Methods and means for influencing expression of heteroallelic genes or alleles in plants by modification of untranslated regions

Abstract

Compositions and methods are provided for influencing expression of endogenous genes or alleles in plants only in a heteroallelic state, such as in hybrid crops, while retaining unaffected expression of the endogenous genes or alleles in a homozygous state, by editing untranslated regions of the endogenous genes through genomic editing techniques.

Description

Methods and means for influencing expression of heteroallelic genes or alleles in plants by modification of untranslated regions. FIELD

[0001] The present disclosure relates to compositions and methods for influencing expression of endogenous genes or alleles in plants only in a heteroallelic state, such as in hybrid crops, while retaining unaffected expression of the endogenous genes or alleles in a homozygous state, by editing untranslated regions of the endogenous genes through genomic editing techniques. The compositions and methods can also be used to produce moderate, beneficial, phenotypes based on alleles of genes in plants with strong, potentially detrimental or non-viable phenotypes, particularly when present in homozygous state. CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the priority of U.S. Provisional Application Serial No. 63 / 520,915, and 63 / 520,898 filed on August 21, 2023, the entire disclosure of which is incorporated herein by reference. INCORPORATION OF SEQUENCE LISTING

[0003] A sequence listing contained in the file named “BCS236339.XML” which is 493 kilobytes (measured in MS-Windows®) containing 235 sequences and created on August 17th, 2024, is filed electronically herewith and incorporated by reference in its entirety. BACKGROUND

[0004] Variant alleles of plant endogenous genes have been described which produce phenotypes of potentially commercial interest, such as reduced plant height or reduced seed shattering, but which may also be detrimental or even non-viable, or may produce unwanted phenotypes on plant development or reproduction, particularly when present in homozygous state.

[0005] Such variant alleles would hamper the commercial production of hybrid crops comprising such alleles, since parent plants used for the production of hybrid seeds inherently would contain these variant alleles in homozygous state, thereby reducing the ability to produce high quality hybrid seeds and / or sufficient quantities thereof during large scale seed production. For example, known dominant mutant alleles for reduced plant height, such as reduced cornplant height, result in an excessively short phenotype and exhibit reproductive off-types when present in a plant in the homozygous state. As another example, known mutant alleles affecting the development of a dehiscence zone in Brassica plants, can reduce seed or pod shattering when present in heterozygous state while still allowing opening of pods and harvesting of seeds using conventional harvesting equipment, but when present in homozygous state in the parent plants used for hybrid seed production result in pods that cannot be opened any longer using conventional harvesting equipment.

[0006] There thus remains a need for methods and compositions to produce variant alleles with moderate phenotypes in hybrid plants based on variant alleles in plants with a strong, potentially detrimental phenotype, without occurrence of a mutant phenotype that would detrimentally impact seed or plant production on a large scale, when such alleles are present in a homozygous state. The problem has been solved as hereinafter described, including the following different embodiments, claims and examples. SUMMARY OF THE INVENTION

[0007] In summary, various aspects of the invention are described in the following numbered embodiments.

[0008] Embodiment 1. A method for editing the genome of a plant cell to modify an endogenous gene, comprising the steps of a) generating a first double-stranded break and a second double stranded break using a targeted editing technique targeting at least one untranslated region of said endogenous gene, in said plant cell without the perturbance of the coding region; b) isolating a modified plant cell comprising a modified allele of said endogenous gene wherein the modified allele comprises an inverted DNA sequence of at least part of said at least one untranslated region of said endogenous gene and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said at least one untranslated region, wherein the modified allele does not comprise a sense sequence complementary to the antisense sequence of part of said at least one untranslated region.

[0009] Embodiment 2) The method of embodiment 1, wherein transcription of the modified allele does not yield an RNA molecule comprising a stem-loop structure.

[0010] Embodiment 3) The method of any one of embodiments 1 or 2, wherein said modified allele of said endogenous gene when present in the cell in homozygous state does not result in reduced expression of said modified allele of said endogenous gene.

[0011] Embodiment 4) The method of any one of embodiments 1 to 3, wherein said inverted DNA sequence of at least part of said untranslated region of said endogenous gene encodes an antisense RNA sequence that is at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of said untranslated region of said gene.

[0012] Embodiment 5) The method of any one of embodiments 1 to 4, wherein reduced expression of said gene results in a phenotype of interest in a plant comprising or consisting essentially of said modified plant cells.

[0013] Embodiment 6) The method of any one of embodiments 1 to 5, wherein said untranslated region of said endogenous gene is a 5’ untranslated region or a 3’ untranslated region or both.

[0014] Embodiment 7) The method of any one of embodiments 1 to 5, wherein said untranslated region is an intron sequence of said endogenous gene.

[0015] Embodiment 8) The method of any one of embodiments 1 to 7, wherein said modified allele of said endogenous gene is homozygously present in said plant cell.

[0016] Embodiment 9) The method of any one of embodiments 1 to 7, wherein said modified allele of said endogenous gene is heterozygously present in said plant cell.

[0017] Embodiment 10) The method of any one of embodiments 1 to 7, wherein said plant cell comprises a modified allele and an unmodified allele of said endogenous gene.

[0018] Embodiment 11) The method of any one of embodiments 1 to 7, wherein said modified allele of said endogenous gene is present in the plant cell in heteroallelic state and wherein said cell further comprises an unmodified allele of said gene, wherein expression of the modified and unmodified allele of said gene is reduced.

[0019] Embodiment 12) The method of embodiment 11, wherein said modified allele and said unmodified allele are transcribed to a mRNA and wherein said RNA from said modified allele and said RNA from said unmodified allele are capable of producing a double stranded RNA region of at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides.

[0020] Embodiment 13) The method of any one of embodiments 9 to 12, wherein expression of said modified and said unmodified allele is reduced.

[0021] Embodiment 14) The method of embodiment 13, wherein said reduced expression results in a phenotype of interest.

[0022] Embodiment 15) The method of any one of embodiments 1 to 14, comprising a further step of regenerating a plant from said modified plant cell.

[0023] Embodiment 16) The method of embodiment 14 comprising a further step of crossing said plant comprising said modified allele of said endogenous gene in homozygous state with another plant comprising an unmodified allele of said endogenous gene in homozygous state and harvesting hybrid seeds. The hybrid seeds contain a modified allele of said endogenous gene and an unmodified allele of said endogenous gene.

[0024] Embodiment 17) The method of any one of embodiments 1 to 16, wherein said plant is oilseed rape and said endogenous gene is an indehiscence gene from oilseed rape.

[0025] Embodiment 18) The method of any one of embodiments 1 to 16, wherein said plant is corn and said endogenous gene is selected from GA20 oxidase or GA3 oxidase.

[0026] Embodiment 19) The method of embodiment 18, wherein said GA20 oxidase is selected from GA20 oxidase_5 or GA20 oxidase_3.

[0027] Embodiment 20) The method of embodiment 18, wherein said GA3 oxidase is selected from GA3 oxidase_1, GA3 oxidase_2 or GA3 oxidase_3.

[0028] Embodiment 21) The method of any one of embodiments 1 to 16, wherein said plant is corn and said endogenous gene is selected from Anther Ear1 (GRMZM2G081554) dwarf 4 (GRMZM2G065635) brs1 - brassinosteroid synthesis1, nana plant 1 (GRMZM2G057000), brassinosteroid receptor ZmBRI1a / ZmBRI1b (GRMZM2G048294 / GRMZM2G449830), the meristem development gene compact plant 2 (GRMZM2G064732) and ZMWRKY60.

[0029] Embodiment 22) The method of any one of embodiments 1 to 16, wherein the endogenous gene is Agamous, Bri1, Dwarf1, Pin 1 from Arabidopsis, or an orthologous gene from another plant.

[0030] Embodiment 23) The method of any one of embodiments 1 to 16, wherein said endogenous gene encodes a protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 9, 15, 30, 33, 173, and 214 - 217.

[0031] Embodiment 24) The method of any one of embodiments 1 to 16, wherein said inverted DNA sequence comprises a nucleotide sequence having at least 90% sequence identity orcomplementarity to a nucleotide sequence of at least 18 nucleotides, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of: nucleotides 1-29 of SEQ ID NO: 36, nucleotides 1664-1788 of SEQ ID NO: 36, nucleotides 1- 38 of SEQ ID NO: 37, nucleotides 1446-1698 of SEQ ID NO: 37, nucleotides 3001-3161 of SEQ ID NO: 168, nucleotides 4796-5406 of SEQ ID NO: 168, nucleotides 3001-3056 of SEQ ID NO: 169, nucleotides 4464-4581 of SEQ ID NO: 169, nucleotides 3001-3130 of SEQ ID NO: 170, nucleotides 4275-4332 of SEQ ID NO: 170, nucleotides 7621-8029 of SEQ ID NO: 174, nucleotides 9672-10276 of SEQ ID NO: 174, nucleotides 7386-7831 of SEQ ID NO: 175, nucleotides 8862-8967 of SEQ ID NO: 175, nucleotides 7547-7751 of SEQ ID NO: 176, nucleotides 8904-9178 of SEQ ID NO: 176, nucleotides 1-1060 of SEQ ID NO: 204, nucleotides 5418-5648 of SEQ ID NO: 204, nucleotides 1-165 of SEQ ID NO: 211, nucleotides 3757-4167 of SEQ ID NO: 211, nucleotides 664-699 of SEQ ID NO: 212, nucleotides 2482- 2700 of SEQ ID NO: 212, nucleotides 1-99 of SEQ ID NO: 213, or nucleotides 3205-3506 of SEQ ID NO: 213.

[0032] Embodiment 25) The method of any one of embodiments 1 to 24, wherein said targeted editing technique involves use of a RNA guided effector protein or a TALE protein or a custom meganuclease.

[0033] Embodiment 26) The method of embodiment 25, wherein said RNA guided effector protein is a CRISPR-Cas effector protein , selected from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR-Cas system, or a CRISPR-Cas effector protein derived therefrom, optionally a CRISPR-Cas effector protein comprising one or more nuclear localization signals.

[0034] Embodiment 27) The method of any one of embodiments 25 or 26, wherein said RNA guided endonuclease is a CRISPR-Cas effector protein selected from a Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c effector protein.

[0035] Embodiment 28) The method of any one of embodiments 25 to 28, wherein said RNA guided effector protein is a Cas12a effector protein or a Cas12a derived effector protein.

[0036] Embodiment 29) The method according to embodiment 28, wherein said Cas12a effector protein is selected from FnCas12a, LbCas12a, ErCas12a or AsCas12a or variants thereof.

[0037] Embodiment 30) The method according to embodiment 29, wherein the Cas12a effector protein has an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 194 and 199.

[0038] Embodiment 30) The method according to any one of embodiments 25 to 29, wherein said targeted gene editing technique involves use of one or more guideRNA comprising a nucleotide sequence selected from the group of SEQ ID NOs: 177, 178, 179, 180, 205, 206, 207, 208 and 209.

[0039] Embodiment 31) A method for modifying expression of an endogenous gene in a hybrid plant while leaving the expression of said gene unaffected in a parent plant or plants, comprising the steps of a) Identifying an endogenous gene in a plant wherein expression of a variant allele of said gene result in unwanted phenotypes when present in homozygous state; b) providing a first plant comprising a modified allele of said gene comprising a nucleic acid region which is an inversion of a part of said gene whereby the inversion does not affect translation of said modified allele, and wherein said first plant comprises said modified allele of said gene homozygously; c) crossing said first plant with a second plant comprising an unmodified allele of said gene not comprising said inversion not affecting translation of said gene, wherein the unmodified gene is in homozygous state; d) obtaining a hybrid seed comprising the modified and unmodified allele of said gene in heterozygous or heteroallelic form.

[0040] Embodiment 32) The method of embodiment 31, wherein upon transcription of said modified allele and said unmodified allele into RNA molecule, a double stranded RNA region can be formed by base-pairing between the nucleic acid region which is an inversion of part of an untranslated region of gene in the RNA transcript of said modified allele and the nucleic acid region and the RNA transcript of said unmodified allele, and wherein the double stranded RNA region is capable of inhibiting expression of said modified allele and said unmodified allele by RNA silencing mechanisms, such as stalling of RNA translation, stalling of RNA transcription,destabilization of the resulting RNA molecules or post-transcriptional degradation of the transcribed RNA molecules.

[0041] Embodiment 33) The method of any one of embodiments 31 or 32, wherein transcription of the modified allele yields an RNA molecule not comprising a stem-loop structure.

[0042] Embodiment 34) The method of any one of embodiments 31 to 33, wherein said modified allele of said endogenous gene when present in the cell in homozygous state in said first plant does not result in reduced expression of said modified allele of said endogenous gene.

[0043] Embodiment 35) The method of any one of embodiments 31 to 34, wherein said nucleic acid region which is an inversion of a part of said gene results upon transcription in an antisense RNA sequence that is at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of said untranslated region of said gene.

[0044] Embodiment 36) The method of any one of embodiments 31 to 35, wherein expression of said modified allele and said unmodified allele of said endogenous gene is reduced.

[0045] Embodiment 37) The method of any one of embodiments 31 to 36, wherein said untranslated region of said endogenous gene is a 5’ untranslated region or a 3’ untranslated region.

[0046] Embodiment 37) The method of any one of embodiments 31 to 36, wherein said untranslated region is an intron sequence of said endogenous gene.

[0047] Embodiment 38) The method of any one of embodiments 31 to 37, wherein said plant is oilseed rape and said endogenous gene is an indehiscence gene from oilseed rape.

[0048] Embodiment 39) The method of any one of embodiments 31 to 37, wherein said plant is corn and said endogenous gene is selected from GA20 oxidase or GA3 oxidase.

[0049] Embodiment 40) The method of embodiment 39, wherein said GA20 oxidase is selected from GA20 oxidase_5 or GA20 oxidase_3.

[0050] Embodiment 41) The method of embodiment 39, wherein said GA3 oxidase is selected from GA3 oxidase_1, GA3 oxidase_2 or GA3 oxidase_3.

[0051] Embodiment 42) The method of any one of embodiments 31 to 37, wherein said plant is corn and said endogenous gene is selected from Anther Ear1 (GRMZM2G081554) dwarf 4 (GRMZM2G065635) brs1 brassinosteroid synthesis1, nana plant 1 (GRMZM2G057000), brassinosteroid receptor ZmBRI1a / ZmBRI1b (GRMZM2G048294 / GRMZM2G449830), the meristem development gene compact plant 2 (GRMZM2G064732), and ZMWRKY60.

[0052] Embodiment 43) The method of any one of embodiments 31 to 37, wherein the endogenous gene is Agamous, Bri1, Dwarf1, Pin 1 from Arabidopsis, or an orthologous gene from another plant.

[0053] Embodiment 44) The method of any one of embodiments 31 to 37, wherein said endogenous gene encodes a protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID Nos: 9, 15, 30, 33, 173, and 214 - 217

[0054] Embodiment 45) The method of any one of embodiments 31 to 37, wherein said inverted DNA sequence comprises a nucleotide sequence having at least 90% sequence identity or complementarity to a nucleotide sequence of at least 18 nucleotides, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of: nucleotides 1-29 of SEQ ID NO: 36, nucleotides 1664-1788 of SEQ ID NO: 36, nucleotides 1- 38 of SEQ ID NO: 37, nucleotides 1446-1698 of SEQ ID NO: 37, nucleotides 3001-3161 of SEQ ID NO: 168, nucleotides 4796-5406 of SEQ ID NO: 168, nucleotides 3001-3056 of SEQ ID NO: 169, nucleotides 4464-4581 of SEQ ID NO: 169, nucleotides 3001-3130 of SEQ ID NO: 170, nucleotides 4275-4332 of SEQ ID NO: 170, nucleotides 7621-8029 of SEQ ID NO: 174, nucleotides 9672-10276 of SEQ ID NO: 174, nucleotides 7386-7831 of SEQ ID NO: 175, nucleotides 8862-8967 of SEQ ID NO: 175, nucleotides 7547-7751 of SEQ ID NO: 176, nucleotides 8904-9178 of SEQ ID NO: 176, nucleotides 1-1060 of SEQ ID NO : 204, nucleotides 5418-5648 of SEQ ID NO: 204, nucleotides 1-165 of SEQ ID NO: 211, nucleotides 3757-4167 of SEQ ID NO: 211, nucleotides 664-699 of SEQ ID NO: 212, nucleotides 2482- 2700 of SEQ ID NO: 212, nucleotides 1-99 of SEQ ID NO: 213, or nucleotides 3205-3506 of SEQ ID NO: 213.

[0055] Embodiment 46) The method of any one of embodiments 31 to 45, wherein the modified allele in said first plant is obtained by a targeted editing technique, such as a targeted editing technique involving the use of a RNA guided effector protein or a TALE protein or a custom meganuclease.

[0056] Embodiment 47) The method of embodiment 46, wherein said RNA guided effector protein is a CRISPR-Cas effector protein , selected from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR-Cas system, or a CRISPR-Cas effector proteinderived therefrom, optionally a CRISPR-Cas effector protein comprising one or more nuclear localization signals.

[0057] Embodiment 48) The method of any one of embodiments 46 or 47, wherein said RNA guided endonuclease is a CRISPR-Cas effector protein selected from a Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c effector protein.

[0058] Embodiment 49) The method of any one of embodiments 46 to 48, wherein said RNA guided effector protein is a Cas12a effector protein or a Cas12a derived effector protein.

[0059] Embodiment 50) The method according to embodiment 49, wherein said Cas12a effector protein is selected from FnCas12a, LbCas12a, ErCas12a or AsCas12a or variants thereof.

[0060] Embodiment 51) The method according to embodiment 50, wherein the Cas12a effector protein has an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 194 and 199.

[0061] Embodiment 52) The method according to any one of embodiments 46 to 51, wherein said targeted gene editing technique involves use of one or more guide RNAs comprising a nucleotide sequence selected from the group of SEQ ID NOs: 177, 178, 179, 180, 205, 206, 207, 208 and 209.

[0062] Embodiment 53) A plant cell, plant or part or seed thereof comprising a modified allele of an endogenous gene wherein the modified allele comprises an inverted DNA sequence of at least part of an untranslated region of said endogenous gene, and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said untranslated region, wherein the modified allele does not comprise a sense nucleotide sequence of more than 17 nucleotides complementary to the antisense sequence of part of said untranslated region.

[0063] Embodiment 54) The plant cell, plant or part or seed thereof of embodiment 53, wherein said plant cell, plant or part thereof is non-transgenic.

[0064] Embodiment 55) The plant cell, plant or part or seed thereof of any one of embodiments 53 or 54, wherein said modified allele of said endogenous gene is obtained by a targeted editing technique.

[0065] Embodiment 56) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 55, wherein transcription of the modified allele yields an RNA molecule not comprising a stem-loop structure.

[0066] Embodiment 57) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 56, wherein said inverted DNA sequence of at least part of an untranslated region of said endogenous gene results upon transcription in an antisense RNA sequence that is at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of said untranslated region of said gene.

[0067] Embodiment 58) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 57, wherein said untranslated region of said endogenous gene is a 5’ untranslated region or a 3’ untranslated region.

[0068] Embodiment 59) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 57, wherein said untranslated region is an intron sequence of said endogenous gene.

[0069] Embodiment 60) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 59, wherein said plant is oilseed rape and said endogenous gene is an indehiscence gene from oilseed rape.

[0070] Embodiment 61) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 59, wherein said plant is corn and said endogenous gene is selected from GA20 oxidase or GA3 oxidase.

[0071] Embodiment 62) The plant cell, plant or part or seed thereof embodiment 61, wherein said GA20 oxidase is selected from GA20 oxidase_5 or GA20 oxidase_3.

[0072] Embodiment 63) The plant cell, plant or part or seed thereof embodiment 61, wherein said GA3 oxidase is selected from GA3 oxidase_1, GA3 oxidase_2 or GA3 oxidase_3.

[0073] Embodiment 64) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 59, wherein said plant is corn and said endogenous gene is selected from Anther Ear1 (GRMZM2G081554) dwarf 4 (GRMZM2G065635) brs1 - brassinosteroid synthesis1, nana plant 1 (GRMZM2G057000), brassinosteroid receptor ZmBRI1a / ZmBRI1b(GRMZM2G048294 / GRMZM2G449830), the meristem development gene compact plant 2 (GRMZM2G064732) or ZMWRKY60.

[0074] Embodiment 65) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 59, wherein the endogenous gene is Agamous, Bri1, Dwarf1, Pin 1 from Arabidopsis, or an orthologous gene from another plant.

[0075] Embodiment 66) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 59, wherein said endogenous gene encodes a protein having an amino acid sequence with at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs : 9, 15, 30, 33, 173, and 214 – 217.

[0076] Embodiment 67) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 59, wherein said inverted DNA sequence comprises a nucleotide sequence having at least 90% sequence identity or complementarity to a nucleotide sequence of at least 18 nucleotides, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of : nucleotides 1-29 of SEQ ID NO: 36, nucleotides 1664-1788 of SEQ ID NO: 36, nucleotides 1-38 of SEQ ID NO: 37, nucleotides 1446-1698 of SEQ ID NO: 37, nucleotides 3001-3161 of SEQ ID NO: 168, nucleotides 4796-5406 of SEQ ID NO: 168, nucleotides 3001-3056 of SEQ ID NO: 169, nucleotides 4464-4581 of SEQ ID NO: 169, nucleotides 3001-3130 of SEQ ID NO: 170, nucleotides 4275-4332 of SEQ ID NO: 170, nucleotides 7621-8029 of SEQ ID NO: 174, nucleotides 9672-10276 of SEQ ID NO: 174, nucleotides 7386-7831 of SEQ ID NO: 175, nucleotides 8862-8967 of SEQ ID NO: 175, nucleotides 7547-7751 of SEQ ID NO: 176, nucleotides 8904-9178 of SEQ ID NO: 176, nucleotides 1-1060 of SEQ ID NO : 204, nucleotides 5418-5648 of SEQ ID NO: 204, nucleotides 1-165 of SEQ ID NO: 211, nucleotides 3757-4167 of SEQ ID NO: 211, nucleotides 664-699 of SEQ ID NO: 212, nucleotides 2482-2700 of SEQ ID NO: 212, nucleotides 1-99 of SEQ ID NO: 213, or nucleotides 3205-3506 of SEQ ID NO: 213.

[0077] Embodiment 68) The plant cell, plant or part or seed thereof of any one of embodiments 53 to 67, wherein the modified allele in said first plant is obtained by a targeted editing technique, such as a targeted editing technique involving the use of a RNA guided effector protein or a TALE protein or a custom meganuclease.

[0078] Embodiment 69) The plant cell, plant or part or seed thereof of embodiment 68, wherein said RNA guided effector protein is a CRISPR-Cas effector protein , selected from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a TypeIV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR-Cas system, or a CRISPR-Cas effector protein derived therefrom, optionally a CRISPR-Cas effector protein comprising one or more nuclear localization signals.

[0079] Embodiment 70) The plant cell, plant or part or seed thereof of any one of embodiments 68 or 69, wherein said RNA guided endonuclease is a CRISPR-Cas effector protein selected from a Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c effector protein.

[0080] Embodiment 71) The plant cell, plant or part or seed thereof of any one of embodiments 68 to 70, wherein said RNA guided effector protein is a Cas12a effector protein or a Cas12a derived effector protein.

[0081] Embodiment 72) The plant cell, plant or part or seed thereof according to embodiment 71, wherein said Cas12a effector protein is selected from FnCas12a, LbCas12a, ErCas12a or AsCas12a or variants thereof.

[0082] Embodiment 73) The plant cell, plant or part or seed thereof according to embodiment 72, wherein the Cas12a effector protein has an amino acid sequence having at least 95% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NOs: 194 and 199.

[0083] Embodiment 74) The plant cell, plant or part or seed thereof according to any one of embodiments 68 to 73, wherein said targeted gene editing technique involves use of one or more guideRNA comprising a nucleotide sequence selected from the group of SEQ ID NOs: 177, 178, 179, 180, 205, 206, 207, 208 and 209.

[0084] Embodiment 75) The plant cell, plant or part or seed thereof according to any one of embodiments 53 to 74, wherein said modified allele is present homozygously.

[0085] Embodiment 76) The plant cell, plant or part or seed thereof according to any one of embodiments 53 to 74, wherein said modified allele is present heterozygously or in heteroallelic form.

[0086] Embodiment 77) The plant cell, plant or part or seed thereof according to embodiment 78, wherein said plant cell, plant or part or seed thereof further comprises an unmodified allele of said endogenous gene.

[0087] Embodiment 78) The plant cell, plant or part or seed thereof according to embodiment 77, wherein said modified allele and said unmodified allele are transcribed into an RNA and wherein said RNA from said modified allele and said RNA from said unmodified allele are capable of producing a double stranded RNA region of at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides.

[0088] Embodiment 79) The plant cell, plant or part or seed thereof according to embodiment 78, wherein the double stranded RNA region is capable of inhibiting expression of said modified allele and said unmodified allele by RNA silencing mechanisms, such as stalling of RNA translation, stalling of RNA transcription, destabilization of the resulting RNA molecules or post-transcriptional degradation of the transcribed RNA molecules.

[0089] Embodiment 80) The plant cell, plant or part or seed thereof according to embodiment 75, wherein expression of said modified allele is not reduced.

[0090] Embodiment 81) The plant cell, plant or part or seed thereof according to any one of embodiments 76 to 79, wherein said plant cell, plant or part or seed thereof exhibit a phenotype of interest.

[0091] Embodiment 82) The plant according to embodiment 81 wherein the phenotype of interest is a short stature compared to a plant not comprising said modified an unmodified allele of said endogenous gene.

[0092] Embodiment 83) The plant cell, plant or part or seed thereof according to embodiment 53, wherein the modified allele of the endogenous gene comprising an inverted DNA sequence is operably linked to its native or homologous promoter.

[0093] Embodiment 84) A plant regenerated from a plant cell according to any one of embodiments 53 to 83.

[0094] Embodiment 85) A plant comprising or consisting essentially of a plant cell as described in any one of embodiments 53 to 83.

[0095] Embodiment 86) A plant or a seed obtained by the methods according to embodiment 15 or 16.

[0096] Embodiment 87) A plant according to any one of embodiments 53 to 86, wherein said plant is a plant selected from a monocotyledonous species, a dicotyledonous species, an angiosperm species or a gymnosperm species.

[0097] Embodiment 88) A plant according to embodiment 87, wherein said plant is selected from a corn plant, a rice plant, a sorghum plant, a wheat plant, an alfalfa plant, a barley plant, a millet plant, a rye plant, a sugarcane plant, a cotton plant, a soybean plant, a canola plant, a tomato plant, an onion plant, a cucumber plant, an Arabidopsis plant, or a potato plant.

[0098] Embodiment 89) A plant according to embodiment 88, wherein said plant is a corn plant with short stature.

[0099] Embodiment 90) The method according to any one of embodiments 1 to 30, wherein said modified allele of said endogenous gene comprises an inverted DNA sequence of at least part of two untranslated regions.

[0100] Embodiment 91) A method for editing the genome of a plant cell to modify an endogenous gene, comprising the steps of a. generating a double-stranded DNA break or a single-stranded DNA break (nick) using a targeted editing technique targeting at least one untranslated region of said endogenous gene, in said plant cell without the perturbance of the coding region; b. providing at least one template nucleic acid to said plant cell wherein said template nucleic acid comprises a portion of said at least one untranslated region of said endogenous gene in inverted orientation; c. isolating a modified plant cell comprising a modified allele of said endogenous gene wherein the modified allele comprises an inverted DNA sequence of at least part of said at least one untranslated region of said endogenous gene and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said untranslated region, wherein the modified allele does not comprise a sense sequence complementary to the antisense sequence of part of said untranslated region.

[0101] Embodiment 92) The method according to embodiment 91, wherein said template nucleic acid comprising said portion of said at least one untranslated region of said endogenous gene in inverted orientation, is inserted in said targeted untranslated region of said endogenous gene by non-homologous end-joining.

