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 related to the expression of inducible nucleases and guide nucleic acids in flower cells and flower 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 on March 29, 2022, the entire content of which is incorporated herein by reference.

[0003] Incorporation of sequence listing 132 which is in kilobytes (measured in MS - Windows®), 2025 March 10 and is named "P34740WO00_SL 2 .XML" and is electronically submitted herewith and incorporated herein by reference in its entirety.

Background Art

[0004] CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) nucleases (e.g., Cas12a, CasX, Cas9) are proteins that are guided by guide RNAs to target nucleic acid molecules, and the nuclease can cleave one or both strands of the target nucleic acid molecule.

Prior Art Documents

Patent Documents

[0005] [[ID=4l]]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

Patent Document 5

[0007] In one embodiment, the disclosure provides a plant comprising (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter or a floral cell-preferential promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the plant genome.

[0008] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize 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-strand break in a target sequence in the at least one flower cell.

[0009] In one embodiment, the present disclosure provides a method for 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 producing alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize 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 at least one embryo, and the ribonucleoprotein produces at least one double-strand break in a target sequence in at least one embryo.

[0010] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising the steps of: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing 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 at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one embryo.

[0011] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome; and (B) a heterogeneous second promoter capable of hybridizing to first and second sites flanking the nucleic acid sequence encoding the gene of interest. The method comprises (iii) introducing a second nucleic acid sequence encoding one or more movably linked guide nucleic acids; (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest being incorporated into the target site in at least one flower cell.

[0012] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell containing (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter; and (ii) one or more guide nucleic acids operably linked to a heterogeneous second promoter, the guide nucleic acids being capable of (A) hybridizing to a target sequence in the plant genome; and (B) hybridizing to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method comprises (iii) introducing a second nucleic acid sequence encoding the gene of interest; (b) regenerating at least one plant from the plant cells 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 at least one guide RNA form a ribonucleoprotein in at least one embryo, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest is incorporated into the target site in at least one embryo.

[0013] In one embodiment, the disclosure provides a recombinant DNA construct comprising (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably ligated to a heterogeneous floral tissue-preferential promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably ligated to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the plant genome.

[0014] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize 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-strand break in a target sequence in the at least one flower cell.

[0015] In one embodiment, the present disclosure provides a method for 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 producing alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; and (b) obtaining at least one offspring 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 floral cell, and the ribonucleoprotein produces at least one double-strand break in a target sequence in at least one floral cell.

[0016] In one embodiment, the present disclosure provides a method for 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 producing alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; and (b) obtaining at least one offspring 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 produces at least one double-strand break in a target sequence in at least one flower cell.

[0017] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell having (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter; and (ii) one or more guide nucleic acids operably linked to a heterogeneous second promoter, which can (A) hybridize to a target sequence in the plant genome and (B) hybridize to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method comprises (iii) introducing a second nucleic acid sequence encoding one or more guide nucleic acids linked to a target sequence molecule; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generating double-strand breaks in the target sequence molecule, a first site, and a second site, and the gene of interest is incorporated into the target site in at least one flower cell.

[0018] In one embodiment, the present disclosure is a method for generating two or more offspring plants having unique editing from a single transformed plant cell, comprising: (a) a plant cell comprising: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cuts 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 can hybridize to a target sequence in the genome. The present invention provides a method comprising: (b) introducing a nucleic acid sequence 2; (c) regenerating a first plant from the plant cells of step (a), wherein an inducible nuclease and 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-strand break in a target sequence in at least one flower cell; (d) pollinating the first plant of step (b); and (e) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing.

[0019] In one embodiment, the present disclosure is a method for generating two or more progeny plants having unique editing from a single transformed plant cell, comprising: (a) a plant cell comprising: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating 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 can hybridize to a target sequence in the genome. The present invention provides a method comprising: (b) introducing a nucleic acid sequence 2; (c) regenerating a first plant from the plant cells of step (a), wherein an inducible nuclease and 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-strand break in a target sequence in at least one flower cell; (d) pollinating the first plant of step (b); and (e) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing.

[0020] In one embodiment, the present disclosure provides a method for producing two or more offspring plants having unique editing from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of producing alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize 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) such that the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, the ribonucleoprotein producing at least one double-strand break in the target sequence in the floral tissue; and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing.

[0021] In one embodiment, the present disclosure provides a method for producing two or more offspring plants having unique editing from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of producing alternating breaks in a double-stranded DNA molecule operably linked to a first 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 can hybridize 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) such that the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, the ribonucleoprotein producing at least one double-strand break in the target sequence in the floral tissue; and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing. [Modes for carrying out the invention]

[0022] Unless otherwise defined, all technical and scientific terms used have the same meaning as those generally understood by those skilled in the art to which this disclosure belongs. Where a term is provided in the singular form, the inventors also intend aspects of this disclosure described by that term in the plural form. Where there are differences 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 common meanings 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 limit themselves to mechanisms or modes of operation. The references are provided for illustrative purposes only.

[0023] Unless otherwise noted, the implementation of this disclosure includes, but is not limited to, prior art in biochemistry, chemistry, molecular biology, microbiology, cell biology, plant biology, genomics, biotechnology, and genetics, which are within the scope of the art in this field. For example, 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 and T. Tzfira (eds.) (2011) Plant Transformation Technologies See Wiley-Blackwell and RH Smith (2013) Plant Tissue Culture: Techniques and Experiments (Academic Press, Inc.).

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

[0025] When a set of options is presented, all possible combinations of the members constituting that set of options are specifically envisioned. For example, if an item is selected from a group consisting of A, B, C, and D, the inventors specifically envision 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 singular forms of “a,” “an,” and “the” refer to multiple subjects, for example, unless otherwise explicitly indicated by the text.

[0027] Any composition, nucleic acid molecule, polypeptide, cell, plant, etc., provided herein is specifically intended for use in conjunction with any method provided herein.

[0028] In one embodiment, the disclosure provides a recombinant DNA construct comprising: (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the plant genome.

[0029] In one embodiment, the present disclosure provides a recombinant DNA construct comprising (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage 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, such as a heterologous floral tissue-preferential promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the plant genome.

[0030] In one embodiment, the disclosure provides a plant comprising (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the plant genome.

[0031] In one embodiment, the disclosure provides a plant comprising a recombinant DNA construct comprising (b) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage 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 can hybridize to a target sequence in the plant genome.

[0032] In some embodiments, the disclosure provides a plant comprising (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter or a floral cell-preferential promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the plant genome. In some embodiments, the disclosure provides a plant comprising (a) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage 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 heterogeneous floral tissue-preferential promoter or a floral cell-preferential promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the plant genome.

[0033] In some embodiments, the disclosure provides seeds of any plant provided herein.

[0034] As used herein, “floral tissue” refers to any tissue or cells that give rise to any part of a flower, excluding the apical meristem. Non-limiting examples of floral tissue include branch meristem, axillary bud meristem, inflorescence meristem, flower bud meristem, outer glume, inner glume, scales, pedicel, receptacle, sepals, petals, stigma, style, filament, and anther. As used herein, “floral tissue” does not refer to the ovary, ovule, or pollen.

[0035] In some embodiments, the floral tissue includes structures selected from the group consisting of branch meristems, axillary bud meristems, inflorescence meristems, flower bud meristems, outer glumes, inner glumes, scales, pedicels, receptacles, sepals, petals, stigmas, styles, filaments, and anthers.

[0036] As a plant transitions from vegetative growth to flowering, the apical meristem is transformed into the inflorescence meristem. The inflorescence meristem then produces the flower bud meristem, which ultimately gives rise to all the tissues of the flower, including, but not limited to, the outer glume, inner glume, scales, receptacle, sepals, petals, stigma, style, filaments, and anthers. In some embodiments, the flower tissue includes the inflorescence meristem. In some embodiments, the flower tissue includes the flower bud meristem. In some embodiments, the flower tissue includes the branch meristem. In herbs, the branch meristem is produced by the inflorescence meristem, which produces branches or spikelets in two parallel arrangements, patterning the floral organs in whorled phyllotaxy.

[0037] In some embodiments, the floral tissue includes the pedicel. In some embodiments, the floral tissue includes the outer glume. In some embodiments, the floral tissue includes the inner glume. In some embodiments, the floral tissue includes the scales. In some embodiments, the floral tissue includes the receptacle. In some embodiments, the floral tissue includes the sepals. In some embodiments, the floral tissue includes the petals. In some embodiments, the floral tissue includes the stigma. In some embodiments, the floral tissue includes the style. In some embodiments, the floral tissue includes the filaments. In some embodiments, the floral tissue includes the anthers.

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

[0039] As used herein, “floral cell” refers to any cell in the floral tissue. In some aspects, a floral cell is a branch meristem cell. In some aspects, a floral cell is an inflorescence meristem cell. In some aspects, a floral cell is a bud meristem cell. In some aspects, a floral cell is a pedicel cell. In some aspects, the floral tissue is an exoglacial cell. In some aspects, the floral tissue is an inoglacial cell. In some aspects, the floral cell is a scaly cell. In some aspects, the floral tissue is a receptacle cell. In some aspects, the floral tissue is a sepal cell. In some aspects, the floral cell is a petal cell. In some aspects, the floral tissue is a stigma cell. In some aspects, the floral tissue is a style cell. In some aspects, the floral cell is a filament cell. In some aspects, the floral tissue is an anther cell.

[0040] Nucleic acids and amino acids The use of the terms “polynucleotide” or “nucleic acid molecule” is not intended to limit this disclosure to polynucleotides containing deoxyribonucleic acid (DNA). For example, ribonucleic acid (RNA) molecules are also envisioned. Those skilled in the art will recognize that polynucleotides and nucleic acid molecules may include deoxyribonucleotides, ribonucleotides, or combinations of ribonucleotides and deoxyribonucleotides. Such deoxyribonucleotides and ribonucleotides include both naturally occurring molecules and synthetic analogs. The polynucleotides of this disclosure also encompass, but are not limited to, all forms of sequences, including single-stranded, double-stranded, hairpin, stem-loop structures, etc. In some embodiments, the nucleic acid molecule provided herein is a DNA molecule. In other embodiments, the nucleic acid molecule provided herein is an RNA molecule. In some embodiments, the nucleic acid molecule provided herein is single-stranded. In other embodiments, the nucleic acid molecule provided herein is double-stranded.

[0041] As used herein, the term “recombinant” refers to nucleic acid (DNA or RNA) molecules, proteins, constructs, vectors, etc., which include combinations of polynucleotides or protein sequences that do not naturally exist adjacent to or very close to each other without human intervention, and / or polynucleotide molecules, proteins, constructs, etc., which include at least two polynucleotides or protein sequences that are heterogeneous with respect to each other, which are artificial, not normally found in nature, and / or exist in circumstances in which they are not normally found in nature.

[0042] In one embodiment, the methods and compositions provided herein include a vector. As used herein, the term “vector” refers to a DNA molecule used as a vehicle for transporting exogenous genetic material into a cell.

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

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

[0045] As used herein, the term “polypeptide” refers to a chain of at least two covalently linked amino acids. Polypeptides may be encoded by polynucleotides provided herein. An example of a polypeptide is a protein. Proteins provided herein may be encoded by nucleic acid molecules provided herein.

[0046] Nucleic acids can be isolated using techniques commonplace 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). Common PCR techniques are described, for example, in *PCR Primer: A Laboratory Manual*, edited by Dieffenbach & Dveksler, 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 a single nucleic acid molecule 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 nucleic acids in an expression vector. Furthermore, purified polypeptides can be obtained by chemical synthesis. The purity of polypeptides can be measured using any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0047] Nucleic acids can be detected using hybridization, though not exclusively. Hybridization between nucleic acids is discussed in detail by Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual, 2nd edition, 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 blotting, immunoprecipitation, and immunofluorescence. The antibodies provided herein may be polyclonal or monoclonal antibodies. Antibodies having specific binding affinity to the polypeptides provided herein can be produced using methods known in the art. The antibodies provided herein can be attached to a solid support such as a microtiter plate using methods known in the art.

[0049] As used herein in reference to two or more nucleotide or protein sequences, the term “percent identity” or “percent identical” is calculated by (i) comparing two optimally aligned sequences (nucleotides or proteins) on a comparison window; (ii) determining the number of positions in both sequences where identical nucleic acid bases (for nucleotide sequences) or amino acid residues (for proteins) exist, thereby obtaining the number of matching positions; (iii) dividing the number of matching positions by the total number of positions in the comparison window; and then (iv) multiplying this quotient by 100% to obtain the percentage identity. When “percent identity” is calculated with respect to a reference sequence without specifying a particular comparison window, the percentage identity is determined by dividing the number of matching positions on the alignment region by the total length of the reference sequence. Therefore, for the purposes of this application, when two sequences (query and target) are optimally aligned (allowing for gaps in their alignment), the "percent identity" of the query sequence is equal to the number of identical positions between the two sequences obtained by dividing by the total number of positions in the query sequence over its entire length (or comparison window) and then multiplying by 100%. When the percentage of sequence identity is used in reference to a protein, it is recognized that non-identical residue positions are often distinguished by conservative amino acid substitutions, where the amino acid residue is substituted for another amino acid residue having similar chemical properties (e.g., charge or hydrophobicity), and therefore does not change the functional properties of the molecule. If sequences differ in conservative substitutions, the percentage of sequence identity can be adjusted upward to compensate for the conservative nature of the substitutions. 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 in 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 with or hybridize with the nucleotides of the target sequence, given that the query sequence and the target sequence are linearly arranged and optimally base-pair 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. "Percent 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) on a comparison window, (ii) determining the number of base-pairing positions between the two sequences on 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. Optimal base-pairing of two sequences can be determined based on the pairing of known nucleotide bases such as GC, AT, and AU via hydrogen bonding. When "percent complementarity" is calculated with respect to a reference sequence without specifying a particular comparison window, percentage identity is determined by dividing the number of complementary positions between the two linear sequences by the total length of the reference sequence. Therefore, for the purposes of this application, when the two sequences (query and target) optimally form base pairs (allowing for mismatches or nucleotides that do not form base pairs), the "percent complementarity" of the query sequence is equal to the number of base-paired positions between the two sequences, obtained by dividing the query sequence by the total number of positions in the query sequence over its entire length and then multiplying by 100%.

