Methods and compositions for improving yield characteristics in plants

Abstract

The present invention relates to compositions and methods for modifying the homologous domain-leucine zipper transcription factor (HD-Zip) gene in plants. The invention further relates to plants with improved yield characteristics produced using the methods and compositions of the invention.

Description

[0001] Statement Regarding Electronic File of Sequence Listing

[0002] The sequence listing in XML text format, titled 1499-108_ST26.xml, sized 226,465 bytes, generated on August 28, 2023, and submitted herewith, is hereby incorporated by reference into this specification for its disclosure.

[0003] Claims

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 374,916, filed on September 8, 2022, under 35 U.S.C.§119(e), the entire content of which is incorporated herein by reference. Technical Field

[0005] The present invention relates to compositions and methods for modifying homeodomain-leucine zipper transcription factor (HD-Zip) genes in plants. The present invention further relates to plants having improved yield characteristics produced using the methods and compositions of the present invention. Background Art

[0006] HD-Zip transcription factors can be subdivided into four subfamilies: HD-Zip I to HD-Zip IV, based on different sequence features (DNA-binding domain and additional conserved motifs specific to each subfamily) and different functions of the proteins from each subfamily (Sessa et al., Int J Mol Sci, 19:4047 (2018)).

[0007] The characteristic feature of the HD-Zip gene family is the association of a homeodomain (HD) and a leucine zipper (LZ) motif in a single protein. In other kingdoms, these genes exist as domains of different proteins. The homeodomain of the HD-Zip gene is a DNA-binding domain composed of approximately 60 amino acids and consists of a helix-turn-helix structure that folds into three characteristic α helices. This DNA-binding domain is capable of specifically interacting with DNA. The LZ motif is a dimerization motif and is located immediately after the HD. The LZ motif allows the formation of homodimers and heterodimers, which are required for DNA binding (Sessa et al., Int J Mol Sci, 19:4047 (2018)).

[0008] HD-Zip II transcription factors also contain an ERF-associated amphiphilic repression (EAR) motif of the LxLxL type, which can act as a negative regulator of gene expression. In addition, HOMEOBOX ARABIDOPSIS THALIANA (HAT) 1 and HAT22, two members of the HD-Zip II protein family, interact with the TOPLESS (TPL) co-repressor through the EAR motif. HD-Zip II proteins preferentially bind to the CAAT(C / G)ATTG motif (Sessa et al., Int J Mol Sci, 19:4047 (2018)).

[0009] The present invention relates to improving photosynthesis and yield traits by manipulating class II HD-Zip factors, which can produce crop plants with improved growth characteristics. Summary of the Invention

[0010] One aspect of the present invention provides a plant or plant part comprising at least one mutation in an endogenous homeodomain-leucine zipper transcription factor (HD-Zip) gene encoding an HD-Zip transcription factor (HD-Zip) polypeptide, wherein the mutation alters the function of the HD-Zip polypeptide as a gene expression regulator, optionally wherein the at least one mutation can be a non-natural mutation.

[0011] A second aspect of the present invention provides a plant cell comprising an editing system comprising: (a) a CRISPR-Cas effector protein; and (b) a guide nucleic acid (e.g., gRNA, gDNA, crRNA, crDNA) having a spacer sequence complementary to an endogenous target gene encoding an HD-Zip transcription factor polypeptide.

[0012] A third aspect of the present invention provides a plant cell comprising at least one mutation within an HD-Zip gene, wherein the at least one mutation is a base substitution, base insertion, and / or base deletion, and the base substitution, base insertion, and / or base deletion is introduced using an editing system comprising a nucleic acid binding domain that binds to a target site within the HD-Zip gene, optionally wherein the at least one mutation can be a non-natural mutation.

[0013] A fourth aspect of the present invention provides a method for generating / cultivating a genetically edited plant without transgenes, the method comprising: crossing the plant of the present invention with a plant without transgenes, thereby introducing at least one mutation into the plant without transgenes; and selecting a progeny plant without transgenes that contains the at least one mutation, thereby generating a genetically edited plant without transgenes, optionally wherein the at least one mutation can be a non-natural mutation.

[0014] A fifth aspect of the present invention provides a method for providing a plurality of plants with improved yield traits, the method comprising growing two or more plants of the present invention in a growing area, thereby providing a plurality of plants of the present invention, the plurality of plants of the present invention having improved yield traits compared to a plurality of control plants lacking at least one mutation (optionally an increase in the number of seeds (e.g., the number of grains), an increase in seed weight (e.g., grain weight; 100-seed weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching).

[0015] In a sixth aspect, a method for generating a mutation in an endogenous HD-Zip gene in a plant is provided, the method comprising: (a) targeting a gene editing system to a region of the HD-Zip17-1 gene and / or the HD-Zip17-2 gene that contains a sequence having at least 80% sequence identity with any one of SEQ ID NOs: 72-85 or 91-105; and (b) selecting a plant that contains a modification in a region of the gene that has at least 80% sequence identity with any one of SEQ ID NOs: 72-85 or 91-105.

[0016] A seventh aspect of the present invention provides a method for generating a variation in an HD-Zip gene, the method comprising introducing an editing system into a plant cell, wherein the editing system is targeted to a region of the HD-Zip gene that encodes an HD-Zip polypeptide; and contacting the region of the HD-Zip gene with the editing system, thereby introducing a mutation into the HD-Zip gene and generating a variation in the HD-Zip gene in the plant cell.

[0017] An eighth aspect of the present invention provides a method for detecting a mutant HD-Zip gene (a mutation in an endogenous HD-Zip gene) in a plant, the method comprising detecting in the genome of the plant an HD-Zip gene that has at least one mutation in a region having at least 80% sequence identity with any one of the nucleotide sequences of SEQ ID NOs: 72-85 or 91-105.

[0018] A ninth aspect provides a method for editing a specific site in the genome of a plant cell, the method comprising: cutting a target site within an endogenous HD-Zip gene in the plant cell in a site-specific manner, the endogenous HD-Zip gene: (a) comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprising a region of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO: 72-85 or 91-105; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encoding a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107 or 108.

[0019] A tenth aspect provides a method for producing a plant, the method comprising: (a) contacting a population of plant cells comprising an endogenous HD-Zip gene with a nuclease linked to a nucleic acid binding domain (e.g., an editing system), the nucleic acid binding domain binding to a target site within the endogenous HD-Zip gene, wherein the endogenous gene (i) comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 69, 70, 88 or 89; (ii) comprises a region of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO: 72-85 or 91-105; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; (iv) and / or encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107 or 108; (b) selecting plant cells from the population of plant cells in which the endogenous HD-Zip gene has been mutated, thereby producing plant cells comprising a mutation in the endogenous HD-Zip gene; and (c) growing the selected plant cells into a plant comprising the mutation in the endogenous HD-Zip gene.

[0020] The eleventh aspect provides a method for increasing the number of seeds (e.g., the number of grains), increasing the seed weight (e.g., the grain weight), increasing the number of pods per node, increasing the number of pods per plant, altering the flowering time (e.g., earlier flowering time), shortening the plant height, reducing the number of nodes and / or reducing the branching in a plant, the method comprising (a) contacting a plant cell comprising an endogenous HD-Zip gene with a nuclease linked to a nucleic acid binding domain (e.g., an editing system), the nucleic acid binding domain binding to a target site within the endogenous HD-Zip gene, wherein the endogenous gene (i) comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 69, 70, 88 or 89, (ii) comprises a region of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO: 72-85 or 91-105; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; (iv) and / or encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107 or 108; and (b) growing the plant cell comprising the mutated endogenous HD-Zip gene into a plant, thereby producing a plant having a mutated HD-Zip gene and exhibiting a phenotype of increased number of seeds (e.g., the number of grains), increased seed weight (e.g., the grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of nodes and / or reduced branching.

[0021] A twelfth aspect provides a method for generating a plant or a part thereof, the plant or part thereof comprising at least one cell having an endogenous HD-Zip gene that has been mutated, the method comprising: contacting a target site within the endogenous HD-Zip gene in the plant or plant part with a nuclease, the nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site within the endogenous HD-Zip gene, wherein the endogenous HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 69, 70, 88 or 89, (b) comprises a region of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO: 72-85 or 91-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107 or 108, thereby generating a plant or a part thereof, the plant or part thereof comprising at least one cell having a mutation in the endogenous HD-Zip gene.

[0022] The thirteenth aspect of the present invention provides a method for generating a plant or a part thereof, the plant or the part thereof comprising a mutated endogenous HD-Zip gene and exhibiting a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching. The method comprises contacting a target site within the endogenous HD-Zip gene in the plant or plant part with a nuclease, the nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site within the HD-Zip gene, and wherein the HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity with any one of SEQ ID NO: 69, 70, 88, or 89; (b) comprises a region consisting of contiguous nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region consisting of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107, or 108, thereby generating a plant or a part thereof, the plant or the part thereof comprising a mutated endogenous HD-Zip gene and exhibiting a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching.

[0023] In a fourteenth aspect, there is provided a method for modifying an endogenous HD-Zip gene in a plant or a part thereof to increase the number of seeds (e.g., the number of grains), increase the seed weight (e.g., the grain weight), increase the number of pods per plant, change the flowering time (e.g., earlier flowering time), reduce the plant height, reduce the number of stem nodes, and / or reduce the branching in the plant or the part thereof, the method comprising modifying a target site within the endogenous HD-Zip gene in the plant or the part thereof, wherein the endogenous HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity with any one of SEQ ID NO: 69, 70, 88 or 89, (b) comprises a region consisting of consecutive nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region consisting of consecutive amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, whereby modifying the endogenous HD-Zip gene and improving the yield traits in the plant or the part thereof.

[0024] A fifteenth aspect provides a guide nucleic acid that binds within a target site within an HD-Zip gene, the target site comprising a sequence having at least 80% identity with any one or more nucleotide sequences of the nucleotide sequences of SEQ ID NO: 72-85 or 91-105.

[0025] In a sixteenth aspect, there is provided a system comprising the guide nucleic acid of the present invention and a CRISPR-Cas effector protein, the CRISPR-Cas effector protein being associated with the guide nucleic acid.

[0026] A seventeenth aspect provides a gene editing system comprising a CRISPR-Cas effector protein, the CRISPR-Cas effector protein being associated with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to an endogenous HD-Zip gene.

[0027] In an eighteenth aspect, there is provided a complex comprising a guide nucleic acid and a CRISPR-Cas effector protein, the CRISPR-Cas effector protein comprising a cleavage domain, wherein the guide nucleic acid binds to a target site within an endogenous HD-Zip gene (a) comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 69, 70, 88 or 89, (b) comprising a region of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO: 72-85 or 91-105; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encoding a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107 or 108, and the cleavage domain cleaves the target strand in the HD-Zip gene.

[0028] In a nineteenth aspect, there is provided an expression cassette comprising (a) a polynucleotide encoding a CRISPR-Cas effector protein, the CRISPR-Cas effector protein comprising a cleavage domain, and (b) a guide nucleic acid that binds to a target site within an endogenous HD-Zip gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (i) a portion of a nucleic acid encoding an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 90; (ii) a portion of a nucleic acid encoding an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107 or 108; (iii) a portion of a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: 69, 70, 88 or 89; and / or (iii) a portion of a sequence having at least 80% sequence identity to any one nucleotide sequence of the nucleotide sequences of SEQ ID NO: 72-85 or 91-105.

[0029] In a further aspect, there is provided a nucleic acid encoding an HD-Zip polypeptide having a mutated ethylene response element binding factor-associated amphiphilic repression (EAR) motif, wherein the mutated EAR motif comprises a mutation that alters its function as a gene expression regulator, optionally wherein the nucleic acid encoding the HD-Zip polypeptide has the gene identification number (SoyBase Database) Glyma.20g014400 (HD-Zip17-1) or Glyma.07g218000 (HD-Zip17-2), optionally wherein the mutation can be a non-natural mutation.

[0030] There is further provided a modified HD-Zip gene comprising at least 90% sequence identity with SEQ ID NO: 113 and / or encoding a mutated HD-Zip polypeptide having at least 90% sequence identity with SEQ ID NO: 115.

[0031] There is additionally provided a soybean plant or a plant part thereof comprising a mutation in at least one endogenous HD-Zip gene having the gene identification number (SoyBase Database) Glyma.20g014400 (HD-Zip17-1) or Glyma.07g218000 (HD-Zip17-2).

[0032] A further aspect provides a guide nucleic acid that binds to a target nucleic acid in an endogenous HD-Zip gene having the gene identification number Glyma.20g014400 or Glyma.07g218000.

[0033] There is further provided a plant that comprises in its genome one or more mutated HD-Zip genes generated by the method of the present invention, as well as polypeptides, polynucleotides, nucleic acid constructs, expression cassettes, and vectors for preparing the plant of the present invention.

[0034] These and other aspects of the present invention are set forth in more detail in the description of the present invention below.

[0035] Brief Description of the Sequences

[0036] SEQ ID NOs: 1-17 are exemplary Cas12a amino acid sequences that can be used in the present invention.

[0037] SEQ ID NOs: 18-20 are exemplary Cas12a nucleotide sequences that can be used in the present invention.

[0038] SEQ ID NO: 21-22 are exemplary regulatory sequences encoding promoters and introns.

[0039] SEQ ID NO: 23-29 are exemplary cytosine deaminase sequences that can be used in the present invention.

[0040] SEQ ID NO: 30-40 are exemplary adenine deaminase amino acid sequences that can be used in the present invention.

[0041] SEQ ID NO: 41 is an exemplary uracil-DNA glycosylase inhibitor (UGI) sequence that can be used in the present invention.

[0042] SEQ ID NO: 42-44 provide exemplary peptide tags and affinity polypeptides that can be used in the present invention.

[0043] SEQ ID NO: 45-55 provide exemplary RNA recruitment motifs and corresponding affinity polypeptides that can be used in the present invention.

[0044] SEQ ID NO: 56-57 are exemplary Cas9 polypeptide sequences that can be used in the present invention.

[0045] SEQ ID NO: 58-68 are exemplary Cas9 polynucleotide sequences that can be used in the present invention.

[0046] SEQ ID NO: 69 and SEQ ID NO: 88 are exemplary HD-Zip genomic sequences.

[0047] SEQ ID NO: 70 and SEQ ID NO: 89 are the exemplary HD-Zip coding (cds) sequences of SEQ ID NO: 69 and SEQ ID NO: 88, respectively.

[0048] SEQ ID NO: 71 and SEQ ID NO: 90 are the exemplary HD-Zip polypeptide sequences encoded by SEQ ID NO: 69 and SEQ ID NO: 70, and SEQ ID NO: 88 and SEQ ID NO: 89, respectively.

[0049] SEQ ID NO: 72-83 are exemplary target regions of the HD-Zip genomic sequence (SEQ ID NO: 69) that can be used in the present invention.

[0050] SEQ ID NO: 91-102 are exemplary target regions of the HD-Zip genomic sequence (SEQ ID NO: 88) that can be used in the present invention.

[0051] SEQ ID NOs: 84 - 85 and 105 are example sequences containing the EAR motif of the HD-Zip genomic sequence SEQ ID NO: 69.

[0052] SEQ ID NOs: 103 - 105 are example sequences containing the EAR motif of the HD-Zip genomic sequence SEQ ID NO: 88.

[0053] SEQ ID NOs: 86, 87, and 108 are example peptide sequences containing the EAR motif of the HD-Zip polypeptide sequence SEQ ID NO: 71.

[0054] SEQ ID NOs: 106 - 108 are example peptide sequences containing the EAR motif of the HD-Zip polypeptide sequence SEQ ID NO: 90.

[0055] SEQ ID NOs: 109 - 112 are example spacer sequences of the guide nucleic acid that can be used in the present invention.

[0056] SEQ ID NO: 113 is an example mutated HD-Zip genomic sequence edited as described herein.

[0057] SEQ ID NO: 114 is 21 consecutive nucleotides of the mutated nucleic acid sequence deleted from SEQ ID NO: 69 to generate SEQ ID NO: 113.

[0058] SEQ ID NO: 115 is an example mutated HD-Zip polypeptide encoded by SEQ ID NO: 113.

[0059] SEQ ID NO: 116 is 7 consecutive amino acids of the mutated polypeptide deleted from SEQ ID NO: 71 to generate SEQ ID NO: 115. Detailed Description

[0060] The present invention will now be described with reference to the following examples, in which embodiments of the present invention are shown. This description is not intended to be an exhaustive catalog of all the different ways in which the present invention can be implemented or of all the features that can be added to the present invention. For example, features shown with respect to one embodiment can be incorporated into other embodiments, and features shown with respect to a particular embodiment can be deleted from that embodiment. Thus, the present invention contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein can be excluded or omitted. Additionally, many variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the present disclosure, and such variations and additions do not depart from the present invention. Accordingly, the following description is intended to illustrate some particular embodiments of the present invention and is not intended to exhaustively specify all of its permutations, combinations, and variations.

[0061] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terms used in the description of the present invention are for the purpose of describing particular embodiments only and are not intended to limit the present invention.

[0062] All publications, patent applications, patents, and other references cited herein are incorporated herein by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0063] Unless the context otherwise indicates, the various features of the present invention specifically intended to be described herein can be used in any combination. Additionally, the present invention contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein can be excluded or omitted. By way of illustration, if the specification states that a composition contains components A, B, and C, it is specifically intended that any one of A, B, or C, or any combination thereof, can be omitted and disclaimed, either singly or in any combination.

[0064] As used in the description of the present invention and the appended claims, the singular forms "a", "an", and "the" are also intended to include the plural forms unless the context clearly indicates otherwise.

[0065] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the absence of a combination when interpreted in the alternative form ("or").

[0066] As used herein, when the term "about" refers to a measurable value such as an amount or concentration, it is intended to cover variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value, as well as the specified value. For example, in the case where X is a measurable value, "about X" is intended to include X and variations of X of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1%. The ranges of measurable values provided herein can include any other ranges and / or individual values therein.

[0067] As used herein, phrases such as "between X and Y" and "between about X and Y" shall be interpreted to include X and Y. As used herein, the phrase "between about X and Y" means "between about X and about Y", and the phrase "about X to Y" means "about X to about Y".

[0068] Unless otherwise indicated herein, the recitation of numerical ranges herein is merely intended to be a shorthand method of referring individually to each separate value falling within the range, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if the range 10 to 15 is disclosed, then 11, 12, 13, and 14 are also disclosed.

[0069] As used herein, the terms "comprise", "comprises", and "comprising" specify the presence of the stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0070] As used herein, the transitional phrase "consisting essentially of" means that the scope of the claim should be interpreted to cover the specified materials or steps recited in the claim, as well as materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. Thus, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as equivalent to "comprising".

[0071] As used herein, the terms “increase,” “increasing,” “increased,” “enhance,” “enhanced,” “enhancing,” and “enhancement” (and their grammatical variants) describe an elevation of at least about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more as compared to a control. For example, a plant comprising a mutation in an HD-Zip gene as described herein can exhibit improved yield traits, optionally, the improved yield traits can be a phenotype of an increase in seed number (e.g., an increase in the number of grains), an increase in seed weight (e.g., an increase in grain weight, 100-seed weight), and an increase in the number of pods per plant as compared to a control plant that does not comprise the same HD-Zip mutation. The control plant is generally a plant that is the same as the edited plant, but the control plant has not been similarly edited and thus does not comprise or lack the edit / mutation. The control plant can be an isogenic plant and / or a wild-type plant. Thus, the control plant can be the same breeding line, variety or cultivar as the test plant into which the mutation is introgressed as described herein, but the control breeding line, variety or cultivar does not contain the mutation. In some embodiments, the comparison between the plant of the present invention and the control plant is performed under the same growth conditions, e.g., the same environmental conditions (soil, hydration, light, heat, nutrients, etc.).

[0072] As used herein, the terms “reduce,” “reduced,” “reducing,” “reduction,” “diminish,” and “decrease” (and their grammatical variants) describe a reduction of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% as compared to a control, for example. In certain embodiments, the reduction can result in no or substantially no (i.e., a non-significant amount, e.g., less than about 10% or even 5%) detectable activity or amount. As an example, a plant produced by the method of the present invention can exhibit a phenotype of earlier flowering time (e.g., a shorter time to flowering), reduced height (e.g., shorter plant height), reduced number of stem nodes, and / or reduced branching.

[0073] A "control plant" is typically a plant that is the same as the edited plant, but the control plant has not been similarly edited and thus lacks the mutation. The control plant can be an isogenic plant and / or a wild-type plant. Thus, the control plant can be the same breeding line, variety, or cultivar as the test plant into which the mutation has been introgressed as described herein, but the control breeding line, variety, or cultivar does not contain the mutation. In some embodiments, the comparison between the plant of the invention and the control plant is carried out under the same growth conditions, such as the same environmental conditions (soil, hydration, light, heat, nutrients, etc.).

[0074] As used herein, the terms "express", "expresses", "expressed", or "expression" with respect to a nucleic acid molecule and / or nucleotide sequence (e.g., RNA or DNA) mean that the nucleic acid molecule and / or nucleotide sequence is transcribed and optionally translated. Thus, the nucleic acid molecule and / or nucleotide sequence can express a polypeptide of interest or, for example, a functional untranslated RNA.

[0075] As used herein, the term "heterologous" refers to a nucleotide / polypeptide that is derived from a foreign species or, in the case of being from the same species, is substantially modified from its natural form by intentional human intervention in a composition and / or genomic locus. A "heterologous" or "recombinant" nucleotide sequence is a nucleotide sequence that is not naturally associated with the host cell into which it is introduced, including non-naturally occurring multiple copies of a naturally occurring nucleotide sequence.

[0076] A "natural" or "wild-type" nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide, or amino acid sequence. In some contexts, a "wild-type" nucleic acid is a nucleic acid that has not been edited as described herein and can be different from an "endogenous" gene that can be edited as described herein (e.g., a mutated endogenous gene). In some contexts, a "wild-type" nucleic acid (e.g., unedited) can be heterologous to the organism in which the wild-type nucleic acid is present (e.g., a transgenic organism). As an example, a "wild-type endogenous homeodomain-leucine zipper transcription factor (HD-Zip) gene" is an HD-Zip gene that is naturally present in or endogenous to a reference organism (e.g., a plant (e.g., a soybean plant)) and can be subjected to modifications as described herein, after which such a modified endogenous gene is no longer wild-type.

[0077] As used herein, the term "heterozygous" refers to the genetic state in which different alleles reside at corresponding loci on homologous chromosomes.

[0078] As used herein, the term "homozygous" refers to a genetic state in which identical alleles reside at corresponding loci on homologous chromosomes.

[0079] As used herein, the term "allele" refers to one of two or more different nucleotides or nucleotide sequences that occur at a particular locus.

[0080] A "null allele" is a non-functional allele caused by a gene mutation that results in the complete absence of the corresponding protein or the production of a non-functional protein.

[0081] A "knockout mutation" is a mutation that produces a non-functional protein but may have a detectable transcript or protein.

[0082] A "recessive mutation" is a mutation in a gene that produces a phenotype when homozygous, but the phenotype is not observable when the locus is heterozygous.

[0083] A "dominant mutation" is a mutation in a gene that produces a mutant phenotype in the presence of an unmutated copy of the gene. A dominant mutation can be a loss-of-function or gain-of-function mutation, a hypomorphic mutation, a hypermorphic mutation, or a weak loss or weak gain of function.

[0084] A "dominant negative mutation" is a mutation that alters a gene product (e.g., has an abnormal function relative to the wild type), and the altered gene product adversely affects the function of the wild-type allele or gene product. For example, a "dominant negative mutation" can block the function of the wild-type gene product. A dominant negative mutation can also be referred to as an "antimorphic mutation".

[0085] A "semidominant mutation" is a mutation in which the penetrance of the phenotype in a heterozygous organism is less than the penetrance of the phenotype observed in a homozygous organism.

[0086] A "weak loss-of-function mutation" is a mutation that produces a gene product with partial or reduced function (partial inactivation) compared to the wild-type gene product.

[0087] A "hypomorphic mutation" is a mutation that causes a partial loss of gene function but does not completely abolish function / activity, and the partial loss can occur through reduced expression (e.g., reduced protein and / or reduced RNA) or reduced functional performance (e.g., reduced activity). A "hypomorphic" allele is a semi-functional allele caused by a gene mutation that produces a corresponding protein that functions at any level between 1% and 99% of normal efficiency.

[0088] A "hypermorphic mutation" is a mutation that increases the expression of the gene product and / or increases the activity of the gene product.

[0089] "Gain-of-function" alleles or mutations are mutations that confer new functions to the encoded gene products and / or confer new gene expression patterns. In some embodiments, gain-of-function mutations can be dominant or semi-dominant.

[0090] As used herein, "non-natural mutation" refers to a mutation that is generated by human intervention and is different from the naturally occurring mutations present in the same gene (e.g., naturally occurring and not the result of human modification).

[0091] "Locus" is the position on a chromosome where a gene or a marker or an allele is located. In some embodiments, a locus can encompass one or more nucleotides.

[0092] As used herein, the terms "desired allele", "target allele" and / or "allele of interest" are used interchangeably to refer to an allele associated with a desired trait. In some embodiments, the desired allele can be associated with an increase or decrease (relative to a control) of a given trait, depending on the nature of the desired phenotype.

[0093] A marker is "associated with" a trait when the trait is related to the marker and the presence of the marker is an indication of whether and / or to what extent the desired trait or form of the trait occurs in a plant / germplasm containing the marker. Similarly, a marker is "associated with" an allele or chromosomal interval when the marker is related to the allele or chromosomal interval and the presence of the marker is an indication of whether the allele or chromosomal interval is present in a plant / germplasm containing the marker.

[0094] As used herein, the terms "backcross" and "backcrossing" refer to the process of crossing a progeny plant with one of its parental plants one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.). In a backcrossing scheme, the "donor" parent is the parental plant having the desired gene or locus to be introgressed. The "recipient" parent (used one or more times) or "recurrent" parent (used two or more times) is the parental plant into which the gene or locus is being introgressed. See, e.g., Ragot, M. et al., Marker-assisted Backcrossing: A Practical Example, in TECHNIQUES ET UTILISATIONSDES MARQUEURS MOLECULAIRES LES COLLOQUES, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in BackcrossBreeding, in PROCEEDINGS OF THE SYMPOSIUM “ANALYSIS OF MOLECULAR MARKER DATA”, pp. 41-43 (1994). The initial cross produces the F1 generation. The term "BC1" refers to the second use of the recurrent parent, "BC2" refers to the third use of the recurrent parent, and so on.

[0095] As used herein, the terms "cross" or "crossed" refer to the production of progeny (e.g., cells, seeds, or plants) by the fusion of gametes through pollination. The terms encompass sexual crosses (pollination of one plant by another) and selfing (self-pollination, e.g., when pollen and ovules are from the same plant). The term "crossing" refers to the act of producing progeny by the fusion of gametes through pollination.

[0096] As used herein, the terms "introgression", "introgressing", and "introgressed" refer to the natural and artificial transfer of desired alleles or combinations of desired alleles at one or more genetic loci from one genetic background to another. For example, a desired allele at a designated locus can be transferred to at least one offspring by sexual hybridization between two parents of the same species, where at least one parent has the desired allele in its genome. Alternatively, for example, the transfer of alleles can occur by recombination between two donor genomes, such as in fused protoplasts, where at least one donor protoplast has the desired allele in its genome. The desired alleles can be selected alleles of a marker, QTL, transgene, etc. The offspring containing the desired alleles can be backcrossed one or more times (e.g., 1, 2, 3, 4, or more times) to a line having the desired genetic background, and the desired alleles are selected, resulting in the fixation of the desired alleles in the desired genetic background. For example, a marker associated with increased yield under non-water stress conditions can be introgressed from a donor into a recurrent parent that does not contain the marker and does not exhibit increased yield under non-water stress conditions. The resulting offspring can then be backcrossed one or more times and selected until the offspring possess the genetic marker associated with increased yield under non-water stress conditions in the recurrent parent background.