[0102] Embodiment 93) The method according to embodiment 91, wherein said template nucleic acid comprises at least one, or two homology arms having homology to the nucleic acid sequence flanking the double stranded break, optionally wherein said homology arms are flanking the portion of the at least one untranslated region in inverted orientation.

[0103] Embodiment 94) The method according to embodiment 93, wherein the portion of the untranslated region of said endogenous gene is introduced in inverted orientation by homology dependent repair.

[0104] Embodiment 95) A method for editing the genome of a plant cell to modify an endogenous gene, comprising the steps of a. generating a double-stranded DNA break or single-stranded DNA break using a CRISPR / CAS fusion protein fused to a reverse transcriptase functional domain and a guide RNA targeting at least one untranslated region of said endogenous gene, in said plant cell without the perturbance of the coding region; wherein said guide RNA further comprises a nucleotide sequence acid comprises a portion of said at least one untranslated region of said endogenous gene in inverted orientation; b. isolating a modified plant cell comprising a modified allele of said endogenous gene wherein the modified allele comprises an inverted DNA sequence of at least part of said at least one untranslated region of said endogenous gene and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said untranslated region, wherein the modified allele does not comprise a sense sequence complementary to the antisense sequence of part of said untranslated region. BRIEF DESCRIPTION OF THE FIGURES

[0105] Figure 1. Schematic representation of the methods and composition according to the invention. Panel A on the scheme shows a non-edited structure of gene with 3’UTR oriented in normal direction. To reverse the direction of 3’UTR of the candidate gene, two unique gRNAs are used to excise and insert a section of 3’UTR in reversed orientation in both homologous alleles of the candidate gene (panel B). Such rearrangement of 3’UTR, when present in both alleles, would not affect the function of the UTR therefore no phenotype would be expected in the homozygous stage. However, upon crossing of such homozygous edited plant with wild type plant (where both alleles have 3’UTR in normal directions), the resulting offspring would inherit two alleles with different directions of the 3’UTR. This may generate a double stranded RNA region formation between complementary sequence that would form from both transcripts. Consequently, such rearrangement may lead to generation of siRNA molecules that could affect stability of the mRNA of the gene and suppression of function of the target gene.

[0106] Figure 2. Schematic representation of recombinant nucleic acid constructs used in Example 3. Panel A and B: recombinant nucleic acid constructs expressing Cas12a under control of a plant promoter and expressing two guide RNAs targeting an untranslated region of Agamous. Panel C: a positive control nucleic acid expressing a miRNA having complementarityto Agamous. Panel D: a control nucleic acid expressing agamous1 allele wherein the 5’ UTR has been inverted.

[0107] Figure 3. Schematic representation of recombinant nucleic acid constructs used in Example 4. Panel A: recombinant nucleic acid construct for expressing an inverted 5’UTR of Agamous. Panel B: recombinant nucleic acid construct for expressing an inverted 3’UTR of Agamous. Panel C: recombinant nucleic acid construct for expressing an inverted 5’ UTR and 3’UTR of Agamous.

[0108] Figure 4. Provides an illustration comparing the wild type (WT) and an edited allele of the Zm.GA3ox_1 gene, with the edited allele having a deletion and inversion in the 3’ UTR region. BRIEF DESCRIPTION OF THE SEQUENCE ENTRIES IN THE SEQUENCE LISTING

[0109] SEQ ID NO: 1: nucleotide sequence of GA20 oxidase_1 cDNA from Zea mays.

[0110] SEQ ID NO: 2: nucleotide sequence of GA20 oxidase_1 coding sequence from Zea mays.

[0111] SEQ ID NO: 3: amino acid sequence of GA20 oxidase_1 protein from Zea mays.

[0112] SEQ ID NO: 4: nucleotide sequence of GA20 oxidase_2 cDNA from Zea mays.

[0113] SEQ ID NO: 5: nucleotide sequence of GA20 oxidase_2 coding sequence from Zea mays.

[0114] SEQ ID NO: 6: amino acid sequence of GA20 oxidase_2 protein from Zea mays.

[0115] SEQ ID NO: 7: nucleotide sequence of GA20 oxidase_3 cDNA from Zea mays.

[0116] SEQ ID NO: 8: nucleotide sequence of GA20 oxidase_3 coding sequence from Zea mays.

[0117] SEQ ID NO: 9: amino acid sequence of GA20 oxidase_3 protein from Zea mays.

[0118] SEQ ID NO: 10: nucleotide sequence of GA20 oxidase_4 cDNA from Zea mays.

[0119] SEQ ID NO: 11: nucleotide sequence of GA20 oxidase_4 coding sequence from Zea mays.

[0120] SEQ ID NO: 12: amino acid sequence of GA20 oxidase_4 protein from Zea mays.

[0121] SEQ ID NO: 13: nucleotide sequence of GA20 oxidase_5 cDNA from Zea mays.

[0122] SEQ ID NO: 14: nucleotide sequence of GA20 oxidase_5 coding sequence from Zea mays.

[0123] SEQ ID NO: 15: amino acid sequence of GA20 oxidase_5 protein from Zea mays.

[0124] SEQ ID NO: 16: nucleotide sequence of GA20 oxidase_6 cDNA from Zea mays.

[0125] SEQ ID NO: 17: nucleotide sequence of GA20 oxidase_6 coding sequence from Zea mays.

[0126] SEQ ID NO: 18: amino acid sequence of GA20 oxidase_6 protein from Zea mays.

[0127] SEQ ID NO: 19: nucleotide sequence of GA20 oxidase_7 cDNA from Zea mays.

[0128] SEQ ID NO: 20: nucleotide sequence of GA20 oxidase_7 coding sequence from Zea mays.

[0129] SEQ ID NO: 21: amino acid sequence of GA20 oxidase_7 protein from Zea mays.

[0130] SEQ ID NO: 22: nucleotide sequence of GA20 oxidase_8 cDNA from Zea mays.

[0131] SEQ ID NO: 23: nucleotide sequence of GA20 oxidase_8 coding sequence from Zea mays.

[0132] SEQ ID NO: 24: amino acid sequence of GA20 oxidase_8 protein from Zea mays.

[0133] SEQ ID NO: 25: nucleotide sequence of GA20 oxidase_9 cDNA from Zea mays.

[0134] SEQ ID NO: 26: nucleotide sequence of GA20 oxidase_9 coding sequence from Zea mays.

[0135] SEQ ID NO: 27: amino acid sequence of GA20 oxidase_9 protein from Zea mays.

[0136] SEQ ID NO: 28: nucleotide sequence of GA3 oxidase_1 cDNA from Zea mays.

[0137] SEQ ID NO: 29: nucleotide sequence of GA3 oxidase_1 coding sequence from Zea mays.

[0138] SEQ ID NO: 30: amino acid sequence of GA3 oxidase_1 protein from Zea mays.

[0139] SEQ ID NO: 31: nucleotide sequence of GA3 oxidase_2 cDNA from Zea mays.

[0140] SEQ ID NO: 32: nucleotide sequence of GA3 oxidase_2 coding sequence from Zea mays.

[0141] SEQ ID NO: 33: amino acid sequence of GA3 oxidase_2 protein from Zea mays.

[0142] SEQ ID NO: 34: nucleotide sequence of GA20 oxidase_3 genomic sequence from Zea mays.

[0143] SEQ ID NO: 35: nucleotide sequence of GA20 oxidase_5 genomic sequence from Zea mays.

[0144] SEQ ID NO: 36: nucleotide sequence of GA3 oxidase_1 genomic sequence from Zea mays.

[0145] SEQ ID NO: 37: nucleotide sequence of GA3 oxidase_2 genomic sequence from Zea mays.

[0146] SEQ ID NO: 38: nucleotide sequence of GA20 oxidase_4 genomic sequence from Zea mays.

[0147] SEQ ID NO: 39: nucleotide sequence of GA20 oxidase_3 / 5-1 cDNA target sequence.

[0148] SEQ ID NO: 40: nucleotide sequence of GA20 oxidase_3 / 5-1 miRNA targeting sequence..

[0149] SEQ ID NO: 41: nucleotide sequence of GA20 oxidase_3 / 5-2 cDNA target sequence.

[0150] SEQ ID NO: 42: nucleotide sequence of GA20 oxidase_3 / 5-2 miRNA targeting sequence.

[0151] SEQ ID NO: 43: nucleotide sequence of GA20 oxidase_3 / 5-3 cDNA target sequence.

[0152] SEQ ID NO: 44: nucleotide sequence of GA20 oxidase_3 / 5-3 miRNA targeting sequence.

[0153] SEQ ID NO: 45: nucleotide sequence of GA20 oxidase_3 / 5-4 cDNA target sequence

[0154] SEQ ID NO: 46: nucleotide sequence of GA20 oxidase_3 / 5-4 miRNA targeting sequence.

[0155] SEQ ID NO: 47: nucleotide sequence of GA20 oxidase_1 / 2 cDNA target sequence.

[0156] SEQ ID NO: 48: nucleotide sequence of GA20 oxidase_1 / 2 miRNA targeting sequence.

[0157] SEQ ID NO: 49: nucleotide sequence of GA20 oxidase_3 / 9 cDNA target sequence.

[0158] SEQ ID NO: 50: nucleotide sequence of GA20 oxidase_3 / 9 miRNA targeting sequence.

[0159] SEQ ID NO: 51: nucleotide sequence of GA20 oxidase_7 / 8 cDNA target sequence.

[0160] SEQ ID NO: 52: nucleotide sequence of GA20 oxidase_7 / 8 miRNA targeting sequence.

[0161] SEQ ID NO: 53: nucleotide sequence of GA20 oxidase_3 individual cDNA target sequence.

[0162] SEQ ID NO: 54: nucleotide sequence of GA20 oxidase_3 individual miRNA targeting sequence.

[0163] SEQ ID NO: 55: nucleotide sequence of GA20 oxidase_5 individual cDNA target sequence.

[0164] SEQ ID NO: 56: nucleotide sequence of GA20 oxidase_5 individual miRNA targeting sequence.

[0165] SEQ ID NO: 57: nucleotide sequence of GA3 oxidase_1 cDNA target sequence.

[0166] SEQ ID NO: 58: nucleotide sequence of GA3 oxidase_1 miRNA targeting sequence.

[0167] SEQ ID NO: 59: nucleotide sequence of GA3 oxidase_2 cDNA target sequence.

[0168] SEQ ID NO: 60: nucleotide sequence of GA3 oxidase_2 miRNA targeting sequence.

[0169] SEQ ID NO: 61: nucleotide sequence of GA20 oxidase_4 / 6-4 cDNA target sequence.

[0170] SEQ ID NO: 62: nucleotide sequence of GA20 oxidase_4 / 6-4 miRNA targeting sequence.

[0171] SEQ ID NO: 63: nucleotide sequence of GA20 oxidase_4 / 6-6 cDNA target sequence.

[0172] SEQ ID NO: 64: nucleotide sequence of GA20 oxidase_4 / 6-6 miRNA targeting sequence.

[0173] SEQ ID NO: 65: nucleotide sequence of a Rice tungro bacilliform virus promoter.

[0174] SEQ ID NO: 66: nucleotide sequence of a truncated Rice tungro bacilliform virus promoter.

[0175] SEQ ID NO: 67: nucleotide sequence of a sucrose synthase (Sus1) promoter from Zea mays.

[0176] SEQ ID NO: 68: nucleotide sequence of a sucrose synthase (Sus1) promoter from Zea mays.

[0177] SEQ ID NO: 69: nucleotide sequence of a sucrose synthase 1 (Sus1) promoter from Oryza sativa.

[0178] SEQ ID NO: 70: nucleotide sequence of a sucrose transporter (Sut1) promoter from Oryza sativa.

[0179] SEQ ID NO: 71: nucleotide sequence of a YSL2 promoter from Oryza sativa.

[0180] SEQ ID NO: 72: nucleotide sequence of a PPDK promoter from Zea mays.

[0181] SEQ ID NO: 73: nucleotide sequence of a FDA promoter from Zea mays.

[0182] SEQ ID NO: 74: nucleotide sequence of a Nadh-Gogat promoter from Oryza sativa.

[0183] SEQ ID NO: 75: nucleotide sequence of an Actin 1 promoter 1 from Oryza sativa.

[0184] SEQ ID NO: 76: nucleotide sequence of an Actin 1 promoter 2 from Oryza sativa.

[0185] SEQ ID NO: 77: nucleotide sequence of an Actin 2 promoter 1 from Oryza sativa.

[0186] SEQ ID NO: 78: nucleotide sequence of an Actin 2 promoter 2 from Oryza sativa.

[0187] SEQ ID NO: 79: nucleotide sequence of a Cauliflower mosaic virus 35S promoter.

[0188] SEQ ID NO: 80: nucleotide sequence of a polubiquitin promoter from Coix lacryma- jobi.

[0189] SEQ ID NO: 81: nucleotide sequence of an Gos2 promoter 2 from Oryza sativa.

[0190] SEQ ID NO: 82: nucleotide sequence of a promoter from Mirabilis mosaic caulimovirus.

[0191] SEQ ID NO: 83: nucleotide sequence of a promoter from Peanut chlorotic streak caulimovirus.

[0192] SEQ ID NO: 84: nucleotide sequence of GA20 oxidase 2 cDNA from Sorghum bicolor.

[0193] SEQ ID NO: 85: nucleotide sequence of GA20 oxidase 2 coding sequence from Sorghum bicolor.

[0194] SEQ ID NO: 86: amino acid sequence of GA20 oxidase 2 from Sorghum bicolor.

[0195] SEQ ID NO: 87: genomic nucleotide sequence of GA20 oxidase 2 from Sorghum bicolor.

[0196] SEQ ID NO: 88: nucleotide sequence of GA20 oxidase 2-like cDNA from Setarica italica.

[0197] SEQ ID NO: 89: nucleotide sequence of GA20 oxidase 2-like coding sequence from Setarica italica.

[0198] SEQ ID NO: 90: amino acid sequence of GA20 oxidase 2-like from Setarica italica.

[0199] SEQ ID NO: 91: genomic nucleotide sequence of GA20 oxidase 2-like from Setarica italica.

[0200] SEQ ID NO: 92: nucleotide sequence of GA20 oxidase 2 cDNA from Oryza sativa.

[0201] SEQ ID NO: 93: nucleotide sequence of GA20 oxidase 2 coding sequence from Oryza sativa.

[0202] SEQ ID NO: 94: amino acid sequence of GA20 oxidase 2 gene from Oryza sativa.

[0203] SEQ ID NO: 95: genomic nucleotide sequence of GA20 oxidase 2 from Oryza sativa.

[0204] SEQ ID NO: 96: nucleotide sequence of GA20 oxidase-D2 coding sequence from Triticum aestivum.

[0205] SEQ ID NO: 97: amino acid sequence of GA20 oxidase-D2 from Triticum aestivum.

[0206] SEQ ID NO: 98: genomic nucleotide sequence of GA20 oxidase-D2 from Triticum aestivum.

[0207] SEQ ID NO: 99: nucleotide sequence of Fe2OG dioxygenase cDNA from Hordeum vulgare.

[0208] SEQ ID NO: 100: nucleotide sequence of Fe2OG dioxygenase coding sequence from Hordeum vulgare.

[0209] SEQ ID NO: 101: amino acid sequence of Fe2OG dioxygenase from Hordeum vulgare.

[0210] SEQ ID NO: 102: nucleotide sequence of a probable 2-ODD cDNA from Sorghum bicolor.

[0211] SEQ ID NO: 103: nucleotide sequence of a probable 2-ODD coding sequence from Sorghum bicolor.

[0212] SEQ ID NO: 104: amino acid sequence of a probable 2-ODD gene from Sorghum bicolor.

[0213] SEQ ID NO: 105: genomic nucleotide sequence of a probable 2-ODD gene from Sorghum bicolor.

[0214] SEQ ID NO: 106: nucleotide sequence of a flavonol synthase / flavanone 3- hydroxylase-like cDNA from Setarica italica.

[0215] SEQ ID NO: 107: nucleotide sequence of a flavonol synthase / flavanone 3- hydroxylase-like coding sequence from Setarica italica.

[0216] SEQ ID NO: 108: amino acid sequence of a flavonol synthase / flavanone 3- hydroxylase-like gene from Setarica italica.

[0217] SEQ ID NO: 109: genomic nucleotide sequence of a flavonol synthase / flavanone 3- hydroxylase-like gene from Setarica italica.

[0218] SEQ ID NO: 110: nucleotide sequence of a naringenin, 2-oxoglutarate 3-dioxygenase cDNA from Oryza sativa.

[0219] SEQ ID NO: 111: nucleotide sequence of a naringenin, 2-oxoglutarate 3-dioxygenase coding sequence from Oryza sativa.

[0220] SEQ ID NO: 112: amino acid sequence of a naringenin, 2-oxoglutarate 3-dioxygenase gene from Oryza sativa.

[0221] SEQ ID NO: 113: genomic nucleotide sequence of a naringenin, 2-oxoglutarate 3- dioxygenase gene from Oryza sativa.

[0222] SEQ ID NO: 114: nucleotide sequence of a Fe2OG dioxygenase cDNA from Triticum aestivum.

[0223] SEQ ID NO: 115: nucleotide sequence of a Fe2OG dioxygenase coding sequence from Triticum aestivum.

[0224] SEQ ID NO: 116: amino acid sequence of a Fe2OG dioxygenase gene from Triticum aestivum.

[0225] SEQ ID NO: 117: genomic nucleotide sequence of a Fe2OG dioxygenase gene from Triticum aestivum.

[0226] SEQ ID NO: 118: amino acid sequence of a Fe2OG dioxygenase gene from Hordeum vulgare.

[0227] SEQ ID NO: 119: nucleotide sequence of a GA3-beta-dioxygenase 2-2 cDNA from Sorghum bicolor.

[0228] SEQ ID NO: 120: nucleotide sequence of a GA3-beta-dioxygenase 2-2 coding sequence from Sorghum bicolor.

[0229] SEQ ID NO: 121: amino acid sequence of a GA3-beta-dioxygenase 2-2 gene from Sorghum bicolor.

[0230] SEQ ID NO: 122: genomic nucleotide sequence of a GA3-beta-dioxygenase 2-2 gene from Sorghum bicolor.

[0231] SEQ ID NO: 123: nucleotide sequence of a GA3-beta-dioxygenase 2-2-like cDNA from Setarica italica.

[0232] SEQ ID NO: 124: nucleotide sequence of a GA3-beta-dioxygenase 2-2-like coding sequence from Setarica italica.

[0233] SEQ ID NO: 125: amino acid sequence of a GA3-beta-dioxygenase 2-2-like gene from Setarica italica.

[0234] SEQ ID NO: 126: genomic nucleotide sequence of a GA3-beta-dioxygenase 2-2-like gene from Setarica italica.

[0235] SEQ ID NO: 127: nucleotide sequence of a GA3-beta-dioxygenase 2-3 cDNA from Oryza sativa.

[0236] SEQ ID NO: 128: nucleotide sequence of a GA3-beta-dioxygenase 2-3 coding sequence from Oryza sativa.

[0237] SEQ ID NO: 129: amino acid sequence of a GA3-beta-dioxygenase 2-3 gene from Oryza sativa.

[0238] SEQ ID NO: 130: genomic nucleotide sequence of a GA3-beta-dioxygenase 2-3 gene from Oryza sativa.

[0239] SEQ ID NO: 131: nucleotide sequence of a GA3-beta-hydroxylase cDNA from Hordeum vulgare.

[0240] SEQ ID NO: 132: nucleotide sequence of a GA3-beta-hydroxylase coding sequence from Hordeum vulgare.

[0241] SEQ ID NO: 133: amino acid sequence of a GA3-beta-hydroxylase gene from Hordeum vulgare.

[0242] SEQ ID NO: 134: nucleotide sequence of a GA3ox-D2 protein cDNA from Triticum aestivum.

[0243] SEQ ID NO: 135: nucleotide sequence of a GA3ox-D2 protein coding sequence from Triticum aestivum.

[0244] SEQ ID NO: 136: amino acid sequence of a GA3ox-D2 protein gene from Triticum aestivum.

[0245] SEQ ID NO: 137: genomic nucleotide sequence of a GA3ox-D2 protein gene from Triticum aestivum.

[0246] SEQ ID NO: 138: nucleotide sequence of synthetic construct GA20 oxidase_3-A.

[0247] SEQ ID NO: 139: nucleotide sequence of synthetic construct GA20 oxidase_3-B.

[0248] SEQ ID NO: 140: nucleotide sequence of synthetic construct GA20 oxidase_3-C.

[0249] SEQ ID NO: 141: nucleotide sequence of synthetic construct GA20 oxidase_3-D.

[0250] SEQ ID NO: 142: nucleotide sequence of synthetic construct GA20 oxidase_3-E.

[0251] SEQ ID NO: 143: nucleotide sequence of synthetic construct GA20 oxidase_3-F.

[0252] SEQ ID NO: 144: nucleotide sequence of synthetic construct GA20 oxidase_3-G.

[0253] SEQ ID NO: 145: nucleotide sequence of synthetic construct GA20 oxidase_3-H.

[0254] SEQ ID NO: 146: nucleotide sequence of synthetic construct GA20 oxidase_3-I.

[0255] SEQ ID NO: 147: nucleotide sequence of synthetic construct GA20 oxidase_3-J.

[0256] SEQ ID NO: 148: nucleotide sequence of synthetic construct GA20 oxidase_5-A.

[0257] SEQ ID NO: 149: nucleotide sequence of synthetic construct GA20 oxidase_5-B.

[0258] SEQ ID NO: 150: nucleotide sequence of synthetic construct GA20 oxidase_5-C.

[0259] SEQ ID NO: 151: nucleotide sequence of synthetic construct GA20 oxidase_5-D.

[0260] SEQ ID NO: 152: nucleotide sequence of synthetic construct GA20 oxidase_5-E.

[0261] SEQ ID NO: 153: nucleotide sequence of synthetic construct GA20 oxidase_5-F.

[0262] SEQ ID NO: 154: nucleotide sequence of synthetic construct GA20 oxidase_5-G.

[0263] SEQ ID NO: 155: nucleotide sequence of synthetic construct GA20 oxidase_5-H.

[0264] SEQ ID NO: 156: nucleotide sequence of synthetic construct GA20 oxidase_5-I.

[0265] SEQ ID NO: 157: nucleotide sequence of synthetic construct GA20 oxidase_5-J.

[0266] SEQ ID NO: 158: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-A.

[0267] SEQ ID NO: 159: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-B.

[0268] SEQ ID NO: 160: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-C.

[0269] SEQ ID NO: 161: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-D.

[0270] SEQ ID NO: 162: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-E.

[0271] SEQ ID NO: 163: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-F.

[0272] SEQ ID NO: 164: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-G.

[0273] SEQ ID NO: 165: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-H.

[0274] SEQ ID NO: 166: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-I.

[0275] SEQ ID NO: 167: nucleotide sequence of synthetic construct GA20 oxidase_3 / 5-J.

[0276] SEQ ID NO: 168: nucleotide sequence of genomic sequence of GA3 oxidase_1 of Zea mays inclusive 3 kb promoter and 3kb downstream of 3' UTR.

[0277] SEQ ID NO: 169: nucleotide sequence of genomic sequence of GA3 oxidase_2 of Zea mays inclusive 3 kb promoter and 3kb downstream of 3' UTR.

[0278] SEQ ID NO: 170: nucleotide sequence of genomic sequence of GA3 oxidase_3 of Zea mays inclusive 3 kb promoter and 3kb downstream of 3' UTR.

[0279] SEQ ID NO: 171: nucleotide sequence of cDNA sequence of GA3 oxidase_3 of Zea mays.

[0280] SEQ ID NO: 172: nucleotide sequence of coding sequence of GA3 oxidase_3 of Zea mays.

[0281] SEQ ID NO: 173: amino acid sequence of GA3 oxidase_3 protein of Zea mays.

[0282] SEQ ID NO: 174: nucleotide sequence of genomic sequence of GA3 oxidase_1 of Zea mays 01DKD2.

[0283] SEQ ID NO: 175: nucleotide sequence of genomic sequence of GA3 oxidase_2 of Zea mays 01DKD2.

[0284] SEQ ID NO: 176: nucleotide sequence of genomic sequence of GA3 oxidase_3 of Zea mays 01DKD2.

[0285] SEQ ID NO: 177: nucleotide sequence of SP1 (GA3ox13’UTR).

[0286] SEQ ID NO: 178: nucleotide sequence of SP2 (GA3ox13’UTR).

[0287] SEQ ID NO: 179: nucleotide sequence of SP3 (GA3ox15’UTR).

[0288] SEQ ID NO: 180: nucleotide sequence of SP4 (GA3ox15’UTR).

[0289] SEQ ID NO: 181: nucleotide sequence of SP5 (GA3ox1 promoter).

[0290] SEQ ID NO: 182: nucleotide sequence of SP6 (GA3ox1 promoter).

[0291] SEQ ID NO: 183: nucleotide sequence of SP7 (GA3ox1 promoter).

[0292] SEQ ID NO: 184: nucleotide sequence of SP8 (GA3ox1 promoter).

[0293] SEQ ID NO: 185: nucleotide sequence of SP9 (GA3ox1 promoter).

[0294] SEQ ID NO: 186: nucleotide sequence of SP10 (GA3ox1 promoter).

[0295] SEQ ID NO: 187: nucleotide sequence of SP11 (GA3ox1 promoter).

[0296] SEQ ID NO: 188: nucleotide sequence of SP12 (GA3ox1 promoter).

[0297] SEQ ID NO: 189: nucleotide sequence of SP13 (GA3ox2 upstream).

[0298] SEQ ID NO: 190: nucleotide sequence of SP14 (GA3ox2 downstream).

[0299] SEQ ID NO: 191: nucleotide sequence of SP15 (GA3ox3 upstream).

[0300] SEQ ID NO: 192: nucleotide sequence of SP16 (GA3ox3 downstream).

[0301] SEQ ID NO: 193: nucleotide sequence of a maize reproductive tissue preferred promoter (Zea mays).

[0302] SEQ ID NO: 194: amino acid sequence of a Cpf1 protein from Lachnospiraceae bacterium.

[0303] SEQ ID NO: 195: amino acid sequence of an NLS signal from Solanum lycopersicum (HSFA1).

[0304] SEQ ID NO: 196: nucleotide sequence of a synthetic POL III promoter (GSP2262).

[0305] SEQ ID NO: 197: nucleotide sequence of Scaffold RNA SC1 derived from Lachnospiraceae bacterium.

[0306] SEQ ID NO: 198: nucleotide sequence of a constitutive maize ubiquitin promoter (Zea mays).

[0307] SEQ ID NO: 199: amino acid sequence of a Cpf1 protein from Francisella tularensis subsp. novicida.

[0308] SEQ ID NO: 200: amino acid sequence of an NLS signal from Solanum tuberosum (NLS5).

[0309] SEQ ID NO: 201: amino acid sequence of an NLS signal from Solanum lycopersicum (HSFA1).

[0310] SEQ ID NO: 202: nucleotide sequence of Scaffold RNA SC1 derived from Francisella tularensis.

[0311] SEQ ID NO: 203: nucleotide sequence of a synthetic POL III promoter (GSP2269).

[0312] SEQ ID NO: 204: nucleotide sequence of AtAG1 gene from Arabidopsis thaliana.

[0313] SEQ ID NO: 205: nucleotide sequence of SP17 (AtAG1).

[0314] SEQ ID NO: 206: nucleotide sequence of SP18 (AtAG1).

[0315] SEQ ID NO: 207: nucleotide sequence of SP19 (AtAG1).

[0316] SEQ ID NO: 208: nucleotide sequence of SP20 (AtAG1).

[0317] SEQ ID NO: 209: nucleotide sequence of SP21 (AtAG1).