[0051] Various pairwise or multi-sequence alignment algorithms and programs, such as ClustalW or Basic Local Alignment Search Tool (BLAST®), are known in the art and can be used to compare the sequence identity or similarity between two or more nucleotide or protein sequences for their optimal alignment to calculate the percentage identity of sequences. Although other alignment and comparison methods are known in the art, the alignment and percentage identity (including the percentage identity range described above) between two sequences 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 See 215:403–410 (1990) (the entire contents and disclosures of these are incorporated herein by reference).

[0052] As used herein, a first nucleic acid molecule can "hybridize" to a second nucleic acid molecule by non-covalent interactions (e.g., Watson-Crick base pairing) in a sequence-specific, antiparallel manner (i.e., nucleic acid specifically binds to complementary nucleic acid) under appropriate in vitro and / or in vivo conditions of temperature and solution ionic strength. As is known in the art, standard Watson-Crick base pairings 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 contribute to 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, guanine in the protein-binding segment (dsRNA double helix) of the target DNA-targeting RNA molecule is considered complementary to uracil, and vice versa. Therefore, if a G / U base pair can be constructed at a given nucleotide position on the protein-binding segment (dsRNA double helix) of the target DNA-targeting RNA molecule, that position is not considered non-complementary, but rather 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), particularly in Chapter 11 and Table 11.1; and in Sambrook, J. and Russell, W., Molecular Cloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (2001). Temperature and ionic strength conditions determine the "stringency" of hybridization.

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

[0055] It is understood in the art that the sequence of a polynucleotide does not need to be specifically hybridizable or 100% complementary to the sequence of its target nucleic acid that is hybridizable. Furthermore, a polynucleotide may hybridize across one or more segments (e.g., loop or hairpin structures) such that intervening or adjacent segments do not participate in the hybridization event. For example, if 18 of the 20 nucleotides in an antisense compound are complementary to the target region, and therefore the antisense nucleic acid that specifically hybridizes represents 90 percent complementarity. In this example, the remaining non-complementary nucleotides may cluster with complementary nucleotides or be scattered, and do not need to be contiguous with each other or with complementary nucleotides. The percentage complementarity between specific ranges of nucleic acid sequences within a nucleic acid can be routinely determined using the BLAST® program (basic local alignment search tools) and the PowerBLAST program (see Altschul et al., J. Mol. Biol., 1990, 215, pp. 403-410; Zhang and Madden, Genome Res., 1997, 7, pp. 649-656), which are known in the art, or by using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, pp. 482-489) and the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) with default settings.

[0056] Edit generation In some embodiments, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous flower tissue-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize 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-strand break in a target sequence in at least one flower cell.

[0057] In some embodiments, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks 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 flower tissue preferential promoter, wherein the at least one guide nucleic acid can hybridize 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-strand break in a target sequence in at least one flower cell.

[0058] In some embodiments, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize 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-strand break in a target sequence in the at least one flower cell.

[0059] In some embodiments, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks 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 can hybridize 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-strand break in a target sequence in at least one flower cell.

[0060] In one embodiment, the present disclosure provides a method for editing the genome of a plant cell, comprising the steps of (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 producing alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize 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 at least one embryo, and the ribonucleoprotein produces at least one double-strand break in a target sequence in at least one embryo.

[0061] In one embodiment, the present disclosure provides a method for editing the genome of a plant cell, comprising the steps of (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 producing alternating breaks in a double-stranded DNA molecule operably linked to a heterologous promoter, and the second plant comprises 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 can hybridize 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 at least one embryo, and the ribonucleoprotein produces at least one double-strand break in a target sequence in at least one embryo.

[0062] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising the steps of (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing 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 at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one embryo.

[0063] In one embodiment, the present disclosure provides a method for editing a plant genome, comprising the steps of (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks 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 can hybridize to a target sequence in the genome; (b) regenerating at least one plant from the plant cell of step (a); and (c) fertilizing 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 at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one embryo.

[0064] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell containing (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome; and (B) a heterogeneous second promoter capable of hybridizing to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method provides comprising the steps of (iii) introducing a second nucleic acid sequence encoding one or more operably linked guide nucleic acids; and (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest being incorporated into the target site in at least one flower cell.

[0065] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell comprising (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome; and (B) a first and second site flanking the nucleic acid sequence encoding the gene of interest, wherein the second promoter is floral tissue-preferential. The present invention provides a method comprising: (iii) introducing a second nucleic acid sequence encoding one or more movably linked guide nucleic acids; and (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest being incorporated into the target site in at least one flower cell.

[0066] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell comprising: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome; and (B) a heterogeneous second promoter capable of hybridizing to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method provides comprising the steps of (iii) introducing a second nucleic acid sequence encoding one or more operably linked guide nucleic acids; and (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest being incorporated into the target site in at least one flower cell.

[0067] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell comprising: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome; and (B) a first and second site flanking the nucleic acid sequence encoding the gene of interest, wherein the second promoter is flower cell-preferential. The present invention provides a method comprising: (iii) introducing a second nucleic acid sequence encoding one or more movably linked guide nucleic acids; and (iii) introducing a third nucleic acid sequence encoding a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest being incorporated into the target site in at least one flower cell.

[0068] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell containing (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter; and (ii) one or more guide nucleic acids operably linked to a heterogeneous second promoter, the guide nucleic acids being capable of (A) hybridizing to a target sequence in the plant genome; and (B) hybridizing to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method comprises (iii) introducing a second nucleic acid sequence encoding a target gene; (b) regenerating at least one plant from the plant cells 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 at least one guide RNA form a ribonucleoprotein in at least one embryo, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the target gene is incorporated into the target site in at least one embryo.

[0069] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell, (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterologous promoter; and (ii) one or more guide nucleic acids encoding one or more guide nucleic acids operably linked to a floral tissue-preferential promoter, which can (A) hybridize to a target sequence in the plant genome; and (B) hybridize to first and second sites flanking the nucleic acid sequence encoding the gene of interest. The method comprises (iii) introducing a second nucleic acid sequence; and (b) introducing a third nucleic acid sequence encoding the gene of interest; (c) regenerating at least one plant from the plant cells of step (a); and (b) fertilizing at least one plant derived from step (b) to produce at least one embryo; wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one embryo, the ribonucleoprotein generating double-strand breaks in the target sequence, a first site, and a second site, and the gene of interest is incorporated into the target site in at least one embryo.

[0070] In some embodiments, the present disclosure provides a method for editing a plant genome, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize 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-strand break in a target sequence in the at least one flower cell.

[0071] In some embodiments, the present disclosure provides a method for 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 producing alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; and (b) obtaining at least one offspring 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 floral cell, and the ribonucleoprotein produces at least one double-strand break in a target sequence in at least one floral cell.

[0072] In some embodiments, the present disclosure provides a method for 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 producing alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome; and (b) obtaining at least one offspring 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 produces at least one double-strand break in a target sequence in at least one flower cell.

[0073] In one embodiment, the present disclosure is a method for generating site-directed integration in a plant, comprising: (a) a plant cell having (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter; and (ii) one or more guide nucleic acids capable of hybridizing to (A) a target sequence in the plant genome and (B) a heterogeneous second promoter capable of hybridizing to a first and second site flanking the nucleic acid sequence encoding the gene of interest. The method comprises (iii) introducing a second nucleic acid sequence encoding one or more guide nucleic acids linked to a gene of interest; and (b) regenerating at least one plant from the plant cells of step (a); wherein the inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one flower cell of a plant, the ribonucleoprotein generating double-strand breaks in the target sequence molecule, a first site, and a second site, and the gene of interest is incorporated into the target site in at least one flower cell.

[0074] In one embodiment, the present disclosure is a method for generating two or more progeny plants having unique edits from a single transformed plant cell, comprising: (a) a plant cell comprising: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cuts in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize to a target sequence in the genome. The present invention provides a method comprising: (b) introducing a nucleic acid sequence 2; (c) regenerating a first plant from the plant cells of step (a), wherein an inducible nuclease and 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-strand break in a target sequence in at least one flower cell; (d) pollinating the first plant of step (b); and (e) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing.

[0075] In one embodiment, the present disclosure is a method for generating two or more progeny plants having unique edits from a single transformed plant cell, comprising: (a) a plant cell comprising: (i) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating 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 can hybridize to a target sequence in the genome. The present invention provides a method comprising: (b) introducing a nucleic acid sequence 2; (c) regenerating a first plant from the plant cells of step (a), wherein an inducible nuclease and 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-strand break in a target sequence in at least one flower cell; (d) pollinating the first plant of step (b); and (e) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing.

[0076] In some embodiments, the present disclosure provides a method for producing two or more offspring plants having unique editing from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of producing alternating breaks in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter; and (ii) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, wherein the at least one guide nucleic acid can hybridize 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) such that the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, the ribonucleoprotein producing at least one double-strand break in the target sequence in the floral tissue; and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing.

[0077] In some embodiments, the present disclosure provides a method for producing two or more offspring plants having unique editing from a single transformed plant cell, comprising: (a) introducing into a plant cell a first nucleic acid sequence encoding an inducible nuclease capable of producing alternating breaks in a double-stranded DNA molecule operably linked to a first 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 can hybridize 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) such that the inducible nuclease and the at least one guide nucleic acid form a ribonucleoprotein in the floral tissue, the ribonucleoprotein producing at least one double-strand break in the target sequence in the floral tissue; and (d) germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing.

[0078] promoter As is commonly 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 that assists or promotes the transcription and expression of a bound, transcribed polynucleotide sequence and / or gene (or transgene). Promoters can be synthetically produced, known, or naturally occurring promoter sequences, or modified or induced from other promoter sequences. Promoters may also include a leader, a 5'UTR, and introns. Promoters may also include chimeric promoters that include a combination of two or more heterologous sequences. Accordingly, promoters of this application may include variants of promoter sequences that are similar in composition but are not identical to known or other promoter sequences provided herein. Promoters can be classified according to various criteria related to the expression pattern of the associated code or transcribed sequence or gene (including transgenes) operably linked to the promoter, such as constitutive, developmental, tissue-specific, cell cycle-specific, and inducible.

[0079] As used herein, “operably linked” refers to a functional link between two or more elements. For example, an operably linked link between a polynucleotide of interest and a regulatory element (e.g., a promoter) is a functional link that enables the expression of the polynucleotide of interest. The operably linked elements may be continuous or discontinuous.

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

[0081] A promoter that is expressed in specific tissues of an organism but not in other tissues is called a “tissue-specific” promoter. A promoter that drives enhanced expression in certain tissues of an organism compared to other tissues of the organism is called a “tissue-preferential” promoter. Thus, a “tissue-preferential” promoter will cause relatively high or preferential expression in certain tissues of a plant, but will show lower levels of expression in other tissues of the plant. In another embodiment, the promoters provided herein are tissue-specific promoters. In yet another embodiment, the promoters provided herein are tissue-preferential promoters. In some embodiments, a tissue-preferential promoter includes a tissue-specific promoter.

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

[0083] Flowers are organized into concentric spirals of sepals, petals, stamens (e.g., structures containing filaments and anthers), and carpels (e.g., structures containing the ovary, stigma, and often the style), and each of these floral organ types plays a unique role in reproduction. The genes involved in establishing floral structure are homeotic MADS domain transcription factors (with the exception of APATELA2, which is gene A; see below) classified as A, B, C, D, or E genes by the ABCDE model of florogenesis, which has been used to describe how floral structure is genetically determined. See, for example, Coen and Meyerowitz, Nature, 353: pp. 31-37 (1991); and Murai, Plants, 2: pp. 379-395 (2013). Under the ABCDE model, sepals are formed when genes A and E are expressed simultaneously; carpels are formed when genes C and E are expressed simultaneously; petals are formed when genes A, B, and E are expressed simultaneously; stamens are formed when genes B, C, and E are expressed simultaneously; and ovules are formed when genes D and E are expressed simultaneously.

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

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

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

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

[0088] In one embodiment, the flower tissue-preferential promoter includes a promoter 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.

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

[0090] In one embodiment, the flower cell-preferential promoter includes a promoter 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.

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

[0092] In one embodiment, the floral tissue preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4It includes a sequence or functional fragment thereof that is at least 70% identical to a sequence selected from group 9. In one embodiment, the floral tissue preferential promoter is selected from the group consisting of sequence numbers 1 to 30, or sequence numbers 1 to 16, 18 to 19, 21 to 30 and bi4 It includes a sequence or functional fragment thereof that is at least 75% identical to a sequence selected from group 9. In one embodiment, the floral tissue preferential promoter is selected from the group consisting of sequence numbers 1 to 30, or sequence numbers 1 to 16, 18 to 19, 21 to 30 and bi4 It includes a sequence or functional fragment thereof that is at least 80% identical to a sequence selected from group 9. In one embodiment, the floral tissue preferential promoter is selected from the group consisting of sequence numbers 1 to 30, or sequence numbers 1 to 16, 18 to 19, 21 to 30 and bi4 It includes a sequence or functional fragment thereof that is at least 85% identical to a sequence selected from group 9. In one embodiment, the floral tissue preferential promoter is selected from the group consisting of sequence numbers 1 to 30, or sequence numbers 1 to 16, 18 to 19, 21 to 30 and bi4 It includes a sequence or functional fragment thereof that is at least 90% identical to a sequence selected from group 9. In one embodiment, the floral tissue preferential promoter is selected from the group consisting of sequence numbers 1 to 30, or sequence numbers 1 to 16, 18 to 19, 21 to 30 and bi4 It includes a sequence or functional fragment thereof that is at least 95% identical to a sequence selected from group 9. In one embodiment, the floral tissue preferential promoter is selected from the group consisting of sequence numbers 1 to 30, or sequence numbers 1 to 16, 18 to 19, 21 to 30 and bi4 It includes a sequence or functional fragment thereof that is at least 99% identical to a sequence selected from group 9. In one embodiment, the floral tissue preferential promoter is selected from the group consisting of sequence numbers 1 to 30, or sequence numbers 1 to 16, 18 to 19, 21 to 30 and bi4 It includes a sequence or functional fragment thereof that is 100% identical to a sequence selected from the 9 groups.