[0097] A "genetic map" is a description of the genetic linkage relationships between loci on one or more chromosomes within a given species, typically depicted in a diagram or table. For each genetic map, the distance between loci is measured by the frequency of recombination between them. A variety of markers can be used to detect recombination between loci. A genetic map is a product of the mapping population, the type of markers used, and the polymorphism potential of each marker between different populations. The order and genetic distance between loci can vary depending on the genetic map.

[0098] As used herein, the term "genotype" refers to the genetic constitution of an individual (or population of individuals) at one or more genetic loci, as contrasted with the observable and / or detectable and / or manifested traits (phenotypes). A genotype is defined by the alleles at one or more known loci that an individual inherits from its parents. The term genotype can be used to refer to the genetic constitution of an individual at a single locus, multiple loci, or more generally, the term genotype can be used to refer to the genetic makeup of all the genes in an individual's genome. A genotype can be indirectly characterized, for example, using markers and / or directly characterized by nucleic acid sequencing.

[0099] As used herein, the term "germplasm" refers to the genetic material of an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety, or family), or a clone derived from a line, variety, species, or culture, or the genetic material from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety, or family), or a clone derived from a line, variety, species, or culture. Germplasm can be part of an organism or cell or can be separate from an organism or cell. Generally, germplasm provides genetic material with a specific genetic constitution that underlies some or all of the genetic qualities of an organism or cell culture. As used herein, germplasm includes cells, seeds, or tissues that can grow into new plants, as well as plant parts (e.g., leaves, stems, buds, roots, pollen, cells, etc.) that can be cultured into whole plants.

[0100] As used herein, the terms "cultivar" and "variety" refer to a group of similar plants that can be distinguished from other varieties within the same species by structural or genetic characteristics and / or performance.

[0101] As used herein, the terms "exotic", "exotic line", and "exotic germplasm" refer to any plant, line, or germplasm that is not elite. Generally, exotic plants / germplasm do not originate from any known elite plants or germplasm but are selected to introduce one or more desired genetic elements into a breeding program (e.g., to introduce new alleles into a breeding program).

[0102] As used herein, the term "hybrid" in the context of plant breeding refers to a plant that is the offspring of genetically distinct parents produced by crossing plants of different lines, varieties, or species (including but not limited to crosses between two inbred lines).

[0103] As used herein, the term "inbred line" refers to a plant or variety that is substantially homozygous. The term can refer to a plant or plant variety that is substantially homozygous throughout the genome or a plant or plant variety that is substantially homozygous with respect to a part of the genome of particular interest.

[0104] A "haplotype" is the genotype of an individual at multiple genetic loci, i.e., the combination of alleles. Generally, the genetic loci that define a haplotype are physically and genetically linked, i.e., on the same chromosomal segment. The term "haplotype" can refer to polymorphisms at a specific locus (such as a single marker locus) or polymorphisms at multiple loci along a chromosomal segment.

[0105] Plants in which at least one (e.g., one or more, e.g., 1, 2, 3, or 4 or more) endogenous HD-Zip genes are modified (e.g., contain the modifications as described herein) can have improved yield traits compared to plants that do not contain (lack) modifications in at least one endogenous HD-Zip gene. As used herein, "improved yield traits" refers to any plant trait related to growth, such as biomass, yield, nitrogen use efficiency (NUE), inflorescence size / weight, fruit yield, fruit quality, fruit size, seed size (e.g., seed area, seed size), seed number, leaf tissue weight, nodule number, nodule mass, nodule activity, seed head number, tiller number, branch number, flower number, tuber number, tuber mass, bulb mass, seed number, total seed mass, leaf emergence rate, tiller / branch emergence rate, emergence rate, root length, root number, size and / or weight of the root system, and / or any combination thereof. In some aspects, "improved yield traits" can include, but are not limited to, increased inflorescence yield, increased fruit yield (e.g., increased number, weight, and / or size of fruits; e.g., increased number, weight, and / or length of spikes, e.g., for maize), improved fruit quality, increased number, size, and / or weight of roots, increased meristem size, increased seed size (e.g., seed area and / or seed weight), increased biomass, increased leaf size, and / or increased nitrogen use efficiency compared to a control plant or a part thereof (e.g., a plant that does not contain the mutated endogenous HD-Zip nucleic acid as described herein). In some aspects, the improved yield traits can be expressed as the number of grains / seeds produced per unit area of land (e.g., bushels per acre of land). In some embodiments, one or more improved yield traits can be any one or more of the following: increased flower number, increased flower structure size, increased spike length, and / or increased number of kernel rows, optionally wherein when the number of kernel rows increases, the spike length does not decrease significantly.

[0106] As used herein, "control plant" means a plant that does not contain an edited HD-Zip gene or a gene as described herein that confers an enhanced / improved trait (e.g., a yield trait) or an altered phenotype (e.g., an increase in the number of seeds (e.g., an increase in the number of grains), an increase in seed weight (e.g., an increase in grain weight, an increase in the weight of 100 seeds), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), shorter plant height, a decrease in the number of internodes, and / or a decrease in branching). Control plants are used to identify and select plants edited as described herein that have an enhanced trait or an altered phenotype compared to the control plants. Suitable control plants can be the parental line plants used to generate the plants containing the mutated HD-Zip gene, e.g., wild-type plants or isogenic plants lacking the edit in the endogenous HD-Zip gene as described herein. Suitable control plants can also be plants containing recombinant nucleic acids conferring other traits, e.g., transgenic plants with enhanced herbicide tolerance. In some cases, suitable control plants can be the progeny of a heterozygous or hemizygous transgenic plant line lacking the mutated HD-Zip gene as described herein, referred to as a negative segregant or a negative isogenic line.

[0107] Enhanced traits (e.g., improved yield traits) can include, for example, a decrease in the number of days from planting to maturity, an increase in stalk size, an increase in the number of leaves, an increase in ear size, an increase in the dry weight of the ear per plant, an increase in the number of kernels per ear, an increase in the weight of each kernel, an increase in the number of kernels per plant, a decrease in empty kernels in the ear, an extension of the grain filling period, a decrease in plant height, an increase in the number of root branches, an increase in the total root length, an increase in yield, an increase in nitrogen use efficiency, and / or an increase in water use efficiency compared to a control plant. Altered phenotypes can be, for example, plant height, biomass, canopy area, anthocyanin content, chlorophyll content, amount of water applied, water content, and water use efficiency.

[0108] In some embodiments, the plants of the present invention may comprise one or more improved yield traits, including but not limited to, in some embodiments, one or more improved yield traits including higher yield (bushels per acre), reduced plant height, increased stem diameter, increased leaf area, increased flower number, increased number of kernel rows (optionally where ear length is not significantly reduced), increased number of kernels, increased kernel size, increased ear length, increased tiller number, reduced number of tassel branches, increased pod number (including increased number of pods per plant, increased number of seeds per pod), increased seed number, increased seed size, and / or increased seed weight (e.g., increased 100-seed weight) compared to a control plant lacking at least one mutation. In some embodiments, the plants of the present invention may comprise one or more improved yield traits compared to a control plant or a part thereof, including but not limited to, optionally increased yield (bushels per acre), seed size (including kernel size), seed weight (including kernel weight), increased number of kernel rows (optionally where ear length is not significantly reduced), increased pod number, increased number of seeds per pod, and increased ear length.

[0109] As used herein, a "trait" is a physiological, morphological, biochemical, or physical characteristic of a plant or a specific plant material or cell. In some cases, this characteristic is visible to the human eye and can be mechanically measured, such as the size, weight, shape, morphology, length, height, growth rate, and developmental stage of a seed or plant, or can be measured by biochemical techniques, such as detecting the protein, starch, certain metabolites, or oil content of a seed or leaf, or by observing metabolic or physiological processes, e.g., by measuring tolerance to water deficit or specific salt or sugar concentrations, or by measuring the expression level of one or more genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression arrays, or reporter gene expression systems, or by agricultural observations such as tolerance to osmotic stress or yield. However, any technique can be used to measure the amount, comparative level, or difference of any selected chemical compound or macromolecule in a transgenic plant.

[0110] As used herein, "enhanced trait" means a characteristic of a plant resulting from a mutation in an HD-Zip gene as described herein. Such traits include, but are not limited to, enhanced agronomic traits characterized by plant morphology, physiology, growth and development, increased yield, enhanced nutrition, disease or pest resistance, or environmental or chemical tolerance. In some embodiments, the enhanced trait / altered phenotype can be, for example, a reduction in the number of days from planting to maturity, an increase in stalk size, an increase in the number of leaves, an increase in ear size, an increase in the dry weight of the ear per plant, an increase in the number of kernels per ear, an increase in the weight per kernel, an increase in the number of kernels per plant, a reduction in ear blanking, an extended grain filling period, a reduction in plant height (optionally further showing no significant change in yield or showing an increase in yield with a reduction in plant height), an increase in the number of root branches, an increase in total root length, drought tolerance, improved water use efficiency, cold tolerance, improved nitrogen use efficiency, and / or an increase in yield. In some embodiments, the trait is an increase in yield under non-stress conditions or an increase in yield under environmental stress conditions. Stress conditions can include biotic and abiotic stresses such as drought, shade, fungal diseases, viral diseases, bacterial diseases, insect infestations, nematode infestations, cold exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability, and high plant density. "Yield" can be affected by many characteristics, including but not limited to plant height, plant biomass, number of pods, pod position on the plant, number of internodes, incidence of pod shattering, grain size, ear size, ear tip fill, kernel abortion, nodulation and nitrogen fixation efficiency, nutrient assimilation efficiency, biotic and abiotic stress resistance, carbon assimilation, plant architecture, lodging resistance, percentage of seed germination, seedling vigor, and juvenile traits. Yield can also be affected by the following factors: germination efficiency (including germination under stress conditions), growth rate (including growth rate under stress conditions), flowering time and duration, number of ears, ear size, ear weight, number of seeds per ear or per pod, seed size, seed composition (starch, oil, protein), and characteristics of seed filling.

[0111] Also as used herein, the term "trait modification" encompasses altering a naturally occurring trait by generating a detectable difference in characteristics in a plant containing a mutation in an endogenous HD-Zip gene as described herein relative to a plant that does not contain the mutation (such as a wild-type plant or a negative segregant). In some cases, trait modification can be evaluated quantitatively. For example, compared to a control plant, trait modification can result in an increase or decrease in the observed trait characteristics or phenotype. It is well known that modified traits may have natural variants. Thus, compared to a control plant, the observed trait modification can cause a change in the normal distribution and magnitude of the trait characteristics or phenotype in the plant.

[0112] The present disclosure relates to plants having improved economically relevant traits, and more specifically, plants with reduced plant height (optionally further showing no significant change in yield or an increase in yield), increased flower number, increased flower structure size, and / or increased spike length. More specifically, the present disclosure relates to a plant comprising a mutation in an HD-Zip gene as described herein, wherein the plant shows reduced plant height (optionally further showing no significant change in yield or an increase in yield), increased number of flowers, increased flower structure size, and / or increased spike length compared to a control plant lacking the mutation. In some embodiments, the plants of the present disclosure exhibit further improved traits related to yield, including but not limited to increased nitrogen use efficiency, increased nitrogen stress tolerance, increased water use efficiency, and / or increased drought tolerance, as defined and discussed below.

[0113] Yield can be defined as the measurable product of economic value from a crop. Yield can be defined in terms of quantity and / or quality. Yield can directly depend on several factors, such as the number and size of organs (e.g., flower number), plant architecture (such as the number of branches, plant biomass, e.g., increased root biomass, steeper root angle, and / or longer roots, etc.), flowering time and duration, and grain filling period. Root architecture and development, photosynthetic efficiency, nutrient uptake, stress tolerance, early vigor, delayed senescence, and functional stay-green phenotypes can be factors determining yield. Therefore, optimizing the above factors can contribute to increasing crop yield.

[0114] The increase / improvement in yield-related traits mentioned herein can also be considered to refer to an increase in the biomass (weight) of one or more parts of the plant, and the one or more parts can include above-ground and / or below-ground (harvestable) plant parts. Specifically, such harvestable parts are seeds, and the implementation of the method of the present disclosure results in a plant with increased seed yield, particularly increased seed yield, relative to a suitable control plant. The term "yield" of a plant can relate to the vegetative biomass (root and / or shoot biomass), reproductive organs, and / or propagules (such as seeds) of the plant.

[0115] The increase in yield of the plants of the present disclosure can be measured in various ways, including test weight, number of seeds per plant, seed weight, number of seeds per unit area (e.g., number of seeds or seed weight per acre), bushels per acre, tons per acre, or kilograms per hectare. The increase in yield can be attributed to increased utilization of key biochemical compounds such as nitrogen, phosphorus, and carbohydrates, or to improved responses to environmental stresses such as cold, heat, drought, salt, shade, high plant density, and pest or pathogen attack.

[0116] "Yield increase" can be manifested as one or more of the following: (i) an increase in the plant biomass (weight) of one or more parts of the plant, particularly the above-ground (harvestable) parts of the plant, an increase in root biomass (an increase in root number, an increase in root thickness, an increase in root length), or an increase in the biomass of any other harvestable part; or (ii) an increase in early vigor, as defined herein as an increase in the above-ground area of the seedlings at about three weeks after germination.

[0117] "Early vigor" refers to the active and healthy growth of plants, especially in the early stages of plant growth, and can result from an increase in plant fitness caused by, for example, the plant better adapting to its environment (e.g., optimizing the utilization of energy sources, the absorption of nutrients, and the distribution of carbon between the shoot and the root). For example, early vigor can be a combination of the ability of the seeds to germinate and emerge after sowing and the ability of the young plants to grow and develop after emergence. Plants with early vigor also exhibit an increase in seedling survival rate and better crop establishment, which generally results in a highly uniform field where most plants reach each developmental stage substantially simultaneously, thus generally leading to an increase in yield. Therefore, early vigor can be determined by measuring various factors, such as kernel weight, germination percentage, emergence percentage, seedling growth, seedling height, root length, root and shoot biomass, canopy size and color, etc.

[0118] In addition, yield increase can also be manifested as an increase in the total seed yield, which can be attributed to one or more of the following: an increase in seed biomass (seed weight) due to an increase in seed weight per plant and / or per individual seed, an increase in the number of, for example, flowers / cones per plant; an increase in the number of pods; a change in the number of stem nodes; an increase in the number of flowers per cone / plant ("florets"); an increase in the seed filling rate; an increase in the number of filled seeds; an increase in seed size (length, width, area, perimeter, and / or weight), which can also affect the composition of the seeds; and / or an increase in seed volume, which can also affect the composition of the seeds. In one embodiment, the yield increase can be an increase in seed yield, for example, an increase in seed weight; an increase in the number of filled seeds; and / or an increase in the harvest index.

[0119] Yield increase can also cause a change in architecture, or can occur due to a change in plant architecture.

[0120] Yield increase can also be manifested as an increase in the harvest index, which is expressed as the ratio of the yield of the harvestable part (such as seeds) to the total biomass.

[0121] The present disclosure also extends to the harvestable parts of plants, such as but not limited to seeds, leaves, fruits, flowers, pods, legumes, siliques, nuts, stems, rhizomes, tubers, and bulbs. The present disclosure also relates to products derived from the harvestable parts of such plants, such as dry granules, powders, oils, fats and fatty acids, starches, or proteins.

[0122] The present disclosure provides a method for increasing the "yield" of a plant or the "acre yield" of a plant or plant part, which is defined as the plant part that can be harvested per unit area, such as the number of seeds or seed weight per acre, pounds per acre, bushels per acre, tons per acre, tons per acre, kilograms per hectare.

[0123] As used herein, "nitrogen use efficiency" is the process of increasing plant yield, biomass, vigor, and growth rate per unit of nitrogen applied. These processes can include nitrogen uptake, assimilation, accumulation, signaling, sensing, remobilization (within the plant), and utilization by the plant.

[0124] As used herein, "enhanced nitrogen use efficiency" refers to the ability of a plant to grow, develop, or produce faster or better than normal when subjected to the same available / applied amount of nitrogen as under normal or standard conditions; the ability of a plant to grow, develop, or produce normally, or faster or better, when subjected to less than optimal available / applied amounts of nitrogen or under nitrogen-limiting conditions.

[0125] As used herein, "nitrogen-limiting conditions" refers to growth conditions or environments that provide less than the optimal amount of nitrogen required for a plant's full or successful metabolism, growth, reproductive success, and / or survival.

[0126] As used herein, "enhanced nitrogen stress tolerance" refers to the ability of a plant to grow, develop, or produce normally, or faster or better, when subjected to less than optimal available / applied amounts of nitrogen or under nitrogen-limiting conditions.

[0127] Improved plant nitrogen use efficiency can translate in the field to harvesting similar amounts of yield while supplying less nitrogen, or to obtaining increased yields by supplying optimal / sufficient amounts of nitrogen. Enhanced nitrogen use efficiency can improve plant nitrogen stress tolerance and also improve crop quality and the biochemical composition of seeds, such as protein yield and oil yield. The terms "enhanced nitrogen use efficiency", "enhanced nitrogen use effectiveness", and "nitrogen stress tolerance" are used interchangeably in the present disclosure to refer to plants with increased productivity under nitrogen-limiting conditions.

[0128] As used herein, "water use efficiency" refers to the amount of carbon dioxide assimilated by the leaves per unit of transpired water vapor. It constitutes one of the most important traits controlling plant productivity in dry environments. "Drought tolerance" refers to the degree to which a plant can adapt to dry or arid conditions. Physiological responses of plants to water shortage include leaf wilting, reduced leaf area, leaf abscission, and stimulation of root growth by redirecting nutrients to the underground parts of the plant. Generally, plants are more vulnerable to drought during flowering and seed development (reproductive stage) because the plant's resources are biased towards supporting root growth. Additionally, abscisic acid (ABA) is a plant stress hormone that induces the closure of leaf stomata (microscopic pores involved in gas exchange), thereby reducing water loss due to transpiration and decreasing the photosynthesis rate. These responses increase the plant's water use efficiency in the short term. The terms "increased water use efficiency", "enhanced water use efficiency", and "increased drought tolerance" are used interchangeably in this disclosure to refer to plants with increased productivity under water-limited conditions.

[0129] As used herein, "increased water use efficiency" refers to the ability of a plant to grow, develop, or produce faster or better than normal when subjected to the same available / applied amount of water as under normal or standard conditions; the ability of a plant to grow, develop, or produce normally, or faster or better, when subjected to a reduced available / applied amount of water (water input) or under conditions of water stress or water deficit stress.

[0130] As used herein, "increased drought tolerance" refers to the ability of a plant to grow, develop, or produce normally, or faster or better than normal, when subjected to a reduced available / applied amount of water and / or under conditions of short-term or long-term drought; the ability of a plant to grow, develop, or produce normally when subjected to a reduced available / applied amount of water (water input) or under conditions of water deficit stress or short-term or long-term drought.

[0131] As used herein, "drought stress" refers to a dry period (short-term or long-term / extended) that results in water shortage and stresses the plant and / or causes damage to plant tissues and / or has a negative impact on grain / crop yield; a dry period (short-term or long-term / extended) that results in water shortage and / or increased temperature and stresses the plant and / or causes damage to plant tissues and / or has a negative impact on grain / crop yield.

[0132] As used herein, "water deficit" refers to a condition or environment that provides less than the optimal amount of water required for full / successful growth and development of a plant.

[0133] As used herein, "water stress" refers to a condition or environment that provides an inappropriate amount of water (less / insufficient or more / excessive) relative to the amount of water required for adequate / successful growth and development of the plant / crop, thereby stressing the plant and / or causing damage to plant tissue and / or negatively affecting grain / crop yield.

[0134] As used herein, "water stress" refers to a condition or environment that provides less / insufficient water than required for adequate / successful growth and development of a plant / crop, thereby stressing the plant and / or causing damage to plant tissue and / or negatively affecting grain yield.

[0135] As used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleotide sequence" and "polynucleotide" refer to linear or branched, single-stranded or double-stranded RNA or DNA, or hybrids thereof. The term also encompasses RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. may also be used for antisense, dsRNA and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications may also be made, such as modifications to the 2'-hydroxyl in the phosphodiester backbone or the RNA ribose group.

[0136] As used herein, the term "nucleotide sequence" refers to a heteropolymer of nucleotides or a sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule, and includes DNA or RNA molecules, including cDNA, DNA fragments or parts, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA and antisense RNA, any of which can be single-stranded or double-stranded. The terms "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid construct", "oligonucleotide" and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides. Nucleic acid molecules and / or nucleotide sequences provided herein are presented in the 5' to 3' direction from left to right herein, and are represented by the standard code for representing nucleotide characters specified in the U.S. sequence rules 37CFR§§1.821-1.825 and the World Intellectual Property Organization (WIPO) standard ST.25. As used herein, "5' district" can represent the polynucleotide district closest to the 5' end of a polynucleotide. Therefore, for example, the elements in the 5' district of a polynucleotide can be located at any position from the first nucleotide located at the 5' end of a polynucleotide to the nucleotide located in the middle of a polynucleotide. As used herein, "3' region" may refer to the region of a polynucleotide closest to the 3' end of a polynucleotide. Thus, for example, elements in the 3' region of a polynucleotide may be located anywhere from the first nucleotide at the 3' end of the polynucleotide to a nucleotide in the middle of the polynucleotide.

[0137] As used herein with respect to nucleic acids, the term "fragment" or "portion" refers to a nucleic acid that is reduced in length relative to a reference nucleic acid (e.g., reduced by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, or 900 or more nucleotides or any range or value therein) and that contains a nucleotide sequence of contiguous nucleotides that is the same as or substantially the same as (e.g., 70%, 97%, 98%, 99%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96% the same) the corresponding portion of the reference nucleic acid, consists essentially of, and / or consists of such. In appropriate instances, such a nucleic acid fragment may be included within a larger polynucleotide and is a component of the larger polynucleotide. As an example, the repeat sequence of a guide nucleic acid of the invention may contain a "portion" of a wild-type CRISPR-Cas repeat sequence (e.g., a wild-type CRISPR-Cas repeat sequence; e.g., a repeat sequence from a CRISPR Cas system such as, for example, Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, and / or Cas14c).

[0138] In some embodiments, the nucleic acid fragment can comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 70, 75, 80, 85, 90, 95, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 120, 121, 122, 123, 124, 125, 130, 135, 140, 141, 142, 143, 144, 145, 150, 151, 152, 153, 154, 155, 160, 165, 170, 175, 180, 185, 190, 191, 192, 193, 194, 195, 200, 205, 210, 215, 220, 221, 222, 223, 224, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 271, 272, 273, 274, 275, 280, 285, 290, 295, 300, 305, 310, 320, 330, 340, 350, 360, 370, 380, 390, 395, 400, 410, 415, 420, 425, 430, 435, 440, 445, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2500, 3000, 3500 or 4000 or more consecutive nucleotides or any range or value therein, consisting essentially of or consisting of the same,Optionally, the length of the fragment of the HD-Zip polynucleotide can be about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 120, 125, 130, 135, 140, 141, 142, 143, 144, 145, 150 consecutive nucleotides to about 155, 160, 165, 170, 175, 180, 181, 182, 183, 184, 185, 190, 195, 200, 205, 210, 215, 220, 221, 222, 223, 224, 225, 230, 240, 245, 250, 255, 260, 265, 270, 271, 272, 273, 274, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395 or 400 or more consecutive nucleotides or any range or value therein (e.g., SEQ ID NO:69, SEQ ID NO:70, SEQ ID NO:88 or SEQ ID NO:89 (e.g.,Any of the contiguous nucleotides in SEQ ID NO: 72-85 or 91-105 is optionally to a fragment or portion of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105).

[0139] As used herein with respect to polypeptides, the terms "fragment" or "portion" can refer to an amino acid sequence that is reduced in length relative to a reference polypeptide and contains contiguous amino acids that are the same as or nearly the same as (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) the corresponding portion of the reference polypeptide, consists essentially of, and / or consists of such a polypeptide. Optionally, such polypeptide fragments can be included within a larger polypeptide and are a component of the larger polypeptide. In some embodiments, a polypeptide fragment contains at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, or 290 or more contiguous amino acids, consists essentially of, or consists of the corresponding portion of the reference polypeptide. In some embodiments, an HD-Zip polypeptide fragment can contain about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 60, 70, 71, 72, 73, 74, 75, 80, 90, 100, 125, 150, 175, 200, or 210 contiguous amino acid residues or any range or value therein, consists essentially of, or consists of such residues, optionally the fragment can contain about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 contiguous amino acid residues or any range or value therein (e.g., SEQ ID NO:71 or SEQ ID NO:90, respectively, e.g., see fragments or portions of SEQ ID NO:86, 87, or 108 or SEQ ID NO:106 - 108), consists essentially of, or consists of such residues. In some embodiments, a fragment of an HD-Zip polypeptide or HD-Zip polynucleotide can be the result of a deletion in the HD-Zip gene that produces a truncated polypeptide, optionally wherein the amount of the HD-Zip polypeptide is reduced or undetectable. The deletion can produce an in-frame deletion allele or an out-of-frame deletion allele. The HD-Zip gene can be edited at more than one location, thereby providing an HD-Zip gene that contains more than one mutation.

[0140] In some embodiments, a "portion" or "region" can relate to the number of amino acids deleted from a polypeptide. Thus, for example, a deleted "portion" or "region" of an HD-Zip polypeptide can comprise at least one amino acid residue (e.g., at least 1, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 or more contiguous amino acid residues, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 to about 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 residues or any range or value therein) deleted from the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:90 (or from a sequence having at least 80% sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity) to SEQ ID NO:71 or SEQ ID NO:90). In some embodiments, the percentage identity can be at least 85%. In some embodiments, the percentage identity can be at least 90%. In some embodiments, the percentage identity can be at least 95%. In some embodiments, the percentage identity can be 100%.

[0141] In some embodiments, a "portion" or "region" with respect to a nucleic acid means at least 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 240, 250, 260, 270, 280, 285, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 or more contiguous nucleotides (e.g., contiguous nucleotides from an HD-Zip gene),Optionally, the length of a "portion" or "region" of an HD-Zip gene can be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 115, 120, 125, 130, 135, 140, 141, 142, 143, 144, 145, or 150 consecutive nucleotides to about 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 221, 222, 223, 224, 225, 230, 235, 240, 245, 250, 255, 255, 260, 265, 270, 271, 273, 274, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 326, 327, 328, 329, 330, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, or 450 or more consecutive nucleotides or any range or value therein (e.g., a portion or region consisting of consecutive nucleotides from SEQ ID NO:69 or SEQ ID NO:88 (e.g., see SEQ ID NO:72-85 or 91-105, optionally any one of SEQ ID NO:72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, or 103-105)).

[0142] A "region" of a polynucleotide or polypeptide refers to a portion of the polynucleotide or polypeptide consisting of contiguous nucleotides or contiguous amino acid residues. For example, regions of an HD-Zip polynucleotide sequence can include, but are not limited to, SEQ ID NO: 72-85 or 91-105, optionally any one of the nucleic acid sequences of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105. In some embodiments, a region can be a target region or target site for modification in an HD-Zip polynucleotide.

[0143] In some embodiments, a "sequence-specific nucleic acid binding domain" (e.g., a sequence-specific DNA binding domain) can bind to an HD-Zip gene (e.g., SEQ ID NO: 69 or SEQ ID NO: 88) and / or one or more fragments, portions or regions of an HD-Zip nucleic acid (e.g., SEQ ID NO: 72-85 or 91-105, optionally any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105) as described herein.

[0144] As used herein with respect to nucleic acids, the term "functional fragment" refers to a nucleic acid encoding a functional fragment of a polypeptide. A "functional fragment" of a polypeptide is a polypeptide fragment that retains one or more activities of the native reference polypeptide.

