Gene editing using developmental promoters and increasing site-specific integration events

Abstract

The present disclosure provides methods and compositions for increasing genome editing and site-specific integration events using inducible endonucleases and floral cell-preferred or floral tissue-preferred promoters.

Description

[Technical field]

[0001] The present disclosure relates to compositions and methods relating to the expression of inducible nucleases and guide nucleic acids in floral cells and tissues in plants.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 63 / 324,880, filed March 29, 2022, the entire contents of which are incorporated herein by reference.

[0003] Inclusion of sequence listing The Sequence Listing, contained under file name "P34740WO00_SL.XML", which is 121 kilobytes (measured in MS-Windows®) and was created on March 27, 2023, has been submitted electronically herewith and is incorporated by reference in its entirety. [Background technology]

[0004] CRISPR (clustered regularly interspaced short palindromic repeats) nucleases (e.g., Cas12a, CasX, Cas9) are proteins that are guided by a guide RNA to a target nucleic acid molecule, and the nuclease can cleave one or both strands of the target nucleic acid molecule. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 6,437,217 [Patent Document 2] U.S. Patent No. 5,641,876 [Patent Document 3] U.S. Patent No. 6,426,446 [Patent Document 4] U.S. Patent No. 6,429,362 [Patent Document 5] U.S. Patent No. 6,232,526 [Patent Document 6] U.S. Patent No. 6,177,611 [Patent Document 7] U.S. Patent No. 5,322,938 [Patent Document 8] U.S. Patent No. 5,352,605 [Patent Document 9] U.S. Patent No. 5,359,142 [Patent Document 10] U.S. Patent No. 5,530,196 [Patent Document 11] U.S. Patent No. 6,433,252 [Patent Document 12] U.S. Patent No. 6,429,357 [Patent Document 13] U.S. Patent No. 5,837,848 [Patent Document 14] U.S. Patent No. 6,294,714 [Patent Document 15] U.S. Patent No. 6,140,078 [Patent Document 16] U.S. Patent No. 6,252,138 [Patent Document 17] U.S. Patent No. 6,175,060 [Patent Document 18] U.S. Patent No. 6,635,806 [Patent Document 19] U.S. Patent Application No. 09 / 757,089 [Patent Document 20] U.S. Patent No. 6,051,753 [Patent Document 21] U.S. Patent No. 5,378,619 [Patent Document 22] U.S. Patent No. 5,850,019 [Patent Document 23] U.S. Patent No. 5,106,739 [Patent Document 24] WO 2019 / 084148 [Patent Document 25] U.S. Patent No. 5,550,318 [Patent Document 26] U.S. Patent No. 5,538,880 [Patent Document 27] U.S. Patent No. 6,160,208 [Patent Document 28] U.S. Patent No. 6,399,861 [Patent Document 29] U.S. Patent No. 6,153,812 [Patent Document 30] U.S. Patent No. 5,159,135 [Patent Document 31] U.S. Patent No. 5,824,877 [Patent Document 32] U.S. Patent No. 5,591,616 [Patent Document 33] U.S. Patent No. 6,384,301 [Patent Document 34] U.S. Patent No. 5,750,871 [Patent Document 35] U.S. Patent No. 5,463,174 [Patent Document 36] U.S. Patent No. 5,188,958 [Patent Document 37] U.S. Patent No. 5,049,386 [Patent Document 38] U.S. Patent No. 4,946,787 [Patent Document 39] U.S. Patent No. 4,897,355 [Patent Document 40] WO91 / 17424 [Patent Document 41] WO91 / 16024 [Patent Document 42] WO2014 / 093622 [Patent Document 43] U.S. Patent No. 6,194,636 [Patent Document 44] US Patent Application Publication No. 2004 / 0216189 [Non-patent literature]

[0006] [Non-Patent Document 1] “The American Heritage Science Dictionary” (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York) [Non-Patent Document 2] "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th edition, 2002, McGraw-Hill, New York) [Non-Patent Document 3] "Oxford Dictionary of Biology" (6th edition, 2008, Oxford University Press, Oxford and New York) [Non-Patent Document 4] Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed. (2012) [Non-Patent Document 5] Current Protocols In Molecular Biology (eds. FM Ausubel et al. (1987)) [Non-Patent Document 6] Plant Breeding Methodology (NF Jensen, Wiley-Interscience (1988)) [Non-Patent Document 7] Methods In Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor (eds.) (1995)) [Non-Patent Document 8] Harlow and Lane (eds.) (1988) Antibodies, A Laboratory Manual [Non-Patent Document 9] Animal Cell Culture (RI Freshney (ed.) (1987)) [Non-Patent Document 10] Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences [Non-Patent Document 11] CN Stewart, A. Touraev, V. Citovsky, T. Tzfira (eds.) (2011) Plant Transformation Technologies (Wiley-Blackwell) [Non-Patent Document 12] RH Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.) [Non-Patent Document 13] PCR Primer: A Laboratory Manual, Dieffenbach & Dveksler (eds.), Cold Spring Harbor Laboratory Press, 1995 [Non-Patent Document 14] Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) [Non-Patent Document 15] Chenna R. et al., "Multiple sequence alignment with the Clustal series of programs", Nucleic Acids Research 31: pp. 3497-3500 (2003) [Non-Patent Document 16] Thompson JD, "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 pages (1994)

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[0007] In one aspect, the disclosure provides a plant comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter or a floral cell-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome of the plant.

[0008] In one aspect, the disclosure provides a method of editing the genome of a plant, comprising: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0009] In one aspect, the disclosure provides a method of editing the genome of a plant cell, the method comprising: (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one embryo from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one embryo.

[0010] In one aspect, the disclosure provides a method of editing the genome of a plant, comprising the steps of: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant to produce at least one embryo, wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one embryo.

[0011] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferential promoter; and (ii) one or more guide nucleic acids operably linked to a heterologous second promoter that (A) is capable of hybridizing to a target sequence in the genome of the plant; and (B) is capable of hybridizing to first and second sites that flank a nucleic acid sequence encoding a gene of interest. (iii) a second nucleic acid sequence encoding one or more operably linked guide nucleic acids; (b) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cell of step (a); wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generates a double-stranded break within the target sequence, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one flower cell.

[0012] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) administering to a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; (ii) one or more guide nucleic acids operably linked to a heterologous second promoter that (A) is capable of hybridizing to a target sequence within the genome of the plant; and (B) is capable of hybridizing to a first site and a second site that flank a nucleic acid sequence encoding a gene of interest. (iii) introducing a second nucleic acid sequence encoding a gene of interest; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant derived from step (b) to produce at least one embryo; wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in the at least one embryo, and the ribonucleoprotein generates double-stranded breaks in the target sequence, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one embryo.

[0013] In one aspect, the disclosure provides a recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome of a plant.

[0014] In one aspect, the disclosure provides a method of editing the genome of a plant, comprising the steps of: (a) introducing into a plant cell (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferential promoter, and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0015] In one aspect, the disclosure provides a method of editing the genome of a plant cell, the method comprising: (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one progeny plant from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0016] In one aspect, the disclosure provides a method of editing the genome of a plant cell, the method comprising: (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferred promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one progeny plant from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one flower cell, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0017] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) administering to a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferential promoter; (ii) one or more guide nucleic acids operably linked to a heterologous second promoter that (A) is capable of hybridizing to a target sequence in the genome of the plant; and (B) is capable of hybridizing to first and second sites that flank a nucleic acid sequence encoding a gene of interest. and (iii) a second nucleic acid sequence encoding one or more guide nucleic acids linked to the first nucleic acid sequence; and (b) regenerating at least one plant from the plant cell of step (a); wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates a double-stranded break in the target sequence molecule, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one flower cell.

[0018] In one aspect, the disclosure provides a method of generating two or more progeny plants with unique edits from a single transformed plant cell, comprising: (a) administering to the plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome. (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the first plant, and the ribonucleoprotein generates at least one double-stranded break within the target sequence in the at least one flower cell; (c) pollinating the first plant of step (b); and (d) germinating two or more seeds produced from step (c) to produce two or more progeny plants with unique edits.

[0019] In one aspect, the disclosure provides a method of generating two or more progeny plants with unique edits from a single transformed plant cell, comprising: (a) administering to the plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous first promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous flower cell preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome. (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the first plant, and the ribonucleoprotein generates at least one double-stranded break within the target sequence in the at least one flower cell; (c) pollinating the first plant of step (b); and (d) germinating two or more seeds produced from step (c) to produce two or more progeny plants with unique edits.

[0020] In one aspect, the disclosure provides a method of generating two or more progeny plants with unique edits from a single transformed plant cell, the method comprising: (a) introducing into the plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, where the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; (b) regenerating a first plant from the plant cell of step (a); (c) pollinating the first plant of step (b), where the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the floral tissue; and (d) germinating two or more seeds produced from step (c) to generate two or more progeny plants with unique edits.

[0021] In one aspect, the disclosure provides a method of generating two or more progeny plants with unique edits from a single transformed plant cell, the method comprising: (a) introducing into the plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous first promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral tissue preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in a genome; (b) regenerating a first plant from the plant cell of step (a); (c) pollinating the first plant of step (b), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the floral tissue; and (d) germinating the two or more seeds produced from step (c) to generate two or more progeny plants with unique edits. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Unless otherwise defined, all technical and scientific terms used have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Where a term is provided in the singular, the inventors also contemplate aspects of the present disclosure described by the plural of that term. Where there are discrepancies in terms and definitions used in references 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 industry in which they are used, as exemplified by various industry-specific dictionaries, such as "The American Heritage® Science Dictionary" (Editors of the American Heritage Dictionaries, 2011, Houghton Mifflin Harcourt, Boston and New York), "McGraw-Hill Dictionary of Scientific and Technical Terms" (6th Edition, 2002, McGraw-Hill, New York), or "Oxford Dictionary of Biology" (6th Edition, 2008, Oxford University Press, Oxford and New York). The inventors do not intend to be limited by mechanism or mode of action. That reference is provided for illustrative purposes only.

[0023] The practice of the present disclosure involves, 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, e.g., Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); Current Protocols In Molecular Biology (FM Ausubel et al., eds. (1987)); Plant Breeding Methodology (NF Jensen, Wiley-Interscience (1988)); Methods In Enzymology series (Academic Press, Inc.): PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor, eds. (1995)); Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual; Animal Cell Culture (RI Freshney, ed. (1987)); Recombinant Protein Purification: Principles And Methods, 18-1142-75, GE Healthcare Life Sciences; CN Stewart, A. Touraev, V. Citovsky, T. Tzfira, eds. (2011) Plant Transformation Technologies (Wiley-Blackwell); and RH Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.).

[0024] All references cited herein, including, for example, all patents, published patent applications, and non-patent publications, are hereby incorporated by reference in their entirety.

[0025] When a group of options is presented, any and all combinations of the members of the group are specifically contemplated. For example, if an item is selected from the group consisting of A, B, C, and D, the inventors specifically contemplate each option individually (e.g., A only, B only, etc.) as well as combinations such as A, B, and D; A and C; B and C, etc.

[0026] As used herein, singular terms and the singular forms "a," "an," and "the," for example, include plural referents unless the context clearly dictates otherwise.

[0027] Any of the compositions, nucleic acid molecules, polypeptides, cells, plants, etc. provided herein are specifically contemplated for use with any of the methods provided herein.

[0028] In one aspect, the disclosure provides a recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome of a plant.

[0029] In one aspect, the disclosure provides a recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a floral tissue-preferred promoter, such as a heterologous floral tissue-preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome of a plant.

[0030] In one aspect, the disclosure provides a plant comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome of the plant.

[0031] In one aspect, the disclosure provides a plant comprising a recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered cut in a double-stranded DNA molecule operably linked to a heterologous promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a floral tissue preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome of the plant.

[0032] In one aspect, the present disclosure provides a plant comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered cut in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter or a flower cell-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome of the plant. In one aspect, the present disclosure provides a plant comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered cut in a double-stranded DNA molecule operably linked to a first promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral tissue-preferred promoter or a flower cell-preferred promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome of the plant.

[0033] In one aspect, the present disclosure provides a seed of any of the plants provided herein.

[0034] As used herein, "floral tissue" refers to any tissue or cell that gives rise to any part of a flower, except the shoot apical meristem. Non-limiting examples of floral tissues include branch meristems, axillary meristems, inflorescence meristems, floral bud meristems, glumes, glumes, lodices, pedicels, receptacles, sepals, petals, stigmas, styles, filaments, and anthers. As used herein, "floral tissue" does not refer to ovaries, ovules, or pollen.

[0035] In some aspects, the floral tissue comprises a structure selected from the group consisting of a branch meristem, an axillary meristem, an inflorescence meristem, a floral bud meristem, a glume, a glume, a lodicule, a pedicel, a receptacle, a sepal, a petal, a stigma, a style, a filament, and an anther.

[0036] When a plant transitions from vegetative growth to flowering, the shoot apical meristem is transformed into an inflorescence meristem. The inflorescence meristem then produces a floral bud meristem, which ultimately gives rise to all the tissues of the flower, including but not limited to, glumes, glumes, lodices, receptacles, sepals, petals, stigma, style, filaments, and anthers. In some embodiments, the floral tissue comprises an inflorescence meristem. In some embodiments, the floral tissue comprises a floral bud meristem. In some embodiments, the floral tissue comprises a branch meristem. In grasses, the branch meristem is produced by the inflorescence meristem, which produces branches or spikelets in two rows to pattern the floral organs in a whorl of leaves.

[0037] In some embodiments, the floral tissue comprises a pedicel. In some embodiments, the floral tissue comprises a glume. In some embodiments, the floral tissue comprises a lodicule. In some embodiments, the floral tissue comprises a receptacle. In some embodiments, the floral tissue comprises a sepal. In some embodiments, the floral tissue comprises a petal. In some embodiments, the floral tissue comprises a stigma. In some embodiments, the floral tissue comprises a style. In some embodiments, the floral tissue comprises a filament. In some embodiments, the floral tissue comprises an anther.

[0038] In some embodiments, the floral tissue does not include a shoot apical meristem. In some embodiments, the floral cells do not include shoot apical meristem cells. In some embodiments, the floral tissue does not include an ovary. In some embodiments, the floral tissue does not include an ovule. In some embodiments, the floral tissue does not include pollen.

[0039] As used herein, "floral cell" refers to a cell of any floral tissue. In some embodiments, the floral cell is a branch meristem cell. In some embodiments, the floral cell is an inflorescence meristem cell. In some embodiments, the floral cell is a flower bud meristem cell. In some embodiments, the floral cell is a pedicel cell. In some embodiments, the floral tissue is a glume cell. In some embodiments, the floral tissue is a lodicule cell. In some embodiments, the floral tissue is a receptacle cell. In some embodiments, the floral tissue is a sepal cell. In some embodiments, the floral cell is a petal cell. In some embodiments, the floral tissue is a stigma cell. In some embodiments, the floral tissue is a style cell. In some embodiments, the floral cell is a filament cell. In some embodiments, the floral tissue is an anther cell.

[0040] Nucleic acids and amino acids 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 contemplated. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules may comprise deoxyribonucleotides, ribonucleotides, or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. Polynucleotides of the present disclosure also encompass sequences of any form, including, but not limited to, single-stranded forms, double-stranded forms, hairpins, stem-loop structures, and the like. In one embodiment, the nucleic acid molecules provided herein are DNA molecules. In another embodiment, the nucleic acid molecules provided herein are RNA molecules. In one embodiment, the nucleic acid molecules provided herein are single-stranded. In another embodiment, the nucleic acid molecules provided herein are double-stranded.

[0041] As used herein, the term "recombinant" with reference to nucleic acid (DNA or RNA) molecules, proteins, constructs, vectors, etc., refers to a nucleic acid or amino acid molecule or sequence that is man-made and not normally found in nature and / or exists in a context in which it is not normally found in nature, including nucleic acid (DNA or RNA) molecules, proteins, constructs, etc. that include combinations of polynucleotide or protein sequences that do not naturally occur contiguous or in close proximity without human intervention, and / or polynucleotide molecules, proteins, constructs, etc. that include at least two polynucleotide or protein sequences that are heterologous with respect to each other.

[0042] In one aspect, the methods and compositions provided herein include a vector. As used herein, the term "vector" refers to a DNA molecule used as a vehicle to transport exogenous genetic material into a cell.

[0043] In some embodiments, the one or more polynucleotide sequences derived from the vector are stably integrated into the genome of the plant. In some embodiments, the one or more polynucleotide sequences derived from the vector are not stably integrated into the genome of the plant cell.

[0044] In one aspect, the first and second nucleic acid sequences are provided in a single vector, hi another aspect, the first nucleic acid sequence is provided in a first vector and the second nucleic acid sequence is provided in a second vector.

[0045] As used herein, the term "polypeptide" refers to a chain of at least two covalently linked amino acids. A polypeptide may be encoded by a polynucleotide provided herein. An example of a polypeptide is a protein. A protein provided herein may be encoded by a nucleic acid molecule provided herein.

[0046] Nucleic acids can be isolated using techniques routine in the art. For example, nucleic acids can be isolated using any method, including but not limited to recombinant nucleic acid techniques and / or 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 nucleic acids. Isolated nucleic acids can also be chemically synthesized as single nucleic acid molecules or as a series of oligonucleotides. Polypeptides can be purified from natural sources (e.g., biological samples) by known methods such as DEAE ion exchange, gel filtration, and hydroxyapatite chromatography. Polypeptides can also be purified, for example, by expressing a nucleic acid in an expression vector. Additionally, purified polypeptides can be obtained by chemical synthesis. Purity of a polypeptide can be measured using any suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0047] Without being limited thereto, 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).

[0048] Polypeptides can be detected using antibodies. Techniques for detecting polypeptides using antibodies include enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation and immunofluorescence. The antibodies provided herein may be polyclonal or monoclonal. Antibodies having specific binding affinity for the polypeptides provided herein can be generated using methods well known in the art. The antibodies provided herein can be attached to a solid support, such as a microtiter plate, using methods well known in the art.

[0049] The term "percent identity" or "percent identical" as used herein with reference to two or more nucleotide or protein sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or protein) over a comparison window, (ii) determining the number of positions where identical nucleic acid bases (for nucleotide sequences) or amino acid residues (for proteins) occur in both sequences to obtain the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the comparison window, and then (iv) multiplying this quotient by 100% to obtain the percent identity. When "percent identity" is calculated with respect to a reference sequence without specifying a particular comparison window, 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. Thus, for the purposes of this application, when two sequences (query and subject) are optimally aligned (allowing for gaps in the alignment), the "percent identity" with respect to 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 entire length (or comparison window), then multiplied by 100%. When percentages of sequence identity are used in reference to proteins, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions, in which amino acid residues are replaced with other amino acid residues that have similar chemical properties (e.g., charge or hydrophobicity), thus not changing 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".

[0050] The term "percent sequence complementarity" or "percent complementarity," as used herein with reference to two nucleotide sequences, is similar to the concept of percent identity, but refers to the percentage of nucleotides in the query sequence that optimally base-pair or hybridize to the nucleotides of the subject sequence when the query and subject sequences are arranged linearly and optimally base-paired without secondary folding structures such as loops, stems, or hairpins. Such percent complementarity may be between two DNA strands, between two RNA strands, or between a DNA strand and an RNA strand. "Percentage complementarity" can be calculated by (i) optimally base-pairing or hybridizing two nucleotide sequences in a linear and fully extended configuration (i.e., without folding or secondary structure) over a comparison window, (ii) determining the number of base-pairing positions between the two sequences over the comparison window to obtain the number of complementary positions, (iii) dividing the number of complementary positions by the total number of positions in the comparison window, and (iv) multiplying this quotient by 100% to obtain the percentage complementarity of the two sequences. The optimal base-pairing of two sequences can be determined based on known nucleotide base pairing, such as GC, AT, and AU, through hydrogen bonds. When "percentage complementarity" is calculated with respect to a reference sequence without specifying a specific comparison window, the percentage identity is determined by dividing the number of complementary positions between two linear sequences by the total length of the reference sequence. Thus, for the purposes of this application, when two sequences (query and subject) are optimally base-paired (allowing for mismatched or unpaired nucleotides), the "percent complementarity" with respect to a 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 entire length, then multiplied by 100%.

[0051] A variety of pairwise or multiple sequence alignment algorithms and programs are known in the art, such as ClustalW or Basic Local Alignment Search Tool (BLAST®), which can be used to compare sequence identity or similarity between two or more nucleotide or protein sequences for optimal alignment thereof to calculate percent identity of the 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 listed above) can be determined by the ClustalW algorithm. For example, 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: pp. 4673-4680 (1994); Larkin MA et al., "Clustal W and Clustal 215:403-410 (1990), the entire contents and disclosures of which are incorporated herein by reference.