[0093] In one embodiment, the flower cell-preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4The sequence includes a sequence or a functional fragment thereof that is at least 70% identical to a sequence selected from group 9. In one embodiment, the flower cell preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 It includes a sequence or functional fragment thereof that is at least 75% identical to a sequence selected from group 9. In one embodiment, the flower cell preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 It includes a sequence or functional fragment thereof that is at least 80% identical to a sequence selected from group 9. In one embodiment, the flower cell preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 It includes a sequence or functional fragment thereof that is at least 85% identical to a sequence selected from group 9. In one embodiment, the flower cell preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 It includes a sequence or functional fragment thereof that is at least 90% identical to a sequence selected from group 9. In one embodiment, the flower cell preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 It includes a sequence or functional fragment thereof that is at least 95% identical to a sequence selected from group 9. In one embodiment, the flower cell preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 It includes a sequence or functional fragment thereof that is at least 99% identical to a sequence selected from group 9. In one embodiment, the flower cell preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 It includes a sequence or functional fragment thereof that is 100% identical to a sequence selected from the 9 groups.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

[0126] In some embodiments, the ZMM7 / SEP-like promoter is operably ligated to a nucleic acid encoding an inducible nuclease. In some embodiments, the ZMM7 / SEP-like promoter is operably ligated to a nucleic acid encoding a Cas12a nuclease. In some embodiments, the ZMM7 / SEP-like promoter is operably ligated to a nucleic acid encoding a CasX nuclease. In some embodiments, the ZMM7 promoter is operably ligated to a nucleic acid encoding a MAD7® nuclease. In some embodiments, the ZMM7 / SEP-like promoter is operably ligated to a nucleic acid encoding a guide nucleic acid. In some embodiments, the ZMM7 / SEP-like promoter is operably ligated to a nucleic acid encoding a guide RNA. In some embodiments, the ZMM7 / SEP-like promoter is operably ligated 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 SEQ ID NOs: 1 to 16, 18 to 19, 21 to 30 and bi4 A promoter, or a functional fragment thereof, selected from the group of 9, is operably ligated to a nucleic acid encoding an inducible nuclease. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 A promoter, or a functional fragment thereof, selected from the group of 9, is operably ligated to a nucleic acid encoding Cas12a nuclease. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 A promoter, or a functional fragment thereof, selected from the group of 9, is operably ligated 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 SEQ ID NOs: 1-16, 18-19, 21-30 and bi4A promoter selected from the group of 9, or a functional fragment thereof, is operably ligated to a nucleic acid encoding the MAD7® nuclease. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 A promoter, or a functional fragment thereof, selected from the group of 9, is operably ligated 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 SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 A promoter, or a functional fragment thereof, selected from the group of 9, is operably ligated to a nucleic acid encoding a guide RNA. In one embodiment, a promoter selected from the group consisting of SEQ ID NOs. 1-30, or SEQ ID NOs. 1-16, 18-19, 21-30 and bi4 A promoter, or a functional fragment thereof, selected from the nine groups, is operably ligated to a nucleic acid encoding a single guide RNA.

[0128] As used herein, “floral tissue-preferential promoter” refers to a promoter that exhibits higher or preferential expression in floral tissue compared to other cell or tissue types of the plant. A floral tissue-preferential promoter may exhibit expression in any floral tissue, and, but is not limited to, neighboring cells or tissues such as stem cells, vascular cells, and trichrome cells. A floral tissue-preferential promoter may also exhibit expression in other plant tissues or cells, but is 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, “floral tissue-specific promoter” refers to a promoter that is expressed only in floral tissue. In some embodiments, a floral tissue-preferential promoter includes a floral tissue-specific promoter.

[0130] As used herein, “flower cell-preferential promoter” refers to a promoter that exhibits higher or preferential expression in flower cells compared to other cell or tissue types of the plant. A flower cell-preferential promoter may exhibit expression in any flower cell, and, but is not limited to, neighboring cells or tissues such as stem cells, vascular cells, and trichrome cells. A flower cell-preferential promoter may also exhibit expression in other plant tissues or cells, but is 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, “flower cell-specific promoter” refers to a promoter that is expressed only in flower cells. In some embodiments, a flower cell-preferential promoter includes a flower cell-specific promoter.

[0132] It is understood in the art that a fragment of a promoter sequence can function to drive the transcription of a operably linked nucleic acid molecule. For example, 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 the “functional fragment.” It is understood that a promoter may also be a variant. As used herein, the term “variant” means a second DNA molecule, such as a regulatory element, having a composition similar to but not identical to a first DNA molecule, where the second DNA molecule still maintains overall functionality, i.e., the same or similar expression pattern, for example, by more or less equivalent transcriptional activity to the first DNA molecule. A variant may be a shorter, longer, or truncated version of the first DNA molecule, or a modified version of the first DNA molecule, such as having different restriction enzyme sites and / or internal deletions, substitutions, or insertions. The “variant” may also encompass a regulatory element having a nucleotide sequence comprising substitution, deletion, or insertion of one or more nucleotides in a reference sequence, wherein the derivative regulatory element has higher, lower, or equivalent transcriptional or translational activity than the corresponding parent regulatory molecule.

[0133] Promoter that drives expression in all or many tissues of a plant is called a “constitutive” promoter. Promoter that drives expression during a specific period or stage of development is called a “developmental” promoter. “Inducible” promoters are promoters that initiate transcription in response to environmental stimuli such as heat, cold, drought, or light, or other stimuli such as wounds or chemical application. Promoter can also be classified in terms of their origin, such as heterogeneous, homogeneous, chimeric, or synthetic.

[0134] As used herein, the term “heterogeneous” refers to a promoter that has a different origin from its associated transcriptable DNA sequence, coding sequence, or gene (or transgene) and / or is not naturally occurring in the plant to be transformed. More broadly, the term “heterogeneous” may refer to a promoter and a combination of two or more DNA molecules or sequences, such as an associated transcriptable DNA sequence, coding sequence, or gene, in which case such a combination is artificial and not typically found in nature.

[0135] In some embodiments, the promoters provided herein are constitutive promoters. In yet another embodiment, the promoters provided herein are inductive promoters. In some embodiments, the promoters provided herein are selected from the group consisting of constitutive promoters, tissue-specific promoters, tissue-preferential promoters, and inductive promoters.

[0136] The expression of non-protein-coding RNA molecules can be driven using an RNA polymerase III (PolIII) promoter. In one embodiment, the promoter provided herein is a PolIII promoter. In another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding non-protein-coding RNA. In yet another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding a guide nucleic acid. In yet another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding a single guide RNA. In yet another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding CRISPR RNA (crRNA). In yet another embodiment, the PolIII promoter provided herein is operably ligated to a nucleic acid molecule encoding tracer RNA (tracrRNA).

[0137] Non-limiting examples of PolIII promoters include the 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: pp. 2593–2620, which is incorporated herein by reference in its entirety. In one embodiment, the PolIII promoter provided herein is selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another embodiment, the guide RNA provided herein is operably ligated to a promoter selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another embodiment, the single guide RNA provided herein is operably ligated to a promoter selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another embodiment, the CRISPR RNA provided herein is operably ligated to a promoter selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter. In another embodiment, the tracer RNA provided herein is operably ligated to a promoter selected from the group consisting of the U6 promoter, H1 promoter, 5S promoter, adenovirus type 2 (Ad2) VAI promoter, tRNA promoter, and 7SK promoter.

[0138] In one embodiment, the promoter provided herein is a dahlia mosaic virus (DaMV) promoter. In another embodiment, the promoter provided herein is a U6 promoter. In yet another embodiment, the promoter provided herein is an actin promoter. In one embodiment, the promoter provided herein is a cauliflower mosaic virus (CaMV) 35S promoter. In one embodiment, the promoter provided herein is a ubiquitin promoter.

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

[0140] Examples of promoters that may be used herein include, but are not limited to, U.S. Patent No. 6,437,217 (Maize RS81 Promoter), U.S. Patent No. 5,641,876 (Comeactin Promoter), U.S. Patent No. 6,426,446 (Maize RS324 Promoter), U.S. Patent No. 6,429,362 (Maize PR-1 Promoter), U.S. Patent No. 6,232,526 (Maize A3 Promoter), U.S. Patent No. 6,177,611 (Constitutive Maize Promoter), U.S. Patents No. 5,322,938, No. 5,352,605, No. 5,359,142 and No. 5,530,196 (35S Promoter), U.S. Examples include Japanese Patent No. 6,433,252 (Maize L3 Oleosin Promoter), U.S. Patent No. 6,429,357 (Comeactin 2 Promoter and Comeactin 2 Intron), U.S. Patent No. 5,837,848 (Root-Specific Promoter), U.S. Patent No. 6,294,714 (Photo-Inducible Promoter), U.S. Patent No. 6,140,078 (Salt-Inducible Promoter), U.S. Patent No. 6,252,138 (Pathogen-Inducible Promoter), U.S. Patent No. 6,175,060 (Phosphorus Deficiency-Inducible Promoter), U.S. Patent No. 6,635,806 (Gamma-Coixin Promoter), and U.S. Patent Application No. 09 / 757,089 (Maize Chloroplast Aldolase Promoter).Further promoters that may be useful include the nopalin synthase (NOS) promoter (Ebert et al., 1987), the octopine synthase (OCS) promoter (supported on the tumor-inducing plasmid of Agrobacterium tumefaciens), caulimovirus promoters, such as the cauliflower mosaic virus (CaMV) 19S promoter (Lawton et al., Plant Molecular Biology (1987) 9: pp. 315-324), the CaMV 35S promoter (Odell et al., Nature (1985) 313: pp. 810-812), the scrophularia mosaic virus 35S promoter (US Patent No. 6,051,753; US Patent No. 5,378,619), and the sucrose synthase promoter (Yang and Russell, Proceedings of the National Academy of Sciences, USA). These include the promoters of the R gene complex (1990) 87: pp. 4144-4148), the chlorophyll a / b binding protein gene promoter, PC1SV (US Patent No. 5,850,019), and the AGRtu.nos (GenBank accession number V00087; Depicker et al., Journal of Molecular and Applied Genetics (1982) 1: pp. 561-573; Bevan et al., 1983).

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

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

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

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

[0145] Transcription Activator-like Effector (TALE) Activator-like effectors (TALEs) are transcription factors that contain a C-terminal activation domain and, once bound to a TALE binding site in or near a promoter, can activate / increase the expression of operably linked transcriptionable polynucleotides. While not limited by any theory, it has been previously shown that TALE proteins can induce high expression of genes operably linked to TALE binding sites, and that expression can be modulated depending on how many TALE binding sites are present in the regulatory region. In some embodiments, promoters selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30, and bi4 A promoter, or a functional fragment or variant thereof, selected from the group consisting of nine elements, is operably ligated to the nucleic acid encoding TALE.

[0146] In some embodiments, TALE binding sites are 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 Inducible nucleases are nucleases that form a complex (e.g., ribonucleoprotein) with a guide nucleic acid molecule (e.g., guide RNA) and then guide that complex to a target site within a target sequence. One non-limiting example of an inducible nuclease is the CRISPR nuclease.

[0148] CRISPR (clustered, short-interval, palindromic repeating structure) nucleases (e.g., Cas9, CasX, Cas12a (also known as Cpf1), CasY, MAD7®) are proteins found in bacteria that are guided to target nucleic acid molecules by guide RNA ("gRNA"), and the endonuclease can then cleave one or two strands of the target nucleic acid molecule. Although CRISPR nucleases originate in bacteria, many CRISPR nucleases have been shown to function in eukaryotic cells.

[0149] While not limited by any particular scientific theory, CRISPR nucleases form a complex with a guide RNA (gRNA) that hybridizes with a complementary target site, thereby guiding the CRISPR nuclease to the target site. In a Class II CRISPR-Cas system, a CRISPR array containing spacers is transcribed upon encounter with recognized invading DNA and processed into small interfering CRISPR RNA (crRNA). The crRNA contains a repetitive 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 the eukaryotic genome.

[0150] CRISPR nucleases bind to their corresponding crRNAs in their active form. Similar to the class II endonuclease Cas9, CasX requires another non-coding RNA component, called trans-activating crRNA (tracrRNA), to have functional activity. The nucleic acid molecules provided herein can combine crRNA and tracrRNA into a single nucleic acid molecule, referred herein to as “single guide RNA” (sgRNA). Cas12a or MAD7® does not require the tracrRNA to be guided 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 an RNA-induced CRISPR nuclease and a guide RNA form a complex, the entire system is called a "ribonucleoprotein." The ribonucleoproteins provided herein may also include further nucleic acids or proteins.