[0318] SEQ ID NO: 210: nucleotide sequence of a CaMV35S promoter.

[0319] SEQ ID NO: 211: nucleotide sequence of BRI1 gene from Arabidopsis thaliana.

[0320] SEQ ID NO: 212: nucleotide sequence of Dwarf1 gene from Arabidopsis thaliana.

[0321] SEQ ID NO: 213: nucleotide sequence of PIN1 gene from Arabidopsis thaliana.

[0322] SEQ ID NO: 214: amino acid sequence of AtAG1 gene from Arabidopsis thaliana.

[0323] SEQ ID NO: 215: amino acid sequence of BRI1 gene from Arabidopsis thaliana.

[0324] SEQ ID NO: 216: amino acid sequence of Dwarf1 gene from Arabidopsis thaliana.

[0325] SEQ ID NO: 217: amino acid sequence of PIN1 gene from Arabidopsis thaliana.

[0326] SEQ ID NO: 218: nucleotide sequence, codon optimized, coding for LbCPf1.

[0327] SEQ ID NO: 219: nucleotide sequence, codon optimized, coding for FnCPf1.

[0328] SEQ ID NO: 220: nucleotide sequence of promoter, leader sequence and intron of ubiquitin gene from Medicago truncatula.

[0329] SEQ ID NO: 221: nucleotide sequence of a terminator sequence from Medicago truncatula.

[0330] SEQ ID NO: 222: nucleotide sequence of U6 promoter from Arabidopsis thaliana.

[0331] SEQ ID NO: 223: nucleotide sequence of inverted 5’ UTR of AtAG1.

[0332] SEQ ID NO: 224: nucleotide sequence of ORF of AtAG1.

[0333] SEQ ID NO: 225: nucleotide sequence of terminator of FbL2 gene from Gossypium barbadense.

[0334] SEQ ID NO: 226: nucleotide sequence of inverted 3’ UTR of AtAG1.

[0335] SEQ ID NO: 227: nucleotide sequence of SP22 targeting a region in At.BRI1.

[0336] SEQ ID NO: 228: nucleotide sequence of SP23 targeting a region in At.BRI1.

[0337] SEQ ID NO: 229: nucleotide sequence of inverted 5’ UTR of AtBRI1.

[0338] SEQ ID NO: 230: nucleotide sequence of ORF of AtBRI1.

[0339] SEQ ID NO: 231: nucleotide sequence of inverted 3’ UTR of AtBRI1.

[0340] SEQ ID NO: 232: nucleotide sequence of GA3ox1 genomic seq from Zea mays comprising the 2000bp of promoter sequence upstream of transcription start site, the 5’UTR sequence, the coding sequence and 3’ UTR sequence.

[0341] SEQ ID NO: 233: nucleotide sequence of the 3’UTR of GA3ox1 gene from Zea mays.

[0342] SEQ ID NO: 234: nucleotide sequence of the genomic sequence of the edited allele (S049) of GA3ox1 from Zea mays comprising the 2000bp of promoter sequence upstream of transcription start site, the 5’UTR sequence, the coding sequence and the edited 3’ UTR sequence .

[0343] SEQ ID NO: 235: nucleotide sequence of the 3’UTR region of the edited allele (S049) of GA3ox1 gene from Zea mays. DETAILED DESCRIPTION OF THE INVENTION

[0344] Unless defined otherwise, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosurebelongs. Where a term is provided in the singular, the inventors also contemplate aspects of the disclosure described by the plural of that term. Where there are discrepancies in terms and definitions used in references that are incorporated by reference, the terms used in this application shall have the definitions given herein. Other technical terms used have their ordinary meaning in the art in which they are used, as exemplified by various art-specific dictionaries, for example, “The American Heritage® Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), the “McGraw-Hill Dictionary of Scientific and Technical Terms” (6th edition, 2002, McGraw-Hill, New York), or the “Oxford Dictionary of Biology” (6th edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited to a mechanism or mode of action. Reference thereto is provided for illustrative purposes only.

[0345] The practice of this disclosure includes, unless otherwise indicated, conventional techniques of biochemistry, chemistry, molecular biology, microbiology, cell biology, plant biology, genomics, biotechnology, and genetics, which are within the skill of the art. See, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th edition (2012); Current Protocols In Molecular Biology (F. M. Ausubel, et al. eds., (1987)); Plant Breeding Methodology (N.F. Jensen, Wiley-Interscience (1988)); the series Methods In Enzymology (Academic Press, Inc.): PCR 2: A Practical Approach (M. J. MacPherson, B. D. Hames and G. R. Taylor eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (R. I. Freshney, ed. (1987)); Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences; C. N. Stewart, A. Touraev, V. Citovsky, T. Tzfira eds. (2011) Plant Transformation Technologies (Wiley-Blackwell); and R. H. Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.).

[0346] Any references cited herein, including, e.g., all patents, published patent applications, and non-patent publications, are incorporated herein by reference in their entirety.

[0347] When a grouping of alternatives is presented, any and all combinations of the members that make up that grouping of alternatives is specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision each alternative individually (e.g., A alone, B alone, etc.), as well as combinations such as A, B, and D; A and C; B and C; etc.

[0348] As used herein, terms in the singular and the singular forms “a,” “an,” and “the,” for example, include plural referents unless the content clearly dictates otherwise.

[0349] Any composition, nucleic acid molecule, polypeptide, cell, plant, etc. provided herein is specifically envisioned for use with any method provided herein.

[0350] As used herein, the term "heterozygous" refers to a genetic status wherein different alleles reside at corresponding loci on homologous chromosomes.

[0351] As used herein, the term "homozygous" refers to a genetic status wherein identical alleles reside at corresponding loci on homologous chromosomes.

[0352] As used herein, the term "allele" refers to one of two or more different nucleotides or 30 nucleotide sequences that occur at a specific locus.

[0353] As used herein, the term “heteroallelic” refers to the presence of two different alleles at the same genetic locus.

[0354] The term "gene expression" refers to the process of converting genetic information encoded in genomic DNA into RNA (e.g., mRNA, rRNA, tRNA, or 25 snRNA) through transcription of the gene via the enzymatic action of an RNA polymerase, and into protein, through translation of mRNA.

[0355] As used herein, the phrases "inhibition of gene expression" or "gene suppression" or "silencing a target gene" and similar terms and phrases refer to the absence or observable reduction in the level of protein and / or mRNA product from the target gene. The consequences of inhibition, suppression, or silencing can be confirmed by phenotypes of a cell or organism or by biochemical techniques.

[0356] As used herein the term "dsRNA" or “dsRNA region” or “double stranded RNA region” relates to two strands of anti-parallel poly-ribonucleic acids held together by base pairing. The dsRNA molecule may be formed by intramolecular hybridization or intermolecular hybridization. In some embodiments, the dsRNA may comprise a single strand of RNA that self-hybridizes to form a hairpin or stem loop structure having an at least partially double- stranded structure including at least one segment that will hybridize to an RNA transcribed from the gene targeted for suppression. In some embodiments, the dsRNA may comprise two separate strands of RNA that hybridize through complementary base pairing. The RNA strands may or may not be polyadenylated; the RNA strands may or may not be capable of being translated into a polypeptide by a cell's translational apparatus. The two strands can be of identical length or of different lengths provided there is enough sequence homology between the two strands that a double stranded structure is formed with at least 80%, 90%, 95% or 100% complementarity over the entire length.

[0357] As used herein, “an inverted DNA sequence” may refer, depending on the context, to either the DNA sequence prior to the inversion according to the methods in the description, or the DNA sequence resulting after the inversion. Thus, any reference to the nucleotide sequence of the inverted DNA region may refer to a nucleotide sequence having at least acertain percentage of sequence identity to at least part of a non-coding sequence, such as an untranslated region (UTR) or intron sequence or to a nucleotide sequence having at least a certain percentage of sequence complementarity to at least part of a non-coding sequence such as an untranslated region (UTR) or intron sequence. Both methods of reference are used interchangeably. When referring to a sequence of the RNA transcribed from the inverted DNA sequence it is usually stated that such transcribed RNA comprises an antisense RNA sequence which has a certain percentage of complementarity.

[0358] The current disclosure enables development of an RNAi (RNA interference) based editing system for dominant suppression of loci, which may result in strong and sometimes unwanted phenotypes in hybrid crops, without the occurrence of phenotype linked to the edited loci in the homozygous state.

[0359] This disclosure allows the use of mutant alleles exhibiting strong, unwanted phenotypes in commercial pipelines due to the absence of phenotypes in the production pipeline (in the case described would be excessively short homozygous parent plants in seed production fields) while also mitigating the strong phenotypes to a moderate level in hybrid / heterozygous plants due the mitigated levels of RNAi suppression that result from antisense UTR pairing in the mRNA transcripts.

[0360] Without intending to limit the invention to a particular mode of action, it is thought that the inversion of a part or the entirety of at least one untranslated region of a single gene / locus (and resultant mRNA) without the perturbance of the coding region, such as inversion of a part or all of the 5’ or 3’ untranslated region (or both), results in a silent mutation within the mRNAs when present in homozygous state. Interaction of the mRNAs determines the outcome in that edited mRNAs with the inverted UTRs will not interact with each other – thus in a uniform pool of edited mRNAs (in a homozygous edit plant) – no interactions will occur and no phenotype will be produced. Only in a pool of edited mRNA and mRNAs from alleles without the inversion, will antisense base pairing occur between species of mRNAs to produce double stranded RNA region, triggering RNA silencing mechanisms (ribosome stalling, RNA transcription stalling, post-transcriptional degradation, destabilization etc.) that result in the reduction of viable transcript levels – thus producing a knock down phenotype or reduced expression in the plant (see e.g. Roy B, Jacobson A. The intimate relationships of mRNA decay and translation. Trends Genet.2013;29:691–699).

[0361] The methods and compositions disclosed herein can also be used to produce moderate phenotypes from previously described alleles with strong unwanted phenotypes in ahybrid dominant mechanism without the occurrence of any mutant / edit phenotype in the homozygous state that would detrimentally impact seed or plant production at larger scale.

[0362] In one aspect, a method is provided for editing the genome of a plant cell to modify an endogenous gene, comprising the steps of a) generating a first double-stranded break and a second double stranded break using a targeted editing technique targeting an untranslated region of said endogenous gene, in the plant cell without the perturbance of the coding region; and b) isolating a modified plant cell comprising a modified allele of the endogenous gene wherein the modified allele comprises an inverted DNA sequence of at least part of the untranslated region of the endogenous gene and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said untranslated region, wherein the modified allele does not comprise a sense sequence complementary to the antisense sequence of part of said untranslated region. In one aspect, the transcription of the modified allele does not yield an RNA molecule comprising a stem-loop structure or intramolecular double stranded RNA region. In one aspect, the inverted DNA sequence of at least part of the untranslated region of the endogenous gene encodes an antisense RNA sequence that is at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of the untranslated region of the gene. In one aspect, the modified allele does not comprise a sense nucleotide sequence of more than 17 nucleotides complementary to the antisense sequence of inverted part of the untranslated region.

[0363] In another aspect, a method is provided for modifying expression of an endogenous gene in a hybrid plant while leaving the expression of the gene unaffected in a parent plant or plants, comprising the steps of a) identifying an endogenous gene in a plant wherein expression of a variant allele of said gene result in unwanted phenotypes when present in homozygous state; b) providing a first plant comprising a modified allele of the gene comprising a nucleic acid region which is an inversion of a part of the gene whereby the inversion does not affect translation of the modified allele, and wherein the first plant comprises the modified allele of the gene homozygously; c) crossing said first plant with a second plant comprising an unmodified allele of the gene and not comprising the inversion not affecting translation of said gene wherein the unmodified gene is in homozygous state; and d) obtaining a hybrid seed comprising the modified and unmodified allele of said gene in heterozygous or heteroallelic form. In one aspect of this method, upon transcription of the modified allele andthe unmodified allele into an RNA molecule, a double stranded RNA region can be formed by base-pairing between the nucleic acid region which is an inversion of part of an untranslated region of the gene in the RNA transcript of the modified allele and the nucleic acid region in the RNA transcript of the unmodified allele, and wherein the double stranded RNA region is capable of inhibiting expression of the modified allele and the unmodified allele by RNA silencing mechanisms, such as stalling of RNA translation, stalling of RNA transcription, destabilization of the resulting RNA molecules or post-transcriptional degradation of the transcribed RNA molecules. In one aspect, said nucleic acid region which is an inversion of a part of said gene results upon transcription in an antisense RNA sequence that is at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of the untranslated region of the gene.

[0364] In yet another aspect, a plant cell, plant or part or seed thereof comprising a modified allele of an endogenous gene is provided wherein the modified allele comprises an inverted DNA sequence of at least part of an untranslated region of said endogenous gene, and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said untranslated region, wherein the modified allele does not comprise a sense nucleotide sequence of more than 17 nucleotides complementary to the antisense sequence of part of said untranslated region. In an aspect, the plant cell, plant or part or seed thereof comprises a modified allele of an endogenous gene as herein described, in homozygous state. In one aspect, the expression of the modified allele is not reduced. In another aspect the modified allele is present in heterozygous state. In yet another aspect, the modified allele is present in heteroallelic state. In an aspect, the other allele is an unmodified version of the endogenous gene, and does not comprise the inverted DNA sequence of the untranslated region of the endogenous gene. In an aspect, expression of the modified allele and the unmodified allele is reduced in heteroallelic plants. In one aspect, the heteroallelic plant exhibits a commercially interesting phenotype. In one aspect, the plant is corn and the commercially interesting phenotype is short stature.

[0365] The various embodiments of this disclosure have several features in common which will be described in more detail hereafter. It will be clear that the following description of the features can be combined with each of the main aspects of the current disclosure.

[0366] A shared feature of all aspects of the current disclosure is that the modified allele of the endogenous gene comprises an inversion of part or all of at least one untranslated region of the endogenous gene. As used herein an “untranslated region” is a region of a gene which is transcribed into RNA but is not translated into a polypeptide. Polypeptide encoding endogenous gene contain a region with is transcribed and translated (“coding region”) but may contain a DNA sequence located upstream or 5’ of the coding region which is transcribed but not translated (“5’UTR”) or a DNA sequence located downstream or 3’ of the coding region, which is transcribed but not translated (“3’UTR”). Other untranslated regions of endogenous genes which may be suitable for the various aspects of the current disclosure are introns. As used herein, “Introns” are nucleotide sequences in genomic DNA and transcribed RNA (as referred to as heteronuclear RNA or hnRNA) of genes, particularly eukaryotic genes, that do not directly code for proteins, and are removed during the precursor messenger RNA (pre-mRNA) stage of maturation of mRNA by RNA splicing. Introns may be located in the 5’ or 3’ UTRs of an endogenous gene. The methods of the current disclosure are applicable to introns also, particularly if the intron wherein part or all of the intron is inverted in the modified allele, is retained during the RNA maturation process e.g. due to interference with the splice signals or because the inversion results in an intron no longer recognized as such during the RNA splicing process. The inversion of part or all of an untranslated region in a modified allele of an endogenous gene may result upon transcription in an RNA molecule comprising an antisense RNA sequence that is at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of the untranslated region of the gene. In one aspect, the inversion does not result in formation of an intramolecular double stranded RNA region or stem-loop structure in the transcribed RNA molecule. In other words, the antisense sequence in the modified allele of the endogenous gene does not have a complementary sense nucleotide of more than 17 nucleotides in the modified allele of the endogenous gene.

[0367] Another shared feature of all aspects of the current disclosure is that the modified allele of the endogenous gene comprises an inversion of part or all of an untranslated region of the endogenous gene may be obtained by targeted genome editing techniques through generation of a first and second double-stranded break in the untranslated region and isolation of a modified allele of the endogenous gene wherein the DNA sequence located between the first and second double stranded break, or part thereof, is re-inserted in inverted orientationthrough non-homologous end joining. Alternatively, a template or donor nucleic acid may be used wherein part or all of the untranslated region is present in antisense orientation. Targeted gene editing techniques are well known in the art and include use of a RNA guided effector protein and RNA guides or a TALE protein or a custom meganuclease or Zn-finger protein.

[0368] RNA guided effector protein include a CRISPR-Cas effector protein , selected from a Type I CRISPR-Cas system, a Type II CRISPR-Cas system, a Type III CRISPR-Cas system, a Type IV CRISPR-Cas system, Type V CRISPR-Cas system, or a Type VI CRISPR- Cas system, or a CRISPR-Cas effector protein derived therefrom, optionally a CRISPR-Cas effector protein comprising one or more nuclear localization signals, such as a CRISPR-Cas effector protein selected from a Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c effector protein as elaborated below. Guide nucleic acids for use with RNA guided effector proteins are also described in more detail below.

[0369] In one aspect, the inversion of at least part of at least one untranslated region of an endogenous gene may result from templated editing, whereby a single DNA break, such as mediated by a RNA guided endonuclease and guide RNA, is sufficient to allow the introduction of the inverted portion of the untranslated region of the endogenous gene provided via a template or donor nucleic acid wherein part or all of the untranslated region is present in antisense orientation. Parameters for the inverted portion of the untranslated region are as described elsewhere in this document. The template nucleic acid or the inverted portion may be inserted at the DNA break, via non-homologous end-joining or via homology dependent repair mechanisms. In the latter case, the presence of at least one homology arm, and preferably two homology arms in the template nucleic acid is preferred, whereby the homology arms flank the inverted portion of the untranslated region, and whereby the homology arms have sufficient sequence identity or complementarity to allow hybridization with the nucleotide sequence of the untranslated region, such as the nucleotide sequences of the untranslated region flanking the DNA break.

[0370] Another example of templated editing allowing the creation of inversions of at least a portion of an untranslated region of an endogenous gene as herein described, involvesprime -editing, whereby the inverted portion of the untranslated region is introduced through reverse transcription of an extended guide RNA specifying the target site and further comprising a template nucleotide sequence comprising a nucleotide sequence corresponding to the untranslated region wherein a part of the untranslated region is in inverted orientation, as described elsewhere in this document. Such reverse transcription and introduction into the target gene can be achieved by simultaneous action of a RNA guided endonuclease, such as Cas9 or Cas12 or a nickase generating single stranded DNA breaks or nicks, derived from such RNA guided endonuclease, and a polymerase such as a reverse transcriptase, optionally fused into one protein, as reviewed by Chen and Liu, 2023 (Prime editing for precise and highly versatile genome manipulation, Nat. Rev. Genetic.24(3); 161-177) or as described by Kim et al., 2022 (A novel mechanistic framework for precise sequence replacement using reverse transcriptase and diverse CRISPR-Cas systems DOI:10.1101 / 2022.12.13.520319). These prime-editing methods are also described in WO2020191153, WO2020191171, WO2020191233, WO2020191234, WO2020191239, WO2020191241, WO2020191242, WO2020191243, WO2020191245, WO2020191246, WO2020191248, WO2020191249, WO2021 / 092130 and WO2022 / 047135 (all herein incorporated by reference).

[0371] Accordingly, in one aspect, a method is provided for editing the genome of a plant cell to modify an endogenous gene, comprising the steps of a) generating a double-stranded break or single stranded break using a targeted editing technique targeting at least one untranslated region of said endogenous gene, in said plant cell without the perturbance of the coding region; b) providing at least one template nucleic acid to said plant cell wherein said template nucleic acid comprises a portion of said at least one untranslated region of said endogenous gene in inverted orientation; and c) isolating a modified plant cell comprising a modified allele of said endogenous gene wherein the modified allele comprises an inverted DNA sequence of at least part of said at least one untranslated region of said endogenous gene and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said untranslated region, wherein the modified allele does not comprise a sense sequence complementary to the antisense sequence of part of said untranslated region.

[0372] The template nucleic acid may comprise a portion of at least one untranslated region of an endogenous gene in inverted orientation, and can be inserted the targeted untranslated region of the endogenous gene by non-homologous end-joining. The templatenucleic acid may otherwise comprises at least one, or two homology arms having homology to the nucleic acid sequence flanking the double stranded break, optionally wherein the homology arms are flanking the portion of the at least one untranslated region in inverted orientation and can be introduced into the untranslated region in inverted orientation by homology dependent repair.

[0373] In another aspect, a method is provided for prime-editing the genome of a plant cell to modify an endogenous gene, comprising the steps of a) generating a double-stranded break or a single-stranded break (“nick”) using a CRISPR / CAS fusion protein fused to a reverse transcriptase functional domain and a guide RNA targeting at least one untranslated region of said endogenous gene, in said plant cell without the perturbance of the coding region, wherein said guide RNA further comprises a nucleotide sequence acid comprises a portion of said at least one untranslated region of said endogenous gene in inverted orientation; and b) isolating a modified plant cell comprising a modified allele of said endogenous gene wherein the modified allele comprises an inverted DNA sequence of at least part of said at least one untranslated region of said endogenous gene and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said untranslated region, wherein the modified allele does not comprise a sense sequence complementary to the antisense sequence of part of said untranslated region.

[0374] As used herein, a “template nucleic acid molecule” refers to a nucleic acid molecule that comprises a nucleic acid sequence that is to be inserted into a target DNA molecule. In an aspect, a template nucleic acid molecule comprises single-stranded DNA. In another aspect, a template nucleic acid molecule comprises double-stranded DNA. In a further aspect, a template nucleic acid molecule comprises single-stranded RNA. In yet another aspect, a template nucleic acid molecule comprises double-stranded RNA. In another aspect, a template nucleic acid molecule comprises DNA and RNA.

[0375] In an aspect, a ribonucleoprotein comprises at least one template nucleic acid molecule. In another aspect, a ribonucleoprotein comprises at least two template nucleic acid molecules.

[0376] In an aspect, a template nucleic acid molecule comprises at least 10 nucleotides. In another aspect, a template nucleic acid molecule comprises at least 25 nucleotides. In another aspect, a template nucleic acid molecule comprises at least 50 nucleotides. In another aspect, atemplate nucleic acid molecule comprises at least 75 nucleotides. In another aspect, a template nucleic acid molecule comprises at least 100 nucleotides. In another aspect, a template nucleic acid molecule comprises at least 250 nucleotides. In another aspect, a template nucleic acid molecule comprises at least 500 nucleotides. In another aspect, a template nucleic acid molecule comprises at least 750 nucleotides. In another aspect, a template nucleic acid molecule comprises at least 1000 nucleotides. In another aspect, a template nucleic acid molecule comprises at least 2500 nucleotides.

[0377] In an aspect, a template nucleic acid molecule comprises between 10 nucleotides and 2500 nucleotides. In another aspect, a template nucleic acid molecule comprises between 25 nucleotides and 2500 nucleotides. In another aspect, a template nucleic acid molecule comprises between 50 nucleotides and 2500 nucleotides. In another aspect, a template nucleic acid molecule comprises between 75 nucleotides and 2500 nucleotides. In another aspect, a template nucleic acid molecule comprises between 100 nucleotides and 2500 nucleotides. In another aspect, a template nucleic acid molecule comprises between 250 nucleotides and 2500 nucleotides. In another aspect, a template nucleic acid molecule comprises between 500 nucleotides and 2500 nucleotides. In another aspect, a template nucleic acid molecule comprises between 25 nucleotides and 1000 nucleotides. In another aspect, a template nucleic acid molecule comprises between 25 nucleotides and 500 nucleotides. In another aspect, a template nucleic acid molecule comprises between 25 nucleotides and 250 nucleotides.

[0378] Template nucleic acids may be tethered to the gene editing component, particularly a CRISPR / Cas effector protein using various methods as described in the art. In one aspect, template nucleic acids can be tethered to the gene editing component, using HUH endonucleases as described in WO2021 / 025999 (herein incorporated by reference).

[0379] Template nucleic acids suitable for homology dependent recombination mediated knock-in of inverted repeats of the untranslated regions of an endogenous gene as described herein, may be single stranded template nucleic acids, double stranded template nucleic acids or circular template nucleic acids, as described in the art.

[0380] As elaborated elsewhere in this disclosure, the methods described herein are particularly suited for reduction of expression of endogenous genes having variants which produce phenotypes of potentially commercial interest, such as reduced plant height or reduced seed shattering, but which may also be detrimental or even non-viable, or may produce unwanted phenotypes on plant development or reproduction, particularly when present in homozygous state.

[0381] One example of a gene which can be used in the herein described methods and compositions is ind (indehiscence) involved in the normal development of a dehiscence zone of pods in Brassica plants. Nucleotide sequences, coding region, genomic sequences and untranslated regions of ind genes can be found in WO2004 / 113542, WO2009 / 068313 or WO2010 / 006732 (herein incorporated by reference).

[0382] Other examples of suitable genes are GA20 oxidase genes, including GA20 oxidase subtype 3 or 5, involved in gibberellin biosynthesis and plant height determination, such as GA20 oxidase genes from corn, as those disclosed in WO2018 / 035354, WO2020 / 243361 or WO2020 / 243363 (herein incorporated by reference).

[0383] Yet other examples of suitable genes are GA3 oxidase genes, including GA3 oxidase subtype 1, 2 or 3 involved in gibberellin biosynthesis and plant height determination, such as GA20 oxidase genes from corn, as disclosed herein or as disclosed in PCT / US2023 / 062985 (herein incorporated by reference).

[0384] Several of the GA oxidases in cereal plants consist of a family of related GA oxidase genes. For example, corn has a family of at least nine GA20 oxidase genes that includes GA20 oxidase_1, GA20 oxidase_2, GA20 oxidase_3, GA20 oxidase_4, GA20 oxidase_5, GA20 oxidase_6, GA20 oxidase_7, GA20 oxidase_8, and GA20 oxidase_9. However, there are three known or potential GA3 oxidase genes in corn, GA3 oxidase_1, GA3 oxidase_2, and GA3 oxidase_3. The DNA and protein sequences by SEQ ID NOs for each of these GA20 oxidase genes are provided in Table 1, and the DNA and protein sequences by SEQ ID NOs for each of these GA3 oxidase genes are provided in Table 2. Table 1. DNA and protein sequences by sequence identifier for GA20 oxidase genes in corn.Table 2. DNA and protein sequences by sequence identifier for GA3 oxidase genes in corn.

[0385] The genomic DNA sequence of GA20 oxidase_3 is provided in SEQ ID NO: 34, and the genomic DNA sequence of GA20 oxidase_5 is provided in SEQ ID NO: 35. For the GA20 oxidase_3 gene, SEQ ID NO: 34 provides 3000 nucleotides upstream of the GA20 oxidase_3 5’-UTR (nucleotides 1-3000); nucleotides 3001-3096 correspond to the 5’-UTR; nucleotides 3097-3665 correspond to the first exon; nucleotides 3666-3775 correspond to the first intron; nucleotides 3776-4097 correspond to the second exon; nucleotides 4098-5314 correspond to the second intron; nucleotides 5315-5584 correspond to the third exon; and nucleotides 5585-5800 correspond to the 3’-UTR. SEQ ID NO: 34 also provides 3000 nucleotides downstream of the end of the 3’-UTR (nucleotides 5801-8800). For the GA20 oxidase_5 gene, SEQ ID NO: 35 provides 3000 nucleotides upstream of the GA20 oxidase_5 start codon (nucleotides 1-3000); nucleotides 3001-3791 correspond to the first exon; nucleotides 3792-3906 correspond to the first intron; nucleotides 3907-4475 correspond to the second exon; nucleotides 4476-5197 correspond to the second intron; nucleotides 5198-5473correspond to the third exon; and nucleotides 5474-5859 correspond to the 3’-UTR. SEQ ID NO: 35 also provides 3000 nucleotides downstream of the end of the 3’-UTR (nucleotides 5860- 8859).