[0052] As used herein, a first nucleic acid molecule can "hybridize" to a second nucleic acid molecule in a sequence-specific, antiparallel manner (i.e., the nucleic acid specifically binds to a complementary nucleic acid) through non-covalent interactions (e.g., Watson-Crick base pairing) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As known in the art, standard Watson-Crick base pairs include adenine pairing with thymine, adenine pairing with uracil, and guanine (G) pairing with cytosine (C) [DNA, RNA]. Furthermore, for hybridization between two RNA molecules (e.g., dsRNA), it is also known in the art that guanine bases pair with uracil. For example, G / U base pairs are responsible in part for the degeneracy (i.e., redundancy) of the genetic code in the context of tRNA anticodons that base pair with codons in mRNA. In the context of this disclosure, a guanine of the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule is considered to be complementary to a uracil, and vice versa. Therefore, if a G / U base pair can be made at a given nucleotide position of the protein-binding segment (dsRNA duplex) of a subject DNA-targeting RNA molecule, that position is not considered to be non-complementary, but instead is considered to be complementary.

[0053] Hybridization and washing conditions are well known and are exemplified in Sambrook, J., Fritsch, EF and Maniatis, T. Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989), especially Chapter 11 and Table 11.1 therein; and Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). The conditions of temperature and ionic strength determine the "stringency" of hybridization.

[0054] Hybridization requires that the two nucleic acids contain complementary sequences, although mismatches between bases are acceptable. Appropriate conditions for hybridization between two nucleic acids depend on the length of the nucleic acids and the degree of complementarity, variables well known in the art. The higher the degree of complementarity between two nucleotide sequences, the higher the melting temperature (Tm) value for hybrids of nucleic acids with these sequences. For hybridization between nucleic acids with short ranges of complementarity (e.g., complementarity over 35 nucleotides or less), the position of mismatches becomes important (see Sambrook et al.). Typically, the length of a hybridizable nucleic acid is at least 10 nucleotides. Examples of minimum lengths for hybridizable nucleic acids 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, one skilled in the art will recognize that temperature and salt concentration of washing solutions can be adjusted as necessary depending on factors such as the length of the complementary region and the degree of complementarity.

[0055] It is understood in the art that the sequence of a polynucleotide does not have to be 100% complementary to the sequence of its target nucleic acid to be specifically hybridizable or hybridizable. Furthermore, a polynucleotide may hybridize over one or more segments (e.g., loop or hairpin structures) such that intervening or adjacent segments are not involved in the hybridization event. For example, an antisense nucleic acid in which 18 of 20 nucleotides of an antisense compound are complementary to a target region and thus specifically hybridize represents 90 percent complementarity. In this example, the remaining non-complementary nucleotides may be clustered or interspersed with complementary nucleotides and do not have to be contiguous with each other or with complementary nucleotides. Percent complementarity between particular stretches of nucleic acid sequences within a nucleic acid can be routinely determined 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 algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482-489) using the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, Inc., University Research Park, Madison Wis.) using default settings.

[0056] Generate edits In one aspect, the disclosure provides a method of editing the genome of a plant, comprising: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0057] In one aspect, the disclosure provides a method of editing the genome of a plant, comprising: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral tissue preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0058] In one aspect, the disclosure provides a method of editing the genome of a plant, comprising: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0059] In one aspect, the disclosure provides a method of editing the genome of a plant, comprising: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a flower cell preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0060] In one aspect, the disclosure provides a method of editing the genome of a plant cell, comprising: (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence within the genome; and (b) obtaining at least one embryo from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one embryo.

[0061] In one aspect, the disclosure provides a method of editing the genome of a plant cell, the method comprising: (a) crossing a first plant with a second plant, the first plant comprising a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous promoter, and the second plant comprising a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a floral tissue preferential promoter, the at least one guide nucleic acid being capable of hybridizing to a target sequence within the genome; and (b) obtaining at least one embryo from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one embryo.

[0062] In one aspect, the disclosure provides a method of editing the genome of a plant, comprising the steps of: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant to produce at least one embryo, wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one embryo.

[0063] In one aspect, the disclosure provides a method of editing the genome of a plant, comprising the steps of: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a floral tissue preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant to produce at least one embryo, wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one embryo.

[0064] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) administering to a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; and (ii) one or more guide nucleic acids operably linked to a heterologous second promoter that (A) is capable of hybridizing to a target sequence in the genome of the plant; and (B) is capable of hybridizing to first and second sites that flank a nucleic acid sequence encoding a gene of interest. and (iii) a third nucleic acid sequence encoding a gene of interest; (b) regenerating at least one plant from the plant cell of step (a); wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generates double-stranded breaks within the target sequence, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one flower cell.

[0065] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) one or more guide nucleic acids operably linked to a floral tissue-preferential second promoter that (A) is capable of hybridizing to a target sequence in the genome of the plant; and (B) is capable of hybridizing to a first site and a second site that flank a nucleic acid sequence encoding a gene of interest. and (iii) a third nucleic acid sequence encoding a gene of interest; (b) regenerating at least one plant from the plant cell of step (a); wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generates double-stranded breaks within the target sequence, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one flower cell.

[0066] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) administering to a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferred promoter; and (ii) one or more guide nucleic acids operably linked to a heterologous second promoter that (A) is capable of hybridizing to a target sequence in the genome of the plant; and (B) is capable of hybridizing to first and second sites that flank a nucleic acid sequence encoding a gene of interest. and (iii) a third nucleic acid sequence encoding a gene of interest; (b) regenerating at least one plant from the plant cell of step (a); wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generates double-stranded breaks within the target sequence, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one flower cell.

[0067] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) introducing into a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) one or more guide nucleic acids operably linked to a flower cell-preferential second promoter that (A) is capable of hybridizing to a target sequence in the genome of the plant; and (B) is capable of hybridizing to first and second sites that flank a nucleic acid sequence encoding a gene of interest. and (iii) a third nucleic acid sequence encoding a gene of interest; (b) regenerating at least one plant from the plant cell of step (a); wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generates double-stranded breaks within the target sequence, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one flower cell.

[0068] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) administering to a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; (ii) one or more guide nucleic acids operably linked to a heterologous second promoter that (A) is capable of hybridizing to a target sequence within the genome of the plant; and (B) is capable of hybridizing to a first site and a second site that flank a nucleic acid sequence encoding a gene of interest. and (iii) a third nucleic acid sequence encoding a gene of interest; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant derived from step (b) to produce at least one embryo; wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in the at least one embryo, and the ribonucleoprotein generates double-stranded breaks in the target sequence, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one embryo.

[0069] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) administering to a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered cut in a double-stranded DNA molecule operably linked to a heterologous promoter; (ii) one or more guide nucleic acids encoding one or more guide nucleic acids operably linked to a floral tissue-preferred promoter that (A) is capable of hybridizing to a target sequence in the genome of the plant; and (B) a first site and a second site that flank a nucleic acid sequence encoding a gene of interest. and (iii) a third nucleic acid sequence encoding a gene of interest; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant derived from step (b) to produce at least one embryo; wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in the at least one embryo, and the ribonucleoprotein generates double-stranded breaks within the target sequence, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one embryo.

[0070] In one aspect, the disclosure provides a method of editing the genome of a plant, comprising the steps of: (a) introducing into a plant cell (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferential promoter, and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0071] In one aspect, the disclosure provides a method of editing the genome of a plant cell, the method comprising: (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one progeny plant from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0072] In one aspect, the disclosure provides a method of editing the genome of a plant cell, the method comprising: (a) crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferred promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one progeny plant from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one flower cell, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

[0073] In one aspect, the disclosure provides a method of generating site-specific integration in a plant, comprising: (a) administering to a plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferential promoter; (ii) one or more guide nucleic acids operably linked to a heterologous second promoter that (A) is capable of hybridizing to a target sequence within the genome of the plant; and (B) is capable of hybridizing to a first site and a second site that flank a nucleic acid sequence encoding a gene of interest. and (iii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to the target sequence molecule; and (b) regenerating at least one plant from the plant cell of step (a); wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates double-stranded breaks in the target sequence molecule, the first site, and the second site, and the gene of interest is integrated into the target site in the at least one flower cell.

[0074] In one aspect, the disclosure provides a method of generating two or more progeny plants with unique edits from a single transformed plant cell, comprising: (a) administering to the plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome. (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the first plant, and the ribonucleoprotein generates at least one double-stranded break within the target sequence in the at least one flower cell; (c) pollinating the first plant of step (b); and (d) germinating two or more seeds produced from step (c) to produce two or more progeny plants with unique edits.

[0075] In one aspect, the disclosure provides a method of generating two or more progeny plants with unique edits from a single transformed plant cell, comprising: (a) administering to the plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts in a double-stranded DNA molecule operably linked to a heterologous first promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous flower cell preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome. (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the first plant, and the ribonucleoprotein generates at least one double-stranded break within the target sequence in the at least one flower cell; (c) pollinating the first plant of step (b); and (d) germinating two or more seeds produced from step (c) to produce two or more progeny plants with unique edits.

[0076] In one aspect, the disclosure provides a method of generating two or more progeny plants with unique edits from a single transformed plant cell, the method comprising: (a) introducing into the plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, where the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; (b) regenerating a first plant from the plant cell of step (a); (c) pollinating the first plant of step (b), where the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the floral tissue; and (d) germinating two or more seeds produced from step (c) to generate two or more progeny plants with unique edits.

[0077] In one aspect, the disclosure provides a method of generating two or more progeny plants with unique edits from a single transformed plant cell, the method comprising: (a) introducing into the plant cell: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous first promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral tissue preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in a genome; (b) regenerating a first plant from the plant cell of step (a); (c) pollinating the first plant of step (b), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the floral tissue; and (d) germinating two or more seeds produced from step (c) to generate two or more progeny plants with unique edits.

[0078] promoter As generally understood in the art, the term "promoter" refers to a DNA sequence that contains an RNA polymerase binding site, a transcription initiation site, and / or a TATA box and aids or facilitates the transcription and expression of an associated transcribable polynucleotide sequence and / or gene (or transgene). Promoters can be synthetically produced, altered, or derived from known or naturally occurring promoter sequences or other promoter sequences. Promoters may also include leaders, 5'UTRs, and introns. Promoters may also include chimeric promoters that include combinations of two or more heterologous sequences. Thus, promoters of the present application may include variants of promoter sequences that are similar in composition but not identical to other promoter sequences known or provided herein. Promoters can be classified according to various criteria related to the expression pattern of the associated coding or transcribable sequence or gene (including transgene) operably linked to the promoter, such as constitutive, developmental, tissue-specific, cell cycle-specific, inducible, etc.

[0079] As used herein, "operably linked" refers to a functional link between two or more elements. For example, an operable link between a polynucleotide of interest and a regulatory sequence (e.g., a promoter) is a functional link that allows expression of the polynucleotide of interest. The operably linked elements may be contiguous or non-contiguous.

[0080] In one embodiment, the recombinant nucleic acid provided herein comprises at least one promoter. In another embodiment, the polynucleotide encoding the inducible nuclease is operably linked to at least one promoter. In another embodiment, the polynucleotide encoding the Cas12a nuclease is operably linked to at least one promoter. In another embodiment, the polynucleotide encoding the CasX nuclease is operably linked to at least one promoter. In another embodiment, the polynucleotide encoding the MAD7® nuclease is operably linked to at least one promoter. In another embodiment, the polynucleotide encoding the guide nucleic acid is operably linked to at least one promoter.

[0081] A promoter that expresses in a particular tissue of an organism and not in other tissues is referred to as a "tissue-specific" promoter. A promoter that drives enhanced expression in a particular tissue of an organism compared to other tissues of the organism is referred to as a "tissue-preferred" promoter. Thus, a "tissue-preferred" promoter causes relatively high or preferential expression in a particular tissue of a plant, but shows lower levels of expression in other tissues of the plant. In another aspect, the promoter provided herein is a tissue-specific promoter. In a further aspect, the promoter provided herein is a tissue-preferred promoter. In an aspect, a tissue-preferred promoter comprises a tissue-specific promoter.

[0082] Determining promoter activity can be performed using any method standard in the art, for example, but not limited to, a promoter of interest can be used to drive expression of a fluorophore or other reporting molecule, and the concentration of the expressed molecule can be used to determine promoter activity in different cell or tissue types.

[0083] Flowers are organized into concentric whorls of sepals, petals, stamens (e.g., the structures including filaments and anthers), and carpels (e.g., the structures including the ovary, stigma, and often the style), with each of these floral organ types having a unique role in reproduction. Genes involved in the establishment of floral structures are homeotic MADS domain transcription factors (with the exception of APATELA2, which is an A gene; see below) classified as A genes, B genes, C genes, D genes, or E genes according to the ABCDE model of flower development, which has been used to describe how floral structures are genetically specified. See, e.g., Coen and Meyerowitz, Nature, 353:31-37 (1991); and Murai, Plants, 2:379-395 (2013). Under the ABCDE model, sepals are formed when A and E genes are co-expressed; carpels are formed when C and E genes are co-expressed; petals are formed when A, B, and E genes are co-expressed; stamens are formed when B, C, and E genes are co-expressed; and ovules are formed when D and E genes are co-expressed.

[0084] Non-limiting examples of A genes in Arabidopsis and soybean include APETALA1 (AP1) and APETALA2 (AP2), which are ERENUCLEOTIDE transcription factors. Non-limiting examples of B genes in Arabidopsis and soybean include APETALA3 (AP3) and PISTILLATA (PI). Non-limiting examples of C genes in Arabidopsis and soybean include AGAMOUS (AG). Non-limiting examples of D genes in Arabidopsis and soybean include AGAMOUS-LIKE 11 / SEEDSTICK (AGL11 / STK), AGAMOUS-LIKE 1 / SHATTERPROOF1 (AGL1 / SHP1), AGAMOUS-LIKE 5 / SHATTERPROOF2 (AGL5 / SHP2). Non-limiting examples of E genes in Arabidopsis and soybean include SEPALLATA1 (SEP1), SEPALLATA2 (SEP2), SEPALLATA3 (SEP3), and SEPALLATA4 (SEP4).

[0085] Non-limiting examples of A genes in maize include ZEA APETALA HOMOLOG1 (ZAP1). Non-limiting examples of B genes in maize include ZEA MAYS MADS16 (ZMM16) and ZEA MAYS MADS18 (ZMM18). Non-limiting examples of C genes in maize include ZEA AGAMOUS HOMOLOG1 (ZAG1), ZEA MAYS MADS2 (ZMM2), and ZEA MAYS MADS23 (ZMM23). Non-limiting examples of D genes in maize include ZEA AGAMOUS HOMOLOG2 (ZAG2) and ZEA MAYS MADS1 (ZMM1). Non-limiting examples of E genes in maize include ZEA AGAMOUS HOMOLOG3 (ZAG3) and ZEA MAYS MADS7 / SEPALLATA-LIKE (ZMM7 / SEP-like).

[0086] In some embodiments, the floral tissue preferred promoter is the A gene promoter. In some embodiments, the floral tissue preferred promoter is the B gene promoter. In some embodiments, the floral tissue preferred promoter is the C gene promoter. In some embodiments, the floral tissue preferred promoter is the D gene promoter. In some embodiments, the floral tissue preferred promoter is the E gene promoter.

[0087] In some embodiments, the flower cell preferred promoter is the A gene promoter. In some embodiments, the flower cell preferred promoter is the B gene promoter. In some embodiments, the flower cell preferred promoter is the C gene promoter. In some embodiments, the flower cell preferred promoter is the D gene promoter. In some embodiments, the flower cell preferred promoter is the E gene promoter.

[0088] In one aspect, the floral tissue-preferred promoter comprises a promoter selected from the group consisting of an AP1 promoter, an AP2 promoter, a ZAP1 promoter, an AP3 promoter, a PI promoter, a ZMM16 promoter, a ZMM18 promoter, an AG promoter, a ZAG1 promoter, a ZMM2 promoter, a ZMM23 promoter, an AGL11 / STK promoter, an AGL1 / SHP1 promoter, an AGL5 / SHP2 promoter, a ZAG2 promoter, a ZMM1 promoter, a SEP1 promoter, a SEP2 promoter, a SEP3 promoter, a SEP4 promoter, a ZAG3 promoter, and a ZMM7 / SEP-like promoter.

[0089] In some embodiments, the floral tissue preferred promoter comprises an AP1 promoter. In some embodiments, the floral tissue preferred promoter comprises an AP2 promoter. In some embodiments, the floral tissue preferred promoter comprises a ZAP1 promoter. In some embodiments, the floral tissue preferred promoter comprises an AP3 promoter. In some embodiments, the floral tissue preferred promoter comprises a PI promoter. In some embodiments, the floral tissue preferred promoter comprises a ZMM16 promoter. In some embodiments, the floral tissue preferred promoter comprises a ZMM18 promoter. In some embodiments, the floral tissue preferred promoter comprises an AG promoter. In some embodiments, the floral tissue preferred promoter comprises a ZAG1 promoter. In some embodiments, the floral tissue preferred promoter comprises a ZMM2 promoter. In some embodiments, the floral tissue preferred promoter comprises a ZMM23 promoter. In some embodiments, the floral tissue preferred promoter comprises an AGL11 / STK promoter. In some embodiments, the floral tissue preferred promoter comprises an AGL1 / SHP1 promoter. In some embodiments, the floral tissue preferred promoter comprises an AGL5 / SHP2 promoter. In some embodiments, the floral tissue preferred promoter comprises a ZAG2 promoter. In some embodiments, the floral tissue preferred promoter comprises a ZMM1 promoter. In some embodiments, the floral tissue preferred promoter comprises a SEP1 promoter. In some embodiments, the floral tissue preferred promoter comprises a SEP2 promoter. In some embodiments, the floral tissue preferred promoter comprises a SEP3 promoter. In some embodiments, the floral tissue preferred promoter comprises a SEP4 promoter. In some embodiments, the floral tissue preferred promoter comprises a ZAG3 promoter. In some embodiments, the floral tissue preferred promoter comprises a ZMM7 / SEP-like promoter.

[0090] In one aspect, the floral cell-preferred promoter comprises a promoter selected from the group consisting of an AP1 promoter, an AP2 promoter, a ZAP1 promoter, an AP3 promoter, a PI promoter, a ZMM16 promoter, a ZMM18 promoter, an AG promoter, a ZAG1 promoter, a ZMM2 promoter, a ZMM23 promoter, an AGL11 / STK promoter, an AGL1 / SHP1 promoter, an AGL5 / SHP2 promoter, a ZAG2 promoter, a ZMM1 promoter, a SEP1 promoter, a SEP2 promoter, a SEP3 promoter, a SEP4 promoter, a ZAG3 promoter, and a ZMM7 / SEP-like promoter.

[0091] In some embodiments, the flower cell preferred promoter comprises an AP1 promoter. In some embodiments, the flower cell preferred promoter comprises an AP2 promoter. In some embodiments, the flower cell preferred promoter comprises a ZAP1 promoter. In some embodiments, the flower cell preferred promoter comprises an AP3 promoter. In some embodiments, the flower cell preferred promoter comprises a PI promoter. In some embodiments, the flower cell preferred promoter comprises a ZMM16 promoter. In some embodiments, the flower cell preferred promoter comprises a ZMM18 promoter. In some embodiments, the flower cell preferred promoter comprises an AG promoter. In some embodiments, the flower cell preferred promoter comprises a ZAG1 promoter. In some embodiments, the flower cell preferred promoter comprises a ZMM2 promoter. In some embodiments, the flower cell preferred promoter comprises a ZMM23 promoter. In some embodiments, the flower cell preferred promoter comprises an AGL11 / STK promoter. In some embodiments, the flower cell preferred promoter comprises an AGL1 / SHP1 promoter. In some embodiments, the flower cell preferred promoter comprises an AGL5 / SHP2 promoter. In some embodiments, the flower cell preferred promoter comprises a ZAG2 promoter. In some embodiments, the flower cell preferred promoter comprises a ZMM1 promoter. In some embodiments, the flower cell preferred promoter comprises a SEP1 promoter. In some embodiments, the flower cell preferred promoter comprises a SEP2 promoter. In some embodiments, the flower cell preferred promoter comprises a SEP3 promoter. In some embodiments, the flower cell preferred promoter comprises a SEP4 promoter. In some embodiments, the flower cell preferred promoter comprises a ZAG3 promoter. In some embodiments, the flower cell preferred promoter comprises a ZMM7 / SEP-like promoter.