[0152] In one embodiment, the inducible nuclease and guide nucleic acid form ribonucleoproteins in flower cells. In another embodiment, the inducible nuclease and guide nucleic acid form ribonucleoproteins in flower tissue. In one embodiment, the Cas12a nuclease and guide nucleic acid form ribonucleoproteins in flower cells. In one embodiment, the Cas12a nuclease and guide nucleic acid form ribonucleoproteins in flower tissue. In one embodiment, the CasX nuclease and guide nucleic acid form ribonucleoproteins in flower cells. In another embodiment, the CasX nuclease and guide nucleic acid form ribonucleoproteins in flower tissue. In one embodiment, the MAD7® nuclease and guide nucleic acid form ribonucleoproteins in flower cells. In another embodiment, the MAD7® nuclease and guide nucleic acid form ribonucleoproteins in flower tissue. In one embodiment, the inducible nuclease and guide RNA form ribonucleoproteins in flower cells. In another embodiment, the inducible nuclease and guide RNA form ribonucleoproteins in flower tissue. In one embodiment, the Cas12a nuclease and guide RNA form ribonucleoproteins in flower cells. In another embodiment, the CasX nuclease and guide RNA form ribonucleoproteins in flower tissue. In one embodiment, the inducible nuclease and single guide RNA form ribonucleoproteins in flower cells. In another embodiment, the inducible nuclease and single guide RNA form ribonucleoproteins in flower tissue. In another embodiment, the CasX nuclease and single guide RNA form ribonucleoproteins in flower tissue. In another embodiment, the MAD7® nuclease and single guide RNA form ribonucleoproteins in flower tissue.

[0153] In one embodiment, the ribonucleoprotein generates at least one double-strand break at a target site in a flower cell. In another embodiment, the ribonucleoprotein generates at least one double-strand break at a target site in a flower tissue. In another embodiment, the ribonucleoprotein generates at least one single-strand break at a target site in a flower cell. In another embodiment, the ribonucleoprotein generates at least one single-strand break at a target site in a flower tissue.

[0154] A prerequisite for cleavage of a target site by CRISPR ribonucleoproteins 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 required only for type I and type II CRISPR-related proteins, and different CRISPR endonucleases recognize different PAM sites. While not limited to these, 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 thought 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 a class II, type V CRISPR / Cas system RNA-induced nuclease. When Cas12a nucleases cleave double-stranded DNA molecules, they produce alternating breaks. These alternating breaks result in a single-stranded DNA overhang of at least one nucleotide. This is in contrast to blunt-end breaks (such as those produced by Cas9), which do not result in a single-stranded DNA overhang when cleaving double-stranded DNA.

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

[0157] In one embodiment, Cas12a nuclease, or the nucleic acid encoding Cas12a nuclease, is derived from Streptococcus, Campylobacter, Nitratifractor, Staphylococcus, Parvibaculum, Roseburia, Neisseria, Gluconacetobacter, and Azospirillum. Lactobacillus, Eubacterium, Corynebacter, Carnobacterium, Rhodobacter, Listeria, Paludibacter, Clostridium, Lachnospiraceae, Clostridia Idium), Leptotrichia, Francisella, Legionella, Alicyclobacillus, Methanomethyophilus, Porphyromonas, Prevotella, Bacteroidetes, Helcococcus, Letospira, Desulfov 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 containing a sequence that is at least 80% identical to a polynucleotide selected from the group consisting of SEQ ID NOs. 32 and SEQ ID NOs. 36. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 85% identical to a polynucleotide selected from the group consisting of SEQ ID NOs. 32 and SEQ ID NOs. 36. In yet another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 90% identical to a polynucleotide selected from the group consisting of SEQ ID NOs. 32 and SEQ ID NOs. 36. In yet another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 95% identical to a polynucleotide selected from the group consisting of SEQ ID NOs. 32 and SEQ ID NOs. 36. In yet another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 96% identical to a polynucleotide selected from the group consisting of SEQ ID NOs. 32 and SEQ ID NOs. 36. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 97% identical to a polynucleotide selected from the group consisting of SEQ ID NOs. 32 and SEQ ID NOs. 36. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 98% identical to a polynucleotide selected from the group consisting of SEQ ID NOs. 32 and SEQ ID NOs. 36. In another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is at least 99% identical to a polynucleotide selected from the group consisting of SEQ ID NOs. 32 and SEQ ID NOs. 36. In yet another embodiment, the Cas12a nuclease is encoded by a polynucleotide containing a sequence that is 100% identical to a polynucleotide selected from the group consisting of SEQ ID NOs. 32 and SEQ ID NOs. 36.

[0159] CasX is a type of class II CRISPR-Cas nuclease identified in the phylum Bacteria, specifically in the Deltaproteobacteria and Planctomyces. Similar to Cas12a, CasX nucleases produce alternating cleavage when cleaving double-stranded DNA molecules. However, unlike Cas12a, CasX nucleases require crRNA and tracrRNA, or a single guide RNA, to target and cleave the target nucleic acid.

[0160] In one embodiment, the CasX nuclease provided herein is a CasX nuclease derived from the phylum Deltaproteobacteria. In another embodiment, the CasX nuclease provided herein is a CasX nuclease derived from the phylum Planctomyces. Further preferred CasX nucleases, though not limited thereto, are those described in WO 2019 / 084148, which are incorporated herein by reference in their entirety.

[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 standard Cas12a nucleases. The MAD7® nuclease produces alternating cleavage when cleaving double-stranded DNA molecules. The MAD7® nuclease was first identified in Eubacterium rectale. It requires only standard Cas12a, such as crRNA. The nucleotide sequence encoding ErCas12a / MAD7® can be found in supplemental data (sequence S1) provided in Lin et al., Journal of Genetics and Genomics, 48:444-451 (2021).

[0162] In one embodiment, the inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule is selected from the group consisting of Cas12a;MAD7(registered trademark) and CasX. In one embodiment, the inducible nuclease is selected from the group consisting of Cas12a, MAD7(registered trademark) and CasX.

[0163] In one embodiment, the inducible nuclease is an RNA-inducible 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 molecules provided herein encode a nuclear localization signal. In another embodiment, the nucleic acid molecules provided herein encode two or more nuclear localization signals.

[0165] In one embodiment, the Cas12a nuclease provided herein includes a nuclear localization signal. In one embodiment, the nuclear localization signal is located at the N-terminus of the Cas12a nuclease. In a further embodiment, the nuclear localization signal is located at the C-terminus of the Cas12a nuclease. In yet another embodiment, 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 nuclease provided herein includes 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 another embodiment, the nuclear localization signal is located on both the N-terminus and the C-terminus of the CasX nuclease.

[0167] In some embodiments, the MAD7® nuclease provided herein includes a nuclear localization signal. In some embodiments, the nuclear localization signal is located on the N-terminus of the MAD7® nuclease. In further embodiments, the nuclear localization signal is located on the C-terminus of the MAD7® nuclease. In yet another embodiment, 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 includes at least one nuclear localization signal. In another embodiment, the ribonucleoprotein includes at least two nuclear localization signals.

[0169] In one embodiment, the nuclear localization signals provided herein are encoded by SEQ ID NO: 33 or 34.

[0170] Various species exhibit specific biases for certain codons of particular amino acids. Codon bias (differences in codon use between organisms) often correlates with the efficiency of messenger RNA (mRNA) translation, and is thought to depend, in particular, on the characteristics of the translated codon and the availability of specific 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. Therefore, genes can be tuned 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 available for codon-optimizing specific sequences for expression in specific plant cells, such as Gene Forge (Aptagen; Jacobus, PA).

[0171] As used herein, “codon optimization” refers to the process of modifying a nucleic acid sequence to enhance expression in a target plant cell by replacing at least one codon in a given sequence (e.g., at least 1, 2, 3, 4, 5, 10, 15, 20, 25, 50 or more codons) with a codon that is more frequently or most frequently used in the gene of the plant cell, while maintaining the original amino acid sequence (e.g., by introducing silent mutations).

[0172] In one 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 an 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 a Cas12a nuclease, CasX nuclease, or MAD7® nuclease correspond to the most frequently used codon for a particular amino acid. For codon use in plants, see Campbell and Gowri, 1990, Plant Physiol., 92: pp. 1-11; and Murray et al., 1989, Nucleic Acids Res., 17: pp. 477-98, respectively, which are incorporated herein by reference in their entirety.

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

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

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

[0176] Guide nucleic acids As used herein, “guide nucleic acid” refers to a nucleic acid that, after forming a ribonucleoprotein (e.g., a complex) with an inducible nuclease (e.g., 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 some embodiments, the ribonucleoprotein provided herein comprises at least one guide nucleic acid.

[0177] In one embodiment, the guide nucleic acid includes DNA. In another embodiment, the guide nucleic acid includes RNA. In one embodiment, the guide nucleic acid includes DNA, RNA, or a combination thereof. In one embodiment, the guide nucleic acid is single-stranded. In another embodiment, the guide nucleic acid is at least partially double-stranded.

[0178] If the guide nucleic acid contains RNA, it can also be called "guide RNA." In another embodiment, the guide nucleic acid contains both DNA and RNA. In another embodiment, the guide RNA is single-stranded. In yet another embodiment, the guide RNA is double-stranded. In yet another embodiment, the guide RNA is partially double-stranded.

[0179] In one embodiment, the guide nucleic acid includes guide RNA. In another embodiment, the guide nucleic acid includes at least one guide RNA. In another embodiment, the guide nucleic acid includes at least two guide RNAs. In another embodiment, the guide nucleic acid includes at least three guide RNAs. In another embodiment, the guide nucleic acid includes at least five guide RNAs. In another embodiment, the guide nucleic acid includes at least ten guide RNAs.

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

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

[0182] In some embodiments, the guide nucleic acid includes at least 70% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 75% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 80% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 85% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 90% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 91% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 92% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 93% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 94% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 95% sequence complementarity to the target site. In some embodiments, the guide nucleic acid includes at least 96% sequence complementarity to the target site. In one embodiment, the guide nucleic acid contains at least 97% sequence complementarity to the target site. In another embodiment, the guide nucleic acid contains at least 98% sequence complementarity to the target site. In another embodiment, the guide nucleic acid contains at least 99% sequence complementarity to the target site. In another embodiment, the guide nucleic acid contains 100% sequence complementarity to the target site. In yet another embodiment, the guide nucleic acid contains 70% to 100% sequence complementarity to the target site. In yet another embodiment, the guide nucleic acid contains 80% to 100% sequence complementarity to the target site. In yet another embodiment, the guide nucleic acid contains 90% to 100% sequence complementarity to the target site.

[0183] In some embodiments, the guide nucleic acid can hybridize to the target site.

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

[0185] In one embodiment, the guide nucleic acid includes crRNA. In another embodiment, the guide nucleic acid includes tracrRNA. In yet another embodiment, the guide nucleic acid includes sgRNA.

[0186] target site As used herein, “target sequence” refers to a selected sequence or region of a DNA molecule that is to be modified (e.g., cleaved, site-directed integration). The target sequence includes the target site.

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

[0188] In one embodiment, the target site is 100% complementary to the guide nucleic acid. In another embodiment, the target site is 99% complementary to the guide nucleic acid. In another embodiment, the target site is 98% complementary to the guide nucleic acid. In another embodiment, the target site is 97% complementary to the guide nucleic acid. In another embodiment, the target site is 96% complementary to the guide nucleic acid. In another embodiment, the target site is 95% complementary to the guide nucleic acid. In another embodiment, 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 one embodiment, the target site includes at least one PAM site. In another embodiment, the target site is adjacent to a nucleic acid sequence including at least one PAM site. In yet another embodiment, the target site is within 5 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 10 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 15 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 20 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 25 nucleotides of at least one PAM site. In yet another embodiment, the target site is within 30 nucleotides of at least one PAM site.

[0190] In one embodiment, the target site is located within gene DNA. In another embodiment, the target site is located within a gene. In another embodiment, the target site is located within the target gene. In another embodiment, the target site is located within an exon of a gene. In another embodiment, the target site is located within an intron of a gene. In another embodiment, the target site is located within the promoter of a gene. In another embodiment, the target site is located within the 5'-UTR of a gene. In another embodiment, the target site is located within the 3'-UTR of a gene. In another embodiment, the target site is located within intergenetic 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 embodiments, the target sequence includes genomic DNA. In some embodiments, the target sequence is located within the nuclear genome. In some embodiments, the target sequence includes chromosomal DNA. In some embodiments, the target sequence includes plasmid DNA. In some embodiments, the target sequence is located within a plasmid. In some embodiments, the target sequence includes mitochondrial DNA. In some embodiments, the target sequence is located within the mitochondrial genome. In some embodiments, the target sequence includes plastid DNA. In some embodiments, the target sequence is located within the plastid genome. In some embodiments, the target sequence includes chloroplast DNA. In some embodiments, the target sequence is located within the chloroplast genome. In some embodiments, the target sequence is located within a genome selected from the group consisting of the nuclear genome, the mitochondrial genome, and the plastid genome.

[0193] In one embodiment, the target sequence includes gene DNA. As used herein, “gene DNA” means DNA that codes for one or more genes. In another embodiment, the target sequence includes intergenetic DNA. In contrast to gene DNA, “intergenetic DNA” includes non-coding DNA and lacks DNA that codes for genes. In one embodiment, intergenetic DNA is located between two genes.

[0194] In one embodiment, the target sequence encodes a gene. As used herein, “gene” means a polynucleotide capable of producing a functional unit (e.g., 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 embodiment, the gene encodes a non-protein-coding RNA molecule or its precursor. In another embodiment, the gene encodes a protein. In some embodiments, the target sequence is selected from the group consisting of promoters, enhancer sequences, leader sequences, transcription start sites, transcription stop sites, polyadenylation sites, exons, introns, splice sites, 5'-UTR, 3'-UTR, protein-coding sequences, non-protein-coding sequences, miRNAs, pre-miRNAs, and miRNA-binding sites.