[0386] The genomic DNA sequence of GA3 oxidase_1 is provided in SEQ ID NO: 36, 168 and 174, the genomic DNA sequence of GA3 oxidase_2 is provided in SEQ ID NO: 37, 169 and 175, and the genomic DNA sequence of GA3 oxidase_3 is provided in SEQ ID NO: 170 and 176. While SEQ ID NOs: 36 and 37 provide 5’-UTR, exon, intron and 3’-UTR sequences for the GA3 oxidase_1 and GA3 oxidase_2 genes, respectively, SEQ ID NOs: 168 and 174 and SEQ ID NOs: 169 and 175 further provide upstream and downstream genomic sequences and additional 5’ and 3’ UTR sequences for the GA3 oxidase_1 and GA3 oxidase_2 genes, respectively.

[0387] For the GA3 oxidase_1 gene, nucleotides 1-29 of SEQ ID NO: 36 correspond to the 5’-UTR; nucleotides 30-514 of SEQ ID NO: 36 correspond to the first exon; nucleotides 515-879 of SEQ ID NO: 36 correspond to the first intron; nucleotides 880-1038 of SEQ ID NO: 36 correspond to the second exon; nucleotides 1039-1158 of SEQ ID NO: 36 correspond to the second intron; nucleotides 1159-1663 of SEQ ID NO: 36 correspond to the third exon; and nucleotides 1664-1788 of SEQ ID NO: 36 correspond to the 3’-UTR. Alternatively for the GA3 oxidase_1 gene, SEQ ID NO: 168 provides 3000 nucleotides upstream of the GA3 oxidase_1 5’-UTR (nucleotides 1-3000); nucleotides 3001-3161 of SEQ ID NO: 168 correspond to the 5’-UTR; nucleotides 3162-3646 of SEQ ID NO: 168 correspond to the first exon; nucleotides 3647-4011 of SEQ ID NO: 168 correspond to the first intron; nucleotides 4012-4170 of SEQ ID NO: 168 correspond to the second exon; nucleotides 4171-4290 of SEQ ID NO: 168 correspond to the second intron; nucleotides 4291-4795 of SEQ ID NO: 168 correspond to the third exon; and nucleotides 4796-5406 of SEQ ID NO: 168 correspond to the 3’-UTR. SEQ ID NO: 168 also provides 3000 nucleotides downstream of the end of the 3’- UTR (nucleotides 5407-8406). Alternatively for the GA3 oxidase_1 gene, SEQ ID NO: 174 provides 7620 nucleotides upstream of the GA3 oxidase_15’-UTR (nucleotides 1-5620 of SEQ ID NO: 174 correspond to upstream intergenic sequence, and nucleotides 5621-7620 of SEQ ID NO: 174 correspond to the GA3 oxidase_1 promoter region); nucleotides 7621-8029 of SEQ ID NO: 174 correspond to the 5’-UTR; nucleotides 8030-8514 of SEQ ID NO: 174 correspond to the first exon; nucleotides 8515-8887 of SEQ ID NO: 174 correspond to the first intron; nucleotides 8888-9046 of SEQ ID NO: 174 correspond to the second exon; nucleotides 9047- 9166 of SEQ ID NO: 174 correspond to the second intron; nucleotides 9167-9671 of SEQ ID NO: 174 correspond to the third exon; and nucleotides 9672-10276 of SEQ ID NO: 174correspond to the 3’-UTR. SEQ ID NO: 174 also provides 3951 nucleotides of intergenic sequence downstream of the end of the 3’-UTR (nucleotides 10277-14227 of SEQ ID NO: 174).

[0388] For the GA3 oxidase_2 gene, nucleotides 1-38 of SEQ ID NO: 37 correspond to the 5-UTR; nucleotides 39-532 of SEQ ID NO: 37 correspond to the first exon; nucleotides 533-692 of SEQ ID NO: 37 correspond to the first intron; nucleotides 693-851 of SEQ ID NO: 37 correspond to the second exon; nucleotides 852-982 of SEQ ID NO: 37 correspond to the second intron; nucleotides 983-1445 of SEQ ID NO: 37 correspond to the third exon; and nucleotides 1446-1698 of SEQ ID NO: 37 correspond to the 3’-UTR. Alternatively for the GA3 oxidase_2 gene, SEQ ID NO: 169 provides 3000 nucleotides upstream of the GA3 oxidase_2 5’-UTR (nucleotides 1-3000); nucleotides 3001-3056 of SEQ ID NO: 169 correspond to the 5’-UTR; nucleotides 3057-3550 of SEQ ID NO: 169 correspond to the first exon; nucleotides 3551-3710 of SEQ ID NO: 169 correspond to the first intron; nucleotides 3711-3869 of SEQ ID NO: 169 correspond to the second exon; nucleotides 3870-3991 of SEQ ID NO: 169 correspond to the second intron; nucleotides 3992-4463 of SEQ ID NO: 169 correspond to the third exon; and nucleotides 4464-4581 of SEQ ID NO: 169 correspond to the 3’-UTR. SEQ ID NO: 169 also provides 3000 nucleotides downstream of the end of the 3’- UTR (nucleotides 4582-7581). Alternatively for the GA3 oxidase_2 gene, SEQ ID NO: 175 provides 7285 nucleotides upstream of the GA3 oxidase_25’-UTR (nucleotides 1-5385 of SEQ ID NO: 175 correspond to upstream intergenic sequence, and nucleotides 5386-7385 of SEQ ID NO: 175 correspond to the GA3 oxidase_2 promoter region); nucleotides 7386-7831 of SEQ ID NO: 175 correspond to the 5’-UTR; nucleotides 7832-7926 of SEQ ID NO: 175 correspond to the first exon; nucleotides 7927-8086 of SEQ ID NO: 175 correspond to the first intron; nucleotides 8087-8245 of SEQ ID NO: 175 correspond to the second exon; nucleotides 8246- 8371 of SEQ ID NO: 175 correspond to the second intron; nucleotides 8372-8861 of SEQ ID NO: 175 correspond to the third exon; and nucleotides 8862-8967 of SEQ ID NO: 175 correspond to the 3’-UTR. SEQ ID NO: 175 also provides 7630 nucleotides of intergenic sequence downstream of the end of the 3’-UTR (nucleotides 8968-16597 of SEQ ID NO: 175).

[0389] For the GA3 oxidase_3 gene, SEQ ID NO: 170 provides 3000 nucleotides upstream of the GA3 oxidase_35’-UTR (nucleotides 1-3000); nucleotides 3001-3130 of SEQ ID NO: 170 correspond to the 5’-UTR; nucleotides 3131-3483 of SEQ ID NO: 170 correspond to the first exon; nucleotides 3484-3582 of SEQ ID NO: 170 correspond to the first intron; nucleotides 3583-3907 of SEQ ID NO: 170 correspond to the second exon; nucleotides 3908- 3998 of SEQ ID NO: 170 correspond to the second intron; nucleotides 3999-4274 of SEQ ID NO: 170 correspond to the third exon; and nucleotides 4275-4332 of SEQ ID NO: 170correspond to the 3’-UTR. SEQ ID NO: 170 also provides 3000 nucleotides downstream of the end of the 3’-UTR (nucleotides 4333-7332). Alternatively for the GA3 oxidase_3 gene, SEQ ID NO: 176 provides 7546 nucleotides upstream of the GA3 oxidase_3 5’-UTR (nucleotides 1-5546 of SEQ ID NO: 176 correspond to upstream intergenic sequence, and nucleotides 5547-7546 of SEQ ID NO: 176 correspond to the GA3 oxidase_3 promoter region); nucleotides 7547-7751 of SEQ ID NO: 176 correspond to the 5’-UTR; nucleotides 7752-8104 of SEQ ID NO: 176 correspond to the first exon; nucleotides 8105-8205 of SEQ ID NO: 176 correspond to the first intron; nucleotides 8206-8530 of SEQ ID NO: 176 correspond to the second exon; nucleotides 8531-8621 of SEQ ID NO: 176 correspond to the second intron; nucleotides 8622-8903 of SEQ ID NO: 176 correspond to the third exon; and nucleotides 8904- 9178 of SEQ ID NO: 176 correspond to the 3’-UTR. SEQ ID NO: 176 also provides 6176 nucleotides of intergenic sequence downstream of the end of the 3’-UTR (nucleotides 9179- 15354 of SEQ ID NO: 176).

[0390] Note that for SEQ ID NOs: 174, 175 and 176, the nucleotide boundary between the upstream promoter and intergenic regions may not be exactly according to the coordinates above and that promoter, expression (e.g., enhancer or repressor) and / or regulatory element(s) for transcription also may be present in the upstream intergenic sequence of the respective gene.

[0391] Still other examples of suitable genes include Anther Ear1 (GRMZM2G081554) dwarf 4 (GRMZM2G065635) brs1 - brassinosteroid synthesis1, nana plant 1 (GRMZM2G057000), brassinosteroid receptor ZmBRI1a / ZmBRI1b (GRMZM2G048294 / GRMZM2G449830), the meristem development gene compact plant 2 (GRMZM2G064732) and ZMWRKY60 (as disclosed in CN116217684, herein incorporated by reference).

[0392] Other examples of genes which can be used in the methods and compositions of the current disclosure are Agamous, Bri1, Dwarf1, Pin1 from Arabidopsis (as herein disclosed), or an orthologous gene from other plants.

[0393] The genomic DNA sequence of Arabidopsis AGAMOUS (AG) gene (AT4G18960) is provided in SEQ ID NO: 204 and the protein sequence is provided in SEQ ID NO: 214. The genomic DNA sequence of Arabidopsis BRASSINOSTEROID INSENSITIVE 1 (Bri1) gene is provided in SEQ ID NO: 211 and the protein sequence is provided in SEQ ID NO: 215. The genomic DNA sequence of Arabidopsis DWARF1 gene (AT3G19820) is provided in SEQ ID NO:212 and the protein sequence is provided in SEQ ID NO: 216. The genomic DNA sequence of Arabidopsis PIN-FORMED1 gene (Pin1) (AT1G73590) is provided in SEQ ID NO:213 and the protein sequence is provided in SEQ ID NO: 217.

[0394] For the AG gene, nucleotides 1-501 and 1057-1060 of SEQ ID NO: 204 correspond to the 5-UTR; and nucleotides 5418-5648 of SEQ ID NO: 204 correspond to the 3’ UTR. For the Bri1 gene, nucleotides 1-165 of SEQ ID NO: 211 correspond to the 5-UTR; and nucleotides 3757-4167 of SEQ ID NO: 211 correspond to the 3’ UTR. For the Dwarf1 gene, nucleotides 664-699 of SEQ ID NO: 212 correspond to the 5-UTR; and nucleotides 2482-2700 of SEQ ID NO: 212 correspond to the 3’ UTR. For the Pin1 gene, nucleotides 1-99 of SEQ ID NO: 213 correspond to the 5-UTR; and nucleotides 3205-3506 of SEQ ID NO: 213 correspond to the 3’ UTR. Nucleic acids and amino acids.

[0395] The use of the term “polynucleotide” or “nucleic acid molecule” is not intended to limit the present disclosure to polynucleotides comprising deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also envisioned. Those of ordinary skill in the art will recognize that polynucleotides and nucleic acid molecules can comprise deoxyribonucleotides, ribonucleotides, or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogues. The polynucleotides of the present disclosure also encompass all forms of sequences including, but not limited to, single-stranded forms, double- stranded forms, hairpins, stem-and-loop structures, and the like. In an aspect, a nucleic acid molecule provided herein is a DNA molecule. In another aspect, a nucleic acid molecule provided herein is an RNA molecule. In an aspect, a nucleic acid molecule provided herein is single-stranded. In another aspect, a nucleic acid molecule provided herein is double-stranded.

[0396] As used herein, the term “recombinant” in reference to a nucleic acid (DNA or RNA) molecule, protein, construct, vector, etc., refers to a nucleic acid or amino acid molecule or sequence that is man-made and not normally found in nature, and / or is present in a context in which it is not normally found in nature, including a nucleic acid molecule (DNA or RNA) molecule, protein, construct, etc., comprising a combination of polynucleotide or protein sequences that would not naturally occur contiguously or in close proximity together without human intervention, and / or a polynucleotide molecule, protein, construct, etc., comprising at least two polynucleotide or protein sequences that are heterologous with respect to each other.

[0397] As used herein, the term "heterologous" refers to a nucleotide / polypeptide that originates from a foreign species, or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. A"heterologous" or a "recombinant" nucleotide sequence is a nucleotide sequence not naturally associated with a host cell into which it is introduced, including non- naturally occurring multiple copies of a naturally occurring nucleotide sequence

[0398] As used herein, the term “homologous” refers to nucleotide / polypeptide that is normally occurring in a particular species, normally operably linked to other components as they occur in such species, without deliberate human intervention.

[0399] In one aspect, methods and compositions provided herein comprise a vector. As used herein, the term “vector” refers to a DNA molecule used as a vehicle to carry exogenous genetic material into a cell.

[0400] In an aspect, one or more polynucleotide sequences from a vector are stably integrated into a genome of a plant. In an aspect, one or more polynucleotide sequences from a vector are stably integrated into a genome of a plant cell.

[0401] In an aspect, a first nucleic acid sequence and a second nucleic acid sequence are provided in a single vector. In another aspect, a first nucleic acid sequence is provided in a first vector, and a second nucleic acid sequence is provided in a second vector.

[0402] As used herein, the term “polypeptide” refers to a chain of at least two covalently linked amino acids. Polypeptides can be encoded by polynucleotides provided herein. An example of a polypeptide is a protein. Proteins provided herein can be encoded by nucleic acid molecules provided herein.

[0403] Nucleic acids can be isolated using techniques routine in the art. For example, nucleic acids can be isolated using any method including, without limitation, recombinant nucleic acid technology, and / or the polymerase chain reaction (PCR). General PCR techniques are described, for example in PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory Press, 1995. Recombinant nucleic acid techniques include, for example, restriction enzyme digestion and ligation, which can be used to isolate a nucleic acid. Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule or as a series of oligonucleotides. Polypeptides can be purified from natural sources (e.g., a biological sample) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. A polypeptide also can be purified, for example, by expressing a nucleic acid in an expression vector. In addition, a purified polypeptide can be obtained by chemical synthesis. The extent of purity of a polypeptide can be measured using any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0404] Without being limiting, nucleic acids can be detected using hybridization. Hybridization between nucleic acids is discussed in detail in Sambrook et. al. (1989, Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).

[0405] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence. An antibody provided herein can be a polyclonal antibody or a monoclonal antibody. An antibody having specific binding affinity for a polypeptide provided herein can be generated using methods well known in the art. An antibody provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.

[0406] The terms “percent identity” or “percent identical” as used herein in reference to two or more nucleotide or protein sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or protein) over a window of comparison, (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity. If the “percent identity” is being calculated in relation to a reference sequence without a particular comparison window being specified, then the percent identity is determined by dividing the number of matched positions over the region of alignment by the total length of the reference sequence. Accordingly, for purposes of the present application, when two sequences (query and subject) are optimally aligned (with allowance for gaps in their alignment), the “percent identity” for the query sequence is equal to the number of identical positions between the two sequences divided by the total number of positions in the query sequence over its length (or a comparison window), which is then multiplied by 100%. When percentage of sequence identity is used in reference to proteins it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. When sequences differ in conservative substitutions, the percent sequence identity can be adjusted upwards to correct for the conservative nature of the substitution. Sequences that differ by such conservative substitutions are said to have “sequence similarity” or “similarity.”

[0407] The terms “percent sequence complementarity” or “percent complementarity” as used herein in reference to two nucleotide sequences is similar to the concept of percent identity but refers to the percentage of nucleotides of a query sequence that optimally base-pair or hybridize to nucleotides a subject sequence when the query and subject sequences are linearly arranged and optimally base paired without secondary folding structures, such as loops, stems or hairpins. Such a percent complementarity can be between two DNA strands, two RNA strands, or a DNA strand and a RNA strand. The “percent complementarity” can be calculated by (i) optimally base-pairing or hybridizing the two nucleotide sequences in a linear and fully extended arrangement (i.e., without folding or secondary structures) over a window of comparison, (ii) determining the number of positions that base-pair between the two sequences over the window of comparison to yield the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the window of comparison, and (iv) multiplying this quotient by 100% to yield the percent complementarity of the two sequences. Optimal base pairing of two sequences can be determined based on the known pairings of nucleotide bases, such as G-C, A-T, and A-U, through hydrogen binding. If the “percent complementarity” is being calculated in relation to a reference sequence without specifying a particular comparison window, then the percent identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Thus, for purposes of the present application, when two sequences (query and subject) are optimally base-paired (with allowance for mismatches or non-base- paired nucleotides), the “percent complementarity” for the query sequence is equal to the number of base-paired positions between the two sequences divided by the total number of positions in the query sequence over its length, which is then multiplied by 100%.

[0408] For optimal alignment of sequences to calculate their percent identity, various pair-wise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool (BLAST®), etc., that can be used to compare the sequence identity or similarity between two or more nucleotide or protein sequences. Although other alignment and comparison methods are known in the art, the alignment and percent identity between two sequences (including the percent identity ranges described above) can be as determined by the ClustalW algorithm, see, e.g., Chenna R. et. al., “Multiple sequence alignment with the Clustal series of programs,” Nucleic Acids Research 31: 3497-3500 (2003); Thompson JD et. al., “Clustal W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice,” Nucleic Acids Research 22: 4673-4680 (1994); LarkinMA et. al., “Clustal W and Clustal X version 2.0,” Bioinformatics 23: 2947-48 (2007); and Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol.215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.

[0409] As used herein, a first nucleic acid molecule can “hybridize” a second nucleic acid molecule via non-covalent interactions (e.g., Watson-Crick base-pairing) in a sequence- specific, antiparallel manner (i.e., a nucleic acid specifically binds to a complementary nucleic acid) under the appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base-pairing includes: adenine (A) pairing with thymine (T), adenine (A) pairing with uracil (U), and guanine (G) pairing with cytosine (C). In addition, it is also known in the art that for hybridization between two RNA molecules (e.g., dsRNA), guanine base pairs with uracil. For example, G / U base-pairing is partially responsible for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anti-codon base-pairing with codons in mRNA. In the context of this disclosure, a guanine of a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered complementary to an uracil, and vice versa. As such, when a G / U base- pair can be made at a given nucleotide position a protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, the position is not considered to be non- complementary, but is instead considered to be complementary.

[0410] Hybridization and washing conditions are well known and exemplified in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), particularly Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of the hybridization.

[0411] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are possible. The conditions appropriate for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementation, variables well known in the art. The greater the degree of complementation between two nucleotide sequences, the greater the value of the melting temperature (Tm) for hybrids of nucleic acids having those sequences. For hybridizations between nucleic acids with short stretches of complementarity (e.g. complementarity over 35 or fewer nucleotides) the position of mismatches becomes important (see Sambrook et. al. ). Typically, the length for a hybridizable nucleic acid is at least 10 nucleotides. Illustrativeminimum lengths for a hybridizable nucleic acid are: at least 15 nucleotides; at least 18 nucleotides; at least 20 nucleotides; at least 22 nucleotides; at least 25 nucleotides; and at least 30 nucleotides). Furthermore, the skilled artisan will recognize that the temperature and wash solution salt concentration may be adjusted as necessary according to factors such as length of the region of complementation and the degree of complementation.

[0412] It is understood in the art that the sequence of polynucleotide need not be 100% complementary to that of its target nucleic acid to be specifically hybridizable or hybridizable. Moreover, a polynucleotide may hybridize over one or more segments such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure or hairpin structure). For example, an antisense nucleic acid in which 18 of 20 nucleotides of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleotides may be clustered or interspersed with complementary nucleotides and need not be contiguous to each other or to complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within nucleic acids can be determined routinely using BLAST® programs (basic local alignment search tools) and PowerBLAST programs known in the art (see Altschul et. al., J. Mol. Biol., 1990, 215, 403- 410; Zhang and Madden, Genome Res., 1997, 7, 649-656) or by using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489). Promoters

[0413] In some aspects, guide RNAs and RNA guided effector proteins are provided to the plant cells as recombinant nucleic acids, wherein the coding regions are operably linked to plant -expressible promoters. In one aspect, the RNA guided effector proteins are expressed from a nucleic acid operably linked to a POL II promoters. In one aspect, the RNA guide molecules are transcribed from a nucleic acid operably linked to a POL II promoter or a POL III promoter.

[0414] As used herein, a "promoter" is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) that is operably associated with the promoter. The coding sequence controlled or regulated by a promoter may encode a polypeptide and / or a functional RNA. A "promoter" may refer to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. Ingeneral, promoters are found 5', or upstream, relative to the start of the coding region of the corresponding coding sequence. A promoter may comprise other elements that act as regulators of gene expression; e.g., a promoter region. These include a TATA box consensus sequence, and often a CAAT box consensus sequence (Breathnach and Chambon (1981) Annu. Rev. Biochem.50:349). In plants, the CAAT box may be substituted by the AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp.211-227).

[0415] Promoters useful with this invention can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue- preferred and / or tissue-specific promoters for use in the preparation of recombinant nucleic acid molecules, e.g., "synthetic nucleic acid constructs" or "protein-RNA complex." These various types of promoters are known in the art.

[0416] The choice of promoter may vary depending on the temporal and spatial requirements for expression, and also may vary based on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge present in the art, the appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about promoters upstream of highly constitutively expressed genes in model organisms and such knowledge can be readily accessed and implemented in other systems as appropriate.

[0417] In some embodiments, a promoter functional in a plant may be used with the constructs of this invention. Non-limiting examples of a promoter useful for driving expression in a plant include the promoter of the RubisCo small subunit gene 1 (PrbcS1), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr) and the promoter of duplicated carbonic anhydrase gene 1 (Pdca1) (See, Walker et al. (2005) Plant Cell Rep. 23:727-735; Li et al. (2007) Gene 403:132-142; Li et al. (2010) Mol Biol. Rep.37:1143-1154). PrbcS1 and Pactin are constitutive promoters and Pnr and Pdca1 are inducible promoters. Pnr is induced by nitrate and repressed by ammonium (Li et al. (2007) Gene 403:132-142) and Pdca1 is induced by salt (Li et al. (2010) Mol Biol. Rep.37:1143-1154). In some embodiments, a promoter useful with this invention is RNA polymerase II (Pol II) promoter.

[0418] Examples of constitutive promoters useful for plants include, but are not limited to, cestrum virus promoter (cmp) (US Patent No.7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol.12:3399-3406; as well as US Patent No.5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), sucrose synthase promoter (Yang & Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148), and the ubiquitin promoter. The constitutive promoter derived from ubiquitin accumulates in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al. (1991) Plant Science 79: 87-94), maize (Christensen et al. (1989) Plant Molec. Biol.12: 619-632), and Arabidopsis (Norris et al. (1993) Plant Molec. Biol. 21:895-906). The maize ubiquitin promoter (UbiP) has been developed in transgenic monocot systems and its sequence and vectors constructed for monocot transformation are disclosed in the patent publication EP 0342926. The ubiquitin promoter is suitable for the expression of the nucleotide sequences of the invention in transgenic plants, especially monocotyledons. Further, the promoter expression cassettes described by McElroy et al. ((1991) Mol. Gen. Genet.231: 150-160) can be easily modified for the expression of the nucleotide sequences of the invention and are particularly suitable for use in monocotyledonous hosts.

[0419] In some embodiments, tissue specific / tissue preferred promoters can be used for expression of a heterologous polynucleotide in a plant cell. Tissue specific or preferred expression patterns include, but are not limited to, green tissue specific or preferred, root specific or preferred, stem specific or preferred, flower specific or preferred or pollen specific or preferred. Promoters suitable for expression in green tissue include many that regulate genes involved in photosynthesis and many of these have been cloned from both monocotyledons and dicotyledons. In one embodiment, a promoter useful with the invention is the maize PEPC promoter from the phosphoenol carboxylase gene (Hudspeth & Grula (1989) Plant Molec. Biol. 12:579-589). Non-limiting examples of tissue-specific promoters include those associated with genes encoding the seed storage proteins (such as β-conglycinin, cruciferin, napin and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase and fatty acid desaturases (fad 2-1)), and other nucleic acids expressed during embryo development (such as Bce4, see, e.g., Kridl et al. (1991) Seed Sci. Res. 1:209-219; as well as EP Patent No. 255378). Tissue- specific or tissue-preferential promoters useful for the expression of the nucleotide sequences of the invention in plants, particularly maize, include but are not limited to those that direct expression in root, pith, leaf, or pollen. Such promoters are disclosed, for example, in WO 93 / 07278, herein incorporated by reference in its entirety. Other non-limiting examples of tissue specific or tissue preferred promoters useful with the invention the cotton rubisco promoter disclosed in US Patent No.6,040,504; the rice sucrose synthase promoter disclosed in US PatentNo. 5,604,121; the root specific promoter described by de Framond ((1991) FEBS 290:103- 106; EP 0452269 to Ciba- Geigy); the stem specific promoter described in US Patent No. 5,625,136 (to Ciba-Geigy) and which drives expression of the maize trpA gene; the cestrum yellow leaf curling virus promoter disclosed in WO 01 / 73087; and pollen specific or preferred promoters including, but not limited to, ProOsLPS10 and ProOsLPS11 from rice (Nguyen et al. (2015) Plant Biotechnol. Reports 9(5):297-306), ZmSTK2_USP from maize (Wang et al. (2017) Genome 60(6):485-495), LAT52 and LAT59 from tomato (Twell et al. (1990) Development 109(3):705-713), Zm13 (US Patent No. 10,421,972), PLA2-δ promoter from Arabidopsis (US Patent No.7,141,424), and / or the ZmC5 promoter from maize (International PCT Publication No. WO1999 / 042587.

[0420] Additional examples of plant tissue-specific / tissue preferred promoters include, but are not limited to, the root hair-specific cis-elements (RHEs) (Kim et al. (2006) The Plant Cell 18:2958-2970), the root-specific promoters RCc3 (Jeong et al. (2010) Plant Physiol. 153:185-197) and RB7 (US Patent No.5459252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), S- adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology 37(8):1108-1115), corn light harvesting complex promoter (Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658), corn heat shock protein promoter (O'Dell et al. (1985) EMBO J.5:451-458; and Rochester et al. (1986) EMBO J.5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore, "Nuclear genes encoding the small subunit of ribulose- l,5-bisphosphate carboxylase" pp. 29-39 In: Genetic Engineering of Plants (Hollaender ed., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219- 3223), Ti plasmid nopaline synthase promoter (Langridge et al. (1989) supra), petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBO J.7:1257-1263), bean glycine rich protein 1 promoter (Keller et al. (1989) Genes Dev. 3:1639-1646), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810-812), potato patatin promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), maize zein promoter (Kriz et al. (1987) Mol. Gen. Genet.207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acids Res.18:6425; Reina et al. (1990) Nucleic Acids Res.18:7449; and Wandelt et al. (1989) Nucleic Acids Res.17:2354), globulin- 1 promoter (Belanger et al. (1991) Genetics 129:863-872), α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet.215:431-440), PEPCase promoter (Hudspeth & Grula (1989) PlantMol. Biol. 12:579-589), R gene complex-associated promoters (Chandler et al. (1989) Plant Cell 1:1175-1183), and chalcone synthase promoters (Franken et al. (1991) EMBO J.10:2605- 2612).

[0421] Useful for seed-specific expression is the pea vicilin promoter (Czako et al. (1992) Mol. Gen. Genet.235:33-40; as well as the seed-specific promoters disclosed in US Patent No. 5,625,136. Useful promoters for expression in mature leaves are those that are switched at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al. (1995) Science 270:1986-1988).

[0422] Plant-expressible promoters useful for the methods and compositions herein described also include egg cell-preferred or embryo-tissue preferred promoters as described in WO2022 / 056139 (incorporated herein in its entirety), such as a DSUL1 promoter, an EA1 promoter, an ES4 promoter, a DMC1 promoter, a Mps1 promoter, an Adf1 promoter or an EAL promoter.

[0423] Other plant-expressible promoters useful for the invention include floral-tissue preferred or floral cell-preferred promoter as described in PCT / US2023 / 065042 (incorporated herein in its entirety).