[0092] In one embodiment, the floral tissue-preferred promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence at least 70% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the floral tissue-preferred promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence at least 75% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the floral tissue-preferred promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence at least 80% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the floral tissue-preferred promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence that is at least 85% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the floral tissue-preferred promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence that is at least 90% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the floral tissue-preferred promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence that is at least 95% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the floral tissue-preferred promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the floral tissue-preferred promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof.

[0093] In one embodiment, the flower cell preferential promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence at least 70% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the flower cell preferential promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence at least 75% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the flower cell preferential promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence at least 80% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the flower cell preferential promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence at least 85% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the flower cell preferential promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence at least 90% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the flower cell preferential promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence at least 95% identical to a sequence selected from the group of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the flower cell-preferential promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence that is at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof. In one embodiment, the flower cell-preferential promoter comprises a sequence selected from the group consisting of SEQ ID NOs: 1-30, or a sequence that is 100% identical to a sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof.

[0094] In some embodiments, the promoters provided herein are operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the promoters provided herein are operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the promoters provided herein are operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the promoters provided herein are operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the promoters provided herein are operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the promoters provided herein are operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the promoters provided herein are operably linked to a nucleic acid encoding a single guide RNA.

[0095] In some aspects, the floral tissue preferred promoter provided herein is operably linked to a nucleic acid encoding an inducible nuclease. In some aspects, the floral tissue preferred promoter provided herein is operably linked to a nucleic acid encoding a Cas12a nuclease. In some aspects, the floral tissue preferred promoter provided herein is operably linked to a nucleic acid encoding a CasX nuclease. In some aspects, the floral tissue preferred promoter provided herein is operably linked to a nucleic acid encoding a MAD7® nuclease. In some aspects, the floral tissue preferred promoter provided herein is operably linked to a nucleic acid encoding a guide nucleic acid. In some aspects, the floral tissue preferred promoter provided herein is operably linked to a nucleic acid encoding a guide RNA. In some aspects, the floral tissue preferred promoter provided herein is operably linked to a nucleic acid encoding a single guide RNA.

[0096] In some aspects, the floral tissue specific promoter provided herein is operably linked to a nucleic acid encoding an inducible nuclease. In some aspects, the floral tissue specific promoter provided herein is operably linked to a nucleic acid encoding a Cas12a nuclease. In some aspects, the floral tissue specific promoter provided herein is operably linked to a nucleic acid encoding a CasX nuclease. In some aspects, the floral tissue specific promoter provided herein is operably linked to a nucleic acid encoding a MAD7® nuclease. In some aspects, the floral tissue specific promoter provided herein is operably linked to a nucleic acid encoding a guide nucleic acid. In some aspects, the floral tissue specific promoter provided herein is operably linked to a nucleic acid encoding a guide RNA. In some aspects, the floral tissue specific promoter provided herein is operably linked to a nucleic acid encoding a single guide RNA.

[0097] In some aspects, the flower cell preferential promoter provided herein is operably linked to a nucleic acid encoding an inducible nuclease. In some aspects, the flower cell preferential promoter provided herein is operably linked to a nucleic acid encoding a Cas12a nuclease. In some aspects, the flower cell preferential promoter provided herein is operably linked to a nucleic acid encoding a CasX nuclease. In some aspects, the flower cell preferential promoter provided herein is operably linked to a nucleic acid encoding a MAD7® nuclease. In some aspects, the flower cell preferential promoter provided herein is operably linked to a nucleic acid encoding a guide nucleic acid. In some aspects, the flower cell preferential promoter provided herein is operably linked to a nucleic acid encoding a guide RNA. In some aspects, the flower cell preferential promoter provided herein is operably linked to a nucleic acid encoding a single guide RNA.

[0098] In some aspects, the flower cell specific promoter provided herein is operably linked to a nucleic acid encoding an inducible nuclease. In some aspects, the flower cell specific promoter provided herein is operably linked to a nucleic acid encoding a Cas12a nuclease. In some aspects, the flower cell specific promoter provided herein is operably linked to a nucleic acid encoding a CasX nuclease. In some aspects, the flower cell specific promoter provided herein is operably linked to a nucleic acid encoding a MAD7® nuclease. In some aspects, the flower cell specific promoter provided herein is operably linked to a nucleic acid encoding a guide nucleic acid. In some aspects, the flower cell specific promoter provided herein is operably linked to a nucleic acid encoding a guide RNA. In some aspects, the flower cell specific promoter provided herein is operably linked to a nucleic acid encoding a single guide RNA.

[0099] In some embodiments, the A gene promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the A gene promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the A gene promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the A gene promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the A gene promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the A gene promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the A gene promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0100] In some embodiments, the B gene promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the B gene promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the B gene promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the B gene promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the B gene promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the B gene promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the B gene promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0101] In some embodiments, the C gene promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the C gene promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the C gene promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the C gene promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the C gene promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the C gene promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the C gene promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0102] In some embodiments, the D gene promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the D gene promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the D gene promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the D gene promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the D gene promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the D gene promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the D gene promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0103] In some embodiments, the E gene promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the E gene promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the E gene promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the E gene promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the E gene promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the E gene promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the E gene promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0104] In some embodiments, the AP1 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the AP1 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the AP1 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the AP1 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the AP1 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the AP1 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the AP1 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0105] In some embodiments, the AP2 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the AP2 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the AP2 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the AP2 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the AP2 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the AP2 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the AP2 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0106] In some embodiments, the ZAP1 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZAP1 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZAP1 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZAP1 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZAP1 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZAP1 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZAP1 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0107] In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0108] In some embodiments, the PI promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the PI promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the PI promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the PI promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the PI promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the PI promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the PI promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0109] In some embodiments, the ZMM16 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZMM16 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZMM16 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZMM16 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZMM16 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZMM16 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZMM16 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0110] In some embodiments, the ZMM18 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZMM18 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZMM18 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZMM18 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZMM18 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZMM18 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZMM18 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0111] In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the AP3 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0112] In some embodiments, the AG promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the AG promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the AG promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the AG promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the AG promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the AG promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the AG promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0113] In some embodiments, the ZAG1 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZAG1 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZAG1 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZAG1 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZAG1 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZAG1 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZAG1 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0114] In some embodiments, the ZMM2 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZMM2 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZMM2 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZMM2 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZMM2 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZMM2 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZMM2 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0115] In some embodiments, the ZMM3 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZMM3 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZMM3 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZMM3 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZMM3 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZMM3 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZMM3 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0116] In some embodiments, the AGL11 / STK promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the AGL11 / STK promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the AGL11 / STK promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the AGL11 / STK promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the AGL11 / STK promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the AGL11 / STK promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the AGL11 / STK promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0117] In some embodiments, the AGL1 / SHP1 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the AGL1 / SHP1 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the AGL1 / SHP1 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the AGL1 / SHP1 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the AGL1 / SHP1 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the AGL1 / SHP1 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the AGL1 / SHP1 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0118] In some embodiments, the AGL5 / SHP2 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the AGL5 / SHP2 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the AGL5 / SHP2 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the AGL5 / SHP2 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the AGL5 / SHP2 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the AGL5 / SHP2 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the AGL5 / SHP2 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0119] In some embodiments, the ZAG2 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZAG2 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZAG2 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZAG2 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZAG2 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZAG2 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZAG2 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0120] In some embodiments, the ZMM1 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZMM1 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZMM1 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZMM1 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZMM1 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZMM1 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZMM1 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0121] In some embodiments, the SEP1 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the SEP1 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the SEP1 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the SEP1 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the SEP1 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the SEP1 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the SEP1 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0122] In some embodiments, the SEP2 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the SEP2 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the SEP2 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the SEP2 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the SEP2 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the SEP2 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the SEP2 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0123] In some embodiments, the SEP3 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the SEP3 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the SEP3 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the SEP3 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the SEP3 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the SEP3 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the SEP3 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0124] In some embodiments, the SEP4 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the SEP4 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the SEP4 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the SEP4 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the SEP4 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the SEP4 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the SEP4 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0125] In some embodiments, the ZAG3 promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZAG3 promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZAG3 promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZAG3 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZAG3 promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZAG3 promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZAG3 promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0126] In some embodiments, the ZMM7 / SEP-like promoter is operably linked to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZMM7 / SEP-like promoter is operably linked to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZMM7 / SEP-like promoter is operably linked to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZMM7 promoter is operably linked to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZMM7 / SEP-like promoter is operably linked to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZMM7 / SEP-like promoter is operably linked to a nucleic acid encoding a guide RNA. In some embodiments, the ZMM7 / SEP-like promoter is operably linked to a nucleic acid encoding a single guide RNA.

[0127] In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1 to 30, or a promoter selected from the group consisting of SEQ ID NOs: 1 to 16, 18 to 19, 21 to 30, and 45 to 49, or a functional fragment thereof, is operably linked to a nucleic acid encoding an inducible nuclease. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1 to 30, or a promoter selected from the group consisting of SEQ ID NOs: 1 to 16, 18 to 19, 21 to 30, and 45 to 49, or a functional fragment thereof, is operably linked to a nucleic acid encoding a Cas12a nuclease. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1 to 30, or a promoter selected from the group consisting of SEQ ID NOs: 1 to 16, 18 to 19, 21 to 30, and 45 to 49, or a functional fragment thereof, is operably linked to a nucleic acid encoding a CasX nuclease. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1-30, or a promoter selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof, is operably linked to a nucleic acid encoding MAD7 (registered trademark) nuclease. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1-30, or a promoter selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof, is operably linked to a nucleic acid encoding a guide nucleic acid. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1-30, or a promoter selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof, is operably linked to a nucleic acid encoding a guide RNA. In one aspect, a promoter selected from the group consisting of SEQ ID NOs: 1-30, or a promoter selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment thereof, is operably linked to a nucleic acid encoding a single guide RNA.

[0128] As used herein, "floral tissue-preferred promoter" refers to a promoter that exhibits higher or preferential expression in floral tissues compared to other cell or tissue types of a plant. A floral tissue-preferred promoter may exhibit expression in any floral tissue and neighboring cells or tissues, such as, but not limited to, stem cells, vascular cells, and trichrome cells. A floral tissue-preferred promoter may also exhibit expression in other plant tissues or cells, such as, but not limited to, root cells, egg cells, endosperm cells, cotyledon cells, seed coat cells, leaf cells, vascular cells, embryo cells, and shoot apical meristem cells.

[0129] As used herein, a "floral tissue specific promoter" refers to a promoter that exhibits expression only in floral tissue. In some embodiments, a floral tissue preferential promoter comprises a floral tissue specific promoter.

[0130] As used herein, a "flower cell-preferred promoter" refers to a promoter that exhibits higher or preferential expression in flower cells compared to other cell or tissue types of a plant. A flower cell-preferred promoter may exhibit expression in any flower cell and neighboring cells or tissues, such as, but not limited to, stem cells, vascular cells, and trichrome cells. A flower cell-preferred promoter may also exhibit expression in other plant tissues or cells, such as, but not limited to, root cells, egg cells, endosperm cells, cotyledon cells, seed coat cells, leaf cells, vascular cells, embryo cells, and shoot apical meristem cells.

[0131] As used herein, a "flower cell specific promoter" refers to a promoter that exhibits expression only in flower cells. In some aspects, a flower cell preferential promoter comprises a flower cell specific promoter.

[0132] It is understood in the art that a fragment of a promoter sequence can function to drive transcription of an operably linked nucleic acid molecule. For example, but not limited to, if a 1000 nucleotide promoter is truncated to 500 nucleotides and the 500 nucleotide fragment can drive transcription, the 500 nucleotide fragment is referred to as a "functional fragment". It is understood that the promoter may be a variant. As used herein, the term "variant" refers to a second DNA molecule, such as a regulatory element, that has a similar but not identical composition to a first DNA molecule, where the second DNA molecule still maintains the overall functionality, i.e., the same or similar expression pattern, for example, by a more or less equivalent transcription activity of the first DNA molecule. A variant may be a shorter, longer, or truncated version of the first DNA molecule, or an altered version of the sequence of the first DNA molecule, such as one with a different restriction enzyme site and / or internal deletion, substitution, or insertion. "Variant" may also encompass a regulatory element having a nucleotide sequence that contains one or more nucleotide substitutions, deletions, or insertions of a reference sequence, where the derivative regulatory element has greater, lesser, or equivalent transcriptional or translational activity than the corresponding parent regulatory molecule.

[0133] A promoter that drives expression in all or many tissues of a plant is referred to as a "constitutive" promoter. A promoter that drives expression during a specific period or stage of development is referred to as a "developmental" promoter. An "inducible" promoter is a promoter that initiates transcription in response to an environmental stimulus such as heat, cold, drought, light, or other stimuli such as wounding or application of a chemical. Promoters can also be classified in terms of their origin, such as being heterologous, homologous, chimeric, synthetic, etc.

[0134] As used herein, the term "heterologous" in reference to a promoter is a promoter sequence that has a different origin to its associated transcribable DNA sequence, coding sequence or gene (or transgene) and / or does not naturally occur in the plant to be transformed. The term "heterologous" may refer more broadly to a combination of two or more DNA molecules or sequences, such as a promoter and an associated transcribable DNA sequence, coding sequence or gene, where such a combination is man-made and not normally found in nature.

[0135] In some aspects, the promoters provided herein are constitutive promoters. In yet other aspects, the promoters provided herein are inducible promoters. In some aspects, the promoters provided herein are selected from the group consisting of constitutive promoters, tissue-specific promoters, tissue-preferred promoters, and inducible promoters.

[0136] RNA polymerase III (PolIII) promoters can be used to drive expression of non-protein-coding RNA molecules. In one aspect, the promoters provided herein are PolIII promoters. In another aspect, the PolIII promoters provided herein are operably linked to a nucleic acid molecule encoding a non-protein-coding RNA. In yet another aspect, the PolIII promoters provided herein are operably linked to a nucleic acid molecule encoding a guide nucleic acid. In yet another aspect, the PolIII promoters provided herein are operably linked to a nucleic acid molecule encoding a single guide RNA. In a further aspect, the PolIII promoters provided herein are operably linked to a nucleic acid molecule encoding a CRISPR RNA (crRNA). In another aspect, the PolIII promoters provided herein are operably linked to a nucleic acid molecule encoding a tracer RNA (tracrRNA).

[0137] Non-limiting examples of PolIII promoters include U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. See, for example, Schramm and Hernandez, 2002, Genes & Development, 16:2593-2620, which is incorporated herein by reference in its entirety. In one aspect, the PolIII promoter provided herein is selected from the group consisting of U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another aspect, the guide RNA provided herein is operably linked to a promoter selected from the group consisting of U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another aspect, the single guide RNA provided herein is operably linked to a promoter selected from the group consisting of U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another aspect, the CRISPR RNA provided herein is operably linked to a promoter selected from the group consisting of U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another aspect, the tracer RNA provided herein is operably linked to a promoter selected from the group consisting of U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter.

[0138] In one aspect, the promoter provided herein is a Dahlia Mosaic Virus (DaMV) promoter. In another aspect, the promoter provided herein is a U6 promoter. In another aspect, the promoter provided herein is an actin promoter. In one aspect, the promoter provided herein is a Cauliflower Mosaic Virus (CaMV) 35S promoter. In one aspect, the promoter provided herein is a ubiquitin promoter.

[0139] In one embodiment, the constitutive promoter is selected from the group consisting of a CaMV 35S promoter, an actin promoter, and a ubiquitin promoter.

[0140] Examples describing promoters that can be used herein include, but are not limited to, U.S. Pat. No. 6,437,217 (maize RS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter), U.S. Pat. No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362 (maize PR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat. No. 6,177,611 (constitutive maize promoters), U.S. Pat. Nos. 5,322,938, 5,352,605, 5,359,142, and 5,530,196 (35S promoter), U.S. Pat. No. 6,433,252 (maize L3 oleosin promoter), U.S. Pat. No. 6,429,357 (rice actin 2 promoter and rice actin 2 intron), U.S. Pat. No. 5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714 (light inducible promoter), U.S. Pat. No. 6,140,078 (salt inducible promoter), U.S. Pat. No. 6,252,138 (pathogen inducible promoter), U.S. Pat. No. 6,175,060 (phosphorus deficiency inducible promoter), U.S. Pat. No. 6,635,806 (gamma-coixin promoter), and U.S. Patent Application Serial No. 09 / 757,089 (maize chloroplast aldolase promoter).Further promoters which may be useful are the nopaline synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (carried on the tumor-inducing plasmid of Agrobacterium tumefaciens), caulimovirus promoters, e.g. the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Molecular Biology (1987) 9: 315-324), the CaMV 35S promoter (Odell et al., Nature (1985) 313: 810-812), the Scrophulariac mosaic virus 35S-promoter (U.S. Pat. Nos. 6,051,753; 5,378,619), the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA (1990) 87: 4144-4148), the R gene complex promoter (Chandler et al., Plant Cell (1989) 1: 1175-1183), and the chlorophyll a / b binding protein gene promoter, PC1SV (U.S. Pat. No. 5,850,019), and the AGRtu.nos (GenBank Accession No. V00087; Depicker et al., Journal of Molecular and Applied Genetics (1982) 1: 561-573; Bevan et al., 1983) promoter.

[0141] Promoter hybrids can also be used and constructed to enhance transcriptional activity (see U.S. Pat. No. 5,106,739) or combine desired transcriptional activity, inducibility and tissue or developmental specificity. Promoters that function in plants include, but are not limited to, promoters that are inducible, viral, synthetic, constitutive, temporally regulated, spatially regulated, and spatial-temporal regulated. Other promoters that are tissue-enhanced, tissue-specific, or developmentally regulated are also known in the art and are envisioned to be useful in the practice of the present disclosure.

[0142] In some embodiments, the constitutive promoter is operably linked to a nucleic acid sequence encoding an inducible nuclease. In some embodiments, the constitutive promoter is operably linked to a nucleic acid sequence encoding a Cas12a nuclease. In some embodiments, the constitutive promoter is operably linked to a nucleic acid sequence encoding a CasX nuclease. In some embodiments, the constitutive promoter is operably linked to a nucleic acid sequence encoding a MAD7® nuclease. In some embodiments, the constitutive promoter is operably linked to a nucleic acid sequence encoding a guide nucleic acid. In some embodiments, the constitutive promoter is operably linked to a nucleic acid sequence encoding a guide RNA. In some embodiments, the constitutive promoter is operably linked to a nucleic acid sequence encoding a single guide RNA.

[0143] In some embodiments, the inducible promoter is operably linked to a nucleic acid sequence encoding an inducible nuclease. In some embodiments, the inducible promoter is operably linked to a nucleic acid sequence encoding a Cas12a nuclease. In some embodiments, the inducible promoter is operably linked to a nucleic acid sequence encoding a CasX nuclease. In some embodiments, the inducible promoter is operably linked to a nucleic acid sequence encoding a MAD7® nuclease. In some embodiments, the inducible promoter is operably linked to a nucleic acid sequence encoding a guide nucleic acid. In some embodiments, the inducible promoter is operably linked to a nucleic acid sequence encoding a guide RNA. In some embodiments, the inducible promoter is operably linked to a nucleic acid sequence encoding a single guide RNA.

[0144] In some embodiments, the developmental promoter is operably linked to a nucleic acid sequence encoding an inducible nuclease. In some embodiments, the developmental promoter is operably linked to a nucleic acid sequence encoding a Cas12a nuclease. In some embodiments, the developmental promoter is operably linked to a nucleic acid sequence encoding a CasX nuclease. In some embodiments, the developmental promoter is operably linked to a nucleic acid sequence encoding a MAD7® nuclease. In some embodiments, the developmental promoter is operably linked to a nucleic acid sequence encoding a guide nucleic acid. In some embodiments, the developmental promoter is operably linked to a nucleic acid sequence encoding a guide RNA. In some embodiments, the developmental promoter is operably linked to a nucleic acid sequence encoding a single guide RNA.