[0145] As used herein, the term "gene" refers to a nucleic acid molecule capable of being used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxynucleotides (AMOs), etc. A gene may or may not be capable of being used to produce a functional protein or gene product. A gene can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences and / or 5' and 3' untranslated regions). A gene can be "isolated", meaning that the nucleic acid is substantially or largely free of components that are normally associated with the nucleic acid in its natural state. Such components include other cellular materials, media from recombinant production and / or various chemicals used in the chemical synthesis of nucleic acids.

[0146] The term "mutation" refers to point mutations (e.g., missense or nonsense, or insertion or deletion of a single base pair that causes a frameshift), insertions, deletions, and / or truncations. When a mutation is a substitution of one residue for another within an amino acid sequence, or a deletion or insertion of one or more residues within the sequence, the mutation is typically described by identifying the original residue, followed by identifying the position of the residue within the sequence and the identity of the newly substituted residue. Truncation can include truncation at the C-terminus of the polypeptide or at the N-terminus of the polypeptide. Truncation of a polypeptide can be the result of a deletion of the corresponding 5' or 3' end of the gene encoding the polypeptide. A frameshift mutation occurs when one or more base pairs are deleted or inserted into a gene. A frameshift mutation in a gene can produce a polypeptide that is longer, shorter, or the same length as the wild-type polypeptide, depending on when the first stop codon appears after the mutated region of the gene.

[0147] As used herein, the terms "complementary" or "complementarity" refer to the natural binding of polynucleotides through base pairing under permissive salt and temperature conditions. For example, the sequence "A-G-T" (5' to 3') binds to the complementary sequence "T-C-A" (3' to 5'). Complementarity between two single-stranded molecules can be "partial", where only some of the nucleotides bind, or it can be complete when there is full complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant effect on the efficiency and strength of hybridization between the nucleic acid strands.

[0148] As used herein, "complementary" can mean 100% complementarity to a comparison nucleotide sequence, or it can mean less than 100% complementarity to a comparison nucleotide sequence (e.g., complementarity of about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc.).

[0149] Different nucleic acids or proteins having homology are referred to herein as "homologs". The term homologs includes homologous sequences from the same species and other species as well as orthologous sequences from the same species and other species. "Homology" refers to the level of similarity between two or more nucleic acid and / or amino acid sequences, expressed as a percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Thus, the compositions and methods of the present invention further comprise homologs of the nucleotide sequences and polypeptide sequences of the present invention. As used herein, "orthologous" refers to homologous nucleotide sequences and / or amino acid sequences in different species that arose from a common ancestral gene during speciation. Homologs of the nucleotide sequences of the present invention have substantial sequence identity with the nucleotide sequences of the present invention (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%).

[0150] As used herein, "sequence identity" refers to the degree of identity of two optimally aligned polynucleotide or polypeptide sequences over the entire component (e.g., nucleotide or amino acid) alignment window. "Identity" can be readily calculated by known methods, including but not limited to the methods described in Computational Molecular Biology (Lesk, A.M. ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, D.W. ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, A.M. and Griffin, H.G. eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G. ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. eds.) Stockton Press, New York (1991).

[0151] As used herein, the term "percent sequence identity" or "identity percent" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percent sequence identity" may refer to the percentage of identical amino acids in an amino acid sequence compared to a reference polypeptide. With respect to the HD-Zip gene, the sequence can have at least 80% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 88, and / or 89. In some embodiments, the HD-Zip gene can have at least 85% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 88, and / or 89. In some embodiments, the HD-Zip gene can have at least 90% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 88, and / or 89. In some embodiments, the HD-Zip gene can have at least 95% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 88, and / or 89, optionally wherein the HD-Zip gene can have 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 88, and / or 89. The HD-Zip polypeptide as described herein can have at least 80% sequence identity with the polypeptide sequence of any one of SEQ ID NOs: 71 and / or SEQ ID NO: 90. In some embodiments, the HD-Zip polypeptide can have at least 85% sequence identity with the polypeptide sequence of any one of SEQ ID NOs: 71 and / or SEQ ID NO: 90. In some embodiments, the HD-Zip polypeptide can have at least 90% sequence identity with the polypeptide sequence of any one of SEQ ID NOs: 71 and / or SEQ ID NO: 90. In some embodiments, the HD-Zip polypeptide can have at least 95% sequence identity with the polypeptide sequence of any one of SEQ ID NOs: 71 and / or SEQ ID NO: 90, optionally wherein the HD-Zip polypeptide can have 100% sequence identity with the polypeptide sequence of any one of SEQ ID NOs: 71 and / or SEQ ID NO: 90. With respect to a region or portion of the HD-Zip gene, the region or portion can have at least 80% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 72-85 and / or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, and / or 103-105.In some embodiments, a region or portion of an HD-Zip gene can have at least 85% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 72-85 and / or 91-105, optionally having at least 85% sequence identity with any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, and / or 103-105. In some embodiments, a region or portion of an HD-Zip gene can have at least 90% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 72-85 and / or 91-105, optionally having at least 90% sequence identity with any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, and / or 103-105. With respect to a region or portion of an HD-Zip polypeptide, the region or portion can have at least 80% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 86, 87, 106, 107, and / or 108. In some embodiments, a region or portion of an HD-Zip polypeptide can have at least 85% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 86, 87, 106, 107, and / or 108. In some embodiments, a region or portion of an HD-Zip polypeptide can have at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 86, 87, 106, 107, and / or 108. In some embodiments, a region or portion of an HD-Zip polypeptide can have at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 86, 87, 106, 107, and / or 108. In some embodiments, a region or portion of an HD-Zip polypeptide can have 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 86, 87, 106, 107, and / or 108. In some embodiments, a mutated HD-Zip gene can have at least 90% sequence identity with a mutated HD-Zip gene having the nucleotide sequence of SEQ ID NO: 113. In some embodiments, a mutated HD-Zip gene can have at least 95% sequence identity with a mutated HD-Zip gene having the nucleotide sequence of SEQ ID NO: 113. In some embodiments, a mutated HD-Zip gene can have 100% sequence identity with a mutated HD-Zip gene having the nucleotide sequence of SEQ ID NO: 113. In some embodiments, a mutated HD-Zip polypeptide can have at least 90% sequence identity with a mutated HD-Zip polypeptide having the amino acid sequence of SEQ ID NO: 115.In some embodiments, the mutated HD-Zip polypeptide may have at least 95% sequence identity with the mutated HD-Zip polypeptide having the amino acid sequence of SEQ ID NO: 115. In some embodiments, the mutated HD-Zip polypeptide may have 100% sequence identity with the mutated HD-Zip polypeptide having the amino acid sequence of SEQ ID NO: 115.

[0152] As used herein, in the context of two nucleic acid molecules, nucleotide sequences, or polypeptide sequences, the phrases "substantially identical" or "substantial identity" refer to two or more sequences or subsequences that have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence, such as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the invention, substantial identity exists in regions of the nucleotide sequences of the invention consisting of contiguous nucleotides, the length of said regions being from about 10 nucleotides to about 20 nucleotides, from about 10 nucleotides to about 25 nucleotides, from about 10 nucleotides to about 30 nucleotides, from about 15 nucleotides to about 25 nucleotides, from about 30 nucleotides to about 40 nucleotides, from about 50 nucleotides to about 60 nucleotides, from about 70 nucleotides to about 80 nucleotides, from about 90 nucleotides to about 100 nucleotides, from about 100 nucleotides to about 200 nucleotides, from about 100 nucleotides to about 300 nucleotides, from about 100 nucleotides to about 400 nucleotides, from about 100 nucleotides to about 500 nucleotides, from about 100 nucleotides to about 600 nucleotides, from about 100 nucleotides to about 800 nucleotides, from about 100 nucleotides to about 900 nucleotides or more nucleotides, or any range therein, up to the full length of the sequence. In some embodiments, the nucleotide sequences may be substantially identical over at least about 20 nucleotides (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70 or 80 nucleotides or more).In some embodiments, two or more HD-Zip genes can be substantially identical to each other over at least about 30 or more contiguous nucleotides of any one of SEQ ID NO: 69, 70, 88, or 89 (see, e.g., SEQ ID NO: 72 - 85 or 91 - 105) (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 54, 56, 57, 58, 59, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, 520, 540, 560, 580, 600 or more contiguous nucleotides).

[0153] In some embodiments of the present invention, substantial identity exists within regions of the polypeptides of the present invention that consist of contiguous amino acid residues, and the length of such regions is from about 3 amino acid residues to about 20 amino acid residues, from about 5 amino acid residues to about 25 amino acid residues, from about 7 amino acid residues to about 30 amino acid residues, from about 10 amino acid residues to about 25 amino acid residues, from about 15 amino acid residues to about 30 amino acid residues, from about 20 amino acid residues to about 40 amino acid residues, from about 25 amino acid residues to about 40 amino acid residues, from about 25 amino acid residues to about 50 amino acid residues, from about 30 amino acid residues to about 50 amino acid residues, from about 40 amino acid residues to about 50 amino acid residues, from about 40 amino acid residues to about 70 amino acid residues, from about 50 amino acid residues to about 70 amino acid residues, from about 60 amino acid residues to about 80 amino acid residues, from about 70 amino acid residues to about 80 amino acid residues, from about 90 amino acid residues to about 100 amino acid residues or more amino acid residues, and any range therein, up to the full length of the sequence. In some embodiments, the polypeptide sequences can be substantially identical to each other over at least about 8 to about 350 contiguous amino acid residues (e.g., lengths of about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 130, 140, 150, 175, 200 or 225 or more amino acids or more contiguous amino acid residues of SEQ ID NO:71 or SEQ ID NO:90).In some embodiments, two or more HD-Zip polypeptides may be identical or substantially identical over at least 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 or more consecutive amino acids (e.g., at least 70% to 99.9% identical, e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical, or any range or value therein). In some embodiments, substantially identical nucleotide or protein sequences may perform substantially the same functions as their substantially identical nucleotides (or encoded protein sequences).

[0154] For sequence comparison, typically one sequence acts as a reference sequence to which the test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are designated if necessary, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the percentage of sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

[0155] Optimal alignment of sequences for comparison windows is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally by computerized implementations of these algorithms, such as GAP, BESTFIT, FASTA, and TFASTA, which are available as Wisconsin Obtained in part from (Accelrys Inc., San Diego, CA). The "identity score" for an alignment segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment (e.g., the entire reference sequence or a smaller defined portion of the reference sequence). The percent sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more polynucleotide sequences can be with a full-length polynucleotide sequence or a portion thereof, or with a longer polynucleotide sequence. For the purposes of the present invention, BLASTX version 2.0 for translating nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences can also be used to determine the "percent identity".

[0156] Two nucleotide sequences are also considered to be substantially complementary when they hybridize to each other under stringent conditions. In some embodiments, two nucleotide sequences that are considered to be substantially complementary hybridize to each other under highly stringent conditions.

[0157] In the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations, "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. Extensive guidelines for nucleic acid hybridization are available in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology - Hybridization with Nucleic Acid Probes, Part I, Chapter 2, "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be about 5 °C lower than the thermal melting temperature (T m ) of a particular sequence at a defined ionic strength and pH.

[0158] T m is the temperature (at a defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are selected to be equal to the T of a particular probe m。In Southern or Northern blotting, examples of stringent hybridization conditions for a complementary nucleotide sequence having more than 100 complementary residues to hybridize on a filter membrane are hybridization overnight at 42 °C with 50% formamide and 1 mg heparin. Examples of highly stringent washing conditions are washing for about 15 minutes at 72 °C with 0.15 M NaCl. An example of stringent washing conditions is washing for 15 minutes at 65 °C with 0.2x SSC (for the description of SSC buffer, see Sambrook below). Generally, low-stringency washing is performed before high-stringency washing to remove background probe signals. An example of medium-stringency washing for a duplex of more than 100 nucleotides is washing for 15 minutes at 45 °C with 1x SSC. An example of low-stringency washing for a duplex of more than 100 nucleotides is washing for 15 minutes at 40 °C with 4 - 6x SSC. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions generally involve a salt concentration of less than about 1.0 M Na ions, usually about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is usually at least about 30 °C. The addition of destabilizers (such as formamide) can also achieve stringent conditions. Generally, in a particular hybridization assay, a signal-to-noise ratio 2-fold (or higher) than that observed for an unrelated probe indicates the detection of specific hybridization. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially the same if the proteins encoded by the nucleotide sequences are substantially the same. This occurs, for example, when copies of a nucleotide sequence are generated using the maximum codon degeneracy permitted by the genetic code.

[0159] The polynucleotides and / or recombinant nucleic acid constructs (e.g., expression cassettes and / or vectors) of the present invention can be codon-optimized for expression. In some embodiments, the polynucleotides, nucleic acid constructs, expression cassettes, and / or vectors of the editing system of the present invention (e.g., comprising / encoding a sequence-specific nucleic acid binding domain (e.g., a sequence-specific nucleic acid binding domain from: a polynucleotide-guided endonuclease, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an Argonaute protein, and / or a CRISPR-Cas endonuclease (e.g., a CRISPR-Cas effector protein) (e.g., a type I CRISPR-Cas effector protein, a type II CRISPR-Cas effector protein, a type III CRISPR-Cas effector protein, a type IV CRISPR-Cas effector protein, a type V CRISPR-Cas effector protein, or a type VI CRISPR-Cas effector protein)), a nuclease (e.g., an endonuclease (e.g., Fok1), a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., a CRISPR-Cas effector protein), a zinc finger nuclease, and / or a transcription activator-like effector nuclease (TALEN)), a deaminase protein / domain (e.g., an adenine deaminase, a cytosine deaminase), a polynucleotide encoding a reverse transcriptase protein or domain, a polynucleotide encoding a 5'-3' exonuclease polypeptide, and / or an affinity polypeptide, peptide tag, etc.) can be codon-optimized for expression in plants. In some embodiments, the codon-optimized nucleic acids, polynucleotides, expression cassettes, and / or vectors of the present invention have about 70% to about 99.9% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) identity or greater identity with the non-codon-optimized reference nucleic acids, polynucleotides, expression cassettes, and / or vectors.

[0160] In the embodiments described herein, the polynucleotides or nucleic acid constructs of the invention can be operably associated with a variety of promoters and / or other regulatory elements for expression in plants and / or plant cells. Thus, in some embodiments, the polynucleotides or nucleic acid constructs of the invention can also comprise one or more promoters, introns, enhancers, and / or terminators operably linked to one or more nucleotide sequences. In some embodiments, a promoter can be operably associated with an intron (e.g., the Ubi1 promoter and intron). In some embodiments, a promoter associated with an intron can be referred to as a "promoter region" (e.g., the Ubi1 promoter and intron) (see, for example, SEQ ID NO:21 and SEQ ID NO:22).

[0161] As used herein, "operably linked" or "operably associated" in reference to polynucleotides means that the indicated elements are functionally related to each other and are generally also physically related. Thus, as used herein, the terms "operably linked" or "operably associated" refer to nucleotide sequences that are functionally associated on a single nucleic acid molecule. Thus, a first nucleotide sequence operably linked to a second nucleotide sequence refers to a situation where the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, if a promoter affects the transcription or expression of a nucleotide sequence, the promoter is operably associated with the nucleotide sequence. Those skilled in the art will understand that a control sequence (e.g., a promoter) need not be adjacent to the nucleotide sequence with which it is operably associated, so long as the control sequence functions to direct its expression. Thus, for example, there can be intervening untranslated but transcribed nucleic acid sequences between a promoter and a nucleotide sequence, and the promoter can still be considered to be "operably linked" to the nucleotide sequence.

[0162] As used herein, the term "linked" in reference to polypeptides refers to the joining of one polypeptide to another polypeptide. Polypeptides can be joined directly (e.g., via a peptide bond) or via a linker to another polypeptide (at the N-terminus or C-terminus).

[0163] The term "linker" is well recognized in the art and refers to a chemical group or molecule that joins two molecules or moieties, such as two domains of a fusion protein, such as, for example, a nucleic acid-binding polypeptide or domain and a peptide tag and / or a reverse transcriptase and an affinity polypeptide that binds to the peptide tag; or a DNA endonuclease polypeptide or domain and a peptide tag and / or a reverse transcriptase and an affinity polypeptide that binds to the peptide tag. A linker can comprise a single linking molecule or can comprise more than one linking molecule. In some embodiments, a linker can be an organic molecule, group, polymer, or chemical moiety, such as a divalent organic moiety. In some embodiments, a linker can be an amino acid or can be a peptide. In some embodiments, the linker is a peptide.

[0164] In some embodiments, the length of the peptide linker useful in the present invention can be from about 2 to about 100 or more amino acids. For example, the length can be about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (e.g., the length can be from about 2 to about 40, from about 2 to about 50, from about 2 to about 60, from about 4 to about 40, from about 4 to about 50, from about 4 to about 60, from about 5 to about 40, from about 5 to about 50, from about 5 to about 60, from about 9 to about 40, from about 9 to about 50, from about 9 to about 60, from about 10 to about 40, from about 10 to about 50, from about 10 to about 60 or from about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 amino acids to about 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids) (e.g.,The length is about 105, 110, 115, 120, 130, 140, 150 or more amino acids). In some embodiments, the peptide linker can be a GS linker.,

[0165] As used herein, the term "ligation" or "fusion" with respect to polynucleotides refers to the joining of one polynucleotide to another polynucleotide. In some embodiments, two or more polynucleotide molecules can be joined by a linker, which can be an organic molecule, group, polymer or chemical moiety, such as a divalent organic moiety. Polynucleotides can be joined or fused to another polynucleotide (at the 5' end or 3' end) by covalent or non-covalent bonds or associations (including, for example, by Watson-Crick base pairing or by one or more linker nucleotides). In some embodiments, a polynucleotide motif of a certain structure can be inserted into another polynucleotide sequence (e.g., an extension of the hairpin structure in a guide RNA). In some embodiments, the linker nucleotides can be naturally occurring nucleotides. In some embodiments, the linker nucleotides can be non-naturally occurring nucleotides.

[0166] A "promoter" is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) operably associated with the promoter. The coding sequence controlled or regulated by the promoter can encode a polypeptide and / or a functional RNA. Generally, a "promoter" refers to a nucleotide sequence that contains a binding site for RNA polymerase II and directs the initiation of transcription. Generally, the promoter is located 5' or upstream of the coding region start point relative to the corresponding coding sequence. A promoter can contain other elements that act as regulators of gene expression; for example, a promoter region. These include the TATA box consensus sequence and usually also the CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50:349). In plants, the CAAT box can be replaced by the AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).

[0167] Promoters that can be used in the present invention can include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and / or tissue-specific promoters for preparing recombinant nucleic acid molecules, such as "synthetic nucleic acid constructs" or "protein-RNA complexes". These different types of promoters are known in the art.

[0168] The choice of promoter can vary depending on the temporal and spatial requirements of expression, and also on the host cell to be transformed. Promoters from many different organisms are well known in the art. Based on the extensive knowledge present in the art, an appropriate promoter can be selected for the particular host organism of interest. Thus, for example, much is known about the promoters upstream of genes that are highly constitutively expressed in model organisms, and this knowledge can be readily obtained and implemented in other systems as appropriate.

[0169] In some embodiments, promoters functional in plants can be used with the constructs of the present invention. Non-limiting examples of promoters that can be used to drive expression in plants include the promoter of the RubisCo small subunit gene 1 (PrbcS1), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr), and the promoter of the repetitive carbonic anhydrase gene 1 (Pdca1) (see Walker et al., Plant Cell Rep. 23:727-735 (2005); Li et al., Gene 403:132-142 (2007); Li et al., Mol Biol.Rep. 37:1143-1154 (2010)). PrbcS1 and Pactin are constitutive promoters, and Pnr and Pdca1 are inducible promoters. Pnr is induced by nitrate and repressed by ammonium (Li et al., Gene 403:132-142 (2007)), and Pdca1 is induced by salt (Li et al., Mol Biol.Rep. 37:1143-1154 (2010)). In some embodiments, the promoter that can be used in the present invention is an RNA polymerase II (Pol II) promoter. In some embodiments, the U6 promoter or 7SL promoter from Zea mays can be used in the constructs of the present invention. In some embodiments, the U6c promoter and / or 7SL promoter from maize can be used to drive the expression of the guide nucleic acid. In some embodiments, the U6c promoter, U6i promoter, and / or 7SL promoter from Glycine max can be used in the constructs of the present invention. In some embodiments, the U6c promoter, U6i promoter, and / or 7SL promoter from soybean can be used to drive the expression of the guide nucleic acid.

[0170] Examples of constitutive promoters useful in plants include, but are not limited to, the cestrum virus promoter (cmp) (U.S. Patent No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; and U.S. Patent No. 5,641,876), the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), the CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), the nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci USA 84:5745-5749), the Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:6624-6629), the sucrose synthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148), and ubiquitin promoters. Constitutive promoters derived from ubiquitin accumulate in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, e.g., sunflower (Binet et al., 1991. Plant Science 79:87-94), maize (Christensen et al., 1989. Plant Molec. Biol. 12:619-632), and Arabidopsis (Norris et al., 1993. Plant Molec. Biol. 21:895-906). The maize ubiquitin promoter (UbiP) has been developed in transgenic monocot systems, and its sequence and vectors constructed for monocot transformation are disclosed in patent publication EP 0 342 926. The ubiquitin promoter is suitable for expressing the nucleotide sequences of the present invention in transgenic plants, especially monocots. In addition, the promoter expression cassette described by McElroy et al. (Mol. Gen. Genet. 231:150-160 (1991)) can be readily modified for the expression of the nucleotide sequences of the present invention and is particularly suitable for monocot hosts.

[0171] In some embodiments, tissue-specific / tissue-preferred promoters can be used to express heterologous polynucleotides in plant cells. Tissue-specific or preferred expression patterns include, but are not limited to, green tissue-specific or preferred, root-specific or preferred, stem-specific or preferred, flower-specific or preferred, or pollen-specific or preferred. Promoters suitable for expression in green tissue include many promoters that regulate genes involved in photosynthesis, many of which have been cloned from monocotyledonous and dicotyledonous plants. In one embodiment, a promoter useful in the present invention is the maize PEPC promoter from the phosphoenolpyruvate carboxylase gene (Hudspeth and Grula, Plant Molec. Biol. 12:579-589 (1989)). Non-limiting examples of tissue-specific promoters include those associated with genes encoding seed storage proteins such as β-conglycinin, cruciferin, napin, and phaseolin, zein, or oleosins such as oleosin, or proteins involved in fatty acid biosynthesis including acyl carrier protein, stearoyl-ACP desaturase, and fatty acid desaturase (fad 2-1), and other nucleic acids expressed during embryo development such as Bce4, see for example Kridl et al. (1991) Seed Sci. Res. 1:209-219; and EP patent No. 255378). Tissue-specific or tissue-preferred promoters useful for expressing the nucleotide sequences of the present invention in plants, particularly maize, include, but are not limited to, those that direct expression in roots, pith, leaves, or pollen. Such promoters are disclosed, for example, in WO 93 / 07278, which is incorporated herein by reference in its entirety.Other non-limiting examples of tissue-specific or tissue-preferred promoters useful in the present invention are the cotton rubisco promoter disclosed in U.S. Patent 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Patent 5,604,121; the root-specific promoter described by de Framond (FEBS 290:103-106 (1991); EP 0 452 269 to Ciba-Geigy); the stem-specific promoter described in U.S. Patent 5,625,136 (to Ciba-Geigy) that drives the expression of the maize trpA gene; the Cestrum yellow leaf curling virus promoter disclosed in WO 01 / 73087; and pollen-specific or pollen-preferred promoters, including but not limited to ProOsLPS10 and ProOsLPS11 from rice (Nguyen et al., Plant Biotechnol. Reports 9(5):297-306 (2015)), ZmSTK2_USP from maize (Wang et al., Genome 60(6):485-495 (2017)), LAT52 and LAT59 from tomato (Twell et al., Development 109(3):705-713 (1990)), Zm13 (U.S. Patent No. 10,421,972), and the PLA - δ promoter from Arabidopsis (U.S. Patent No. 7,141,424) and / or the ZmC5 promoter from maize (International PCT Publication WO1999 / 042587). 2 -δ promoter (U.S. Patent No. 7,141,424) and / or the ZmC5 promoter from maize (International PCT Publication WO1999 / 042587).

[0172] Additional examples of plant tissue-specific / tissue-preferred promoters include, but are not limited to, the root hair-specific cis element (RHE) (Kim et al., The Plant Cell 18:2958-2970 (2006)), the root-specific promoter RCc3 (Jeong et al., Plant Physiol. 153:185-197 (2010)) and RB7 (U.S. Patent No. 5,459,252), the lectin promoter (Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), the maize alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), S-adenosyl-L-methionine synthase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115), the maize light-harvesting complex promoter (Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658), the maize heat shock protein promoter (O'Dell et al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J. 5:451-458), the pea small subunit RuBP carboxylase promoter (Cashmore, “Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase” pp. 29-39, in: Genetic Engineering of Plants (Hollaender ed.), Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), the Ti plasmid mannosine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), the Ti plasmid nopaline synthase promoter (Langridge et al. (1989), supra), the petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), the legume glycine-rich protein 1 promoter (Keller et al. (1989) Genes Dev.3:1639 - 1646), the truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810 - 812), the potato glycoprotein promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347 - 354), the root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), the zein promoter (Kriz et al. (1987) Mol. Gen. Genet. 207:90 - 98; Langridge et al. (1983) Cell 34:1015 - 1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), the legumin - 1 promoter (Belanger et al. (1991) Genetics 129:863 - 872), the α - tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet. 215:431 - 440), the PEPCase promoter (Hudspeth and Grula (1989) Plant Mol. Biol. 12:579 - 589), the R - gene complex - associated promoter (Chandler et al. (1989) Plant Cell 1:1175 - 1183), and the chalcone synthase promoter (Franken et al. (1991) EMBO J. 10:2605 - 2612).

[0173] The legumin promoter (Czako et al. (1992) Mol. Gen. Genet. 235:33 - 40) can be used for seed - specific expression; and the seed - specific promoter disclosed in U.S. Patent No. 5,625,136. Promoters that can be used for expression in mature leaves are those that switch at the onset of senescence, such as the SAG promoter from Arabidopsis (Gan et al. (1995) Science 270:1986 - 1988).

[0174] In addition, promoters that are functional in chloroplasts can be used. Non - limiting examples of such promoters include the bacteriophage T3 gene 9 5'UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters that can be used in the present invention include, but are not limited to, the S - E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0175] Additional regulatory elements useful in the present invention include, but are not limited to, introns, enhancers, termination sequences, and / or 5' and 3' untranslated regions.

[0176] Introns useful in the present invention can be introns that are identified in plants and isolated from plants and then inserted into an expression cassette for plant transformation. As understood by those skilled in the art, introns can contain the sequences required for self-excision and be incorporated in-frame into a nucleic acid construct / expression cassette. Introns can be used as spacers to separate multiple protein-coding sequences in a nucleic acid construct, or an intron can be used within a protein-coding sequence, for example, to stabilize mRNA. If they are used within a protein-coding sequence, they are inserted "in-frame" and include the excision site. Introns can also be associated with promoters to improve or alter expression. As an example, promoter / intron combinations useful in the present invention include, but are not limited to, the promoter / intron combination of the maize Ubi1 promoter and intron (see, for example, SEQ ID NO:21 and SEQ ID NO:22).

[0177] Non-limiting examples of introns useful in the present invention include introns from the following genes: ADHI gene (e.g., Adh1-S introns 1, 2, and 6), ubiquitin gene (Ubi1), RuBisCO small subunit (rbcS) gene, RuBisCO large subunit (rbcL) gene, actin gene (e.g., actin-1 intron), pyruvate dehydrogenase kinase gene (pdk), nitrate reductase gene (nr), repetitive carbonic anhydrase gene 1 (Tdca1), psbA gene, atpA gene, or any combination thereof.