[0424] In addition, promoters functional in chloroplasts can be used. Non-limiting examples of such promoters include the bacteriophage T3 gene 95' UTR and other promoters disclosed in US Patent No. 7,579,516. Other promoters useful with the invention include but are not limited to the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0425] In some embodiments, the differing promoters of the at least two recombinant constructs or cassettes expressing the guide RNAs comprising the spacer sequence complementary to a target site, such as a genomic target site, as described herein, may be selected from RNA polymerase III (Pol III) promoters. In some aspects, the POL III promoter may be a U6 promoter, an H1 promoter, a 5S promoter, an Adenovirus 2 (Ad2) VAI promoter, a tRNA promoter, and a 7SK promoter. See, for example, Schramm and Hernandez, 2002, Genes & Development, 16:2593-2620, which is incorporated by reference herein in its entirety.

[0426] In some aspects, the POL III promoters may be derived from small nuclear RNA (snRNA) encoding genes. In some aspects, the POL III promoters may be selected from the corn, tomato and soybean U6, U3, U2, U5 and 7SL snRNA promoters disclosed in WO2015 / 131101 (incorporated herein by reference in its entirety) including the snRNA promoter sequences of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20; SEQ ID NOs: 146- 149, SEQ ID NOs: 160-166, SEQ ID NOs: 201 or SEQ ID NO: 283, included therein in the accompanying sequence listing.

[0427] In some aspects, the POL III promoters may be synthetic snRNA promoters, such as the snRNA promoters described in WO2022 / 232407 (incorporated herein by reference in its entirety) including the snRNA promoter sequences of SEQ ID Nos: 1-10 included therein in the accompanying sequence listing.

[0428] In some aspects, the POL III promoters may be chimeric POL III promoters. In some aspects, the POL III promoters may be variants of the POL III promoters. In some aspects, a variant of a POL III promoters comprising a sequence that, when optimally aligned to the reference sequence has at least about 85 percent identity, at least about 86 percent identity, at least about 87 percent identity, at least about 88 percent identity, at least about 89 percent identity, at least about 90 percent identity, at least about 91 percent identity, at least about 92 percent identity, at least about 93 percent identity, at least about 94 percent identity, at least about 95 percent identity, at least about 96 percent identity, at least about 97 percent identity, at least about 98 percent identity, or at least about 99 percent identity to the reference sequence and having promoter activity as disclosed herein are provided. Regulatory elements

[0429] Additional regulatory elements useful with this invention include, but are not limited to, introns, enhancers, termination sequences and / or 5' and 3' untranslated regions.

[0430] An intron useful with this invention can be an intron identified in and isolated from a plant and then inserted into an expression cassette to be used in transformation of a plant. As would be understood by those of skill in the art, introns can comprise the sequences required for self-excision and are incorporated into nucleic acid constructs / expression cassettes in frame. An intron can be used either as a spacer to separate multiple protein-coding sequences in one nucleic acid construct, or an intron can be used inside one protein-coding sequence to, for example, stabilize the mRNA. If they are used within a protein-coding sequence, they are inserted "in-frame" with the excision sites included. Introns may also be associated with promoters to improve or modify expression. As an example, a promoter / intron combination useful with this invention includes but is not limited to that of the maize Ubi1 promoter and intron.

[0431] Non-limiting examples of introns useful with the present invention include introns from the ADHI gene (e.g., Adh1-S introns 1, 2 and 6), the ubiquitin gene (Ubi1), the RuBisCO small subunit (rbcS) gene, the RuBisCO large subunit (rbcL) gene, the actin gene (e.g., actin-1 intron), the pyruvate dehydrogenase kinase gene (pdk), the nitrate reductase gene (nr), the duplicated carbonic anhydrase gene 1 (Tdca1), the psbA gene, the atpA gene, or any combination thereof. Guide Nucleic Acids

[0432] As used herein, a “guide nucleic acid” refers to a nucleic acid that forms a ribonucleoprotein (e.g., a complex) with a guided nuclease (e.g., without being limiting, Cas12a, CasX) and then guides the ribonucleoprotein to a specific sequence in a target nucleic acid molecule, where the guide nucleic acid and the target nucleic acid molecule share complementary sequences. In an aspect, a ribonucleoprotein provided herein comprises at least one guide nucleic acid.

[0433] In an aspect, a guide nucleic acid comprises DNA. In another aspect, a guide nucleic acid comprises RNA. In an aspect, a guide nucleic acid comprises DNA, RNA, or a combination thereof. In an aspect, a guide nucleic acid is single-stranded. In another aspect, a guide nucleic acid is at least partially double-stranded.

[0434] When a guide nucleic acid comprises RNA, it can be referred to as a “guide RNA.” In another aspect, a guide nucleic acid comprises DNA and RNA. In another aspect, a guide RNA is single-stranded. In another aspect, a guide RNA is double-stranded. In a further aspect, a guide RNA is partially double-stranded.

[0435] A "guide nucleic acid," "guide RNA," "gRNA," "CRISPR RNA / DNA" "crRNA" or "crDNA" as used herein means a nucleic acid that comprises at least one spacer sequence, which is complementary to (and hybridizes to) a target DNA (e.g., protospacer), and at least one repeat sequence (e.g., a repeat of a Type V Cas12a CRISPR-Cas system, or a fragment or portion thereof; a repeat of a Type II Cas9 CRISPR-Cas system, or fragment thereof; a repeat of a Type V C2c1 CRISPR Cas system, or a fragment thereof; a repeat of a CRISPR-Cas system of, for example, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3’, Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and / or Csf5, or a fragment thereof), wherein the repeat sequence may be linked to the5’ end and / or the 3’ end of the spacer sequence. The design of a gRNA of this invention may be based on a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR-Cas system.

[0436] In some embodiments, a Cas12a gRNA may comprise, from 5’ to 3’, a repeat sequence (full length or portion thereof ("handle"); e.g., pseudoknot-like structure) and a spacer sequence.

[0437] In some embodiments, a guide nucleic acid may comprise more than one repeat sequence-spacer sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more repeat-spacer sequences) (e.g., repeat-spacer-repeat, e.g., repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer-repeat- spacer, and the like). The guide nucleic acids of this invention are synthetic, human-made and not found in nature. A gRNA can be quite long and may be used as an aptamer (like in the MS2 recruitment strategy) or other RNA structures hanging off the spacer. A guide RNA may comprise a donor template for introducing specific modifications in the target sequence.

[0438] A "repeat sequence" as used herein, refers to, for example, any repeat sequence of a wild-type CRISPR Cas locus (e.g., a Cas9 locus, a Cas12a locus, a C2c1 locus, etc.) or a repeat sequence of a synthetic crRNA that is functional with the CRISPR-Cas effector protein encoded by the nucleic acid constructs of the invention. A repeat sequence useful with this invention can be any known or later identified repeat sequence of a CRISPR-Cas locus (e.g., Type I, Type II, Type III, Type IV, Type V or Type VI) or it can be a synthetic repeat designed to function in a Type I, II, III, IV, V or VI CRISPR-Cas system. A repeat sequence may comprise a hairpin structure and / or a stem loop structure. In some embodiments, a repeat sequence may form a pseudoknot-like structure at its 5’ end (i.e., "handle"). Thus, in some embodiments, a repeat sequence can be identical to or substantially identical to a repeat sequence from wild-type Type I CRISPR-Cas loci, Type II, CRISPR-Cas loci, Type III, CRISPR-Cas loci, Type IV CRISPR-Cas loci, Type V CRISPR-Cas loci and / or Type VI CRISPR-Cas loci. A repeat sequence from a wild-type CRISPR-Cas locus may be determined through established algorithms, such as using the CRISPRfinder offered through CRISPRdb (see, Grissa et al. (2007) Nucleic Acids Res. 35(Web Server issue):W52-7). In some embodiments, a repeat sequence or portion thereof is linked at its 3’ end to the 5’ end of a spacer sequence, thereby forming a repeat-spacer sequence (e.g., guide nucleic acid, guide RNA / DNA, crRNA, crDNA).

[0439] In some embodiments, a repeat sequence comprises, consists essentially of, or consists of at least 10 nucleotides depending on the particular repeat and whether the guide nucleic acid comprising the repeat is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides, or any range or value therein). In some embodiments, a repeat sequence comprises, consists essentially of, or consists of about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100 or more nucleotides.

[0440] A repeat sequence linked to the 5’ end of a spacer sequence can comprise a portion of a repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more contiguous nucleotides of a wild type repeat sequence). In some embodiments, a portion of a repeat sequence linked to the 5’ end of a spacer sequence can be about five to about ten consecutive nucleotides in length (e.g., about 5, 6, 7, 8, 9, 10 nucleotides) and have at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more) to the same region (e.g., 5’ end) of a wild type CRISPR Cas repeat nucleotide sequence. In some embodiments, a portion of a repeat sequence may comprise a pseudoknot-like structure at its 5’ end (e.g., "handle").

[0441] A "spacer sequence" as used herein is a nucleotide sequence that is complementary to portion of a target nucleic acid (e.g., target DNA) (e.g., protospacer).. A spacer sequence can be fully complementary or substantially complementary (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to a target nucleic acid. In some embodiments, the spacer sequence can have one, two, three, four, or five mismatches as compared to the target nucleic acid, which mismatches can be contiguous or noncontiguous. In some embodiments, the spacer sequence can have 70% complementarity to a target nucleic acid. In other embodiments, the spacer nucleotide sequence can have 80% complementarity to a target nucleic acid. In still other embodiments, the spacer nucleotide sequence can have 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementarity, and the like, to the target nucleic acid (protospacer). In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid. A spacer sequence may have a length from about 15 nucleotides to about 30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any range or value therein). Thus, in some embodiments, a spacer sequence may have complete complementarity or substantial complementarity over a region of a target nucleic acid (e.g., protospacer) that is at least about 15 nucleotides to about 30 nucleotides in length. In some embodiments, the spacer is about 20 nucleotides in length. In some embodiments, the spacer is about 21, 22, or 23nucleotides in length. In some embodiments, a spacer sequence may comprise any one of the sequences of SEQ ID NOs:88-90, or any combination thereof.

[0442] In some embodiments, the 5’ region of a spacer sequence of a guide nucleic acid may be identical to a target DNA, while the 3’ region of the spacer may be substantially complementary to the target DNA (such as a spacer of a Type V CRISPR-Cas system), or the 3’ region of a spacer sequence of a guide nucleic acid may be identical to a target DNA, while the 5’ region of the spacer may be substantially complementary to the target DNA (such as a spacer of a Type II CRISPR-Cas system), and therefore, the overall complementarity of the spacer sequence to the target DNA may be less than 100%. Thus, for example, in a guide for a Type V CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 5’ region (i.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA. In some embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, nucleotides, and any range therein) of the 5’ end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3’ region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more)) to the target DNA.

[0443] As a further example, in a guide for a Type II CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 3’ region (i.e., seed region) of, for example, a 20 nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 70% complementary) to the target DNA. In some embodiments, the first 1 to 10 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, and any range therein) of the 3’ end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5’ region of the spacer sequence are substantially complementary (e.g., at least about 50% complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more or any range or value therein)) to the target DNA.

[0444] In some embodiments, a seed region of a spacer may be about 8 to about 10 nucleotides in length, about 5 to about 6 nucleotides in length, or about 6 nucleotides in length.

[0445] In an aspect, a guide nucleic acid comprises a guide RNA. In another aspect, a guide nucleic acid comprises at least one guide RNA. In another aspect, a guide nucleic acid comprises at least two guide RNAs. In another aspect, a guide nucleic acid comprises at least three guide RNAs. In another aspect, a guide nucleic acid comprises at least five guide RNAs. In another aspect, a guide nucleic acid comprises at least ten guide RNAs.

[0446] In another aspect, a guide nucleic acid comprises at least 10 nucleotides. In another aspect, a guide nucleic acid comprises at least 11 nucleotides. In another aspect, a guide nucleic acid comprises at least 12 nucleotides. In another aspect, a guide nucleic acid comprises at least 13 nucleotides. In another aspect, a guide nucleic acid comprises at least 14 nucleotides. In another aspect, a guide nucleic acid comprises at least 15 nucleotides. In another aspect, a guide nucleic acid comprises at least 16 nucleotides. In another aspect, a guide nucleic acid comprises at least 17 nucleotides. In another aspect, a guide nucleic acid comprises at least 18 nucleotides. In another aspect, a guide nucleic acid comprises at least 19 nucleotides. In another aspect, a guide nucleic acid comprises at least 20 nucleotides. In another aspect, a guide nucleic acid comprises at least 21 nucleotides. In another aspect, a guide nucleic acid comprises at least 22 nucleotides. In another aspect, a guide nucleic acid comprises at least 23 nucleotides. In another aspect, a guide nucleic acid comprises at least 24 nucleotides. In another aspect, a guide nucleic acid comprises at least 25 nucleotides. In another aspect, a guide nucleic acid comprises at least 26 nucleotides. In another aspect, a guide nucleic acid comprises at least 27 nucleotides. In another aspect, a guide nucleic acid comprises at least 28 nucleotides. In another aspect, a guide nucleic acid comprises at least 30 nucleotides. In another aspect, a guide nucleic acid comprises at least 35 nucleotides. In another aspect, a guide nucleic acid comprises at least 40 nucleotides. In another aspect, a guide nucleic acid comprises at least 45 nucleotides. In another aspect, a guide nucleic acid comprises at least 50 nucleotides.

[0447] In another aspect, a guide nucleic acid comprises between 10 nucleotides and 50 nucleotides. In another aspect, a guide nucleic acid comprises between 10 nucleotides and 40 nucleotides. In another aspect, a guide nucleic acid comprises between 10 nucleotides and 30 nucleotides. In another aspect, a guide nucleic acid comprises between 10 nucleotides and 20 nucleotides. In another aspect, a guide nucleic acid comprises between 16 nucleotides and 28 nucleotides. In another aspect, a guide nucleic acid comprises between 16 nucleotides and 25 nucleotides. In another aspect, a guide nucleic acid comprises between 16 nucleotides and 20 nucleotides.

[0448] In an aspect, a guide nucleic acid comprises at least 70% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 75%sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 80% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 85% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 90% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 91% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 92% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 93% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 94% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 95% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 96% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 97% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 98% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises at least 99% sequence complementarity to a target site. In an aspect, a guide nucleic acid comprises 100% sequence complementarity to a target site. In another aspect, a guide nucleic acid comprises between 70% and 100% sequence complementarity to a target site. In another aspect, a guide nucleic acid comprises between 80% and 100% sequence complementarity to a target site. In another aspect, a guide nucleic acid comprises between 90% and 100% sequence complementarity to a target site.

[0449] In an aspect, a guide nucleic acid is capable of hybridizing to a target site.

[0450] As noted above, some guided nucleases, such as CasX and Cas9, require another non-coding RNA component, referred to as a trans-activating crRNA (tracrRNA), to have functional activity. Guide nucleic acid molecules provided herein can combine a crRNA and a tracrRNA into one nucleic acid molecule in what is herein referred to as a “single guide RNA” (sgRNA). The gRNA guides the active CasX complex to a target site within a target sequence, where CasX can cleave the target site. In other embodiments, the crRNA and tracrRNA are provided as separate nucleic acid molecules.

[0451] In an aspect, a guide nucleic acid comprises a crRNA. In another aspect, a guide nucleic acid comprises a tracrRNA. In a further aspect, a guide nucleic acid comprises a sgRNA.

[0452] It is expected that the methods and compositions disclosed herein are useful to increase the editing efficiency of a guide RNA which results in poor editing or has a low editing efficiency at the target site when expressed as a single guide RNA, or from a single recombinant construct, in combination with an RNA guided effector protein. As used herein “a guide RNA with low editing efficiency” results in a cutting efficiency of less than 30%, less than 25% orless than 20% when assayed, e.g. in an assay involving introduction of such guide RNA or a nucleic acid construct encoding such guide RNA in plant protoplast comprising a target site recognized by the guide RNA and determination of the percentage of protoplasts comprising insertions / deletions at the target site e.g. by determination of the number of reads by sequencing. The potential editing efficiency of a particular guide RNA can be estimated in silico as described in US2023091138 (herein incorporated in its entirety). RNA guided nucleases

[0453] Guided nucleases are nucleases that form a complex (e.g., a ribonucleoprotein) with a guide nucleic acid molecule (e.g., a guide RNA), which then guides the complex to a target site within a target sequence. One non-limiting example of guided nucleases are CRISPR nucleases.

[0454] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleases (e.g., Cas9, CasX, Cas12a (also referred to as Cpf1), CasY, MAD7®) are proteins found in bacteria that are guided by guide RNAs (“gRNAs”) to a target nucleic acid molecule, where the endonuclease can then cleave one or two strands the target nucleic acid molecule. Although the origins of CRISPR nucleases are bacterial, many CRISPR nucleases have been shown to function in eukaryotic cells.

[0455] While not being limited by any particular scientific theory, a CRISPR nuclease forms a complex with a guide RNA (gRNA), which hybridizes with a complementary target site, thereby guiding the CRISPR nuclease to the target site. In class II CRISPR-Cas systems, CRISPR arrays, including spacers, are transcribed during encounters with recognized invasive DNA and are processed into small interfering CRISPR RNAs (crRNAs). The crRNA comprises a repeat sequence and a spacer sequence which is complementary to a specific protospacer sequence in an invading pathogen. The spacer sequence can be designed to be complementary to target sequences in a eukaryotic genome.

[0456] CRISPR nucleases associate with their respective crRNAs in their active forms. CasX, similar to the class II endonuclease Cas9, requires another non-coding RNA component, referred to as a trans-activating crRNA (tracrRNA), to have functional activity. Nucleic acid molecules provided herein can combine a crRNA and a tracrRNA into one nucleic acid molecule in what is herein referred to as a “single guide RNA” (sgRNA). Cas12a or MAD7® do not require a tracrRNA to be guided to a target site; a crRNA alone is sufficient for Cas12aor MAD7®. The gRNA guides the active CRISPR nuclease complex to a target site, where the CRISPR nuclease can cleave the target site.

[0457] When an RNA-guided CRISPR nuclease and a guide RNA form a complex, the whole system is called a “ribonucleoprotein.” Ribonucleoproteins provided herein can also comprise additional nucleic acids or proteins.

[0458] A prerequisite for cleavage of the target site by a CRISPR ribonucleoprotein is the presence of a conserved Protospacer Adjacent Motif (PAM) near the target site. Depending on the CRISPR nuclease, cleavage can occur within a certain number of nucleotides (e.g., between 18-23 nucleotides for Cas12a) from the PAM site. PAM sites are only required for type I and type II CRISPR associated proteins, and different CRISPR endonucleases recognize different PAM sites. Without being limiting, Cas12a can recognize at least the following PAM sites: TTTN, and YTN; CasX can recognize at least the following PAM sites: TTCN, TTCA, and TTC and MAD7® nuclease recognizes T-rich PAM sequences YTTN and seems to prefer TTTN to CTTN PAMs (where T is thymine; C is cytosine; A is adenine; Y is thymine or cytosine; and N is thymine, cytosine, guanine, or adenine).

[0459] Cas12a is an RNA-guided nuclease of a class II, type V CRISPR / Cas system. Cas12a nucleases generate staggered cuts when cleaving a double-stranded DNA molecule. Staggered cuts of double-stranded DNA produce a single-stranded DNA overhang of at least one nucleotide. This is in contrast to a blunt-end cut (such as those generated by Cas9), which does not produce a single-stranded DNA overhang when cutting double-stranded DNA.

[0460] In an aspect, a Cas12a nuclease provided herein is a Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease. In another aspect, a Cas12a nuclease provided herein is a Francisella novicida Cas12a (FnCas12a) nuclease. In an aspect, a Cas12a nuclease is selected from the group consisting of LbCas12a and FnCas12a.

[0461] In an aspect, a Cas12a nuclease, or a nucleic acid encoding a Cas12a nuclease, is derived from a bacteria genus selected from the group consisting of Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiaridium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, Acidaminococcus, Peregrinibacteria, Butyrivibrio, Parcubacteria, Smithella, Candidatus, Moraxella, and Leptospira.

[0462] In an aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 80% identical to a polynucleotide selected from SEQ ID NO: 218 or SEQ ID NO:219. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 85% identical to a polynucleotide selected from SEQ ID NO: 218 or SEQ ID NO:219. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 90% identical to a polynucleotide selected from SEQ ID NO: 218 or SEQ ID NO:219. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 95% identical to a polynucleotide selected from SEQ ID NO: 218 or SEQ ID NO:219. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 96% identical to a polynucleotide selected from SEQ ID NO: 218 or SEQ ID NO:219. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 97% identical to a polynucleotide selected from SEQ ID NO: 218 or SEQ ID NO:219. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 98% identical to a polynucleotide selected from SEQ ID NO: 218 or SEQ ID NO:219. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 99% identical to a polynucleotide selected from SEQ ID NO: 218 or SEQ ID NO:219. In another aspect, a Cas12a nuclease is encoded by a polynucleotide comprising a sequence 100% identical to a polynucleotide selected from SEQ ID NO: 218 or SEQ ID NO:219.

[0463] In an aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 80% identical to an amino acid sequence selected from SEQ ID NO:194 or SEQ ID NO:199 In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 85% identical to an amino acid sequence selected from SEQ ID NO:194 or SEQ ID NO:199 In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 90% identical to an amino acid sequence selected from SEQ ID NO:194 or SEQ ID NO:199 In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 95% identical to an amino acid sequence selected from SEQ ID NO:194 or SEQ ID NO:199 In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 96% identical to an amino acid sequence selected from SEQ ID NO:194 or SEQ ID NO:199 In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 97% identical to an amino acid sequence selected from SEQ ID NO:194 or SEQ ID NO:199. In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 98% identical to an amino acid sequence selected from SEQ ID NO:194 or SEQ ID NO:199 Inanother aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at least 99% identical to an amino acid sequence selected from SEQ ID NO:194 or SEQ ID NO:199 In another aspect, a Cas12a nuclease provided herein comprises an amino acid sequence having at 100% identity to an amino acid sequence selected from SEQ ID NO:194 or SEQ ID NO:199.

[0464] In an aspect, a Cas12a provided herein is a variant Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease with enhanced DNA cleavage activities at non-canonical TTTT protospacer adjacent motifs such as described in US2021 / 0348144 (incorporated herein by reference in its entirety) In another aspect, a Cas12a provided herein is a variant Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease with enhanced activity as described in US20230040148 (incorporated herein by reference in its entirety) such as the LbCas12a-ultra having an N527R and E795L substitution in its amino acid sequence (reference amino acid sequence is SEQ ID NO: 194).

[0465] In an aspect, a Cas12a provided herein provided herein is a variant Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease recognizing a PAM variant TYCV having a G532R and K595R substitution in its amino acid sequence (reference amino acid sequence is SEQ ID NO: 4) or a variant Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease recognizing a PAM variant TATT having a G532R, K538R and Y524R substitution in its amino acid sequence (reference amino acid sequence is SEQ ID NO: 194) as disclosed in WO2016205711 (herein incorporated by reference in its entirety).

[0466] CasX is a type of class II CRISPR-Cas nuclease that has been identified in the bacterial phyla Deltaproteobacteria and Planctomycetes. Similar to Cas12a, CasX nucleases generate staggered cuts when cleaving a double-stranded DNA molecule. However, unlike Cas12a, CasX nucleases require a crRNA and a tracrRNA, or a single-guide RNA, in order to target and cleave a target nucleic acid.

[0467] In an aspect, a CasX nuclease provided herein is a CasX nuclease from the phylum Deltaproteobacteria. In another aspect, a CasX nuclease provided herein is a CasX nuclease from the phylum Planctomycetes. Without being limiting, additional suitable CasX nucleases are those set forth in WO 2019 / 084148, which is incorporated by reference herein in its entirety.

[0468] MAD7® (also known as ErCas12a) is an engineered nuclease of the Class 2 type V-A CRISPR-Cas (Cas12a / Cpf1) family with a low level of homology to canonical Cas12a nucleases. MAD7® nucleases generate staggered cuts when cleaving a double-stranded DNA molecule.MAD7® nuclease was initially identified in Eubacterium rectale. It only requires a crRNA like canonical Cas12a. An ErCas12a / MAD7® encoding nucleotide sequence can befound in the supplementary data (sequences S1) provided with Lin et al., 2021, Journal of Genetics and Genomics 48, pages 444-451)

[0469] In an aspect, a guided nuclease capable of generating a staggered cut in a double- stranded DNA molecule is selected from the group consisting of Cas12a; MAD7® and CasX. In an aspect, a guided nuclease is selected from the group consisting of Cas12a, MAD7® and CasX.

[0470] In an aspect, a guided nuclease is a RNA-guided nuclease. In another aspect, a guided nuclease is a CRISPR nuclease. In another aspect, a guided nuclease is a Cas12a nuclease. In another aspect, a guided nuclease is a CasX nuclease. In another aspect, a guided nuclease is a MAD7® nuclease.

[0471] As used herein, a “nuclear localization signal” (NLS) refers to an amino acid sequence that “tags” a protein for import into the nucleus of a cell. In an aspect, a nucleic acid molecule provided herein encodes a nuclear localization signal. In another aspect, a nucleic acid molecule provided herein encodes two or more nuclear localization signals.

[0472] In an aspect, a Cas12a nuclease provided herein comprises a nuclear localization signal. In an aspect, a nuclear localization signal is positioned on the N-terminal end of a Cas12a nuclease. In a further aspect, a nuclear localization signal is positioned on the C-terminal end of a Cas12a nuclease. In yet another aspect, a nuclear localization signal is positioned on both the N-terminal end and the C-terminal end of a Cas12a nuclease.

[0473] In an aspect, a CasX nuclease provided herein comprises a nuclear localization signal. In an aspect, a nuclear localization signal is positioned on the N-terminal end of a CasX nuclease. In a further aspect, a nuclear localization signal is positioned on the C-terminal end of a CasX nuclease. In yet another aspect, a nuclear localization signal is positioned on both the N-terminal end and the C-terminal end of a CasX nuclease.

[0474] In an aspect, a MAD7® nuclease provided herein comprises a nuclear localization signal. In an aspect, a nuclear localization signal is positioned on the N-terminal end of a MAD7® nuclease. In a further aspect, a nuclear localization signal is positioned on the C- terminal end of a MAD7® nuclease. In yet another aspect, a nuclear localization signal is positioned on both the N-terminal end and the C-terminal end of a MAD7® nuclease

[0475] In an aspect, a ribonucleoprotein comprises at least one nuclear localization signal. In another aspect, a ribonucleoprotein comprises at least two nuclear localization signals.

[0476] Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which is in turn believed to be dependenton, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at www[dot]kazusa[dot]or[dot]jp[forwards slash]codon and these tables can be adapted in a number of ways. See Nakamura et al., 2000, Nucl. Acids Res. 28:292. Computer algorithms for codon optimizing a particular sequence for expression in a particular plant cell are also available, such as Gene Forge (Aptagen; Jacobus, PA), are also available.

[0477] As used herein, “codon optimization” refers to a process of modifying a nucleic acid sequence for enhanced expression in a plant cell of interest by replacing at least one codon (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of a sequence with codons that are more frequently or most frequently used in the genes of the plant cell while maintaining the original amino acid sequence (e.g., introducing silent mutations).

[0478] In an aspect, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a guided nuclease correspond to the most frequently used codon for a particular amino acid. In another aspect, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cas12a nuclease or a CasX nuclease or a MAD7® nuclease correspond to the most frequently used codon for a particular amino acid. As to codon usage in plants, reference is made to Campbell and Gowri, 1990, Plant Physiol., 92: 1-11; and Murray et al., 1989, Nucleic Acids Res., 17:477-98, each of which is incorporated herein by reference in their entireties.