[0145] Transcription activator-like effector (TALE) Transcription Activator-Like Effectors (TALEs) are transcription factors that contain a C-terminal activation domain and can activate / increase expression of an operably linked transcribable polynucleotide once the TALE binds to a TALE binding site at or near the promoter. Without being limited by any theory, it has previously been shown that TALE proteins can induce high expression of genes operably linked to TALE binding sites and can modulate expression depending on how many TALE binding sites are present in the regulatory region. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1-30, or a promoter selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, and 45-49, or a functional fragment or variant thereof, is operably linked to a nucleic acid encoding a TALE.

[0146] In some embodiments, the TALE binding site is operably linked to the promoter. In some embodiments, at least two TALE binding sites are operably linked to the promoter. In some embodiments, at least three TALE binding sites are operably linked to the promoter. In some embodiments, at least four TALE binding sites are operably linked to the promoter. In some embodiments, at least five TALE binding sites are operably linked to the promoter. In some embodiments, at least six TALE binding sites are operably linked to the promoter. In some embodiments, at least seven TALE binding sites are operably linked to the promoter. In some embodiments, at least eight TALE binding sites are operably linked to the promoter. In some embodiments, at least nine TALE binding sites are operably linked to the promoter. In some embodiments, at least ten TALE binding sites are operably linked to the promoter.

[0147] Inducible nuclease An inducible nuclease is a nuclease that forms a complex (e.g., a ribonucleoprotein) with a guide nucleic acid molecule (e.g., a guide RNA) and then guides the complex to a target site within a target sequence. One non-limiting example of an inducible nuclease is a CRISPR nuclease.

[0148] CRISPR (clustered regularly interspaced short palindromic repeats) nucleases (e.g., Cas9, CasX, Cas12a (also called Cpf1), CasY, MAD7®) are proteins found in bacteria that are guided by a guide RNA ("gRNA") to a target nucleic acid molecule, and the endonuclease can then cleave one or both strands of the target nucleic acid molecule. Although CRISPR nucleases originate from bacteria, many CRISPR nucleases have been shown to function in eukaryotic cells.

[0149] Without being limited by any particular scientific theory, CRISPR nuclease forms a complex with a guide RNA (gRNA) that hybridizes with a complementary target site, thereby guiding CRISPR nuclease to the target site. In class II CRISPR-Cas systems, a CRISPR array containing a spacer is transcribed during an encounter with a recognized invading DNA and processed into a small interfering CRISPR RNA (crRNA). The crRNA contains a repeat sequence and a spacer sequence that is complementary to a specific protospacer sequence in the invading pathogen. The spacer sequence can be designed to be complementary to a target sequence in a eukaryotic genome.

[0150] In its active form, CRISPR nuclease binds to its corresponding crRNA. Similar to the class II endonuclease Cas9, CasX requires another non-coding RNA component, termed trans-activating crRNA (tracrRNA), to have functional activity. The nucleic acid molecules provided herein can combine crRNA and tracrRNA into one nucleic acid molecule, termed herein "single guide RNA" (sgRNA). Cas12a or MAD7® is not required to guide tracrRNA to the target site; for Cas12a or MAD7®, crRNA alone is sufficient. The gRNA guides the active CRISPR nuclease complex to the target site, where the CRISPR nuclease can cleave the target site.

[0151] When the RNA-guided CRISPR nuclease and the guide RNA form a complex, the entire system is called a "ribonucleoprotein." The ribonucleoproteins provided herein may also include additional nucleic acids or proteins.

[0152] In some embodiments, the inducible nuclease and guide nucleic acid form a ribonucleoprotein in a flower cell. In other embodiments, the inducible nuclease and guide nucleic acid form a ribonucleoprotein in a flower tissue. In some embodiments, the Cas12a nuclease and guide nucleic acid form a ribonucleoprotein in a flower cell. In some embodiments, the Cas12a nuclease and guide nucleic acid form a ribonucleoprotein in a flower tissue. In some embodiments, the CasX nuclease and guide nucleic acid form a ribonucleoprotein in a flower cell. In other embodiments, the CasX nuclease and guide nucleic acid form a ribonucleoprotein in a flower tissue. In some embodiments, the MAD7® nuclease and guide nucleic acid form a ribonucleoprotein in a flower cell. In other embodiments, the MAD7® nuclease and guide nucleic acid form a ribonucleoprotein in a flower tissue. In some embodiments, the inducible nuclease and guide RNA form a ribonucleoprotein in a flower cell. In another embodiment, the inducible nuclease and the guide RNA form a ribonucleoprotein in the flower tissue. In an embodiment, the Cas12a nuclease and the guide RNA form a ribonucleoprotein in the flower cell. In another embodiment, the CasX nuclease and the guide RNA form a ribonucleoprotein in the flower tissue. In an embodiment, the inducible nuclease and the single guide RNA form a ribonucleoprotein in the flower cell. In another embodiment, the inducible nuclease and the single guide RNA form a ribonucleoprotein in the flower tissue. In another embodiment, the CasX nuclease and the single guide RNA form a ribonucleoprotein in the flower tissue. In another embodiment, the MAD7® nuclease and the single guide RNA form a ribonucleoprotein in the flower tissue.

[0153] In some embodiments, the ribonucleoprotein generates at least one double-stranded break within the target site in a flower cell. In some embodiments, the ribonucleoprotein generates at least one double-stranded break within the target site in a flower tissue. In some embodiments, the ribonucleoprotein generates at least one single-stranded break within the target site in a flower cell. In some embodiments, the ribonucleoprotein generates at least one single-stranded break within the target site in a flower tissue.

[0154] A prerequisite for cleavage of a 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 from the PAM site (e.g., between 18 and 23 nucleotides for Cas12a). The PAM site is only required for type I and type II CRISPR-associated proteins, and different CRISPR endonucleases recognize different PAM sites. Without limitation, 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 the T-rich PAM sequence YTTN and is believed to prefer TTTN over CTTN PAM (where T is thymine, C is cytosine, A is adenine, Y is thymine or cytosine, and N is thymine, cytosine, guanine, or adenine).

[0155] Cas12a is an RNA-guided nuclease of the class II type V CRISPR / Cas system. Cas12a nuclease generates staggered cuts when cleaving double-stranded DNA molecules. Staggered cuts in double-stranded DNA result in single-stranded DNA overhangs of at least one nucleotide. This is in contrast to blunt-end cuts (such as those generated by Cas9), which do not result in single-stranded DNA overhangs when cleaving double-stranded DNA.

[0156] In one aspect, the Cas12a nuclease provided herein is a Lachnospiraceae bacterium Cas12a (LbCas12a) nuclease. In another aspect, the Cas12a nuclease provided herein is a Francisella novicida Cas12a (FnCas12a) nuclease. In one aspect, the Cas12a nuclease is selected from the group consisting of LbCas12a and FnCas12a.

[0157] In some embodiments, the Cas12a nuclease, or a nucleic acid encoding a Cas12a nuclease, is selected from the group consisting of Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, Azospirillum, and the like. llum, Sphaerochaeta, Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridiar idium, Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfovibrio ibrio, Desulfonatronum, Opitutaceae, Tuberibacillus, Bacillus, Brevibacilus, Methylobacterium, Acidaminococcus, Peregrinibacteria, Butyrivibrio, Parcubacteria,It is derived from a bacterial genus selected from the group consisting of Smithella, Candidatus, Moraxella, and Leptospira.

[0158] In one embodiment, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 80% identical to a polynucleotide selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:36. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 85% identical to a polynucleotide selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:36. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 90% identical to a polynucleotide selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:36. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 95% identical to a polynucleotide selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:36. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 96% identical to a polynucleotide selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:36. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 97% identical to a polynucleotide selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:36. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 98% identical to a polynucleotide selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:36. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 99% identical to a polynucleotide selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:36. In another aspect, the Cas12a nuclease is encoded by a polynucleotide comprising a sequence at least 90% identical to a polynucleotide selected from the group consisting of SEQ ID NO:32 and SEQ ID NO:36.

[0159] CasX is a type of class II CRISPR-Cas nuclease identified in the bacterial phyla Deltaproteobacteria and Planctomycetes. Similar to Cas12a, CasX nuclease generates staggered cuts when cleaving double-stranded DNA molecules. However, unlike Cas12a, CasX nuclease requires a crRNA and a tracrRNA, or a single guide RNA, to target and cleave the target nucleic acid.

[0160] In one aspect, the CasX nuclease provided herein is a CasX nuclease from the phylum Deltaproteobacteria. In another aspect, the CasX nuclease provided herein is a CasX nuclease from the phylum Planctomycetes. Additional suitable CasX nucleases include, but are not limited to, those described in WO 2019 / 084148, the entire contents of which are incorporated herein by reference.

[0161] MAD7® (also known as ErCas12a) is an engineered nuclease of the class 2V-A type CRISPR-Cas (Cas12a / Cpf1) family with low levels of homology to the canonical Cas12a nuclease. MAD7® nuclease generates staggered cuts when cleaving double-stranded DNA molecules. MAD7® nuclease was first identified in Eubacterium rectale. It requires only the canonical Cas12a as crRNA. The nucleotide sequence encoding ErCas12a / MAD7® can be found in the supplemental data (Sequence S1) provided in Lin et al., Journal of Genetics and Genomics, 48:444-451 (2021).

[0162] In some embodiments, the inducible nuclease capable of generating staggered cuts in double stranded DNA molecules is selected from the group consisting of Cas12a; MAD7® and CasX. In some embodiments, the inducible nuclease is selected from the group consisting of Cas12a, MAD7® and CasX.

[0163] In one embodiment, the inducible nuclease is an RNA-guided nuclease. In another embodiment, the inducible nuclease is a CRISPR nuclease. In another embodiment, the inducible nuclease is a Cas12a nuclease. In another embodiment, the inducible nuclease is a CasX nuclease. In another embodiment, the inducible nuclease is a MAD7® nuclease.

[0164] As used herein, "nuclear localization signal" (NLS) refers to an amino acid sequence that "tags" a protein for transport to the nucleus of a cell. In one embodiment, the nucleic acid molecule provided herein encodes a nuclear localization signal. In another embodiment, the nucleic acid molecule provided herein encodes two or more nuclear localization signals.

[0165] In some embodiments, the Cas12a nucleases provided herein comprise a nuclear localization signal. In some embodiments, the nuclear localization signal is located at the N-terminus of the Cas12a nuclease. In further embodiments, the nuclear localization signal is located at the C-terminus of the Cas12a nuclease. In yet other embodiments, the nuclear localization signal is located at both the N-terminus and the C-terminus of the Cas12a nuclease.

[0166] In some embodiments, the CasX nucleases provided herein comprise a nuclear localization signal. In some embodiments, the nuclear localization signal is located at the N-terminus of the CasX nuclease. In further embodiments, the nuclear localization signal is located at the C-terminus of the CasX nuclease. In yet other embodiments, the nuclear localization signal is located on both the N-terminus and the C-terminus of the CasX nuclease.

[0167] In certain aspects, the MAD7® nucleases provided herein comprise a nuclear localization signal. In certain aspects, the nuclear localization signal is located on the N-terminus of the MAD7® nuclease. In further aspects, the nuclear localization signal is located on the C-terminus of the MAD7® nuclease. In yet other aspects, the nuclear localization signal is located on both the N-terminus and the C-terminus of the MAD7® nuclease.

[0168] In one embodiment, the ribonucleoprotein comprises at least one nuclear localization signal, hi another embodiment, the ribonucleoprotein comprises at least two nuclear localization signals.

[0169] In certain aspects, the nuclear localization signal provided herein is encoded by SEQ ID NO:33 or 34.

[0170] Various species show a particular bias towards certain codons for certain amino acids. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA), which in turn is believed to depend, among other things, on the properties of the codon being translated and on the availability of certain transfer RNA (tRNA) molecules. The dominance of selected tRNAs in a cell is generally a reflection of the codons most frequently used in peptide synthesis. Thus, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, in the "Codon Usage Database" available at www(dot)kazusa(dot)or(dot)jp(forwards slash)codon, and these tables can be adapted in several ways. See Nakamura et al., 2000, Nucl. Acids Res. 28:292. Computer algorithms are also available for codon-optimizing a particular sequence for expression in a particular plant cell, such as Gene Forge (Aptagen; Jacobus, Pa.).

[0171] As used herein, "codon optimization" refers to the 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 a codon that is more frequently or most frequently used in genes of the plant cell, while maintaining the original amino acid sequence (e.g., introducing silent mutations).

[0172] In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all of the codons) in the sequence encoding the inducible nuclease correspond to the most frequently used codon for a particular amino acid. In another embodiment, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more, or all of the codons) in the sequence encoding the Cas12a nuclease or CasX nuclease or MAD7® nuclease correspond to the most frequently used codon for a particular amino acid. For codon usage in plants, see 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 its entirety.

[0173] In some embodiments, the nucleic acid molecule encodes an inducible nuclease that is codon-optimized for plants. In some embodiments, the nucleic acid molecule encodes a Cas12a nuclease that is codon-optimized for plants. In some embodiments, the nucleic acid molecule encodes a CasX nuclease that is codon-optimized for plants. In some embodiments, the nucleic acid molecule encodes a MAD7® nuclease that is codon-optimized for plants.

[0174] In another aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for a plant cell. In another aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for a monocotyledonous plant species. In another aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for a dicotyledonous plant species. In a further aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for a gymnosperm species. In a further aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for angiosperm species. In a further aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for a corn cell. In a further aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for a soybean cell. In a further aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for a rice cell. In a further aspect, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for wheat cells. In a further aspect, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for cotton cells. In a further aspect, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for sorghum cells. In a further aspect, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for alfalfa cells. In a further aspect, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for sugarcane cells. In a further aspect, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for Arabidopsis cells. In a further aspect, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for tomato cells. In a further aspect, the nucleic acid molecule provided herein encodes an inducible nuclease that is codon-optimized for cucumber cells.In a further aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for potato cells. In a further aspect, the nucleic acid molecules provided herein encode an inducible nuclease that is codon-optimized for onion cells.

[0175] In another aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for plant cells. In another aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for monocotyledonous plant species. In another aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for dicotyledonous plant species. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for gymnosperm species. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for angiosperm species. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for maize cells. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for soybean cells. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for rice cells. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for wheat cells. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for cotton cells. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for sorghum cells. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for alfalfa cells. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for sugarcane cells. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for Arabidopsis cells. In a further aspect, the nucleic acid molecules provided herein encode a Cas12a nuclease that is codon-optimized for tomato cells.In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for a cucumber cell. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for a potato cell. In a further aspect, the nucleic acid molecule provided herein encodes a Cas12a nuclease that is codon-optimized for an onion cell. In another aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a plant cell. In another aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a monocotyledonous plant species. In another aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a dicotyledonous plant species. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a gymnosperm species. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for an angiosperm species. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a corn cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a soybean cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a rice cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a wheat cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a cotton cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a sorghum cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for an alfalfa cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a sugarcane cell.In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for an Arabidopsis thaliana cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a tomato cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a cucumber cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for a potato cell. In a further aspect, the nucleic acid molecule provided herein encodes a CasX nuclease that is codon-optimized for an onion cell. In another aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for a plant cell. In another aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for a monocotyledonous plant species. In another aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for a dicotyledonous plant species. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that has been codon optimized for a gymnosperm species. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that has been codon optimized for an angiosperm species. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that has been codon optimized for a corn cell. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that has been codon optimized for a soybean cell. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that has been codon optimized for a rice cell. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that has been codon optimized for a wheat cell. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that has been codon optimized for a cotton cell.In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for sorghum cells. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for alfalfa cells. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for sugarcane cells. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for Arabidopsis cells. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for tomato cells. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for cucumber cells. In a further aspect, the nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for potato cells. In a further aspect, a nucleic acid molecule provided herein encodes a MAD7® nuclease that is codon-optimized for onion cells.

[0176] Guide Nucleic Acid As used herein, a "guide nucleic acid" refers to a nucleic acid that, after forming a ribonucleoprotein (e.g., complex) with a guided nuclease (e.g., but not limited to, Cas12a, CasX, MAD7®), 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 a complementary sequence. In certain aspects, the ribonucleoproteins provided herein comprise at least one guide nucleic acid.

[0177] In some embodiments, the guide nucleic acid comprises DNA. In other embodiments, the guide nucleic acid comprises RNA. In some embodiments, the guide nucleic acid comprises DNA, RNA, or a combination thereof. In some embodiments, the guide nucleic acid is single stranded. In other embodiments, the guide nucleic acid is at least partially double stranded.

[0178] When the guide nucleic acid comprises RNA, it can also be referred to as a "guide RNA". In another embodiment, the guide nucleic acid comprises DNA and RNA. In another embodiment, the guide RNA is single-stranded. In another embodiment, the guide RNA is double-stranded. In a further embodiment, the guide RNA is partially double-stranded.

[0179] In some embodiments, the guide nucleic acid comprises a guide RNA. In other embodiments, the guide nucleic acid comprises at least one guide RNA. In other embodiments, the guide nucleic acid comprises at least two guide RNAs. In other embodiments, the guide nucleic acid comprises at least three guide RNAs. In other embodiments, the guide nucleic acid comprises at least five guide RNAs. In other embodiments, the guide nucleic acid comprises at least ten guide RNAs.

[0180] In another embodiment, the guide nucleic acid comprises at least 10 nucleotides. In another embodiment, the guide nucleic acid comprises at least 11 nucleotides. In another embodiment, the guide nucleic acid comprises at least 12 nucleotides. In another embodiment, the guide nucleic acid comprises at least 13 nucleotides. In another embodiment, the guide nucleic acid comprises at least 14 nucleotides. In another embodiment, the guide nucleic acid comprises at least 15 nucleotides. In another embodiment, the guide nucleic acid comprises at least 16 nucleotides. In another embodiment, the guide nucleic acid comprises at least 17 nucleotides. In another embodiment, the guide nucleic acid comprises at least 18 nucleotides. In another embodiment, the guide nucleic acid comprises at least 19 nucleotides. In another embodiment, the guide nucleic acid comprises at least 20 nucleotides. In another embodiment, the guide nucleic acid comprises at least 21 nucleotides. In another embodiment, the guide nucleic acid comprises at least 22 nucleotides. In another embodiment, the guide nucleic acid comprises at least 23 nucleotides. In another embodiment, the guide nucleic acid comprises at least 24 nucleotides. In another embodiment, the guide nucleic acid comprises at least 25 nucleotides. In another embodiment, the guide nucleic acid comprises at least 26 nucleotides. In another embodiment, the guide nucleic acid comprises at least 27 nucleotides. In another embodiment, the guide nucleic acid comprises at least 28 nucleotides. In another embodiment, the guide nucleic acid comprises at least 30 nucleotides. In another embodiment, the guide nucleic acid comprises at least 35 nucleotides. In another embodiment, the guide nucleic acid comprises at least 40 nucleotides. In another embodiment, the guide nucleic acid comprises at least 45 nucleotides. In another embodiment, the guide nucleic acid comprises at least 50 nucleotides.

[0181] In another embodiment, the guide nucleic acid comprises 10 to 50 nucleotides. In another embodiment, the guide nucleic acid comprises 10 to 40 nucleotides. In another embodiment, the guide nucleic acid comprises 10 to 30 nucleotides. In another embodiment, the guide nucleic acid comprises 10 to 20 nucleotides. In another embodiment, the guide nucleic acid comprises 16 to 28 nucleotides. In another embodiment, the guide nucleic acid comprises 16 to 25 nucleotides. In another embodiment, the guide nucleic acid comprises 16 to 20 nucleotides.

[0182] In certain embodiments, the guide nucleic acid comprises at least 70% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 75% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 80% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 85% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 90% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 91% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 92% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 93% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 94% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 95% sequence complementarity to the target site. In certain embodiments, the guide nucleic acid comprises at least 96% sequence complementarity to the target site. In some embodiments, the guide nucleic acid comprises at least 97% sequence complementarity to the target site. In some embodiments, the guide nucleic acid comprises at least 98% sequence complementarity to the target site. In some embodiments, the guide nucleic acid comprises at least 99% sequence complementarity to the target site. In some embodiments, the guide nucleic acid comprises 100% sequence complementarity to the target site. In other embodiments, the guide nucleic acid comprises 70%-100% sequence complementarity to the target site. In other embodiments, the guide nucleic acid comprises 80%-100% sequence complementarity to the target site. In other embodiments, the guide nucleic acid comprises 90%-100% sequence complementarity to the target site.

[0183] In some aspects, the guide nucleic acid is capable of hybridizing to the target site.

[0184] As mentioned above, some inducible nucleases, such as CasX and Cas9, require another non-coding RNA component, termed trans-activating crRNA (tracrRNA), to have functional activity. The guide nucleic acid molecules provided herein can combine crRNA and tracrRNA into one nucleic acid molecule, termed herein "single guide RNA" (sgRNA). The gRNA guides the active CasX complex to a target site within the target sequence, where CasX can cleave the target site. In other embodiments, the crRNA and tracrRNA are provided as separate nucleic acid molecules.