[0195] Non-protein-coding RNA molecules include, but are not limited to, microRNAs (miRNAs), miRNA precursors (premiRNAs), small interfering RNAs (siRNAs), small RNAs (18-26 nucleotides in length) and their encoding precursors, heterochromatin siRNAs (hc-siRNAs), Piwi-binding RNAs (piRNAs), hairpin double-stranded RNAs (hairpin dsRNAs), trans-acting siRNAs (ta-siRNAs), naturally occurring antisense siRNAs (nat-siRNAs), CRISPR RNAs (crRNAs), tracer RNAs (tracrRNAs), guide RNAs (gRNAs), and single guide RNAs (sgRNAs). In some embodiments, non-protein-coding RNA molecules include miRNAs. In some embodiments, non-protein-coding RNA molecules include siRNAs. In some embodiments, non-protein-coding RNA molecules include ta-siRNAs. In some embodiments, non-protein-coding RNA molecules are selected from the group consisting of miRNAs, siRNAs, and ta-siRNAs.

[0196] As used herein, “the gene of interest” means a polynucleotide sequence encoding a protein or non-protein-coding RNA molecule to be incorporated into a target sequence, or an endogenous polynucleotide sequence encoding a protein or non-protein-coding RNA molecule to be edited by a ribonucleoprotein. In some embodiments, the gene of interest encodes a protein. In other embodiments, the gene of interest encodes a non-protein-coding RNA molecule. In some embodiments, the gene of interest is exogenous with respect to the target DNA molecule. In some embodiments, the gene of interest replaces an endogenous gene in the target DNA molecule.

[0197] mutation In some embodiments, the ribonucleoprotein or method provided herein generates at least one mutation in a target sequence.

[0198] In one embodiment, seeds produced from the plants provided herein contain at least one mutation in the gene of interest, including the target site, compared to seeds from a control plant of the same lineage or variety that lacks a first nucleic acid sequence encoding an inducible nuclease operably linked to a flower cell-preferential promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter. In one embodiment, seeds produced from the plants provided herein contain at least one mutation in the gene of interest, including the target site, compared to seeds from a control plant of the same lineage or variety that lacks a first nucleic acid sequence encoding an inducible nuclease operably linked to a flower tissue-preferential promoter or a second nucleic acid encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter.

[0199] In one embodiment, seeds produced from the plants provided herein contain at least one mutation in the gene of interest, including the target site, compared to seeds from a control plant of the same lineage 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 flower cell-preferential promoter. In one embodiment, seeds produced from the plants provided herein contain at least one mutation in the gene of interest, including the target site, compared to seeds from a control plant of the same lineage 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 flower tissue-preferential promoter.

[0200] As used herein, “mutation” refers to a modification to a nucleic acid or amino acid sequence that does not exist in nature, compared to a naturally occurring reference nucleic acid or amino acid sequence from the same organism. When identifying mutations, 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 when determining whether a difference between two sequences constitutes a mutation, comparisons should not be made between homologous sequences of two different species or between homologous sequences of two different varieties of a single species. Rather, comparisons should be made between an edited (e.g., mutated) sequence and an endogenous, unedited (e.g., “wild-type”) sequence from the same organism.

[0201] Several types of mutations are known in the art. In some embodiments, a mutation includes an insertion. "Insertion" means the addition of one or more nucleotides or amino acids to a given polynucleotide or amino acid chain sequence, compared to an endogenous reference polynucleotide or amino acid sequence. In other embodiments, a mutation includes a deletion. "Deletion" means the removal of one or more nucleotides or amino acids to a given polynucleotide or amino acid chain sequence, compared to an endogenous reference polynucleotide or amino acid sequence. In other embodiments, a mutation includes a substitution. "Substitution" means the replacement of one or more nucleotides or amino acids to a given polynucleotide or amino acid chain sequence, compared to an endogenous reference polynucleotide or amino acid sequence. In other embodiments, a mutation includes an inversion. "Inversion" means that a segment of a polynucleotide or amino acid sequence is reversed from end to end. In some embodiments, the mutations provided herein include mutations selected from the group consisting of insertions, deletions, substitutions, and inversions.

[0202] In one embodiment, a plant or seed contains at least one mutation in the gene of interest, the at least one mutation resulting 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 embodiment, a plant or seed contains at least one mutation in the gene of interest, the at least one mutation resulting in one or more amino acid substitutions in the protein encoded by the gene of interest compared to the wild-type protein.

[0204] In one embodiment, a plant or seed contains at least one mutation in the gene of interest, the at least one mutation resulting in the insertion of one or more amino acids into 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 some embodiments, this disclosure provides mutations that result in the truncation of a protein or polypeptide. As used herein, a “truncated” protein or polypeptide contains at least one fewer amino acid than an endogenous control protein or polypeptide. For example, if endogenous protein A contains 100 amino acids, the truncated protein A may contain 1 to 99 amino acids.

[0206] While not limited by any scientific theory, one method for causing truncation of a protein or polypeptide is by introducing an immature stop codon into the mRNA transcript of an endogenous gene. In some embodiments, this disclosure provides mutations that result in an immature stop codon in the mRNA transcript of an endogenous gene. As used herein, “stop codon” refers to a nucleotide triplet in an mRNA transcript that signals the termination of protein translation. “Immature stop codon” refers to a stop codon located earlier (e.g., 5') than the normal stop codon position in the endogenous mRNA transcript. Several stop codons are known in the art, including, but are not limited to, “UAG”, “UAA”, “UGA”, “TAG”, “TAA”, and “TGA”.

[0207] In one embodiment, the seed or plant contains at least one mutation, the mutation resulting in the introduction of an immature stop codon into the messenger RNA encoded by the gene of interest, compared to the wild-type messenger RNA.

[0208] In some embodiments, the mutations provided herein include null mutations. As used herein, “null mutation” means a mutation that results in complete loss of function for a protein encoded by the gene containing the mutation, or a mutation that results in complete loss of function for a small RNA encoded by a genomic locus. A null mutation may result in 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 an endogenous gene. In one embodiment, the mutations provided herein are located within an exon of the endogenous gene. In another embodiment, the mutations provided herein are located within an intron of the endogenous gene. In yet another embodiment, the mutations provided herein are located within the 5' untranslated region of the endogenous gene. In yet another embodiment, the mutations provided herein are located within the 3' untranslated region of the endogenous gene. In yet another embodiment, the mutations provided herein are located within the promoter of the endogenous gene.

[0210] In some embodiments, mutations are located at splice sites within a gene. Mutations at splice sites can disrupt 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, lost exons, or both, derived from the mature mRNA sequence. Typically, but not always, proper splicing requires a "GU" sequence at the 5' end of an intron and an "AG" sequence at the 3' end of an intron. If either of these splice sites is mutated, splicing can be perturbed.

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

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

[0213] As used herein, “site-directed integration” means all or part of a desired sequence (e.g., an exogenous gene, an edited endogenous gene) that is inserted into or integrated into a desired site or locus (e.g., a target sequence) in the plant genome. As used herein, “desired sequence” means a DNA molecule containing a nucleic acid sequence to be integrated into the genome of a plant or plant cell. The desired sequence may include a transgene or a construct. In some embodiments, the nucleic acid molecule containing the desired sequence includes one or two homology arms flanking the desired sequence to facilitate an insertion event targeted by homologous recombination and / or homology-directed repair.

[0214] In some embodiments, the methods provided herein include site-specific insertion of a desired sequence into a target sequence.

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

[0216] For site-directed integration, a double-strand break (DSB) or nick can be first created at 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 homology arms of the desired sequence and the target sequence, or by non-homologous end joining (NHEJ), resulting in site-directed integration of all or part of the desired sequence into the target sequence and creating a targeted insertion event at the site of the DSB or nick.

[0217] In one embodiment, site-directed integration includes the use of an endogenous NHEJ repair mechanism for the cell. In another embodiment, site-directed integration includes the use of an endogenous HR repair mechanism for the cell.

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

[0219] In some embodiments, the mutation includes the incorporation of at least five consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least ten consecutive nucleotides of the desired sequence molecule into the target sequence. In some embodiments, the mutation includes the incorporation of at least fifteen consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least twenty consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least twenty-five consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least fifty consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least one hundred consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least two hundred consecutive nucleotides of the desired sequence into the target sequence. In some embodiments, the mutation includes the incorporation of at least two hundred consecutive nucleotides of the desired sequence into the target sequence. In one embodiment, the mutation includes the incorporation of at least 1,000 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of at least 2,000 consecutive nucleotides of the desired sequence into the target sequence.

[0220] In one embodiment, the mutation includes the incorporation of 5 to 3500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 2500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 1500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 750 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation includes the incorporation of 5 to 250 consecutive nucleotides of the desired sequence into the target sequence. In yet another embodiment, the mutation includes the incorporation of 5 to 150 consecutive nucleotides of the desired sequence into the target sequence. In one embodiment, the mutation involves the incorporation of 25 to 2500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 25 to 1500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 25 to 750 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 50 to 2500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 50 to 1500 consecutive nucleotides of the desired sequence into the target sequence. In another embodiment, the mutation involves the incorporation of 50 to 750 consecutive nucleotides of the desired sequence into the target sequence. In yet another embodiment, the mutation involves the incorporation of 100 to 2500 consecutive nucleotides of the desired sequence into the target sequence. In one embodiment, the mutation involves the incorporation of 100 to 1500 consecutive nucleotides of a desired sequence into a target sequence.In one embodiment, the mutation involves the incorporation of 100 to 750 consecutive nucleotides of a desired sequence into a target sequence.

[0221] In some embodiments, the methods provided herein further include detecting edits or mutations in a target sequence. Screening and selection of mutagenic or edited plants or plant cells may be by any method known to those skilled in the art. Examples of screening and selection methods, but not limited to, include Southern spectroscopy, PCR amplification for the detection of polynucleotides, Northern blotting, RNase protection, primer extension, RT-PCR amplification for the detection of RNA transcripts, Sanger sequencing, next-generation sequencing techniques (e.g., Illumina, PacBio, Ion Torrent, 454), enzyme assays for detecting the enzymatic or ribozyme activity of polypeptides and polynucleotides, protein gel electrophoresis, Western blotting, immunoprecipitation, and enzyme-conjugated immunoassays for detecting polypeptides. The presence or expression of polypeptides and / or polynucleotides can also be detected using other techniques such as in situ hybridization, enzyme staining, and immunostaining. Methods for carrying out all the techniques referenced above 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 ribozymes are autocleavable ribozymes. Autocleavable ribozymes are known in the art. See, for example, Jimenez et al., Trends Biochem. Sci., 40:648-661 (2015).

[0223] In one embodiment, the sequence encoding at least one guide nucleic acid is flanked by a self-cleaving ribozyme. In another embodiment, the sequence encoding at least one guide nucleic acid is in immediate proximity to the sequence encoding the ribozyme (for example, the outermost 5' nucleotide of the guide nucleic acid is adjacent to the outermost 3' nucleotide of the ribozyme, or the outermost 3' nucleotide of the guide nucleic acid is adjacent to the outermost 5' nucleotide of the ribozyme). In another embodiment, 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 in conjunction with the methods and compositions provided herein.

[0225] In certain embodiments, the plant is selected from the group consisting of corn plants, rice plants, sorghum plants, wheat plants, alfalfa plants, barley plants, millet plants, rye plants, sugarcane plants, cotton plants, soybean plants, canola plants, tomato plants, onion plants, cucumber plants, Arabidopsis plants, and potato plants. In certain embodiments, the plant is an angiosperm. In certain embodiments, the plant is a gymnosperm. In certain embodiments, the plant is a monocotyledon. In certain embodiments, the plant is a dicotyledon. In certain embodiments, the plant is a plant of a family selected from the group consisting of Alliaceae, Anacardiaceae, Apiaceae, Arecaceae, Asteraceae, Brassicaceae, Caesalpiniaceae, Cucurbitaceae, Ericaceae, Fabaceae, Juglandaceae, Malvaceae, Mimosaceae, Moraceae, Musaceae, Orchidaceae, Papilionaceae, Pinaceae, Poaceae, Rosaceae, Rutaceae, Rubiaceae, and Solanaceae.

[0226] In certain aspects, the plant cell is selected from the group consisting of maize cells, rice cells, sorghum cells, wheat cells, alfalfa cells, barley cells, millet cells, rye cells, sugarcane cells, cotton cells, soybean cells, canola cells, tomato cells, onion cells, cucumber cells, Arabidopsis thaliana cells, and potato cells. In certain aspects, the plant cell is an angiosperm cell. In certain aspects, the plant cell is a gymnosperm cell. In certain aspects, the plant cell is a monocotyledonous plant cell. In certain aspects, the plant cell is a dicotyledonous plant cell. In certain aspects, the plant cell is a plant cell of a family selected from the group consisting of the Alliaceae family, the Urticaceae family, the Apiaceae family, the Arecaceae family, the Asteraceae family, the Brassicaceae family, the Rosaceae family, the Cucurbitaceae family, the Ericaceae family, the Fabaceae family, the Juglandaceae family, the Malvaceae family, the Moraceae family, the Urticaceae family, the Cannabaceae family, the Orchidaceae family, the Faboideae subfamily, the Pinaceae family, the Poaceae family, the Rosaceae family, the Rutaceae family, the Rubiaceae family, and the Solanaceae family.

[0227] As used herein, the term "variety" refers to a group of plants within a species (such as, but not limited to, Zea mays) that share certain genetic traits that distinguish them from other possible varieties within that species. A variety may be an inbred line or a hybrid, although commercially available plants are often hybrids to take advantage of heterosis. Individuals within a cultivar of a hybrid are of the same species, are nearly genetically identical, and many loci are in a heterozygous state.

[0228] As used herein, the term "inbred line" means a strain that has been bred for genetic uniformity. In certain aspects, the seeds provided herein are inbred line seeds. In certain aspects, the plants provided herein are inbred line plants.

[0229] As used herein, the term “hybrid” means the offspring of a cross between at least two genetically dissimilar parents. Examples of hybrid schemes, though not limited to, include single hybrids, modified single hybrids, double modified single hybrids, triline hybrids, modified triline hybrids, and double hybrids, where at least one parent in the modified hybrid is an offspring of a cross between sister lines. In some embodiments, the seeds provided herein are hybrid seeds. In some embodiments, the plants provided herein are hybrid plants.

