory gene involved in allergen synthesis and / or fatty acid metabolism is edited by deletion, silencing, downregulation, deregulation, inactivation, knockout, loss of function, reduced expression, at least partial deletion, at least partial silencing, at least partial inactivation, removal, partial removal, duplication, inversion, missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication of the regulatory region or the coding region of the gene or any combination thereof.

[0034] It is another object of the present invention to disclose a method as defined in any above, wherein at least one genetic modification in a regulatory gene involved in allergen synthesis is generated in a plant via the introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NOs: 3-340, SEQ ID NOs: 343-685, SEQ ID NOs: 688-907, SEQ ID NOs: 910-1160, SEQ ID NOs: 1163-1347, SEQ ID NOs: 1350-1513, SEQ ID NOs: 1516-1689, SEQ ID NOs: 1692-1959, SEQ ID NOs: 1962-2207, SEQ ID NOs: 1 : (a) a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NOs: 3-340, SEQ ID NOs: 343-685, SEQ ID NOs: 688-907, SEQ ID NOs: 910-1160, SEQ ID NOs: 1163-1347, SEQ ID NOs: 1350-1513, SEQ ID NOs: 1516-1689, SEQ ID NOs: 1521-1693, SEQ ID NOs: 1523-1694, SEQ ID NOs: 1527-1695, SEQ ID NOs: 1529-1696, SEQ ID NOs: 1530-1697, SEQ ID NOs: 1531-1698, SEQ ID NOs: 1532-1699, SEQ ID NOs: 1533-1691, SEQ ID NOs: 1534-1693, SEQ ID NOs: 1535-1694, SEQ ID NOs: 1536-1695, SEQ ID NOs: 1537-1696, SEQ ID NOs: 1538-1697, SEQ ID NOs: 1539-1698, SEQ ID NOs: 1540-1699, SEQ ID NOs: 1541-1699, SEQ ID NOs: 1543-1693, SEQ ID NOs: 1544-1694, SEQ ID NOs: ID NO: 1692-1959, SEQ ID NO: 1962-2207, SEQ ID NO: 2210-2475, SEQ ID NO: 2478-2664, SEQ ID NO: 2667-2853 and SEQ ID NO: 2856-3064, or any combination thereof.

[0035] It is another object of the present invention to disclose a method as defined in any of the above, wherein at least one genetic modification in a regulatory gene involved in fatty acid metabolism is generated in a plant via the introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NOs: 3067-3409, SEQ ID NOs: 3412-3808, SEQ ID NOs: 3811-4104, SEQ ID NOs: 4107-4400, or any combination thereof, for FAD2-1–FAD2-4, respectively, or (b) a ribonucleoprotein (RNP) complex comprising a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NOs: 3067-3409, SEQ ID NOs: 3412-3808, SEQ ID NOs: 3811-4104, SEQ ID NOs: 4107-4400, or any combination thereof, for FAD2-1–FAD2-4, respectively.

[0036] It is another object of the present invention to disclose the method as defined in any above, wherein the gRNA sequence comprises a 3' NGG protospacer adjacent motif (PAM).

[0037] It is another object of the present invention to disclose a method as defined in any of the above, wherein the method comprises the following steps: (a) selecting at least one regulatory gene and / or promoter of a regulatory gene involved in allergen synthesis and / or fatty acid metabolism of a peanut plant from the peanut plant genome; (b) synthesizing or designing at least one guide RNA (gRNA) comprising a nucleotide sequence complementary to at least one selected peanut gene; (c) transforming a peanut plant cell, cell nucleus, tissue, seed or progeny cell of the peanut plant with a construct comprising (a) a Cas nucleotide sequence operably linked to at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising a Cas protein and at least one gRNA; (d) culturing peanut plant cells, cell nuclei, tissues, seeds or progeny thereof; (e) selecting peanut cells, cell nuclei, tissues, seeds or progeny thereof expressing a desired mutation / genomic modification in at least one selected peanut regulatory gene and / or a promoter of a regulatory gene, (f) regenerating peanut plants from the transformed plant cells, cell nuclei, or tissues, seeds or progeny thereof carrying the desired mutation / genomic modification; and (g) screening the regenerated peanut plants for peanut plants having at least one improved phenotypic trait associated with at least one regulatory gene and / or a promoter of a regulatory gene involved in allergen synthesis and / or at least one regulatory gene and / or a promoter of a regulatory gene involved in fatty acid metabolism.

[0038] It is another object of the present invention to disclose a method as defined in any above, wherein the method comprises administering a nucleic acid composition comprising: a) a first nucleotide sequence encoding a gRNA molecule and b) a second nucleotide sequence encoding a Cas molecule.

[0039] It is another object of the present invention to disclose the method as defined in any above, wherein said CRISPR / Cas system is delivered to the cells by Agrobacterium infiltration, virus-based plasmids for delivery of genome editing molecules and mechanical insertion of DNA (PEG-mediated DNA transformation, biolistics, etc.).

[0040] It is another object of the present invention to disclose the method as defined in any above, wherein the Cas protein is selected from the group including but not limited to Cpf1, Cas9, Cas12, Cas13, Cas14, CasX or CasY.

[0041] It is another object of the present invention to disclose the modified peanut plant, plant part, plant cell, plant progeny, plant tissue, plant seed, plant pod or tissue culture produced by the method as defined in any above.

[0042] It is another object of the present invention to disclose a plant part, plant cell, plant progeny, plant tissue, plant pod, plant seed or tissue culture of a modified peanut plant produced by a method as defined in any above.

[0043] It is another object of the present invention to disclose the tissue culture of regenerable cells, protoplasts or callus obtained from the modified peanut plant produced by the method as defined in any above.

[0044] It is another object of the present invention to disclose a modified peanut plant exhibiting at least one improved phenotypic trait relative to a corresponding peanut plant lacking the mutation, wherein the modified plant comprises a targeted genome editing mutation in at least one regulatory gene involved in allergen synthesis and / or at least one regulatory gene involved in fatty acid metabolism.

[0045] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein said genetic modification is introduced by targeted genome editing.

[0046] It is another object of the present invention to disclose the modified peanut plant as defined in any of the above, wherein at least one regulatory gene involved in allergen synthesis is chosen from Ara h (peanut) regulatory genes, and at least one regulatory gene involved in fatty acid metabolism is chosen from FAD (fatty acid desaturase) regulatory genes.

[0047] Another object of the present invention is to disclose a modified peanut plant as defined in any of the above, wherein at least one regulatory gene involved in allergen synthesis is selected from Ara h1 having a genomic nucleotide sequence as shown in SEQ ID NO: 1 or a functional variant thereof, Arah1.1 having a genomic nucleotide sequence as shown in SEQ ID NO: 341 or a functional variant thereof, Ara h2 having a genomic nucleotide sequence as shown in SEQ ID NO: 686 or a functional variant thereof, Ara h3 having a genomic nucleotide sequence as shown in SEQ ID NO: 908 or a functional variant thereof, Ara h5 having a genomic nucleotide sequence as shown in SEQ ID NO: 1161 or a functional variant thereof, Ara h6 having a genomic nucleotide sequence as shown in SEQ ID NO: 1348 or a functional variant thereof, Ara h7 having a genomic nucleotide sequence as shown in SEQ ID NO: 1514 or a functional variant thereof, Ara h8 having a genomic nucleotide sequence as shown in SEQ ID NO: 1690 or a functional variant thereof, Arah9 having a genomic nucleotide sequence as shown in SEQ ID NO: 1960 or a functional variant thereof, Ara h10 having a genomic nucleotide sequence as shown in SEQ ID NO: 2208 or a functional variant thereof, Ara h11 having a genomic nucleotide sequence as shown in SEQ ID NO: 2476 or a functional variant thereof, Ara h14 having a genomic nucleotide sequence as shown in SEQ ID NO: 2665 or a functional variant thereof, Ara h15 having a genomic nucleotide sequence as shown in SEQ ID NO: 2854 or a functional variant thereof, or any combination thereof.

[0048] Another object of the present invention is to disclose a modified peanut plant as defined in any of the above, wherein at least one regulatory gene involved in allergen synthesis encodes a polypeptide sequence selected from the group consisting of Ara h1 having a polypeptide sequence as shown in SEQ ID NO: 2 or a functional variant thereof, Arah1.1 having a polypeptide sequence as shown in SEQ ID NO: 342 or a functional variant thereof, Ara h2 having a polypeptide sequence as shown in SEQ ID NO: 687 or a functional variant thereof, Ara h3 having a polypeptide sequence as shown in SEQ ID NO: 909 or a functional variant thereof, Ara h5 having a polypeptide sequence as shown in SEQ ID NO: 1162 or a functional variant thereof, Arah6 having a polypeptide sequence as shown in SEQ ID NO: 1349 or a functional variant thereof, Ara h7 having a polypeptide sequence as shown in SEQ ID NO: 1515 or a functional variant thereof, Ara h8 having a polypeptide sequence as shown in SEQ ID NO: 1691 or a functional variant thereof, Ara h9 having a polypeptide sequence as shown in SEQ ID NO: 1961 or a functional variant thereof, Arah10 having a polypeptide sequence as shown in SEQ ID NO: 2209 or a functional variant thereof, Ara h11 having a polypeptide sequence as shown in SEQ ID NO: 2477 or a functional variant thereof, Ara h14 having a polypeptide sequence as shown in SEQ ID NO: 2666 or a functional variant thereof, Ara h15 having a polypeptide sequence as shown in SEQ ID NO: 2855 or a functional variant thereof, or any combination thereof.

[0049] It is another object of the present invention to disclose a modified peanut plant as defined in any of the above, wherein the functional variant has at least 75% sequence identity with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665, SEQ ID NO: 2854, and / or with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 342, SEQ ID NO: 687, SEQ ID NO: 909, SEQ ID NO: 1162, SEQ ID NO: 1349, SEQ ID NO: 1514, SEQ ID NO: 1691, SEQ ID NO: 1961, SEQ ID NO: 2209, SEQ ID NO: 2477, SEQ ID NO: 2665, SEQ ID NO: 2854. NO: 2666, the Ara h polypeptide sequence of SEQ ID NO: 2855 having at least 75% sequence identity, or any combination thereof.

[0050] Another object of the present invention is to disclose a modified peanut plant as defined in any of the above, wherein at least one regulatory gene involved in fatty acid metabolism is selected from FAD2-1 having a genomic nucleotide sequence as shown in SEQ ID NO:3065 or a functional variant thereof, FAD2-2 having a genomic nucleotide sequence as shown in SEQ ID NO:3410 or a functional variant thereof, FAD2-3 having a genomic nucleotide sequence as shown in SEQ ID NO:3809 or a functional variant thereof, FAD2-4 having a genomic nucleotide sequence as shown in SEQ ID NO:4105 or a functional variant thereof, or any combination thereof.

[0051] Another object of the present invention is to disclose a modified peanut plant as defined in any of the above, wherein at least one regulatory gene involved in fatty acid metabolism encodes a polypeptide selected from the group consisting of: FAD2-1 having a polypeptide sequence as shown in SEQ ID NO: 3066 or a functional variant thereof, FAD2-2 having a polypeptide sequence as shown in SEQ ID NO: 3411 or a functional variant thereof, FAD2-3 having a polypeptide sequence as shown in SEQ ID NO: 3810 or a functional variant thereof, FAD2-4 having a polypeptide sequence as shown in SEQ ID NO: 4106 or a functional variant thereof, or any combination thereof.

[0052] Another object of the present invention is to disclose a modified peanut plant as defined in any of the above, wherein the functional variant has at least 75% sequence identity with a FAD nucleotide sequence selected from the group consisting of SEQ ID NO: 3065, SEQ ID NO: 3410, SEQ ID NO: 3809, SEQ ID NO: 4105, and / or has at least 75% sequence identity with a FAD polypeptide sequence selected from the group consisting of SEQ ID NO: 3066, SEQ ID NO: 3411, SEQ ID NO: 3810, SEQ ID NO: 4106, or any combination thereof.

[0053] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein the genetic modification in the Arah (peanut) regulatory gene confers altered allergen levels, different allergen expression, modified or mutated allergen molecule expression and / or lower, reduced, decreased and / or eliminated allergic response.

[0054] It is a further object of the present invention to disclose the modified peanut plant as defined in any above, wherein said altered allergen levels are lowered, reduced, decreased and / or eliminated.

[0055] It is another object of the present invention to disclose the modified peanut plant as defined in any of the above, wherein said altered allergen levels, different allergen expression, modified or mutated allergen molecule expression confers lower, reduced, decreased and / or eliminated allergic response.

[0056] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein the genetic modification in the FAD (fatty acid desaturase) regulatory gene confers an altered fatty acid content level or ratio.

[0057] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein said altered fatty acid content levels or ratios are characterized by altered fatty acid transport and / or altered fatty acid conversion.

[0058] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein said altered fatty acid content level or ratio is selected from the group consisting of oleic acid:linoleic acid ratio with high oleic acid / linoleic acid ratio.

[0059] It is another object of the present invention to disclose the modified peanut plant as defined in any of the above, wherein said at least one phenotypic trait is selected from the group consisting of lower allergic response, reduced allergic response, decreased allergic response, eliminated allergic response, longer shelf life, high oleic acid / linoleic acid ratio, healthier fatty acid profile, and any combination thereof.

[0060] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein said peanut plant is an Ara h / FAD double mutant.

[0061] It is another object of the present invention to disclose the modified peanut plant as defined in any of the above, wherein the step of introducing the genetic modification is using mutagenesis, small interfering RNA (siRNA), micro RNA (miRNA), artificial miRNA (amiRNA), DNA gene introgression, endonuclease or any combination thereof.

[0062] It is another object of the present invention to disclose a modified peanut plant as defined in any of the above, wherein the targeted genome editing is selected from the group consisting of meganucleases, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes (CRISPR / Cas), transcription activator-like effector nucleases (TALENs) or zinc finger nucleases (ZFNs) or any combination thereof.

[0063] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein the Cas protein is selected from the group including but not limited to Cpf1, Cas9, Cas12, Cas13, Cas14, CasX or CasY.

[0064] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein the mutated regulatory gene is a CRISPR / Cas9 induced heritable mutant allele.

[0065] It is another object of the present invention to disclose a modified peanut plant as defined in any of the above, wherein at least one regulatory gene involved in allergen synthesis and / or fatty acid metabolism is edited by deletion, silencing, downregulation, deregulation, inactivation, knockout, loss of function, reduced expression, at least partial deletion, at least partial silencing, at least partial inactivation, removal, partial removal, duplication, inversion, missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication of the regulatory region or the coding region of the gene or any combination thereof.