[0185] In one embodiment, the guide nucleic acid comprises a crRNA. In another embodiment, the guide nucleic acid comprises a tracrRNA. In a further embodiment, the guide nucleic acid comprises a sgRNA.

[0186] target site As used herein, "target sequence" refers to a selected sequence or region of a DNA molecule where modification (e.g., cleavage, site-specific integration) is desired. The target sequence includes the target site.

[0187] As used herein, "target site" refers to the portion of the target sequence that is cleaved by a CRISPR nuclease. In contrast to non-target nucleic acid (e.g., non-target ssDNA) or non-target region, the target site contains significant complementarity to the guide nucleic acid or guide RNA.

[0188] In some embodiments, the target site is 100% complementary to the guide nucleic acid. In other embodiments, the target site is 99% complementary to the guide nucleic acid. In other embodiments, the target site is 98% complementary to the guide nucleic acid. In other embodiments, the target site is 97% complementary to the guide nucleic acid. In other embodiments, the target site is 96% complementary to the guide nucleic acid. In other embodiments, the target site is 95% complementary to the guide nucleic acid. In other embodiments, the target site is 94% complementary to the guide nucleic acid. In another embodiment, the target site is 93% complementary to the guide nucleic acid. In another embodiment, the target site is 92% complementary to the guide nucleic acid. In another embodiment, the target site is 91% complementary to the guide nucleic acid. In another embodiment, the target site is 90% complementary to the guide nucleic acid. In another embodiment, the target site is 85% complementary to the guide nucleic acid. In another embodiment, the target site is 80% complementary to the guide nucleic acid.

[0189] In some embodiments, the target site comprises at least one PAM site. In some embodiments, the target site is adjacent to a nucleic acid sequence comprising at least one PAM site. In other embodiments, the target site is within 5 nucleotides of at least one PAM site. In further embodiments, the target site is within 10 nucleotides of at least one PAM site. In other embodiments, the target site is within 15 nucleotides of at least one PAM site. In other embodiments, the target site is within 20 nucleotides of at least one PAM site. In other embodiments, the target site is within 25 nucleotides of at least one PAM site. In other embodiments, the target site is within 30 nucleotides of at least one PAM site.

[0190] In one embodiment, the target site is located within genomic DNA. In another embodiment, the target site is located within a gene. In another embodiment, the target site is located within a gene of interest. In another embodiment, the target site is located within an exon of the gene. In another embodiment, the target site is located within an intron of the gene. In another embodiment, the target site is located within a promoter of the gene. In another embodiment, the target site is located within the 5'-UTR of the gene. In another embodiment, the target site is located within the 3'-UTR of the gene. In another embodiment, the target site is located within intergenic DNA.

[0191] In one embodiment, the target DNA molecule is single stranded. In another embodiment, the target DNA molecule is double stranded.

[0192] In some aspects, the target sequence comprises genomic DNA. In some aspects, the target sequence is located in a nuclear genome. In some aspects, the target sequence comprises chromosomal DNA. In some aspects, the target sequence comprises plasmid DNA. In some aspects, the target sequence is located in a plasmid. In some aspects, the target sequence comprises mitochondrial DNA. In some aspects, the target sequence is located in a mitochondrial genome. In some aspects, the target sequence comprises plastid DNA. In some aspects, the target sequence is located in a plastid genome. In some aspects, the target sequence comprises chloroplast DNA. In some aspects, the target sequence is located in a chloroplast genome. In some aspects, the target sequence is located in a genome selected from the group consisting of a nuclear genome, a mitochondrial genome, and a plastid genome.

[0193] In one embodiment, the target sequence comprises genomic DNA. As used herein, "genomic DNA" refers to DNA that encodes one or more genes. In another embodiment, the target sequence comprises intergenic DNA. In contrast to genomic DNA, "intergenic DNA" comprises non-coding DNA and lacks DNA that encodes genes. In one embodiment, intergenic DNA is located between two genes.

[0194] In one aspect, the target sequence encodes a gene. As used herein, "gene" refers to a polynucleotide capable of producing a functional unit (such as, but not limited to, a protein, or a non-coding RNA molecule). A gene may include a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription 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" may include a polynucleotide sequence encoding a promoter, an enhancer sequence, a leader sequence, a transcription start site, a transcription 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 transcription start site, a transcription 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, an miRNA, a pre-miRNA, and an miRNA binding site.

[0195] Non-limiting examples of non-protein-coding RNA molecules include microRNA (miRNA), miRNA precursor (pre-miRNA), small interfering RNA (siRNA), small RNA (18-26 nucleotides long) and its encoding precursor, heterochromatic siRNA (hc-siRNA), Piwi-binding RNA (piRNA), hairpin double-stranded RNA (hairpin dsRNA), trans-acting siRNA (ta-siRNA), naturally occurring antisense siRNA (nat-siRNA), CRISPR RNA (crRNA), tracer RNA (tracrRNA), guide RNA (gRNA), and single guide RNA (sgRNA). In some embodiments, the non-protein-coding RNA molecule comprises a miRNA. In some embodiments, the non-protein-coding RNA molecule comprises a siRNA. In some embodiments, the non-protein-coding RNA molecule comprises a ta-siRNA. In some embodiments, the non-protein-coding RNA molecule is selected from the group consisting of a miRNA, an siRNA, and a ta-siRNA.

[0196] As used herein, "gene of interest" refers to a polynucleotide sequence that encodes a protein or a non-protein-coding RNA molecule to be integrated into a target sequence, or alternatively, an endogenous polynucleotide sequence that encodes a protein or a non-protein-coding RNA molecule to be edited by a ribonucleoprotein. In one embodiment, the gene of interest encodes a protein. In another embodiment, the gene of interest encodes a non-protein-coding RNA molecule. In one embodiment, the gene of interest is exogenous to the target DNA molecule. In one embodiment, the gene of interest replaces an endogenous gene in the target DNA molecule.

[0197] mutation In certain aspects, the ribonucleoprotein or methods provided herein generate at least one mutation in the target sequence.

[0198] In some aspects, seeds produced from plants provided herein contain at least one mutation in a gene of interest that includes a target site, compared to seeds from a control plant of the same line or variety that lacks a first nucleic acid sequence encoding an inducible 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 some aspects, seeds produced from plants provided herein contain at least one mutation in a gene of interest that includes a target site, compared to seeds from a control plant of the same line or variety that lacks a first nucleic acid sequence encoding an inducible 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.

[0199] In some aspects, seeds produced from plants provided herein contain at least one mutation in a gene of interest that includes a target site, as compared to seeds from a control plant of the same line or variety that lacks a first nucleic acid sequence encoding an inducible 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-preferential promoter. In some aspects, seeds produced from plants provided herein contain at least one mutation in a gene of interest that includes a target site, as compared to seeds from a control plant of the same line or variety that lacks a first nucleic acid sequence encoding an inducible 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-preferential promoter.

[0200] As used herein, "mutation" refers to a non-naturally occurring alteration to a nucleic acid or amino acid sequence compared to a naturally occurring reference nucleic acid or amino acid sequence derived from the same organism. When identifying a mutation, it will be understood that the reference sequence should be derived from the same nucleic acid (e.g., gene, non-coding RNA) or amino acid (e.g., protein). It will be understood in the art that in determining whether a difference between two sequences contains a mutation, a 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, a comparison should be made between an edited (e.g., mutated) sequence of the same organism and an endogenous, unedited (e.g., "wild type") sequence.

[0201] Several types of mutations are known in the art. In one aspect, the mutation includes an insertion. An "insertion" refers to the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid sequence, respectively, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, the mutation includes 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, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, the mutation includes 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, compared to an endogenous reference polynucleotide or amino acid sequence. In another aspect, the mutation includes an inversion. An "inversion" refers to when a segment of a polynucleotide or amino acid sequence is reversed from end to end. In one aspect, the mutations provided herein include a mutation selected from the group consisting of an insertion, a deletion, a substitution, and an inversion.

[0202] In one aspect, the plant or seed comprises at least one mutation in a gene of interest, wherein the at least one mutation results in the deletion of one or more amino acids from the protein encoded by the gene of interest compared to the wild-type protein.

[0203] In one aspect, the plant or seed comprises at least one mutation in a gene of interest, the at least one mutation resulting in the substitution of one or more amino acids in the protein encoded by the gene of interest compared to the wild-type protein.

[0204] In one aspect, the plant or seed comprises at least one mutation in a gene of interest, wherein the at least one mutation results in an insertion of one or more amino acids in the protein encoded by the gene of interest compared to the wild-type protein.

[0205] Mutations in the coding region of a gene (e.g., exon mutations) can result in a truncated protein or polypeptide when the mutated messenger RNA (mRNA) is translated into a protein or polypeptide. In certain aspects, the present disclosure provides mutations that result in truncation of a protein or polypeptide. As used herein, a "truncated" protein or polypeptide contains at least one less amino acid compared to an endogenous control protein or polypeptide. For example, if endogenous protein A contains 100 amino acids, then a truncated form of protein A may contain 1-99 amino acids.

[0206] Without being limited by any scientific theory, one way to cause truncation of a protein or polypeptide is by the introduction of a premature stop codon into the mRNA transcript of an endogenous gene. In certain aspects, the present disclosure provides a mutation that results in a premature stop codon in the mRNA transcript of an endogenous gene. As used herein, a "stop codon" refers to a nucleotide triplet in an mRNA transcript that signals the termination of protein translation. A "premature stop codon" refers to a stop codon that is located earlier (e.g., 5') than the normal stop codon position in an endogenous mRNA transcript. Several stop codons are known in the art, including, but not limited to, "UAG", "UAA", "UGA", "TAG", "TAA", and "TGA".

[0207] In one aspect, the seed or plant comprises at least one mutation, which results in the introduction of a premature stop codon in a messenger RNA encoded by the gene of interest compared to a wild-type messenger RNA.

[0208] In some embodiments, the mutations provided herein include null mutations. As used herein, "null mutation" refers to a mutation that causes a complete loss of function for the protein encoded by the gene containing the mutation, or alternatively, a complete loss of function for the small RNA encoded by the genomic locus. A null mutation can cause a lack of mRNA transcript production, a lack of small RNA transcript production, a lack of protein function, or a combination thereof.

[0209] The mutations provided herein may be located in any part of the endogenous gene. In one embodiment, the mutations provided herein are located in an exon of the endogenous gene. In another embodiment, the mutations provided herein are located in an intron of the endogenous gene. In a further embodiment, the mutations provided herein are located in the 5' untranslated region of the endogenous gene. In yet another embodiment, the mutations provided herein are located in the 3' untranslated region of the endogenous gene. In yet another embodiment, the mutations provided herein are located in the promoter of the endogenous gene.

[0210] In some embodiments, the mutation is located at a splice site in a gene. Mutations at splice sites can inhibit the splicing of exons during mRNA processing. Splicing can be perturbed if one or more nucleotides are inserted, deleted, or substituted at a splice site. Perturbed splicing can result in unspliced ​​introns, missing exons, or both, from the mature mRNA sequence. Typically, but not always, a "GU" sequence is required at the 5' end of an intron and an "AG" sequence is required at the 3' end of an intron for proper splicing. If any of these splice sites are mutated, splicing perturbation can occur.

[0211] In one embodiment, the seed or plant comprises at least one mutation, wherein the at least one mutation comprises a deletion of one or more splice sites from the gene of interest. In another embodiment, the seed or plant comprises at least one mutation, wherein the at least one mutation is located within one or more splice sites from the gene of interest.

[0212] In some embodiments, the mutation comprises site-specific integration, hi some embodiments, the site-specific integration comprises insertion of all or part of a desired sequence into a target sequence.

[0213] As used herein, "site-specific integration" refers to all or a portion of a desired sequence (e.g., an exogenous gene, an edited endogenous gene) being inserted or integrated into a desired site or locus (e.g., a target sequence) within a plant genome. As used herein, "desired sequence" refers to a DNA molecule comprising a nucleic acid sequence to be integrated into the genome of a plant or plant cell. The desired sequence may comprise a transgene or construct. In some embodiments, the nucleic acid molecule comprising the desired sequence comprises one or two homology arms flanking the desired sequence to facilitate targeted insertion events by homologous recombination and / or homology-directed repair.

[0214] In some aspects, the methods provided herein involve site-specific integration of a desired sequence into a target sequence.

[0215] Any site or locus within the genome of the plant can be selected for site-specific integration of the transgene or construct of the present disclosure. In one aspect, the target sequence is located within the B, or extra chromosome.

[0216] For site-specific integration, a double-strand break (DSB) or nick can first be created in the target sequence via an inducible nuclease or ribonucleoprotein provided herein. Then, in the presence of the desired sequence, the DSB or nick can be repaired by homologous recombination (HR) between the homology arms of the desired sequence and the target sequence, or by non-homologous end joining (NHEJ), resulting in site-specific integration of all or part of the desired sequence into the target sequence, creating a targeted insertion event at the site of the DSB or nick.

[0217] In one embodiment, the site-specific integration involves the use of NHEJ repair machinery endogenous to the cell, hi another embodiment, the site-specific integration involves the use of HR repair machinery endogenous to the cell.

[0218] In one embodiment, repair of the double-stranded break generates at least one mutation in the gene of interest compared to a control plant of the same line or variety.

[0219] In some embodiments, the mutation comprises the incorporation of at least 5 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of at least 10 contiguous nucleotides of the desired sequence molecule into the target sequence. In some embodiments, the mutation comprises the incorporation of at least 15 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of at least 20 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of at least 25 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of at least 50 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of at least 100 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of at least 250 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of at least 500 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of at least 1000 contiguous nucleotides of the desired sequence into the target sequence, hi some embodiments, the mutation comprises the incorporation of at least 2000 contiguous nucleotides of the desired sequence into the target sequence.

[0220] In some embodiments, the mutation comprises incorporation of 5 contiguous nucleotides to 3500 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises incorporation of 5 contiguous nucleotides to 2500 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises incorporation of 5 contiguous nucleotides to 1500 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises incorporation of 5 contiguous nucleotides to 750 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises incorporation of 5 contiguous nucleotides to 500 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises incorporation of 5 contiguous nucleotides to 250 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises incorporation of 5 contiguous nucleotides to 150 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of 25 contiguous nucleotides to 2500 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of 25 contiguous nucleotides to 1500 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of 25 contiguous nucleotides to 750 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of 50 contiguous nucleotides to 2500 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of 50 contiguous nucleotides to 1500 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of 50 contiguous nucleotides to 750 contiguous nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation comprises the incorporation of 100 contiguous nucleotides to 2500 contiguous nucleotides of the desired sequence into the target sequence. In one embodiment, the mutation involves the incorporation of between 100 and 1500 contiguous nucleotides of a desired sequence into the target sequence.In one embodiment, the mutation involves the incorporation of between 100 contiguous nucleotides and 750 contiguous nucleotides of a desired sequence into the target sequence.

[0221] In some embodiments, the methods provided herein further include detecting editing or mutations in the target sequence. Screening and selection of mutagenized or edited plants or plant cells may be by any method known to those skilled in the art. Examples of screening and selection methods include, but are not limited to, Southern analysis, PCR amplification for detection of polynucleotides, Northern blots, RNase protection, primer extension, RT-PCR amplification for detection of RNA transcripts, Sanger sequencing, next generation sequencing techniques (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting enzyme or ribozyme activity of polypeptides and polynucleotides, protein gel electrophoresis, Western blots, immunoprecipitation, and enzyme-linked immunoassays for detecting polypeptides. Other techniques such as in situ hybridization, enzyme staining, and immunostaining can also 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.

[0222] In some embodiments, the sequences provided herein encode at least one ribozyme. In some embodiments, the sequences provided herein encode at least two ribozymes. In some embodiments, the ribozyme is a self-cleaving ribozyme. Self-cleaving ribozymes are known in the art. See, for example, Jimenez et al., Trends Biochem. Sci., 40:648-661 (2015).

[0223] In some embodiments, the sequence encoding at least one guide nucleic acid is flanked by self-cleaving ribozymes. In some embodiments, the sequence encoding at least one guide nucleic acid is immediately adjacent to the sequence encoding the ribozyme (e.g., the 5'-most nucleotide of the guide nucleic acid is adjacent to the 3'-most nucleotide of the ribozyme, or the 3'-most nucleotide of the guide nucleic acid is adjacent to the 5'-most nucleotide of the ribozyme). In some embodiments, the sequence encoding at least one guide nucleic acid is 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 10,000 nucleotides away from the sequence encoding the ribozyme.

[0224] plant Any plant or plant cell can be used with the methods and compositions provided herein.

[0225] In some aspects, the plant is selected from the group consisting of a corn plant, a rice plant, a sorghum plant, a wheat plant, 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 some aspects, the plant is an angiosperm. In some aspects, the plant is a gymnosperm. In some aspects, the plant is a monocotyledonous plant. In some aspects, the plant is a dicotyledonous plant. In some embodiments, the plant is from the family Alliaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Brassicaceae, Caesalpiniaceae, Cucurbitaceae, Ericaceae, Fabaceae, Juglandaceae, Aster ... The plant is a plant of a family selected from the group consisting of Malvaceae, Mimosaceae, Moraceae, Musaceae, Orchidaceae, Papilionaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae, and Solanaceae.

[0226] In some aspects, the 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 some aspects, the plant cell is an angiosperm cell. In some aspects, the plant cell is a gymnosperm cell. In some aspects, the plant cell is a monocotyledonous plant cell. In some aspects, the plant cell is a dicotyledonous plant cell. In one aspect, the plant cell is a plant cell of a family selected from the group consisting of Liliaceae, Anacardiaceae, Apiaceae, Palmaceae, Asteraceae, Brassicaceae, Caesalpinaceae, Cucurbitaceae, Ericaceae, Fabaceae, Juglandaceae, Malvaceae, Mimosa, Mulberry, Musaceae, Orchidaceae, Fabaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae, and Solanaceae.

[0227] As used herein, "variety" refers to a group of plants within a species (including but not limited to corn (Zea mays)) that share certain genetic traits that separate them from other possible varieties within that species. Varieties may be inbred or hybrids, although commercially available plants are often hybrids to take advantage of hybrid vigor. Individuals within a hybrid cultivar are homogeneous and nearly genetically identical, with many loci in the heterozygous state.

[0228] As used herein, the term "inbred" refers to a line bred for genetic uniformity. In some aspects, the seeds provided herein are inbred seeds. In some aspects, the plants provided herein are inbred plants.

[0229] As used herein, the term "cross" refers to the offspring of cross between at least two genetically dissimilar parents. Non-limiting examples of crossing schemes include single cross, modified single cross, double modified single cross, three-way cross, modified three-way cross, and double cross, where at least one parent in the modified cross is the offspring of cross between sister lines. In some aspects, the seed provided herein is a cross seed. In some aspects, the plant provided herein is a cross plant.

[0230] Transformation The methods may include transient transformation or stable integration of any of the nucleic acid molecules provided herein into any plant or plant cell.

[0231] As used herein, "stable integration" or "stably integrated" refers to the transfer of DNA into the genomic DNA of a target cell or plant that allows the target cell or plant to pass on the transferred DNA to the next generation of the transformed organism. Stable transformation requires the integration of the transferred DNA into the germ cells of the transformed organism. As used herein, "transiently transformed" or "transient transformation" refers to the transfer of DNA into a cell that is not transferred to the next generation of the transformed organism. In transient transformation, the transformed DNA is typically not integrated into the genomic DNA of the transformed cell. In one aspect, the method stably transforms a plant cell or plant with one or more nucleic acid molecules provided herein. In another aspect, the method transiently transforms a plant cell or plant with one or more nucleic acid molecules provided herein.

[0232] In some embodiments, the nucleic acid molecule encoding an inducible nuclease is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding a Cas12a nuclease is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding a CasX nuclease is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding a MAD7® nuclease is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding a guide nucleic acid is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding a guide RNA is stably integrated into the genome of the plant. In some embodiments, the nucleic acid molecule encoding a single guide RNA is stably integrated into the genome of the plant.

[0233] Several methods for transforming cells with recombinant nucleic acid molecules or constructs are known in the art and can be used in accordance with the methods of the present application. Any suitable method or technique for transforming cells known in the art can be used in accordance with the methods of the present invention. Effective methods for plant transformation include bacteria-mediated transformation, such as Agrobacterium- or Rhizobium-mediated transformation, and microprojectile bombardment-mediated transformation. Various methods are known in the art for transforming explants with transformation vectors by bacteria-mediated transformation or microprojectile bombardment, and then culturing or otherwise culturing these explants to regenerate or generate transgenic plants.