[0178] In some embodiments, the polynucleotides and / or nucleic acid constructs of the present invention can be an "expression cassette" or can be contained within an expression cassette. As used herein, an "expression cassette" refers to a recombinant nucleic acid molecule that includes, for example, one or more polynucleotides of the present invention (e.g., a polynucleotide encoding a sequence-specific nucleic acid (e.g., DNA) binding domain, a polynucleotide encoding a deaminase protein or domain, a polynucleotide encoding a reverse transcriptase protein or domain, a polynucleotide encoding a 5'-3' exonuclease polypeptide or domain, a guide nucleic acid, and / or a reverse transcriptase (RT) template), wherein the polynucleotide is operably associated with one or more control sequences (e.g., a promoter, a terminator, etc.). Thus, in some embodiments, one or more expression cassettes can be provided that are designed to express, for example, the nucleic acid constructs of the present invention (e.g., a polynucleotide encoding a sequence-specific nucleic acid binding domain, a polynucleotide encoding a nuclease polypeptide / domain, a polynucleotide encoding a deaminase protein / domain, a polynucleotide encoding a reverse transcriptase protein / domain, a polynucleotide encoding a 5'-3' exonuclease polypeptide / domain, a polynucleotide encoding a peptide tag, and / or a polynucleotide encoding an affinity polypeptide, etc., or including a guide nucleic acid, an extended guide nucleic acid, and / or an RT template, etc.). When the expression cassette of the present invention contains more than one polynucleotide, the polynucleotides can be operably linked to a single promoter driving the expression of all the polynucleotides, or the polynucleotides can be operably linked to one or more separate promoters (e.g., three polynucleotides can be driven by one, two, or three promoters in any combination). When two or more separate promoters are used, the promoters can be the same promoter, or they can be different promoters. Thus, when contained within a single expression cassette, the polynucleotide encoding a sequence-specific nucleic acid binding domain, the polynucleotide encoding a nuclease protein / domain, the polynucleotide encoding a CRISPR-Cas effector protein / domain, the polynucleotide encoding a deaminase protein / domain, the polynucleotide encoding a reverse transcriptase polypeptide / domain (e.g., an RNA-dependent DNA polymerase), and / or the polynucleotide encoding a 5'-3' exonuclease polypeptide / domain, a guide nucleic acid, an extended guide nucleic acid, and / or an RT template can each be operably linked to a single promoter or any combination of separate promoters.

[0179] An expression cassette containing the nucleic acid construct of the present invention can be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components (e.g., a promoter from a host organism is operably linked to a polynucleotide of interest to be expressed in the host organism, where the polynucleotide of interest is from an organism different from the host or does not normally coexist with the promoter). The expression cassette can also be naturally occurring but has been obtained in a recombinant form useful for heterologous expression.

[0180] The expression cassette may optionally include a transcription and / or translation termination region (i.e., a termination region) and / or an enhancer region functional in a selected host cell. A variety of transcription terminators and enhancers are known in the art and can be used in the expression cassette. The transcription terminator is responsible for terminating transcription and correct mRNA polyadenylation. The termination region and / or enhancer region may be native to the transcription initiation region, may be native to, for example, a gene encoding a sequence-specific nucleic acid binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase, etc., or may be native to the host cell, or may be native to another source (e.g., foreign or heterologous to, for example, a promoter, a gene encoding a sequence-specific nucleic acid binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase, etc., or to the host cell, or any combination thereof).

[0181] The expression cassette of the present invention may further include a polynucleotide encoding a selectable marker, which can be used to select transformed host cells. As used herein, "selectable marker" refers to a polynucleotide sequence that, when expressed, confers a unique phenotype on the host cell expressing the marker, thereby allowing such transformed cells to be distinguished from cells without the marker. Such a polynucleotide sequence can encode a selectable or screenable marker, depending on whether the marker confers a trait that can be selected by chemical means (such as by using a selection agent (e.g., an antibiotic, etc.)) or whether the marker is a trait that can be identified only by observation or testing (such as by screening (e.g., fluorescence)). Many examples of suitable selectable markers are known in the art and can be used in the expression cassette described herein.

[0182] In addition to expression cassettes, the nucleic acid molecules / constructs and polynucleotide sequences described herein can also be used in combination with vectors. The term "vector" refers to a composition used to transfer, deliver, or introduce nucleic acid(s) into a cell. A vector contains a nucleic acid construct (e.g., an expression cassette) that includes the nucleotide sequence to be transferred, delivered, or introduced. Vectors for transforming host organisms are well known in the art. Non-limiting examples of general classes of vectors include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, phages, artificial chromosomes, microcircles, or Agrobacterium binary vectors in double-stranded or single-stranded linear or circular form, which may or may not be self-transferable or mobilizable. In some embodiments, viral vectors can include, but are not limited to, retroviral, lentiviral, adenoviral, adeno-associated viral, or herpes simplex viral vectors. A vector as defined herein can transform a prokaryotic or eukaryotic host by integration into the cell genome or by being present extrachromosomally (e.g., an autonomously replicating plasmid with an origin of replication). Also included are shuttle vectors, which are DNA vectors capable of replicating in two different host organisms either naturally or intentionally, and the host organisms can be selected from actinomycetes and related species, bacteria, and eukaryotes (e.g., higher plants, mammals, yeast, or fungal cells). In some embodiments, the nucleic acid in the vector is under the control of an appropriate promoter or other regulatory element and is operably linked to the appropriate promoter or other regulatory element for transcription in the host cell. The vector can be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, this can contain its own promoter and / or other regulatory elements, while in the case of cDNA, this can be under the control of an appropriate promoter and / or other regulatory elements for expression in the host cell. Thus, the nucleic acids or polynucleotides of the present invention and / or the expression cassettes containing the nucleic acids or polynucleotides can be contained in vectors as described herein and known in the art.

[0183] As used herein, "contact", "contacting", "contacted" and their grammatical variants refer to bringing together the components of a desired reaction under conditions suitable for the desired reaction (e.g., transformation, transcriptional control, genome editing, nicking and / or cleavage). As an example, a target nucleic acid can be contacted with a sequence-specific nucleic acid-binding protein (e.g., a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., a CRISPR-Cas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and / or an Argonaute protein) and a deaminase or a nucleic acid construct encoding the same under conditions where the sequence-specific nucleic acid-binding protein, reverse transcriptase, and / or deaminase are expressed, the sequence-specific nucleic acid-binding protein binds to the target nucleic acid, and the reverse transcriptase and / or deaminase can be fused to or recruited to the sequence-specific nucleic acid-binding protein (e.g., via a peptide tag fused to the sequence-specific nucleic acid-binding protein and an affinity tag fused to the reverse transcriptase and / or deaminase), such that the deaminase and / or reverse transcriptase is located near the target nucleic acid to modify the target nucleic acid. Other methods of recruiting reverse transcriptase and / or deaminase using other protein-protein interactions can be used, and RNA-protein interactions and chemical interactions can also be used for protein-protein and protein-nucleic acid recruitment.

[0184] As used herein, "modifying" or "modification" with respect to a target nucleic acid includes editing (e.g., mutating), covalently altering, exchanging / substituting nucleic acid / nucleotide bases, deleting, cleaving, nicking, and / or altering transcriptional control of the target nucleic acid. In some embodiments, the modification can include any type of one or more single-base changes (SNPs).

[0185] In the context of a polynucleotide of interest, "introducing", "introduce", "introduced" (and their grammatical variants) refer to presenting the nucleotide sequence of interest (e.g., a polynucleotide, an RT template, a nucleic acid construct, and / or a guide nucleic acid) to a plant, a plant part, or a cell thereof in such a manner that the nucleotide sequence can enter the interior of the cell.

[0186] The terms "transformation" or "transfection" are used interchangeably and, as used herein, refer to the introduction of a heterologous nucleic acid into a cell. Transformation of a cell can be stable or transient. Thus, in some embodiments, a host cell or a host organism (e.g., a plant) can be stably transformed with a polynucleotide / nucleic acid molecule of the invention. In some embodiments, a host cell or a host organism can be transiently transformed with a polynucleotide / nucleic acid molecule of the invention.

[0187] In the context of a polynucleotide, "transient transformation" means that the polynucleotide is introduced into a cell but does not integrate into the genome of the cell.

[0188] In the context of a polynucleotide being introduced into a cell, "stable introduction" or "being stably introduced" means that the introduced polynucleotide is stably incorporated into the genome of the cell, such that the cell is stably transformed with the polynucleotide.

[0189] As used herein, "stable transformation" or "being stably transformed" means that a nucleic acid molecule is introduced into a cell and integrated into the genome of the cell. Thus, the integrated nucleic acid molecule can be inherited by its progeny, and more specifically, can be inherited by progeny for multiple successive generations. As used herein, "genome" includes nuclear genomes and plastid genomes, and thus includes integration of nucleic acids into, for example, chloroplast or mitochondrial genomes. As used herein, stable transformation can also refer to a transgene maintained episomally, for example, as a minichromosome or plasmid.

[0190] Transient transformation can be detected, for example, by enzyme-linked immunosorbent assay (ELISA) or Western blotting, which can detect the presence of a peptide or polypeptide encoded by one or more transgenes introduced into an organism. Stable transformation of a cell can be detected, for example, by Southern blot hybridization assay of genomic DNA of the cell with a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of the transgene introduced into the organism (e.g., a plant). Stable transformation of a cell can be detected, for example, by Northern blot hybridization assay of RNA of the cell with a nucleic acid sequence that specifically hybridizes to the nucleotide sequence of the transgene introduced into the host organism. Stable transformation of a cell can also be detected by, for example, polymerase chain reaction (PCR) or other amplification reactions known in the art that employ specific primer sequences that hybridize to the target sequence of the transgene, causing amplification of the transgene sequence, and the amplification can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols known in the art.

[0191] Thus, in some embodiments, the nucleotide sequences, polynucleotides, nucleic acid constructs, and / or expression cassettes of the invention can be transiently expressed and / or they can be stably incorporated into the genome of a host organism. Thus, in some embodiments, the nucleic acid constructs of the invention (e.g., one or more expression cassettes comprising a polynucleotide for editing as described herein) can be transiently introduced into a cell together with a guide nucleic acid, and thus, the DNA is not maintained in the cell.

[0192] The nucleic acid constructs of the present invention can be introduced into plant cells by any method known to those skilled in the art. Non-limiting examples of transformation methods include transformation by bacterium-mediated nucleic acid delivery (e.g., by Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, particle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, sonication, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical), and / or biological mechanism that enables the introduction of nucleic acids into plant cells, including any combination thereof. Procedures for transforming eukaryotic and prokaryotic organisms are well-known and conventional in the art and are described in the literature (see, e.g., Jiang et al., 2013. Nat. Biotechnol. 31:233-239; Ran et al., Nature Protocols 8:2281–2308 (2013)). General guidelines for various plant transformation methods known in the art include Miki et al. ("Procedures for Introducing Foreign DNA into Plants", in Methods in Plant Molecular Biology and Biotechnology, Glick, B.R. and Thompson, J.E. eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0193] In some embodiments of the present invention, the transformation of cells can include nuclear transformation. In other embodiments, the transformation of cells can include plastid transformation (e.g., chloroplast transformation). In further embodiments, the nucleic acids of the present invention can be introduced into cells by conventional breeding techniques. In some embodiments, one or more of the polynucleotide, expression cassette, and / or vector can be introduced into plant cells by Agrobacterium transformation.

[0194] Thus, polynucleotides can be introduced into plants, plant parts, and plant cells in any number of ways known in the art. The methods of the invention do not depend on a particular method for introducing one or more nucleotide sequences into a plant, so long as they are capable of entering into the interior of the cell. If more than one polynucleotide is to be introduced, they can be assembled as part of a single nucleic acid construct or as separate nucleic acid constructs, and can be located on the same or different nucleic acid constructs. Thus, the polynucleotides can be introduced into the cell of interest in a single transformation event, or in separate transformation events, or alternatively, the polynucleotides can be incorporated into the plant as part of a breeding program.

[0195] The present invention provides methods and compositions for improving yield traits, optionally wherein the change is a conformational change that can result in increased yield. Specifically, the present invention relates to modifying endogenous homeodomain-leucine zipper transcription factor (HD-Zip) genes in plants, and the HD-Zip transcription factor (HD-Zip) polypeptides. In some embodiments, the improved yield traits can include, but are not limited to, an increase in the number of seeds (e.g., the number of grains), an increase in seed weight (e.g., grain weight, an increase in the weight of 100 seeds), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), shorter plant height (i.e., reduced height), a reduction in the number of stem nodes, and / or a reduction in branching. A reduction in branching allows plants to be planted at higher densities without shading adjacent plants. The reduction in branching and the increased number of seed pods on the main stem provide higher individual plant yields and higher planting densities.

[0196] As used herein, "increased seed number" means an increase in seed number of at least about 10% (e.g., about 10% to about 200%, e.g., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 141%, 142%, 143%, 144%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or 200% or any range or value therein) compared to a control plant lacking a mutation as described herein.

[0197] As used herein, "increased seed weight" can mean seeds having an increased seed weight (e.g., the weight of 100 seeds). In some embodiments, the weight of the seeds can be increased by up to about 50% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 45%, 46%, 47%, 48%, 49% or 50%) compared to seeds from a control plant (e.g., a plant that does not contain a mutation in an endogenous HD-Zip gene as described herein). In some embodiments, an increase in seed size can include an increase in both seed size and seed area. An increase in seed weight can be measured, for example, as the weight of 100 seeds.

[0198] As used herein, "an increase in the number of pods per plant" refers to the number of flowers that result in the formation of at least one seed in the pods produced at the time of pollination. Compared to a control plant lacking the mutation as described herein, the number of pods per plant can be increased by at least about 10% (e.g., from about 10% to about 200%, e.g., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 115%, 120%, 125%, 130%, 135%, 140%, 141%, 142%, 143%, 144%, 145%, 150%, 155%, 160%, 165%, 170%, 175%, 180%, 185%, 190%, 195% or 200% or any range or value therein).

[0199] As used herein, "reduced flowering time" can mean earlier flowering time (a decrease in the time to flowering). A decrease in the time to flowering means a decrease in the number of days from planting to the onset of flowering of about 15% to 40% (e.g., about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39% or 40%, or any range or value therein) compared to a control plant lacking the mutation as described herein. Generally, soybean plants begin to flower about 45 days to 55 days (45 days, 46 days, 47 days, 48 days, 49 days, 50 days, 51 days, 52 days, 53 days or 54 days) after planting. However, some soybean varieties used for double cropping can begin to flower within about 34 days to about 38 days (34 days, 35 days, 36 days, 37 days or 38 days) after planting.

[0200] As used herein, "shorter plant height" refers to a plant with a reduced height. Shorter plant height can mean a reduction in height of about 10% to about 75% (e.g., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74% or 75% or any range or value therein) compared to a control plant lacking the mutation as described herein.

[0201] As used herein, "reduced internode number" and / or "reduced branching" refers to a decrease in the number of lateral branches formed. This can be observed as a decrease in the "bushiness" of the plant and / or a decrease in the total number of primary and secondary branches. Reduced internode number or reduced branching can be a decrease of about 10% to about 100% (e.g., about 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% or any range or value therein) compared to a control plant lacking the mutation as described herein.

[0202] Thus, as described herein, editing techniques are used to target HD-Zip genes in plants to produce plants having improved yield traits such as increased seed number (e.g., number of grains), increased seed weight (e.g., grain weight, increased 100-seed weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced internode number and / or reduced branching, optionally wherein the mutation can be a non-natural mutation. Types of mutations that can be used to produce plants exhibiting improved yield traits include, for example, substitutions, deletions, and insertions. In some aspects, the mutations produced by the editing techniques can be point mutations. In some embodiments, the mutations produced by the editing techniques of the present invention can be dominant negative mutations.

[0203] In some embodiments, the present invention provides a plant or a plant part thereof that comprises at least one mutation (e.g., 1, 2, 3, 4, or 5 or more mutations) in an endogenous homeodomain-leucine zipper transcription factor (HD-Zip) gene, the endogenous HD-Zip gene encoding an HD-Zip protein, wherein the mutation alters the function of the HD-Zip polypeptide as a gene expression regulator. In some embodiments, at least one mutation can be a non-natural mutation. In some embodiments, the HD-Zip gene is an HD-Zip II gene, optionally the HD-Zip17-1 gene and / or the HD-Zip17-2 gene, wherein the HD-Zip17-1 gene has the gene identification number (SoyBase database) Glyma.20g014400 and / or the HD-Zip17-2 gene has the gene identification number (SoyBase database) Glyma.07g218000. In some embodiments, at least one mutation can result in a dominant negative mutation. In some embodiments, the plant or a part thereof comprising the mutated HD-Zip gene comprises a mutated HD-Zip nucleic acid that has at least 90% sequence identity with SEQ ID NO: 113 and / or encodes an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 115.

[0204] The endogenous HD-Zip gene (e.g., endogenous target gene) useful in the present invention encodes an HD-Zip transcription factor (HD-Zip) polypeptide. In some embodiments, the endogenous HD-ZIP gene may (a) comprise a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with SEQ ID NO: 69, 70, 88 or 89, (b) comprise a region having at least 80% sequence identity with any one of the nucleotide sequences of SEQ ID NO: 72-85 or 91-105, optionally any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105, (c) encode an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encode a region having at least 80% sequence identity with SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 85% or at least 90%, or it may be at least 95%, optionally the sequence identity may be 100%.Thus, the plant or plant part of the present invention may comprise at least one mutation (e.g., one or more mutations) in an endogenous HD-ZIP gene, wherein the endogenous HD-ZIP gene (a) comprises a sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprises a region having at least 80% sequence identity with any one nucleotide sequence among the nucleotide sequences of any one of SEQ ID NO: 72-85 or 91-105, optionally any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with any one amino acid sequence among the amino acid sequences of SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encodes a region having at least 80% sequence identity with SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 85% or at least 90%, or it may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, the mutated HD-Zip gene may comprise a nucleotide sequence having at least about 90% sequence identity with any one of the mutated nucleic acid sequences described herein. In some embodiments, the mutated HD-Zip gene may comprise a non-natural mutation.

[0205] Mutations in the HD-Zip genes of plants, plant parts, or plant cells can be any type of mutation, including substitutions, deletions, and / or insertions. In some embodiments, the mutations can be non-natural mutations. In some embodiments, the mutations can comprise base substitutions of A, T, G, or C. In some embodiments, the mutations can be at least one base pair, optionally 1 base pair to about 200 consecutive base pairs or more consecutive base pairs (e.g., 1 base pair, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, or 200 or more consecutive base pairs or any range or value therein), optionally a deletion or insertion of 1 base pair to about 100 consecutive base pairs (e.g., 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 consecutive base pairs or a deletion or insertion of any range or value therein), or a deletion of from 1 base pair to about 21 consecutive base pairs (e.g., a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or 21 base pairs or any range or value therein), optionally wherein the mutation is an in-frame deletion, optionally wherein the base deletion is in the ethylene-responsive element binding factor-related amphiphilic repression (EAR) motif encoded by the HD-Zip gene.,

[0206] In some embodiments, at least one mutation can be an in-frame deletion, optionally an in-frame deletion that causes a dominant negative mutation, optionally wherein the mutation is a non-natural mutation. In some embodiments, at least one mutation can produce a modified HD-Zip polypeptide that is altered in its ability to regulate gene expression. In some embodiments, at least one mutation can be a base deletion that causes the deletion of one or more amino acid residues, optionally wherein the deletion of one or more amino acid residues is in the ethylene-responsive element binding factor-related amphiphilic repression (EAR) motif of the HD-Zip polypeptide.

[0207] In some embodiments, at least one mutation(s) can be a base deletion from a region of an HD-Zip gene, wherein the region is within or adjacent to an EAR motif encoded by the HD-Zip gene. As used herein, "adjacent" to an EAR motif means within about 150 contiguous nucleotides 5' and / or 3' of the EAR motif (TTGGAATTGACCATA SEQ ID NO: 105) (e.g., see SEQ ID NOs: 72-85, 105 (from HD-Zip17-1, SoyBase database gene identification number Glyma.20g014400 (SEQ ID NO: 69)) and / or SEQ ID NOs: 91-105 (from HD-Zip17-2, SoyBase database gene identification number Glyma.07g218000 (SEQ ID NO: 88))). In some embodiments, the deletion in the HD-Zip gene can have one or more nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) at positions 2206 to 2220 of the nucleotide position numbering of reference SEQ ID NO: 69 or at positions 2179 to 2193 of the nucleotide position numbering of reference SEQ ID NO: 88 (e.g., at least one deletion of one or more nucleotides, optionally wherein when more than one, the deletions have two or more contiguous nucleotides), optionally wherein the deletion can be three or more contiguous nucleotides (e.g., 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15). In some embodiments, the deletion can be 3, 6, 9, 12, or 15 contiguous nucleotides from a region of the HD-Zip gene, the region being at positions 2206 to 2220 of the nucleotide position numbering of reference SEQ ID NO: 69 or at positions 2179 to 2193 of the nucleotide position numbering of reference SEQ ID NO: 88. In some embodiments, the base deletion causes a deletion of one or more amino acid residues of the HD-Zip polypeptide, optionally wherein the deletion is in the EAR motif of the HD-Zip polypeptide, optionally causing a deletion of the EAR motif.

[0208] In some embodiments, mutations in endogenous HD-Zip genes can produce a mutated HD-Zip gene having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%) with the nucleotide sequence of SEQ ID NO: 113 and / or encode an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115.

[0209] In some embodiments, a plant comprising at least one mutation (e.g., a non-natural mutation) in an endogenous HD-Zip gene can exhibit one or more improved yield traits compared to a control plant lacking said at least one mutation, optionally an increase in seed number (e.g., number of grains), an increase in seed weight (e.g., grain weight, increase in 100-seed weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), shorter plant height, a decrease in the number of stem nodes and / or a decrease in branching. In some embodiments, a plant comprising at least one mutation in an endogenous HD-Zip gene can exhibit an increase in yield. In some embodiments, a plant can be regenerated from a plant part and / or plant cell of the present invention, wherein the regenerated plant comprises a mutated endogenous HD-Zip gene and exhibits a phenotype of an increase in seed number (e.g., number of grains), an increase in seed weight (e.g., grain weight, increase in 100-seed weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), shorter plant height, a decrease in the number of stem nodes and / or a decrease in branching, optionally the regenerated plant further exhibits an increase in yield. In some embodiments, the at least one mutation can be a non-natural mutation. In some embodiments, a plant comprising at least one mutation in an endogenous HD-Zip gene is not regenerated.

[0210] In some embodiments, a plant cell is provided, the plant cell comprising an editing system, the editing system comprising: (a) a CRISPR-Cas effector protein; and (b) a guide nucleic acid (gRNA, gDNA, crRNA, crDNA, sgRNA, sgDNA), the guide nucleic acid comprising a spacer sequence that is complementary to an endogenous target gene encoding an HD-Zip protein. In some embodiments, the spacer sequence of the guide nucleic acid that is complementary thereto and the endogenous HD-Zip gene may (a) comprise a sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprise a region having at least 80% sequence identity to any one nucleotide sequence of the nucleotide sequences of any one of SEQ ID NO: 72-85 or 91-105, optionally any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105; (c) encode a polypeptide comprising a sequence having at least 80% sequence identity to any one amino acid sequence of the amino acid sequences of SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encode a region having at least 80% sequence identity to SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 85% or at least 90%, or it may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, the spacer sequences useful in the present invention may include, but are not limited to, the nucleotide sequence of any one of SEQ ID NO: 109, 110, 111 or 112, or its reverse complement or a combination thereof. In some embodiments, the endogenous target gene is an HD-Zip II gene, optionally the HD-Zip17-1 gene and / or the HD-Zip17-2 gene. The editing system can be used to generate mutations in an endogenous target gene encoding an HD-Zip protein. In some embodiments, the mutation is a non-natural mutation.

[0211] In some embodiments, a plant cell is provided that comprises at least one mutation within an HD-Zip gene, wherein the mutation is a substitution, insertion, or deletion, and the substitution, insertion, or deletion is introduced using an editing system that comprises a nucleic acid binding domain that binds to a target site within the HD-Zip gene. Optionally, wherein the endogenous HD-Zip gene: (a) comprises a sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88, or 89; (b) comprises a region having at least 80% sequence identity with any one of the nucleotide sequences of SEQ ID NO: 72-85 or 91-105, optionally any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with any one of the amino acid sequences of SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encodes a region having at least 80% sequence identity with SEQ ID NO: 86, 87, 106, 107, or 108. Optionally, the sequence identity of (a), (b), (c), and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, and optionally the sequence identity can be 100%. In some embodiments, the substitution, insertion, or deletion within the HD-Zip gene results in a dominant negative allele. In some embodiments, the mutation is a point mutation. In some embodiments, the target site is within a region of the HD-Zip gene that comprises a sequence having at least 80% sequence identity (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 72-85 or 91-105. In some embodiments, the editing system further comprises a nuclease, and the nucleic acid binding domain binds to a target site within a sequence having at least 80% sequence identity with the nucleotide sequence of any one of SEQ ID NO: 72-85 or 91-105, and upon cleavage by the nuclease, generates at least one mutation within the HD-Zip gene.In some embodiments, the HD-Zip gene of the plant cell comprising at least one mutation is an HD-Zip II gene, optionally the HD-Zip17-1 gene and / or the HD-Zip17-2 gene. In some embodiments, the HD-Zip II gene comprises a sequence having at least 80% sequence identity with any one of the nucleotide sequences of nucleotides 69, 70, 88 or 89, or encodes a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:90. In some embodiments, the at least one mutation is an in-frame deletion that produces a mutated HD-Zip polypeptide, optionally a HD-Zip polypeptide having a deletion within the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif or a deleted EAR motif (e.g., part or all of the EAR motif is absent in the mutated HD-Zip polypeptide). In some embodiments, the mutation can be a non-natural mutation.

[0212] In some embodiments, at least one mutation within the HD-Zip gene in the plant cell can produce a modification in the encoded HD-Zip polypeptide such that it is capable of regulating gene expression, optionally wherein the HD-Zip polypeptide exhibits a reduced ability to inhibit gene expression or is unable to inhibit gene expression. In some embodiments, the methods of the present invention can produce an HD-Zip gene in which the HD-Zip polypeptide comprises a mutated EAR motif or does not comprise an EAR motif. Thus, while not wishing to be bound by any particular theory, the HD-Zip polypeptide encoded by the mutated HD-Zip gene as described herein can comprise a mutation in the EAR motif that can reduce or eliminate the ability of the encoded HD-Zip polypeptide to negatively regulate downstream genes.

[0213] In some embodiments, the mutated HD-Zip gene comprised in the plant cell can have at least 90% sequence identity with SEQ ID NO:113 (optionally the sequence identity can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%) and / or encodes a mutated HD-Zip polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO:115.

[0214] In some embodiments, a plant cell or plant part comprising the mutated HD-Zip gene as described herein can be regenerated into a plant comprising the mutated HD-Zip gene (optionally comprising the mutated HD-Zip polypeptide). In some embodiments, a plant part or plant cell comprising the mutated HD-Zip gene as described herein does not regenerate into a plant.

[0215] The present invention also provides a method for providing a plurality of plants (e.g., soybean plants) having one or more improved yield traits, the method comprising growing two or more plants of the present invention (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 4000, 5000, or 10,000 or more plants) that comprise one or more mutations (e.g., non-natural mutations) in one or more HD-Zip genes and have one or more improved yield traits in a growth area, thereby providing a plurality of plants having one or more improved yield traits as compared to a plurality of control plants lacking the mutations, optionally wherein the improved yield traits can be an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight; 100-seed weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching). The growth area can be any area in which a plurality of plants can be grown together, including but not limited to fields (e.g., cultivated fields, agricultural fields), growth chambers, greenhouses, recreational areas, lawns, and / or roadside, etc.