[0230] Transformation The method may include transient transformation or stable incorporation of any nucleic acid molecule into any plant or plant cell provided herein.

[0231] As used herein, “stable integration” or “stable integration” refers to the transfer of DNA into the genomic DNA of a target cell or plant, enabling the target cell or plant to pass 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, “transient transformation” or “transient transformation” refers to the transfer of DNA into a cell that does not pass 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 embodiment, the method stably transforms plant cells or plants with one or more nucleic acid molecules provided herein. In another embodiment, the method transiently transforms plant cells or plants with one or more nucleic acid molecules provided herein.

[0232] In some embodiments, nucleic acid molecules encoding inducible nucleases are stably integrated into the plant genome. In some embodiments, nucleic acid molecules encoding Cas12a nuclease are stably integrated into the plant genome. In some embodiments, nucleic acid molecules encoding CasX nuclease are stably integrated into the plant genome. In some embodiments, nucleic acid molecules encoding MAD7® nuclease are stably integrated into the plant genome. In some embodiments, nucleic acid molecules encoding guide nucleic acids are stably integrated into the plant genome. In some embodiments, nucleic acid molecules encoding guide RNA are stably integrated into the plant genome. In some embodiments, nucleic acid molecules encoding single guide RNA are stably integrated into the plant genome.

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

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

[0235] In some embodiments, nucleic acid molecules are delivered to cells by a method selected from the group consisting of Agrobacterium-mediated transformation, polyethylene glycol-mediated transformation, bioristic transformation, liposome-mediated transfection, viral transduction, the use of one or more delivery particles, microinjection, and electroporation.

[0236] Other methods for transformation, such as vacuum immersion, pressure, sonication, and silicon carbide fiber agitation, are also known in the art and are intended for use in conjunction with any method 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., bioristic transformation) using recombinant DNA-coated particles can be found in U.S. Patents 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. Patents 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 Compendium of Transgenic Crop Plants (2009) Blackwell Publishing. Plant cells can be transformed with any of the nucleic acid molecules provided herein using any suitable method known to those skilled in the art.

[0238] Lipofection is described, for example, in U.S. Patents 5,049,386, 4,946,787, and 4,897,355, and lipofection reagents are commercially available (e.g., Transfectam® and Lipofectin®). Suitable cationic and neutral lipids for efficient receptor recognition lipofection of polynucleotides include those described in Felgner's WO91 / 17424; WO91 / 16024. Delivery may be to cells (e.g., in vitro or ex vivo administration) or target tissue (e.g., in vivo administration).

[0239] Delivery vehicles, vectors, particles, nanoparticles, formulations and components thereof for the expression of one or more elements of nucleic acid molecules are as used in WO2014 / 093622. In one embodiment, a method for delivering nucleic acid molecules or proteins to cells includes delivery by delivery particles. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes delivery by delivery vesicles. In one embodiment, delivery vesicles are selected from the group consisting of exosomes and liposomes. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes delivery by viral vectors. In one embodiment, viral vectors are selected from the group consisting of adenovirus vectors, lentivirus vectors, and adeno-associated virus vectors. In another embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes delivery by nanoparticles. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes microinjection. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes polycations. In one embodiment, a method for delivering nucleic acid molecules to plant cells or plants includes cationic oligopeptides.

[0240] In some embodiments, the delivery particles are selected from the group consisting of exosomes, adenovirus vectors, lentiviral vectors, adeno-associated virus 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 yet another embodiment, the methods provided herein include the use of three or more delivery particles.

[0241] Suitable agents for facilitating the transfer of nucleic acids into plant cells include agents that increase the permeability to the outside of the plant, or agents that increase the permeability of plant cells to oligonucleotides or polynucleotides. Such agents for facilitating the transfer of a composition into plant cells include chemical agents, physical agents, or combinations thereof. Chemical agents for conditioning include (a) surfactants, (b) organic solvents, aqueous solutions, or aqueous mixtures of organic solvents, (c) oxidizing agents, (e) acids, (f) bases, (g) oils, (h) enzymes, or combinations thereof.

[0242] Useful organic solvents for conditioning plants to permeate 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). Natural or synthetic oils, with or without surfactants or emulsifiers, may be used, such as plant-derived oils, crop oils (such as those listed in the 9th Compendium of Herbicide Adjuvants, which is publicly available online at www.herbicide.dot.adjuvants.com), and oils having 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 animal fats, animal fat amines, or phospholipids) and organosilicon surfactants. Other useful surfactants include nonionic organosilicon surfactants, such as organosilicon surfactants containing trisiloxane ethoxylate surfactants, or silicone polyether copolymers such as the copolymer of polyalkylene oxide-modified heptamethyltrisiloxane and allyloxypolypropylene glycol methyl ether (commercially available as Silwet® L-77).

[0244] Useful physical agents may include (a) abrasives such as carborundum, corundum, sand, calcite, pumice, and garnet; (b) nanoparticles such as carbon nanotubes; or (c) physical forces. Carbon nanotubes are disclosed by Kam et al. (2004) / . Am. Chem. Soc, 126 (22): pp. 6850-6851, Liu et al. (2009) Nano Lett, 9(3): pp. 1007-1010, and Khodakovskaya et al. (2009) ACS Nano, 3(10): pp. 3221-3227. Examples of physical force agents include heating, cooling, application of positive pressure, or sonication. Embodiments of the method may optionally include incubation steps, neutralization steps (e.g., to neutralize acids, bases, or oxidizing agents, or to inactivate enzymes), washing steps, or a combination thereof. The method of the present invention may further include the application of other agents that would have an enhanced effect due to the silencing of certain genes. For example, if a polynucleotide is designed to modulate a gene that provides herbicide resistance, subsequent application of the herbicide may have a dramatic effect on the efficacy of the herbicide.

[0245] Examples of agents used for laboratory conditioning of plant cells for polynucleotide permeability include the application of chemical agents, enzymatic treatment, heating or cooling, treatment using positive or negative pressure, or sonication. Examples of agents used for conditioning plants in the field include chemical agents such as surfactants and salts.