[0234] In some embodiments, the method comprises providing the nucleic acid molecule to the cell by Agrobacterium-mediated transformation. In some embodiments, the method comprises providing the nucleic acid molecule to the cell by polyethylene glycol-mediated transformation. In some embodiments, the method comprises providing the nucleic acid molecule to the cell by biolistic transformation. In some embodiments, the method comprises providing the nucleic acid molecule to the cell by liposome-mediated transfection. In some embodiments, the method comprises providing the nucleic acid molecule to the cell by viral transduction. In some embodiments, the method comprises providing the nucleic acid molecule to the cell by use of one or more delivery particles. In some embodiments, the method comprises providing the nucleic acid molecule to the cell by microinjection. In some embodiments, the method comprises providing the nucleic acid molecule to the cell by electroporation.

[0235] In one aspect, the nucleic acid molecule is provided to the cell by a method selected from the group consisting of Agrobacterium-mediated transformation, polyethylene glycol-mediated transformation, biolistic transformation, liposome-mediated transfection, viral transduction, use of one or more delivery particles, microinjection, and electroporation.

[0236] Other methods for transformation, such as vacuum infiltration, pressure, sonication, and silicon carbide fiber agitation, are also known in the art and are contemplated for use with any of the methods provided herein.

[0237] Methods for transforming cells are well known to those skilled in the art.For example, specific instructions for transforming plant cells by microprojectile bombardment (e.g., biolistic transformation) using particles coated with recombinant DNA can be 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 can be found 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. Further methods for transforming plants can be found, for example, in the Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Any suitable method known to those of skill in the art can be used to transform plant cells with any of the nucleic acid molecules provided herein.

[0238] Lipofection is described, for example, in U.S. Patent Nos. 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam™ and Lipofectin™). Cationic and neutral lipids that are suitable for efficient receptor-recognition lipofection of polynucleotides include those described in Felgner, WO 91 / 17424; WO 91 / 16024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or to target tissues (e.g., in vivo administration).

[0239] 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 WO2014 / 093622. In an embodiment, the method of providing a nucleic acid molecule or protein to a cell comprises delivery by a delivery particle. In an embodiment, the method of providing a nucleic acid molecule to a plant cell or plant comprises delivery by a delivery vesicle. In an embodiment, the delivery vesicle is selected from the group consisting of an exosome and a liposome. In an embodiment, the method of providing a nucleic acid molecule to a plant cell or plant comprises delivery by a viral vector. In an embodiment, the viral vector is selected from the group consisting of an adenoviral vector, a lentiviral vector, and an adeno-associated viral vector. In another embodiment, the method of providing a nucleic acid molecule to a plant cell or plant comprises delivery by a nanoparticle. In an embodiment, the method of providing a nucleic acid molecule to a plant cell or plant comprises microinjection. In an embodiment, the method of providing a nucleic acid molecule to a plant cell or plant comprises a polycation. In an embodiment, the method of providing a nucleic acid molecule to a plant cell or plant comprises a cationic oligopeptide.

[0240] In some embodiments, the delivery particle is selected from the group consisting of exosomes, adenoviral vectors, lentiviral vectors, adeno-associated viral vectors, nanoparticles, polycations, and cationic oligopeptides. In some embodiments, the methods provided herein include the use of one or more delivery particles. In other embodiments, the methods provided herein include the use of two or more delivery particles. In other embodiments, the methods provided herein include the use of three or more delivery particles.

[0241] Suitable agents for facilitating the transfer of nucleic acid into plant cells include agents that increase the permeability of the plant exterior or agents that increase the permeability of plant cells to oligonucleotides or polynucleotides.Such agents for facilitating the transfer of compositions into plant cells include chemical agents, or physical agents, or combinations thereof.Chemical agents for conditioning include (a) detergents, (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.

[0242] Organic solvents useful for conditioning plants to be permeabilized with polynucleotides include DMSO, DMF, pyridine, N-pyrrolidine, hexamethylphosphoramide, acetonitrile, dioxane, polypropylene glycol, and other solvents that are miscible with water or dissolve phosphonucleotides in non-aqueous systems (such as those used in synthesis reactions). Naturally derived or synthetic oils with or without surfactants or emulsifiers can be used, such as oils of plant origin, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, publicly available online at www(dot)herbicide(dot)adjuvants(dot)com), such as paraffin oil, polyol fatty acid esters, or oils with short chain molecules modified with amides or polyamines, such as polyethyleneimine or N-pyrrolidine.

[0243] Examples of useful surfactants include sodium or lithium salts of fatty acids (such as tallow or tallow amines or phospholipids) and organosilicone surfactants. Other useful surfactants include nonionic organosilicone surfactants, such as organosilicone surfactants including trisiloxane ethoxylate surfactants or silicone polyether copolymers, such as copolymers of polyalkylene oxide modified heptamethyltrisiloxane and allyloxy polypropylene glycol methyl ether (commercially available as Silwet® L-77).

[0244] Useful physical agents may include (a) abrasives such as carborundum, corundum, sand, calcite, pumice, garnet, etc., (b) nanoparticles such as carbon nanotubes, or (c) physical forces. 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 include heating, cooling, application of positive pressure, or sonication. Method embodiments may include incubation steps, neutralization steps (e.g., to neutralize acids, bases, or oxidizing agents, or to inactivate enzymes), washing steps, or combinations thereof, as needed. The methods of the invention may further include the application of other agents that may have an enhanced effect due to the silencing of certain genes. For example, if a polynucleotide is designed to regulate a gene that provides herbicide resistance, the subsequent application of the herbicide may have a dramatic effect on the efficacy of the herbicide.

[0245] Agents for laboratory conditioning of plant cells for penetration with polynucleotides include, for example, application of chemical agents, enzyme treatment, heating or cooling, treatment with positive or negative pressure, or ultrasonic treatment. Agents for conditioning plants in the field include chemical agents such as surfactants and salts.

[0246] In one aspect, the cell to be transformed or transfected is a plant cell. Recipient plant cells or explant targets for transformation include, but are not limited to, seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristematic cells, embryo cells, endosperm cells, root cells, shoot cells, stem cells, sheath cells, flower cells, inflorescence cells, stalk cells, pedicel cells, style cells, stigma cells, receptacle cells, petal cells, sepal cells, pollen cells, anther cells, filament cells, ovary cells, ovule cells, pericarp cells, phloem cells, bud cells, or vascular tissue cells. In another aspect, the present disclosure provides a plant chloroplast. In a further aspect, the present 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, the present disclosure provides a protoplast. In another aspect, the present disclosure provides a plant callus cell. Any cell capable of regenerating a fertile plant is contemplated as a useful recipient cell for the practice of the present disclosure. Callus can be initiated from a variety of tissue sources, including, but not limited to, immature embryos or embryo parts, apical meristems of seedlings, microspores, and the like. These cells capable of growing as callus can serve as recipient cells for transformation. Practical transformation methods and materials (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) for producing the transgenic plants of the present disclosure are disclosed, for example, in U.S. Patent Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication No. 2004 / 0216189, all of which are incorporated herein by reference. The transformed explants, cells, or tissues can be subjected to further culture steps, such as callus induction, selection, regeneration, as known in the art. Transformed cells, tissues or explants containing recombinant DNA inserts can be propagated, developed or regenerated into transgenic plants in culture, plugs or soil according to methods known in the art. In one aspect, the present disclosure provides a plant cell that is not reproductive material and does not mediate the natural reproduction of a plant. In another aspect, the present disclosure provides a plant cell that is reproductive material and mediates the natural reproduction of a plant. In another aspect, the present disclosure provides a plant cell that cannot sustain itself by photosynthesis.In another aspect, the present disclosure provides somatic plant cells. Somatic cells, in contrast to germline cells, do not mediate plant reproduction. In one aspect, the present disclosure provides non-reproductive plant cells.

[0247] The following non-limiting embodiments are specifically contemplated: 1. (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter or a floral cell-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome of the plant; or (c) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a first heterologous promoter; and (d) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral tissue-preferred promoter or a floral cell-preferred promoter; or (e) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter or a floral cell-preferred promoter; and (f) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral tissue-preferred promoter or a floral cell-preferred promoter; A plant containing. 2. The plant of embodiment 1, wherein the inducible nuclease is selected from the group consisting of Cas12a, MAD7® and CasX. 3. A plant according to embodiment 2, wherein Cas12a is selected from the group consisting of LbCas12a and FnCas12a. 4. The plant of embodiment 2, wherein the first nucleic acid sequence comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:32 or SEQ ID NO:36. 5. A plant according to any one of embodiments 1 to 4, wherein the first nucleic acid sequence is codon-optimized for the plant. 6. A plant according to any one of embodiments 1 to 5, wherein the first nucleic acid sequence encodes at least one nuclear localization signal. 7. The plant of embodiment 6, wherein at least one nuclear localization signal comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 33 and 34. 8. A plant according to any one of embodiments 1 to 7, wherein the flower cell preferential promoter is a flower cell specific promoter. 9. A plant according to any one of embodiments 1 to 8, wherein the floral tissue preferential promoter is a floral tissue specific promoter. 10. A plant according to any one of embodiments 1 to 9, wherein the flower cell preferential promoter is selected from the group consisting of an A gene promoter, a B gene promoter, a C gene promoter, a D gene promoter, and an E gene promoter. 11. The plant according to any one of embodiments 1 to 10, wherein the floral cell-preferred promoter or floral tissue-preferred promoter is selected from the group consisting of AP1 promoter, AP2 promoter, ZAP1 promoter, AP3 promoter, PI promoter, ZMM16 promoter, ZMM18 promoter, AG promoter, ZAG1 promoter, ZMM2 promoter, ZMM23 promoter, AGL11 / STK promoter, AGL1 / SHP1 promoter, AGL5 / SHP2 promoter, ZAG2 promoter, ZMM1 promoter, SEP1 promoter, SEP2 promoter, SEP3 promoter, SEP4 promoter, ZAG3 promoter, and ZMM7 / SEP-like promoter. 12. The plant according to any one of embodiments 1 to 11, wherein the floral cell-preferred promoter or the floral tissue-preferred promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 30, or a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 16, 18 to 19, 21 to 30, and 45 to 49, or a functional fragment thereof. 13. A plant according to any one of embodiments 1 to 12, wherein the first or second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. 14. A plant according to any one of embodiments 1 to 13, wherein the first or second promoter is a floral cell-preferred promoter or a floral tissue-specific promoter. 15. A plant according to any one of embodiments 1 to 14, wherein the first or second promoter is a floral cell-specific promoter or a floral tissue-specific promoter. 16. A plant according to any one of embodiments 1 to 15, wherein the first or second promoter is selected from the group consisting of an A gene promoter, a B gene promoter, a C gene promoter, a D gene promoter, and an E gene promoter. 17. A plant according to any one of embodiments 1 to 16, wherein the first or second promoter is selected from the group consisting of AP1 promoter, AP2 promoter, ZAP1 promoter, AP3 promoter, PI promoter, ZMM16 promoter, ZMM18 promoter, AG promoter, ZAG1 promoter, ZMM2 promoter, ZMM23 promoter, AGL11 / STK promoter, AGL1 / SHP1 promoter, AGL5 / SHP2 promoter, ZAG2 promoter, ZMM1 promoter, SEP1 promoter, SEP2 promoter, SEP3 promoter, SEP4 promoter, ZAG3 promoter, and ZMM7 / SEP-like promoter. 18. A plant according to any one of embodiments 1 to 17, wherein the first or second promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 30, or a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 16, 18 to 19, 21 to 30 and 45 to 49, or a functional fragment thereof. 19. A plant according to embodiment 13, wherein the constitutive promoter is selected from the group consisting of a DaMV promoter, a CaMV 35S promoter, an actin promoter, a Rab15 promoter, and a ubiquitin promoter. 20. A plant according to any one of the preceding embodiments, wherein the at least one guide nucleic acid comprises at least one guide RNA. 21. A plant according to any one of embodiments 1 to 20, wherein the first nucleic acid sequence, the second nucleic acid sequence, or both, are stably integrated into the genome of the plant. 22. A plant according to any one of the preceding embodiments, wherein the inducible nuclease and at least one RNA form a ribonucleoprotein in the flower cells. 23. The plant of embodiment 22, wherein the ribonucleoprotein generates at least one double-stranded break within the target site in the flower cell. 24. The plant according to any one of embodiments 1 to 23, 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, and a potato plant. 25. A plant according to any one of the preceding embodiments, wherein the genome is selected from the group consisting of a nuclear genome, a mitochondrial genome, and a plastid genome. 26. A seed produced by a plant according to any one of embodiments 1 to 25, optionally comprising an alteration at or near a target sequence in the plant genome. 27. The seed according to embodiment 26, comprising at least one mutation in the gene of interest comprising the target sequence, compared to seeds of a control plant of the same variety lacking the first nucleic acid sequence or the second nucleic acid sequence. 28. The seed according to embodiment 26, wherein at least one mutation in the gene of interest results in the deletion of one or more amino acids from the protein encoded by the gene of interest compared to the wild-type protein. 29. The seed according to embodiment 26, wherein at least one mutation in the gene of interest results in the substitution of one or more amino acids in the protein encoded by the gene of interest compared to the wild-type protein. 30. The seed according to embodiment 26, wherein at least one mutation in the gene of interest results in the introduction of a premature stop codon into the messenger RNA encoded by the gene of interest compared to the wild-type messenger RNA. 31. The seed of embodiment 26, wherein at least one mutation in the gene of interest comprises a deletion of one or more splice sites from the gene of interest. 32. The seed according to any one of embodiments 26 to 31, which is a hybrid seed. 33. The seed of any one of embodiments 26 to 31, which is an inbred seed. 34. A method for editing the genome of a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; introducing (b) regenerating at least one plant from the plant cells of step (a). wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell. 35. A method for editing the genome of a plant cell, comprising: (a) crossing a first plant with a second plant, the first plant comprising a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter, and the second plant comprising a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, the at least one guide nucleic acid being capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one embryo from the mating of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo, and the ribonucleoprotein generates at least one double-stranded break within the target sequence in the at least one embryo. A method comprising: 36. A method for editing the genome of a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; introducing (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant to produce at least one embryo. wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-stranded break within the target sequence in the at least one embryo. 37. A method for generating site-specific integration in a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral cell-preferred promoter; (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterologous second promoter, the one or more guide nucleic acids comprising: (A) capable of hybridizing to a target sequence in the genome of a plant; and (B) a second nucleic acid sequence capable of hybridizing to the first site and the second site that flank the nucleic acid sequence encoding the gene of interest; and (iii) a third nucleic acid sequence encoding a gene of interest. introducing (b) regenerating at least one plant from the plant cells of step (a). wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generates a double stranded break within the target sequence molecule, the first site, and the second site, and a gene of interest is integrated into the target sequence in the at least one flower cell. 38. A method for generating site-specific integration in a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterologous second promoter, the one or more guide nucleic acids comprising: (A) capable of hybridizing to a target sequence in the genome of a plant; and (B) a second nucleic acid sequence capable of hybridizing to the first site and the second site that flank the nucleic acid sequence encoding the gene of interest; (iii) a third nucleic acid sequence encoding a gene of interest. introducing (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing at least one plant derived from step (b) to produce at least one embryo. wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in the at least one embryo, the ribonucleoprotein generates a double stranded break in the target DNA molecule, the first site, and the second site, and a gene of interest is integrated into the target sequence in the at least one embryo. 39. A method for editing the genome of a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous first promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral cell-preferred or floral tissue-preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome. introducing (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell. A method comprising: 40. A method for editing the genome of a plant cell, comprising: (a) crossing a first plant with a second plant, the first plant comprising a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter, and the second plant comprising a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, the at least one guide nucleic acid being capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one progeny plant from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one flower cell, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell. A method comprising: 41. A method for editing the genome of a plant cell, comprising: (a) crossing a first plant with a second plant, the first plant comprising a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferred promoter, the second plant comprising a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, the at least one guide nucleic acid being capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one progeny plant from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one flower cell, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell. A method comprising: 42. A method for generating site-specific integration in a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous first promoter; (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterologous floral cell-preferred or floral tissue-preferred promoter, wherein the one or more guide nucleic acids a. capable of hybridizing to a target sequence within the genome of a plant; and b. a second nucleic acid sequence capable of hybridizing to the first site and the second site that flank the nucleic acid sequence encoding the gene of interest; and (iii) a third nucleic acid sequence encoding a gene of interest. introducing (b) regenerating at least one plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generates double-stranded breaks in the target sequence molecule, the first site, and the second site, and the gene of interest is integrated into the target sequence in the at least one flower cell. A method comprising: 43. The method of any one of embodiments 34 to 42, wherein the target sequence comprises genomic DNA. 44. The method of any one of embodiments 34 to 42, wherein the target sequence comprises intergenic DNA. 45. The method of any one of embodiments 34 to 36 and 39 to 41, wherein the target sequence is within a gene of interest. 46. ​​The method of embodiment 45, wherein the gene of interest encodes a protein or a non-protein-coding RNA. 47. The method of embodiment 46, wherein the non-protein-coding RNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or precursors thereof. 48. The method of any one of embodiments 34 to 42, wherein the inducible nuclease is selected from the group consisting of Cas12a, MAD7® and CasX. 49. The method of embodiment 48, wherein Cas12a is selected from the group consisting of LbCas12a and FnCas12a. 50. The method of any one of embodiments 34, 37, or 39-42, wherein the flower cell preferential promoter is a flower cell specific promoter. 51. The method of any one of embodiments 35, 36, or 38, wherein the floral tissue-preferred promoter is a floral tissue-specific promoter. 52. The method of any one of embodiments 34, 37, or 39-42, wherein the flower cell-preferential promoter is selected from the group consisting of an A gene promoter, a B gene promoter, a C gene promoter, a D gene promoter, and an E gene promoter. 53. The method of any one of embodiments 34, 37, or 39-42, wherein the floral cell-preferential promoter is selected from the group consisting of AP1 promoter, AP2 promoter, ZAP1 promoter, AP3 promoter, PI promoter, ZMM16 promoter, ZMM18 promoter, AG promoter, ZAG1 promoter, ZMM2 promoter, ZMM23 promoter, AGL11 / STK promoter, AGL1 / SHP1 promoter, AGL5 / SHP2 promoter, ZAG2 promoter, ZMM1 promoter, SEP1 promoter, SEP2 promoter, SEP3 promoter, SEP4 promoter, ZAG3 promoter, and ZMM7 / SEP-like promoter. 54. The method of any one of embodiments 34, 37, or 39-42, wherein the flower cell-preferential promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, 45-49, or a functional fragment thereof. 55. The method of any one of embodiments 35, 36, or 38, wherein the floral tissue-preferred promoter is selected from the group consisting of an A gene promoter, a B gene promoter, a C gene promoter, a D gene promoter, and an E gene promoter. 56. The method of any one of embodiments 35, 36, or 38, wherein the floral cell-preferred promoter is selected from the group consisting of AP1 promoter, AP2 promoter, ZAP1 promoter, AP3 promoter, PI promoter, ZMM16 promoter, ZMM18 promoter, AG promoter, ZAG1 promoter, ZMM2 promoter, ZMM23 promoter, AGL11 / STK promoter, AGL1 / SHP1 promoter, AGL5 / SHP2 promoter, ZAG2 promoter, ZMM1 promoter, SEP1 promoter, SEP2 promoter, SEP3 promoter, SEP4 promoter, ZAG3 promoter, and ZMM7 / SEP-like promoter. 57. The method of any one of embodiments 35, 36, or 38, wherein the floral tissue-preferential promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, 45-49, or a functional fragment thereof. 58. The method of any one of embodiments 34 to 42, wherein the first or second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. 59. The method of any one of embodiments 34-42, wherein the first or second promoter is a floral cell-preferred promoter or a floral tissue-preferred promoter. 60. The method of embodiment 59, wherein the floral cell-preferred promoter or floral tissue-preferred promoter is selected from the group consisting of an A gene promoter, a B gene promoter, a C gene promoter, a D gene promoter, and an E gene promoter. 61. The method of embodiment 59 or 60, wherein the floral cell-preferred promoter or floral tissue-preferred promoter is selected from the group consisting of AP1 promoter, AP2 promoter, ZAP1 promoter, AP3 promoter, PI promoter, ZMM16 promoter, ZMM18 promoter, AG promoter, ZAG1 promoter, ZMM2 promoter, ZMM23 promoter, AGL11 / STK promoter, AGL1 / SHP1 promoter, AGL5 / SHP2 promoter, ZAG2 promoter, ZMM1 promoter, SEP1 promoter, SEP2 promoter, SEP3 promoter, SEP4 promoter, ZAG3 promoter, and ZMM7 / SEP-like promoter. 62. The method according to any one of embodiments 59 to 61, wherein the floral cell-preferred promoter or the floral tissue-preferred promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 30, or a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 16, 18 to 19, 21 to 30, 45 to 49, or a functional fragment thereof. 63. The method of embodiment 58, wherein the constitutive promoter is selected from the group consisting of CAMV35S promoter, actin promoter, Rab15 promoter, and ubiquitin promoter. 64. The method of any one of embodiments 34 to 42, wherein the one or more guide nucleic acids comprise at least one guide RNA. 65. The method of any one of embodiments 34 to 42, wherein the first nucleic acid sequence, the second nucleic acid sequence, or both, are stably integrated into the genome of the plant. 66. The method of any one of embodiments 34-42, wherein the plant is selected from the group consisting of corn, rice, sorghum, wheat, alfalfa, barley, millet, rye, sugarcane, cotton, soybean, canola, tomato, and potato. 67. The method of any one of embodiments 34 to 42, wherein the genome is selected from the group consisting of a nuclear genome, a mitochondrial genome, and a plastid genome. 68. The method of any one of embodiments 34-36 or 39-41, wherein repair of the double-stranded break generates at least one mutation in the target sequence compared to a control plant of the same line or variety lacking the first nucleic acid sequence or the second nucleic acid sequence, and optionally the mutation results in a deletion, insertion or substitution of at least one nucleotide in or near the target sequence. 69. The method according to embodiment 68, wherein at least one mutation in the target sequence results in the deletion of one or more amino acids from the protein encoded by the gene of interest compared to the wild-type protein. 70. The method of embodiment 68, wherein at least one mutation in the target sequence results in the substitution of one or more amino acids in the protein encoded by the gene of interest compared to the wild-type protein. 71. The method of embodiment 68, wherein at least one mutation in the target sequence results in the introduction of a premature stop codon into the messenger RNA encoded by the gene of interest compared to the wild-type messenger RNA. 72. The method of embodiment 68, wherein at least one mutation in the target sequence comprises a deletion of one or more splice sites from the gene of interest. 73. A recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of making a staggered cut in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferred or floral tissue-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter; or (c) a first nucleic acid sequence encoding an inducible nuclease capable of making a staggered cut in a double-stranded DNA molecule operably linked to a heterologous promoter; and (d) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous flower cell-preferred or floral tissue-preferred promoter, wherein the at least one guide nucleic acid comprises a second nucleic acid sequence capable of hybridizing to a target sequence in the genome of a plant. 74. The recombinant DNA construct of embodiment 73, wherein the sequence encoding at least one guide nucleic acid is flanked by self-cleaving ribozymes. 75. A method for generating two or more progeny plants with unique edits from a single transformed plant cell, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; introducing (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the first plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell; (c) pollinating the first plant of step (b); and (d) germinating the two or more seeds produced from step (c) to produce two or more progeny plants having unique edits. A method comprising: 76. A method for generating two or more progeny plants with unique edits from a single transformed plant cell, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous first promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous flower cell-preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; introducing (b) regenerating a first plant from the plant cell of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the first plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell; (c) pollinating the first plant of step (b); and (d) germinating the two or more seeds produced from step (c) to produce two or more progeny plants having unique edits. A method comprising: 77. A method for generating two or more progeny plants with unique edits from a single transformed plant cell, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; introducing (b) regenerating a first plant from the plant cell of step (a); (c) pollinating the first plant of step (b), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the floral tissue; and (d) germinating the two or more seeds produced from step (c) to produce two or more progeny plants having unique edits. A method comprising: 78. A method for generating two or more progeny plants with unique edits from a single transformed plant cell, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous first promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral tissue-preferential promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; introducing (b) regenerating a first plant from the plant cell of step (a); (c) pollinating the first plant of step (b), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the floral tissue; and (d) germinating the two or more seeds produced from step (c) to produce two or more progeny plants having unique edits. A method comprising: 79. The method of any one of embodiments 75 to 78, wherein the target sequence comprises genomic DNA. 80. The method of any one of embodiments 75 to 78, wherein the target sequence comprises intergenic DNA. 81. The method of any one of embodiments and 75-78, wherein the target sequence is within a gene of interest. 82. The method of embodiment 81, wherein the gene of interest encodes a protein or a non-protein-coding RNA. 83. The method of embodiment 82, wherein the non-protein-coding RNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or precursors thereof. 84. The method of any one of embodiments 75 to 78, wherein the inducible nuclease is selected from the group consisting of Cas12a, MAD7® and CasX. 85. The method of embodiment 84, wherein Cas12a is selected from the group consisting of LbCas12a and FnCas12a. 86. The method of embodiment 75 or 76, wherein the flower cell-preferred promoter is a flower cell-specific promoter. 87. The method of any one of embodiments 77 or 78, wherein the floral tissue-preferred promoter is a floral tissue-specific promoter. 88. The method of embodiment 75 or 76, wherein the floral cell-preferential promoter is selected from the group consisting of an A gene promoter, a B gene promoter, a C gene promoter, a D gene promoter, and an E gene promoter. 89. The method of embodiment 75 or 76, wherein the floral cell-preferential promoter is selected from the group consisting of AP1 promoter, AP2 promoter, ZAP1 promoter, AP3 promoter, PI promoter, ZMM16 promoter, ZMM18 promoter, AG promoter, ZAG1 promoter, ZMM2 promoter, ZMM23 promoter, AGL11 / STK promoter, AGL1 / SHP1 promoter, AGL5 / SHP2 promoter, ZAG2 promoter, ZMM1 promoter, SEP1 promoter, SEP2 promoter, SEP3 promoter, SEP4 promoter, ZAG3 promoter, and ZMM7 / SEP-like promoter. 90. The method of embodiment 75 or 76, wherein the flower cell-preferential promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, 45-49, or a functional fragment thereof. 91. The method of embodiment 77 or 78, wherein the floral tissue-preferred promoter is selected from the group consisting of an A gene promoter, a B gene promoter, a C gene promoter, a D gene promoter, and an E gene promoter. 92. The method of embodiment 77 or 78, wherein the floral tissue-preferred promoter is selected from the group consisting of AP1 promoter, AP2 promoter, ZAP1 promoter, AP3 promoter, PI promoter, ZMM16 promoter, ZMM18 promoter, AG promoter, ZAG1 promoter, ZMM2 promoter, ZMM23 promoter, AGL11 / STK promoter, AGL1 / SHP1 promoter, AGL5 / SHP2 promoter, ZAG2 promoter, ZMM1 promoter, SEP1 promoter, SEP2 promoter, SEP3 promoter, SEP4 promoter, ZAG3 promoter, and ZMM7 / SEP-like promoter. 93. The method of embodiment 77 or 78, wherein the floral tissue-preferential promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, 45-49, or a functional fragment thereof. 94. The method of embodiment 75 or 77, wherein the second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. 95. The method of embodiment 76 or 79, wherein the first promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. 96. The method of embodiment 94 or 95, wherein the constitutive promoter is selected from the group consisting of CAMV35S promoter, actin promoter, Rab15 promoter, DAMV promoter and ubiquitin promoter. 97. The method of any one of embodiments 75 to 96, wherein the one or more guide nucleic acids comprise at least one guide RNA. 98. The method of any one of embodiments 75 to 97, wherein the first nucleic acid sequence, the second nucleic acid sequence, or both are stably integrated into the genome of the first plant. 99. The method of any one of embodiments 75-98, wherein the plant cell is selected from the group consisting of corn, rice, sorghum, wheat, alfalfa, barley, millet, rye, sugarcane, cotton, soybean, canola, tomato, and potato. 100. The method of any one of embodiments 75 to 99, wherein the genome is selected from the group consisting of a nuclear genome, a mitochondrial genome, and a plastid genome. 101. The method of any one of embodiments 75 to 100, wherein repair of the double-stranded break generates at least one mutation in the target sequence compared to a control plant of the same line or variety lacking the first nucleic acid sequence or the second nucleic acid sequence, and optionally the mutation results in a deletion, insertion or substitution of at least one nucleotide in or near the target sequence. 102. The method of embodiment 101, wherein at least one mutation in the target sequence results in the deletion of one or more amino acids from the protein encoded by the gene of interest compared to the wild-type protein. 103. The method of embodiment 101, wherein at least one mutation in the target sequence results in the substitution of one or more amino acids in the protein encoded by the gene of interest compared to the wild-type protein. 104. The method of embodiment 101, wherein at least one mutation in the target sequence results in the introduction of a premature stop codon into the messenger RNA encoded by the gene of interest, as compared to the wild-type messenger RNA. 105. The method of embodiment 101, wherein at least one mutation in the target sequence comprises a deletion of one or more splice sites from the gene of interest. 106. The method of embodiment 76 or 78, wherein the sequence encoding at least one guide nucleic acid is flanked by self-cleaving ribozymes. 107. The method of any one of embodiments 75-106, wherein the first plant is self-pollinated.