[0216] In some embodiments, a method for generating / cultivating a transgene-free edited plant is provided, the method comprising: crossing a plant of the present invention (e.g., a plant comprising a mutation in an HD-Zip gene and exhibiting a phenotype of an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching) with a transgene-free plant, thereby introducing at least one mutation (e.g., one or more mutations) into the transgene-free plant (e.g., into the progeny plant); and selecting a progeny plant that comprises the at least one mutation and is transgene-free, thereby generating a transgene-free edited (e.g., base-edited) plant. In some embodiments, the at least one mutation can be a non-natural mutation.

[0217] In some embodiments, the present invention provides a method for generating a mutation in an endogenous HD-Zip gene in a plant, the method comprising: (a) targeting a gene editing system to a portion of the HD-Zip gene, the portion (i) comprising a sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89, (ii) comprising a region having at least 80% sequence identity with any nucleotide sequence of the nucleotide sequences of SEQ ID NO: 72-85 or 91-105, optionally any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105; (iii) encoding a polypeptide comprising a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 90, and / or (iv) encoding a region having at least 80% sequence identity with SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%; and (b) selecting a plant comprising a modification in a region of the HD-Zip gene having at least 80% sequence identity with any nucleic acid of the nucleic acids of SEQ ID NO: 72-85 or 91-105, optionally any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105. In some embodiments, the mutation generated produces a nucleic acid having at least 90% sequence identity with SEQ ID NO: 113 and / or produces a polypeptide having at least 90% sequence identity with SEQ ID NO: 115.

[0218] In some embodiments, a method of generating a variant in a region of an HD-Zip polypeptide, the method comprising: introducing an editing system into a plant cell, wherein the editing system is targeted to a region encoding an HD-Zip polypeptide of an HD-Zip gene; and contacting the region of the HD-Zip gene with the editing system, thereby introducing a mutation into the HD-Zip gene and generating a variant in the HD-Zip gene of the plant cell. In some embodiments, the HD-Zip gene: (a) comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprises a region of contiguous nucleotides having at least 80% sequence identity to any one of SEQ ID NO: 72-85 or 91-105, optionally any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, contacting a region of an endogenous HD-Zip gene in a plant cell with an editing system generates a plant cell that contains an edited endogenous HD-Zip gene in its genome, the method further comprising (a) regenerating a plant from the plant cell; (b) selfing the plant to produce progeny plants (E1); (c) determining an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight, 100-seed weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes and / or a decrease in branching in the progeny plants of (b); and (d) selecting the progeny plants that exhibit an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes and / or a decrease in branching compared to control plants.In some embodiments, the method may further comprise: (e) selfing the selected progeny plants of (d) to produce progeny plants (E2); (f) determining an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching in the progeny plants of (e); and (g) selecting the progeny plants that exhibit an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching to produce selected progeny plants that exhibit an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching as compared to control plants, optionally repeating (e) through (g) one or more additional times.

[0219] In some embodiments, a mutated HD-Zip gene produced by the method of the invention may comprise a sequence having at least 90% sequence identity to a mutated HD-Zip gene having the nucleotide sequence of SEQ ID NO: 113 and / or may encode a modified HD-Zip polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 115.

[0220] In some embodiments, a plant may comprise one or more (e.g., at least one, such as 1, 2, 3, 4, 5, 6, or more) mutated HD-Zip genes as described herein, optionally wherein for one or more mutations at any given allele, the edited plant may be heterozygous or homozygous or a combination thereof. In some embodiments, the plant may be heterozygous and may comprise a mutation at a specific locus in one allele of the HD-Zip gene and may be wild-type at the same locus in the second copy of the same gene. In some embodiments, in a specific HD-Zip locus, the plant may comprise different mutations at each allele of a particular HD-Zip gene or may comprise the same mutation at each allele.

[0221] In some embodiments, the present invention provides a method for detecting a mutant HD-Zip gene (a mutation in an endogenous HD-Zip gene) in a plant, the method comprising detecting in the genome of the plant an HD-Zip gene having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, optionally the sequence identity can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%) with any one of the nucleotide sequences of SEQ ID NOs: 72-85 or 91-105, and optionally having at least one mutation in a region having at least 80% sequence identity with any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105. In some embodiments, the detected mutant HD-Zip gene may comprise a mutated nucleotide sequence having at least 90% sequence identity with the mutated HD-Zip gene having the nucleotide sequence of SEQ ID NO: 113, optionally wherein the detected mutation is a non-natural mutation. In some embodiments, a mutated HD-Zip polypeptide as described herein, a mutated HD-Zip polypeptide having at least 90% sequence identity with SEQ ID NO: 115 can be detected, optionally wherein the detected mutation is a non-natural mutation.

[0222] In some embodiments, a method for detecting a mutant HD-Zip gene (a mutation in an endogenous HD-Zip gene) is provided, the method comprising detecting in the genome of a plant an HD-Zip gene having at least 80% sequence identity with any one of the nucleotide sequences of NO: 72-85 or 91-105, and optionally having at least one mutation in a region having at least 80% sequence identity with any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105.

[0223] In some embodiments, provided is a method for detecting a mutant HD-Zip gene (a mutation in an endogenous HD-Zip gene), the method comprising detecting in the genome of a plant an HD-Zip gene having at least one mutation in a nucleic acid encoding the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:90, optionally wherein the mutation is a modification of the EAR motif of the HD-Zip polypeptide, optionally a deletion of the entire EAR motif or a part thereof. In some embodiments, the mutation results in an HD-Zip polypeptide that is modified in its ability to regulate gene expression, optionally wherein the ability of the mutated HD-Zip polypeptide to regulate gene expression is reduced.

[0224] In some embodiments, provided is a method for detecting a mutation in an endogenous HD-Zip gene, the method comprising detecting in the genome of a plant a mutated HD-Zip gene. In some embodiments, the detected mutated HD-Zip gene comprises a sequence having at least 90% sequence identity with SEQ ID NO:113, optionally wherein the mutation detected in the HD-Zip gene comprises a non-natural mutation.

[0225] In some embodiments, provided is a method for editing a specific site in the genome of a plant cell, the method comprising: cutting a target site within an endogenous HD-Zip gene in the plant cell in a site-specific manner, the endogenous HD-Zip gene: (a) comprising a nucleotide sequence having at least 80% sequence identity (e.g., at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater (e.g., 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity)) with SEQ ID NO:69, 70, 88 or 89, (b) comprising a region having at least 80% sequence identity with any one of the nucleotide sequences of SEQ ID NO:72 - 85 or 91 - 105, (c) encoding an amino acid sequence having at least 80% sequence identity with SEQ ID NO:71 or SEQ ID NO:90, and / or (d) encoding a region having at least 80% sequence identity with SEQ ID NO:86, 87, 106, 107 or 108, thereby generating an edit in the endogenous HD-Zip gene of the plant cell and generating a plant cell comprising the edit in the endogenous HD-Zip gene, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, the edit can be located in a region of the endogenous HD-Zip gene that has at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, optionally at least 90% or 95%, optionally 100%) with any one of SEQ ID NO:72 - 85 and / or 91 - 105, optionally having at least 80% sequence identity with any one of SEQ ID NO:72 - 75, 76 - 79, 80 - 83, 84 - 85, 91 - 94, 95 - 98, 99 - 102 and / or 103 - 105; and / or encoding an HD-Zip polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO:86, 87, 106, 107 or 108. In some embodiments, the edit causes a non-natural mutation. In some embodiments, the edit causes a deletion, substitution or insertion, optionally generating a dominant negative allele.In some embodiments, editing causes a deletion within the box, optionally resulting in an HD-Zip polypeptide with a modified EAR motif. In some embodiments, mutations in the EAR motif result in an HD-Zip polypeptide with altered function (e.g., reduced ability to regulate gene expression or inability to regulate gene expression). In some embodiments, the target site comprises a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 72-85 or 91-105 (optionally having at least 80% sequence identity to any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, and / or 103-105). In some embodiments, editing results in a mutated HD-Zip gene having at least 90% sequence identity to SEQ ID NO: 113 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%).

[0226] In some embodiments, the editing method may further comprise regenerating a plant from a plant cell comprising an editing in an endogenous HD-Zip gene, thereby producing a plant that comprises the editing in its endogenous HD-Zip gene and has a phenotype of increased seed number (e.g., number of grains), increased seed weight (e.g., weight of grains), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching when compared to a control plant that does not comprise the editing, optionally wherein the plant exhibits increased yield when compared to a control plant that does not comprise the editing.

[0227] In some embodiments, a method for preparing a plant is provided, the method comprising: (a) contacting a population of plant cells comprising an endogenous HD-Zip gene with a nuclease linked to a nucleic acid binding domain (e.g., an editing system), the nucleic acid binding domain binding to a target site within the endogenous HD-Zip gene, wherein the endogenous gene (i) comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with any one of SEQ ID NO: 69, 70, 88 or 89; (ii) comprises a region of contiguous nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90; and / or (iv) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%; (b) selecting plant cells from the population of plant cells in which the endogenous HD-Zip gene has been mutated, thereby producing plant cells comprising a mutation in the endogenous HD-Zip gene; and (c) growing the selected plant cells into a plant comprising the mutation in the endogenous HD-Zip gene. In some embodiments, the mutation in the endogenous HD-Zip gene can produce a mutated HD-Zip gene having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%) with SEQ ID NO: 113 and / or can produce a mutated HD-Zip polypeptide, a mutated HD-Zip polypeptide having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 115.

[0228] In some embodiments, a method for increasing the number of seeds (e.g., the number of grains), increasing the seed weight (e.g., the grain weight), increasing the number of pods per node, increasing the number of pods per plant, altering the flowering time (e.g., earlier flowering time), shortening the plant height, reducing the number of nodes and / or reducing the branching in a plant, the method comprising (a) contacting a plant cell comprising an endogenous HD-Zip gene with a nuclease linked to a nucleic acid binding domain (e.g., an editing system), the nucleic acid binding domain binding to a target site within the endogenous HD-Zip gene, wherein the endogenous gene (i) comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with any one of the nucleotide sequences of SEQ ID NO: 69, 70, 88 or 89, (ii) comprises a region of contiguous nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105, (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90, and / or (iv) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%; and (b) growing the plant cell comprising the mutated endogenous HD-Zip gene into a plant, thereby producing a plant having a mutated HD-Zip gene and exhibiting a phenotype of increased number of seeds (e.g., number of grains), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of nodes and / or reduced branching.In some embodiments, a plant regenerated from a plant cell comprises a mutated HD-Zip gene having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%) to SEQ ID NO: 113 and / or comprises a mutated HD-Zip polypeptide having at least 90% sequence identity to SEQ ID NO: 115.

[0229] In some embodiments, provided is a method for generating a plant or a part thereof, the plant or part thereof comprising at least one cell (e.g., one or more cells) having an endogenous HD-Zip gene that has been mutated, the method comprising contacting a target site within the endogenous HD-Zip gene in the plant or plant part with a nuclease, the nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site within the endogenous HD-Zip gene, wherein the endogenous HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89, (b) comprises a region of contiguous nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, whereby a plant or a part thereof is generated, the plant or part thereof comprising at least one cell having a mutation in the endogenous HD-Zip gene, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, the generated plant comprises a mutated HD-Zip gene having at least 90% sequence identity (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%) with SEQ ID NO: 113, and / or encodes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115.

[0230] The present invention also provides a method for generating a plant or a part thereof, the plant or the part thereof comprising an endogenous HD-Zip gene that has been mutated and exhibits improved yield traits (one or more improved yield traits), the method comprising contacting a target site within the endogenous HD-Zip gene in the plant or plant part with a nuclease, the nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site within the HD-Zip gene, wherein the HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with any one of SEQ ID NO: 69, 70, 88 or 89, (b) comprises a region consisting of contiguous nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region consisting of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, thereby generating a plant or a part thereof, the plant or the part thereof comprising an endogenous HD-Zip gene that has a mutation and exhibits a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight, 100-seed weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes and / or reduced branching, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, and optionally the sequence identity can be 100%. In some embodiments, the plant may further exhibit increased yield compared to a control plant that does not contain the mutation.In some embodiments, the method can produce a plant or a part thereof that contains a mutated HD-Zip gene having at least 90% sequence identity with SEQ ID NO: 113 (e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%), and / or encodes an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115.

[0231] In some embodiments, the target site can be a region of the HD-Zip gene having at least 80% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 72-85 and / or 91-105 (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, optionally the sequence identity can be at least 85% or can be at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%), optionally having at least 80% sequence identity with any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105 (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, optionally the sequence identity can be at least 85% or can be at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%), or located within said region.

[0232] In some embodiments, the nuclease that can be used in the present invention can cleave the endogenous HD-Zip gene, thereby introducing mutations into the endogenous HD-Zip gene. In some embodiments, the nuclease can include, but is not limited to, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), endonucleases (e.g., Fok1), and / or CRISPR-Cas effector proteins. Similarly, the nucleic acid binding domain that can be used in the present invention (e.g., DNA binding domain, RNA binding domain) can be any nucleic acid binding domain that can be used to edit / modify the target nucleic acid. Such nucleic acid binding domains include, but are not limited to, zinc fingers, transcription activator-like DNA binding domains (TALs), argonaute, and / or CRISPR-Cas effector DNA binding domains. In some embodiments, the mutation can be a non-natural mutation. In some embodiments, the mutation can be a dominant negative mutation. In some embodiments, the mutation can be a deletion, optionally wherein the mutation can be an in-frame deletion. In some embodiments, the mutation can produce a mutated HD-Zip polypeptide with an in-frame deletion, optionally wherein the in-frame deletion causes the deletion of the entire ethylene response element binding factor-related amphiphilic repression (EAR) motif or a part thereof, and the mutated EAR motif contains a mutation that alters its function as a gene expression regulator.

[0233] In some embodiments, a plant or a part thereof comprising at least one cell having a mutation in the endogenous HD-Zip gene as described herein comprises a sequence having at least 90% identity to sequence identity with SEQ ID NO: 113 and / or a modified HD-Zip polypeptide, a modified HD-Zip polypeptide comprising an amino acid sequence having at least 90% sequence identity with SEQ ID NO: 115. In some embodiments, the plant or a part thereof of the present invention comprises the mutated endogenous HD-Zip gene as described herein and has a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching when compared to a control plant that does not contain the mutation, optionally wherein the plant shows increased yield when compared to the control plant.

[0234] In some embodiments, provided is a method of editing an endogenous HD-Zip gene in a plant or a plant part, the method comprising contacting a target site within the HD-Zip gene in the plant or its part with a cytosine base editing system comprising a cytosine deaminase and a nucleic acid binding domain that binds to the target site within the HD-Zip gene, wherein the HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89, (b) comprises a region of contiguous nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105, (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, whereby editing the endogenous HD-Zip gene in the plant or its part and producing a plant or its part comprising at least one cell having a mutation in the endogenous HD-Zip gene, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, the detected nucleic acid can comprise an unnatural mutation.

[0235] In some embodiments, a method of editing an endogenous HD-Zip gene in a plant or a plant part is provided, the method comprising contacting a target site within the HD-Zip gene in the plant or the plant part with an adenosine base editing system comprising an adenosine deaminase and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site within the HD-Zip gene, wherein the HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprises a region consisting of consecutive nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region consisting of consecutive amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, whereby the endogenous HD-Zip gene in the plant or the part thereof is edited and a plant or a part thereof is produced, the plant or the part thereof comprising at least one cell having a mutation in the endogenous HD-Zip gene, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, the detected nucleic acid can comprise non-natural mutations.

[0236] In some embodiments, provided is a method for modifying an endogenous HD-Zip gene in a plant or a part thereof to increase the number of seeds (e.g., the number of grains), increase the seed weight (e.g., the grain weight), increase the number of pods per plant, change the flowering time (e.g., earlier flowering time), reduce the plant height, reduce the number of stem nodes, and / or reduce the number of branches in the plant or the part thereof, the method comprising modifying a target site within the endogenous HD-Zip gene in the plant or the part thereof, wherein the endogenous HD-Zip gene (a) comprises a sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88, or 89; (b) comprises a region having at least 80% sequence identity with any one nucleotide sequence among the nucleotide sequences of any one of SEQ ID NO: 72-85 or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with any one amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a region having at least 80% sequence identity with SEQ ID NO: 86, 87, 106, 107, or 108, whereby the endogenous HD-Zip gene is modified and the number of seeds (e.g., the number of grains) is increased, the seed weight (e.g., the grain weight) is increased, the number of pods per plant is increased, the flowering time is changed (e.g., earlier flowering time), the plant height is reduced, the number of stem nodes is reduced, and / or the number of branches is reduced in the plant or the part thereof, optionally wherein the sequence identity of (a), (b), (c), and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, and optionally the sequence identity can be 100%. In some embodiments, the target site is a region of the HD-Zip gene having at least 80% sequence identity with any one nucleotide sequence of SEQ ID NO: 72-85 and / or 91-105, optionally wherein the sequence identity can be at least 85% or at least 90%, or it can be at least 95%, and optionally the sequence identity can be 100%.

[0237] In some embodiments, mutations in the endogenous HD-Zip gene edited as described herein result in a mutated HD-Zip gene having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 113. In some embodiments, mutations in the endogenous HD-Zip gene edited as described herein result in non-natural mutations.

[0238] In some embodiments, a method of producing a plant is provided, the plant comprising a mutation in an endogenous HD-Zip gene and at least one polynucleotide of interest, the method comprising crossing a plant of the invention (a first plant) comprising at least one mutation in an endogenous HD-Zip gene with a second plant, the second plant comprising at least one polynucleotide of interest; and selecting a progeny plant comprising at least one mutation in the HD-Zip gene and at least one polynucleotide of interest, thereby producing a plant comprising a mutation in an endogenous HD-Zip gene and at least one polynucleotide of interest.

[0239] In some embodiments, the invention provides a method of producing a plant, the plant comprising a mutation in an endogenous HD-Zip gene and at least one polynucleotide of interest, the method comprising introducing at least one polynucleotide of interest into a plant of the invention comprising at least one mutation in an endogenous HD-Zip gene, thereby producing a plant comprising at least one mutation in the HD-Zip gene and at least one polynucleotide of interest.

[0240] In some embodiments, a method of producing a plant is provided, the plant comprising a mutation in an endogenous HD-Zip gene and exhibiting a phenotype of improved yield traits, improved plant architecture, and / or improved defense traits, the method comprising crossing a first plant with a second plant, the first plant being a plant of the invention (e.g., comprising at least one mutation in an endogenous HD-Zip gene), the second plant exhibiting a phenotype of improved yield traits, improved plant architecture, and / or improved defense traits; and selecting a progeny plant comprising a mutation in the HD-Zip gene and a phenotype of improved yield traits, improved plant architecture, and / or improved defense traits, thereby producing the plant comprising a mutation in an endogenous HD-Zip gene and exhibiting a phenotype of improved yield traits, improved plant architecture, and / or improved defense traits as compared to a control plant.

[0241] Further provided is a method of controlling weeds in a container (e.g., a pot or a seed tray, etc.), a growth chamber, a greenhouse, a field, a recreational area, a lawn or beside a road, the method comprising applying a herbicide to one or more (a plurality of) plants of the present invention (e.g., comprising at least one mutation in an endogenous HD-Zip gene) growing in the container, the growth chamber, the greenhouse, the field, the recreational area, the lawn or beside the road, thereby controlling the weeds in the container, the growth chamber, the greenhouse, the field, the recreational area, the lawn or beside the road where the one or more plants grow.

[0242] In some embodiments, provided is a method of reducing insect predation on a plant, the method comprising applying an insecticide to one or more plants of the invention (e.g., comprising at least one mutation in an endogenous HD-Zip gene), thereby reducing insect predation on the one or more plants.

[0243] In some embodiments, provided is a method of reducing fungal diseases on a plant, the method comprising applying a fungicide to one or more plants of the present invention (e.g., comprising at least one mutation in an endogenous HD-Zip gene), thereby reducing fungal diseases on the one or more plants, optionally wherein the one or more plants grow in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn or beside a road.

[0244] In some embodiments, provided is a method of reducing bacterial diseases on a plant, the method comprising applying a bactericide to one or more plants of the present invention (e.g., comprising at least one mutation in an endogenous CT2 gene), thereby reducing bacterial diseases on the one or more plants, optionally wherein the one or more plants grow in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn or beside a road.

[0245] The polynucleotide of interest can be any polynucleotide capable of conferring a desired phenotype to a plant or otherwise altering the phenotype or genotype of a plant. In some embodiments, the polynucleotide of interest can include, but is not limited to, polynucleotides conferring herbicide tolerance, insect resistance, nematode resistance, disease resistance, increased yield, increased nutrient use efficiency, and / or abiotic stress resistance.

[0246] Thus, plants or plant cultivars to be preferentially processed according to the invention include all plants obtained by genetic modification with genetic material which confers on these plants particularly advantageous useful properties ("traits"). Examples of such properties are better plant growth, vigour, stress tolerance, erectness, lodging resistance, nutrient uptake, plant nutrition and / or yield, in particular improved growth, increased tolerance to high or low temperatures, increased tolerance to drought or water or soil salinity levels, enhanced flowering performance, easier harvesting, accelerated ripening, higher yield, higher quality and / or higher nutritional value of the harvested product, better shelf life and / or processability of the harvested product.

[0247] Additional examples of such traits are increased resistance to animal and microbial pests, such as resistance to insects, arachnids, nematodes, mites, slugs, and snails, which is caused, for example, by toxins formed in the plant. Among the DNA sequences encoding proteins conferring tolerance traits to such animal and microbial pests, especially insects, mention will be made in particular of the genetic material encoding Bt proteins from Bacillus thuringiensis, which are widely described in the literature and well known to those skilled in the art. Also mentioned will be proteins extracted from bacteria such as Photorhabdus (WO97 / 17432 and WO98 / 08932). Specifically, mention will be made of Bt Cry or VIP proteins (which include Cry1A, CryIAb, CryIAc, CryIIA, CryIIIA, CryIIIB2, Cry9c Cry2Ab, Cry3Bb, and Cry1F proteins or their toxic fragments, as well as their hybrids or combinations, especially Cry1F proteins or hybrids derived from Cry1F proteins (e.g., hybrid Cry1A-Cry1F proteins or their toxic fragments), Cry1A-type proteins or their toxic fragments (preferably Cry1Ac proteins or hybrids derived from Cry1Ac proteins (e.g., hybrid Cry1AbCry1Ac proteins) or Cry1Ab or Bt2 proteins or their toxic fragments, Cry2Ae, Cry2Af, or Cry2Ag proteins or their toxic fragments, Cry1A.105 proteins or their toxic fragments, VIP3Aa19 proteins, VIP3Aa20 proteins, VIP3A proteins produced in COT202 or COT203 cotton events, VIP3Aa proteins or their toxic fragments as described by Estruch et al. (1996), Proc Natl Acad Sci US A. 28; 93(11):5389-94, Cry proteins as described in WO2001 / 47952, insecticidal proteins from Xenorhabdus (as described in WO98 / 50427), Serratia (especially from S. entomophila), or Photorhabdus strains, such as the Tc protein from Photorhabdus as described in WO98 / 08932. In addition, included herein are any variants or mutants of any of these proteins that differ in some amino acids (1-10, preferably 1-5) from any of the sequences named above, especially the sequences of their toxic fragments, or are fused to a transit peptide, such as a plastid transit peptide, or another protein or peptide.

[0248] Another particularly emphasized instance of such a trait is conferring tolerance to one or more herbicides, such as imidazolinones, sulfonylureas, glyphosate or glufosinate. Among the DNA sequences (i.e., the polynucleotides of interest) encoding proteins that confer the trait of tolerance to certain herbicides to the transformed plant cells and plants, particular mention will be made of the bar or PAT genes or the Streptomyces coelicolor genes described in WO2009 / 152359, which confer tolerance to the glufosinate herbicide; genes encoding a suitable EPSPS (5-enolpyruvylshikimate-3-phosphate synthase), which confer tolerance to herbicides targeting EPSPS (especially herbicides such as glyphosate and its salts); genes encoding glyphosate-N-acetyltransferase, or genes encoding glyphosate oxidoreductase. Further suitable herbicide tolerance traits include at least one ALS (acetolactate synthase) inhibitor (e.g., WO2007 / 024782); mutated Arabidopsis ALS / AHAS genes (e.g., U.S. Patent 6,855,533); genes encoding 2,4-D-monooxygenase, which confer tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid); and genes encoding dicamba monooxygenase, which confer tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid).

[0249] Another instance of such a trait is increased resistance to plant pathogenic fungi, bacteria and / or viruses, which is caused, for example, by systemic acquired resistance (SAR), systemin, phytoalexins, elicitors, and resistance genes and the correspondingly expressed proteins and toxins.