[0066] It is another object of the present invention to disclose a modified peanut plant as defined in any of the above, wherein at least one genetic modification in a regulatory gene involved in allergen synthesis is generated in the plant via the introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NOs: 3-340, SEQ ID NOs: 343-685, SEQ ID NOs: 688-907, SEQ ID NOs: 910-1160, SEQ ID NOs: 1163-1347, SEQ ID NOs: 1350-1513, SEQ ID NOs: 1516-1689, SEQ ID NOs: 1692-1959, SEQ ID NOs: 1730-1731, SEQ ID NOs: 1731-1733, SEQ ID NOs: 1732-1734, SEQ ID NOs: 1733-1735, SEQ ID NOs: 1734-1736, SEQ ID NOs: 1735-1737, SEQ ID NOs: 1736-1738, SEQ ID NOs: 1737-1739, SEQ ID NOs: 1738-1739, SEQ ID NOs: 1739-1733 : (a) a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NOs: 3-340, SEQ ID NOs: 343-685, SEQ ID NOs: 688-907, SEQ ID NOs: 910-1160, SEQ ID NOs: 1163-1347, SEQ ID NOs: 1350-1513, SEQ ID NOs: 1514-1515, SEQ ID NOs: 1516-1517, SEQ ID NOs: 1517-1519, SEQ ID NOs: 1521-1523, SEQ ID NOs: 1524-1527, SEQ ID NOs: 1525-1529, SEQ ID NOs: 1527-1528, SEQ ID NOs: 1529-1530, SEQ ID NOs: 1531-1531, SEQ ID NOs: 1532-1533, SEQ ID NOs: 1533-1534, SEQ ID NOs: 1534-1535, SEQ ID NOs: 1535-1536, SEQ ID NOs: 1536-1537, SEQ ID NOs: 1537-1538, SEQ ID NOs: 1538-1539, SEQ ID NOs: 1539-1530, SEQ ID NOs: 1531-1533 ID NO: 1516-1689, SEQ ID NO: 1692-1959, SEQ ID NO: 1962-2207, SEQ ID NO: 2210-2475, SEQ ID NO: 2478-2664, SEQ ID NO: 2667-2853 and SEQ ID NO: 2856-3064, or any combination thereof.

[0067] Another object of the present invention is to disclose a modified peanut plant as defined in any of the above, wherein at least one genetic modification in a regulatory gene involved in fatty acid metabolism is generated in the plant via the introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NO: 3067 to SEQ ID NO: 3409, SEQ ID NO: 3412 to SEQ ID NO: 3808, SEQ ID NO: 3811 to SEQ ID NO: 4104, SEQ ID NO: 4107 to SEQ ID NO: 4400 for FAD2-1–FAD2-4, respectively, or (b) a ribonucleoprotein (RNP) complex comprising a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NO: 3067 to SEQ ID NO: 3409, SEQ ID NO: 3412 to SEQ ID NO: 3808, SEQ ID NO: 3811 to SEQ ID NO: 4104, SEQ ID NO: 4107 to SEQ ID NO: 4400 for FAD2-1–FAD2-4, respectively. NO:4107 to SEQ ID NO:4400 or any combination thereof.

[0068] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein the gRNA sequence comprises a 3' NGG protospacer adjacent motif (PAM).

[0069] It is another object of the present invention to disclose the modified peanut plant as defined in any above, wherein said peanut is homozygous for the modified genetic locus.

[0070] It is another object of the present invention to disclose a plant part, plant cell, plant progeny, plant tissue, plant pod, plant seed or tissue culture of a modified peanut plant as defined in any above.

[0071] It is another object of the present invention to disclose the tissue culture of regenerable cells, protoplasts or callus obtained from the modified peanut plant as defined in any above.

[0072] It is another object of the present invention to disclose the harvestable parts of the modified peanut plant as defined in any above, wherein said harvestable parts are preferably shoot biomass pods and / or seeds.

[0073] It is another object of the present invention to disclose products derived from a modified peanut plant as defined in any above and / or derived from harvestable parts of a modified peanut plant as defined in any above.

[0074] It is another object of the present invention to disclose a method for producing a modified peanut plant characterized as conferring a reduced allergic response and / or having an altered fatty acid content, the method comprising the steps of genetically modifying at least one regulatory gene involved in allergen synthesis selected from Ara h (peanut) regulatory genes for conferring a reduced allergic response, and genetically modifying at least one regulatory gene involved in fatty acid metabolism selected from FAD (fatty acid desaturase) regulatory genes for conferring an altered fatty acid content using targeted genome editing.

[0075] Another object of the present invention is to disclose a method as defined in any of the above, wherein the at least one regulatory gene involved in allergen synthesis is selected from Arah1 having a genomic nucleotide sequence as shown in SEQ ID NO: 1 or a functional variant thereof, Ara h1.1 having a genomic nucleotide sequence as shown in SEQ ID NO: 341 or a functional variant thereof, Ara h2 having a genomic nucleotide sequence as shown in SEQ ID NO: 686 or a functional variant thereof, Ara h3 having a genomic nucleotide sequence as shown in SEQ ID NO: 908 or a functional variant thereof, Ara h5 having a genomic nucleotide sequence as shown in SEQ ID NO: 1161 or a functional variant thereof, Ara h6 having a genomic nucleotide sequence as shown in SEQ ID NO: 1348 or a functional variant thereof, Ara h7 having a genomic nucleotide sequence as shown in SEQ ID NO: 1514 or a functional variant thereof, Ara h8 having a genomic nucleotide sequence as shown in SEQ ID NO: 1690 or a functional variant thereof, Ara h9 having a genomic nucleotide sequence as shown in SEQ ID NO: 1960 or a functional variant thereof, Ara h10 having a genomic nucleotide sequence as shown in SEQ ID NO: 2208 or a functional variant thereof, Ara h11 having a genomic nucleotide sequence as shown in SEQ ID NO: 2476 or a functional variant thereof, Ara h14 having a genomic nucleotide sequence as shown in SEQ ID NO: 2665 or a functional variant thereof, Ara h15 having a genomic nucleotide sequence as shown in SEQ ID NO: 2854 or a functional variant thereof, or any combination thereof.

[0076] Another object of the present invention is to disclose a method as defined in any of the above, wherein at least one regulatory gene involved in allergen synthesis encodes a polypeptide sequence selected from the group consisting of: Ara h1 having a polypeptide sequence as shown in SEQ ID NO: 2 or a functional variant thereof, Ara h1.1 having a polypeptide sequence as shown in SEQ ID NO: 342 or a functional variant thereof, Ara h2 having a polypeptide sequence as shown in SEQ ID NO: 687 or a functional variant thereof, Ara h3 having a polypeptide sequence as shown in SEQ ID NO: 909 or a functional variant thereof, Ara h5 having a polypeptide sequence as shown in SEQ ID NO: 1162 or a functional variant thereof, Ara h6 having a polypeptide sequence as shown in SEQ ID NO: 1349 or a functional variant thereof, Ara h7 having a polypeptide sequence as shown in SEQ ID NO: 1515 or a functional variant thereof, Ara h8 having a polypeptide sequence as shown in SEQ ID NO: 1691 or a functional variant thereof, Ara h9 having a polypeptide sequence as shown in SEQ ID NO: 1961 or a functional variant thereof, Ara h10 having a polypeptide sequence as shown in SEQ ID NO: 2209 or a functional variant thereof, Ara h11 having a polypeptide sequence as shown in SEQ ID NO: 2477 or a functional variant thereof, Ara h14 having a polypeptide sequence as shown in SEQ ID NO: 2666 or a functional variant thereof, Ara h15 having a polypeptide sequence as shown in SEQ ID NO: 2855 or a functional variant thereof, or any combination thereof.

[0077] It is a further object of the present invention to disclose a method as defined in any of the above, wherein the functional variant has at least 75% sequence identity with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665, SEQ ID NO: 2854, and / or with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 342, SEQ ID NO: 687, SEQ ID NO: 909, SEQ ID NO: 1162, SEQ ID NO: 1349, SEQ ID NO: 1514, SEQ ID NO: 1691, SEQ ID NO: 1961, SEQ ID NO: 2209, SEQ ID NO: 2476, SEQ ID NO: 2666, SEQ ID NO: The Ara h polypeptide sequence of ID NO: 2855 has at least 75% sequence identity, or any combination thereof.

[0078] Another object of the present invention is to disclose a method as defined in any of the above, wherein at least one regulatory gene involved in fatty acid metabolism is selected from FAD2-1 having a genomic nucleotide sequence as shown in SEQ ID NO:3065 or a functional variant thereof, FAD2-2 having a genomic nucleotide sequence as shown in SEQ ID NO:3410 or a functional variant thereof, FAD2-3 having a genomic nucleotide sequence as shown in SEQ ID NO:3809 or a functional variant thereof, FAD2-4 having a genomic nucleotide sequence as shown in SEQ ID NO:4105 or a functional variant thereof, or any combination thereof.

[0079] Another object of the present invention is to disclose a method as defined in any of the above, wherein at least one regulatory gene involved in fatty acid metabolism encodes a polypeptide selected from the group consisting of: FAD2-1 having a polypeptide sequence as shown in SEQ ID NO: 3066 or a functional variant thereof, FAD2-2 having a polypeptide sequence as shown in SEQ ID NO: 3411 or a functional variant thereof, FAD2-3 having a polypeptide sequence as shown in SEQ ID NO: 3810 or a functional variant thereof, FAD2-4 having a polypeptide sequence as shown in SEQ ID NO: 4106 or a functional variant thereof, or any combination thereof.

[0080] It is a further object of the present invention to disclose a method as defined in any of the above, wherein the functional variant has at least 75% sequence identity with a FAD nucleotide sequence selected from the group consisting of SEQ ID NO: 3065, SEQ ID NO: 3410, SEQ ID NO: 3809, SEQ ID NO: 4105, and / or has at least 75% sequence identity with a FAD polypeptide sequence selected from the group consisting of SEQ ID NO: 3066, SEQ ID NO: 3411, SEQ ID NO: 3810, SEQ ID NO: 4106, or any combination thereof.

[0081] It is another object of the present invention to disclose the method as defined in any above, wherein said genetic modification in Ara h (peanut) regulatory genes confers altered allergen levels, different allergen expression, modified or mutated allergen molecule expression and / or lower, reduced, decreased and / or eliminated allergic response.

[0082] It is another object of the present invention to disclose the method as defined in any above, wherein said altered allergen level is lowered, reduced, decreased and / or eliminated.

[0083] It is another object of the present invention to disclose the method as defined in any above, wherein said altered allergen levels, different allergen expression, modified or mutated allergen molecule expression confers lower, reduced, decreased and / or eliminated allergic response.

[0084] It is a further object of the present invention to disclose the method as defined in any above, wherein the genetic modification in the FAD (fatty acid desaturase) regulatory gene confers an altered fatty acid content level or ratio.

[0085] It is another object of the present invention to disclose the method as defined in any above, wherein said altered fatty acid content levels or ratios are characterized by altered fatty acid transport and / or altered fatty acid conversion.

[0086] It is another object of the present invention to disclose the method as defined in any above, wherein said altered fatty acid content level or ratio is selected from oleic acid:linoleic acid ratio of high oleic acid / linoleic acid ratio.

[0087] It is another object of the present invention to disclose the method as defined in any of the above, wherein said at least one phenotypic trait is selected from the group consisting of lower allergic response, reduced allergic response, decreased allergic response, eliminated allergic response, longer shelf life, high oleic acid / linoleic acid ratio, healthier fatty acid profile, and any combination thereof.

[0088] It is another object of the present invention to disclose the use of (a) the nucleotide sequences as shown in SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665 and SEQ ID NO: 2854 and / or (b) the nucleotide sequences as shown in SEQ ID NO: 3-340, SEQ ID NO: 3-340, SEQ ID NO: 343-685, SEQ ID NO: 688-907, SEQ ID NO: 910-1160, SEQ ID NO: 1163-1347, SEQ ID NO: 2 NO:1350-1513, SEQ ID NO:1516-1689, SEQ ID NO:1692-1959, SEQ ID NO:1962-2207, SEQ ID NO:2210-2475, SEQ ID NO:2478-2664, SEQ ID NO:2667-2853, and the nucleotide sequence shown in at least one of SEQ ID NO:2856-3064, and any combination thereof.

[0089] Another object of the present invention is to disclose the use of the following for generating and / or producing modified peanut plants having at least one genetically modified regulatory gene involved in fatty acid metabolism: (a) the nucleotide sequence as shown in SEQ ID NO:3065, SEQ ID NO:3410, SEQ ID NO:3809 and SEQ ID NO:4105 and / or (b) the nucleotide sequence as shown in at least one of SEQ ID NO:3067-3409, SEQ ID NO:3412-3808, SEQ ID NO:3811-4104, SEQ ID NO:4107-4400 and any combination thereof for targeted genome modification of peanut. DETAILED DESCRIPTION OF THE INVENTION

[0091] In the following detailed description, specific details are set forth to facilitate understanding of the present invention; however, it will be appreciated by those skilled in the art that the present invention can be practiced without these specific details. Unless otherwise defined herein, the technical terms used should have the meanings commonly understood by those of ordinary skill in the art. Further, unless the context otherwise requires, singular terms should include plural terms, and plural terms should include the singular. In addition, well-known methods, procedures, and components have been omitted to highlight the present invention.

[0092] The present invention provides a method for producing a modified peanut plant having at least one improved phenotypic trait relative to a corresponding peanut plant lacking the genetic modification, wherein the method comprises the step of genetically modifying at least one regulatory gene involved in allergen synthesis and / or at least one regulatory gene involved in fatty acid metabolism.

[0093] The present invention further provides modified peanut plants, tissues, cells, cell nuclei, seeds, pods or progeny cells thereof produced by the methods disclosed within the present invention.

[0094] According to one embodiment, the method of the present invention allows the production of peanut plants with altered allergen content and / or peanuts with altered fatty acid content that confer reduced allergic reactions, and in particular, it is through gene editing technology. By this method, the present invention enables the production of peanut plants, tissues, cells, cell nuclei, seeds, pods or their progeny cells that confer reduced allergic reactions (peanuts with reduced allergens, modified allergens or allergen-free) and / or have altered fatty acid levels (more oleic acid than linoleic acid).

[0095] According to a specific embodiment, the peanut plants of the present invention have both reduced or altered allergen content and altered fatty acid content generated by gene editing technology.

[0096] It should be noted herein that allergic individuals exposed to peanuts can experience severe, life-threatening reactions. Exposure can occur through ingestion, skin contact, or inhalation, and even small amounts can trigger an allergic reaction. Contact with the smallest amounts of peanut protein can be life-threatening for particularly sensitive individuals.

[0097] It is within the scope of the present invention that the World Health Organization and International Union of Immunological Societies (WHO / IUIS) Allergen Nomenclature Subcommittee has published and named 16 peanut allergens, named Ara h1 to Ara h17 (Ara h4 was renamed Ara h 3.02). The major peanut allergens are Ara h1, Ara h2, Ara h3 and Ara h6, but Ara h4, Ara h5, Ara h7, Ara h8, Ara h9, Arah10, Ara h11 Ara h12, Ara h13, Ara h14, Ara h15, Ara h16 and Ara h17 have also been described.

[0098] Without wishing to be bound by theory, Ara h1 and Ara h3 are seed storage proteins. Both allergens have two β-barrel cupin domains and are members of the cupin superfamily and cupin 1 protein family. Cupin seed storage proteins serve as a source of amino acids and energy during seed germination. In many plant species, cupins are involved in defense against fungi and insects. It is acknowledged herein that Cupin storage proteins play an important role as allergens in primary peanut allergy. Arah2, Ara h6, and Ara h7 are part of the prolamin superfamily, which includes the 2S albumin and the nonspecific lipid transfer protein (nsLTP) family, two families of low molecular weight proteins. These proteins are heavily disulfide bonded, resulting in the high stability of the allergenic peanut 2S albumins Ara h2, Ara h6, and Ara h7. The 2S albumins in seeds store amino acids, but also have antifungal and antibacterial properties. Ara h 9, Ara h 16 and Ara h 17 are part of the non-specific lipid transfer protein (nsLTP) family. They are found in seeds where they play important roles in resistance to biotic and abiotic stresses, plant growth and development.