[0479] In an aspect, a nucleic acid molecule encodes a guided nuclease that is codon optimized for a plant. In an aspect, a nucleic acid molecule encodes a Cas12a nuclease that is codon optimized for a plant. In an aspect, a nucleic acid molecule encodes a CasX nuclease that is codon optimized for a plant. In an aspect, a nucleic acid molecule encodes a MAD7® nuclease that is codon optimized for a plant

[0480] In another aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a plant cell. In another aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a monocotyledonous plant species. In another aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a dicotyledonous plant species. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a gymnosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodesa guided nuclease that is codon optimized for an angiosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a corn cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a soybean cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a rice cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a wheat cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a cotton cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a sorghum cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for an alfalfa cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a sugarcane cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for an Arabidopsis cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a tomato cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a cucumber cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for a potato cell. In a further aspect, a nucleic acid molecule provided herein encodes a guided nuclease that is codon optimized for an onion cell.

[0481] In another aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a plant cell. In another aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a monocotyledonous plant species. In another aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a dicotyledonous plant species. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a gymnosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for an angiosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a corn cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a soybean cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a rice cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a wheat cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a cotton cell. In a further aspect, a nucleic acidmolecule provided herein encodes a Cas12a nuclease that is codon optimized for a sorghum cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for an alfalfa cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a sugar cane cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for an Arabidopsis cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a tomato cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a cucumber cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for a potato cell. In a further aspect, a nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon optimized for an onion cell.

[0482] In another aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a plant cell. In another aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a monocotyledonous plant species. In another aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a dicotyledonous plant species. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a gymnosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for an angiosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a corn cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a soybean cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a rice cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a wheat cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a cotton cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a sorghum cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for an alfalfa cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a sugar cane cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for an Arabidopsis cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a tomato cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a cucumber cell. In a further aspect, anucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for a potato cell. In a further aspect, a nucleic acid molecule provided herein encodes a CasX nuclease that is codon optimized for an onion cell. In another aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a plant cell. In another aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a monocotyledonous plant species. In another aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a dicotyledonous plant species. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a gymnosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for an angiosperm plant species. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a corn cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a soybean cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a rice cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a wheat cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a cotton cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a sorghum cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for an alfalfa cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a sugar cane cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for an Arabidopsis cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a tomato cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a cucumber cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for a potato cell. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon optimized for an onion cell.

[0483] In some aspects the guided nuclease may be selected from Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX,Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c effector protein

[0484] In some aspects, the guided nuclease, such as a CRISPR / Cas effector protein useful with the invention may comprise a mutation in its nuclease active site (e.g., RuvC, HNH, e.g., RuvC site of a Cas12a nuclease domain, e.g., RuvC site and / or HNH site of a Cas9 nuclease domain). A CRISPR-Cas effector protein having a mutation in its nuclease active site, and therefore, no longer comprising nuclease activity, is commonly referred to as "dead," e.g., dCas. In some embodiments, a CRISPR-Cas effector protein domain or polypeptide having a mutation in its nuclease active site may have impaired activity or reduced activity as compared to the same CRISPR-Cas effector protein without the mutation, e.g., a nickase, e.g., Cas9 nickase, Cas12a nickase.

[0485] In some aspects, the guided nuclease may comprise another functional domain than a nuclease, such as a adenine deaminase domain or a cytosine deaminase domain or a reverse transcriptase domain.

[0486] An adenine deaminase (or adenosine deaminase) useful with this invention may be any known or later identified adenine deaminase from any organism (see, e.g., U.S. Patent No. 10,113,163, which is incorporated by reference herein for its disclosure of adenine deaminases). An adenine deaminase can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the adenine deaminase may catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxy-inosine, respectively. In some embodiments, the adenosine deaminase may catalyze the hydrolytic deamination of adenine or adenosine in DNA. In some embodiments, an adenine deaminase encoded by a nucleic acid construct of the invention may generate an A→G conversion in the sense (e.g.,template) strand of the target nucleic acid or a T→C conversion in the antisense (e.g., "˗", complementary) strand of the target nucleic acid.

[0487] In some embodiments, an adenosine deaminase may be a variant of a naturally occurring adenine deaminase. Thus, in some embodiments, an adenosine deaminase may be about 70% to 100% identical to a wild type adenine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical, and any range or value therein, to a naturally occurring adenine deaminase). In some embodiments, the deaminase or deaminase does not occur in nature and may be referred to as an engineered, mutated or evolved adenosine deaminase. Thus, for example, an engineered, mutated or evolvedadenine deaminase polypeptide or an adenine deaminase domain may be about 70% to 99.9% identical to a naturally occurring adenine deaminase polypeptide / domain (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical, and any range or value therein, to a naturally occurring adenine deaminase polypeptide or adenine deaminase domain). In some embodiments, the adenosine deaminase may be from a bacterium, (e.g., Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, Caulobacter crescentus, and the like). In some embodiments, a polynucleotide encoding an adenine deaminase polypeptide / domain may be codon optimized for expression in a plant.

[0488] In some embodiments, an adenine deaminase domain may be a wild type tRNA- specific adenosine deaminase domain, e.g., a tRNA-specific adenosine deaminase (TadA) and / or a mutated / evolved adenosine deaminase domain, e.g., mutated / evolved tRNA-specific adenosine deaminase domain (TadA*). In some embodiments, a TadA domain may be from E. coli. In some embodiments, the TadA may be modified, e.g., truncated, missing one or more N-terminal and / or C-terminal amino acids relative to a full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19, or 20 N-terminal and / or C terminal amino acid residues may be missing relative to a full length TadA. In some embodiments, a TadA polypeptide or TadA domain does not comprise an N-terminal methionine. In some embodiments, a polynucleotide encoding a TadA / TadA* may be codon optimized for expression in a plant.

[0489] A cytosine deaminase catalyzes cytosine deamination and results in a thymidine (through a uracil intermediate), causing a C to T conversion, or a G to A conversion in the complementary strand in the genome. Thus, in some embodiments, the cytosine deaminase encoded by the polynucleotide of the invention generates a C→T conversion in the sense (e.g., template) strand of the target nucleic acid or a G →A conversion in antisense (e.g., "˗", complementary) strand of the target nucleic acid.

[0490] In some embodiments, the adenine deaminase encoded by the nucleic acid construct of the invention generates an A→G conversion in the sensetemplate) strand of the target nucleic acid or a T→C conversion in the antisense (e.g.,complementary) strand of the target nucleic acid.

[0491] The nucleic acid constructs of the invention encoding a base editor comprising a sequence-specific DNA binding protein and a cytosine deaminase polypeptide, and nucleic acid constructs / expression cassettes / vectors encoding the same, may be used in combination with guide nucleic acids for modifying target nucleic acid including, but not limited to, generationof C→T or G →A mutations in a target nucleic acid including, but not limited to, a plasmid sequence; generation of C→T or G →A mutations in a coding sequence to alter an amino acid identity; generation of C→T or G →A mutations in a coding sequence to generate a stop codon; generation of C→T or G →A mutations in a coding sequence to disrupt a start codon; generation of point mutations in genomic DNA to disrupt transcription factor binding; and / or generation of point mutations in genomic DNA to disrupt splice junctions.

[0492] The nucleic acid constructs of the invention encoding a base editor comprising a sequence-specific DNA binding protein and an adenine deaminase polypeptide, and expression cassettes and / or vectors encoding the same may be used in combination with guide nucleic acids for modifying a target nucleic acid including, but not limited to, generation of A→G or T→C mutations in a target nucleic acid including, but not limited to, a plasmid sequence; generation of A→G or T→C mutations in a coding sequence to alter an amino acid identity; generation of A→G or T→C mutations in a coding sequence to generate a stop codon; generation of A→G or T→C mutations in a coding sequence to disrupt a start codon; generation of point mutations in genomic DNA to disrupt function; and / or generation of point mutations in genomic DNA to disrupt splice junctions. Target sites

[0493] As used herein, a “target sequence” refers to a selected sequence or region of a DNA molecule in which a modification (e.g., cleavage, site-directed integration) is desired. A target sequence comprises a target site.

[0494] As used herein, a “target site” refers to the portion of a target sequence that is cleaved by a guided nuclease such as CRISPR nuclease. In contrast to a non-target nucleic acid (e.g., non-target ssDNA) or non-target region, a target site comprises significant complementarity to a guide nucleic acid or a guide RNA.

[0495] In an aspect, a target site is 100% complementary to a guide nucleic acid. In another aspect, a target site is 99% complementary to a guide nucleic acid. In another aspect, a target site is 98% complementary to a guide nucleic acid. In another aspect, a target site is 97% complementary to a guide nucleic acid. In another aspect, a target site is 96% complementary to a guide nucleic acid. In another aspect, a target site is 95% complementary to a guide nucleic acid. In another aspect, a target site is 94% complementary to a guide nucleic acid. In another aspect, a target site is 93% complementary to a guide nucleic acid. In another aspect, a target site is 92% complementary to a guide nucleic acid. In another aspect, a target site is 91% complementary to a guide nucleic acid. In another aspect, a target site is 90% complementaryto a guide nucleic acid. In another aspect, a target site is 85% complementary to a guide nucleic acid. In another aspect, a target site is 80% complementary to a guide nucleic acid.

[0496] In an aspect, a target site comprises at least one PAM site. In an aspect, a target site is adjacent to a nucleic acid sequence that comprises at least one PAM site. In another aspect, a target site is within 5 nucleotides of at least one PAM site. In a further aspect, a target site is within 10 nucleotides of at least one PAM site. In another aspect, a target site is within 15 nucleotides of at least one PAM site. In another aspect, a target site is within 20 nucleotides of at least one PAM site. In another aspect, a target site is within 25 nucleotides of at least one PAM site. In another aspect, a target site is within 30 nucleotides of at least one PAM site.

[0497] In an aspect, a target site is positioned within genic DNA. In another aspect, a target site is positioned within a gene. In another aspect, a target site is positioned within a gene of interest. In another aspect, a target site is positioned within an exon of a gene. In another aspect, a target site is positioned within an intron of a gene. In another aspect, a target site is positioned within the promoter of a gene. In another aspect, a target site is positioned within 5’- UTR of a gene. In another aspect, a target site is positioned within a 3’-UTR of a gene. In another aspect, a target site is positioned within intergenic DNA.

[0498] A "protospacer sequence" refers to the target double stranded DNA and specifically to the portion of the target DNA (e.g., or target region in the genome) that is fully or substantially complementary (and hybridizes) to the spacer sequence of the CRISPR repeat- spacer sequences (e.g., guide nucleic acids, CRISPR arrays, crRNAs).

[0499] In the case of Type V CRISPR-Cas (e.g., Cas12a) systems and Type II CRISPR- Cas (Cas9) systems, the protospacer sequence is flanked by (e.g., immediately adjacent to) a protospacer adjacent motif (PAM). For Type IV CRISPR-Cas systems, the PAM is located at the 5’ end on the non-target strand and at the 3’ end of the target strand (see below, as an example). 5'-NNNNNNNNNNNNNNNNNNN-3' RNA Spacer | | | | | | | | | | | | | | | | | | | | 3'AAANNNNNNNNNNNNNNNNNNN-5' Target strand | | |Non-target strand

[0500] In the case of Type II CRISPR-Cas (e.g., Cas9) systems, the PAM is located immediately 3’ of the target region. The PAM for Type I CRISPR-Cas systems is located 5’ of the target strand. There is no known PAM for Type III CRISPR-Cas systems. Makarova et al. describes the nomenclature for all the classes, types and subtypes of CRISPR systems ((2015)Nature Reviews Microbiology 13:722–736). Guide structures and PAMs are described in by R. Barrangou ((2015) Genome Biol.16:247).

[0501] Canonical Cas12a PAMs are T rich. In some embodiments, a canonical Cas12a PAM sequence may be 5’-TTN, 5’-TTTN, or 5’-TTTV. In some embodiments, canonical Cas9 (e.g., S. pyogenes) PAMs may be 5’-NGG-3’. In some embodiments, non-canonical PAMs may be used but may be less efficient.

[0502] Additional PAM sequences may be determined by those skilled in the art through established experimental and computational approaches. Thus, for example, experimental approaches include targeting a sequence flanked by all possible nucleotide sequences and identifying sequence members that do not undergo targeting, such as through the transformation of target plasmid DNA (Esvelt et al. (2013) Nat. Methods 10:1116-1121; Jiang et al. (2013) Nat. Biotechnol. 31:233-239). In some aspects, a computational approach can include performing BLAST searches of natural spacers to identify the original target DNA sequences in bacteriophages or plasmids and aligning these sequences to determine conserved sequences adjacent to the target sequence (Briner and Barrangou. (2014) Appl. Environ. Microbiol. 80:994-1001; Mojica et al. (2009) Microbiology 155:733-740).

[0503] In an aspect, a target DNA molecule is single-stranded. In another aspect, a target DNA molecule is double-stranded.

[0504] In an aspect, a target sequence comprises genomic DNA. In an aspect, a target sequence is positioned within a nuclear genome. In an aspect, a target sequence comprises chromosomal DNA. In an aspect, a target sequence comprises plasmid DNA. In an aspect, a target sequence is positioned within a plasmid. In an aspect, a target sequence comprises mitochondrial DNA. In an aspect, a target sequence is positioned within a mitochondrial genome. In an aspect, a target sequence comprises plastid DNA. In an aspect, a target sequence is positioned within a plastid genome. In an aspect, a target sequence comprises chloroplast DNA. In an aspect, a target sequence is positioned within a chloroplast genome. In an aspect, a target sequence is positioned within a genome selected from the group consisting of a nuclear genome, a mitochondrial genome, and a plastid genome.

[0505] In an aspect, a target sequence comprises genic DNA. As used herein, “genic DNA” refers to DNA that encodes one or more genes. In another aspect, a target sequence comprises intergenic DNA. In contrast to genic DNA, “intergenic DNA” comprises noncoding DNA, and lacks DNA encoding a gene. In an aspect, intergenic DNA is positioned between two genes.

[0506] In an aspect, a target sequence encodes a gene. As used herein, a “gene” refers to a polynucleotide that can produce a functional unit (e.g., without being limiting, for example, a protein, or a non-coding RNA molecule). A gene can comprise a promoter, an enhancer sequence, a leader sequence, a transcriptional start site, a transcriptional stop site, a polyadenylation site, one or more exons, one or more introns, a 5’-UTR, a 3’-UTR, or any combination thereof. A “gene sequence” can comprise a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcriptional start site, a transcriptional stop site, a polyadenylation site, one or more exons, one or more introns, a 5’-UTR, a 3’-UTR, or any combination thereof. In one aspect, a gene encodes a non-protein-coding RNA molecule or a precursor thereof. In another aspect, a gene encodes a protein. In some embodiments, the target sequence is selected from the group consisting of: a promoter, an enhancer sequence, a leader sequence, a transcriptional start site, a transcriptional stop site, a polyadenylation site, an exon, an intron, a splice site, a 5’-UTR, a 3’-UTR, a protein coding sequence, a non-protein- coding sequence, a miRNA, a pre-miRNA and a miRNA binding site.

[0507] Non-limiting examples of a non-protein-coding RNA molecule include a microRNA (miRNA), a miRNA precursor (pre-miRNA), a small interfering RNA (siRNA), a small RNA (18 to 26 nucleotides in length) and precursor encoding same, a heterochromatic siRNA (hc-siRNA), a Piwi-interacting RNA (piRNA), a hairpin double strand RNA (hairpin dsRNA), a trans-acting siRNA (ta-siRNA), a naturally occurring antisense siRNA (nat-siRNA), a CRISPR RNA (crRNA), a tracer RNA (tracrRNA), a guide RNA (gRNA), and a single guide RNA (sgRNA). In an aspect, a non-protein-coding RNA molecule comprises a miRNA. In an aspect, a non-protein-coding RNA molecule comprises a siRNA. In an aspect, a non-protein- coding RNA molecule comprises a ta-siRNA. In an aspect, a non-protein-coding RNA molecule is selected from the group consisting of a miRNA, a siRNA, and a ta-siRNA.

[0508] As used herein, a “gene of interest” refers to a polynucleotide sequence encoding a protein or a non-protein-coding RNA molecule that is to be integrated into a target sequence, or, alternatively, an endogenous polynucleotide sequence encoding a protein or a non-protein- coding RNA molecule that is to be edited by a ribonucleoprotein. In an aspect, a gene of interest encodes a protein. In another aspect, a gene of interest encodes a non-protein-coding RNA molecule. In an aspect, a gene of interest is exogenous to a targeted DNA molecule. In an aspect, a gene of interest replaces an endogenous gene in a targeted DNA molecule. Mutations

[0509] In an aspect, a ribonucleoprotein or method provided herein generates at least one mutation in a target sequence.

[0510] In an aspect, a seed produced from a plant provided herein comprises at least one mutation in a gene of interest comprising a target site as compared to a seed of a control plant of the same line or variety that lacks a first nucleic acid sequence encoding a guided nuclease operably linked to a floral cell-preferred promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterologous second promoter. In an aspect, a seed produced from a plant provided herein comprises at least one mutation in a gene of interest comprising a target site as compared to a seed of a control plant of the same line or variety that lacks a first nucleic acid sequence encoding a guided nuclease operably linked to a floral tissue- preferred promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterologous second promoter.

[0511] In an aspect, a seed produced from a plant provided herein comprises at least one mutation in a gene of interest comprising a target site as compared to a seed of a control plant of the same line or variety that lacks a first nucleic acid sequence encoding a guided nuclease operably linked to a heterologous promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a floral cell-preferred promoter. In an aspect, a seed produced from a plant provided herein comprises at least one mutation in a gene of interest comprising a target site as compared to a seed of a control plant of the same line or variety that lacks a first nucleic acid sequence encoding a guided nuclease operably linked to a heterologous promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a floral tissue-preferred promoter.

[0512] As used herein, a “mutation” refers to a non-naturally occurring alteration to a nucleic acid or amino acid sequence as compared to a naturally occurring reference nucleic acid or amino acid sequence from the same organism. It will be appreciated that, when identifying a mutation, the reference sequence should be from the same nucleic acid (e.g., gene, non-coding RNA) or amino acid (e.g., protein). In determining if a difference between two sequences comprises a mutation, it will be appreciated in the art that the comparison should not be made between homologous sequences of two different species or between homologous sequences of two different varieties of a single species. Rather, the comparison should be made between the edited (e.g., mutated) sequence and the endogenous, non-edited (e.g., “wildtype”) sequence of the same organism.

[0513] Several types of mutations are known in the art. In an aspect, a mutation comprises an insertion. An “insertion” refers to the addition of one or more nucleotides or amino acids toa given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation comprises a deletion. A “deletion” refers to the removal of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation comprises a substitution. A “substitution” refers to the replacement of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, as compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, a mutation comprises an inversion. An “inversion” refers to when a segment of a polynucleotide or amino acid sequence is reversed end-to-end. In an aspect, a mutation provided herein comprises a mutation selected from the group consisting of an insertion, a deletion, a substitution, and an inversion.

[0514] In an aspect, a plant or seed comprises at least one mutation in a gene of interest, where the at least one mutation results in the deletion of one or more amino acids from a protein encoded by the gene of interest as compared to a wildtype protein.

[0515] In an aspect, a plant or seed comprises at least one mutation in a gene of interest, where the at least one mutation results in the substitution of one or more amino acids within a protein encoded by the gene of interest as compared to a wildtype protein.

[0516] In an aspect, a plant or seed comprises at least one mutation in a gene of interest, where the at least one mutation results in the insertion of one or more amino acids within a protein encoded by the gene of interest as compared to a wildtype protein.

[0517] Mutations in coding regions of genes (e.g., exonic mutations) can result in a truncated protein or polypeptide when a mutated messenger RNA (mRNA) is translated into a protein or polypeptide. In an aspect, this disclosure provides a mutation that results in the truncation of a protein or polypeptide. As used herein, a “truncated” protein or polypeptide comprises at least one fewer amino acid as compared to an endogenous control protein or polypeptide. For example, if endogenous Protein A comprises 100 amino acids, a truncated version of Protein A can comprise between 1 and 99 amino acids.

[0518] Without being limited by any scientific theory, one way to cause a protein or polypeptide truncation is by the introduction of a premature stop codon in an mRNA transcript of an endogenous gene. In an aspect, this disclosure provides a mutation that results in a premature stop codon in an mRNA transcript of an endogenous gene. As used herein, a “stop codon” refers to a nucleotide triplet within an mRNA transcript that signals a termination of protein translation. A “premature stop codon” refers to a stop codon positioned earlier (e.g., on the 5’-side) than the normal stop codon position in an endogenous mRNA transcript. Withoutbeing limiting, several stop codons are known in the art, including “UAG,” “UAA,” “UGA,” “TAG,” “TAA,” and “TGA.”

[0519] In an aspect, a seed or plant comprises at least one mutation, where the at least one mutation results in the introduction of a premature stop codon in a messenger RNA encoded by the gene of interest as compared to a wildtype messenger RNA.

[0520] In an aspect, a mutation provided herein comprises a null mutation. As used herein, a “null mutation” refers to a mutation that confers a complete loss-of-function for a protein encoded by a gene comprising the mutation, or, alternatively, a mutation that confers a complete loss-of-function for a small RNA encoded by a genomic locus. A null mutation can cause lack of mRNA transcript production, a lack of small RNA transcript production, a lack of protein function, or a combination thereof.

[0521] A mutation provided herein can be positioned in any part of an endogenous gene. In an aspect, a mutation provided herein is positioned within an exon of an endogenous gene. In another aspect, a mutation provided herein is positioned within an intron of an endogenous gene. In a further aspect, a mutation provided herein is positioned within a 5’-untranslated region of an endogenous gene. In still another aspect, a mutation provided herein is positioned within a 3’-untranslated region of an endogenous gene. In yet another aspect, a mutation provided herein is positioned within a promoter of an endogenous gene.

[0522] In an aspect, a mutation is positioned at a splice site within a gene. A mutation at a splice site can interfere with the splicing of exons during mRNA processing. If one or more nucleotides are inserted, deleted, or substituted at a splice site, splicing can be perturbed. Perturbed splicing can result in unspliced introns, missing exons, or both, from a mature mRNA sequence. Typically, although not always, a “GU” sequence is required at the 5’ end of an intron and a “AG” sequence is required at the 3’ end of an intron for proper splicing. If either of these splice sites are mutated, splicing perturbations can occur.

[0523] In an aspect, a seed or plant comprises at least one mutation, where the at least one mutation comprises the deletion of one or more splice sites from a gene of interest. In another aspect, a seed or plant comprises at least one mutation, where the at least one mutation is positioned within one or more splice sites from a gene of interest.

[0524] In an aspect, a mutation comprises a site-directed integration. In an aspect, a site- directed integration comprises the insertion of all or part of a desired sequence into a target sequence.

[0525] As used herein, “site-directed integration” refers to all, or a portion, of a desired sequence (e.g., an exogenous gene, an edited endogenous gene) being inserted or integrated ata desired site or locus within the plant genome (e.g., target sequence). As used herein, a “desired sequence” refers to a DNA molecule comprising a nucleic acid sequence that is to be integrated into a genome of a plant or plant cell. The desired sequence can comprise a transgene or construct. In an aspect, a nucleic acid molecule comprising a desired sequence comprises one or two homology arms flanking the desired sequence to promote the targeted insertion event through homologous recombination and / or homology-directed repair.

[0526] In an aspect, a method provided herein comprises site-directed integration of a desired sequence into a target sequence.

[0527] Any site or locus within the genome of a plant can be chosen for site-directed integration of a transgene or construct of the present disclosure. In an aspect, a target sequence is positioned within a B, or supernumerary, chromosome.

[0528] For site-directed integration, a double-strand break (DSB) or nick may first be made at a target sequence via a guided nuclease or ribonucleoprotein provided herein. In the presence of a desired sequence, the DSB or nick can then be repaired by homologous recombination (HR) between the homology arm(s) of the desired sequence and the target sequence, or by non-homologous end joining (NHEJ), resulting in site-directed integration of all or part of the desired sequence into the target sequence to create the targeted insertion event at the site of the DSB or nick.

[0529] In an aspect, site-directed integration comprises the use of NHEJ repair mechanisms endogenous to a cell. In another aspect, site-directed integration comprises the use of HR repair mechanisms endogenous to a cell.

[0530] In an aspect, repair of a double-stranded break generates at least one mutation in a gene of interest as compared to a control plant of the same line or variety.

[0531] In an aspect, a mutation comprises the integration of at least 5 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 10 contiguous nucleotides of a desired sequence molecule into a target sequence. In an aspect, a mutation comprises the integration of at least 15 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 20 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 25 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 50 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 100 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 250contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 1000 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of at least 2000 contiguous nucleotides of a desired sequence into a target sequence.

[0532] In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 3500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 2500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 1500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 750 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 250 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 5 contiguous nucleotides and 150 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 25 contiguous nucleotides and 2500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 25 contiguous nucleotides and 1500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 25 contiguous nucleotides and 750 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 50 contiguous nucleotides and 2500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 50 contiguous nucleotides and 1500 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 50 contiguous nucleotides and 750 contiguous nucleotides of a desired sequence into a target sequence. In an aspect, a mutation comprises the integration of between 100 contiguous nucleotides and 2500 contiguous nucleotides of a desired sequence into a target Sequence. In an aspect, a mutation comprises the integration of between 100 contiguous nucleotides and 1500 contiguous nucleotides of a desired sequence into a target Sequence. In an aspect, a mutation comprises the integration of between 100 contiguous nucleotides and 750 contiguous nucleotides of a desired sequence into a target Sequence.

[0533] In an aspect, a method provided herein further comprises detecting an edit or a mutation in a target sequence. The screening and selection of mutagenized or edited plants or plant cells can be through any methodologies known to those having ordinary skill in the art. Examples of screening and selection methodologies include, but are not limited to, Southern analysis, PCR amplification for detection of a polynucleotide, Northern blots, RNase protection, primer-extension, RT-PCR amplification for detecting RNA transcripts, Sanger sequencing, Next Generation sequencing technologies (e.g., Illumina, PacBio, Ion Torrent, 454) enzymatic assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme- linked immunoassays to detect polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining also can be used to detect the presence or expression of polypeptides and / or polynucleotides. Methods for performing all of the above-referenced techniques are known in the art.

[0534] In an aspect, a sequence provided herein encodes at least one ribozyme. In an aspect, a sequence provided herein encodes at least two ribozymes. In an aspect, a ribozyme is a self-cleaving ribozyme. Self-cleaving ribozymes are known in the art. For example, see Jimenez et al., Trends Biochem. Sci., 40:648-661 (2015).

[0535] In an aspect, a sequence encoding at least one guide nucleic acid is flanked by self-cleaving ribozymes. In an aspect, a sequence encoding at least one guide nucleic acid is immediately adjacent to a sequence encoding a ribozyme (e.g., the 5′-most nucleotide of the guide nucleic acid abuts the 3′-most nucleotide of the ribozyme or the 3′-most nucleotide of the guide nucleic acid abuts the 5′-most nucleotide of the ribozyme). In an aspect, a sequence encoding at least one guide nucleic acid is separated from a sequence encoding a ribozyme by at least 1, at least 2, at least 3, at least 4, at least 5, at least 10, at least 20, at least 30, at least 40, at least 50, at least 100, at least 250, at least 500, or at least 10000 nucleotides. Plants

[0536] Any plant or plant cell can be used with the methods and compositions provided herein. In an aspect, a plant is selected from the group consisting of a corn plant, a rice plant, a sorghum plant, a wheat plant, an alfalfa plant, a barley plant, a millet plant, a rye plant, a sugarcane plant, a cotton plant, a soybean plant, a canola plant, a tomato plant, an onion plant, a cucumber plant, an Arabidopsis plant, and a potato plant. In an aspect, a plant is an angiosperm. In an aspect, a plant is a gymnosperm. In an aspect, a plant is a monocotyledonous plant. In an aspect, a plant is a dicotyledonous plant. In an aspect, a plant is a plant of a familyselected from the group consisting of Alliaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Brassicaceae, Caesalpiniaceae, Cucurbitaceae, Ericaceae, Fabaceae, Juglandaceae, Malvaceae, Mimosaceae, Moraceae, Musaceae, Orchidaceae, Papilionaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae, and Solanaceae.