[0250] Particularly useful transgenic events in transgenic plants or plant cultivars that can be preferentially processed according to the present invention include event 531 / PV-GHBK04 (cotton, insect control, described in WO2002 / 040677); event 1143-14A (cotton, insect control, not deposited, described in WO2006 / 128569); event 1143-51B (cotton, insect control, not deposited, described in WO2006 / 128570); event 1445 (cotton, herbicide tolerance, not deposited, described in US-A 2002-120964 or WO2002 / 034946); event 17053 (rice, herbicide tolerance, deposited as PTA-9843, described in WO2010 / 117737); event 17314 (rice, herbicide tolerance, deposited as PTA-9844, described in WO2010 / 117735); event 281-24-236 (cotton, insect control - herbicide tolerance, deposited as PTA-6233, described in WO2005 / 103266 or US-A2005-216969); event 3006-210-23 (cotton, insect control - herbicide tolerance, deposited as PTA-6233, described in US-A2007-143876 or WO2005 / 103266); event 3272 (maize, quality characteristics, deposited as PTA-9972, described in WO2006 / 098952 or US-A2006-230473); event 33391 (wheat, herbicide tolerance, deposited as PTA-2347, described in WO2002 / 027004); event 40416 (maize, insect control - herbicide tolerance, deposited as ATCC PTA-11508, described in WO 11 / 075593); event 43A47 (maize, insect control - herbicide tolerance, deposited as ATCC PTA-11509, described in WO2011 / 075595); event 5307 (maize, insect control, deposited as ATCC PTA-9561, described in WO2010 / 077816); event ASR-368 (foxtail millet, herbicide tolerance, deposited as ATCC PTA-4816, described in US-A2006-162007 or WO2004 / 053062); event B16 (maize, herbicide tolerance, not deposited, described in US-A2003-126634); event BPS-CV127-9 (soybean, herbicide tolerance, deposited as NCIMB No. 41603, described in WO2010 / 080829); event BLR1 (oilseed rape, restoration of male sterility, deposited as NCIMB 41193, described in WO2005 / 074671);Event CE43-67B (cotton, insect control, deposited as DSMACC2724, described in US-A2009-217423 or WO2006 / 128573); Event CE44-69D (cotton, insect control, not deposited, described in US-A2010-0024077); Event CE44-69D (cotton, insect control, not deposited, described in WO2006 / 128571); Event CE46-02A (cotton, insect control, not deposited, described in WO2006 / 128572); Event COT102 (cotton, insect control, not deposited, described in US-A 2006-130175 or WO2004 / 039986); Event COT202 (cotton, insect control, not deposited, described in US-A 2007-067868 or WO2005 / 054479); Event COT203 (cotton, insect control, not deposited, described in WO2005 / 054480); Event DAS21606-3 / 1606 (soybean, herbicide tolerance, deposited as PTA-11028, described in WO2012 / 033794); Event DAS40278 (maize, herbicide tolerance, deposited as ATCC PTA-10244, described in WO2011 / 022469); Event DAS-44406-6 / pDAB8264.44.06.1 (soybean, herbicide tolerance, deposited as PTA-11336, described in WO2012 / 075426); Event DAS-14536-7 / pDAB8291.45.36.2 (soybean, herbicide tolerance, deposited as PTA-11335, described in WO2012 / 075429); Event DAS-59122-7 (maize, insect control - herbicide tolerance, deposited as ATCC PTA 11384, described in US-A2006-070139); Event DAS-59132 (maize, insect control - herbicide tolerance, not deposited, described in WO2009 / 100188); Event DAS68416 (soybean, herbicide tolerance, deposited as ATCC PTA-10442, described in WO2011 / 066384 or WO2011 / 066360); Event DP-098140-6 (maize, herbicide tolerance, deposited as ATCCPTA-8296, described in US-A2009-137395 or WO 08 / 112019); Event DP-305423-1 (soybean, quality trait, not deposited, described in US-A2008-312082 or WO2008 / 054747);Event DP-32138-1 (maize, hybrid system, deposited with ATCC PTA-9158, described in US-A2009-0210970 or WO2009 / 103049); Event DP-356043-5 (soybean, herbicide tolerance, deposited with ATCC PTA-8287, described in US-A 2010-0184079 or WO2008 / 002872); Event EE-I (eggplant, insect control, not deposited, described in WO 07 / 091277); Event Fil17 (maize, herbicide tolerance, deposited with ATCC 209031, described in US-A 2006-059581 or WO 98 / 044140); Event FG72 (soybean, herbicide tolerance, deposited with PTA-11041, described in WO2011 / 063413); Event GA21 (maize, herbicide tolerance, deposited with ATCC 209033, described in US-A 2005-086719 or WO 98 / 044140); Event GG25 (maize, herbicide tolerance, deposited with ATCC 209032, described in US-A2005-188434 or WO98 / 044140); Event GHB119 (cotton, insect control-herbicide tolerance, deposited with ATCC PTA-8398, described in WO2008 / 151780); Event GHB614 (cotton, herbicide tolerance, deposited with ATCC PTA-6878, described in US-A2010-050282 or W02007 / 017186); Event GJ11 (maize, herbicide tolerance, deposited with ATCC 209030, described in US-A 2005-188434 or WO98 / 044140); Event GM RZ13 (sugar beet, virus resistance, deposited with NCIMB-41601, described in WO2010 / 076212); Event H7-1 (sugar beet, herbicide tolerance, deposited with NCIMB 41158 or NCIMB 41159, described in US-A 2004-172669 or WO 2004 / 074492); Event JOPLIN1 (wheat, disease resistance, not deposited, described in US-A2008-064032); Event LL27 (soybean, herbicide tolerance, deposited with NCIMB41658, described in WO2006 / 108674 or US-A2008-320616); Event LL55 (soybean, herbicide tolerance, deposited with NCIMB 41660, described in WO 2006 / 108675 or US-A2008-196127);Event LLcotton25 (cotton, herbicide tolerance, deposited with ATCC PTA-3343, described in WO2003 / 013224 or USA2003-097687); Event LLRICE06 (rice, herbicide tolerance, deposited with ATCC 203353, described in US 6,468,747 or WO2000 / 026345); Event LLRice62 (rice, herbicide tolerance, deposited with ATCC 203352, described in WO2000 / 026345); Event LLRICE601 (rice, herbicide tolerance, deposited with ATCC PTA-2600, described in US-A2008-2289060 or WO2000 / 026356); Event LY038 (maize, quality trait, deposited with ATCC PTA-5623, described in US-A 2007-028322 or WO2005 / 061720); Event MIR162 (maize, insect control, deposited with PTA-8166, described in US-A2009-300784 or WO2007 / 142840); Event MIR604 (maize, insect control, not deposited, described in US-A2008-167456 or WO2005 / 103301); Event MON15985 (cotton, insect control, deposited with ATCC PTA-2516, described in US-A2004-250317 or WO2002 / 100163); Event MON810 (maize, insect control, not deposited, described in US-A2002-102582); Event MON863 (maize, insect control, deposited with ATCC PTA-2605, described in WO2004 / 011601 or US-A2006-095986); Event MON87427 (maize, pollination control, deposited with ATCC PTA-7899, described in WO2011 / 062904); Event MON87460 (maize, stress tolerance, deposited with ATCC PTA-8910, described in WO2009 / 111263 or US-A2011-0138504); Event MON87701 (soybean, insect control, deposited with ATCCPTA-8194, described in US-A2009-130071 or WO2009 / 064652); Event MON87705 (soybean, quality trait - herbicide tolerance, deposited with ATCC PTA-9241, described in US-A2010-0080887 or WO2010 / 037016); Event MON87708 (soybean, herbicide tolerance, deposited with ATCC PTA-9670, described in WO2011 / 034704);Event MON87712 (soybean, yield, deposited as PTA-10296, described in WO2012 / 051199); Event MON87754 (soybean, quality characteristic, deposited as ATCC PTA-9385, described in WO2010 / 024976); Event MON87769 (soybean, quality characteristic, deposited as ATCC PTA-8911, described in US-A2011-0067141 or WO2009 / 102873); Event MON88017 (maize, insect control-herbicide tolerance, deposited as ATCC PTA-5582, described in US-A 2008-028482 or WO2005 / 059103); Event MON88913 (cotton, herbicide tolerance, deposited as ATCC PTA-4854, described in WO2004 / 072235 or US-A 2006-059590); Event MON88302 (oilseed rape, herbicide tolerance, deposited as PTA-10955, described in WO2011 / 153186); Event MON88701 (cotton, herbicide tolerance, deposited as PTA-11754, described in WO2012 / 134808); Event MON89034 (maize, insect control, deposited as ATCC PTA-7455, described in WO 07 / 140256 or US-A2008-260932); Event MON89788 (soybean, herbicide tolerance, deposited as ATCC PTA-6708, described in US-A 2006-282915 or WO2006 / 130436); Event MS1 1 (oilseed rape, pollination control-herbicide tolerance, deposited as ATCC PTA-850 or PTA-2485, described in WO2001 / 031042); Event MS8 (oilseed rape, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO2001 / 041558 or US-A2003-188347); Event NK603 (maize, herbicide tolerance, deposited as ATCC PTA-2478, US-A2007-292854); Event PE-7 (rice, insect control, not deposited, described in WO2008 / 114282); Event RF3 (oilseed rape, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO2001 / 041558 or US-A2003-188347); Event RT73 (oilseed rape, herbicide tolerance, not deposited, described in WO2002 / 036831 or US-A2008-070260); Event SYHT0H2 / SYN-000H2-5 (soybean, herbicide tolerance, deposited as PTA-11226, described in WO2012 / 082548);Event T227-1 (sugar beet, herbicide tolerance, not deposited, described in WO2002 / 44407 or US-A2009-265817); Event T25 (maize, herbicide tolerance, not deposited, described in US-A 2001-029014 or WO2001 / 051654); Event T304-40 (cotton, insect control - herbicide tolerance, deposited as ATCC PTA-8171, described in US-A 2010-077501 or WO2008 / 122406); Event T342-142 (cotton, insect control, not deposited, described in WO2006 / 128568); Event TC1507 (maize, insect control - herbicide tolerance, not deposited, described in US-A2005-039226 or WO2004 / 099447); Event VIP1034 (maize, insect control - herbicide tolerance, deposited as ATCC PTA-3925, described in WO2003 / 052073); Event 32316 (maize, insect control - herbicide tolerance, deposited as PTA-11507, described in WO2011 / 084632); Event 4114 (maize, insect control - herbicide tolerance, deposited as PTA-11506, described in W02011 / 084621); Event EE-GM3 / FG72 (soybean, herbicide tolerance, ATCC accession No. PTA-11041), optionally stacked with Event EE-GM1 / LL27 or Event EE-GM2 / LL55 (WO2011 / 063413A2); Event DAS-68416-4 (soybean, herbicide tolerance, ATCC accession No. PTA-10442, WO2011 / 066360A1); Event DAS-68416-4 (soybean, herbicide tolerance, ATCC accession No. PTA-10442, WO2011 / 066384A1); Event DP-040416-8 (maize, insect control, ATCC accession No. PTA-11508, WO2011 / 075593A1); Event DP-043A47-3 (maize, insect control, ATCC accession No. PTA-11509, WO2011 / 075595A1); Event DP-004114-3 (maize, insect control, ATCC accession No. PTA-11506, WO2011 / 084621A1); Event DP-032316-8 (maize, insect control, ATCC accession No. PTA-11507, WO2011 / 084632A1); Event MON-88302-9 (oilseed rape, herbicide tolerance, ATCC accession No. PTA-10955, WO2011 / 153186A1);Event DAS-21606-3 (soybean, herbicide tolerance, ATCC accession number PTA-11028, WO2012 / 033794A2); Event MON-87712-4 (soybean, quality trait, ATCC accession N° PTA-10296, WO2012 / 051199A2); Event DAS-44406-6 (soybean, stacked herbicide tolerance, ATCC accession N° PTA-11336, WO2012 / 075426A1); Event DAS-14536-7 (soybean, stacked herbicide tolerance, ATCC accession N° PTA-11335, WO2012 / 075429A1); Event SYN-000H2-5 (soybean, herbicide tolerance, ATCC accession N° PTA-11226, WO2012 / 082548A2); Event DP-061061-7 (rapeseed, herbicide tolerance, no available deposit N°, WO2012071039A1); Event DP-073496-4 (rapeseed, herbicide tolerance, no available deposit N°, US2012131692); Event 8264.44.06.1 (soybean, stacked herbicide tolerance, accession N° PTA-11336, WO2012075426A2); Event 8291.45.36.2 (soybean, stacked herbicide tolerance, accession N° PTA-11335, WO2012075429A2); Event SYHT0H2 (soybean, ATCC accession N° PTA-11226, WO2012 / 082548A2); Event MON88701 (cotton, ATCC accession N° PTA-11754, WO2012 / 134808A1); Event KK179-2 (alfalfa, ATCC accession N° PTA-11833, WO2013 / 003558A1); Event pDAB8264.42.32.1 (soybean, stacked herbicide tolerance, ATCC accession N° PTA-11993, WO2013 / 010094A1); Event MZDT09Y (maize, ATCC accession N° PTA-13025, WO2013 / 012775A1).;

[0251] Genes / events (e.g., polynucleotides of interest) conferring the desired traits under discussion may also be present in transgenic plants in combination with each other. Examples of transgenic plants that may be mentioned are important crop plants such as cereals (wheat, rice, triticale, barley, rye, oats), maize, soybeans, potatoes, sugar beets, sugar cane, tomatoes, peas and other types of vegetables, cotton, tobacco, oilseed rape and fruit plants (fruits such as apples, pears, citrus fruits and grapes), with particular emphasis on maize, soybeans, wheat, rice, potatoes, cotton, sugar cane, tobacco and oilseed rape. Particular emphasis is placed on traits such as increased resistance of plants to insects, arachnids, nematodes, slugs and snails, and increased resistance of plants to one or more herbicides.

[0252] Commercially available examples of such plants, plant parts or plant seeds that can be preferentially processed according to the invention include commercial products such as those sold or distributed under RIB ROUNDUP VT DOUBLE VT TRIPLE BOLLGARD ROUNDUP READY 2 ROUNDUP 2 XTENDTM, INTACTA RR2 VISTIVE and / or XTENDFLEX TM plant seeds sold under trade names.

[0253] HD-Zip genes useful in the present invention include any HD-Zip gene in which a mutation as described herein can confer improved yield traits (one or more improved yield traits) in a plant or a part thereof, optionally wherein the improved yield traits can include, but are not limited to, an increase in seed number (e.g., grain number), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching, compared to a control plant that does not contain the mutation. In some embodiments, the HD-Zip polypeptide comprises an amino acid sequence having at least 80% identity (e.g., about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% sequence identity) to SEQ ID NO:71 or SEQ ID NO:90, or comprises a region consisting of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO:86, 87, 106, 107 or 108. In some embodiments, the HD-Zip gene can comprise a sequence having at least about 80% sequence identity to the nucleotide sequence of NO:69, 70, 88 or 89, or the HD-Zip gene contains within it a sequence (region or portion) having at least 80% identity to any one of the nucleotide sequences of SEQ ID NO:72 - 85 or 91 - 105.

[0254] In some embodiments, at least one mutation generated in an endogenous HD-Zip gene in a plant can be a substitution, deletion, and / or insertion. In some embodiments, at least one mutation in an endogenous HD-Zip gene in a plant can be a substitution, deletion, and / or insertion that causes a dominant negative mutation, optionally wherein a plant comprising the mutation can exhibit a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, reduced branching, and / or increased yield when compared to a control plant that does not contain the edit / mutation. For example, the mutation can be a deletion of one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more amino acids of an HD-Zip polypeptide), or the mutation can be a deletion of at least 1 nucleotide to about 50 consecutive nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 consecutive nucleotides, or any range or value therein, optionally about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 consecutive nucleotides) in the gene encoding the HD-Zip polypeptide. In some embodiments, the deletion can be in a region of the HD-Zip gene encoding an ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, and the deletion causes a deletion of one or more amino acids in the EAR motif of the encoded HD-Zip polypeptide. In some embodiments, the mutation can be a point mutation. In some embodiments, the at least one mutation can be a base substitution of A, T, G, or C. In some embodiments, the mutation or edit can be an in-frame insertion or an in-frame deletion. In some embodiments, the mutation or edit in the HD-Zip gene can cause a modification of the ability of the encoded HD-Zip polypeptide to regulate gene expression. In some embodiments, at least one mutation can be a non-natural mutation.

[0255] In some embodiments, the mutations generated by the methods of the invention generate mutated HD-Zip genes comprising an edited nucleotide sequence having at least 90% sequence identity (e.g., at least 95%, optionally the sequence identity can be 100%) to the nucleotide sequence of SEQ ID NO: 113 and / or generate a modified HD-Zip polypeptide having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 115, optionally wherein the mutation in the HD-Zip gene is a non-natural mutation.

[0256] In some embodiments, the mutation in the endogenous HD-Zip gene can be performed by an editing system after cleavage, the editing system comprising a nuclease and a nucleic acid binding domain (e.g., a DNA binding domain), the nucleic acid binding domain binding to a target site within a target nucleic acid (e.g., the HD-Zip gene), wherein the target nucleic acid: (a) comprises a sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprises a region having at least 80% sequence identity with any one of the nucleotide sequences of SEQ ID NO: 72-85 or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with any one of the amino acid sequences of SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encodes a region having at least 80% sequence identity with SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, the nuclease cleaves the endogenous HD-Zip gene and a mutation is introduced into the endogenous HD-Zip gene. In some embodiments, the mutation performed by the editing system can result in a deletion or an insertion. In some embodiments, the mutation can modify the EAR motif of the HD-Zip polypeptide, thereby altering the ability of the HD-Zip polypeptide to act as a gene expression regulator. In some embodiments, the mutation can be an in-frame mutation (e.g., an in-frame deletion or insertion). In some embodiments, at least one mutation can be a non-natural mutation. In some embodiments, the cleavage results in a mutated endogenous HD-Zip gene having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 113, optionally wherein the percentage of sequence identity with SEQ ID NO: 113 can be at least 95%, or it can be 100%.

[0257] The nucleases useful in the present invention can cleave endogenous HD-Zip genes, thereby introducing mutations into the endogenous HD-Zip genes. Such nucleases include, but are not limited to, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), endonucleases (e.g., Fok1) and / or CRISPR-Cas effector proteins. Similarly, the nucleic acid binding domains useful in the present invention (e.g., DNA binding domains, RNA binding domains) include any nucleic acid binding domain that can be used to edit / modify target nucleic acids. Such nucleic acid binding domains include, but are not limited to, zinc fingers, transcription activator-like DNA binding domains (TALs), argonaute and / or CRISPR-Cas effector DNA binding domains.

[0258] The present disclosure further provides guide nucleic acids (e.g., gRNA, gDNA, crRNA, crDNA) that bind to target sites within HD-Zip genes, wherein the endogenous HD-Zip genes: (a) comprise a sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity) to the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 88, or 89; (b) comprise a region having at least 80% sequence identity to any nucleotide sequence within the nucleotide sequences of any one of SEQ ID NOs: 72-85 or 91-105, optionally having at least 80% sequence identity to any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, or 103-105; (c) encode a polypeptide comprising a sequence having at least 80% sequence identity to any amino acid sequence of SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encode a region having at least 80% sequence identity to SEQ ID NOs: 86, 87, 106, 107, or 108, optionally wherein the sequence identity of (a), (b), (c), and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, the target site can be within any nucleotide sequence of SEQ ID NOs: 72-85 or 91-105, optionally within a region of any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, or 103-105. In some embodiments, a guide nucleic acid is provided that binds to a target nucleic acid within an HD-Zip gene in a plant, wherein the HD-Zip gene has gene identification number (SoyBase database) Glyma.20g014400 (HD-Zip17-1, SEQ ID NO: 69) or Glyma.07g218000 (HD-Zip17-2, SEQ ID NO: 88). In some embodiments, the guide nucleic acid comprises a spacer having the nucleotide sequence of any one of SEQ ID NOs: 109-112.

[0259] In some embodiments, the target sites to which the guide nucleic acid of the present invention can bind may comprise a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, optionally the sequence identity may be at least 85% or at least 90%, or it may be at least 95%, optionally the sequence identity may be 100%) with any one of the nucleotide sequences of SEQ ID NO: 72-85 or 91-105 or a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity, optionally the sequence identity may be at least 85% or at least 90%, or it may be at least 95%, optionally the sequence identity may be 100%) with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105 or a portion thereof.

[0260] Exemplary spacer sequences for the guide sequences useful in the present invention can include complementarity to any of the nucleotide sequences of SEQ ID NO: 69, 70, 88, and / or 89 having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity, optionally the sequence identity can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100), optionally to any of the nucleotide sequences of SEQ ID NO: 72 - 85 or 91 - 105 (optionally any one of SEQ ID NO: 72 - 85 and / or 91 - 105) having at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity (optionally at least 85% sequence identity or at least 90% sequence identity or at least 95% sequence identity, optionally wherein the sequence identity is 100%) of a fragment or portion of a nucleotide sequence; or a fragment or portion of a nucleotide sequence encoding a polypeptide comprising a sequence having at least 80% (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity, optionally the sequence identity can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%) sequence identity to any of the amino acid sequences of SEQ ID NO: 71 and / or SEQ ID NO: 90 or a fragment or portion of a nucleotide sequence encoding a polypeptide comprising a sequence having at least 80% (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity, optionally the sequence identity can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%) sequence identity to any of the amino acid sequences of SEQ ID NO: 86, 87, 106, 107, and / or 108.

[0261] In some embodiments, the guide nucleic acid comprises a spacer having the nucleotide sequence of SEQ ID NO: 109, 110, 111, or 112, or the reverse complement thereof, or any combination thereof.

[0262] In some embodiments, a system is provided that comprises a guide nucleic acid of the invention and a CRISPR-Cas effector protein, the CRISPR-Cas effector protein being associated with the guide nucleic acid. In some embodiments, a system is provided that comprises a guide nucleic acid and a CRISPR-Cas effector protein, the guide nucleic acid comprising a spacer having a nucleotide sequence of any one of SEQ ID NOs: 109-112, the CRISPR-Cas effector protein being associated with the guide nucleic acid. In some embodiments, the system may further comprise a tracr nucleic acid, the tracr nucleic acid being associated with the guide nucleic acid and the CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

[0263] As used herein, a "CRISPR-Cas effector protein associated with a guide nucleic acid" refers to a complex formed between the CRISPR-Cas effector protein and the guide nucleic acid to direct the CRISPR-Cas effector protein to a target site within a gene.

[0264] In some embodiments, a gene editing system is further provided, which comprises a CRISPR-Cas effector protein, the CRISPR-Cas effector protein is associated with a guide nucleic acid, and the guide nucleic acid comprises a spacer sequence that binds to an HD-Zip gene, wherein the HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with any one of the nucleotide sequences of SEQ ID NO: 69, 70, 88 or 89; (b) comprises a region consisting of consecutive nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105, optionally having at least 80% sequence identity with any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 and / or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region consisting of consecutive amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, the spacer sequence of the guide nucleic acid can comprise the nucleotide sequence of any one of SEQ ID NO: 109-112. In some embodiments, the gene editing system can further comprise a tracr nucleic acid, the tracr nucleic acid is associated with the guide nucleic acid and the CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

[0265] In some embodiments, the guide nucleic acid of the gene editing system can comprise a spacer sequence that is complementary to a region, portion, or segment of a nucleotide sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity) with any one of the nucleotide sequences of SEQ ID NO: 69, 70, 88, and / or 89 (e.g., SEQ ID NOs: 72-85 and / or 91-105, optionally any one of SEQ ID NOs: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102, and / or 103-105), or can encode a region, portion, or segment of a sequence having at least 80% sequence identity with any one of the amino acid sequences of SEQ ID NO: 71 or SEQ ID NO: 90, optionally wherein the sequence identity with any one of SEQ ID NO: 69, 70, 72-85, 88, 89, and / or 91-105 can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, the gene editing system can further comprise a tracr nucleic acid that associates with the guide nucleic acid and the CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

[0266] In some embodiments, a complex is provided that comprises a guide nucleic acid and a CRISPR-Cas effector protein, the CRISPR-Cas effector protein comprising a cleavage domain, wherein the guide nucleic acid binds to a target site within an endogenous HD-Zip gene, wherein the endogenous HD-Zip gene: (a) comprises a sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprises a region having at least 80% sequence identity with any one of the nucleotide sequences of any one of SEQ ID NO: 72-85 or 91-105, optionally any one of SEQ ID NO: 72-75, 76-79, 80-83, 84-85, 91-94, 95-98, 99-102 or 103-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encodes a region having at least 80% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%, and the cleavage domain cleaves the target strand in the HD-Zip gene. In some embodiments, the cleavage domain cleaves the target strand in the HD-Zip gene, thereby creating a mutation in the endogenous HD-Zip gene that comprises a sequence having at least 90% identity with any one of the mutated HD-Zip nucleic acids described herein. In some embodiments, the mutation in the endogenous HD-Zip gene is a non-natural mutation.

[0267] In some embodiments, an expression cassette is provided that comprises (a) a polynucleotide encoding a CRISPR-Cas effector protein that comprises a cleavage domain; and (b) a guide nucleic acid that binds to a target site within an endogenous HD-Zip gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to (i) a portion of a nucleic acid encoding an amino acid sequence having at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:90; (ii) a portion of a nucleic acid encoding an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO:86, 87, 106, 107 or 108; (iii) a portion of a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:69, 70, 88 or 89; and / or (iii) a portion of a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:72-85 or 91-105, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) can be at least 85% or at least 90%, or it can be at least 95%, optionally the sequence identity can be 100%, optionally wherein the provided expression cassette comprises (a) a polynucleotide encoding a CRISPR-Cas effector protein that comprises a cleavage domain; and (b) a guide nucleic acid that binds to a target site within an endogenous HD-Zip gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to a portion of a nucleic acid having at least 80% sequence identity to any one of SEQ ID NO:69, 70, 72-85, 88, 89 and / or 91-105.

[0268] The present disclosure also provides a nucleic acid that encodes a mutated HD-Zip gene, which, when present in a plant (or a part thereof), produces a plant having a phenotype including an increase in the number of seeds (e.g., the number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching, as compared to a control plant or a plant part lacking the mutation. In some embodiments, the mutated HD-Zip gene comprises a nucleic acid encoding a dominant negative mutation. In some embodiments, the mutated HD-Zip gene may comprise a sequence having at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99%, or 100% sequence identity, optionally the sequence identity may be at least 95%, optionally the sequence identity may be 100%) with SEQ ID NO: 113 and / or may encode a mutated HD-Zip polypeptide having at least 90% sequence identity with SEQ ID NO: 115. Also provided is a modified HD-Zip polypeptide comprising the modified amino acid sequence of SEQ ID NO: 115.

[0269] The nucleic acid constructs of the present invention (e.g., constructs comprising a sequence-specific nucleic acid binding domain, a CRISPR-Cas effector domain, a deaminase domain, a reverse transcriptase (RT), an RT template, and / or a guide nucleic acid, etc.) and expression cassettes / vectors comprising the nucleic acid constructs can be used as an editing system of the present invention for modifying a target nucleic acid (e.g., an endogenous HD-Zip gene) and / or its expression.

[0270] Any plant comprising an endogenous HD-Zip gene that, when modified as described herein (e.g., mutated, e.g., base edited, cleaved, nicked, etc.), is capable of conferring one or more improved yield traits such as an increase in the number of seeds (e.g., the number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching, as compared to a control plant or a plant part lacking the mutation, can be used with the present invention to generate the mutated HD-Zip gene of the present invention (e.g., using the polypeptide, polynucleotide, RNP, nucleic acid construct, expression cassette, and / or vector of the present invention) to provide one or more improved yield traits in the plant.

[0271] In some embodiments, a plant or a plant part thereof is provided, which comprises at least one mutation in at least one endogenous homeodomain-leucine zipper transcription factor (HD-Zip) gene having Gene Identification Number (SoyBase database) Glyma.20g014400 (HD-Zip17-1) or Glyma.07g218000 (HD-Zip17-2), wherein the mutated endogenous HD-Zip gene comprises a nucleic acid sequence having at least 90% sequence identity with any one of the mutated HD-Zip nucleic acid sequences described herein, optionally wherein the at least one mutation is a non-natural mutation.

[0272] The editing system that can be used in the present invention can be any site-specific (sequence-specific) genome editing system known now or developed later, and the system can introduce mutations in a target-specific manner. For example, the editing system (e.g., site-specific or sequence-specific editing system) can include, but is not limited to, CRISPR-Cas editing system, meganuclease editing system, zinc finger nuclease (ZFN) editing system, transcription activator-like effector nuclease (TALEN) editing system, base editing system, and / or prime editing system, each of which can comprise one or more polypeptides and / or one or more polynucleotides that can modify (mutate) a target nucleic acid in a sequence-specific manner when expressed as a system in a cell. In some embodiments, the editing system (e.g., site-specific or sequence-specific editing system) can comprise one or more polynucleotides and / or one or more polypeptides, including but not limited to a nucleic acid binding domain (DNA binding domain), a nuclease, and / or other polypeptides and / or polynucleotides.

[0273] In some embodiments, an editing system can comprise one or more sequence-specific nucleic acid binding domains (DNA binding domains), which can be from, for example, polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or Argonaute proteins. In some embodiments, an editing system can comprise one or more cleavage domains (e.g., nucleases), including but not limited to endonucleases (e.g., Fok1), polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, and / or transcription activator-like effector nucleases (TALENs). In some embodiments, an editing system can comprise one or more polypeptides, including but not limited to deaminases (e.g., cytosine deaminase, adenine deaminase), reverse transcriptase, Dna2 polypeptide, and / or 5'-flap endonuclease (FEN). In some embodiments, an editing system can comprise one or more polynucleotides, including but not limited to CRISPR arrays (CRISPR guides) nucleic acids, extended guide nucleic acids, and / or reverse transcriptase templates.

[0274] In some embodiments, a method of modifying or editing an HD-Zip gene can comprise contacting a target nucleic acid (e.g., a nucleic acid encoding an HD-Zip polypeptide) with a base editing fusion protein (e.g., a sequence-specific nucleic acid binding protein, a sequence-specific DNA binding protein (e.g., a CRISPR-Cas effector protein or domain)) fused to a deaminase domain (e.g., adenine deaminase and / or cytosine deaminase) and a guide nucleic acid, wherein the guide nucleic acid is capable of guiding / targeting the base editing fusion protein to the target nucleic acid, thereby editing a locus within the target nucleic acid. In some embodiments, the base editing fusion protein and the guide nucleic acid can be comprised in one or more expression cassettes. In some embodiments, the target nucleic acid can be contacted with the base editing fusion protein and an expression cassette comprising the guide nucleic acid. In some embodiments, the sequence-specific nucleic acid binding fusion protein and the guide sequence can be provided as ribonucleoprotein (RNP). In some embodiments, a cell can be contacted with more than one base editing fusion protein and / or one or more guide nucleic acids, which can target one or more target nucleic acids in the cell.