[0099] Additionally, Ara h10, Ara h11, Ara h14, and Ara h15 have been identified as oleosins, to which all patients with severe allergic reactions had IgE antibodies. Ara h12 and Ara h13 are peanut defensins (peptides involved in seed and reproductive tissue protection), to which not all patients with severe allergic reactions had IgE antibodies.

[0100] It is further acknowledged herein that Ara h5 is a peanut profilin (highly conserved in higher plants) involved in turnover and remodeling of the actin cytoskeleton. Ara h8 is a peanut Bet v 1 homolog that binds lipids via its hydrophobic cavity, thereby protecting the protein from degradation. Ara h8 has been linked to pollen-related peanut allergy, with most mild or moderate symptoms, and may therefore also be responsible for severe allergic reactions.

[0101] Interestingly, it is noted that although peanut allergens are important for seed storage, development and resistance to biotic and abiotic stresses, they are associated with severe allergic symptoms (e.g. due to their high stability). This problem is addressed and solved by the present invention to produce modified peanut plants having targeted genetic modifications in at least one regulatory gene involved in allergen synthesis and / or at least one regulatory gene involved in fatty acid metabolism.

[0102] With regard to fatty acid metabolism, it is acknowledged herein that the bioconversion of oleic acid to linoleic acid is facilitated by the fatty acid desaturase 2 enzyme, which is further classified into FAD2-1, FAD2-2, FAD2-3, and FAD2-4. It should be noted that the second double bond in the linoleic acid molecule renders it much more sensitive to oxidation. Therefore, harvested peanuts suffer from relatively short shelf life storage due to their fatty acid content, which is sensitive to rapid oxidation. Therefore, this 'oxidative rancidity' generates off-flavors in peanut products and thus limits the shelf life of the product in retail stores.

[0103] The present invention solves this problem by producing improved (genome-edited) peanut plants with a higher oleic acid (which has many health-related advantages) / linoleic acid ratio, thus conferring enhanced shelf life (due to high oleic acid content and low linoleic acid levels).

[0104] According to aspects of the present invention, the oxidative stability of oleic acid is 10 times that of linoleic acid; therefore, the peanuts and their products having a high oleic acid / linoleic acid ratio provided by the present invention have a longer shelf life. In addition, compared with wild-type peanuts, the high oleic peanuts and their products of the present invention are beneficial to the health of the cardiovascular system.

[0105] By knocking out the FAD2 gene using targeted genome editing, the present invention provides peanuts with high oleic acid levels (above 30%, preferably in the range of 30%-80%, more preferably above 45%). These modified peanuts have lower linoleic acid levels (below 40%, preferably in the range of 40%-5%, more preferably below 30%).

[0106] As used herein, the term "about" refers to a measurable value, such as an amount, length of time, and the like, and is intended to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% from the specified value, as such variations are appropriate for performing the disclosed methods.

[0107] As used herein, the term "substantially" means essentially, i.e., basically or fundamentally, and it is generally used to identify or emphasize the basic or essential characteristics or properties of things, or to say that the description is essentially true or accurate. In other aspects, it means related to the most important characteristics of things. In the present disclosure, it can also further mean most of the time, or basically or fundamentally. It may refer to a description that is true or accurate in at least 50%, preferably at least 60%, at least 70%, at least 80%, at least 90% or at least 95% of the cases.

[0108] As used herein, the term "mean" refers to an average value as obtained by measuring a predetermined parameter in each plant in a certain plant population, and calculating the average value based on the number of plants in the population.

[0109] As used herein, the term "similar" means a range of correspondence or similarity of about ±20%, particularly ±15%, more particularly about ±10% and even more particularly about ±5%.

[0110] As used herein, the term "corresponding" generally means similar, analogous, identical, same, same kind, parallel, equivalent, like, or comparable. In a further aspect, it means having or participating in the same relationship (e.g., type or species, kind, degree, position, correspondence, or function). It further means related or accompanying. In some embodiments of the invention, it refers to plants or sibling plants of the same peanut species or variety or variety, or one or more individuals having one or two common parents.

[0111] The term "plant" refers to, but is not limited to, whole plants, grafted plants, ancestors and descendants of plants, or any part or derivative thereof. According to the present invention, a non-limiting list of plant parts includes plant cells, plant tissues, plant protoplasts, plant organs, anthers, flowers, flower parts, scions, fruits, leaves, cuticles, indumentum, stomata, stomatal cells, epidermal cells, canopies, cotyledons, pistils, stems, anthers, pods, seeds, seed coats, cuttings, seed coats, roots, root tips, rootstocks, branches, shoots, buds, meristems, suspension cultures, plant cells or tissue cultures from which plants can be regenerated, plant calli or callus, meristems, meristematic cells, gametophytes, sporophytes, microspores, embryos, immature embryos, pollen, ovules, egg cells, zygotes, etc. According to the present invention, the plant is a peanut plant, tissue, cell, or a descendant cell thereof.

[0112] As used herein, the term "plant cell" refers to, but is not limited to, the structural and physiological unit of a plant, including a protoplast and a cell wall. A plant cell may be in the form of an isolated single cell or a cultured cell, or as a part of a higher-order organized unit such as a plant tissue, a plant organ, or a whole plant.

[0113] As used herein, the term "plant cell culture" refers to, but is not limited to, cultures of plant units, such as protoplasts, regenerable cells, cell cultures, cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development, leaves, roots, root tips, anthers, meristems, microspores, flowers, cotyledons, pistils, fruits, seeds, seed coats, or any combination thereof.

[0114] As used herein, the term "plant material" or "plant part" refers to, but is not limited to, leaves, stems, pods, roots, root tips, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, seed coats, cuttings, cell or tissue cultures, or any other part or product of a plant, or a combination thereof.

[0115] As used herein, the term "plant organ" refers to, but is not limited to, a unique and distinctly structured and differentiated part of a plant, such as a root, stem, leaf, flower, flower bud, or embryo.

[0116] The term "plant tissue" refers to, but is not limited to, a group of plant cells organized into structural and functional units. Any plant tissue in a plant or in culture is included. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, protoplasts, meristematic cells, callus, and any group of plant cells organized into structural and / or functional units. The use of the term in conjunction with or in the absence of any specific type of plant tissue as listed above or otherwise encompassed by this definition is not intended to exclude any other type of plant tissue.

[0117] The term "in planta" in the context of the present invention means within a plant or plant cell. More specifically, it means introducing the CRISPR / Cas complex into a tissue culture comprising several cells, into plant material of a whole plant, or into a single plant cell without introducing foreign genes or mutant genes. It is also used to describe conditions that exist in non-laboratory environments (e.g., in vivo).

[0118] The term "phenotype" refers to, but is not limited to, a characteristic distinguishable from a genetically controlled trait, which may be a physical characteristic. Within the scope, the term phenotype refers to a plant characteristic resulting from gene expression. As used herein, the term "phenotype" is interchangeable with "characteristic" or "phenotypic characteristic" or trait. Thus, the term encompasses any characteristic or phenotypic characteristic or trait of a plant. A non-limiting list of phenotypes including characteristics (e.g., physical characteristics or properties) is selected from conferring lower allergic reactions, reduced allergic reactions, reduced allergic reactions, eliminated allergic reactions, longer shelf life, higher linoleic acid: oleic acid ratios, healthier fatty acid properties. In the context of the present invention, improved phenotypic traits are relative to corresponding peanut plants lacking genetic modification.

[0119] According to a further aspect of the present invention, the modified plant exhibits at least one improved phenotypic trait, wherein the modified plant comprises a targeted genome editing mutation in at least one regulatory gene involved in allergen synthesis and / or at least one regulatory gene involved in fatty acid metabolism, the improved phenotypic trait being relative to a corresponding peanut plant lacking the mutation.

[0120] According to a further aspect of the present invention, the modified plant, plant part, plant cell, plant progeny, plant tissue, or plant seed or pod is from peanut and is produced by the methods disclosed herein, wherein the modified plant does not contain a transgene. According to a further aspect of the present invention, a tissue culture of regenerable cells, protoplasts or callus is obtained from the modified peanut plant produced by the methods disclosed herein.

[0121] According to a further aspect of the present invention, the method disclosed herein comprises a) selecting at least one regulatory gene and / or promoter of a regulatory gene involved in allergen synthesis and / or fatty acid metabolism of a peanut plant from the peanut plant genome; b) synthesizing or designing at least one guide RNA (gRNA) comprising a nucleotide sequence complementary to at least one selected peanut gene; c) transforming a peanut plant cell, cell nucleus, tissue, seed or progeny cell of the peanut plant with a construct comprising (a) a Cas nucleotide sequence operably linked to at least one gRNA, or (b) a Cas protein and at least one gRNA. d) culturing peanut plant cells, cell nuclei, tissues, seeds or progeny thereof; e) selecting peanut cells, cell nuclei, tissues, seeds or progeny thereof expressing the desired mutation / genomic modification in the editing target region, and f) regenerating peanut plants from the transformed plant cells, cell nuclei, or tissues, seeds or progeny thereof carrying the desired mutation / genomic modification; and g) screening the regenerated peanut plants for peanut plants having at least one improved phenotypic trait, the phenotypic trait being related to at least one regulatory gene involved in allergen synthesis and / or at least one regulatory gene involved in fatty acid metabolism.

[0122] The term "peanut" refers to, but is not limited to, a plant member of the Leguminosae family, the genus Arachis, and is widely distributed as a food, and more specifically, to peanut (Arachis hypogaea). Peanut is a plant of the Leguminosae family, a plant family that includes soybeans, green beans, and lentils. Further within the scope of the present invention is peanut (Arachis hypogaea), also known as groundnut, goober, pindar, or monkey nut, a leguminous crop plant grown primarily for its edible seeds. According to some aspects of the present invention, the peanut is an Ara h and / or FAD mutant.

[0123] The term "food allergen" refers to, but is not limited to, food proteins / glycoproteins that trigger allergic reactions via IgE responses in individuals. The most common allergens are found in peanuts, tree nuts, fish and shellfish. These food proteins are taken up by specialized epithelial cells (M cells), transferred to antigen presenting cells such as dendritic cells, and processed into peptide fragments that are presented on the cell surface by class II MHC molecules. The peptides are then presented to naive T helper (Th) cells via MHC / T cell receptor interactions, resulting in Th cell priming and activation. In the context of the present invention, this event initiates the humoral and cellular events associated with peanut allergy. Allergic reactions caused by allergens are generally composed of systemic symptoms such as airway obstruction, urticaria, hypotension and arrhythmias; and local symptoms such as itching, swelling, nausea, vomiting, cramps and diarrhea.

[0124] The term "allergen" refers to, but is not limited to, any protein or protein fragment, molecule or any mixture thereof known to induce, cause, elicit or trigger an allergic reaction, such as an IgE-mediated immune response, in an individual, such as a human. Therefore, allergens are often referred to as antigens. Allergens are typically proteins or polypeptides.

[0125] According to various aspects of the present invention, the food allergen is selected from peanut Ara h allergen. According to further aspects of the present invention, the peanut Ara h allergen is selected from Ara h1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15 or any combination thereof.

[0126] The term "fatty acid" refers to, but is not limited to, carboxylic acids (or organic acids) that often have long aliphatic tails that are saturated or unsaturated. Typically, fatty acids have carbon-carbon bonded chains of at least 8 carbon atoms in length, more often at least 12 carbon atoms in length. Most naturally occurring fatty acids have an even number of carbon atoms because their biosynthesis involves acetic acid with two carbon atoms. Fatty acids can be in a free state (non-esterified) or in an esterified form, such as a triglyceride, diacylglycerol, monoacylglycerol, acyl-CoA (thioester) binding or other binding form. Fatty acids can be esterified to phospholipids, such as phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol or diphosphatidylglycerol forms. Peanut oil is primarily composed of triglycerides of eight fatty acids. In one embodiment, the fatty acid is oleic acid or linoleic acid or palmitic acid. In one embodiment, oleic acid, linoleic acid and palmitic acid account for 90% of the fatty acids in peanuts.

[0127] The term "oleic acid" refers to, but is not limited to, a fatty acid found in animals and plants. Oleic acid is also known as a monounsaturated omega-9 fatty acid. In one embodiment, oleic acid is derived from the peanut plant. In one embodiment, oleic acid is derived from peanut seeds. It is characterized by the formula CH3-(CH2)7-CH=CH-(CH2)7-COOH.

[0128] The term "linoleic acid" refers to, but is not limited to, a polyunsaturated ω-6 fatty acid. In one embodiment, linoleic acid is derived from a peanut plant. In one embodiment, linoleic acid is derived from peanut seeds. The bioconversion of oleic acid to linoleic acid is facilitated by the fatty acid desaturase 2 enzyme (FAD2). FAD2 is localized in the endoplasmic reticulum with cytochrome b5 as an electron donor. Six members of the FAD2 family were cloned from peanut and designated FAD2-1 to FAD2-6. FAD2-1, FAD2-2, FAD2-3, and FAD2-4 encode endoplasmic reticulum ω-3 fatty acid desaturases, while FAD2-5 and FAD2-6 encode chloroplast ω-6 fatty acid desaturases. It is characterized by the formula HOOC(CH2)7CH=CHCH2CH=CH(CH2)4CH3.

[0129] According to various aspects of the present invention, fatty acid metabolism is associated with FAD enzymes. According to further aspects of the present invention, the FAD enzyme is selected from FAD2-1, FAD2-2, FAD2-3, FAD2-4 or any combination thereof.

[0130] According to some embodiments of the invention, the modified peanut plant has an altered fatty acid content level or ratio compared to wild-type peanut or peanut that is not mutated in one or more FAD2 genes, wherein the oleic acid:linoleic acid ratio is a high oleic acid / linoleic acid ratio. According to some embodiments of the invention, oleic acid is in the range of 30% to 80%, such as above 40%, preferably 50% to 90%, and linoleic acid is in the range of 5% to 40%, such as below 25%, preferably 2% to 30%.

[0131] The term "gene" refers to, but is not limited to, the basic unit of heredity. Genes are passed from parents to offspring and contain information required to specify physical and biological traits. Most genes encode specific proteins or protein segments, which have different functions in vivo. Within the scope, as used herein, the term "gene" covers the sequence of the regulatory region (e.g., promoter region) of a gene, and / or the coding sequence (CDS) or region of a gene. Therefore, the present invention provides methods for targeted genome editing of regulatory sequences (e.g., promoters) of genes and the coding sequence of targeted genes.

[0132] According to some aspects of the invention, the gene is a regulatory gene involved in allergen synthesis and / or a regulatory gene involved in fatty acid metabolism. According to further aspects of the invention, the regulatory gene involved in allergen synthesis is selected from Ara h regulatory genes, and the regulatory gene involved in fatty acid metabolism is selected from FAD regulatory genes.

[0133] The term "gene coding" sequence refers to, but is not limited to, the information encoded in a gene, which is used to make RNA molecules that encode proteins or to make non-coding RNA molecules that perform other functions.

[0134] The term "genetic modification" refers to, but is not limited to, genetic manipulation or regulation, which is the direct manipulation of the genes of an organism using biotechnology. It also refers to a group of techniques used to alter the genetic makeup of a cell (including gene transfer within and across species, targeted mutagenesis) and genome editing techniques to produce modified organisms. According to a primary embodiment of the present invention, a modified peanut plant with improved traits is generated using a targeted genome editing mechanism. This technology allows for in-plant modification of specific genes that are associated with and / or control fatty acid conversion and allergen-related proteins in peanut plants.