[0248] Having now generally described the present disclosure, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended to be limitations of the disclosure, unless specifically stated. EXAMPLES

[0249] Example 1 Expression of Cas12a in floral cells / tissues to generate germline mutations or targeted integration of template DNA Several Agrobacterium T-DNA vectors were generated to preferentially express Cas12a in corn or soybean floral tissues. See Table 1. A control vector was generated to constitutively express Cas12a, where Cas12a is operably linked to a modified promoter comprising the DaMV promoter (SEQ ID NO:31) operably fused to an enhancer region (SEQ ID NO:43) derived from Banana Streak Virus strain Acuminata Vietnam.

[0250] [Table 1A]

[0251] [Table 1B]

[0252] The plant codon-optimized LbCas12a sequence (SEQ ID NO: 32) in these cassettes is flanked by NLS sequences (nuclear localization signals) at the 5' and 3' ends and operably linked to a transcription termination sequence. Each vector also contains an expression cassette encoding a Cas12a gRNA targeted to a unique maize genomic site (ZmTS1) or soybean genomic site (GmTS1) and operably linked to a plant PolIII promoter; an expression cassette for a gene of interest (GOI) flanked by ZmTS1 or GmTS1 gRNA target sites; and an expression cassette for a selection marker. Maize or soybean embryos are transformed with the above vectors by Agrobacterium-mediated transformation, and R0 plants are regenerated from the transformed cells. DNA is extracted from leaf samples derived from R0 seedlings generated from each construct. Genomic target sites are sequenced and analyzed for the presence, number, and type of mutations observed.

[0253] Several R0 lines containing each transformed construct are grown to maturity and self-pollinated. Several R1 lines are then selected and several seedlings per R1 line are grown and screened for mutations within the target site. The number and type of mutations produced using constructs containing floral tissue-specific promoters (constructs 1-30) are compared to mutations observed in transformed maize plants produced using a control construct 31. Without being bound by any theory, it is predicted that co-expression of Cas12a and its cognate gRNA in flower cells will generate double-stranded breaks at the target site, with subsequent incomplete DNA repair generating unique mutations in flower tissues. Restriction of editing to developing reproductive structures may result in unrelated editing events at multiple locations within a single plant in these tissues resulting in the organ or cell type from which the gametes are derived. These edited gametes can then pass the mutation to the next generation. Thus, it is predicted that a single R0 plant can produce many R1 progeny, each with a unique target site edit.

[0254] Testing site-specific integration (SDI) of T-DNA at target sites in plants expressing Cas12a In addition to the Cas12a and gRNA expression cassettes, each vector also contains an expression cassette for a gene of interest (GOI) flanked by ZmTS1 or GmTS1 gRNA target sequences. Without being limited by any theory, it is expected that expression of Cas12a and gRNA in flower tissues creates double-stranded breaks on both sides of the GOI cassette, releasing it from the T-DNA. This released DNA can serve as a donor for targeted insertion at the genomic target site. When the CRISPR-Cas12a complex cleaves the target site in the genome, the non-homologous end joining (NHEJ) DNA repair pathway can insert the donor GOI cassette into the genomic target site. This form of SDI is also known as trans-fragment targeting (TFT).

[0255] To test for SDI by TFT, putative targeted insertions are identified using a flank PCR assay similar to that described in WO 2019 / 084148, the entirety of which is incorporated herein by reference. Primers are designed to PCR amplify the expected insertion flanking sequences. Four separate PCRs are performed: left flank PCR and right flank PCR for potential insertions located in the sense orientation, and left flank PCR and right flank PCR for insertions located in the antisense orientation. R0 and R1 lines are screened to identify putative flank PCR positive plants, which are further sequenced to confirm the targeted insertion of the GOI cassette at the GmTS1 or ZmTS1 genomic site.

[0256] Example 2 Expression of gRNAs in floral cells / tissues where Cas12a is constitutively expressed to generate germline mutations Several constructs are generated to preferentially express guide RNAs (gRNAs) complementary to target sites under the control of the PolII promoter in flower cells. See Table 2. After transcription, PolII products are rapidly modified with 5' caps and polyA tails and exported from the nucleus. These modifications and localization changes may prevent efficient use of gRNAs. To optimize gRNA availability and performance, self-cleaving ribozymes are incorporated into the gRNA cassette design. Self-cleaving ribozymes have been reported to facilitate cleavage / processing of gRNA transcripts from PolII-expressed transcripts to produce precise guide molecules (see, e.g., Wang et al., J. of Integrative Plant Biol, 60:626-631 (2018)).

[0257] [Table 2A]

[0258] [Table 2B]

[0259] The constructs described in Table 2 are stably introduced into maize or soybean cells using transformation methods routinely used in the art. In addition, a construct ("Cas12a construct") containing a plant codon-optimized nucleic acid sequence encoding Cas12a protein flanked by 5' and 3' NLS sequences and under the control of a ubiquitous promoter (e.g., ZmUbqM1 promoter (SEQ ID NO: 35) or Ubq2 promoter (SEQ ID NO: 44) of Medicago truncatula) is co-introduced with each of the constructs provided in Table 2. The resulting transformed cells contain one of constructs 32-62 as well as the Cas12a construct. Plants are regenerated from the transformed cells, grown to maturity, and pollinated. Seeds resulting from pollination are screened for mutations at the target site, and the number and type of mutations produced using constructs 32-61 are compared to mutations in transformed plants produced using control construct 62. Selective expression of the gRNA is expected to generate one or more unique mutations in the floral tissue.

[0260] Example 3 Expression of Cas12a and gRNA as a single transcript in flower cells Several constructs were generated to preferentially express LbCas12a and a guide RNA (gRNA) complementary to the target site as a single transcript (see Table 3).

[0261] [Table 3A]

[0262] [Table 3B]

[0263] Each of the constructs described in Table 3 is stably introduced into corn or soybean cells using biolistic or Agrobacterium-mediated transformation methods routinely used in the art. The resulting transformed corn cells contain one of constructs 63-93. Plants are regenerated from the transformed cells and grown to maturity. LbCas12a and gRNA are transcribed as part of a single transcript in flower cells where the promoter is expressed. Ribozyme-mediated cleavage then occurs, releasing the gRNA segment. LbCas12a protein transcribed from the transcript forms a ribonucleoprotein (RNP) with the gRNA. The RNP generates a double-stranded break at the target site, and subsequent repair will generate one or more unique mutations in each flower cell. Mature plants are pollinated and the resulting seeds from pollination are screened for mutations at the target site, and the number and type of mutations produced using constructs 63-92 are compared to transformed corn plants produced using the control construct 93.

[0264] Example 4 Generating mutations by crossing Transgenic maize or soybean plants containing one of the LbCas12a cassettes described in Table 1 are generated and grown to flowering. Additional transgenic maize or soybean plants containing the gRNA cassettes described in Example 1 are also generated and grown to flowering. Plants containing LbCas12a are crossed with plants containing the gRNA construct to generate progeny plants containing Cas12a and gRNA expressed in floral tissue.

[0265] Alternatively, transgenic maize or soybean plants containing one of the constructs (see Example 2, Table 2) are generated and grown to flowering. Additional transgenic maize or soybean plants containing the Cas12a construct of Example 2 are also generated and grown to flowering. The transgenic plants containing one of the constructs are crossed with plants containing the Cas12a construct to generate progeny plants containing Cas12a and gRNA expressed in floral tissue.

[0266] Without being limited by any theory, co-expression of Cas12a and gRNA in flower tissue generates a double-stranded break within the target site, thereby generating a unique mutation in each cell in which both components of the CRISPR system are expressed. The plants are crossed or self-pollinated, and the resulting progeny are screened to identify mutations at the target site.

[0267] Example 5 TALE-induced tissue / cell-specific expression of LbCas12a A potential downside of tissue / cell-preferred promoters is that they tend not to be expressed robustly. This example describes constructs that have been generated to overcome this limitation and direct robust expression of a transcribable polynucleotide, such as Cas12a, in a tissue / cell-preferred manner.

[0268] We generate several constructs for preferential and robust TALE-induced Cas12a expression in floral cells / tissues. Transcription activator-like effectors (TALEs) are transcription factors that contain a C-terminal activation domain and can activate / increase expression of an operably linked transcribable polynucleotide once the TALE binds to a TALE binding site at or near the promoter. It has previously been shown that TALE proteins can induce high expression of genes operably linked to TALE binding sites and can modulate expression depending on how many TALE binding sites are present in the regulatory region. We generate constructs that contain a plant codon-optimized LbCas12a coding sequence operably linked to a transcription termination sequence and a minimal 35S(-46) promoter, flanked by NLS sequences at the 5' and 3' ends, with one, three, or six TALE binding sites. We also generate expression constructs that contain a TALE coding sequence operably linked to a promoter that is preferentially or solely expressed in floral tissues / cells. Non-limiting examples of promoters and regulatory sequences for driving preferential cellular expression are provided in Table 1. An expression cassette containing a TALE coding sequence operably linked to a constitutive ubiquitin promoter is generated as a control. Maize or soybean embryos are transformed with the above expression cassette and a vector containing an expression cassette encoding a Cas12a gRNA complementary to a maize genome target site (ZmTS1) or a soybean genome target site (GmTS1) under the control of a plant PolIII promoter and an expression cassette for a selection marker by Agrobacterium-mediated transformation, and R0 plants are generated from the transformed cells. Several R0 lines derived from each transformed construct are grown to maturity and pollinated. Several R1 lines are selected, seedlings are grown, and screened for LbCas12a-induced editing in the target site, and the editing rate is calculated.When plants containing the above Cas12a, gRNA and TALE expression vectors reach reproductive stage, the TALE preferentially expressed in flower tissues / cells is expected to bind to the TALE protein binding site upstream of 35S(-46):Lb.Cas12a and induce preferential robust expression of the nuclease in flower cells. Expression of LbCas12a and gRNA is expected to result in mutations within the target site. R1 plants generated from the transformed R0 lines are expected to show a significant number of unique mutations at the target site.

[0269] Example 6 Preferential expression of Cas12a in floral tissues / cells to generate germline mutations This example describes the use of the Arabidopsis thaliana meristem-preferred promoter AtERL1 to drive Cas12a expression to generate diverse mutations in the R0 generation and beyond. Two Agrobacterium T-DNA constructs were generated, as shown in Table 4. Each construct contained an LbCas12a nuclease cassette, a gRNA array cassette, and a selection marker cassette. The vectors are similar in design, except that in construct 94, the LbCas12a cassette is driven by the Arabidopsis thaliana ERL1 (SEQ ID NO: 11) meristem-preferred promoter, while in control construct 95, LbCas12a is driven by the strong constitutive promoter DaMV.

[0270] [Table 4]

[0271] The plant codon-optimized LbCas12a sequence (SEQ ID NO:36) in these cassettes is flanked by 5' and 3' end NLS sequences (SEQ ID NO:33 and SEQ ID NO:34) and operably linked to a transcription terminator sequence (SEQ ID NO:37) derived from the Medicago sativa gene. The gRNA array expression cassette contains a PolIII promoter operably linked to four guide RNAs, each of which targets a 27 nucleotide sequence within the 864 nucleotide E1 gene sequence (SEQ ID NO:38) in the Glycine max genome (see Table 5). The T-DNA vector also contains an expression cassette for a selection marker that confers resistance to the antibiotic spectinomycin. Soybean A3555 cultivar embryos were transformed with the above vectors by Agrobacterium-mediated transformation, and R0 plants were regenerated from the transformed soybean cells.