[0275] In some embodiments, a method of modifying or editing an HD-Zip gene can comprise contacting a target nucleic acid (e.g., a nucleic acid encoding an HD-Zip polypeptide) with a sequence-specific nucleic acid-binding fusion protein fused to a peptide tag (e.g., a sequence-specific DNA-binding protein (e.g., a CRISPR-Cas effector protein or domain)), a deaminase fusion protein comprising a deaminase domain (e.g., an adenine deaminase and / or a cytosine deaminase) fused to an affinity polypeptide capable of binding to the peptide tag, and a guide nucleic acid, wherein the guide nucleic acid is capable of guiding / targeting the sequence-specific nucleic acid-binding fusion protein to the target nucleic acid, and the sequence-specific nucleic acid-binding fusion protein is capable of recruiting the deaminase fusion protein to the target nucleic acid via peptide tag-affinity polypeptide interaction, thereby editing a locus within the target nucleic acid. In some embodiments, the sequence-specific nucleic acid-binding fusion protein can be fused to an affinity polypeptide that binds to the peptide tag, and the deaminase can be fused to the peptide tag, thereby recruiting the deaminase to the sequence-specific nucleic acid-binding fusion protein and the target nucleic acid. In some embodiments, the sequence-specific binding fusion protein, the deaminase fusion protein, and the guide nucleic acid can be comprised in one or more expression cassettes. In some embodiments, the target nucleic acid can be contacted with the sequence-specific binding fusion protein, the deaminase fusion protein, and an expression cassette comprising the guide nucleic acid. In some embodiments, the sequence-specific nucleic acid-binding fusion protein, the deaminase fusion protein, and the guide sequence can be provided as ribonucleoprotein (RNP).

[0276] In some embodiments, methods such as prime editing can be used to generate mutations in an endogenous HD-Zip gene. In prime editing, an RNA-dependent DNA polymerase (reverse transcriptase, RT) and a reverse transcriptase template (RT template) are used in combination with a sequence-specific nucleic acid-binding domain that confers the ability to recognize and bind to a target in a sequence-specific manner and also to introduce a nick in the PAM-containing strand within the target. The nucleic acid-binding domain can be a CRISPR-Cas effector protein, and in this case, the CRISPR array or guide RNA can be an extended guide sequence that comprises an extension portion that contains a primer binding site (PSB) and the edit to be incorporated into the genome (template). Similar to base editing, prime editing can utilize various methods to recruit proteins for target site editing, such methods including non-covalent and covalent interactions between proteins and nucleic acids used in the selected genome editing process.

[0277] In some embodiments, the sequence-specific nucleic acid binding domain (sequence-specific DNA binding domain) that can be used in the editing system of the present invention can be derived from, for example, polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or Argonaute proteins.

[0278] In some embodiments, the sequence-specific nucleic acid binding domain (e.g., sequence-specific DNA binding domain) can be a CRISPR-Cas effector protein. In some embodiments, the CRISPR-Cas effector protein can be derived from a type I CRISPR-Cas system, a type II CRISPR-Cas system, a type III CRISPR-Cas system, a type IV CRISPR-Cas system, a type V CRISPR-Cas system, or a type VI CRISPR-Cas system. In some embodiments, the CRISPR-Cas effector protein of the present invention can be derived from a type II CRISPR-Cas system or a type V CRISPR-Cas system. In some embodiments, the CRISPR-Cas effector protein can be a type II CRISPR-Cas effector protein, such as a Cas9 effector protein. In some embodiments, the CRISPR-Cas effector protein can be a type V CRISPR-Cas effector protein, such as a Cas12 effector protein.

[0279] As used herein, "CRISPR-Cas effector protein" is a protein or polypeptide or a domain thereof that cleaves or cuts nucleic acids, binds nucleic acids (e.g., target nucleic acids and / or guide nucleic acids), and / or discriminates, identifies, or binds a guide nucleic acid as defined herein. In some embodiments, the CRISPR-Cas effector protein can be an enzyme (e.g., nuclease, endonuclease, nickase, etc.) or a portion thereof and / or can act as an enzyme. In some embodiments, the CRISPR-Cas effector protein refers to a CRISPR-Cas nuclease polypeptide or a domain thereof, the CRISPR-Cas nuclease polypeptide or a domain thereof containing nuclease activity or wherein the nuclease activity has been reduced or eliminated, and / or containing nickase activity or wherein the nickase activity has been reduced or eliminated, and / or containing single-stranded DNA cleavage activity (ssDNA enzyme activity) or wherein the ssDNA enzyme activity has been reduced or eliminated, and / or containing self-processing RNA enzyme activity or wherein the self-processing RNA enzyme activity has been reduced or eliminated. The CRISPR-Cas effector protein can bind to a target nucleic acid.

[0280] In some embodiments, the CRISPR-Cas effector protein can include, but is not limited to, Cas9, C2c1, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Casl, CaslB, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and / or Csf5 nuclease, optionally wherein the CRISPR-Cas effector protein can be a Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b, and / or Cas14c effector protein.

[0281] In some embodiments, the CRISPR-Cas effector protein useful in the present invention can contain mutations in its nuclease active site (e.g., RuvC, HNH, e.g., the RuvC site of the Cas12a nuclease domain, e.g., the RuvC site and / or HNH site of the Cas9 nuclease domain). The CRISPR-Cas effector protein having a mutation in its nuclease active site and thus no longer containing nuclease activity is generally referred to as "dead", e.g., dCas. In some embodiments, the CRISPR-Cas effector protein domain or polypeptide having a mutation in its nuclease active site can have impaired activity or reduced activity compared to the same CRISPR-Cas effector protein (e.g., nickase, e.g., Cas9 nickase, Cas12a nickase) without such mutation.

[0282] The CRISPR Cas9 effector protein or CRISPR Cas9 effector domain useful in the present invention can be any known or later identified Cas9 nuclease. In some embodiments, the CRISPR Cas9 polypeptide can be a Cas9 polypeptide from, for example, Streptococcus spp. (e.g., S. pyogenes, S. thermophilus), Lactobacillus spp., Bifidobacterium spp., Kandleria spp., Leuconostoc spp., Oenococcus spp., Pediococcus spp., Weissella spp., and / or Olsenella spp. Exemplary Cas9 sequences include, but are not limited to, the amino acid sequences of SEQ ID NO:56 and SEQ ID NO:57 or the nucleotide sequences of SEQ ID NO:58-68.

[0283] In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus pyogenes and recognize the PAM sequence motifs NGG, NAG, NGA (Mali et al., Science 2013; 339(6121):823-826). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus thermophilus and recognize the PAM sequence motifs NGGNG and / or NNAGAAW (W = A or T) (see, e.g., Horvath et al., Science, 2010; 327(5962):167-170, and Deveau et al., J Bacteriol 2008; 190(4):1390-1400). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus mutans and recognize the PAM sequence motifs NGG and / or NAAR (R = A or G) (see, e.g., Deveau et al., J BACTERIOL 2008; 190(4):1390-1400). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus aureus and recognize the PAM sequence motif NNGRR (R = A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 protein derived from S. aureus that recognizes the PAM sequence motif N GRRT (R = A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus aureus that recognizes the PAM sequence motif NGRRV (R = A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Neisseria meningitidis and recognize the PAM sequence motifs NGATT or NGCTT (R = A or G, V = A, G or C) (see, e.g., Hou et al., PNAS 2013, 1-6). In the above embodiments, N can be any nucleotide residue, such as any one of A, G, C or T. In some embodiments, the CRISPR-Cas effector protein can be a Cas13a protein derived from Leptotrichia shahii that recognizes a protospacer flanking sequence (PFS) (or RNA PAM (rPAM)) sequence motif of a single 3'A, U or C that can be located within the target nucleic acid).

[0284] In some embodiments, the CRISPR-Cas effector protein can be derived from Cas12a, which is a type V clustered regularly interspaced short palindromic repeat (CRISPR)-Cas nuclease (see, e.g., the amino acid sequences of SEQ ID NO: 1-17, the nucleic acid sequences of SEQ ID NO: 18-20). Cas12a differs from the better-known type II CRISPR Cas9 nuclease in several respects. For example, Cas9 recognizes a guanine-rich protospacer adjacent motif (PAM) (3'-NGG) located 3' of its guide RNA (gRNA, sgRNA, crRNA, crDNA, CRISPR array) binding site (protospacer, target nucleic acid, target DNA), while Cas12a recognizes a thymine-rich PAM (5'-TTN, 5'-TTTN) located 5' of the target nucleic acid. In fact, the orientation of Cas9 and Cas12a binding to their guide RNAs is almost opposite with respect to their N and C termini. In addition, the Cas12a enzyme uses a single guide RNA (gRNA, CRISPR array, crRNA), rather than the dual guide RNA (sgRNA (e.g., crRNA and tracrRNA)) found in the native Cas9 system, and Cas12a processes its own gRNA. In addition, Cas12a nuclease activity produces staggered DNA double-strand breaks, rather than blunt ends produced by Cas9 nuclease activity, and Cas12a relies on a single RuvC domain to cleave both DNA strands, while Cas9 utilizes an HNH domain and an RuvC domain to cleave.

[0285] The CRISPR Cas12a effector protein / domain useful in the present invention can be any known or later identified Cas12a polypeptide (formerly known as Cpf1) (see, e.g., U.S. Patent No. 9,790,490, the disclosure of which regarding Cpf1 (Cas12a) sequences is incorporated herein by reference). The terms "Cas12a", "Cas12a polypeptide" or "Cas12a domain" refer to an RNA-guided nuclease comprising a Cas12a polypeptide or a fragment thereof, the fragment comprising the guide nucleic acid binding domain of Cas12a and / or the active, inactive or partially active DNA cleavage domain of Cas12a. In some embodiments, the Cas12a useful in the present invention can comprise a mutation in the nuclease active site (e.g., the RuvC site of the Cas12a domain). A Cas12a domain or Cas12a polypeptide having a mutation in its nuclease active site and thus no longer comprising nuclease activity is generally referred to as dead Cas12a (e.g., dCas12a). In some embodiments, a Cas12a domain or Cas12a polypeptide having a mutation in its nuclease active site can have impaired activity, e.g., can have nickase activity.

[0286] Any deaminase domain / polypeptide that can be used for base editing can be used in the present invention. In some embodiments, the deaminase domain can be a cytosine deaminase domain or an adenine deaminase domain. The cytosine deaminase (or cytidine deaminase) that can be used in the present invention can be any known or later identified cytosine deaminase from any organism (see, for example, U.S. Patent No. 10,167,457 and Thuronyi et al., Nat. Biotechnol. 37:1070–1079 (2019), the disclosures of their respective cytosine deaminases are incorporated herein by reference). The cytosine deaminase can catalyze the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. Thus, in some embodiments, the deaminase or deaminase domain that can be used in the present invention can be a cytidine deaminase domain that catalyzes the hydrolytic deamination of cytosine to uracil. In some embodiments, the cytosine deaminase can be a variant of a naturally occurring cytosine deaminase, including but not limited to primates (e.g., human, monkey, chimpanzee, gorilla), dog, cow, rat, or mouse. Thus, in some embodiments, the cytosine deaminase that can be used in the present invention can be about 70% to about 100% identical to the wild-type cytosine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the naturally occurring cytosine deaminase, as well as any range or value therein).

[0287] In some embodiments, the cytosine deaminase useful in the present invention may be a deaminase of the apolipoprotein B mRNA editing complex (APOBEC) family. In some embodiments, the cytosine deaminase may be APOBEC1 deaminase, APOBEC2 deaminase, APOBEC3A deaminase, APOBEC3B deaminase, APOBEC3C deaminase, APOBEC3D deaminase, APOBEC3F deaminase, APOBEC3G deaminase, APOBEC3H deaminase, APOBEC4 deaminase, human activation-induced deaminase (hAID), rAPOBEC1, FERNY, and / or CDA1, optionally pmCDA1, atCDA1 (e.g., At2g19570), and their evolved forms (e.g., SEQ ID NO:27, SEQ ID NO:28, or SEQ ID NO:29). In some embodiments, the cytosine deaminase may be APOBEC1 deaminase having the amino acid sequence of SEQ ID NO:23. In some embodiments, the cytosine deaminase may be APOBEC3A deaminase having the amino acid sequence of SEQ ID NO:24. In some embodiments, the cytosine deaminase may be CDA1 deaminase, optionally CDA1 having the amino acid sequence of SEQ ID NO:25. In some embodiments, the cytosine deaminase may be FERNY deaminase, optionally FERNY having the amino acid sequence of SEQ ID NO:26. In some embodiments, the cytosine deaminase useful in the present invention may be about 70% to about 100% identical to the amino acid sequence of a naturally occurring cytosine deaminase (e.g., an evolved deaminase) (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical).In some embodiments, the cytosine deaminase that can be used in the present invention may be about 70% to about 99.5% identical to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26 (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical) (e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29). In some embodiments, the polynucleotide encoding the cytosine deaminase may be codon-optimized for expression in plants, and the codon-optimized polypeptide may be about 70% to 99.5% identical to the reference polynucleotide.

[0288] In some embodiments, the nucleic acid constructs of the invention may further encode a uracil glycosylase inhibitor (UGI) (e.g., uracil-DNA glycosylase inhibitor) polypeptide / domain. Thus, in some embodiments, a nucleic acid construct encoding a CRISPR-Cas effector protein and a cytosine deaminase domain (e.g., encoding a fusion protein comprising a CRISPR-Cas effector protein domain fused to a cytosine deaminase domain, and / or a CRISPR-Cas effector protein domain fused to a peptide tag or an affinity polypeptide capable of binding a peptide tag, and / or a deaminase protein domain fused to a peptide tag or an affinity polypeptide capable of binding a peptide tag) may further encode a uracil-DNA glycosylase inhibitor (UGI), optionally wherein the UGI may be codon-optimized for expression in plants. In some embodiments, the invention provides a fusion protein comprising a CRISPR-Cas effector polypeptide, a deaminase domain, and a UGI, and / or one or more polynucleotides encoding the same, optionally wherein the one or more polynucleotides may be codon-optimized for expression in plants. In some embodiments, the invention provides a fusion protein wherein the CRISPR-Cas effector polypeptide, the deaminase domain, and the UGI may be fused to any combination of peptide tags and affinity polypeptides as described herein, thereby recruiting the deaminase domain and the UGI to the CRISPR-Cas effector polypeptide and the target nucleic acid. In some embodiments, the guide nucleic acid may be linked to a recruitment RNA motif, and one or more of the deaminase domain and / or the UGI may be fused to an affinity polypeptide capable of interacting with the recruitment RNA motif, thereby recruiting the deaminase domain and the UGI to the target nucleic acid.

[0289] The "uracil glycosylase inhibitor" useful in the present invention can be any protein capable of inhibiting the base excision repair enzyme uracil-DNA glycosylase. In some embodiments, the UGI domain comprises wild-type UGI or a fragment thereof. In some embodiments, the UGI domain useful in the present invention may be about 70% to about 100% identical to the amino acid sequence of a naturally occurring UGI domain (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical, and any range or value therein). In some embodiments, the UGI domain may comprise the amino acid sequence of SEQ ID NO: 41 or a polypeptide having about 70% to about 99.5% sequence identity with the amino acid sequence of SEQ ID NO: 41 (e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to the amino acid sequence of SEQ ID NO: 41). For example, in some embodiments, the UGI domain may comprise a fragment that is 100% identical to a portion of the amino acid sequence of SEQ ID NO: 41 consisting of consecutive nucleotides (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides; e.g., about 10, 15, 20, 25, 30, 35, 40, 45 to about 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides) of the amino acid sequence of SEQ ID NO: 41. In some embodiments, the UGI domain may be a variant of a known UGI (e.g., SEQ ID NO: 41) having about 70% to about 99.5% sequence identity with the known UGI (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity, and any range or value therein). In some embodiments, the polynucleotide encoding UGI may be codon-optimized for expression in a plant (e.g., a plant), and the codon-optimized polypeptide may be about 70% to about 99.5% identical to the reference polynucleotide.

[0290] The adenine deaminase (or adenosine deaminase) useful in the present invention can be any known or later identified adenine deaminase from any organism (see, e.g., U.S. Patent No. 10,113,163, the disclosure of which regarding adenine deaminase is incorporated herein by reference). Adenine deaminase can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, adenine deaminase can catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, adenosine deaminase can catalyze the hydrolytic deamination of adenine or adenosine in DNA. In some embodiments, the adenine deaminase encoded by the nucleic acid construct of the present invention can result in an A→G conversion in the sense (e.g., "+", template) strand of the target nucleic acid or a T→C conversion in the antisense (e.g., "-", complementary) strand of the target nucleic acid.

[0291] In some embodiments, the adenosine deaminase can be a variant of a naturally occurring adenine deaminase. Thus, in some embodiments, the adenosine deaminase can be about 70% to 100% identical to the wild-type adenine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to the naturally occurring adenine deaminase, and any ranges or values therein). In some embodiments, the deaminase or deaminase is not naturally occurring and can be referred to as an engineered, mutated or evolved adenosine deaminase. Thus, for example, an engineered, mutated or evolved adenine deaminase polypeptide or adenine deaminase domain can be about 70% to 99.9% identical to the naturally occurring adenine deaminase polypeptide / domain (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical to the naturally occurring adenine deaminase polypeptide or adenine deaminase domain, and any ranges or values therein). In some embodiments, the adenosine deaminase can be from bacteria (e.g., Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, Caulobacter crescentus, etc.). In some embodiments, the polynucleotide encoding the adenine deaminase polypeptide / domain can be codon-optimized for expression in plants.

[0292] In some embodiments, the adenine deaminase domain can be a wild-type tRNA-specific adenosine deaminase domain, such as tRNA-specific adenosine deaminase (TadA), and / or a mutated / evolved adenosine deaminase domain, such as a mutated / evolved tRNA-specific adenosine deaminase domain (TadA*). In some embodiments, the TadA domain can be from Escherichia coli (E. coli). In some embodiments, TadA can be modified, such as truncated, and / or one or more N-terminal and / or C-terminal amino acids may be lost relative to full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N-terminal and / or C-terminal amino acid residues may be lost relative to full-length TadA). In some embodiments, the TadA polypeptide or TadA domain does not contain an N-terminal methionine. In some embodiments, wild-type E. coli TadA contains the amino acid sequence of SEQ ID NO:30. In some embodiments, the mutated / evolved E. coli TadA* contains the amino acid sequence of SEQ ID NOs: 31-40 (e.g., SEQ ID NO:31, 32, 33, 34, 35, 36, 37, 38, 39, or 40). In some embodiments, the polynucleotide encoding TadA / TadA* can be codon-optimized for expression in plants.

[0293] Cytosine deaminase catalyzes the deamination of cytosine and produces thymidine (through a uracil intermediate), causing a C to T conversion in the complementary strand of the genome or a G to A conversion. Thus, in some embodiments, the cytosine deaminase encoded by the polynucleotide of the present invention produces a C→T conversion in the sense (e.g., "+", template) strand of the target nucleic acid or a G→A conversion in the antisense (e.g., "-", complementary) strand of the target nucleic acid.

[0294] In some embodiments, the adenine deaminase encoded by the nucleic acid construct of the present invention produces an A→G conversion in the sense (e.g., "+", template) strand of the target nucleic acid or a T→C conversion in the antisense (e.g., "-", complementary) strand of the target nucleic acid.

[0295] The nucleic acid constructs of the present invention encoding base editors comprising a sequence-specific nucleic acid binding protein and a cytosine deaminase polypeptide, and nucleic acid constructs / expression cassettes / vectors encoding the same, can be used in combination with a guide nucleic acid for modifying a target nucleic acid, including but not limited to generating C→T or G→A mutations in the target nucleic acid (including but not limited to plasmid sequences); generating C→T or G→A mutations in a coding sequence to alter amino acid identity; generating C→T or G→A mutations in a coding sequence to generate a stop codon; generating C→T or G→A mutations in a coding sequence to disrupt a start codon; generating point mutations in genomic DNA to disrupt function; and / or generating point mutations in genomic DNA to disrupt a splice junction.

[0296] The nucleic acid constructs of the present invention encoding base editors comprising a sequence-specific nucleic acid binding protein and an adenine deaminase polypeptide, and expression cassettes and / or vectors encoding the nucleic acid constructs, can be used in combination with a guide nucleic acid for modifying a target nucleic acid, including but not limited to generating A→G or T→C mutations in the target nucleic acid (including but not limited to plasmid sequences); generating A→G or T→C mutations in a coding sequence to alter amino acid identity; generating A→G or T→C mutations in a coding sequence to generate a stop codon; generating A→G or T→C mutations in a coding sequence to disrupt a start codon; generating point mutations in genomic DNA to disrupt function; and / or generating point mutations in genomic DNA to disrupt a splice junction.

[0297] The nucleic acid constructs of the present invention comprising a CRISPR-Cas effector protein or a fusion protein thereof can be used in combination with a guide RNA (gRNA, CRISPR array, CRISPR RNA, crRNA), which is designed to function together with the encoded CRISPR-Cas effector protein or domain. The guide nucleic acid useful in the present invention comprises at least one spacer sequence and at least one repeat sequence. The guide nucleic acid is capable of forming a complex with the CRISPR-Cas nuclease domain encoded and expressed by the nucleic acid constructs of the present invention, and the spacer sequence is capable of hybridizing to the target nucleic acid, thereby guiding the complex (e.g., a CRISPR-Cas effector fusion protein (e.g., a CRISPR-Cas effector domain fused to a deaminase domain and / or a CRISPR-Cas effector domain fused to a peptide tag or an affinity polypeptide to recruit the deaminase domain and optionally UGI)) to the target nucleic acid, wherein the target nucleic acid can be modified (e.g., cleaved or edited) or regulated (e.g., transcriptionally regulated) by the deaminase domain.

[0298] As an example, a nucleic acid construct encoding a Cas9 domain (e.g., a fusion protein) linked to a cytosine deaminase domain can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the cytosine deaminase domain of the fusion protein deaminates a cytosine base in the target nucleic acid, thereby editing the target nucleic acid. In another example, a nucleic acid construct encoding a Cas9 domain (e.g., a fusion protein) linked to an adenine deaminase domain can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the adenine deaminase domain of the fusion protein deaminates an adenosine base in the target nucleic acid, thereby editing the target nucleic acid.

[0299] Similarly, a nucleic acid construct encoding a Cas12a domain (or other selected CRISPR-Cas nuclease, e.g., C2c1, C2c3, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5) linked to a cytosine deaminase domain or an adenine deaminase domain (e.g., a fusion protein) can be used in combination with a Cas12a guide nucleic acid (or a guide nucleic acid of other selected CRISPR-Cas nuclease) to modify a target nucleic acid, wherein the cytosine deaminase domain or the adenine deaminase domain of the fusion protein deaminates a cytosine base in the target nucleic acid, thereby editing the target nucleic acid.

[0300] As used herein, "guide nucleic acid", "guide RNA", "gRNA", "CRISPR RNA / DNA", "crRNA", or "crDNA" means a nucleic acid comprising at least one spacer sequence and at least one repeat sequence (e.g., a repeat sequence of a type V Cas12a CRISPR-Cas system or a fragment or portion thereof; a repeat sequence of a type II Cas9 CRISPR-Cas system or a fragment thereof; a repeat sequence of a type V C2c1 CRISPR Cas system or a fragment thereof; e.g., a repeat sequence of a CRISPR-Cas system of C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG), and / or Csf5 or a fragment thereof), wherein the at least one spacer sequence is complementary (and hybridizes) to a target DNA (e.g., a protospacer), and wherein the repeat sequence may be linked to the 5'-end and / or 3'-end of the spacer sequence. The design of the gRNA of the present invention can be based on type I, type II, type III, type IV, type V, or type VI CRISPR-Cas systems.

[0301] In some embodiments, a Cas12a gRNA can comprise, from 5' to 3', a repeat sequence (full-length or a portion thereof ("stem"); e.g., a pseudoknot-like structure) and a spacer sequence.

[0302] In some embodiments, a guide nucleic acid can comprise more than one repeat sequence-spacer sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeat sequence-spacer sequences) (e.g., repeat sequence-spacer-repeat sequence, e.g., repeat sequence-spacer-repeat sequence-spacer-repeat sequence-spacer-repeat sequence-spacer-repeat sequence-spacer-repeat sequence-spacer, etc.). The guide nucleic acid of the present invention is synthetic, artificial and does not exist in nature. The gRNA can be long and can be used as an aptamer (as in the MS2 recruitment strategy) or other RNA structures with hanging spacers.

[0303] As used herein, "repeat sequence" refers to any repeat sequence of, for example, a wild-type CRISPR Cas locus (e.g., Cas9 locus, Cas12a locus, C2c1 locus, etc.) or a repeat sequence of a synthetic crRNA that functions with a CRISPR-Cas effector protein encoded by a nucleic acid construct of the present invention. The repeat sequences useful in the present invention can be any known or later identified repeat sequences of a CRISPR-Cas locus (e.g., type I, type II, type III, type IV, type V, or type VI), or it can be a synthetic repeat sequence designed to function in a type I, II, III, IV, V, or VI CRISPR-Cas system. The repeat sequence can comprise a hairpin structure and / or a stem-loop structure. In some embodiments, the repeat sequence can form a pseudoknot-like structure (i.e., "handle") at its 5' end. Thus, in some embodiments, the repeat sequence can be the same as or substantially the same as a repeat sequence from a wild-type type I CRISPR-Cas locus, type II CRISPR-Cas locus, type III CRISPR-Cas locus, type IV CRISPR-Cas locus, type V CRISPR-Cas locus, and / or type VI CRISPR-Cas locus. The repeat sequences from wild-type CRISPR-Cas loci can be determined by established algorithms, such as using CRISPRfinder provided by CRISPRdb (see, Grissa et al., Nucleic Acids Res. 35(Web Server issue):W52-7). In some embodiments, the repeat sequence or a portion thereof is linked to the 5' end of a spacer sequence at its 3' end, thereby forming a repeat sequence-spacer sequence (e.g., guide nucleic acid, guide RNA / DNA, crRNA, crDNA).

[0304] In some embodiments, the repeat sequence comprises at least 10 nucleotides, consists essentially of, or consists of, depending on the particular repeat sequence and whether the guide nucleic acid comprising the repeat sequence is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides, or any range or value therein). In some embodiments, the repeat sequence comprises about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100 or more nucleotides, consists essentially of, or consists of.

[0305] The repeat sequence linked to the 5' end of the spacer sequence can comprise a portion of the repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive nucleotides of the wild-type repeat sequence). In some embodiments, the length of a portion of the repeat sequence linked to the 5' end of the spacer sequence can be about five to about ten consecutive nucleotides (e.g., about 5, 6, 7, 8, 9, 10 nucleotides) and have at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater (e.g., 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%)) with the same region (e.g., the 5' end) of the wild-type CRISPR Cas repeat nucleotide sequence. In some embodiments, a portion of the repeat sequence can comprise a pseudoknot-like structure (e.g., a "stem") at its 5' end.