[0135] According to some aspects of the invention, the genetic modification is selected from altered allergen levels, different allergen expression, altered fatty acid content levels, and any combination thereof. According to further aspects of the invention, the altered allergen levels are lower, reduced, decreased, and / or eliminated. According to further aspects of the invention, the different or modified allergen expression results in lower, reduced, decreased, and / or eliminated allergic reactions. According to further aspects of the invention, the altered fatty acid content levels are characterized by altered fatty acid transport and / or altered fatty acid conversion.

[0136] Mutations within the scope of the present invention, i.e. mutations generated by genome editing in the Ara h / FAD sequence, include but are not limited to deletion, silencing, downregulation, deregulation, inactivation, knockout, loss of function, reduced expression, at least partial deletion, at least partial silencing, at least partial inactivation, removal, partial removal, duplication, inversion, missense mutation, nonsense mutation, insertion, indel, substitution or duplication of the regulatory or coding region of the gene, or any combination thereof.

[0137] As used herein, the term "gene knockdown" refers hereinafter to an experimental technique by which the expression of one or more genes of an organism is reduced. Reduction can occur by genetic modification (i.e., targeted genome editing) or with a reagent (e.g., a short DNA or RNA oligonucleotide with a sequence complementary to a gene or mRNA transcript). Reduced expression can be at the RNA level or protein level. Within the scope of the present invention, the term gene knockout also refers to a loss-of-function mutation and / or a gene knockout mutation, in which the genes of an organism are unable to function or are non-functional.

[0138] As used herein, the term "gene silencing" hereinafter refers to the regulation of gene expression in a cell to prevent the expression of a certain gene. Gene silencing can occur during transcription or translation. In certain aspects of the invention, gene silencing is considered to have a similar meaning to gene knockout. When genes are silenced, their expression is reduced. In contrast, when genes are knocked out, they are not expressed at all. Gene silencing can be considered a gene knockout mechanism because methods used to silence genes, such as RNAi, CRISPR or siRNA, generally reduce the expression of a gene by at least 70%, but do not completely eliminate it.

[0139] As used herein, the term "loss of function mutation" refers to a type of mutation in which the altered gene product lacks the function of the wild-type gene. A synonym for the term included within the scope of the present invention is a null mutation.

[0140] The term "gene editing" refers to, but is not limited to, the addition, removal or change of genetic material at a specific desired position in a genome.Gene editing can be used to correct, introduce or delete a variety of different types of cells and organisms, such as animals, plants and bacteria in almost any DNA sequence.Gene editing is performed using enzymes, particularly nucleases, which have been modified to target specific DNA sequences, where they introduce nicks into the DNA chain, allowing the removal of existing DNA and the insertion of replacement DNA. A non-limiting list of techniques for genome editing is restriction enzymes, zinc finger nucleases, guide editing and programmable addition via site-specific targeting elements (PASTE), zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and meganucleases and clustered regularly spaced short palindromic repeats (CRISPR / Cas9).CRISPR / Cas9 is an RNA-based system, and it works by cutting DNA sequences at specific gene positions and deleting or inserting DNA sequences, which can change the regulation of single base pairs of DNA, large chromosomes or gene expression levels.Different from previous genetic modification techniques that randomly insert genetic material into the host genome, genome editing will insert a targeted site-specific position. As used herein, the term "gene editing" is interchangeable with "genome modification," "gene modification," "genetic modification," "genome editing," or "genomic engineering."

[0141] According to some aspects of the invention, the genetic modification is introduced using mutagenesis, small interfering RNA (siRNA), micro RNA (miRNA), artificial miRNA (amiRNA), DNA gene introgression, endonucleases, or any combination thereof.

[0142] According to some aspects of the present invention, genome editing technology is selected from meganucleases, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-related (Cas) genes (CRISPR / Cas), transcription activator-like effector nucleases (TALENs) or zinc finger nucleases (ZFNs) or any combination thereof. According to a further aspect of the present invention, wherein the Cas protein is selected from a group including but not limited to Cpf1, Cas9, Cas12, Cas13, Cas14, CasX or CasY.

[0143] According to further aspects of the invention, the CRISPR / Cas system is delivered to cells via Agrobacterium infiltration, virus-based plasmids for delivery of genome editing molecules, and mechanical insertion of DNA (PEG-mediated DNA transformation, biolistics, etc.).

[0144] According to a further aspect of the present invention, the mutated regulatory gene is a CRISPR / Cas9-induced heritable mutant allele.

[0145] According to a further aspect of the present invention, the regulatory genes involved in allergen synthesis and / or fatty acid metabolism are edited by deletion, silencing, downregulation, deregulation, inactivation, knockout, loss of function, reduced expression, at least partial deletion, at least partial silencing, at least partial inactivation, removal, partial removal, duplication, inversion, missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication of the regulatory genes involved in allergen synthesis and / or fatty acid metabolism or any combination thereof.

[0146] The term gene editing also includes and encompasses the use of base editing techniques as known to those skilled in the relevant art.

[0147] It is within the scope of the present invention that common methods for such editing use modified nucleases or "molecular scissors". These nucleases produce site-specific double-strand breaks (DSBs) at the desired position in the genome. The induced double-strand breaks are repaired by non-homologous end joining (NHEJ) or homologous recombination (HR), resulting in targeted mutations ('editing'). The family of modified nucleases used by the present invention includes, but is not limited to: meganucleases, zinc finger nucleases (ZFNs), nucleases based on transcription activator-like effectors (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR / Cas9) systems.

[0148] Reference is now made to exemplary genome editing terms used throughout the current disclosure:

[0149] Glossary of Genome Editing Terms

[0150]

[0151]

[0152] According to a specific aspect of the present invention, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-related (Cas) genes are used for the first time to generate genome modifications in targeted genes in peanut plants. It is acknowledged herein that the functions of CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and CRISPR-related (Cas) genes are crucial in adaptive immunity in selected bacteria and archaea, enabling organisms to respond to and eliminate invading genetic material. These repeats were first discovered in Escherichia coli in the 1980s. Without being bound by theory, reference is now made to a class of CRISPR mechanisms in which invading DNA from viruses or plasmids is cut into small fragments and incorporated into a CRISPR locus containing a series of short repeats (about 20 bp). The locus is transcribed, and the transcript is then processed to generate small RNAs (crRNAs, i.e., CRISPR RNAs) that are used to guide effector endonucleases that target invading DNA based on sequence complementarity.

[0153] According to a further aspect of the present invention, Cas proteins, such as Cas9 (also referred to as Csn1), are necessary for gene silencing. Cas9 participates in the processing of crRNA and is responsible for the destruction of target DNA. The function of Cas9 in these two steps depends on the presence of two nuclease domains, i.e., the RuvC-like nuclease domain located at the amino terminus and the HNH-like nuclease domain located in the middle region of the protein. In order to achieve site-specific DNA recognition and cutting, Cas9 is compounded with crRNA and a separate trans-activated crRNA (tracrRNA or trRNA), which is partially complementary to crRNA. TracrRNA is necessary for the maturation of crRNA from the primary transcript encoding multiple crRNA precursors. This occurs in the presence of RNA enzyme III and Cas9.

[0154] Without wishing to be bound by theory, it is acknowledged herein that, during the destruction of target DNA, the HNH and RuvC-like nuclease domains cleave both DNA strands, generating double-strand breaks (DSBs) at sites defined by a 20-nucleotide target sequence within the associated crRNA transcript. The HNH domain cleaves the complementary strand, while the RuvC domain cleaves the non-complementary strand.

[0155] It is further noted that the double-stranded endonuclease activity of Cas9 also requires a short conserved sequence (2–5 nt), called the protospacer adjacent motif (PAM), immediately following the 3′-terminal end of the crRNA complementary sequence.

[0156] According to a further aspect of the invention, a two-component system can be used by the present invention to combine trRNA and crRNA into a single synthetic single guide RNA (sgRNA) for guiding targeted genetic alterations.

[0157] Further within the scope are Cas9 nuclease variants including wild-type Cas9, Cas9D10A, and nuclease-deficient Cas9 (dCas9).

[0158] Reference is now made to the CRISPR / Cas9 mechanism of action as described by Xie, Kabin and Yinong Yang. "RNA-guided genome editing inplants using a CRISPR–Cas system." Molecular plant 6.6 (2013): 1975-1983, which schematically presents an example of the CRISPR / Cas9 mechanism. As shown in this publication, the Cas9 endonuclease forms a complex with a chimeric RNA (called a guide RNA or gRNA), replaces the crRNA–transcrRNA heteroduplex, and the gRNA can be programmed to target a specific site. The gRNA-Cas9 should contain at least 15 base pairs (gRNA seed region) without mismatches between the 5' end of the modified gRNA and the targeted genomic site, as well as a motif called a pre-spacer adjacent motif or PAM, which follows the base pairing region in the complementary strand of the targeted DNA. The commonly used Cas9 (SpCas9) from Streptococcus pyogenes recognizes the PAM sequence 5'-NGG-3' (where "N" can be any nucleotide base).

[0159] As used herein, the term "meganuclease" hereinafter refers to an endodeoxyribonuclease characterized by a large recognition site (a double-stranded DNA sequence of 12 to 40 base pairs); as a result, this site generally occurs only once in any given genome. Meganucleases are therefore considered the most specific naturally occurring restriction enzymes.

[0160] As used herein, the term "protospacer adjacent motif" or "PAM" hereinafter refers to a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. The PAM is a component of invading viruses or plasmids, but not a component of the bacterial CRISPR locus. The PAM is an essential targeting component that distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by the nuclease.

[0161] According to some aspects of the invention, the gRNA sequence comprises a 3' NGG pre-spacer adjacent motif (PAM) represented herein for each regulatory gene Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15 and FAD2-1–FAD2-4 in Tables 2-14 and 16-19.

[0162] As used herein, the term "next generation sequencing" or "NGS" hereinafter refers to a large-scale, parallel, high-throughput or deep sequencing technology platform that performs sequencing of millions of small fragments of DNA in parallel. Bioinformatics analysis is used to piece these fragments together by mapping individual reads to a reference genome.

[0163] The term "microRNA" or "miRNA" refers hereinafter to small non-coding RNAs found in most eukaryotes. They are involved in the regulation of gene expression at the post-transcriptional level in a sequence-specific manner. MiRNA is produced by its precursors through the Dicer-dependent small RNA biosynthetic pathway. MiRNA is a candidate for studying gene function using different RNA-based gene silencing techniques. For example, artificial miRNA (amiRNA) targeting one or several genes of interest is a potential tool in functional genomics.

[0164] As used herein, the term "ortholog" hereinafter refers to one of two or more homologous gene sequences found in different species.

[0165] As used herein, the term "functional variant" or "functional variant of a nucleic acid or amino acid sequence" refers to, but is not limited to, a variant of a sequence or a portion of a sequence that retains the biological function of the intact non-variant allele (e.g., Ara h1 or FAD2 allele) and thus has the activity of the gene or protein expressed by Ara h1 or FAD2. Functional variants also include variants of a gene of interest encoding a polypeptide having sequence changes, such as in non-conserved residues, that do not affect the function of the resulting protein. Variants that are substantially identical to the wild-type nucleic acid or amino acid sequence of the allele as shown herein (i.e., having only some sequence changes, such as in non-conserved residues), and are biologically active, are also encompassed.

[0166] As used herein, the term "variety" or "cultivar" means a group of similar plants that can be distinguished from other varieties of the same species by structural characteristics and properties.

[0167] The term "allele" used herein refers to but is not limited to any of one or more alternative or variant forms of a gene or genetic unit at a specific locus, and all of the alleles are related to a trait or characteristic at a specific locus. In the diploid cells of a organism, the allele of a given gene is located at a specific position or locus (multiple loci) on a chromosome. The alternative or variant form of an allele may be the result of a single nucleotide polymorphism, insertion, inversion, translocation or deletion, or the consequence of gene regulation caused by, for example, chemical or structural modification, transcriptional regulation or post-translational modification / regulation. The allele related to a qualitative trait can comprise the alternative or variant form of various genetic units, including those genetic units identical or related to a single gene or multiple genes or their products or even destroying genetic factors or genes controlled by genetic factors, and the genetic factors facilitate the phenotype represented by the locus. According to a further embodiment, the term "allele" is represented in any of one or more alternative forms of a gene at a specific locus. Heterozygous alleles are two different alleles at the same locus. Homozygous alleles are two identical alleles at a specific locus. A wild-type allele is a naturally occurring allele. In the context of the present invention, the term allele refers to the Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Arah9, Ara h10, Ara h11, Ara h14, Ara h15 and FAD2-1–FAD2-4 regulatory genes identified in peanut, i.e., having the sequences of SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665, SEQ ID NO: 2854, SEQ ID NO: 3065, SEQ ID NO: 3410, SEQ ID NO: 3809 and SEQ ID NO: 3810, respectively. Ara h 1, Ara h1.1, Ara h2, Ara h3, Arah5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15 and FAD2-1–FAD2-4 of the genomic nucleotide sequence shown in NO:4105.

[0168] As used herein, the term "locus" (locuses) means one or more specific locations or regions or sites on a chromosome, for example, where a gene or genetic marker element or factor is found. In specific embodiments, such genetic elements contribute to traits.

[0169] As used herein, the term "homozygous" refers to the genetic condition or configuration that exists when two identical or identical alleles are located at a particular locus, but individually placed on corresponding pairs of homologous chromosomes in a cell of a diploid organism.

[0170] In specific embodiments, the peanut plants of the present invention comprise a homozygous configuration of at least one of a mutated Ara h gene (i.e., Ara h1, Arah1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Arah14, Ara h15) and / or a mutated FAD2 gene (i.e., FAD2-1, FAD2-2, FAD2-3, FAD2-4).

[0171] In contrast, as used herein, the term "heterozygous" refers to the genetic state or configuration that exists when two different or dissimilar alleles are located at a particular locus, but are individually placed on corresponding homologous chromosome pairs in a cell of a diploid organism.

[0172] As used herein, the phrase "genetic marker" or "molecular marker" or "biomarker" refers to a feature in the genome of an individual, such as a nucleotide or polynucleotide sequence associated with one or more loci or traits of interest. In some embodiments, depending on the context, the genetic marker is polymorphic in the population of interest, or a locus occupied by a polymorphism. Genetic markers or molecular markers include, for example, single nucleotide polymorphisms (SNPs), insertions / deletions (i.e., insertions and deletions), simple sequence repeats (SSRs), restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAFDs), enzyme-digested amplified polymorphic sequences (CAPS) markers, diversity array technology (DArT) markers, and amplified fragment length polymorphisms (AFLPs) or combinations thereof, and many other examples such as DNA sequences themselves. For example, genetic markers can be used to locate genetic loci containing alleles on chromosomes that contribute to the variability of phenotypic traits. The phrase "genetic marker" or "molecular marker" or "biomarker" can also refer to polynucleotide sequences that are complementary or corresponding to a genomic sequence, such as nucleic acid sequences used as probes or primers.

[0173] As used herein, the term "germplasm" refers to the genotype population of a population or other group of individuals (e.g., species). The term "germplasm" may also refer to plant material; for example, a group of plants that serve as a reservoir for various alleles. Such germplasm genotypes or populations include, for example, plant material that demonstrates genetic superiority for a given environment or geographic region; and plant material with unknown or unproven genetic value (which is not part of an established breeding population and has no known relationship to members of an established breeding population).