[0272] [Table 5]

[0273] DNA is extracted from leaf samples from 20 R0 seedlings for each of constructs 94 and 95. The GmE1 site is sequenced and analyzed for the presence of targeted mutations. Co-expression of Cas12a with its cognate gRNA is expected to generate double-strand breaks at the target site, followed by incomplete DNA repair to generate unique mutations. In plants carrying AtErl1:LbCas12a (construct 94), 6 target site edits were identified, and in plants transformed with the DaMV:LbCas12a construct (construct 95), 15 target site edits were observed (see Table 6). Since AtERL1 is predicted to be a weak promoter preferentially expressed in the axillary meristem, a low mutation rate from construct 94 in newly transformed (or R0) plants is expected.

[0274] [Table 6]

[0275] Several R0 lines from each transformed construct were grown to maturity, and at least one ear from each transformed soybean plant was self-pollinated. Several hundred R1 lines were selected, grown, and screened for mutations. As shown in Table 7, the R1 progeny from the AtERL1:LbCas12a transgenic event carried many more novel mutations when compared to the event expressing Cas12a from the constitutive promoter DaMV.

[0276] [Table 7]

[0277] Sequence analysis of the edits showed that the newly generated mutations detected in the R1 plants of the AtERL1:LbCas12a transgenic events were highly diverse. A wide range of mutations was identified in approximately 40 R1 plants of each event (see Table 8). In addition, some edits were identified in R1 plants whose parent R0 plants were not edited.

[0278] [Table 8A]

[0279] [Table 8B]

[0280] [Table 8C]

[0281] [Table 8D]

[0282] [Table 8E]

[0283] Closer analysis of the segregation patterns of edits / mutations indicates that, unlike those occurring in the R1 progeny of DAMV:LbCas12a, the segregation patterns of edits identified in the AtERL1:LbCas12a R0 events may or may not follow classical Mendelian patterns in the R1 progeny depending on when the mutations were introduced into the R0 plants (Table 9). Without being limited by any theory for events showing atypical segregation patterns, the mutations most likely arise from the meristems after regenerating the R0 plants. Thus, the R1 seeds are a mixture of seeds derived from chimeric R0 plants.

[0284] [Table 9]

[0285] Taken together, the data show that LbCas12a can achieve reproductive editing when expressed under the control of the axillary meristem promoter AtERL1. Furthermore, the data demonstrate that a single R0 plant can produce many R1 progeny, each with a unique target site edit. This suggests that this promoter can be used to drive expression of the nuclease to increase the frequency of unique edits produced per transformed plant.

[0286] Example 7 Preferential expression of Cas12a in floral tissues / cells to generate germline mutations This example describes the use of soybean meristem-preferred promoters derived from GmAP1-like, GmCYC3-1, GmAP3-like-1 and GmERL1-like genes to drive Cas12a expression to generate diverse mutations in the R0 generation and beyond. GmERL1-like is the soybean homolog of the AtERL1 (SEQ ID NO: 11) gene. Five Agrobacterium T-DNA constructs were generated, as shown in Table 10. Each construct contained an LbCas12a nuclease cassette, a gRNA array cassette and a selection marker cassette. The vectors are similar in design, except that in construct 91, the LbCas12a cassette is driven by a strong constitutive promoter derived from the Medicago sativa Ubq2 gene (SEQ ID NO: 44), while in the others, LbCas12a expression is driven by various meristem-preferred promoters derived from soybean (Glycine max).

[0287] [Table 10]

[0288] In construct 97, LbCas12a expression is driven by a variant of the soybean GmAP1-like-1 promoter (SEQ ID NO: 12) and is disclosed as GmAP1-like-1-var (SEQ ID NO: 45). GmAP1-like-1-var (SEQ ID NO: 45) contains a 5' extension of 7 nucleotides and a 3' extension of 371 nucleotides compared to GmAP1-like-1 (SEQ ID NO: 12). In construct 100, LbCas12a expression is driven by a variant of the soybean GmERL1 promoter (SEQ ID NO: 27) and is disclosed as GmERL1-var (SEQ ID NO: 48). GmERL1-var (SEQ ID NO: 48) contains a 5' deletion of 261 nucleotides and a 3' extension of 972 nucleotides compared to GmERL1 (SEQ ID NO: 27).

[0289] The plant codon-optimized LbCas12a sequence (SEQ ID NO:32) in these cassettes is flanked by 5' and 3' end NLS sequences (SEQ ID NO:33 and SEQ ID NO:34) and operably linked to a transcription terminator sequence (SEQ ID NO:50) derived from the Medicago sativa gene. The gRNA array expression cassette contains a PolIII promoter operably linked to one guide RNA targeting a 27 nucleotide sequence within the 1542 nucleotide Tawny coding sequence (SEQ ID NO:51) in the soybean genome (see Table 11). The T-DNA vector also contains an expression cassette for a selection marker that confers resistance to the antibiotics spectinomycin and streptomycin. Soybean A3555 cultivar embryos were transformed with the above vectors by Agrobacterium-mediated transformation, and R0 plants were regenerated from the transformed soybean cells.

[0290] [Table 11]

[0291] DNA is extracted from leaf samples derived from 20 R0 seedlings for each of constructs 96-100. GmTawny sites are sequenced and analyzed for the presence of targeted mutations. Co-expression of Cas12a with its cognate gRNA is expected to generate double-stranded breaks at the target site, with subsequent incomplete DNA repair generating unique mutations. Ten target site edits were identified in plants carrying the MtUbq2::LbCas12a construct (construct 1) (see Table 12). Total edits in transformations showing tissue-specific expression vary but are of the same magnitude as for constitutive transformations. Mutation rates are governed by both tissue specificity and differences in expression magnitude.

[0292] [Table 12]

[0293] Several R0 lines from each transformed construct were grown to maturity and seeds were harvested. Several hundred R1 progeny seeds were selected, grown, and screened for mutations. As shown in Table 13, R1 progeny from GmAP3like:LbCas12a and GmERL1like:LbCas12a transgenic events carried a higher frequency of new mutations when compared to events expressing Cas12a from the constitutive promoter Ubq2.

[0294] [Table 13]

[0295] Sequence analysis of the edits showed that the newly generated mutations detected in the R1 plants of the GmAP3-like-1::LbCas12a and GmERL1-var::LbCas12a transgenic events were highly diverse. A wide range of mutations was identified from approximately 40 R1 plants of each event (see Table 14). In addition, some edits were identified in the R1 plants whose parent R0 plants were not edited.

[0296] [Table 14A]

[0297] [Table 14B]

[0298] [Table 14C]

[0299] [Table 14D]

[0300] Example 8 Preferential expression of Cas12a in meristems using a promoter variant of AtERl1 This example describes the use of a variant of the Arabidopsis meristem-preferred promoter AtERl1 to drive Cas12a expression to generate diverse mutations in the R0 generation and beyond.

[0301] The AtERL1 promoter sequence disclosed as SEQ ID NO: 11 contains a stretch of 34 Ts starting at nucleotide position 2021 to 2054. Long stretches of the same nucleotides can create problems during DNA sequencing. To overcome this potential problem, a variant of the promoter AtERL1 (AtERL1-var) is generated. AtERL1-var (SEQ ID NO: 49) contains T to C substitutions at positions 2031 and 2043. These substitutions are not expected to significantly alter the expression activity of the AtERL1 promoter.

[0302] Generate Agrobacterium T-DNA construct 101, which is similar to construct 94, except that the LbCas12a cassette is driven by the AtERL1-var promoter (SEQ ID NO:49).

[0303] [Table 15]

[0304] Soybean A3555 cultivar embryos are transformed with the above vectors by Agrobacterium-mediated transformation to regenerate R0 plants. DNA is extracted from leaf samples of R0 seedlings for each of constructs 94 and 101. The GmE1 site is sequenced and analyzed for the presence of targeted mutations. Co-expression of Cas12a and its cognate gRNA is expected to generate double-strand breaks at the target site, and subsequent incomplete DNA repair generates unique mutations. Several R0 lines derived from each transformed construct are grown to maturity, and at least one ear from each transformed soybean plant is self-pollinated. Several hundred R1 lines are selected, grown, and screened for mutations. Editing in R1 progeny derived from constructs 94 and 95 transgenic events is compared.

[0305] Example 9 Use of AtERL1 promoter variants does not affect the specificity or strength of reporter gene expression The AtERL1 promoter variant (SEQ ID NO: 49) described in Example 8 was operably linked to the β-glucuronidase reporter gene (GUS) to generate an Agrobacterium T-DNA construct. As a control, the unmodified AtERL1 promoter (SEQ ID NO: 11) was operably linked to the β-glucuronidase reporter gene (GUS) and used to generate an Agrobacterium T-DNA construct. Soybean A3555 cultivar embryos were transformed with the above vectors by Agrobacterium-mediated transformation to regenerate transgenic plants.

[0306] Quantitative GUS analysis was performed on various plant tissues (V5 roots, leaves, petioles) and reproductive tissues (flowers, pollen, immature seeds, pods, seed embryos, seed cotyledons). The spatial expression patterns and expression intensities using the variant AtERL1 promoter were similar to those obtained with the unmodified AtERL1 promoter.

[0307] Various tissues derived from transgenic soybean plants (different events) were subjected to histochemical expression analysis, and expression, indicated by the development of blue spots, was mainly restricted to the meristems even in soybean plants transformed with the GUS gene operably linked to the variant AtERL1 promoter. Some residual blue staining was observed in restricted floral tissues obtained from transgenic plants containing a control construct or a construct with the variant promoter.

[0308] The modifications in the variant AtERL1 promoter did not affect the tissue specificity or expression strength of the promoter.

Claims

1. (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter or a floral cell-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome of the plant; or (c) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a first heterologous promoter; and (d) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral tissue-preferred promoter or a floral cell-preferred promoter; A plant containing.

2. The plant of claim 1, wherein the inducible nuclease is selected from the group consisting of Cas12a, MAD7 (registered trademark) and CasX.

3. The plant of claim 2, wherein the first nucleic acid sequence comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO:32 or SEQ ID NO:

36.

4. 2. The plant of claim 1, wherein the flower cell-preferred promoter is a flower cell-specific promoter or the flower tissue-preferred promoter is a flower tissue-specific promoter.

5. The floral cell-preferred promoter is selected from the group consisting of A gene promoter, B gene promoter, C gene promoter, D gene promoter, and E gene promoter, or the floral cell-preferred promoter or floral tissue-preferred promoter is selected from the group consisting of AP1 promoter, AP2 promoter, ZAP1 promoter, AP3 promoter, PI promoter, ZMM16 promoter, ZMM18 promoter, AG promoter, ZAG1 promoter, ZMM2 promoter, ZMM23 promoter, AGL11 / STK promoter, AGL1 / SHP1 promoter, AGL 2. The plant of claim 1, wherein the floral cell-preferred promoter or the floral tissue-preferred promoter is selected from the group consisting of SEP1 promoter, SEP2 promoter, SEP3 promoter, SEP4 promoter, ZAG3 promoter, and ZMM7 / SEP-like promoter, or the floral cell-preferred promoter or the floral tissue-preferred promoter is selected from the group consisting of SEQ ID NOs: 1-30, or comprises a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, 45-49, or a functional fragment thereof.

6. 2. The plant of claim 1, wherein the first or second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter, or the first or second promoter is a floral cell-preferred promoter or a floral tissue-preferred promoter.

7. 2. The plant of claim 1, wherein the first or second promoter is selected from the group consisting of an A gene promoter, a B gene promoter, a C gene promoter, a D gene promoter, and an E gene promoter; or the first or second promoter is selected from the group consisting of an AP1 promoter, an AP2 promoter, a ZAP1 promoter, an AP3 promoter, a PI promoter, a ZMM16 promoter, a ZMM18 promoter, an AG promoter, a ZAG1 promoter, a ZMM2 promoter, a ZMM23 promoter, an AGL11 / STK promoter, an AGL1 / SHP1 promoter, an AGL5 / SHP2 promoter, a ZAG2 promoter, a ZMM1 promoter, a SEP1 promoter, a SEP2 promoter, a SEP3 promoter, a SEP4 promoter, a ZAG3 promoter, and a ZMM7 / SEP-like promoter; or the first or second promoter is selected from the group consisting of SEQ ID NOs: 1 to 30, or comprises a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1 to 16, 18 to 19, 21 to 30, and 45 to 49, or a functional fragment thereof.

8. The plant of claim 6, wherein the constitutive promoter is selected from the group consisting of a CaMV 35S promoter, an actin promoter, a Rab15 promoter, and a ubiquitin promoter.

9. The plant of claim 1, wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in a flower cell.

10. 2. The plant of claim 1, wherein the 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, and a potato plant.

11. A seed produced by the plant of claim 1, comprising at least one mutation in a gene of interest that contains the target sequence, compared to seeds of a control plant of the same variety that lacks the first nucleic acid sequence or the second nucleic acid sequence.

12. 1. A method for editing the genome of a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral cell-preferred or floral tissue-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; or (b) In a plant cell, (iii) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous first promoter; and (iv) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral cell-preferred or floral tissue-preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome. introducing (c) regenerating at least one plant from the plant cells of step (a) or (b). wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell.

13. 1. A method for editing the genome of a plant cell, comprising: (a) crossing a first plant with a second plant, the first plant comprising a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter, and the second plant comprising a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, the at least one guide nucleic acid being capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one embryo from the mating of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo, and the ribonucleoprotein generates at least one double-stranded break within the target sequence in the at least one embryo. A method comprising:

14. 1. A method for editing the genome of a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; introducing (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing the at least one plant to produce at least one embryo. wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-stranded break within the target sequence in the at least one embryo.

15. 1. A method for generating site-specific integration in a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral cell-preferred or floral tissue-preferred promoter; (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterologous second promoter, the one or more guide nucleic acids comprising: (A) capable of hybridizing to a target sequence in the genome of a plant; and (B) a second nucleic acid sequence capable of hybridizing to the first site and the second site that flank the nucleic acid sequence encoding the gene of interest; and (iii) a third nucleic acid sequence encoding a gene of interest. or (b) In a plant cell, (iv) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous first promoter; (v) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterologous floral cell-preferred or floral tissue-preferred promoter, wherein the one or more guide nucleic acids (C) capable of hybridizing to a target sequence within the genome of a plant; and (D) a second nucleic acid sequence capable of hybridizing to the first site and the second site that flank the nucleic acid sequence encoding the gene of interest; and (vi) a third nucleic acid sequence encoding a gene of interest. introducing (c) regenerating at least one plant from the plant cells of step (a) or (b). wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generates a double stranded break within the target sequence molecule, the first site, and the second site, and a gene of interest is integrated into the target sequence in the at least one flower cell.

16. 1. A method for generating site-specific integration in a plant, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred promoter; (ii) a second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterologous second promoter, the one or more guide nucleic acids comprising: (A) capable of hybridizing to a target sequence in the genome of a plant; and (B) a second nucleic acid sequence capable of hybridizing to the first site and the second site that flank the nucleic acid sequence encoding the gene of interest; (iii) a third nucleic acid sequence encoding a gene of interest. introducing (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing at least one plant derived from step (b) to produce at least one embryo. wherein the inducible nuclease and the at least one guide RNA form a ribonucleoprotein in the at least one embryo, the ribonucleoprotein generates a double stranded break in the target DNA molecule, the first site, and the second site, and a gene of interest is integrated into the target sequence in the at least one embryo.

17. 1. A method for editing the genome of a plant cell, comprising: (a) crossing a first plant with a second plant, the first plant comprising a first nucleic acid sequence encoding an inducible nuclease capable of generating a staggered break in a double-stranded DNA molecule operably linked to a heterologous floral tissue-preferred or floral cell-preferred promoter, and the second plant comprising a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, the at least one guide nucleic acid being capable of hybridizing to a target sequence in the genome; and (b) obtaining at least one progeny plant from the cross of step (a), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the at least one flower cell, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell. A method comprising:

18. 18. The method of any one of claims 12 to 17, wherein the inducible nuclease is selected from the group consisting of Cas12a, MAD7® and CasX.

19. The floral cell-preferred promoter or the floral tissue-preferred promoter is selected from the group consisting of the A gene promoter, the B gene promoter, the C gene promoter, the D gene promoter, and the E gene promoter, or the floral cell-preferred promoter or the floral tissue-preferred promoter is selected from the group consisting of the AP1 promoter, the AP2 promoter, the ZAP1 promoter, the AP3 promoter, the PI promoter, the ZMM16 promoter, the ZMM18 promoter, the AG promoter, the ZAG1 promoter, the ZMM2 promoter, the ZMM23 promoter, the AGL11 / STK promoter, the AGL1 / SHP1 promoter, 18. The method of any one of claims 12 to 17, wherein the floral cell preferential promoter or floral tissue preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, 45-49, or a functional fragment thereof.

20. 18. The method of any one of claims 12 to 17, wherein the first or second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, a flower cell-preferred promoter, a flower tissue-preferred promoter and a constitutive promoter.

21. 18. The method of any one of claims 12 to 17, wherein the plant is selected from the group consisting of corn, rice, sorghum, wheat, alfalfa, barley, millet, rye, sugarcane, cotton, soybean, canola, tomato, and potato.

22. 18. The method of any one of claims 12 to 17, wherein repair of the double-stranded break generates at least one mutation in the target sequence compared to a control plant of the same line or variety lacking the first nucleic acid sequence or the second nucleic acid sequence.

23. A recombinant DNA construct comprising: (a) a first nucleic acid sequence encoding an inducible nuclease capable of making a staggered cut in a double-stranded DNA molecule operably linked to a heterologous flower cell-preferred or floral tissue-preferred promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter; or (c) a first nucleic acid sequence encoding an inducible nuclease capable of making a staggered cut in a double-stranded DNA molecule operably linked to a heterologous promoter; and (d) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous flower cell-preferred or floral tissue-preferred promoter, wherein the at least one guide nucleic acid comprises a second nucleic acid sequence capable of hybridizing to a target sequence in the genome of a plant.

24. 1. A method for generating two or more progeny plants with unique edits from a single transformed plant cell, comprising: (a) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous floral cell-preferred promoter or a floral tissue-preferred promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous second promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; or (b) In a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating staggered breaks in a double-stranded DNA molecule operably linked to a heterologous first promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterologous floral cell-preferred or floral tissue-preferred promoter, wherein the at least one guide nucleic acid is capable of hybridizing to a target sequence in the genome; introducing (c) regenerating a first plant from the plant cell of step (a) or step (b), wherein the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of the first plant, and the ribonucleoprotein generates at least one double-stranded break in the target sequence in the at least one flower cell; (d) pollinating the first plant of step (c); and (e) germinating the two or more seeds produced from step (d) to produce two or more progeny plants having unique edits. A method comprising:

25. 25. The method of claim 24, wherein the inducible nuclease is selected from the group consisting of Cas12a, MAD7® and CasX.

26. The floral cell-preferred promoter or the floral tissue-preferred promoter is selected from the group consisting of A gene promoter, B gene promoter, C gene promoter, D gene promoter, and E gene promoter, or the floral cell-preferred promoter or the floral tissue-preferred promoter is selected from the group consisting of AP1 promoter, AP2 promoter, ZAP1 promoter, AP3 promoter, PI promoter, ZMM16 promoter, ZMM18 promoter, AG promoter, ZAG1 promoter, ZMM2 promoter, ZMM23 promoter, AGL11 / STK promoter, AGL1 / SHP1 promoter, 25. The method of claim 24, wherein the promoter is selected from the group consisting of a motor, an AGL5 / SHP2 promoter, a ZAG2 promoter, a ZMM1 promoter, a SEP1 promoter, a SEP2 promoter, a SEP3 promoter, a SEP4 promoter, a ZAG3 promoter, and a ZMM7 / SEP-like promoter, or the floral cell preferred promoter or the floral tissue preferred promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-30, or a nucleic acid sequence that is at least 90% identical to a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-16, 18-19, 21-30, 45-49, or a functional fragment thereof.

27. 25. The method of claim 24, wherein the first or second promoter is selected from the group consisting of a tissue-preferred promoter, a tissue-specific promoter, an inducible promoter, a floral cell-preferred promoter, a floral tissue-preferred promoter and a constitutive promoter.

28. 25. The method of claim 24, wherein the plant cell is selected from the group consisting of corn, rice, sorghum, wheat, alfalfa, barley, millet, rye, sugarcane, cotton, soybean, canola, tomato, and potato.

29. 25. The method of claim 24, wherein repair of the double-stranded break generates at least one mutation in the target sequence compared to a control plant of the same line or variety lacking the first nucleic acid sequence or the second nucleic acid sequence.

30. 25. The method of claim 24, wherein the first plant is self-pollinated.

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