[0246] In one embodiment, the cells to be transformed or transfected are plant cells. Recipient plant cells or explant targets for transformation include, but are not limited to, seed cells, fruit cells, leaf cells, cotyledon cells, hypocotyl cells, meristem 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 embodiment, the disclosure provides plant chloroplasts. In a further embodiment, the disclosure provides epidermal cells, guard cells, trichome cells, root hair cells, storage root cells, or tuber cells. In another embodiment, the disclosure provides protoplasts. In another embodiment, the disclosure provides plant callus cells. Any cells capable of regenerating fertile plants are intended to be useful recipient cells for the implementation of this disclosure. Callus can be initiated from various tissue sources, including, but not limited to, immature embryos or embryonic portions, seedling apical meristems, microspores, etc. These cells, capable of growing as callus, can serve as recipient cells for transformation. Practical transformation methods and materials for producing transgenic plants of this disclosure (e.g., various media and recipient target cells, transformation of immature embryos, and subsequent regeneration of fertile transgenic plants) are disclosed, for example, in U.S. Patent Nos. 6,194,636 and 6,232,526 and U.S. Patent Application Publication 2004 / 0216189, all of which are incorporated herein by reference. Transformed explants, cells, or tissues can be subjected to further culture steps, such as callus induction, selection, and regeneration, as known in the art. Transformed cells, tissues, or explants containing recombinant DNA insertions can be propagated, developed, or regenerated into transgenic plants in cultures, plugs, or soil according to methods known in the art. In one embodiment, the disclosure provides plant cells that are not reproductive material and do not mediate the natural reproduction of plants. In another embodiment, the disclosure provides plant cells that are reproductive material and mediate the natural reproduction of plants. In yet another embodiment, the disclosure provides plant cells that cannot sustain themselves 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 cuts 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 is capable of hybridizing to a target sequence within the genome of a plant; or (c) A first nucleic acid sequence encoding an inducible nuclease capable of generating staggered cuts 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 flower cell-preferred promoter; or (e) 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 or a flower 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 flower cell-preferred promoter comprising a plant. 2. The plant according to embodiment 1, wherein the inducible nuclease is selected from the group consisting of Cas12a, MAD7®, and CasX. 3. The plant according to embodiment 2, wherein Cas12a is selected from the group consisting of LbCas12a and FnCas12a. 4. The plant according to 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 plants. 6. The 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 according to 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. The plant according to any one of Embodiments 1 to 7, wherein the flower cell-preferential promoter is a flower cell-specific promoter. 9. The plant according to any one of Embodiments 1 to 8, wherein the flower tissue-preferential promoter is a flower tissue-specific promoter. 10. The plant according to any one of Embodiments 1 to 9, wherein the flower cell preferential promoter is selected from the group consisting of gene A promoter, gene B promoter, gene C promoter, gene D promoter, and gene E promoter. 11. A plant according to any one of Embodiments 1 to 10, wherein the flower cell-preferential promoter or flower tissue-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. 12. A flower cell-preferential promoter or flower tissue-preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 A plant according to any one of embodiments 1 to 11, comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of 9. 13. The 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-preferential promoter, a tissue-specific promoter, an inducible promoter, and a constitutive promoter. 14. The plant according to any one of Embodiments 1 to 13, wherein the first or second promoter is a flower cell-preferential promoter or a flower tissue-specific promoter. 15. The plant according to any one of Embodiments 1 to 14, wherein the first or second promoter is a flower cell-specific promoter or a flower tissue-specific promoter. 16. The plant according to any one of Embodiments 1 to 15, wherein the first or second promoter is selected from the group consisting of gene A promoter, gene B promoter, gene C promoter, gene D promoter, and gene E 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. The first or second promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 and bi4 A plant according to any one of Embodiments 1 to 17, comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of 9. 19. The plant according to Embodiment 13, wherein the constitutive promoter is selected from the group consisting of the DaMV promoter, the CaMV 35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. 20. A plant according to any one of Embodiments 1 to 19, wherein 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 incorporated into the plant genome. 22. A plant according to any one of Embodiments 1 to 21, wherein an inducible nuclease and at least one RNA form a ribonucleoprotein in the flower cell. 23. The plant according to Embodiment 22, wherein the ribonucleoprotein generates at least one double-strand break within a target site in a flower cell. 24. A plant according to any one of Embodiments 1 to 23, selected from the group consisting of corn plants, rice plants, sorghum plants, wheat plants, alfalfa plants, barley plants, millet plants, rye plants, sugarcane plants, cotton plants, soybean plants, canola plants, tomato plants, onion plants, and potato plants. 25. The plant according to any one of Embodiments 1 to 24, wherein the genome is selected from the group consisting of a nuclear genome, a mitochondrial genome, and a plastid genome. 26. Seeds produced by any one of Embodiments 1 to 25, which, if necessary, include modifications to or near a target sequence in the plant genome. 27. The seed according to Embodiment 26, which contains at least one mutation in the target gene containing the target sequence, compared to the seed 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 an immature stop codon into the messenger RNA encoded by the gene of interest, compared to wild-type messenger RNA. 31. The seed according to Embodiment 26, wherein at least one mutation in the gene of interest comprises the deletion of one or more splice sites from the gene of interest. 32. A hybrid seed, as described in any one of embodiments 26 to 31. 33. Seeds according to any one of embodiments 26 to 31, which are inbred seeds. 34. A method for editing the genome of a plant, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous flower cell preferential promoter; and (ii) A second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a second heterogeneous promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the genome. The process of introducing; and (b) A step of regenerating at least one plant from the plant cells of step (a). A method comprising an inducible nuclease and at least one guide nucleic acid, wherein the nuclease and at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell of a plant, and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one flower cell. 35. A method for editing the genome of a plant cell, (a) A step of crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of producing alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, the at least one guide nucleic acid capable of hybridizing to a target sequence in the genome; and (b) A step of obtaining at least one embryo from the mating of step (a), wherein the inducible nuclease and at least one guide nucleic acid form a ribonucleoprotein in at least one embryo, and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one embryo. Methods that include... 36. A method for editing the genome of a plant, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue preferential promoter; and (ii) A second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a second heterogeneous promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the genome. The process of introducing; (b) A step of regenerating at least one plant from the plant cells of step (a); and (c) The process of fertilizing at least one plant and producing at least one embryo. A method comprising an inducible nuclease and at least one guide nucleic acid, wherein the inducible nuclease and at least one guide nucleic acid form a ribonucleoprotein in at least one embryo derived from step (c), and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one embryo. 37. A method for generating site-specific integration in plants, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous flower cell preferential promoter; (ii) A second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a second promoter of a different species, wherein one or more guide nucleic acids (A) It can hybridize to target sequences within the plant genome; and (B) A second nucleic acid sequence that can hybridize to the first and second sites flanking the nucleic acid sequence encoding the gene of interest; and (iii) A third nucleic acid sequence encoding the target gene The process of introducing; and (b) A step of regenerating at least one plant from the plant cells of step (a). A method comprising: an inducible nuclease and at least one guide RNA forming a ribonucleoprotein in at least one flower cell of a plant, the ribonucleoprotein generating double-strand breaks in a target sequence molecule, a first site and a second site, and the gene of interest being incorporated into the target sequence in at least one flower cell. 38. A method for generating site-specific integration in plants, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue preferential promoter; (ii) A second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a second promoter of a different species, wherein one or more guide nucleic acids (A) It can hybridize to target sequences within the plant genome; and (B) A second nucleic acid sequence that can hybridize to the first and second sites flanking the nucleic acid sequence encoding the target gene; (iii) A third nucleic acid sequence encoding the gene of interest. The process of introducing; (b) a step of regenerating at least one plant from the plant cells of step (a); and (c) A step of fertilizing at least one plant derived from step (b) to produce at least one embryo. A method comprising: an inducible nuclease and at least one guide RNA forming a ribonucleoprotein in at least one embryo, the ribonucleoprotein generating double-strand breaks in a target DNA molecule, a first site and a second site, and the gene of interest being incorporated into a target sequence in at least one embryo. 39. A method for editing the genome of a plant, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous first promoter; and (ii) A second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous floral cell-preferential or floral tissue-preferential promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the genome. The process of introducing; and (b) A step of regenerating at least one plant from the plant cells of step (a), wherein the inducible nuclease and 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-strand break in a target sequence in at least one flower cell. Methods that include... 40. A method for editing the genome of a plant cell, (a) A step of crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of producing alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue-preferential promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, the at least one guide nucleic acid capable of hybridizing to a target sequence in the genome; and (b) A step of obtaining at least one offspring plant from the cross of step (a), wherein the inducible nuclease and at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell, and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one flower cell. Methods that include... 41. A method for editing the genome of a plant cell, (a) A step of crossing a first plant with a second plant, wherein the first plant comprises a first nucleic acid sequence encoding an inducible nuclease capable of producing alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous flower cell-preferential promoter, and the second plant comprises a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter, the at least one guide nucleic acid capable of hybridizing to a target sequence in the genome; and (b) A step of obtaining at least one offspring plant from the cross of step (a), wherein the inducible nuclease and at least one guide nucleic acid form a ribonucleoprotein in at least one flower cell, and the ribonucleoprotein generates at least one double-strand break in a target sequence in at least one flower cell. Methods that include... 42. A method for generating site-specific integration in plants, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous first promoter; (ii) A second nucleic acid sequence encoding one or more guide nucleic acids operably linked to a heterogeneous floral cell-preferential or floral tissue-preferential promoter, wherein one or more guide nucleic acids a. It can hybridize to target sequences within the plant genome; and b. A second nucleic acid sequence that can hybridize to the first and second sites flanking the nucleic acid sequence encoding the target gene; and (iii) A third nucleic acid sequence encoding the gene of interest. The process of introducing; and (b) A step of regenerating at least one plant from the plant cells of step (a), wherein an inducible nuclease and at least one guide RNA form a ribonucleoprotein in at least one flower cell of the plant, the ribonucleoprotein generates double-strand breaks in a target sequence molecule, a first site, and a second site, and the gene of interest is incorporated into the target sequence in at least one flower cell. Methods that include... 43. The method according to any one of embodiments 34 to 42, wherein the target sequence includes gene DNA. 44. The method according to any one of embodiments 34 to 42, wherein the target sequence includes intergenetic DNA. 45. The method according to any one of embodiments 34-36 and 39-41, wherein the target sequence is located within the gene of interest. 46. ​​The method according to Embodiment 45, wherein the gene of interest encodes a protein or non-protein-coding RNA. 47. The method according to Embodiment 46, wherein the non-protein-coding RNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or a precursor thereof. 48. The method according to any one of embodiments 34 to 42, wherein the inducible nuclease is selected from the group consisting of Cas12a, MAD7(registered trademark), and CasX. 49. The method according to Embodiment 48, wherein Cas12a is selected from the group consisting of LbCas12a and FnCas12a. 50. The method according to any one of embodiments 34, 37, or 39-42, wherein the flower cell-preferential promoter is a flower cell-specific promoter. 51. The method according to any one embodiment of 35, 36, or 38, wherein the floral tissue-preferential promoter is a floral tissue-specific promoter. 52. The method according to any one of Embodiments 34, 37, or 39-42, wherein the flower cell preferential promoter is selected from the group consisting of gene A promoter, gene B promoter, gene C promoter, gene D promoter, and gene E promoter. 53. The method according to any one of embodiments 34, 37, or 39-42, wherein the flower 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 flower cell-preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 、4 The method according to any one of embodiments 34, 37, or 39-42, comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of 9. 55. The method according to any one of Embodiments 35, 36, or 38, wherein the floral tissue preferential promoter is selected from the group consisting of gene A promoter, gene B promoter, gene C promoter, gene D promoter, and gene E promoter. 56. The method according to any one of Embodiments 35, 36, or 38, wherein the flower 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. 57. The floral tissue preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 、4 The method according to any one of Embodiments 35, 36, or 38, comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of 9. 58. The method according to any one of embodiments 34 to 42, wherein the first or second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. 59. The method according to any one of embodiments 34 to 42, wherein the first or second promoter is a flower cell-preferential promoter or a flower tissue-preferential promoter. 60. The method according to Embodiment 59, wherein the flower cell-preferential promoter or flower tissue-preferential promoter is selected from the group consisting of gene A promoter, gene B promoter, gene C promoter, gene D promoter, and gene E promoter. 61. The method according to Embodiment 59 or 60, wherein the flower cell-preferential promoter or flower tissue-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. 62. A flower cell-preferential promoter or flower tissue-preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 、4 The method according to any one of embodiments 59 to 61, comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of 9. 63. The method according to Embodiment 58, wherein the constitutive promoter is selected from the group consisting of the CAMV35S promoter, the actin promoter, the Rab15 promoter, and the ubiquitin promoter. 64. The method according to any one of embodiments 34 to 42, wherein one or more guide nucleic acids comprise at least one guide RNA. 65. The method according to any one of embodiments 34 to 42, wherein the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the genome of a plant. 66. The method according to any one of embodiments 34 to 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 according to 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 according to any one of Embodiments 34-36 or 39-41, wherein the repair of a double-strand break generates at least one mutation in the target sequence compared to a control plant of the same line or variety lacking the first or second nucleic acid sequence, and optionally the mutation results in the 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 according to 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 according to embodiment 68, wherein at least one mutation in the target sequence results in the introduction of an immature stop codon into the messenger RNA encoded by the gene of interest, compared to wild-type messenger RNA. 72. The method according to Embodiment 68, wherein at least one mutation in the target sequence includes the 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 generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral cell-preferential or floral tissue-preferential promoter; and (b) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous second promoter; or (c) a first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous promoter; and (d) a second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous floral cell-preferential or floral tissue-preferential promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the plant genome. 74. The recombinant DNA construct according to Embodiment 73, wherein at least one sequence encoding a guide nucleic acid is flanked by a self-cleaving ribozyme. 75. A method for generating two or more offspring plants having unique editing from a single transformed plant cell, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous flower cell preferential promoter; and (ii) A second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a second heterogeneous promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the genome. The process of introducing; and (b) A step of regenerating a first plant from the plant cells of step (a), wherein an inducible nuclease and 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-strand break in a target sequence in at least one flower cell; (c) the step of pollinating the first plant of step (b); and (d) A process of germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing. Methods that include... 76. A method for generating two or more offspring plants having unique editing from a single transformed plant cell, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous first promoter; and (ii) A second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous flower cell preferential promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the genome. The process of introducing; and (b) A step of regenerating a first plant from the plant cells of step (a), wherein an inducible nuclease and 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-strand break in a target sequence in at least one flower cell; (c) the step of pollinating the first plant of step (b); and (d) A process of germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing. Methods that include... 77. A method for generating two or more offspring plants having unique editing from a single transformed plant cell, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous floral tissue preferential promoter; and (ii) A second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a second heterogeneous promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the genome. The process of introducing; and (b) A step of regenerating a first plant from the plant cells of step (a); (c) A step of pollinating the first plant of step (b), wherein an inducible nuclease and at least one guide nucleic acid form a ribonucleoprotein in the flower tissue, and the ribonucleoprotein generates at least one double-strand break in a target sequence in the flower tissue; and (d) A process of germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing. Methods that include... 78. A method for generating two or more offspring plants having unique editing from a single transformed plant cell, (a) In plant cells, (i) A first nucleic acid sequence encoding an inducible nuclease capable of generating alternating cleavage in a double-stranded DNA molecule operably linked to a heterogeneous first promoter; and (ii) A second nucleic acid sequence encoding at least one guide nucleic acid operably linked to a heterogeneous floral tissue preferential promoter, wherein at least one guide nucleic acid can hybridize to a target sequence in the genome. The process of introducing; and (b) A step of regenerating a first plant from the plant cells of step (a); (c) A step of pollinating the first plant of step (b), wherein an inducible nuclease and at least one guide nucleic acid form a ribonucleoprotein in the flower tissue, and the ribonucleoprotein generates at least one double-strand break in a target sequence in the flower tissue; and (d) A process of germinating two or more seeds produced from step (c) to produce two or more offspring plants having unique editing. Methods that include... 79. The method according to any one of embodiments 75 to 78, wherein the target sequence includes gene DNA. 80. The method according to any one of embodiments 75 to 78, wherein the target sequence includes intergenetic DNA. 81. The embodiment and the method according to any one of claims 75 to 78, wherein the target sequence is located within the gene of interest. 82. The method according to Embodiment 81, wherein the gene of interest encodes a protein or non-protein-coding RNA. 83. The method according to Embodiment 82, wherein the non-protein-coding RNA is selected from the group consisting of microRNA, small interfering RNA (siRNA), trans-acting siRNA, or a precursor thereof. 84. The method according to any one of embodiments 75 to 78, wherein the inducible nuclease is selected from the group consisting of Cas12a, MAD7(registered trademark), and CasX. 85. The method according to Embodiment 84, wherein Cas12a is selected from the group consisting of LbCas12a and FnCas12a. 86. The method according to Embodiment 75 or 76, wherein the flower cell-preferential promoter is a flower cell-specific promoter. 87. The method according to any one embodiment of 77 or 78, wherein the floral tissue-preferential promoter is a floral tissue-specific promoter. 88. The method according to Embodiment 75 or 76, wherein the flower cell preferential promoter is selected from the group consisting of gene A promoter, gene B promoter, gene C promoter, gene D promoter, and gene E promoter. 89. The method according to Embodiment 75 or 76, wherein the flower 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 flower cell-preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 、4 The method according to Embodiment 75 or 76, comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of 9. 91. The method according to Embodiment 77 or 78, wherein the flower tissue preferential promoter is selected from the group consisting of gene A promoter, gene B promoter, gene C promoter, gene D promoter, and gene E promoter. 92. The method according to Embodiment 77 or 78, wherein the floral tissue 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. 93. The floral tissue preferential promoter is selected from the group consisting of SEQ ID NOs: 1-30, or SEQ ID NOs: 1-16, 18-19, 21-30 、4 The method according to Embodiment 77 or 78, comprising a nucleic acid sequence or a functional fragment thereof that is at least 90% identical to a nucleic acid sequence selected from the group consisting of 9. 94. The method according to Embodiment 75 or 77, wherein the second promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. 95. The method according to Embodiment 76 or 79, wherein the first promoter is selected from the group consisting of tissue-preferential promoters, tissue-specific promoters, inductive promoters, and constitutive promoters. 96. The method according to Embodiment 94 or 95, wherein the constitutive promoter is selected from the group consisting of the CAMV35S promoter, the actin promoter, the Rab15 promoter, the DAMV promoter, and the ubiquitin promoter. 97. The method according to any one of embodiments 75 to 96, wherein one or more guide nucleic acids comprise at least one guide RNA. 98. The method according to any one of embodiments 75 to 97, wherein the first nucleic acid sequence, the second nucleic acid sequence, or both are stably incorporated into the genome of a first plant. 99. The method according to any one of embodiments 75 to 98, wherein the plant cells are selected from the group consisting of corn, rice, sorghum, wheat, alfalfa, barley, millet, rye, sugarcane, cotton, soybean, canola, tomato, and potato. 100. The method according to 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 according to any one of Embodiments 75 to 100, wherein the repair of a double-strand break generates at least one mutation in the target sequence compared to a control plant of the same line or variety lacking a first nucleic acid sequence or a second nucleic acid sequence, and optionally the mutation results in the deletion, insertion or substitution of at least one nucleotide in or near the target sequence. 102. The method according to 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 according to 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 according to Embodiment 101, wherein at least one mutation in the target sequence results in the introduction of an immature stop codon into the messenger RNA encoded by the gene of interest, compared to wild-type messenger RNA. 105. The method according to Embodiment 101, wherein at least one mutation in the target sequence comprises the deletion of one or more splice sites from the gene of interest. 106. The method according to embodiment 76 or 78, wherein a sequence encoding at least one guide nucleic acid is flanked by a self-cleaving ribozyme. 107. The method according to any one of embodiments 75 to 106, wherein the first plant is self-pollinating.

[0248] While the disclosure has been described in general terms here, the same will be more readily apparent by referring to the following examples, which are provided as examples and are not intended to be limitations of the disclosure unless otherwise specified. [Examples]

[0249] (Example 1) Cas12a expression in flower cells / tissues to generate germ cell mutations or targeted integration of template DNA. Several Agrobacterium T-DNA vectors were generated to preferentially express Cas12a in maize or soybean flower tissue. See Table 1. A control vector was generated to constitutively express Cas12a, where Cas12a was operably ligated to a modified promoter containing a DaMV promoter (SEQ ID NO: 31) operably fused to an enhancer region (SEQ ID NO: 43) derived from the 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 5' and 3' terminal NLS sequences (nuclear localization signals) and operably ligated to a transcription termination sequence. Each vector also contains an expression cassette encoding a Cas12a gRNA operably ligated to a plant PolIII promoter, targeting a unique maize genomic site (ZmTS1) or soybean genomic site (GmTS1); an expression cassette for a target gene (GOI) flanked by the ZmTS1 or GmTS1 gRNA target site; and an expression cassette for a selection marker. Maize or soybean embryos are transformed using 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 the presence, number, and type of observed mutations are analyzed.