[0174] As used herein, the terms "hybrid," "hybrid plant," and "hybrid progeny" refer to individuals produced from genetically different parents (eg, genetically heterozygous or mostly heterozygous individuals).

[0175] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences refers to the residues in the two sequences that are identical when aligned for maximum correspondence over a specified comparison window. When the percentage of sequence identity is used in reference to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which amino acid residues are substituted with other amino acid residues having similar chemical properties (e.g., charge or hydrophobicity) and thus do not change the functional properties of the molecule. The term further refers hereinafter to the amount of characters that are exactly matched between two different sequences. Thus, gaps are not calculated, and the measurement is relative to the shorter of the two sequences.

[0176] Further within the scope, the terms "similarity" and "identity" also refer to local homology that identifies (in nucleotide and / or amino acid sequences) homologous or similar domains. It is recognized that bioinformatics tools such as BLAST, SSEARCH, FASTA and HMMER calculate local sequence alignments that identify the most similar regions between two sequences. For domains found in different sequence environments in different proteins, alignments should be limited to homologous domains because domain homology provides sequence similarity captured in scoring. According to some aspects, the terms similarity or identity further include sequence motifs, which are widely distributed and have or infer nucleotide or amino acid sequence patterns with biological significance. Proteins can have sequence motifs and / or structural motifs, motifs formed by three-dimensional arrangements of amino acids that may not be adjacent.

[0177] As used herein, the term "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" is intended to include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), naturally occurring, mutated, synthetic DNA or RNA molecules, and analogs of DNA or RNA generated using nucleotide analogs. It can be single-stranded or double-stranded. Such nucleic acids or polynucleotides include, but are not limited to, coding sequences of structural genes, antisense sequences, and non-coding regulatory sequences that do not encode mRNA or protein products. These terms also encompass genes. The terms "gene", "allele" or "gene sequence" are widely used to refer to DNA nucleic acids associated with biological functions. Therefore, a gene can include introns and exons as in a genomic sequence, or can only include coding sequences as in a cDNA, and / or can include cDNA in combination with regulatory sequences. Therefore, according to various aspects of the present invention, genomic DNA, cDNA or coding DNA can be used. In one embodiment, the nucleic acid is a cDNA or coding DNA.

[0178] The terms "peptide," "polypeptide," and "protein" are used interchangeably herein and refer to a polymeric form of amino acids of any length linked together by peptide bonds.

[0179] According to other aspects of the invention, a "modified" or "mutant" plant is a plant that has been altered compared to a naturally occurring wild-type (WT) plant. Specifically, the endogenous nucleic acid sequence of each Ara h or FAD2 homolog in peanut (nucleic acid sequence Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15 and / or FAD2-1-FAD2-4) has been altered using mutagenesis and / or genome editing methods as described herein compared to the wild-type sequence. This results in the inactivation of endogenous Ara h and / or FAD2 genes and thus the inactivation of Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Arah10, Ara h11, Ara h14, Ara h15 and / or FAD2-1-FAD2-4 functions. Such plants have an altered phenotype and display improved traits, such as lower allergen concentrations and / or altered fatty acid levels (i.e., more linoleic acid than oleic acid) compared to wild-type plants. Thus, the improved phenotype is conferred by the presence of at least one mutated endogenous Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Arah11, Ara h14, Ara h15 and / or FAD2–FAD2-4 gene in the peanut plant genome that has been specifically targeted using genome editing technology.

[0180] It is further within the scope of the present invention that Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15 and / or FAD2-1–FAD2-4 mutations that downregulate or disrupt the functional expression of wild-type Ara h 1, Ara h1.1, Arah2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15 and / or FAD2-1–FAD2-4 sequences, respectively, can be recessive such that they can be complemented by expression of the wild-type sequences.

[0181] It is further noted that a wild-type peanut plant is a plant that does not have any mutant Ara h 1, Ara h1.1, Arah2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15 and / or FAD2-1–FAD2-4 alleles.

[0182] The main aspects of the present invention relate to targeted mutagenesis methods, specifically genome editing, and exclude embodiments based solely on generating plants by traditional breeding methods. In a further embodiment of the present invention, as described herein, the improved at least one trait is not due to the presence of a transgene. Thus, according to some aspects of the present invention, the peanut plant does not contain a transgene.

[0183] The inventors have generated mutant peanut lines with mutations that inactivate at least one Ara h regulatory gene among Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Arah7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15 and / or at least one FAD regulatory gene among FAD2-1–FAD2-4, which confer heritable improved traits. In this way, functional Ara h and / or FAD2 proteins are not produced. Therefore, the present invention relates to these mutant peanut lines and related methods.

[0184] It is further within the scope of the present invention to breed peanut cultivars with mutant FAD alleles to extend the shelf life of the resulting plants, resulting in peanuts with a healthier fatty acid profile, and higher linoleic acid content compared to oleic acid. According to a further aspect of the present invention, loss of Ara h function results in peanut plants that are completely free of allergens, or have a different allergen expression, thereby reducing allergic reactions in subjects.

[0185] In order to edit multiple genes and stack the resulting allelic variants simultaneously, a kind of option is to assemble several gRNAs by using the multi-gRNA system of Csy4, to edit several genes into a kind of construct.Then construct is transformed into several wild peanut varieties (accession) via appropriate carrier.

[0186] It is further within the scope of the present invention that the peanut Ara h gene, i.e., Ara h1 having a genomic nucleotide sequence as shown in SEQ.ID.NO:1, is targeted using guide RNAs as shown in SEQ ID NO:3 to SEQ ID NO:340; Ara h1.1 having a genomic nucleotide sequence as shown in SEQ.ID.NO:341 is targeted using guide RNAs as shown in SEQ ID NO:343 to SEQ ID NO:685; Ara h2 having a genomic nucleotide sequence as shown in SEQ.ID.NO:686 is targeted using guide RNAs as shown in SEQ ID NO:688 to SEQ ID NO:907; Ara h3 having a genomic nucleotide sequence as shown in SEQ.ID.NO:908 is targeted using guide RNAs as shown in SEQ ID NO:910 to SEQ ID NO:1160; Arah5 having a genomic nucleotide sequence as shown in SEQ.ID.NO:1161 is targeted using guide RNAs as shown in SEQ ID NO:1163 to SEQ ID NO:1164. NO:1347 for targeting; Ara h6 having a genomic nucleotide sequence as shown in SEQ.ID.NO:1348 is targeted using guide RNAs as shown in SEQ ID NO:1350 to SEQ ID NO:1513; Ara h7 having a genomic nucleotide sequence as shown in SEQ.ID.NO:1514 is targeted using guide RNAs as shown in SEQ ID NO:1516 to SEQ ID NO:1689; Ara h8 having a genomic nucleotide sequence as shown in SEQ.ID.NO:1690 is targeted using guide RNAs as shown in SEQ ID NO:1692 to SEQ ID NO:1959; Ara h9 having a genomic nucleotide sequence as shown in SEQ.ID.NO:1960 is targeted using guide RNAs as shown in SEQ ID NO:1962 to SEQ ID NO:2207; Ara h10 having a genomic nucleotide sequence as shown in SEQ.ID NO:2208 is targeted using guide RNAs as shown in SEQ ID NO:2210 to SEQ IDNO:2475 for targeting; Ara h11 having a genomic nucleotide sequence as shown in SEQ.ID.NO:2476 is targeted using guide RNAs as shown in SEQ ID NO:2478 to SEQ ID NO:2664; having a genomic nucleotide sequence as shown in SEQ.ID.Ara h14 having a genomic nucleotide sequence as shown in SEQ ID NO:2665 is targeted using guide RNAs as shown in SEQ ID NO:2667 to SEQ ID NO:2853; and / or Ara h15 having a genomic nucleotide sequence as shown in SEQ.ID.NO:2854 is targeted using guide RNAs as shown in SEQ ID NO:2856 to SEQ ID NO:3064. .

[0187] It is further within the scope of the present invention that the functional variant has at least 75% sequence identity with the Ara h nucleotide sequence SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665, SEQ ID NO: 2854 or any combination thereof.

[0188] It is further within the scope of the present invention that the peanut FAD gene, namely FAD2-1 having a genomic nucleotide sequence as shown in SEQ.ID NO:3065, is targeted using guide RNAs as shown in SEQ ID NO:3067 to SEQ ID NO:3409;

[0189] FAD2-2 having a genomic nucleotide sequence as shown in SEQ.ID.NO:3410 is targeted using guide RNAs as shown in SEQ ID NO:3412 to SEQ ID NO:3808;

[0190] FAD2-3 having a genomic nucleotide sequence as shown in SEQ.ID.NO:3809 is targeted using a guide RNA as shown in SEQ ID NO:3811 to SEQ ID NO:4104; and / or

[0191] FAD2-4 having a genomic nucleotide sequence as shown in SEQ.ID.NO:4105 was targeted using guide RNAs as shown in SEQ ID NO:4107 to SEQ ID NO:4400.

[0192] It is further within the scope of the present invention that the functional variant has at least 75% sequence identity to the FAD nucleotide sequence SEQ ID NO: 3065, SEQ ID NO: 3410, SEQ ID NO: 3809, SEQ ID NO: 4105 or any combination thereof.

[0193] It is further within the scope of the present invention that the regulatory genes involved in allergen synthesis are generated in the plant via the introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NOs: 3-340, SEQ ID NOs: 343-685, SEQ ID NOs: 688-907, SEQ ID NOs: 910-1160, SEQ ID NOs: 1163-1347, SEQ ID NOs: 1350-1513, SEQ ID NOs: 1516-1689, SEQ ID NOs: 1692-1959, SEQ ID NOs: 1962-2207, SEQ ID NOs: 2210-2475, SEQ ID NOs: 2478-2664, SEQ ID NOs: 2667-2853 and SEQ ID NOs for the regulatory genes. NO:2856-3064 or any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NO:3-340, SEQ ID NO:343-685, SEQ ID NO:688-907, SEQ ID NO:910-1160, SEQ ID NO:1163-1347, SEQ ID NO:1350-1513, SEQ ID NO:1516-1689, SEQ ID NO:1692-1959, SEQ ID NO:1962-2207, SEQ ID NO:2210-2475, SEQ ID NO:2478-2664, SEQ ID NO:2667-2853 and SEQ ID NO:2856-3064 or any combination thereof for regulatory genes.

[0194] It is further within the scope of the present invention that regulatory genes involved in fatty acid metabolism are generated in plants via the introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NO: 3067 to SEQ ID NO: 3409, SEQ ID NO: 3412 to SEQ ID NO: 3808, SEQ ID NO: 3811 to SEQ ID NO: 4104, SEQ ID NO: 4107 to SEQ ID NO: 4400, or any combination thereof, for regulatory genes, or (b) a ribonucleoprotein (RNP) complex comprising a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NO: 3067 to SEQ ID NO: 3409, SEQ ID NO: 3412 to SEQ ID NO: 3808, SEQ ID NO: 3811 to SEQ ID NO: 4104, SEQ ID NO: 4107 to SEQ ID NO: 4400, or any combination thereof, for regulatory genes. Example 1

[0195] Production of peanut plants with improved phenotypic traits by targeted genome editing.

[0196] The production of peanut lines having mutated Ara h, Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Arah14, Ara h15 and / or mutated FAD2-1, FAD2-2, FAD2-3, FAD2-4 genes can be achieved by at least one of the following breeding / cultivation schemes:

[0197] Scenario 1:

[0198] · Line stability through self-pollination

[0199] Generation of F6 parental lines

[0200] Genome editing of parental lines

[0201] Crossing edited parental lines to generate F1 hybrid plants

[0202] Scenario 2:

[0203] Identify genes / alleles of interest

[0204] Design gRNA

[0205] Transformation of plants with Cas9+gRNA constructs

[0206] Screening and identifying editing events

[0207] Genome editing of parental lines

[0208] Note that stabilization can be performed by:

[0209] Inducing male flowers to bloom on female plants

[0210] Self-pollination

[0211] According to some embodiments of the invention, line stabilization requires about 6 selfings (6 generations) and is accomplished by the single seed delivery (SSD) method.

[0212] F1 hybrid seed production: Producing new hybrids through crossing between different peanut varieties.

[0213] According to a further aspect of the present invention, shortening the line stability is performed by double haploid (DH). More specifically, the CRISPR-Cas9 system is transformed into microspores to achieve DH homozygous parental lines. Double haploid (DH) is a genotype formed when haploid cells undergo chromosome doubling. The artificial production of double haploids is important in plant breeding. It is acknowledged herein that conventional inbreeding programs require about six generations to achieve roughly complete homozygosity, and double haploids achieve this in one generation.

[0214] It is within the scope of the present invention to develop and provide genetic markers specific for peanut:

[0215] • Genotyping markers - The germplasm used in the present invention was genotyped using molecular markers in order to allow for a more efficient breeding process and to identify Ara h and / or FAD2 editing events.

[0216] It is further within the scope of the present invention to analyze the alleles and genetic variation for the peanut varieties used.

[0217] Reference is now made to the optional stages for producing mutant Ara h and / or FAD2 peanut plants that have been subjected to genome editing:

[0218] Phase 1: Identification of peanut Ara h and FAD2 orthologs.

[0219] Thirteen Ara h orthologs and four FAD2 orthologs have been identified in peanut plants, namely Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Arah11, Ara h14, Ara h15; and FAD2-1, FAD2-2, FAD2-3, FAD2-4. These homologous genes have been sequenced and mapped.

[0220] Peanut Ara h is as shown in SEQ ID NO:1, SEQ ID NO:341, SEQ ID NO:686, SEQ ID NO:908, SEQ ID NO:1161, SEQ ID NO:1348, SEQ ID NO:1514, SEQ ID NO:1690, SEQ ID NO:1960, SEQ ID NO:2208, SEQ ID NO:2476, SEQ ID NO:2665, SEQ ID NO:2854 for Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Arah7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15, respectively; and these genes encode SEQ ID NO:342, SEQ ID NO:687, SEQ ID NO:909, SEQ ID NO:1162, SEQ ID NO:1349, SEQ ID NO:1514, The amino acid sequences shown in SEQ ID NO:1515, SEQ ID NO:1691, SEQ ID NO:1961, SEQ ID NO:2209, SEQ ID NO:2477, SEQ ID NO:2666, and SEQ ID NO:2855.

[0221] The peanut FAD2 gene is as shown in SEQ ID NO:3065, SEQ ID NO:3410, SEQ ID NO:3809, and SEQ ID NO:4105 for FAD2-1, FAD2-2, FAD2-3, and FAD2-4, respectively; and these genes encode the amino acid sequences shown in SEQ ID NO:3066, SEQ ID NO:3411, SEQ ID NO:3810, and SEQ ID NO:4106, respectively.

[0222] Phase 2: Design and synthesize gRNA molecules corresponding to the sequences targeted for editing (i.e., genes Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, Ara h15; and the respective sequences of FAD2-1, FAD2-2, FAD2-3, and FAD2-4). Note that the editing events preferably target unique restriction site sequences to allow for easier screening of plants carrying editing events within their genomes. According to some aspects of the invention, the nucleotide sequence of the gRNA should be fully compatible with the genomic sequence of the target gene. Thus, for example, suitable gRNA molecules should be constructed for different Ara h and / or FAD2 homologs of different peanut varieties.

[0223] Reference is now made to Table 1, which lists the genomic sequence, amino acid sequence, and gRNA targeting gene sequence or the regulatory sequence upstream of the first ATG of each of the Ara h genes Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Ara h6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, and Ara h15.