[0537] In an aspect, a plant cell is selected from the group consisting of a corn cell, a rice cell, a sorghum cell, a wheat cell, an alfalfa cell, a barley cell, a millet cell, a rye cell, a sugarcane cell, a cotton cell, a soybean cell, a canola cell, a tomato cell, an onion cell, a cucumber cell, an Arabidopsis cell, and a potato cell. In an aspect, a plant cell is an angiosperm plant cell. In an aspect, a plant cell is a gymnosperm plant cell. In an aspect, a plant cell is a monocotyledonous plant cell. In an aspect, a plant cell is a dicotyledonous plant cell. In an aspect, a plant cell is a plant cell of a family selected from the group consisting of Alliaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Brassicaceae, Caesalpiniaceae, Cucurbitaceae, Ericaceae, Fabaceae, Juglandaceae, Malvaceae, Mimosaceae, Moraceae, Musaceae, Orchidaceae, Papilionaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae, and Solanaceae.

[0538] As used herein, a “variety” refers to a group of plants within a species (e.g., without being limiting Zea mays) that share certain genetic traits that separate them from other possible varieties within that species. Varieties can be inbreds or hybrids, though commercial plants are often hybrids to take advantage of hybrid vigor. Individuals within a hybrid cultivar are homogeneous, nearly genetically identical, with most loci in the heterozygous state.

[0539] As used herein, the term “inbred” means a line that has been bred for genetic homogeneity. In an aspect, a seed provided herein is an inbred seed. In an aspect, a plant provided herein is an inbred plant.

[0540] As used herein, the term “hybrid” means a progeny of mating between at least two genetically dissimilar parents. Without limitation, examples of mating schemes include single crosses, modified single cross, double modified single cross, three-way cross, modified three- way cross, and double cross wherein at least one parent in a modified cross is the progeny of a cross between sister lines. In an aspect, a seed provided herein is a hybrid seed. In an aspect, a plant provided herein is a hybrid plant.

[0541] In some jurisdictions, products obtained exclusively by essentially biological processes, such as plant products are excluded from patent protection. Accordingly, the claimed plants, plant parts and cells and their progeny can be defined as directed only to those plants, plant parts and cells and their progeny which are obtained by technical intervention (regardless of any further propagation through crossing and selection). An embodiment of the invention is directed at plants, or plant parts or progeny produced or obtainable using gene editingtechnology herein described. Alternatively, the subject matter excluded from patentability may be disclaimed. An embodiment of the invention is directed at plants, part of plants or progeny thereof comprising the genomic alterations as elsewhere herein described, provided that the plants, parts or plants or progeny are not obtained exclusively through essentially biological processes, wherein essentially biological processes are processes for the production of plants or animals if they consist entirely of natural phenomena such as crossing or selection. Transformation

[0542] Methods can involve transient transformation or stable integration of any nucleic acid molecule into any plant or plant cell provided herein.

[0543] As used herein, “stable integration” or “stably integrated” refers to a transfer of DNA into genomic DNA of a targeted cell or plant that allows the targeted cell or plant to pass the transferred DNA to the next generation of the transformed organism. Stable transformation requires the integration of transferred DNA within the reproductive cell(s) of the transformed organism. As used herein, “transiently transformed” or “transient transformation” refers to a transfer of DNA into a cell that is not transferred to the next generation of the transformed organism. In a transient transformation the transformed DNA does not typically integrate into the transformed cell’s genomic DNA. In one aspect, a method stably transforms a plant cell or plant with one or more nucleic acid molecules provided herein. In another aspect, a method transiently transforms a plant cell or plant with one or more nucleic acid molecules provided herein.

[0544] In an aspect, a nucleic acid molecule encoding a guided nuclease is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a Cas12a nuclease is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a CasX nuclease is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a guide nucleic acid is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a guide RNA is stably integrated into a genome of a plant. In an aspect, a nucleic acid molecule encoding a single-guide RNA is stably integrated into a genome of a plant.

[0545] Numerous methods for transforming cells with a recombinant nucleic acid molecule or construct are known in the art, which can be used according to methods of the present application. Any suitable method or technique for transformation of a cell known in the art can be used according to present methods. Effective methods for transformation of plants include bacterially mediated transformation, such as Agrobacterium-mediated orRhizobium-mediated transformation and microprojectile bombardment-mediated transformation. A variety of methods are known in the art for transforming explants with a transformation vector via bacterially mediated transformation or microprojectile bombardment and then subsequently culturing, etc., those explants to regenerate or develop transgenic plants.

[0546] In an aspect, a method comprises providing a cell with a nucleic acid molecule via Agrobacterium-mediated transformation. In an aspect, a method comprises providing a cell with a nucleic acid molecule via polyethylene glycol-mediated transformation. In an aspect, a method comprises providing a cell with a nucleic acid molecule via biolistic transformation. In an aspect, a method comprises providing a cell with a nucleic acid molecule via liposome- mediated transfection. In an aspect, a method comprises providing a cell with a nucleic acid molecule via viral transduction. In an aspect, a method comprises providing a cell with a nucleic acid molecule via use of one or more delivery particles. In an aspect, a method comprises providing a cell with a nucleic acid molecule via microinjection. In an aspect, a method comprises providing a cell with a nucleic acid molecule via electroporation.

[0547] In an aspect, a nucleic acid molecule is provided to a cell via a method selected from the group consisting of Agrobacterium-mediated transformation, polyethylene glycol- mediated transformation, biolistic transformation, liposome-mediated transfection, viral transduction, the use of one or more delivery particles, microinjection, and electroporation.

[0548] Other methods for transformation, such as vacuum infiltration, pressure, sonication, and silicon carbide fiber agitation, are also known in the art and envisioned for use with any method provided herein.

[0549] Methods of transforming cells are well known by persons of ordinary skill in the art. For instance, specific instructions for transforming plant cells by microprojectile bombardment with particles coated with recombinant DNA (e.g., biolistic transformation) are found in U.S. Patent Nos. 5,550,318; 5,538,880 6,160,208; 6,399,861; and 6,153,812 and Agrobacterium-mediated transformation is described in U.S. Patent Nos.5,159,135; 5,824,877; 5,591,616; 6,384,301; 5,750,871; 5,463,174; and 5,188,958, all of which are incorporated herein by reference. Additional methods for transforming plants can be found in, for example, Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any appropriate method known to those skilled in the art can be used to transform a plant cell with any of the nucleic acid molecules provided herein.

[0550] Lipofection is described in e.g., U.S. Pat. Nos. 5,049,386, 4,946,787; and 4,897,355) and lipofection reagents are sold commercially (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognitionlipofection of polynucleotides include those of Felgner, WO 91 / 17424; WO 91 / 16024. Delivery can be to cells (e.g. in vitro or ex vivo administration) or target tissues (e.g. in vivo administration).

[0551] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for expression of one or more elements of a nucleic acid molecule are as used in WO 2014 / 093622. In an aspect, a method of providing a nucleic acid molecule or a protein to a cell comprises delivery via a delivery particle. In an aspect, a method of providing a nucleic acid molecule to a plant cell or plant comprises delivery via a delivery vesicle. In an aspect, a delivery vesicle is selected from the group consisting of an exosome and a liposome. In an aspect, a method of providing a nucleic acid molecule to a plant cell or plant comprises delivery via a viral vector. In an aspect, a viral vector is selected from the group consisting of an adenovirus vector, a lentivirus vector, and an adeno-associated viral vector. In another aspect, a method providing a nucleic acid molecule to a plant cell or plant comprises delivery via a nanoparticle. In an aspect, a method providing a nucleic acid molecule to a plant cell or plant comprises microinjection. In an aspect, a method providing a nucleic acid molecule to a plant cell or plant comprises polycations. In an aspect, a method providing a nucleic acid molecule to a plant cell or plant comprises a cationic oligopeptide.

[0552] In an aspect, a delivery particle is selected from the group consisting of an exosome, an adenovirus vector, a lentivirus vector, an adeno-associated viral vector, a nanoparticle, a polycation, and a cationic oligopeptide. In an aspect, a method provided herein comprises the use of one or more delivery particles. In another aspect, a method provided herein comprises the use of two or more delivery particles. In another aspect, a method provided herein comprises the use of three or more delivery particles.

[0553] Suitable agents to facilitate transfer of nucleic acids into a plant cell include agents that increase permeability of the exterior of the plant or that increase permeability of plant cells to oligonucleotides or polynucleotides. Such agents to facilitate transfer of the composition into a plant cell include a chemical agent, or a physical agent, or combinations thereof. Chemical agents for conditioning includes (a) surfactants, (b) organic solvents, aqueous solutions, or aqueous mixtures of organic solvents, (c) oxidizing agents, (e) acids, (f) bases, (g) oils, (h) enzymes, or combinations thereof.

[0554] Organic solvents useful in conditioning a plant to permeation by polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, other solvents miscible with water or that will dissolve phosphonucleotides in non-aqueous systems (such as is used in synthetic reactions). Naturallyderived or synthetic oils with or without surfactants or emulsifiers can be used, e. g. , plant- sourced oils, crop oils (such as those listed in the 9thCompendium of Herbicide Adjuvants, publicly available on line at www(dot)herbicide(dot)adjuvants(dot)com) can be used, e. g. , paraffinic oils, polyol fatty acid esters, or oils with short-chain molecules modified with amides or polyamines such as polyethyleneimine or N-pyrrolidine.

[0555] Examples of useful surfactants include sodium or lithium salts of fatty acids (such as tallow or tallowamines or phospholipids) and organosilicone surfactants. Other useful surfactants include organosilicone surfactants including nonionic organosilicone surfactants, e. g. , trisiloxane ethoxylate surfactants or a silicone polyether copolymer such as a copolymer of polyalkylene oxide modified heptamethyl trisiloxane and allyloxypolypropylene glycol methylether (commercially available as Silwet® L-77).

[0556] Useful physical agents can include (a) abrasives such as carborundum, corundum, sand, calcite, pumice, garnet, and the like, (b) nanoparticles such as carbon nanotubes or (c) a physical force. Carbon nanotubes are disclosed by Kam et. al. (2004) Am. Chem. Soc, 126 (22):6850-6851, Liu et. al. (2009) Nano Lett, 9(3): 1007-1010, and Khodakovskaya et. al. (2009) ACS Nano, 3(10):3221-3227. Physical force agents can include heating, chilling, the application of positive pressure, or ultrasound treatment. Embodiments of the method can optionally include an incubation step, a neutralization step (e.g., to neutralize an acid, base, or oxidizing agent, or to inactivate an enzyme), a rinsing step, or combinations thereof. The methods of the invention can further include the application of other agents which will have enhanced effect due to the silencing of certain genes. For example, when a polynucleotide is designed to regulate genes that provide herbicide resistance, the subsequent application of the herbicide can have a dramatic effect on herbicide efficacy.

[0557] Agents for laboratory conditioning of a plant cell to permeation by polynucleotides include, e.g., application of a chemical agent, enzymatic treatment, heating or chilling, treatment with positive or negative pressure, or ultrasound treatment. Agents for conditioning plants in a field include chemical agents such as surfactants and salts.

[0558] In an aspect, a transformed or transfected cell is a plant cell. Recipient plant cell or explant targets for transformation include, but are not limited to, a seed cell, a fruit cell, a leaf cell, a cotyledon cell, a hypocotyl cell, a meristem cell, an embryo cell, an endosperm cell, a root cell, a shoot cell, a stem cell, a pod cell, a flower cell, an inflorescence cell, a stalk cell, a pedicel cell, a style cell, a stigma cell, a receptacle cell, a petal cell, a sepal cell, a pollen cell, an anther cell, a filament cell, an ovary cell, an ovule cell, a pericarp cell, a phloem cell, a bud cell, or a vascular tissue cell. In another aspect, this disclosure provides a plant chloroplast. Ina further aspect, this disclosure provides an epidermal cell, a guard cell, a trichome cell, a root hair cell, a storage root cell, or a tuber cell. In another aspect, this disclosure provides a protoplast. In another aspect, this disclosure provides a plant callus cell. Any cell from which a fertile plant can be regenerated is contemplated as a useful recipient cell for practice of this disclosure. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or parts of embryos, seedling apical meristems, microspores, and the like. Those cells which are capable of proliferating as callus can serve as recipient cells for transformation. Practical transformation methods and materials for making transgenic plants of this disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Patents 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004 / 0216189, all of which are incorporated herein by reference. Transformed explants, cells or tissues can be subjected to additional culturing steps, such as callus induction, selection, regeneration, etc., as known in the art. Transformed cells, tissues or explants containing a recombinant DNA insertion can be grown, developed or regenerated into transgenic plants in culture, plugs or soil according to methods known in the art. In one aspect, this disclosure provides plant cells that are not reproductive material and do not mediate the natural reproduction of the plant. In another aspect, this disclosure also provides plant cells that are reproductive material and mediate the natural reproduction of the plant. In another aspect, this disclosure provides plant cells that cannot maintain themselves via photosynthesis. In another aspect, this disclosure provides somatic plant cells. Somatic cells, contrary to germline cells, do not mediate plant reproduction. In one aspect, this disclosure provides a non-reproductive plant cell.

[0559] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided with respect to certain embodiments herein is intended merely to better illuminate the present disclosure and does not pose a limitation on the scope of the present disclosure otherwise claimed. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the present disclosure.

[0560] Groupings of alternative elements or embodiments of the present disclosure disclosed herein are not to be construed as limitations. Each group member can be referred to and claimed individually or in any combination with other members of the group or otherelements found herein. One or more members of a group can be included in, or deleted from, a group for reasons of convenience or patentability.

[0561] Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent embodiments are possible without departing from the scope of the present disclosure defined in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples.EXAMPLES

[0562] The following examples are included to demonstrate embodiments of the disclosure. It should be appreciated by those of skill in the art that many changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims. Example 1. Editing 5’ and 3’ UTRs for heteroallelic gene modulation.

[0563] Many agronomically relevant traits are often controlled by genes whose mutation can lead to severe agronomic off-types / phenotypes. This example describes the development of an RNAi based editing system for dominant suppression of strong loci in heterozygous state (hybrid state) without the occurrence of phenotype linked to the edited loci in the homozygous state. An important aspect of this methodology is the inversion of a portion of or the entirety of the Untranslated Region (UTR) of a candidate gene / locus (and resultant mRNA) without the perturbance of the coding region.

[0564] Figure 1 provides an illustration of concept for this approach for the 3’UTR. Panel A shows a typical, non-edited structure of a gene comprising the 5’ Untranslated Region (5’UTR), the Coding Sequence (CDS) and the 3’UTR oriented in the normal direction. To generate an inversion within the 3’UTR of the endogenous gene, two functional guide RNAs (gRNAs) for a CRISPR / RNA-guided nuclease system are generated to target the 3’UTR of the gene. See Figure 1 panel B. Each of the two guide RNAs are selected to be unique to the target sites in the respective UTR region without any known off-target sequence within the targeted genome (off-target sequences being defined in this case as not having three or fewer mismatches relative to the 23-mer sequence of the gRNAs). Co delivery of the guide RNAs along with the cognate nuclease system is expected to lead to CRISPR-mediated double stranded break (DSB) at or around the 3’UTR sequence. Following double-strand breaks (DSBs) at each of the two target sites, in a majority of outcomes, the intervening region between the target sites is deleted or excised out and non-homologous end-joining (NHEJ) repair mechanisms join the flanking regions. Less frequently, in some events, the excised region reinserts into the genome in a reverse, antisense or opposite orientation by rejoining with the two excised ends of the flankinggenomic DNA. Although the excised sequence could potentially be perfectly rejoined with the flanking genomic DNA in the opposite orientation without any sequence changes (apart from the inversion), one or more nucleotide insertions or deletions (Indels) may also occur at or near one or both of the junctions where the excised fragment and flanking genomic DNA are rejoined relative to the sequence(s) at or near the two target sites prior to excision.

[0565] An inversion of or within the 3’UTR, when present on both homozygous alleles, would not be expected to affect the function of the protein, therefore no phenotype would be expected in the homozygous stage as shown in Figure 1, panel B. However, upon crossing of such a homozygous edited plant with a wild-type plant, the resulting hybrid offspring would inherit two alleles with different directions of the 3’UTR (see Figure 1, panel C). The edited allele would lead to the production of an RNA transcript comprising a sequence complementary to a portion of the native UTR transcript sequence from the wild type allele. Without being bound by any scientific theory, the inverted region of the mRNA from the edited allele and the corresponding region in the mRNA from the wild-type allele are capable of antisense base- pairing. This in turn could lead to double-stranded RNA formation (dsRNA) between the two populations of mRNAs, triggering RNA-mediated suppression or silencing of both copies of the gene thus producing a knock-down phenotype in the hybrid plant. Example 2: Creation of 5’ UTR and 3’ UTR Inversion Edits of GA3 oxidase_1 gene.

[0566] To create an inversion in either the 5’ or 3’ UTR region of the endogenous Zm.GA3ox_1 gene, a segment or portion of the 5’ or 3’ UTR region of the Zm.GA3ox_1 gene can be excised through genome editing using a CRISPR / Cas system (Cpf1 or Cas12a) and reinserted in a reverse or inverted orientation to produce an antisense sequence in the 5’ or 3’ UTR region of the Zm.GA3ox_1 gene. As provided herein, the inverted antisense sequence would be complementary to the sense sequence of the corresponding segment or portion of a wild-type allele of the Zm.GA3ox_1 gene when the edited Zm.GA3ox_1 allele with the UTR inversion is present in a maize plant or maize plant cell that is heterozygous for the edited allele, which could trigger RNA-mediated suppression or silencing of both copies of the Zm.GA3ox_1 gene. However, when a maize plant or maize plant cell is homozygous for the edited Zm.GA3ox_1 allele with the UTR inversion, the maize plant or maize plant cell would not contain a wild-type copy of the Zm.GA3ox_1 gene and thus RNA-mediated suppression or silencing of the Zm.GA3ox_1 gene would not occur due to the absence of a complementary UTR sequence.

[0567] To generate the inversions in one of the 5’ or 3’ UTR regions of the endogenous GA3 oxidase_1 gene in maize, pairs of two 23-mer guide RNAs (gRNAs, or spacers) were selected to target the respective UTR region to excise the intervening DNA sequence and produce an inversion by excision and reinsertion of the excised DNA fragment into the same UTR in the reverse, antisense or opposite orientation. In each case, the guide RNAs were selected to be unique to the target sites in the respective UTR region without any known off- target sequence present in the maize genome (off-target sequences being defined in this case as not having three or fewer mismatches relative to the 23-mer sequence of the gRNAs). By making a double-strand break (DSB) at the each of the two target sites in the UTR region, the intervening sequence can be cut out and excised and then reinserted in a reverse, antisense or opposite orientation by rejoining with the two excised ends of the flanking genomic DNA. Although the excised sequence could potentially be perfectly rejoined with the flanking genomic DNA in the opposite orientation without any sequence changes (apart from the inversion), one or more nucleotide insertions or deletions (Indels) may also occur at or near one or both of the two junctions where the excised fragment and flanking genomic DNA are rejoined relative to the sequence(s) at or near the two target sites prior to excision.

[0568] In this experiment, a pair of two gRNA spacers (SP1 and SP2; see Table 3) were identified and selected to target excision and inversion of an approximately 450 bp long fragment of the 3’ UTR of the Zm.GA3 oxidase_1 gene, and a pair of two gRNA spacers (SP3 and SP4; see Table 3) were identified and selected to target excision and inversion of an approximately 280 bp long fragment of the 5’ UTR of the GA3 oxidase_1 gene. Four plant transformation constructs were made, two of which were designed to create the 5’ UTR inversion edits, and two were designed to create the 3’ UTR inversion edits through genome editing using a CRISPR / Cas12a (Cpf1) system with two guide RNAs targeting sites bordering the intended UTR sequence to be excised and inverted between the two target sites. In this example, the vector constructs generally contain two functional cassettes encoding gene editing machinery for creation of targeted mutations in the GA3 oxidase_1 gene: (i) a first cassette for expression of a Cpf1 (or Cas12a) variant protein, and (ii) a second cassette for expression of the two relevant guide RNAs targeting the 5’ UTR or 3’ UTR according to the editing scheme. Each guide RNA contains a common scaffold sequence compatible with the Cpf1 variant and a unique spacer / targeting sequence complementary to its intended target site in the 5’ UTR or 3’ UTR of the GA3 oxidase_1 gene.

[0569] For construct pM471, the Cpf1 expression cassette comprises a maize reproductive tissue preferred promoter (SEQ ID NO: 193) operably linked to a maize codon-optimized sequence encoding a Lachnospiraceae bacterium Cpf1 RNA-guided endonuclease enzyme (CR-LACba.Cpf1zmCG2:3, SEQ ID NO: 194) fused to a nuclear localization signal at both N- and C-terminal ends of the Cpf1 enzyme. The gRNA expression cassette comprises a synthetic promoter (P-Syn.GSP2262_Pol3:1, SEQ ID No: 196) operably linked to a transcribable sequence encoding (in order) a Cpf1-compatible common scaffold SC1 (GR- LACba.Cpf1:2, SEQ ID NO.197), spacer SP1, scaffold SC1, spacer SP2 and scaffold SC1 in the gRNA. The SC1-SP1-SC1-SP2-SC1 portion of the transcript is a pre-crRNA precursor RNA that can become processed into the two mature SP1 and SP2 guide RNAs.

[0570] Construct pM313 is similar to the pM471 construct described above, except that the Cpf1 expression cassette comprises a constitutive maize ubiquitin promoter (SEQ ID NO: 198), instead of the reproductive tissue preferred promoter.

[0571] For construct pM323, the Cpf1 expression cassette comprises a maize reproductive tissue preferred promoter (SEQ ID NO: 193) operably linked to a soybean codon- optimized sequence encoding Francisella tularensis subsp. novicida Cpf1 RNA-guided endonuclease enzyme (CR-Fn.Cpf1-Gm:5, SEQ ID NO: 199) fused to a nuclear localization signal (SEQ ID NO: 200) at the N-terminal end and another nuclear localization signal (SEQ ID NO: 201) at the C-terminal end of the Cpf1 enzyme. The gRNA expression cassette comprises a synthetic promoter (P-Syn.GSP2262_Pol3:1 SEQ ID No: 196) operably linked to a Cpf1-compatible common scaffold SC2 (GR-Fn.Cpf1repeat:1, SEQ ID NO: 202), spacer SP3, scaffold SC2, spacer SP4 and scaffold SC2. The SC2-SP3-SC2-SP4-SC2 portion of the transcript is a pre-crRNA precursor RNA that can become processed into the two mature SP3 and SP4 guide RNAs.

[0572] Construct pM314 is similar to the pM323 construct described above, except that the Cpf1 expression cassette comprises a constitutive maize ubiquitin promoter (SEQ ID No. 198), instead of the reproductive tissue preferred promoter. Note that the constitutive maize ubiquitin promoter in different constructs may be combined with different leader and intron sequences.Table 3. Constructs and gRNA spacer sequences for generating targeted inversions in 5’ and 3’ UTR regions of the Zm.GA3ox_1 gene.Example 3: Creation of 5’ UTR inversion edits of Arabidopsis AG gene

[0573] The design of this gene-editing approach is based on excision and reverse- insertion of a fragment of the 5’ UTR region from the Arabidopsis thaliana Agamous (AG) gene (AT4G18960).

[0574] The Arabidopsis thaliana floral homeotic gene AGAMOUS (AG) encodes a MADS box transcription factor and plays a central role in reproductive organ (stamen and carpel) development. AG is thought to control developmental pathways by regulating downstream target genes responsible for stamen and carpel identity. As a result, ag mutants produce distinct and scorable floral phenotypes. Specifically, downregulation of the AG gene results in plants with multiple petal layers (see Yanofsky et al., Nature 346, 35–39 (1990)). AG gene knock-out or coding region point mutations result in plants with a double flower phenotype wherein the anthers and stigma fail to develop due to the uncontrolled development of petals in the inner whorls of the flower. As a result, strong ag mutant alleles have flowers in which the third-whorl stamens are converted to petals, while another flower, that will reiterate the same organ pattern, develops in place of the fourth-whorl carpels.

[0575] The genomic sequence for At.AG (AT4G18960, Chr4:10382855..10388539) gene is provided as SEQ ID NOs: 204. The elements and the coordinates within the genomic sequence are described in Table 4.Table 4. Elements and coordinates within the genomic sequence of Arabidopsis Agamous gene (At. AG)

[0576] The 5’UTR region was scanned and five FnCas12a targets sites were identified. Each comprised the TTN PAM site adjacent to a unique spacer sequence without any known off-target sequence present in the Arabidopsis genome (off-target sequences being defined in this case as not having three or fewer mismatches relative to the 23-mer sequence of the gRNAs). The spacer sequences are listed in Table 5. Table 5. gRNA spacer sequences within the 5’ UTR regions of the At.AG gene.

[0577] In this experiment, a pair of gRNA spacers (SP18 and SP21, see Table 6) were identified and selected to target excision and inversion of an approximately 180bp longfragment of the 5’UTR of the At.AG gene. A second pair of gRNA spacers (SP18 and SP20; see Table 6) were identified and selected to target excision and inversion of an approximately 780 bp long fragment of the 5’ UTR of the At.AG gene. Table 6. Constructs and gRNA spacer sequences for generating targeted inversions in 5’ UTR regions of the At.AG gene.

[0578] Two plant transformation gene editing test constructs were made, each designed to create the 5’ UTR inversion edits through genome editing using a CRISPR / Cas12a (Cpf1) system with two guide RNAs targeting sites bordering the intended UTR sequence to be excised and inverted between the two target sites. In this example, the vector constructs generally contain two functional cassettes encoding gene editing machinery for creation of targeted mutations in the At.AG gene: (i) a first cassette for expression of a Cpf1 or Cas12a variant protein, and (ii) a second cassette for expression of the two relevant guide RNAs targeting the 5’ UTR according to each editing scheme. Each guide RNA contains a common scaffold compatible with the Cpf1 enzyme, and a unique spacer / targeting sequence complementary to its intended target site. See Figure 2.

[0579] For the gene-editing Construct pM-01, the Cpf1 expression cassette comprised a Medicago truncatula Ubiquitin promoter (SEQ ID NO: 220) operably linked to a dicot codon- optimized sequence encoding a Francisella tularensis subsp. novicida Cpf1 RNA-guided endonuclease enzyme (SEQ ID NO: 199) fused to a sequence encoding a nuclear localization signal (SEQ ID NO: 200) at the N-terminal end and another nuclear localization signal (SEQ ID NO: 201) at the C-terminal end of the Cpf1 enzyme followed by a Medicago trunctula terminator sequence (SEQ ID NO: 221). The gRNA expression cassette comprised an Arabidopsis plant Pol III promoter (SEQ ID NO: 222) operably linked to Fn.Cpf1-compatible common scaffold SC2 (GR-FnCba.Cpf1:2, SEQ ID No.202), spacer SP18, scaffold SC2, spacer SP21 and scaffold SC2. See Panel A of Figure 2. The gene editing Construct pM-02 is similar to the pM-01 construct described above, except that the guide RNA comprised SP18 and SP20spacers. See Panel B of Figure 2. In addition to the test constructs described above, two additional control vectors are generated. An miRNA vector control construct pM-03 comprises a functional cassette comprising a 35S promoter (SEQ ID NO: 210) operably linked to a synthetic micro RNA (miRNA) targeting the 5’UTR region within the At.AG gene for suppression. This constructed is expected to serve as a positive control for gene silencing. See Figure 2, panel C.