[0253] Several R0 lines containing each transformed construct were grown to maturity and self-pollinated. Subsequently, several R1 lines were selected, and several seedlings were grown from each R1 line to screen for mutations within the target site. The number and type of mutations produced using constructs containing flower tissue-specific promoters (constructs 1-30) were compared to the mutations observed in transformed maize plants produced using control construct 31. Although not bound by any theory, it is predicted that co-expression of Cas12a and its allogeneic gRNA in flower cells will generate double-strand breaks at the target site, and subsequent incomplete DNA repair will produce unique mutations in the flower tissue. Restriction of editing on reproductive structures during development may result in unrelated editing events at multiple locations within a single plant in these tissues, which give rise to the organs or cell types from which the gametes originate. These edited gametes can then pass on the mutations to the next generation. Therefore, it is predicted that a single R0 plant can produce many R1 offspring, each with unique target site edits.

[0254] A study on site-directed 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 target gene (GOI) flanked by a ZmTS1 or GmTS1 gRNA target sequence. While not limited by any theory, it is expected that Cas12a and gRNA expression in floral tissue will create double-strand breaks on both sides of the GOI cassette, releasing them from the T-DNA. This released DNA can then act as a donor for targeted insertion at a genomic target site. If the CRISPR-Cas12a complex cleaves a 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, identify putative target insertions using a Frank PCR assay similar to that described in WO 2019 / 084148, which is incorporated herein by reference in its entirety. Design primers to PCR amplify the expected insertion flanking sequence. Perform four separate PCRs: left Frank PCR and right Frank PCR for potential insertions located in the sense direction, and left Frank PCR and right Frank PCR for insertions located in the antisense direction. Screen R0 and R1 lines to identify putative Frank PCR-positive plants, and further sequence these to confirm targeted insertions of GOI cassettes at GmTS1 or ZmTS1 genomic sites.

[0256] (Example 2) gRNA expression in flower cells / tissues where Cas12a is constitutively expressed to generate germ cell mutations. Several constructs are generated to preferentially express guide RNA (gRNA) complementary to the target site under the control of the PolII promoter in flower cells. See Table 2. After transcription, PolII products are rapidly modified at the 5' cap and polyA tail and exported from the nucleus. These modifications and alterations in localization can hinder the efficient use of gRNA. 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, thereby producing 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. Furthermore, the Cas12a protein, flanked by 5' and 3' terminal NLS sequences, is encoded and a ubiquitous promoter (e.g., the ZmUbqM1 promoter (SEQ ID NO: 35) or the Ubq2 promoter of Medicago truncatula) is introduced. -)Constructs containing plant codon-optimized nucleic acid sequences under the control of ("Cas12a constructs") are introduced co-introduced along with the respective 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 target sites, 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 gRNA is expected to generate one or more unique mutations in floral tissue.

[0260] (Example 3) Expression of Cas12a and gRNA as single transcripts in flower cells Several constructs are 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 construct listed in Table 3 is stably introduced into maize or soybean cells using a bioristic transformation method or Agrobacterium-mediated transformation method commonly used in the art. The resulting transformed maize 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 the flower cells expressing the promoter. Subsequently, ribozyme-mediated cleavage occurs, releasing the gRNA segment. The LbCas12a protein transcribed from the transcript forms a ribonucleoprotein (RNP) with the gRNA. The RNP generates a double-strand break at the target site, and subsequent repair will produce one or more unique mutations in each flower cell. Mature plants are pollinated, and the resulting seeds are screened for mutations at the target site, comparing the number and type of mutations produced using constructs 63–92 with those of transformed maize plants produced using the control construct 93.

[0264] (Example 4) Generation of mutations through crossbreeding Transgenic maize or soybean plants containing one of the LbCas12a cassettes listed in Table 1 are produced and propagated until flowering. Further transgenic maize or soybean plants containing the gRNA cassette described in Example 1 are also produced and propagated until flowering. Plants containing LbCas12a are crossed with plants containing the gRNA construct to produce offspring plants containing Cas12a and gRNA expressed in the flower tissue.

[0265] Alternatively, a transgenic maize or soybean plant containing one of the constructs (see Example 2, Table 2) is produced and propagated until flowering. Further transgenic maize or soybean plants containing the Cas12a construct from Example 2 are also produced and propagated until flowering. A transgenic plant containing one of the constructs is crossed with a plant containing the Cas12a construct to produce offspring plants containing Cas12a and gRNA expressed in the flower tissue.

[0266] While not limited by any particular theory, the simultaneous expression of Cas12a and gRNA in floral tissue generates double-strand breaks at target sites, thereby producing unique mutations in each cell where both components of the CRISPR system are expressed. Mutations at target sites can be identified by crossbreeding plants or self-pollinating and screening the resulting offspring.

[0267] (Example 5) TALE-induced tissue / cell-specific expression of LbCas12a A potential drawback of tissue / cell-preferential promoters is that they tend not to be robustly expressed. This embodiment overcomes this limitation and describes a construct generated to induce robust expression of a transcriptionable polynucleotide such as Cas12a in a tissue / cell-preferential manner.

[0268] This construct generates several constructs for preferential, robust TALE-inducible Cas12a expression in floral cells / tissues. Activator-like effectors (TALEs) are transcription factors containing a C-terminal activation domain that, once bound to a TALE binding site on or near a promoter, can activate / increase the expression of operably ligated transcriptable polynucleotides. Previous studies have shown that TALE proteins can induce high expression of genes operably ligated to TALE binding sites, and that expression can be modulated depending on how many TALE binding sites are present in the regulatory region. This construct generates a plant codon-optimized LbCas12a coding sequence operably ligated to a transcription termination sequence and a minimum 35S(-46) promoter, flanked by 5' and 3' terminal NLS sequences, along with 1, 3, or 6 TALE binding sites. It also generates expression constructs containing a promoter-operably ligated TALE coding sequence for preferential or standalone expression in floral tissues / cells. Non-limiting examples of promoters and regulatory sequences for driving preferential cell 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 a vector containing the above expression cassette, an expression cassette encoding Cas12a gRNA complementary to a maize genome target site (ZmTS1) or 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 they are 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, TALE, preferentially expressed in floral tissue / cells, is expected to bind to the TALE protein binding site upstream of 35S(-46):Lb.Cas12a, inducing preferential and robust expression of the nuclease in floral cells. The expression of LbCas12a and gRNA is expected to result in mutations within the target site. R1 plants generated from the transformed R0 line are expected to show a significant number of unique mutations at the target site.

[0269] (Example 6) Preferential expression of Cas12a in floral tissue / cells to generate germ cell mutations. This embodiment describes the use of the Arabidopsis thaliana meristem-preferential promoter AtERL1 to drive Cas12a expression for generating 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 were similar in design except that in construct 94, the LbCas12a cassette was driven by the Arabidopsis thaliana ERL1 (SEQ ID NO: 11) meristem-preferential promoter, while in control construct 95, LbCas12a was 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' terminal NLS sequences (SEQ ID NOs: 33 and 34) and operably linked to a transcription termination factor sequence (SEQ ID NO: 37) derived from the Medicago tadpole gene. The gRNA array expression cassette contains a PolIII promoter operably linked to four guide RNAs, each targeting a 27-nucleotide sequence within the 864-nucleotide E1 gene sequence (SEQ ID NO: 38) in the soybean (Glycine max) genome (see Table 5). The T-DNA vector also includes an expression cassette for a select marker conferring resistance to the antibiotic spectinomycin. Soybean A3555 cultivar embryos were transformed using the above vectors via Agrobacterium-mediated transformation, and R0 plants were regenerated from the transformed soybean cells.

[0272] [Table 5]

[0273] DNA was extracted from leaf samples derived from 20 R0 seedlings for each of constructs 94 and 95. The GmE1 site was sequenced and analyzed for the presence of targeted mutations. Co-expression of Cas12a and its allogeneic gRNA was expected to generate double-strand breaks at the target site, and subsequent incomplete DNA repair would generate unique mutations. In plants carrying AtErl1:LbCas12a (construct 94), six 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 axillary meristem, a low mutation rate from construct 94 in newly transformed (or R0) plants is expected.

[0274] [Table 6]

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

[0276] [Table 7]

[0277] Sequence analysis of the edits revealed a highly diverse range of newly generated mutations detected in R1 plants of the AtERL1:LbCas12a transgenic event. A wide range of mutations were identified from approximately 40 R1 plants for each event (see Table 8). Furthermore, several 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 editing / mutation indicates that, unlike when occurring in R1 offspring of DAMV:LbCas12a, the segregation patterns of editing identified in the R0 event of AtERL1:LbCas12a may or may not follow the classical Mendelian pattern in R1 offspring, depending on when the mutation was introduced into the R0 plant (Table 9). While events exhibiting atypical segregation patterns are not limited by any theory, the mutation is most likely to arise from the meristem after the regeneration of the R0 plant. Therefore, R1 seeds are a mixture of seeds derived from chimeric R0 plants.

[0284] [Table 9]

[0285] In summary, the data demonstrate that reproductive editing can be achieved when LbCas12a is expressed under the control of the axillary meristem promoter AtERL1. Furthermore, these data prove that a single R0 plant can produce many R1 offspring, each possessing a unique target site edit. This suggests that this promoter can be used to drive nuclease expression to increase the frequency of unique edits produced per transformed plant.

[0286] (Example 7) Preferential expression of Cas12a in floral tissue / cells to generate germ cell mutations. This example describes the use of soybean meristem-preferential promoters derived from GmAP1-like, GmCYC3-1, GmAP3-like-1, and GmERL1-like genes to drive Cas12a expression for generating diverse mutations in R0 generation and beyond. GmERL1-like is a 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. In construct 91, the LbCas12a cassette was used to promote the expression of the Medicago umbellata Ubq2 gene. to child While both are driven by a powerful constitutive promoter derived from soybeans (Glycine max), their designs are similar in other cases, except that LbCas12a expression is driven by various meristematic-preferential promoters derived from soybeans.

[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 GmAP1-like-1-va r and It is disclosed as follows: GmAP1-like-1-va r is Compared to GmAP1-like-1 (SEQ ID NO: 12), it includes a 5' extension of 7 nucleotides and a 3' extension of 371 nucleotides. In construct 100, LbCas12a expression is driven by a variant of the soy GmERL1 promoter (SEQ ID NO: 27), and GmERL1-va r and It is disclosed as follows: GmERL1-va r is Compared to GmERL1 (SEQ ID NO: 27), it includes a 5' deletion of 261 nucleotides and a 3' extension of 972 nucleotides.

[0289] The plant codon-optimized LbCas12a sequence (SEQ ID NO: 32) in these cassettes is flanked by NLS sequences at the 5' and 3' ends (SEQ ID NOs: 33 and 34), and is a transcription termination factor derived from the Medicago gene. In line It is operable and linked. The gRNA array expression cassette is Tawny coding for 1542 nucleotides in the soybean genome. in column The T-DNA vector contains a PolIII promoter operably ligated to a single guide RNA, targeting a 27-nucleotide sequence (see Table 11). The T-DNA vector also contains an expression cassette for a selection marker conferring resistance to the antibiotics spectinomycin and streptomycin. Soybean A3555 cultivar embryos were transformed using the above vector by Agrobacterium-mediated transformation, and R0 plants were regenerated from the transformed soybean cells.

[0290] [Table 11]

[0291] DNA was extracted from leaf samples derived from 20 R0 seedlings for each of constructs 96–100. The GmTawny site was sequenced and analyzed for the presence of targeted mutations. Co-expression of Cas12a and its allogeneic gRNA was expected to generate double-strand breaks at the target site, and subsequent incomplete DNA repair would generate unique mutations. Ten targeted site edits were identified in plants carrying the MtUbq2::LbCas12a construct (construct 1) (see Table 12). Total editing in tissue-specific transformations varied but was on the same scale as in constitutive transformations. Mutation rates were governed by both tissue specificity and differences in expression scale.

[0292] [Table 12]

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

[0294] [Table 13]

[0295] Sequence analysis of the edits revealed a highly diverse range of newly generated mutations detected in R1 plants of the GmAP3-like-1::LbCas12a and GmERL1-var::LbCas12a transgenic events. A wide range of mutations were identified from approximately 40 R1 plants of each event (see Table 14). Furthermore, several edits were identified in 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 meristematic tissue using AtERl1 promoter variant. This embodiment describes the use of a variant of the Arabidopsis thaliana meristem-preferential promoter AtERl1 to drive Cas12a expression for generating diverse mutations in the R0 generation and beyond.

[0301] The AtERL1 promoter sequence disclosed as SEQ ID NO: 11 contains a sequence of 34 Ts starting at nucleotide position 2021 and extending to 2054. Long extensions of the same nucleotide can create problems during DNA sequencing. To overcome this potential problem, a variant of the AtERL1 promoter (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] This generates 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 using the above vectors via 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 homogeneous gRNA is expected to generate double-strand breaks at the target site, and subsequent incomplete DNA repair will generate unique mutations. Several R0 lines derived from each transformed construct are grown to maturity, and at least one spike from each transformed soybean plant is self-pollinated. Several hundred R1 lines are selected, grown, and screened for mutations. Edits in R1 offspring derived from the transgenic events of constructs 94 and 95 are compared.

[0305] (Example 9) The use of AtERL1 promoter variants does not affect the specificity or intensity of reporter gene expression. The AtERL1 promoter variant (SEQ ID NO: 49) described in Example 8 was operably ligated 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 ligated to the β-glucuronidase reporter gene (GUS), and this was used to generate an Agrobacterium T-DNA construct. Soybean A3555 cultivar embryos were transformed using the above vectors via 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 using the unmodified AtERL1 promoter.

[0307] Histochemical expression analysis of various tissues derived from transgenic soybean plants (different events) revealed that expression, indicated by blue spots, was primarily limited to meristem tissue, even in soybean plants transformed with the GUS gene operably linked to the variant AtERL1 promoter. Some residual blue staining was observed in limited floral tissues obtained from transgenic plants containing either the control construct or the construct with the variant promoter.

[0308] Modifications to the variant AtERL1 promoter did not affect the promoter's tissue specificity or expression intensity.

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.

Images