[0224] Table 1: Genomic sequence, amino acid sequence, and gRNA targeting sequence of each Ara h gene.

[0225] Gene name Genome SEQ ID NO: AA SEQ ID No: gRNA SEQ ID NO: Arah1 1 2 3-340 Arah1.1 341 342 343-685 Arah2 686 687 688-907 Arah3 908 909 910-1160 Arah5 1161 1162 1163-1347 Arah6 1348 1349 1350-1513 Arah7 1514 1515 1516-1689 Arah8 1690 1691 1692-1959 Arah9 1960 1961 1 962-2207 Arah10 2208 2209 2210-2475 Arah11 2476 2477 2478-2664 Arah14 2665 2666 2667-2853 Arah15 2854 2855 2856-3064

[0226] Reference is now made to Table 2-14, which presents the sequences of gRNA molecules targeting Ara h 1, Ara h1.1, Ara h2, Ara h3, Ara h5, Arah6, Ara h7, Ara h8, Ara h9, Ara h10, Ara h11, Ara h14, and Ara h15. The term 'PAM' hereinafter refers to the Protospacer Adjacent Motif, which is a 2-6 base pair DNA sequence that immediately follows the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.

[0227] The gRNA targets the coding sequence of the Ara h gene and the regulatory sequence upstream of the first ATG of the Ara h gene.

[0228] Table 2: Targeting sequences of gRNAs for Ara h1.

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237] Table 3: Targeting sequences of gRNAs for Ara h1.1.

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247] Table 4: Targeting sequences of gRNAs for Ara h2.

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254] Table 5: Targeting sequences of gRNAs for Ara h3.

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262] Table 6 Targeting sequences of gRNAs for Ara h5.

[0263]

[0264]

[0265]

[0266]

[0267]

[0268] Table 7: Targeting sequences of gRNAs for Ara h6.

[0269]

[0270]

[0271]

[0272]

[0273]

[0274] Table 8: Targeting sequences of gRNAs for Ara h7.

[0275]

[0276]

[0277]

[0278]

[0279]

[0280] Table 9: Targeting sequences of gRNAs for Ara h8.

[0281]

[0282]

[0283]

[0284]

[0285]

[0286]

[0287]

[0288] Table 10: Targeting sequences of gRNAs for Ara h9.

[0289]

[0290]

[0291]

[0292]

[0293]

[0294]

[0295] Table 11: Targeting sequences of gRNAs for Ara h10.

[0296]

[0297]

[0298]

[0299]

[0300]

[0301]

[0302]

[0303] Table 12: Targeting sequences of gRNAs for Ara h11.

[0304]

[0305]

[0306]

[0307]

[0308]

[0309]

[0310] Table 13: Targeting sequences of gRNAs for Ara h14.

[0311]

[0312]

[0313]

[0314]

[0315]

[0316] Table 14: Targeting sequences of gRNAs for Ara h15.

[0317]

[0318]

[0319]

[0320]

[0321]

[0322]

[0323] Reference is now made to Table 15, which lists the genomic sequence, amino acid sequence, and gRNA targeting sequence (regulatory sequence and coding sequence upstream of the first ATG) of each FAD2 gene, FAD2-1, FAD2-2, FAD2-3, and FAD2-4.

[0324] Table 15: Genomic sequence, amino acid sequence, and targeting sequence of gRNA for each FAD2 gene.

[0325] Gene name Genome SEQ ID NO: AA SEQ ID NO: gRNA SEQ ID NO: FAD2-1 3065 3066 3067-3409 FAD2-2 3410 3411 3412-3808 FAD2-3 3809 3810 3811-4104 FAD2-4 4105 4106 4107-4400

[0326] Reference is now made to Tables 16-19, which present targeting gRNA molecules for silencing FAD2-1, FAD2-2, FAD2-3, and FAD2-4, respectively. The term 'PAM' refers hereinafter to the protospacer adjacent motif, which is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.

[0327] The gRNA targets the coding sequence of the FAD2 gene and the regulatory sequence upstream of the first ATG of the FAD2 gene.

[0328] Table 16: Targeting sequences of gRNA for FAD2-1.

[0329]

[0330]

[0331]

[0332]

[0333]

[0334]

[0335]

[0336]

[0337]

[0338] Table 17: Targeting sequences of gRNAs for FAD2-2.

[0339]

[0340]

[0341]

[0342]

[0343]

[0344]

[0345]

[0346]

[0347]

[0348]

[0349] Table 18: Targeting sequences of gRNAs for FAD2-3.

[0350]

[0351]

[0352]

[0353]

[0354]

[0355]

[0356]

[0357]

[0358] Table 19: Targeting sequences of gRNAs for FAD2-4.

[0359]

[0360]

[0361]

[0362]

[0363]

[0364]

[0365]

[0366]

[0367] The gRNA molecules have been cloned into suitable vectors and their sequences have been verified. In addition, different Cas9 forms have been analyzed for optimal compatibility between Cas9 protein activity and gRNA molecules in peanut plants.

[0368] The efficiency of the designed gRNA molecules has been verified by transient transformation of peanut tissue culture. Plasmids carrying the gRNA sequence along with the Cas9 gene have been transformed into peanut protoplasts. Protoplast cells have been grown for a short period of time and then analyzed for the presence of genome editing events. Positive constructs have been subjected to the stable transformation protocol established herein into peanut plant tissues for the generation of peanut plants with genome editing in the Ara h and / or FAD2 genes.

[0369] Phase 3: Transformation of peanut plants using Agrobacterium or biolistics (gene gun) methods. For Agrobacterium and biolistics, a DNA plasmid carrying (Cas9 + gene-specific gRNA) can be used. A vector containing a selection marker, the Cas9 gene, and the relevant gene-specific gRNA is constructed. For biolistics, a ribonucleoprotein (RNP) complex carrying (Cas9 protein + gene-specific gRNA) is used. The RNP complex is created by mixing the Cas9 protein with the relevant gene-specific gRNA.

[0370] According to some embodiments of the invention, transformation of various peanut tissues is performed using particle bombardment of:

[0371] DNA vector

[0372] Ribonucleoprotein complex (RNP)

[0373] According to a further embodiment of the present invention, transformation of various peanut tissues is performed using Agrobacterium (Agrobacterium tumefaciens) by:

[0374] Regeneration-based transformation

[0375] Flower soaking transformation

[0376] Seedling transformation

[0377] The transformation efficiency by Agrobacterium tumefaciens has been compared with the bombardment method by transient GUS transformation experiments.After transformation, GUS staining of the transformants has been performed.

[0378] Screening for CRISPR / Cas9 gene editing events has been performed using at least one of the following assays:

[0379] Restriction fragment length polymorphism (RFLP)

[0380] Next Generation Sequencing (NGS)

[0381] PCR fragment analysis

[0382] Screening based on fluorescent tags

[0383] High-resolution melting curve analysis (HRMA)

[0384] Stage 4: Selection of transformed peanut plants exhibiting Ara h and FAD2 related phenotypes as described above.

[0385] It will be appreciated that embodiments formed from combinations of features shown in separate embodiments are also within the scope of the present invention.

[0386] While certain features of the invention have been illustrated and described herein, modifications, substitutions, and equivalents continue to fall within the scope of this invention.

[0387] The terms used herein are only used for the purpose of describing specific embodiments and are not intended to be limitations of the present invention. As used herein, unless the context clearly indicates otherwise, the singular forms "one", "a kind of" and "the / said" are also intended to include plural forms. It should be further understood that when used in this specification, the term "comprises" or "comprising" specifies the presence of the features, integers, steps, operations, elements, components and / or groups or combinations thereof, but does not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups or combinations thereof. As used herein, the terms "comprises", "comprising", "includes", "including", "having" and their word forms mean "including but not limited to". The term "consisting of" means "including but not limited to".

[0388] As used herein, the term "and / or" includes any and all possible combinations or one or more of the associated listed items, as well as the lack of combination when interpreted in the alternative ("or").

[0389] Unless otherwise defined, all terms (including technical and scientific terms) used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention belongs. It should be further understood that terms such as those defined in commonly used dictionaries should be interpreted as having a meaning consistent with their meaning in the context of the specification and claims, and should not be interpreted in an idealized or overly formal sense unless explicitly defined as such herein. For the sake of brevity and / or clarity, well-known functions or structures may not be described in detail.

[0390] It should be understood that although the terms first, second, etc. may be used herein to describe various elements, components, regions, layers and / or sections, these elements, components, regions, layers and / or sections should not be limited by these terms. On the contrary, these terms are only used to distinguish one element, component, region, layer and / or section from another element, component, region, layer and / or section.

[0391] For the sake of clarity, certain features of the invention described in the context of separate embodiments may also be provided in combination with a single embodiment. Conversely, for the sake of brevity, various features of the invention described in the context of a single embodiment may also be provided separately or in any suitable sub-combination or as appropriate in any other described embodiment of the invention. Certain features described in the context of various embodiments should not be considered essential features of these embodiments unless the embodiment is inoperable without these elements.

[0392] Throughout this application, various embodiments of the present invention may be presented in the form of ranges. It should be understood that the description in the form of ranges is only for convenience and brevity, and should not be interpreted as a hard limit to the scope of the invention. Accordingly, the description of the range should be deemed to have specifically disclosed all possible sub-ranges and each numerical value within the range. For example, a description such as a range from 1 to 6 should be deemed to have specifically disclosed a sub-range, such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6, etc., and each number within the range, such as 1, 2, 3, 4, 5 and 6. This applies regardless of the breadth of the scope.

[0393] Whenever a numerical range is indicated herein, it is intended to include any cited numeral (fractional or integer) within the indicated range. The phrases "a range between / between a first indicated numeral and a second indicated numeral" and "a range from / to a first indicated numeral" are used interchangeably herein and are intended to include the first indicated numeral and the second indicated numeral and all fractions and integers therebetween.

[0394] All publications, patent applications, patents and other references mentioned in the disclosures of these publications are hereby incorporated by reference in their entirety into the present application, so as to more fully describe the prior art to which the present invention relates. In the event of a conflict, the patent specification shall prevail, including definitions. In addition, materials, methods and examples are only illustrative and are not intended to be restrictive. Various publications, disclosed patent applications and disclosed patents are mentioned throughout the present application.

[0395] It will be appreciated by those skilled in the art that the present invention is not limited to what is particularly shown and described above. On the contrary, the scope of the present invention is defined by the appended claims, and includes combinations and sub-combinations of the various features described above and variations and modifications thereof that would occur to those skilled in the art after reading the foregoing specification. Although certain features of the present invention have been illustrated and described herein, many modifications, substitutions, changes and equivalents may occur to those skilled in the art. Therefore, it will be appreciated that the appended claims are intended to encompass all modifications and changes that fall within the true spirit of the present invention. Various embodiments have been presented. These embodiments may each of course include features from other embodiments presented, and embodiments that are not specifically described may include various features described herein.

[0396] References

[0397] Xie, Kabin and Yinong Yang. "RNA-guided genome editing in plants using aCRISPR–Cas system." Molecular plant 6.6(2013):1975-1983.

Claims

1. A method for producing a modified peanut plant having at least one improved phenotypic trait relative to a corresponding peanut plant lacking said genetic modification, wherein said method comprises the step of genetically modifying at least one regulatory gene involved in allergen synthesis and / or at least one regulatory gene involved in fatty acid metabolism.

2. The method of claim 1, wherein the genetic modification is introduced by targeted genome editing.

3. The method according to any one of claims 1 and 2, wherein at least one regulatory gene involved in allergen synthesis is selected from the group consisting of Ara h (peanut (Arachis hypogaea)) regulatory genes, and at least one regulatory gene involved in fatty acid metabolism is selected from the group consisting of FAD (fatty acid desaturase) regulatory genes.

4. The method according to any one of claims 1 to 3, wherein the at least one regulatory gene involved in allergen synthesis is selected from Ara h1 having a genomic nucleotide sequence as shown in SEQ ID NO: 1 or a functional variant thereof, Ara h1.1 having a genomic nucleotide sequence as shown in SEQ ID NO: 341 or a functional variant thereof, Ara h2 having a genomic nucleotide sequence as shown in SEQ ID NO: 686 or a functional variant thereof, Ara h3 having a genomic nucleotide sequence as shown in SEQ ID NO: 908 or a functional variant thereof, Ara h5 having a genomic nucleotide sequence as shown in SEQ ID NO: 1161 or a functional variant thereof, Ara h6 having a genomic nucleotide sequence as shown in SEQ ID NO: 1348 or a functional variant thereof, Ara h7 having a genomic nucleotide sequence as shown in SEQ ID NO: 1514 or a functional variant thereof, Ara h8 having a genomic nucleotide sequence as shown in SEQ ID NO: 1690 or a functional variant thereof, Ara h9 having a genomic nucleotide sequence as shown in SEQ ID NO: 1960 or a functional variant thereof, Ara h10 having a genomic nucleotide sequence as shown in SEQ ID NO: 2208 or a functional variant thereof, Ara h11 having a genomic nucleotide sequence as shown in SEQ ID NO: 2476 or a functional variant thereof, Ara h14 having a genomic nucleotide sequence as shown in SEQ ID NO: 2665 or a functional variant thereof, Ara h15 having a genomic nucleotide sequence as shown in SEQ ID NO: 2854 or a functional variant thereof, or any combination thereof.

5. The method according to any one of claims 1 to 4, wherein the at least one regulatory gene involved in allergen synthesis encodes a polypeptide sequence selected from the group consisting of: Ara h1 having a polypeptide sequence as shown in SEQ ID NO: 2 or a functional variant thereof, Ara h1.1 having a polypeptide sequence as shown in SEQ ID NO: 342 or a functional variant thereof, Ara h2 having a polypeptide sequence as shown in SEQ ID NO: 687 or a functional variant thereof, Ara h3 having a polypeptide sequence as shown in SEQ ID NO: 909 or a functional variant thereof, Ara h5 having a polypeptide sequence as shown in SEQ ID NO: 1162 or a functional variant thereof, Ara h6 having a polypeptide sequence as shown in SEQ ID NO: 1349 or a functional variant thereof, Ara h7 having a polypeptide sequence as shown in SEQ ID NO: 1515 or a functional variant thereof, Ara h8 having a polypeptide sequence as shown in SEQ ID NO: 1691 or a functional variant thereof, Ara h9 having a polypeptide sequence as shown in SEQ ID NO: 1961 or a functional variant thereof, Ara h10 having a polypeptide sequence as shown in SEQ ID NO: 2209 or a functional variant thereof, Ara h11 having a polypeptide sequence as shown in SEQ ID NO: 2477 or a functional variant thereof, Ara h14 having a polypeptide sequence as shown in SEQ ID NO: 2666 or a functional variant thereof, Ara h15 having a polypeptide sequence as shown in SEQ ID NO: 2855 or a functional variant thereof, or any combination thereof.

6. The method according to any one of claims 4 and 5, wherein the functional variant has at least 75% sequence identity with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665, SEQ ID NO: 2854, and / or with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 342, SEQ ID NO: 687, SEQ ID NO: 909, SEQ ID NO: 1162, SEQ ID NO: 1349, SEQ ID NO: 1515, SEQ ID NO: 1691, SEQ ID NO: 1961, SEQ ID NO: 2209, SEQ ID NO: 2477, SEQ ID NO: 2666, SEQ ID The Ara h polypeptide sequence of NO:2855 has at least 75% sequence identity, or any combination thereof.