[0580] 5’UTR transgenic test control construct pM-04 comprises a functional cassette comprising a 35S promoter (SEQ ID NO: 210) operably linked to a modified At.AG transcribable sequence comprising an inverted fragment of the 5’UTR of At.AG followed by the AG coding sequence and the 3’UTR in the native orientation. See Figure 2, panel D.

[0581] Arabidopsis plants are transformed with the constructs described above via the floral dip method of transformation (see Clough SJ, Bent AF. Plant J.1998 Dec;16(6):735-43). Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants.

[0582] In plants expressing pMON-01 and pMON-02, upon expression of the gRNAs and the Cpf1 nuclease, the gRNAs guide the nuclease to each of the two target sites with the respective UTRs of the AG gene, where the nuclease creates a double-stranded break at each target site. In the majority of events, the region between the target sites is deleted and non- homologous end-joining repair mechanisms joins the flanking regions. Less frequently, the released UTR DNA fragment can be re-oriented and reintegrate into the cleavage site via NHEJ (Non Homologous End Joining) and native DNA repair mechanisms resulting in an inversion of the DNA sequence between the two guide RNA target sites. Suitable methods known in the art (e.g., PCR, DNA hybridization, sequencing) are used to identify transformation events comprising a complete inversion of the UTRs.

[0583] The expected phenotypes for the plants that are homozygous for the edited UTR inversion are expected to be equivalent to the wild-type Arabidopsis in that they should exhibit normal flowers with four petals and normal anther and stigma development. The expected phenotype for the heterozygous plants with edited UTR (5’ UTR edit allele paired with the wild type allele) is that they would exhibit an intermediate mutant phenotype - more than 4 petals, reduced or eliminated anthers or stigma- when compared to the homozygous state of the AG gene knock-out or coding region point mutation which has a complete double flower wherein the anthers and stigma fail to develop due to the uncontrolled development of petals in the inner whorls of the flower.

[0584] Following phenotypic characterization, small RNA sequencing is performed on several individuals with wildtype and altered phenotypes to detect presence of AG1 specific small RNAs. Without being bound by any theory, it is predicted that plants showing altered phenotypes will have detectable levels of AG1 specific small RNAs. Example 4: Creation and characterization of Arabidopsis AG1 Transgenic UTR inversion plants

[0585] Three AG1 UTR transgenic control constructs were generated. The 5’iUTR(inverted UTR) transgenic test control construct pM-04 comprised a modified At.AG transcribable sequence. The functional cassette comprised a 35S promoter (SEQ ID NO: 210) operably linked to an inverted fragment of the 5’UTR of At.AG (SEQ ID NO: 223) followed by the AG coding sequence (SEQ ID NO: 224) operably linked to a 3’ transcription terminator sequence from cotton (SEQ ID NO: 225). See Figure 3, panel A.

[0586] The 3’iUTR transgenic test control construct pM-05 comprised a functional cassette comprising a 35S promoter (SEQ ID NO: 210) operably linked to the AG coding sequence (SEQ ID NO: 224) followed by an inverted fragment of the 3’UTR of At.AG (SEQ ID NO: 226) and a 3’ transcription terminator sequence from cotton (SEQ ID NO: 225). See Figure 3, panel B.

[0587] The 5’and 3’ iUTR transgenic test control construct pM-06 comprised a functional cassette comprised a 35S promoter (SEQ ID NO: 210) operably linked to an inverted fragment of the 5’UTR of At.AG (SEQ ID NO: 223) followed by the AG coding sequence (SEQ ID NO: 224) operably linked an inverted fragment of the 3’UTR of At.AG (SEQ ID NO: 226) and a 3’ transcription terminator sequence from cotton (SEQ ID NO: 225). See Figure 3, panel C. All constructs also comprised a cassette with the adenylyltransferase (AAD) marker gene that confers resistance to spectinomycin.

[0588] Arabidopsis plants were transformed with the constructs described above via the floral dip method of transformation (see Clough SJ, Bent AF. Plant J.1998 Dec;16(6):735-43). Treated plants were allowed to set seed which were then plated on spectinomycin selective medium to screen for transformants. Surviving R0 seedlings were advanced and Taqman assays were performed to determine the presence and copy number of the LbCas12a expression cassette which is also indicative of the number of copies of the iUTR cassette.

[0589] The iUTR plants comprise a native wild-type copy of the AG1 gene therefore the expected phenotype for these plants (iUTR transcripts paired with the wild type allele transcripts) is that they would exhibit altered or mutant phenotypes with severity influenced bythe copy number of the transgene. Some of the expected altered phenotypes are: more than 4 petals, reduced or eliminated anthers or stigma. The homozygous state of the AG gene knock- out or coding region point mutation has been reported to lead to a complete double flower wherein the anthers and the stigma fail to develop due to the uncontrolled development of petals in the inner whorls of the flower.

[0590] R0 transformants displaying abnormal phenotypes were characterized and summarized as shown in Table 7. The severity of the altered floral phenotypes correlated with copy number wherein multicopy plants tended to display stronger alterations. Table 7. AG iUTR transgenic plants with altered floral phenotypes. WT indicates WT floral phenotype. ALT indicates Altered floral phenotype.

[0591] The data from transgenes shows that inversions in either the 3’UTR and / or the 5’UTR without alterations to the coding sequence is sufficient to produce altered phenotypes.

[0592] Following phenotypic characterization small RNA sequencing is performed on several individuals to detect presence of AG1 specific small RNAs. Without being bound by any theory, it is predicted that plants showing altered phenotypes will have detectable levels of AG1 specific small RNAs. Example 5: Creation of 5’ UTR inversion edits of Arabidopsis BRI1 gene

[0593] The design of this gene-editing approach is based on excision and reverse- insertion of a fragment of the 3’UTR region from the Arabidopsis thaliana Brassinosteroid insensitive 1(BRI1) gene (AT4G39400).

[0594] The Arabidopsis thaliana BRI1 gene encodes a leucine-rich repeat receptor kinase involved in plant hormone brassinosteroid signal transduction. BRI1 is the major receptor ofbrassinosteroid (see Wang ZY et al., Nature., 410 , 6826 , March 2001). It plays very important roles in plant development, especially in the control of cell elongation and for the tolerance of environmental stresses. BRI1 enhances cell elongation, promotes pollen development, controls vasculature development and promotes chilling and freezing tolerance. As a result, bri1 null mutants exhibit extreme dwarfism, dark green downward curling leaves, male sterility, delayed flowering, altered vascular morphology and reduced apical dominance, particularly in older plants (see review in Clouse, The Arabidopsis Book , 2011 https: / / doi.org / 10.1199 / tab.0151)

[0595] The genomic sequence for At.BRI1 (AT4G39400, Chr4 : 18324660 -18328826 ) gene is provided as SEQ ID NO: 211. The elements and the coordinates within the genomic sequence are described in Table 7. Table 8. Elements and coordinates within the genomic sequence of Arabidopsis Brassinosteroid insensitive 1 gene (At. BRI1)

[0596] The 3’UTR region was scanned and two FnCas12a targets sites were identified. Each comprised the TTN PAM site adjacent to a unique spacer sequence without any known off-target sequence present in the Arabidopsis genome (off-target sequences being defined in this case as not having three or fewer mismatches relative to the 23-mer sequence of the gRNAs). The spacer sequences are listed in Table 8. Table 9. gRNA spacer sequences within the 5’ UTR regions of the At.AG gene.

[0597] In this experiment, a pair of gRNA spacers (SP22 and SP23 see Table 8) were identified and selected to target excision and inversion of an approximately 360 bp long fragment of the 3’UTR of the At.BRI1 gene.Table 10. Constructs and gRNA spacer sequences for generating targeted inversions in 3’ UTR region of the At.BRI1 gene.

[0598] A plant transformation gene editing test construct was designed to create the 3’ UTR inversion edit through genome editing using a CRISPR / Cas12a (Cpf1) system with two guide RNAs targeting sites bordering the intended UTR sequence to be excised and inverted between the two target sites. In this example, the vector construct comprised two functional cassettes encoding gene editing machinery for creation of targeted mutations in the At.BRI1 gene: (i) a first cassette for expression of a Cpf1 or Cas12a variant protein, and (ii) a second cassette for expression of the two relevant guide RNAs targeting the 3’ UTR according to the editing scheme. Each guide RNA contained a common scaffold compatible with the Cpf1 enzyme, and a unique spacer / targeting sequence complementary to its intended target site.

[0599] For the gene-editing Construct pM-07, the Cpf1 expression cassette comprised a Medicago truncatula Ubiquitin promoter (SEQ ID NO: 220) operably linked to a dicot codon- optimized sequence encoding a Francisella tularensis subsp. novicida Cpf1 RNA-guided endonuclease enzyme (SEQ ID NO: 199) fused to a sequence encoding a nuclear localization signal (SEQ ID NO: 200) at the N-terminal end and another nuclear localization signal (SEQ ID NO: 201) at the C-terminal end of the Cpf1 enzyme followed by a Medicago trunctula terminator sequence (SEQ ID NO: 221). The gRNA expression cassette comprised an Arabidopsis plant Pol III promoter (SEQ ID NO: 222) operably linked to Fn.Cpf1-compatible common scaffold SC2 (GR-FnCba.Cpf1:2, SEQ ID No.202), spacer SP22, scaffold SC2, spacer SP23 and scaffold SC2.

[0600] In addition to the test construct described above, two additional control vectors were generated and described in Example 6. Arabidopsis plants are transformed with the constructs described above via the floral dip method of transformation (see Clough SJ, Bent AF. Plant J. 1998 Dec;16(6):735-43). Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants.

[0601] In plants expressing pMON-07, upon expression of the gRNAs and the Cpf1 nuclease, the gRNAs guide the nuclease to each of the two target sites within the respective UTR of the At. BRI1 gene, where the nuclease creates a double-stranded break at each targetsite. In the majority of events, the region between the target sites is deleted and non-homologous end-joining repair mechanisms joins the flanking regions. Less frequently, the released UTR DNA fragment can be re-oriented and reintegrate into the cleavage site via NHEJ (Non Homologous End Joining) and native DNA repair mechanisms resulting in an inversion of the DNA sequence between the two guide RNA target sites. Suitable methods known in the art (e.g., PCR, DNA hybridization, sequencing) are used to identify transformation events comprising a complete inversion of the UTRs.

[0602] The expected phenotypes for the plants that are homozygous for the edited UTR inversion are expected to be equivalent to the wild-type Arabidopsis. The expected phenotype for the heterozygous edited UTR plants (3’ UTR edit allele paired with the wild type allele) is that they would exhibit an intermediate mutant phenotype of leaves with curling and crinkles, shorter stature, decreased apical dominance (i.e. increased number of stems / bolts), delayed flowering, and reduced fertility when compared to the homozygous state of the BRI1 gene knock-out or coding region point mutation which has a severe stunted phenotype.

[0603] Following phenotypic characterization small RNA sequencing is performed on several individuals to detect presence of BRI1 specific small RNAs. Without being bound by any theory, it is predicted that plants showing altered phenotypes will have detectable levels of BRI1 specific small RNAs. Example 6: Creation and characterization of Arabidopsis BRI1 Transgenic UTR inversion plants

[0604] Two BRI1 UTR transgenic control constructs were generated. The 5’iUTR(inverted UTR) transgenic test control construct pM-08 comprised a modified At.BRI1 transcribable sequence. The functional cassette comprised a 35S promoter (SEQ ID NO: 210) operably linked to an inverted fragment of the 5’UTR of At.BRI1 (SEQ ID NO: 229) followed by the BRI1coding sequence (SEQ ID NO: 230) operably linked to a 3’ transcription terminator sequence from cotton (SEQ ID NO: 225).

[0605] The 3’iUTR transgenic test control construct pM-09 comprised a functional cassette comprising a 35S promoter (SEQ ID NO: 210) operably linked to the BRI1coding sequence (SEQ ID NO: 230) followed by an inverted fragment of the 3’UTR of At.BRI1 (SEQ ID NO: 231). Both constructs also comprised a cassette with the adenylyltransferase (AAD) marker gene that confers resistance to spectinomycin.

[0606] Arabidopsis plants were transformed with the constructs described above via the floral dip method of transformation (see Clough SJ, Bent AF. Plant J.1998 Dec;16(6):735-43). Treated plants were allowed to set seed which were then plated on spectinomycin selective medium to screen for transformants. Surviving R0 seedlings were advanced and Taqman assays were performed to determine the presence and copy number of the LbCas12a expression cassette.

[0607] The iUTR plants comprise a native wt copy of the BRI1 gene therefore the expected phenotype for these transgene containing plants ( iUTR transcripts paired with the wild type allele transcripts) is that they would exhibit altered or mutant phenotypes with severity influenced by the copy number of the transgenes . Some of the expected altered phenotypes are : altered phenotype of leaves with curling and crinkles, shorter stature, decreased apical dominance (i.e. increased number of stems / bolts), delayed flowering, and reduced fertility. It is likely that stronger phenotypic alterations could be observed with the trangenic plants as compared to the gene edited UTR inversions due to higher expression from CaMV 35S promoter than native BRI1 promoter expression.

[0608] R0 transformants displaying abnormal phenotypes were characterized and summarized as shown in Table 8. The severity of the altered phenotypes correlated with copy number wherein multicopy plants tended to display stronger alterations. Table 11. BRI1 iUTR transgenic plants with altered phenotypes. WT indicates WT phenotype. ALT indicates Altered phenotype.

[0609] The data from transgenes shows that inversions in either the 3’UTR or the 5’UTR of BRI1 without alterations to the coding sequence is sufficient to produce altered phenotypes.

[0610] Following phenotypic characterization small RNA sequencing is performed on several individuals to detect presence of BRI1 specific small RNAs. Without being bound byany theory, it is predicted that plants showing altered phenotypes will have detectable levels of BRI1 specific small RNAs. Example 7: Creation of UTR inversion edits of Arabidopsis PIN1 and D gene

[0611] Similar methods and processes described in Examples 2, 3 and 5 are used to generate and characterize edited Arabidopsis plants comprising UTR inversions in the Dwarf1 gene (At.Dwf1, At3g19820) (SEQ ID NO: 212) and Pin-formed 1 gene ( At.PIN1, At1g73590) (SEQ ID NO: 213). At.DWF1 protein is involved in brassinosteroid hormone synthesis which in turn regulates cell elongation. dwf1 mutants as the name indicates have a dwarf phenotype (see Clouse (2011) The Arabidopsis Book https: / / doi.org / 10.1199 / tab.0151). Pin formed 1 encodes an auxin efflux carrier involved in shoot and root development. Loss of function severely affects organ initiation and pin1 mutants are characterized by an inflorescence meristem that does not initiate any flowers, resulting in the formation of a naked inflorescence stem (see Okada, et.al. The Plant Cell, Volume 3, Issue 7, July 1991, pg 677–684.)

[0612] Briefly, the 5’ and 3’ UTR regions region of At.Dwf1 and At.PIN1 genes are scanned and FnCas12a target sites are identified. Pairs of gRNAs spacers are identified and selected to target excision and inversion of an approximately 150bp long fragment of the 5’UTR of the At.Dwf1 gene, an approximately 65 bp long fragment of the 5’UTR of At.PIN1 or approximately 150bp long fragment of the 3’UTR of At.PIN1. Plant transformation gene editing plant transformation gene editing test constructs are made, each designed to create the 5’ UTR inversion edits through genome editing using a CRISPR / Cas12a (Cpf1) system with two guide RNAs targeting sites bordering the intended UTR sequence to be excised and inverted between each of the two target sites.

[0613] Arabidopsis plants are transformed with the constructs described above via the floral dip method of transformation (see Clough SJ, Bent AF. Plant J.1998 Dec;16(6):735-43). Treated plants are allowed to set seed which are then plated on a selective medium to screen for transformants. Suitable methods known in the art (e.g., PCR, DNA hybridization, sequencing) are used to identify transformation events comprising a complete inversion of the targeted UTRs. Phenotypes of plants homozygous and heterozygous for the inversions are characterized and small RNA sequencing is performed on plants with wild-type phenotype and altered phenotypes to detect the presence of specific small RNAs that target the gene of interest. Example 8: Generation and Confirmation of Edits in 3’ UTR Region of Zm.GA3ox_1 gene and Zygosity of Genome-Edited Plants.

[0614] An inbred wild-type corn line was transformed via Agrobacterium-mediated transformation with the pM313 vector described in Example 2 above. The transformed plant tissue was grown to produce mature R0 plants. R0 plants having one or more unique genome edits were self-crossed to produce R1 plants. R1 plants that were homozygous for alleles comprising edited Zm.GA3ox_1 gene with a 3’ UTR inversion and lacking the T-DNA sequences were self-crossed to produce R2 plants.

[0615] To determine whether the edits were made in the Zm.GA3ox_13’ UTR region, an amplicon sequencing technique was used to produce mutant sequences for the 750 kb 3’ UTR region for comparison with the wild-type sequence. Amplicon sequencing involves the generation of one or more unique PCR products across the genomic region of interest for next- generation sequencing. Sequence data from each sample is then mapped to a reference sequence to identify differences in the consensus sequences. Plants with unique inversions were selected. Individual R1 plants produced by selfing R0 plants having the edits were assayed to confirm the edits. For illustration, one edited plant with allele S049 of the GA3ox_1 gene that was produced using the pM313 transformation vector was selected and characterized as described in Table 12. In Table 12, “Allele Name” is the identifier for a unique allele, and wild-type (WT) refers to the unedited plant with a WT GA3ox_1 gene. The start and end nucleotide positions of the edit inversion and deletion are in reference to the 4800bp GA3ox_1 wild type genomic sequence (SEQ ID NO: 232) which begins with 2000bp of promoter sequence upstream of transcription start site and ends with the 3’ UTR sequence at nucleotide positions 4052 to 4800 (SEQ ID NO: 233). The WT and edited allele sequences and their relationship is illustrated in FIG.4. The editing process converted the 4800bp WT sequence of SEQ ID NO: 232 into a 4642 bp edited allele genomic sequence of SEQ ID NO: 234, as WT 3’ UTR sequence of SEQ ID NO: 233 was converted into a shorter UTR sequence of SEQ ID NO: 235.

[0616] The edited allele zygosity of each of the R1 plants was further tested and determined to be homozygous or heterozygous. Plants of homozygous edit were selected for further selfing to produce inbred plants or crossed with another corn line to produce hybrid plants. Table 12. Description of the edited allele within 3’ UTR region of Zm.GA3ox_1

Claims

CLAIMS 1. A method for editing the genome of a plant cell to modify an endogenous gene, comprising the steps of a) generating a first double-stranded break and a second double stranded break using a targeted editing technique targeting at least one untranslated region of said endogenous gene, in said plant cell without perturbance of the coding region; b) isolating a modified plant cell comprising a modified allele of said endogenous gene wherein the modified allele comprises an inverted DNA sequence of at least part of said untranslated region of said endogenous gene and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said untranslated region, wherein the modified allele does not comprise a sense sequence complementary to the antisense sequence of part of said untranslated region. 2) The method of claim 1, wherein said inverted DNA sequence of at least part of said untranslated region of said endogenous gene encodes an antisense RNA sequence that is at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of said untranslated region of said gene. 3) The method of claim 1, wherein said untranslated region of said endogenous gene is a 5’ untranslated region or a 3’ untranslated region or both. 4) The method of claim 1, wherein said plant cell comprises a modified allele and an unmodified allele of said endogenous gene. 5) The method of claim 4, wherein said modified allele and said unmodified allele are transcribed to a mRNA and wherein said RNA from said modified allele and said RNA from said unmodified allele are capable of producing a double stranded RNA region of at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides. 6) The method of claim 5, wherein expression of said modified and said unmodified allele is reduced.7) The method of claim 1, comprising a further step of regenerating a plant from said modified plant cell. 8) The method of claim 7 comprising a further step of crossing said plant comprising said modified allele of said endogenous gene in homozygous state with another plant comprising an unmodified allele of said endogenous gene in homozygous state and harvesting hybrid seeds. 9) The method of claim 1, wherein said plant is corn and said endogenous gene is selected from GA20 oxidase or GA3 oxidase. 10) The method of claim 1, wherein said inverted DNA sequence comprises a nucleotide sequence having at least 90% sequence identity or complementarity to a nucleotide sequence of at least 18 nucleotides, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of: nucleotides 1-29 of SEQ ID NO: 36, nucleotides 1664-1788 of SEQ ID NO: 36, nucleotides 1-38 of SEQ ID NO: 37, nucleotides 1446-1698 of SEQ ID NO: 37, nucleotides 3001-3161 of SEQ ID NO: 168, nucleotides 4796- 5406 of SEQ ID NO: 168, nucleotides 3001-3056 of SEQ ID NO: 169, nucleotides 4464-4581 of SEQ ID NO: 169, nucleotides 3001-3130 of SEQ ID NO: 170, nucleotides 4275-4332 of SEQ ID NO: 170, nucleotides 7621-8029 of SEQ ID NO: 174, nucleotides 9672-10276 of SEQ ID NO: 174, nucleotides 7386-7831 of SEQ ID NO: 175, nucleotides 8862-8967 of SEQ ID NO: 175, nucleotides 7547-7751 of SEQ ID NO: 176, nucleotides 8904-9178 of SEQ ID NO: 176, nucleotides 1-1060 of SEQ ID NO : 204, nucleotides 5418-5648 of SEQ ID NO: 204, nucleotides 1-165 of SEQ ID NO: 211, nucleotides 3757-4167 of SEQ ID NO: 211, nucleotides 664-699 of SEQ ID NO: 212, nucleotides 2482-2700 of SEQ ID NO: 212, nucleotides 1-99 of SEQ ID NO: 213, or nucleotides 3205-3506 of SEQ ID NO:

213. 11) The method of claim 1, wherein said targeted editing technique involves use of a RNA guided effector protein wherein said RNA guided endonuclease is a CRISPR-Cas effector protein selected from a Cas9, C2c1, C2c3, Cas12a (also referred to as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Csnl, Csx12, Cas10, Csyl, Csy2, Csy3, Csel, Cse2, 30 Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), Csf5 nuclease, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, Cas14c effector protein.12) The method of claim 11, wherein said RNA guided effector protein is a Cas12a effector protein or a Cas12a derived effector protein. 13) The method according to claim 12, wherein said targeted gene editing technique involves use of one or more guide RNAs comprising a nucleotide sequence selected from the group of SEQ ID Nos: 177, 178, 179, 180, 205, 206, 207, 208 and 209. 14) A method for modifying expression of an endogenous gene in a hybrid plant while leaving the expression of said gene unaffected in a parent plant or plants, comprising the steps of a) identifying an endogenous gene in a plant wherein expression of a variant allele of said gene results in unwanted phenotypes when present in homozygous state; b) providing a first plant comprising a modified allele of said gene comprising at least one nucleic acid region which is an inversion of a part of said gene whereby the inversion does not affect translation of said modified allele, and wherein said first plant comprises said modified allele of said gene homozygously; c) crossing said first plant with a second plant comprising an unmodified allele of said gene not comprising said inversion not affecting translation of said gene wherein the unmodified gene is in homozygous state; d) obtaining a hybrid seed comprising the modified and unmodified allele of said gene in heterozygous or heteroallelic form. 15) The method of claim 14, wherein upon transcription of said modified allele and said unmodified allele into RNA molecule, a double stranded RNA region can be formed by base- pairing between the nucleic acid region which is an inversion of part of an untranslated region of gene in the RNA transcript of said modified allele and the nucleic acid region and the RNA transcript of said unmodified allele, and wherein the double stranded RNA regions is capable of inhibiting expression of said modified allele and said unmodified allele by RNA silencing mechanisms, such as stalling of RNA translation, stalling of RNA transcription, destabilization of the resulting RNA molecules or post-transcriptional degradation of the transcribed RNA molecules. 16) The method of claim 14, wherein said nucleic acid region which is an inversion of a part of said gene results upon transcription in an antisense RNA sequence that is at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of said untranslated region of said gene. 17) The method of claim 14, wherein said GA20 oxidase is selected from GA20 oxidase_5 or GA20 oxidase_3. 18) A plant cell, plant or part or seed thereof comprising a modified allele of an endogenous gene wherein the modified allele comprises an inverted DNA sequence of at least part of an untranslated region of said endogenous gene, and wherein the modified allele produces an RNA transcript comprising an antisense sequence of part of said untranslated region, wherein the modified allele does not comprise a sense nucleotide sequence of more than 17 nucleotides complementary to the antisense sequence of part of said untranslated region. 19) The plant cell, plant or part or seed thereof of claim 18, wherein said inverted DNA sequence of at least part of an untranslated region of said endogenous gene results upon transcription in an antisense RNA sequence that is at least 70%, at least 75%, at least 80%, at least 85% at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% complementary to at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of said untranslated region of said gene. 20) The plant cell, plant or part or seed thereof of claim 18, wherein said plant is corn and said endogenous gene is selected from GA20 oxidase or GA3 oxidase. 21) The plant cell, plant or part or seed thereof of claim 18, wherein said inverted DNA sequence comprises a nucleotide sequence having at least 90% sequence identity or complementarity to a nucleotide sequence of at least 18 nucleotides, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides of: nucleotides 1-29 of SEQ ID NO: 36, nucleotides 1664-1788 of SEQ ID NO: 36, nucleotides 1- 38 of SEQ ID NO: 37, nucleotides 1446-1698 of SEQ ID NO: 37, nucleotides 3001-3161 of SEQ ID NO: 168, nucleotides 4796-5406 of SEQ ID NO: 168, nucleotides 3001-3056 of SEQ ID NO: 169, nucleotides 4464-4581 of SEQ ID NO: 169, nucleotides 3001-3130 of SEQ ID NO: 170, nucleotides 4275-4332 of SEQ ID NO: 170, nucleotides 7621-8029 of SEQ ID NO: 174,nucleotides 9672-10276 of SEQ ID NO: 174, nucleotides 7386-7831 of SEQ ID NO: 175, nucleotides 8862-8967 of SEQ ID NO: 175, nucleotides 7547-7751 of SEQ ID NO: 176, nucleotides 8904-9178 of SEQ ID NO: 176, nucleotides 1-1060 of SEQ ID 204, nucleotides5418-5648 of SEQ ID 204, nucleotides 1-165 of SEQ ID 211, nucleotides 3757-4167 of SEQ ID 211, nucleotides 664-699 of SEQ ID 212, nucleotides 2482-2700 of SEQ ID 212, nucleotides 1-99 of SEQ ID 213, or nucleotides 3205-3506 of SEQ ID 213. 22) The plant cell, plant or part or seed thereof according to claim 18, wherein said plant cell, plant or part or seed thereof further comprises an unmodified allele of said endogenous gene. 23) The plant cell, plant or part or seed thereof according to claim 22, wherein said modified allele and said unmodified allele are transcribed into an RNA and wherein said RNA from said modified allele and said RNA from said unmodified allele are capable of producing a double stranded RNA region of at least 18, at least 19, at least 20, at least 25, at least 30, at least 40, at least 50, at least 75, at least 100, at least 150, at least 200, at least 300, at least 400, at least 500, at least 750, or at least 1000 consecutive nucleotides and wherein the double stranded RNA regions is capable of inhibiting expression of said modified allele and said unmodified allele by RNA silencing mechanisms, such as stalling of RNA translation, stalling of RNA transcription, destabilization of the resulting RNA molecules or post-transcriptional degradation of the transcribed RNA molecules. 24) A plant according to claim 118, wherein said plant is a plant selected from a monocotyledonous species, a dicotyledonous species, an angiosperm species or a gymnosperm species. 25) A plant according to claim 18, wherein said plant is selected from a corn plant, a rice plant, a sorghum plant, a wheat plant, an alfalfa plant, a barley plant, a millet plant, a rye plant, a sugarcane plant, a cotton plant, a soybean plant, a canola plant, a tomato plant, an onion plant, a cucumber plant, an Arabidopsis plant, or a potato plant.

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