7. The method according to any one of claims 1 to 3, wherein at least one regulatory gene involved in fatty acid metabolism is selected from FAD2-1 having a genomic nucleotide sequence as shown in SEQ ID NO: 3065 or a functional variant thereof, FAD2-2 having a genomic nucleotide sequence as shown in SEQ ID NO: 3410 or a functional variant thereof, FAD2-3 having a genomic nucleotide sequence as shown in SEQ ID NO: 3809 or a functional variant thereof, FAD2-4 having a genomic nucleotide sequence as shown in SEQ ID NO: 4105 or a functional variant thereof, or any combination thereof.

8. The method according to any one of claims 1 to 3 and 7, wherein at least one regulatory gene involved in fatty acid metabolism encodes a polypeptide selected from the following: FAD2-1 having a polypeptide sequence as shown in SEQ ID NO: 3066 or a functional variant thereof, FAD2-2 having a polypeptide sequence as shown in SEQ ID NO: 3411 or a functional variant thereof, FAD2-3 having a polypeptide sequence as shown in SEQ ID NO: 3810 or a functional variant thereof, FAD2-4 having a polypeptide sequence as shown in SEQ ID NO: 4106 or a functional variant thereof, or any combination thereof.

9. The method according to any one of claims 7 and 8, wherein the functional variant has at least 75% sequence identity with a FAD nucleotide sequence selected from SEQ ID NO: 3065, SEQ ID NO: 3410, SEQ ID NO: 3809, SEQ ID NO: 4105, and / or has at least 75% sequence identity with a FAD polypeptide sequence selected from SEQ ID NO: 3066, SEQ ID NO: 3411, SEQ ID NO: 3810, SEQ ID NO: 4106, or any combination thereof.

10. The method of any one of claims 3 to 6, wherein the genetic modification in the Ara h (peanut) regulatory gene confers altered allergen levels, different allergen expression, expression of modified or mutated allergen molecules and / or lower, reduced, decreased and / or eliminated allergic responses.

11. The method of claim 10, wherein the altered allergen levels are lowered, reduced, decreased and / or eliminated.

12. The method according to any one of claims 10 and 11, wherein the altered allergen levels, different allergen expression, modified or mutated allergen molecule expression confers a lower, reduced, decreased and / or eliminated allergic response.

13. The method according to any one of claims 3 and 7 to 9, wherein the genetic modification in the FAD (fatty acid desaturase) regulatory gene confers an altered fatty acid content level or ratio.

14. The method of claim 13, wherein the altered fatty acid content levels or ratios are characterized by altered fatty acid transport and / or altered fatty acid conversion.

15. The method of claim 13, wherein the altered fatty acid content level or ratio is selected from an oleic acid:linoleic acid ratio of a high oleic acid / linoleic acid ratio.

16. The method of any one of claims 1 to 15, wherein the at least one phenotypic trait is selected from lower allergic reactions, reduced allergic reactions, decreased allergic reactions, eliminated allergic reactions, longer shelf life, high oleic acid / linoleic acid ratio, healthier fatty acid profile, and any combination thereof.

17. The method according to any one of claims 1 to 16, wherein the peanut plant is an Ara h / FAD double mutant.

18. The method of claim 1, wherein the step of introducing the genetic modification is performed using mutagenesis, small interfering RNA (siRNA), micro RNA (miRNA), artificial miRNA (amiRNA), DNA gene introgression, endonucleases, or any combination thereof.

19. The method of claim 2, wherein the targeted genome editing is selected from a meganuclease, a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) gene (CRISPR / Cas), a transcription activator-like effector nuclease (TALEN) or a zinc finger nuclease (ZFN) or any combination thereof.

20. The method of claim 19, wherein the Cas protein is selected from the group consisting of but not limited to Cpf1, Cas9, Cas12, Cas13, Cas14, CasX or CasY.

21. The method of any one of claims 1 to 20, wherein the mutated regulatory gene is a CRISPR / Cas9-induced heritable mutant allele.

22. The method according to any one of claims 1 to 21, wherein at least one regulatory gene involved in allergen synthesis and / or fatty acid metabolism is edited by deletion, silencing, downregulation, deregulation, inactivation, knockout, loss of function, reduced expression, at least partial deletion, at least partial silencing, at least partial inactivation, removal, partial removal, duplication, inversion, missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication of the regulatory region or the coding region of the gene or any combination thereof.

23. The method according to any one of claims 1 to 22, wherein at least one genetic modification in a regulatory gene involved in allergen synthesis is generated in the plant via the introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NOs: 3-340, SEQ ID NOs: 343-685, SEQ ID NOs: 688-907, SEQ ID NOs: 910-1160, SEQ ID NOs: 1163-1347, SEQ ID NOs: 1350-1513, SEQ ID NOs: 1516-1689, SEQ ID NOs: 1692-1959, SEQ ID NOs: 1962-2207, SEQ ID NOs: 1 : (a) a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NOs: 3-340, SEQ ID NOs: 343-685, SEQ ID NOs: 688-907, SEQ ID NOs: 910-1160, SEQ ID NOs: 1163-1347, SEQ ID NOs: 1350-1513, SEQ ID NOs: 1516-1689, SEQ ID NOs: 1521-1693, SEQ ID NOs: 1523-1694, SEQ ID NOs: 1527-1695, SEQ ID NOs: 1529-1696, SEQ ID NOs: 1530-1697, SEQ ID NOs: 1531-1698, SEQ ID NOs: 1532-1699, SEQ ID NOs: 1533-1691, SEQ ID NOs: 1534-1693, SEQ ID NOs: 1535-1694, SEQ ID NOs: 1536-1695, SEQ ID NOs: 1537-1696, SEQ ID NOs: 1538-1697, SEQ ID NOs: 1539-1698, SEQ ID NOs: 1540-1699, SEQ ID NOs: 1541-1699, SEQ ID NOs: 1542-1691, SEQ ID NOs: 1543-1693, SEQ ID NOs: 1544-1694, SEQ ID NOs: ID NO: 1692-1959, SEQ ID NO: 1962-2207, SEQ ID NO: 2210-2475, SEQ ID NO: 2478-2664, SEQ ID NO: 2667-2853 and SEQ ID NO: 2856-3064, or any combination thereof.

24. The method according to any one of claims 1 to 22, wherein at least one genetic modification in a regulatory gene involved in fatty acid metabolism is generated in a plant via the introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NOs: 3067-3409, SEQ ID NOs: 3412-3808, SEQ ID NOs: 3811-4104, SEQ ID NOs: 4107-4400, or any combination thereof, for FAD2-1-FAD2-4, respectively, or (b) a ribonucleoprotein (RNP) complex comprising a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NOs: 3067-3409, SEQ ID NOs: 3412-3808, SEQ ID NOs: 3811-4104, SEQ ID NOs: 4107-4400, or any combination thereof, for FAD2-1-FAD2-4, respectively.

25. The method of any one of claims 23 and 24, wherein the gRNA sequence comprises a 3' NGG pre-spacer adjacent motif (PAM).

26. The method according to any one of claims 1 to 25, wherein the method comprises the following steps: a) selecting at least one regulatory gene and / or a promoter of a regulatory gene involved in allergen synthesis and / or fatty acid metabolism of peanut plants from the peanut plant genome; b) synthesizing or designing at least one guide RNA (gRNA) comprising a nucleotide sequence complementary to at least one selected peanut gene; c) transforming a peanut plant cell, a cell nucleus, a tissue, a seed or a progeny cell of the peanut plant with a construct comprising (a) a Cas nucleotide sequence operably linked to at least one gRNA, or (b) a ribonucleoprotein (RNP) complex comprising a Cas protein and at least one gRNA; d) culturing the peanut plant cell, cell nucleus, tissue, seed or progeny cell thereof; e) selecting peanut cells, cell nuclei, tissues, seeds or progeny cells thereof that express a desired mutation / genomic modification in at least one selected peanut regulatory gene and / or promoter of a regulatory gene, and f) regenerating peanut plants from transformed plant cells, cell nuclei, or tissues, seeds, or plant progeny cells thereof, carrying the desired mutation / genomic modification; and g) screening the regenerated peanut plants for peanut plants having at least one improved phenotypic trait associated with at least one regulatory gene and / or promoter of a regulatory gene involved in allergen synthesis and / or at least one regulatory gene and / or promoter of a regulatory gene involved in fatty acid metabolism.

27. The method of claim 26, wherein the method comprises administering a nucleic acid composition comprising: a) a first nucleotide sequence encoding a gRNA molecule and b) a second nucleotide sequence encoding a Cas molecule.

28. The method of any one of claims 26 and 27, wherein the CRISPR / Cas system is delivered to cells by Agrobacterium infiltration, virus-based plasmids for delivery of genome editing molecules, and mechanical insertion of DNA (PEG-mediated DNA transformation, biolistics, etc.).

29. The method according to any one of claims 23 to 25, wherein the Cas protein is selected from the group including but not limited to Cpf1, Cas9, Cas12, Cas13, Cas14, CasX or CasY.

30. A modified peanut plant, plant part, plant cell, plant progeny, plant tissue, plant seed, plant pod or tissue culture produced by the method of any one of claims 1 to 29.

31. A plant part, plant cell, plant progeny, plant tissue, plant pod, plant seed or tissue culture of a modified peanut plant produced by the method of any one of claims 1 to 29.

32. A tissue culture of regenerable cells, protoplasts or callus obtained from a modified peanut plant produced by the method of any one of claims 1 to 29.

33. A modified peanut plant exhibiting at least one improved phenotypic trait relative to a corresponding peanut plant lacking said mutation, wherein said modified plant comprises a targeted genome editing mutation in at least one regulatory gene involved in allergen synthesis and / or at least one regulatory gene involved in fatty acid metabolism.

34. The modified peanut of claim 33, wherein the genetic modification is introduced by targeted genome editing.

35. The modified peanut according to any one of claims 33 and 34, wherein at least one regulatory gene involved in allergen synthesis is selected from the group consisting of Ara h (peanut) regulatory genes, and at least one regulatory gene involved in fatty acid metabolism is selected from the group consisting of FAD (fatty acid desaturase) regulatory genes.

36. The modified peanut according to any one of claims 33 to 35, wherein the at least one regulatory gene involved in allergen synthesis is selected from Ara h1 having a genomic nucleotide sequence as shown in SEQ ID NO: 1 or a functional variant thereof, Ara h1.1 having a genomic nucleotide sequence as shown in SEQ ID NO: 341 or a functional variant thereof, Ara h2 having a genomic nucleotide sequence as shown in SEQ ID NO: 686 or a functional variant thereof, Ara h3 having a genomic nucleotide sequence as shown in SEQ ID NO: 908 or a functional variant thereof, Ara h5 having a genomic nucleotide sequence as shown in SEQ ID NO: 1161 or a functional variant thereof, Ara h6 having a genomic nucleotide sequence as shown in SEQ ID NO: 1348 or a functional variant thereof, Ara h7 having a genomic nucleotide sequence as shown in SEQ ID NO: 1514 or a functional variant thereof, Ara h8 having a genomic nucleotide sequence as shown in SEQ ID NO: 1690 or a functional variant thereof, Ara h9 having a genomic nucleotide sequence as shown in SEQ ID NO: 1960 or a functional variant thereof, Ara h10 having a genomic nucleotide sequence as shown in SEQ ID NO: 2208 or a functional variant thereof, Ara h11 having a genomic nucleotide sequence as shown in SEQ ID NO: 2476 or a functional variant thereof, Ara h14 having a genomic nucleotide sequence as shown in SEQ ID NO: 2665 or a functional variant thereof, Ara h15 having a genomic nucleotide sequence as shown in SEQ ID NO: 2854 or a functional variant thereof, or any combination thereof.

37. The modified peanut according to any one of claims 33 to 36, wherein the at least one regulatory gene involved in allergen synthesis encodes a polypeptide sequence selected from the group consisting of: Ara h1 having a polypeptide sequence as shown in SEQ ID NO: 2 or a functional variant thereof, Ara h1.1 having a polypeptide sequence as shown in SEQ ID NO: 342 or a functional variant thereof, Ara h2 having a polypeptide sequence as shown in SEQ ID NO: 687 or a functional variant thereof, Ara h3 having a polypeptide sequence as shown in SEQ ID NO: 909 or a functional variant thereof, Ara h5 having a polypeptide sequence as shown in SEQ ID NO: 1162 or a functional variant thereof, Ara h6 having a polypeptide sequence as shown in SEQ ID NO: 1349 or a functional variant thereof, Ara h7 having a polypeptide sequence as shown in SEQ ID NO: 1515 or a functional variant thereof, Ara h8 having a polypeptide sequence as shown in SEQ ID NO: 1691 or a functional variant thereof, Ara h9 having a polypeptide sequence as shown in SEQ ID NO: 1961 or a functional variant thereof, Ara h10 having a polypeptide sequence as shown in SEQ ID NO: 2209 or a functional variant thereof, Ara h11 having a polypeptide sequence as shown in SEQ ID NO: 2477 or a functional variant thereof, Ara h14 having a polypeptide sequence as shown in SEQ ID NO: 2666 or a functional variant thereof, Ara h15 having a polypeptide sequence as shown in SEQ ID NO: 2855 or a functional variant thereof, or any combination thereof.

38. The modified peanut according to any one of claims 36 and 37, wherein the functional variant has at least 75% sequence identity with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665, SEQ ID NO: 2854, and / or with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 342, SEQ ID NO: 687, SEQ ID NO: 909, SEQ ID NO: 1162, SEQ ID NO: 1349, SEQ ID NO: 1515, SEQ ID NO: 1691, SEQ ID NO: 1961, SEQ ID NO: 2209, SEQ ID NO: 2477, SEQ ID NO: NO: 2666, the Ara h polypeptide sequence of SEQ ID NO: 2855 having at least 75% sequence identity, or any combination thereof.

39. The modified peanut according to any one of claims 33 to 35, wherein the at least one regulatory gene involved in fatty acid metabolism is selected from FAD2-1 having a genomic nucleotide sequence as shown in SEQ ID NO: 3065 or a functional variant thereof, FAD2-2 having a genomic nucleotide sequence as shown in SEQ ID NO: 3410 or a functional variant thereof, FAD2-3 having a genomic nucleotide sequence as shown in SEQ ID NO: 3809 or a functional variant thereof, FAD2-4 having a genomic nucleotide sequence as shown in SEQ ID NO: 4105 or a functional variant thereof, or any combination thereof.

40. The modified peanut according to any one of claims 33 to 35 and 39, wherein at least one regulatory gene involved in fatty acid metabolism encodes a polypeptide selected from the group consisting of: FAD2-1 having a polypeptide sequence as shown in SEQ ID NO: 3066 or a functional variant thereof, FAD2-2 having a polypeptide sequence as shown in SEQ ID NO: 3411 or a functional variant thereof, FAD2-3 having a polypeptide sequence as shown in SEQ ID NO: 3810 or a functional variant thereof, FAD2-4 having a polypeptide sequence as shown in SEQ ID NO: 4106 or a functional variant thereof, or any combination thereof.

41. The modified peanut according to any one of claims 39 and 40, wherein the functional variant has at least 75% sequence identity with a FAD nucleotide sequence selected from the group consisting of SEQ ID NO: 3065, SEQ ID NO: 3410, SEQ ID NO: 3809, SEQ ID NO: 4105, and / or has at least 75% sequence identity with a FAD polypeptide sequence selected from the group consisting of SEQ ID NO: 3066, SEQ ID NO: 3411, SEQ ID NO: 3810, SEQ ID NO: 4106, or any combination thereof.

42. The modified peanut of any one of claims 35 to 38, wherein the genetic modification in the Ara h (peanut) regulatory gene confers altered allergen levels, different allergen expression, expression of modified or mutated allergen molecules, and / or lower, reduced, decreased and / or eliminated allergic responses.

43. The modified peanut of claim 42, wherein the altered allergen levels are lowered, reduced, decreased and / or eliminated.

44. The modified peanut according to any one of claims 42 and 43, wherein the altered allergen levels, different allergen expression, modified or mutated allergen molecule expression confers lower, reduced, decreased and / or eliminated allergic response.

45. The modified peanut according to any one of claims 35 and 39 to 41, wherein the genetic modification in the FAD (fatty acid desaturase) regulatory gene confers an altered fatty acid content level or ratio.

46. ​​The modified peanut of claim 45, wherein the altered fatty acid content levels or ratios are characterized by altered fatty acid transport and / or altered fatty acid conversion.

47. The modified peanut of claim 45, wherein the altered fatty acid content level or ratio is selected from a high oleic acid / linoleic acid ratio of oleic acid:linoleic acid.

48. The modified peanut of any one of claims 33 to 47, wherein the at least one phenotypic trait is selected from lower allergic response, reduced allergic response, decreased allergic response, eliminated allergic response, longer shelf life, high oleic acid / linoleic acid ratio, healthier fatty acid profile, and any combination thereof.

49. The modified peanut of any one of claims 33 to 48, wherein the peanut plant is an Ara h / FAD double mutant.

50. The modified peanut of claim 33, wherein the step of introducing the genetic modification is performed using mutagenesis, small interfering RNA (siRNA), micro RNA (miRNA), artificial miRNA (amiRNA), DNA introgression, endonucleases, or any combination thereof.

51. The modified peanut of claim 34, wherein the targeted genome editing is selected from a meganuclease, a clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) gene (CRISPR / Cas), a transcription activator-like effector nuclease (TALEN), or a zinc finger nuclease (ZFN), or any combination thereof.

52. The modified peanut according to claim 51, wherein the Cas protein is selected from the group including but not limited to Cpf1, Cas9, Cas12, Cas13, Cas14, CasX or CasY.

53. The modified peanut of any one of claims 33 to 52, wherein the mutated regulatory gene is a CRISPR / Cas9-induced heritable mutant allele.

54. The modified peanut of any one of claims 33 to 53, wherein at least one regulatory gene involved in allergen synthesis and / or fatty acid metabolism is edited by deletion, silencing, downregulation, deregulation, inactivation, knockout, loss of function, reduced expression, at least partial deletion, at least partial silencing, at least partial inactivation, removal, partial removal, duplication, inversion, missense mutation, nonsense mutation, insertion, deletion, indel, substitution or duplication of the regulatory or coding region of the gene, or any combination thereof.

55. The modified peanut according to any one of claims 33 to 54, wherein at least one genetic modification in a regulatory gene involved in allergen synthesis is generated in the plant via introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NOs: 3-340, SEQ ID NOs: 343-685, SEQ ID NOs: 688-907, SEQ ID NOs: 910-1160, SEQ ID NOs: 1163-1347, SEQ ID NOs: 1350-1513, SEQ ID NOs: 1516-1689, SEQ ID NOs: 1692-1959, SEQ ID NOs: 1930-1960, SEQ ID NOs: 1961-1963, SEQ ID NOs: 1970-1971, SEQ ID NOs: 1971-1973, SEQ ID NOs: 1971-1973, SEQ ID NOs: 1970-1973, SEQ ID NOs: 1970-1973, SEQ ID NOs: 1970-1973, SEQ ID NOs: 1970-1973, SEQ ID NOs: 1970-1973 NO: 1962-2207, SEQ ID NO: 2210-2475, SEQ ID NO: 2478-2664, SEQ ID NO: 2667-2853 and SEQ ID NO: 2856-3064 or any combination thereof, or (b) a ribonucleoprotein (RNP) complex comprising a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NO: 3-340, SEQ ID NO: 343-685, SEQ ID NO: 688-907, SEQ ID NO: 910-1160, SEQ ID NO: 1163-1347, SEQ ID NO: 1350-1513, SEQ ID NO: 1513-1514, SEQ ID NO: 1515-1516, SEQ ID NO: 1517-1518, SEQ ID NO: 1519-1520, SEQ ID NO: 1521-1523, SEQ ID NO: 1524-1525 ID NO: 1516-1689, SEQ ID NO: 1692-1959, SEQ ID NO: 1962-2207, SEQ ID NO: 2210-2475, SEQ ID NO: 2478-2664, SEQ ID NO: 2667-2853 and SEQ ID NO: 2856-3064 or any combination thereof.

56. The modified peanut according to any one of claims 33 to 54, wherein at least one genetic modification in a regulatory gene involved in fatty acid metabolism is generated in the plant via the introduction of a construct comprising (a) a Cas DNA and a guide RNA (gRNA) sequence selected from the group consisting of SEQ ID NO: 3067 to SEQ ID NO: 3409, SEQ ID NO: 3412 to SEQ ID NO: 3808, SEQ ID NO: 3811 to SEQ ID NO: 4104, SEQ ID NO: 4107 to SEQ ID NO: 4400 for FAD2-1 - FAD2-4, respectively, or (b) a ribonucleoprotein (RNP) complex comprising a Cas protein and a gRNA sequence selected from the group consisting of SEQ ID NO: 3067 to SEQ ID NO: 3409, SEQ ID NO: 3412 to SEQ ID NO: 3808, SEQ ID NO: 3811 to SEQ ID NO: 4104, SEQ ID NO: 4107 to SEQ ID NO: 4400 for FAD2-1 - FAD2-4, respectively. NO:4107 to SEQ ID NO:4400 or any combination thereof.

57. The modified peanut of any one of claims 55 and 56, wherein the gRNA sequence comprises a 3' NGG protospacer adjacent motif (PAM).

58. The modified peanut of any one of claims 33 to 57, wherein the peanut is homozygous for the modified genetic locus.

59. A plant part, plant cell, plant progeny, plant tissue, plant pod, plant seed or tissue culture of a modified peanut plant according to any one of claims 33 to 58.

60. A tissue culture of regenerable cells, protoplasts or callus obtained from the modified peanut plant according to any one of claims 33 to 58.

61. Harvestable parts of the modified peanut plant according to any one of claims 33 to 58, wherein the harvestable parts are preferably shoot biomass pods and / or seeds.

62. Products derived from a modified peanut plant according to any one of claims 33 to 58 and / or from harvestable parts of a modified peanut plant according to any one of claims 33 to 58.

63. A method for producing a modified peanut plant characterized as conferring reduced allergic response and / or having altered fatty acid content, the method comprising the steps of: Targeted genome editing is used to genetically modify at least one regulatory gene involved in allergen synthesis selected from the group consisting of Ara h (peanut) regulatory genes for conferring reduced allergic responses, and at least one regulatory gene involved in fatty acid metabolism selected from the group consisting of FAD (fatty acid desaturase) regulatory genes for conferring altered fatty acid content.

64. The method according to claim 63, wherein the at least one regulatory gene involved in allergen synthesis is selected from Ara h1 having a genomic nucleotide sequence as shown in SEQ ID NO: 1 or a functional variant thereof, Ara h1.1 having a genomic nucleotide sequence as shown in SEQ ID NO: 341 or a functional variant thereof, Ara h2 having a genomic nucleotide sequence as shown in SEQ ID NO: 686 or a functional variant thereof, Ara h3 having a genomic nucleotide sequence as shown in SEQ ID NO: 908 or a functional variant thereof, Ara h5 having a genomic nucleotide sequence as shown in SEQ ID NO: 1161 or a functional variant thereof, Ara h6 having a genomic nucleotide sequence as shown in SEQ ID NO: 1348 or a functional variant thereof, Ara h7 having a genomic nucleotide sequence as shown in SEQ ID NO: 1514 or a functional variant thereof, Ara h8 having a genomic nucleotide sequence as shown in SEQ ID NO: 1690 or a functional variant thereof, Ara h9 having a genomic nucleotide sequence as shown in SEQ ID NO: 1960 or a functional variant thereof, Ara h10 having a genomic nucleotide sequence as shown in SEQ ID NO: 2208 or a functional variant thereof, Ara h11 having a genomic nucleotide sequence as shown in SEQ ID NO: 2476 or a functional variant thereof, Ara h14 having a genomic nucleotide sequence as shown in SEQ ID NO: 2665 or a functional variant thereof, Ara h15 having a genomic nucleotide sequence as shown in SEQ ID NO: 2854 or a functional variant thereof, or any combination thereof.

65. The method according to any one of claims 63 and 64, wherein the at least one regulatory gene involved in allergen synthesis encodes a polypeptide sequence selected from the group consisting of Arah1 having a polypeptide sequence as shown in SEQ ID NO: 2 or a functional variant thereof, Ara h1.1 having a polypeptide sequence as shown in SEQ ID NO: 342 or a functional variant thereof, Ara h2 having a polypeptide sequence as shown in SEQ ID NO: 687 or a functional variant thereof, Ara h3 having a polypeptide sequence as shown in SEQ ID NO: 909 or a functional variant thereof, Ara h5 having a polypeptide sequence as shown in SEQ ID NO: 1162 or a functional variant thereof, Ara h6 having a polypeptide sequence as shown in SEQ ID NO: 1349 or a functional variant thereof, Ara h7 having a polypeptide sequence as shown in SEQ ID NO: 1515 or a functional variant thereof, Ara h8 having a polypeptide sequence as shown in SEQ ID NO: 1691 or a functional variant thereof, Ara h9 having a polypeptide sequence as shown in SEQ ID NO: 1961 or a functional variant thereof, Ara h10 having a polypeptide sequence as shown in SEQ ID NO: 2209 or a functional variant thereof, Ara h11 having a polypeptide sequence as shown in SEQ ID NO: 2477 or a functional variant thereof, Ara h14 having a polypeptide sequence as shown in SEQ ID NO: 2666 or a functional variant thereof, Ara h15 having a polypeptide sequence as shown in SEQ ID NO: 2855 or a functional variant thereof, or any combination thereof.

66. The method according to any one of claims 64 and 65, wherein the functional variant has at least 75% sequence identity with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665, SEQ ID NO: 2854, and / or with an Ara h nucleotide sequence selected from the group consisting of SEQ ID NO: 342, SEQ ID NO: 687, SEQ ID NO: 909, SEQ ID NO: 1162, SEQ ID NO: 1349, SEQ ID NO: 1515, SEQ ID NO: 1691, SEQ ID NO: 1961, SEQ ID NO: 2209, SEQ ID NO: 2477, SEQ ID NO: 2666, SEQ ID NO: The Ara h polypeptide sequence of ID NO: 2855 has at least 75% sequence identity, or any combination thereof.

67. The method according to any one of claims 63, wherein at least one regulatory gene involved in fatty acid metabolism is selected from FAD2-1 having a genomic nucleotide sequence as shown in SEQ ID NO: 3065 or a functional variant thereof, FAD2-2 having a genomic nucleotide sequence as shown in SEQ ID NO: 3410 or a functional variant thereof, FAD2-3 having a genomic nucleotide sequence as shown in SEQ ID NO: 3809 or a functional variant thereof, FAD2-4 having a genomic nucleotide sequence as shown in SEQ ID NO: 4105 or a functional variant thereof, or any combination thereof.

68. The method according to any one of claims 63 and 67, wherein at least one regulatory gene involved in fatty acid metabolism encodes a polypeptide selected from the group consisting of: FAD2-1 having a polypeptide sequence as shown in SEQ ID NO: 3066 or a functional variant thereof, FAD2-2 having a polypeptide sequence as shown in SEQ ID NO: 3411 or a functional variant thereof, FAD2-3 having a polypeptide sequence as shown in SEQ ID NO: 3810 or a functional variant thereof, FAD2-4 having a polypeptide sequence as shown in SEQ ID NO: 4106 or a functional variant thereof, or any combination thereof.

69. The method according to any one of claims 67 and 68, wherein the functional variant has at least 75% sequence identity with a FAD nucleotide sequence selected from SEQ ID NO: 3065, SEQ ID NO: 3410, SEQ ID NO: 3809, SEQ ID NO: 4105, and / or has at least 75% sequence identity with a FAD polypeptide sequence selected from SEQ ID NO: 3066, SEQ ID NO: 3411, SEQ ID NO: 3810, SEQ ID NO: 4106, or any combination thereof.

70. The method of any one of claims 63 to 66, wherein the genetic modification in the Ara h (peanut) regulatory gene confers altered allergen levels, different allergen expression, expression of modified or mutated allergen molecules and / or lower, reduced, decreased and / or eliminated allergic responses.

71. The method of claim 70, wherein the altered allergen levels are lowered, reduced, decreased and / or eliminated.

72. The method according to any one of claims 70 and 71, wherein the altered allergen levels, different allergen expression, modified or mutated allergen molecule expression confers a lower, reduced, decreased and / or eliminated allergic response.

73. The method of any one of claims 63 and 67 to 69, wherein the genetic modification in the FAD (fatty acid desaturase) regulatory gene confers an altered fatty acid content level or ratio.

74. The method of claim 73, wherein the altered fatty acid content levels or ratios are characterized by altered fatty acid transport and / or altered fatty acid conversion.

75. The method of claim 73, wherein the altered fatty acid content level or ratio is selected from an oleic acid:linoleic acid ratio of a high oleic acid / linoleic acid ratio.

76. The method of any one of claims 63 to 75, wherein the at least one phenotypic trait is selected from lower allergic reactions, reduced allergic reactions, decreased allergic reactions, eliminated allergic reactions, longer shelf life, high oleic acid / linoleic acid ratio, healthier fatty acid profile, and any combination thereof.

77. Use of the following for generating and / or producing a modified peanut plant having at least one genetically modified regulatory gene involved in allergen synthesis: (a) the nucleotide sequence as shown in SEQ ID NO: 1, SEQ ID NO: 341, SEQ ID NO: 686, SEQ ID NO: 908, SEQ ID NO: 1161, SEQ ID NO: 1348, SEQ ID NO: 1514, SEQ ID NO: 1690, SEQ ID NO: 1960, SEQ ID NO: 2208, SEQ ID NO: 2476, SEQ ID NO: 2665 and SEQ ID NO: 2854 and / or (b) SEQ ID NO: 3-340, SEQ ID NO: 3-340, SEQ ID NO: 343-685, SEQ ID NO: 688-907, SEQ ID NO: 910-1160, SEQ ID NO: 1163-1347, SEQ ID NO: 2 The nucleotide sequence shown in at least one of SEQ ID NO:1350-1513, SEQ ID NO:1516-1689, SEQ ID NO:1692-1959, SEQ ID NO:1962-2207, SEQ ID NO:2210-2475, SEQ ID NO:2478-2664, SEQ ID NO:2667-2853, and SEQ ID NO:2856-3064, and any combination thereof.

78. Use of the following for generating and / or producing a modified peanut plant having at least one genetically modified regulatory gene involved in fatty acid metabolism: (a) the nucleotide sequences as shown in SEQ ID NO:3065, SEQ ID NO:3410, SEQ ID NO:3809 and SEQ ID NO:4105 and / or (b) the nucleotide sequences as shown in at least one of SEQ ID NO:3067-3409, SEQ ID NO:3412-3808, SEQ ID NO:3811-4104, SEQ ID NO:4107-4400 and any combination thereof for targeted genomic modification of peanut.

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