[0306] As used herein, an "spacer sequence" is a nucleotide sequence that is substantially complementary to a target nucleic acid (e.g., target DNA) (e.g., a protospacer), such as being substantially complementary to consecutive nucleotides of a portion / region of an HD-Zip nucleotide sequence, where the portion or region (a) has at least 80% sequence identity (e.g., at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) with the nucleotide sequence of any one of SEQ ID NO: 69, 70, 88 or 89; (b) contains a region having at least 80% sequence identity with any one nucleotide sequence of the nucleotide sequences of SEQ ID NO: 72 - 85 or 91 - 105; (c) encodes an amino acid sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90, and / or (d) encodes a polypeptide containing a region consisting of consecutive amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108, optionally where the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or it can be at least 95%, and optionally the sequence identity can be 100%. In some embodiments, the spacer sequence can include, but is not limited to, the nucleotide sequence of any one of SEQ ID NO: 109 - 112 or its reverse complement or any combination thereof. The spacer sequence can be completely complementary or substantially complementary to the target nucleic acid (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%))). Thus, in some embodiments, compared to the target nucleic acid, the spacer sequence can have one, two, three, four or five mismatches, which can be consecutive or non - consecutive. In some embodiments, the spacer sequence can be 70% complementary to the target nucleic acid. In other embodiments, the spacer nucleotide sequence can be 80% complementary to the target nucleic acid. In still other embodiments, the spacer nucleotide sequence can be 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementary to the target nucleic acid (protospacer), etc. In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid.The length of the spacer subsequence can be from about 15 nucleotides to about 30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides, or any range or value therein). Thus, in some embodiments, the spacer subsequence can have perfect complementarity or substantial complementarity (e.g., at least 70% complementarity) over a region of the target nucleic acid (e.g., the protospacer) that is at least about 15 nucleotides to about 30 nucleotides in length. In some embodiments, the length of the spacer is about 20 nucleotides. In some embodiments, the length of the spacer is about 21, 22, or 23 nucleotides.

[0307] In some embodiments, the 5' region of the spacer subsequence of the guide nucleic acid can be identical to the target DNA, while the 3' region of the spacer can be substantially complementary to the target DNA (see, e.g., the spacer subsequence of type V CRISPR-Cas systems), or the 3' region of the spacer subsequence of the guide nucleic acid can be identical to the target DNA, while the 5' region of the spacer can be substantially complementary to the target DNA (see, e.g., the spacer subsequence of type II CRISPR-Cas systems), and thus the overall complementarity of the spacer subsequence to the target DNA can be less than 100%. Thus, for example, in the guide sequence of a type V CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 5' region (i.e., the seed region) of a 20-nucleotide spacer subsequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer subsequence are substantially complementary to the target DNA (e.g., at least about 70% complementary). In some embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8 nucleotides, and any range therein) at the 5' end of the spacer subsequence are 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer subsequence are substantially complementary to the target DNA (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)).

[0308] As another example, in the guide sequence of a type II CRISPR-Cas system, for example, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 3' region (i.e., the seed region) of a 20-nucleotide spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary to the target DNA (e.g., at least about 70% complementary). In some embodiments, the first 1 to 10 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides and any range therein) at the 3' end of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary to the target DNA (e.g., at least about 50% complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or any range or value therein)).

[0309] In some embodiments, the length of the seed region of the spacer can be about 8 to about 10 nucleotides, the length can be about 5 to about 6 nucleotides, or the length can be about 6 nucleotides.

[0310] As used herein, "target nucleic acid", "target DNA", "target nucleotide sequence", "target region", or "target region in the genome" refers to a region of a plant genome that is completely complementary (100% complementary) or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)) to the spacer sequence in the guide nucleic acid of the present invention. The target region that can be used in the CRISPR-Cas system can be located immediately adjacent to the 3' (e.g., such as for type V CRISPR-Cas systems) or 5' (e.g., such as for type II CRISPR-Cas systems) of the PAM sequence in the genome of an organism (e.g., a plant genome). The target region can be selected from any region composed of at least 15 consecutive nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, etc.) located immediately adjacent to the PAM sequence.

[0311] "Protospacer" refers to a target double-stranded DNA, specifically, a portion of the target DNA (e.g., or a target region in a genome) that is fully or substantially complementary (and hybridizes) to the spacer sequence of a CRISPR repeat-spacer sequence (e.g., a guide nucleic acid, a CRISPR array, a crRNA).

[0312] In the case of type V CRISPR-Cas (e.g., Cas12a) systems and type II CRISPR-Cas (Cas9) systems, the protospacer is flanked by (e.g., immediately adjacent to) a protospacer adjacent motif (PAM). For type IV CRISPR-Cas systems, the PAM is located at the 5' end of the non-target strand and the 3' end of the target strand (see below for examples).

[0313]

[0314] In the case of type II CRISPR-Cas (e.g., Cas9) systems, the PAM is located immediately adjacent to the 3' of the target region. The PAM of type I CRISPR-Cas systems is located at the 5' of the target strand. There is no known PAM for type III CRISPR-Cas systems. The nomenclature for all classes, types, and subtypes of CRISPR systems was described by Makarova et al. (Nature Reviews Microbiology 13:722–736 (2015)). The guide structure and PAM were described by R. Barrangou (Genome Biol. 16:247 (2015)).

[0315] Typical Cas12a PAMs are rich in T. In some embodiments, a typical Cas12a PAM sequence can be 5'-TTN, 5'-TTTN, or 5'-TTTV. In some embodiments, a typical Cas9 (e.g., Streptococcus pyogenes) PAM can be 5'-NGG-3'. In some embodiments, non-typical PAMs can be used, but the efficiency may be lower.

[0316] Those skilled in the art can determine additional PAM sequences through established experimental and computational methods. Thus, for example, experimental methods include targeting sequences flanking all possible nucleotide sequences and identifying sequence members that do not undergo targeting, such as by transformation of target plasmid DNA (Esvelt et al., 2013. Nat. Methods 10:1116 - 1121; Jiang et al., 2013. Nat. Biotechnol. 31:233 - 239). In some aspects, computational methods can include performing BLAST searches on native spacers to identify the original target DNA sequences in phages or plasmids and aligning these sequences to determine conserved sequences adjacent to the target sequences (Briner and Barrangou, 2014. Appl. Environ. Microbiol. 80:994 - 1001; Mojica et al., 2009. Microbiology 155:733 - 740).

[0317] In some embodiments, the present invention provides an expression cassette and / or vector comprising the nucleic acid construct of the present invention (e.g., one or more components of the editing system of the present invention). In some embodiments, an expression cassette and / or vector comprising the nucleic acid construct of the present invention and / or one or more guide nucleic acids can be provided. In some embodiments, the nucleic acid construct of the present invention encoding a base editor (e.g., a construct comprising a CRISPR - Cas effector protein and a deaminase domain (e.g., a fusion protein)) or a component for base editing (e.g., a CRISPR - Cas effector protein fused to a peptide tag or an affinity polypeptide, a deaminase domain fused to a peptide tag or an affinity polypeptide, and / or UGI fused to a peptide tag or an affinity polypeptide) can be comprised on the same or a separate expression cassette or vector as an expression cassette or vector comprising one or more guide nucleic acids. When the nucleic acid construct encoding the base editor or the component for base editing is comprised on an expression cassette or vector separate from the expression cassette or vector comprising the guide nucleic acid, the target nucleic acid can contact (e.g., be provided together with) the expression cassette or vector encoding the base editor or the component for base editing in any order with respect to each other and the guide nucleic acid, e.g., before, simultaneously with, or after providing the expression cassette comprising the guide nucleic acid (e.g., in contact with the target nucleic acid).

[0318] The fusion proteins of the present invention can comprise a sequence - specific nucleic acid - binding domain, a CRISPR - Cas polypeptide, and / or a deaminase domain fused to a peptide tag known in the art or an affinity polypeptide that interacts with a peptide tag for recruiting the deaminase to the target nucleic acid. The recruitment method can also comprise a guide nucleic acid linked to an RNA recruitment motif and a deaminase fused to an affinity polypeptide capable of interacting with the RNA recruitment motif, thereby recruiting the deaminase to the target nucleic acid. Alternatively, chemical interactions can be used to recruit a polypeptide (e.g., a deaminase) to the target nucleic acid.

[0319] Peptide tags (e.g., epitopes) that can be used in the present invention can include, but are not limited to, GCN4 peptide tags (e.g., Sun-Tag), c-Myc affinity tags, HA affinity tags, His affinity tags, S affinity tags, methionine-His affinity tags, RGD-His affinity tags, octapeptides, strep tags or strep tag II, V5 tags, and / or VSV-G epitopes. Any epitope that can be linked to a polypeptide and for which there is a corresponding affinity polypeptide that can be linked to another polypeptide can be used as a peptide tag in the present invention. In some embodiments, the peptide tag can comprise 1 or 2 or more copies of the peptide tag (e.g., repeat units, multimerized epitopes (e.g., tandem repeats)) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more repeat units). In some embodiments, the affinity polypeptide that interacts / binds to the peptide tag can be an antibody. In some embodiments, the antibody can be a scFv antibody. In some embodiments, the affinity polypeptide that binds to the peptide tag can be synthetic (e.g., evolved for an affinity interaction), including but not limited to affibodies, anticalins, monobodies, and / or DARPins (see, e.g., Sha et al., Protein Sci. 26(5):910-924 (2017)); Gilbreth (Curr Opin Struc Biol 22(4):413-420 (2013)); U.S. Patent No. 9,982,053, each of which is incorporated by reference in its entirety for the teachings regarding affibodies, anticalins, monobodies, and / or DARPins). Exemplary peptide tag sequences and their affinity polypeptides include, but are not limited to, the amino acid sequences of SEQ ID NOs: 42-44.

[0320] In some embodiments, the guide nucleic acid can be linked to an RNA recruitment motif, and the polypeptide to be recruited (e.g., a deaminase) can be fused to an affinity polypeptide that binds to the RNA recruitment motif, wherein the guide sequence binds to the target nucleic acid and the RNA recruitment motif binds to the affinity polypeptide, thereby recruiting the polypeptide to the guide sequence and bringing the target nucleic acid into contact with the polypeptide (e.g., a deaminase). In some embodiments, two or more polypeptides can be recruited to the guide nucleic acid, thereby bringing the target nucleic acid into contact with two or more polypeptides (e.g., deaminases). Exemplary RNA recruitment motifs and their affinity polypeptides include, but are not limited to, the sequences of SEQ ID NOs: 45-55.

[0321] In some embodiments, the polypeptide fused to the affinity polypeptide can be a reverse transcriptase, and the guide nucleic acid can be an extended guide nucleic acid linked to an RNA recruitment motif. In some embodiments, the RNA recruitment motif can be located at the 3' end of the extended portion of the extended guide nucleic acid (e.g., 5'-3', repeat-spacer-extension (RT template-prime binding site)-RNA recruitment motif). In some embodiments, the RNA recruitment motif can be embedded within the extended portion.

[0322] In some embodiments of the invention, the extended guide RNA and / or the guide RNA can be linked to one or two or more RNA recruitment motifs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more motifs; e.g., at least 10 to about 25 motifs), optionally where two or more of the RNA recruitment motifs can be the same RNA recruitment motif or different RNA recruitment motifs. In some embodiments, the RNA recruitment motif and the corresponding affinity polypeptide can include but are not limited to the telomerase Ku-binding motif (e.g., Ku-binding hairpin) and the corresponding affinity polypeptide Ku (e.g., Ku heterodimer), the telomerase Sm7-binding motif and the corresponding affinity polypeptide Sm7, the MS2 bacteriophage operator stem-loop and the corresponding affinity polypeptide MS2 coat protein (MCP), the PP7 bacteriophage operator stem-loop and the corresponding affinity polypeptide PP7 coat protein (PCP), the SfMu bacteriophage Com stem-loop and the corresponding affinity polypeptide Com RNA-binding protein, the PUF-binding site (PBS) and the affinity polypeptide Pumilio / fem-3 mRNA-binding factor (PUF), and / or a synthetic RNA aptamer and aptamer ligand as the corresponding affinity polypeptide. In some embodiments, the RNA recruitment motif and the corresponding affinity polypeptide can be the MS2 bacteriophage operator stem-loop and the affinity polypeptide MS2 coat protein (MCP). In some embodiments, the RNA recruitment motif and the corresponding affinity polypeptide can be the PUF-binding site (PBS) and the affinity polypeptide Pumilio / fem-3 mRNA-binding factor (PUF).

[0323] In some embodiments, the components for recruiting polypeptides and nucleic acids can be those that act through chemical interactions, which can include but are not limited to rapamycin-induced FRB-FKBP dimerization; biotin-streptavidin; SNAP tag; Halo tag; CLIP tag; compound-induced DmrA-DmrC heterodimerization; bifunctional ligands (e.g., two protein-binding chemicals fused together; e.g., dihydrofolate reductase (DHFR).

[0324] In some embodiments, the nucleic acid constructs, expression cassettes or vectors of the invention optimized for expression in plants may be about 70% to 100% identical to nucleic acid constructs, expression cassettes or vectors comprising the same polynucleotide but not codon-optimized for expression in plants (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100%).

[0325] Also provided herein are cells comprising one or more polynucleotides, guide nucleic acids, nucleic acid constructs, expression cassettes or vectors of the invention. The target nucleic acids of any plant or plant part (or groupings of plant parts such as genera or higher taxonomic groups) can be modified (e.g., mutated, e.g., base edited, cleaved, nicked, etc.) using the polypeptides, polynucleotides, ribonucleoprotein (RNP), nucleic acid constructs, expression cassettes and / or vectors of the invention, including angiosperms, gymnosperms, monocots, dicots, C3, C4, CAM plants, bryophytes, ferns and / or fern allies, microalgae and / or macroalgae. The plants and / or plant parts that can be modified as described herein can be plants and / or plant parts of any plant species / variety / cultivar. In some embodiments, the plants that can be modified as described herein are monocots. In some embodiments, the plants that can be modified as described herein are dicots.

[0326] As used herein, the term "plant part" includes, but is not limited to, reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower buds, ovules, seeds and embryos); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, culms, seedlings, branches, bark, apical meristems, axillary buds, cotyledons, hypocotyls and leaves); vascular tissues (e.g., phloem and xylem); specialized cells such as epidermal cells, parenchyma cells, collenchyma cells, sclerenchyma cells, stomata, guard cells, cuticle, mesophyll cells; callus; and cuttings. The term "plant part" also includes plant cells, including intact plant cells in plants and / or plant parts, plant protoplasts, plant tissues, plant organs, plant cell cultures, plant callus, plant clumps, etc. As used herein, "seedling" refers to the above-ground part, including leaves and stems. As used herein, the term "tissue culture" encompasses cultures of tissues, cells, protoplasts and callus.

[0327] As used herein, "plant cell" refers to the structural and physiological unit of a plant, which typically includes a cell wall, but also includes protoplasts. The plant cells of the present invention can be in the form of isolated single cells, or can be cultured cells, or can be part of higher tissue units such as, for example, plant tissues (including callus) or plant organs. "Protoplast" is an isolated plant cell without a cell wall or with only a partial cell wall. Thus, in some embodiments of the present invention, the transgenic cells containing the nucleic acid molecules and / or nucleotide sequences of the present invention are cells of any plant or plant part, including but not limited to root cells, leaf cells, tissue culture cells, seed cells, flower cells, fruit cells, pollen cells, etc. In some aspects of the present invention, the plant part can be plant germplasm. In some aspects, the plant cells can be non-reproductive plant cells that no longer generate plants.

[0328] "Plant cell culture" refers to the culture of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various developmental stages.

[0329] As used herein, "plant organ" is a distinct and clearly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo.

[0330] As used herein, "plant tissue" refers to a group of plant cells organized into a structural and functional unit. It includes any plant tissue in situ or in culture. The term includes but is not limited to the whole plant, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into a structural and / or functional unit. The term is not intended to exclude any other type of plant tissue when used in combination with or without any specific type of plant tissue listed above or included in this definition.

[0331] In some embodiments of the present invention, transgenic tissue cultures or transgenic plant cell cultures are provided, wherein the transgenic tissue or cell culture contains the nucleic acid molecule / nucleotide sequence of the present invention. In some embodiments, the transgene can be eliminated from the plants developed from the transgenic tissue or cells by crossing the transgenic plant with a non-transgenic plant and selecting in the progeny the plants that contain the desired gene edit but not the transgene used to generate the edit.

[0332] Any plant containing an endogenous homeodomain-leucine zipper transcription factor (HD-Zip) gene can be modified as described herein to improve one or more yield traits. Non-limiting examples of plants that can be modified as described herein can include, but are not limited to, turfgrasses (e.g., Poa, Agrostis, Lolium, Festuca), feather reed grass, tufted hairgrass, Miscanthus, Arundo donax, switchgrass, vegetable crops, including artichoke, kohlrabi, arugula, leek, asparagus, lettuce (e.g., iceberg lettuce, leaf lettuce, romaine lettuce), taro, melons (e.g., cantaloupe, watermelon, crenshaw melon, honeydew melon, casaba melon), Brassica crops (e.g., brussels sprouts, cabbage, cauliflower, broccoli, kale, collard greens, Chinese cabbage, pak choi), cardoon, carrot, bok choy, okra, onion, celery, parsley, chickpea, parsnip, chicory, pepper, potato, cuc...

Claims

1. A plant or a part thereof, wherein the plant or the part thereof comprises at least one mutation in an endogenous homeodomain-leucine zipper transcription factor (HD-Zip) gene, the gene encoding an HD-Zip transcription factor (HD-Zip) polypeptide, wherein the mutation alters the function of the HD-Zip polypeptide as a gene expression regulator.

2. The plant or the part thereof according to claim 1, wherein the mutation is in the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif of the HD-Zip gene.

3. The plant or the part thereof according to claim 1 or claim 2, wherein the HD-Zip gene is an HD-Zip II gene.

4. The plant or the part thereof according to any one of the preceding claims, wherein the HD-Zip II gene is the HD-Zip17-1 gene and / or the HD-Zip17-2 gene.

5. The plant or the part thereof according to any one of the preceding claims, wherein the HD-Zip gene has a gene identification number (SoyBase Database) Glyma.20g014400 (HD-Zip17-1) or Glyma.07g218000 (HD-Zip17-2).

6. The plant or the part thereof according to any one of the preceding claims, wherein at least one mutation gives rise to a dominant negative allele.

7. The plant or the part thereof according to any one of the preceding claims, wherein the endogenous HD-Zip gene: (a) comprises a nucleotide sequence having at least 80% sequence identity with any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprises a region of contiguous nucleotides having at least 80% identity with any one of SEQ ID NO: 72-85 or 91-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108.

8. The plant or the part thereof according to any one of the preceding claims, wherein at least one mutation is a base substitution, a base deletion and / or a base insertion.

9. The plant or the part thereof according to any one of the preceding claims, wherein at least one mutation comprises a base substitution of A, T, G or C.

10. The plant or the part thereof according to any one of the preceding claims, wherein at least one mutation is a substitution of at least one base pair.

11. The plant or the part thereof according to any one of claims 1 to 8, wherein the at least one mutation in the endogenous HD-Zip gene is a base deletion, optionally a deletion of one or more base pairs, optionally a deletion of about one base pair to about 100 base pairs.

12. The plant or a part thereof according to claim 8 or claim 11, wherein the base deletion is an in-frame deletion.

13. The plant or a part thereof according to any one of claims 1 to 8, 11 or 12, wherein the at least one mutation is a deletion of one or more nucleotides from a region of the HD-Zip gene, optionally wherein the region comprises a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 72-85 or 91-105, optionally wherein the deletion is in the region of the HD-Zip gene at nucleotide position numbers 2206 to 2220 of reference SEQ ID NO: 69 and / or at nucleotide position numbers 2179 to 2193 of reference SEQ ID NO:

88.

14. The plant or a part thereof according to claim 13, wherein the base deletion is a deletion of three or more consecutive nucleotides.

15. The plant or a part thereof according to any one of claims 8 or 11 to 14, wherein the base deletion causes a deletion of one or more amino acids from a region of an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 86, 87, 106, 107 or 108, optionally a deletion of one or more amino acid residues at positions 11 to 15 of the amino acid position numbers of reference SEQ ID NO: 71 or SEQ ID NO: 90 of the HD-Zip polypeptide, optionally a deletion of at least 1, 2, 3, 4 or 5 amino acid residues of SEQ ID NO: 71 or SEQ ID NO:

90.

16. The plant or a part thereof according to claim 15, wherein the deletion is in the EAR motif of the HD-Zip polypeptide, optionally causing a deletion of the EAR motif.

17. The plant or a part thereof according to any one of the preceding claims, wherein the plant or the part thereof comprising the at least one mutation exhibits a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes and / or reduced branching.

18. The plant or a part thereof according to one of the preceding claims, wherein the plant is a dicot.

19. The plant or a part thereof according to claims 1 to 18, wherein the plant is a monocot.

20. A plant or a part thereof according to any one of the preceding claims, wherein the plant is maize, soybean, canola, wheat, rice, cotton, sugarcane, sugar beet, barley, oats, alfalfa, sunflower, safflower, oil palm, sesame, coconut, tobacco, potato, sweet potato, cassava, coffee, apple, plum, apricot, peach, cherry, pear, fig, banana, citrus, cocoa, avocado, olive, almond, walnut, strawberry, watermelon, pepper, grape, tomato, cucumber, blackberry, raspberry, black raspberry or Brassica spp.

21. A plant or a part thereof according to any one of claims 1 to 18, wherein the plant is soybean.

22. A plant or a part thereof according to any one of the preceding claims, wherein the at least one mutation is a non-natural mutation.

23. A plant or a part thereof according to any one of the preceding claims, wherein the at least one mutation gives rise to a mutated HD-Zip gene that has at least 90% sequence identity with SEQ ID NO: 113 and / or encodes a mutated HD-Zip polypeptide that has at least 90% sequence identity with SEQ ID NO:

115.

24. A plant cell comprising an editing system, the editing system comprising: (a) a CRISPR-Cas related effector protein; and (b) a guide nucleic acid (e.g., gRNA, gDNA, crRNA, crDNA) having a spacer sequence that is complementary to an endogenous target gene encoding an HD-Zip transcription factor polypeptide.

25. The plant cell according to claim 24, wherein the endogenous target gene is an HD-Zip II gene, optionally an HD-Zip17-1 gene and / or an HD-Zip17-2 gene.

26. The plant cell according to claim 24 or claim 25, wherein the endogenous target gene: (a) comprises a nucleotide sequence having at least 80% sequence identity with any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprises a region of consecutive nucleotides having at least 80% identity with any one of SEQ ID NO: 72 - 85 or 91 - 105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region of consecutive amino acid residues having at least 90% sequence identity with any one of SEQ ID NO: 86, 87, 106, 107 or 108.

27. The plant cell according to any one of claims 24 to 26, wherein the guide nucleic acid comprises the nucleotide sequence (e.g., spacer sequence) of any one of SEQ ID NO: 109 - 112.

28. The plant cell according to any one of claims 24 to 27, wherein the plant cell is a soybean plant cell.

29. A plant regenerated from a plant part according to any one of claims 1 to 23 or a plant cell according to any one of claims 24 to 28.

30. The plant according to claim 29, wherein the plant exhibits a phenotype of increased seed number (e.g., increased grain number), increased seed weight (e.g., increased grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching.

31. A plant cell comprising at least one mutation within an HD-Zip gene, wherein the at least one mutation is a base substitution, base insertion, and / or base deletion, and the base substitution, base insertion, and / or base deletion is introduced using an editing system comprising a nucleic acid binding domain that binds to a target site within the HD-Zip gene.

32. The plant cell according to claim 31, wherein the at least one mutation is a dominant negative allele.

33. The plant cell according to claim 31 or claim 32, wherein the target site is located within a region of the HD-Zip gene that comprises a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 72 - 85 or 91 - 105.

34. The plant cell according to any one of claims 31 to 33, wherein the editing system further comprises a nuclease, and the at least one mutation within the HD-Zip gene is generated after cleavage by the nuclease.

35. The plant cell according to claim 34, wherein the nuclease is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an endonuclease (e.g., Fok1), or a CRISPR-Cas effector protein.

36. The plant cell according to claim 35, wherein the nucleic acid binding domain of the editing system is derived from a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., a CRISPR-Cas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and / or an Argonaute protein.

37. The plant cell according to any one of claims 31 to 36, wherein the mutation is a deletion, optionally an in-frame deletion.

38. The plant cell according to any one of claims 31 to 37, wherein the mutation is a deletion of all or part of an ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif encoded by the endogenous HD-Zip gene.

39. The plant cell according to claim 38, wherein the HD-Zip gene is an HD-Zip II gene, optionally the HD-Zip17-1 gene and / or the HD-Zip17-2 gene.

40. The plant cell according to any one of claims 31 to 39, wherein the plant cell is a cell from maize, soybean, canola, wheat, rice, cotton, sugarcane, sugar beet, barley, oats, alfalfa, sunflower, safflower, oil palm, sesame, coconut, tobacco, potato, sweet potato, cassava, coffee, apple, plum, apricot, peach, cherry, pear, fig, banana, citrus, cocoa, avocado, olive, almond, walnut, strawberry, watermelon, pepper, grape, tomato, cucumber, blackberry, raspberry, black raspberry or Brassica, optionally wherein the plant cell is a soybean plant cell.

41. The plant cell according to any one of claims 31 to 40, wherein the mutated HD-Zip gene comprises a non-natural mutation.

42. The plant cell according to any one of claims 31 to 41, wherein the plant comprises a mutated HD-Zip gene that has at least 90% sequence identity with SEQ ID NO: 113 and / or encodes a mutated HD-Zip polypeptide that has at least 90% sequence identity with SEQ ID NO:

115.

43. A plant regenerated from the plant cell according to any one of claims 31 to 42, wherein the plant exhibits a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes and / or reduced branching.

44. A method for generating / cultivating a genome-edited plant without transgenes, the method comprising: (a) crossing a plant according to any one of claims 1 to 23, 29, 30 or 43 with a plant without transgenes, thereby introducing the mutation or modification into the plant without transgenes; and (b) selecting progeny plants that contain the mutation or modification but no transgenes, thereby generating a genome-edited plant without transgenes.

45. A method for providing a plurality of plants having a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight, 100-seed weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes and / or reduced branching, the method comprising planting two or more plants according to any one of claims 1 to 23, 29, 30 or 43 in a growing area, thereby providing a plurality of plants having a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes and / or reduced branching compared to a plurality of control plants that do not contain the mutation.

46. A method for generating a mutation in an endogenous HD-Zip gene in a plant, the method comprising: (a) Targeting a gene editing system to a region of the HD-Zip17-1 gene and / or the HD-Zip17-2 gene that contains a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 72-85 or 91-105; and (b) Selecting a plant that contains a modification in a region of the gene that has at least 80% sequence identity to any one of SEQ ID NOs: 72-85 or 91-105.

47. A method for generating a variation in an HD-Zip gene, the method comprising: Introducing an editing system into a plant cell, wherein the editing system is targeted to a region of the HD-Zip gene that encodes an HD-Zip polypeptide, and Contacting the region of the HD-Zip gene with the editing system, thereby introducing a mutation into the HD-Zip gene and generating a variation in the HD-Zip gene of the plant cell.

48. The method according to claim 47, wherein the HD-Zip gene: (a) contains a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 69, 70, 88 or 89, (b) contains a region consisting of contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 72-85 or 91-105; (c) encodes a polypeptide containing a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide containing a region consisting of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NOs: 86, 87, 106, 107 or 108.

49. The method according to claim 47 or claim 48, wherein the targeted region of the HD-Zip gene has at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 72-85 or 91-105.

50. A method according to any one of claims 47 to 49, wherein contacting the region of the endogenous HD-Zip gene in the plant cell with the editing system produces a plant cell that contains an edited endogenous HD-Zip gene in its genome, the method further comprising (a) regenerating a plant from the plant cell; (b) selfing the plant to produce progeny plants (E1); (c) determining an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching in the progeny plants of (b); and (d) selecting the progeny plants that exhibit a phenotype of an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching compared to control plants.

51. The method according to claim 50, which further comprises (e) selfing the selected progeny plants of (d) to produce progeny plants (E2); (f) determining an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching in the progeny plants of (e); and (g) selecting the progeny plants that exhibit a phenotype of an increase in the number of seeds (e.g., number of grains), an increase in seed weight (e.g., grain weight), an increase in the number of pods per plant, a change in flowering time (e.g., earlier flowering time), a shorter plant height, a decrease in the number of stem nodes, and / or a decrease in branching compared to control plants, optionally repeating (e) to (g) one or more additional times.

52. A method for detecting a mutant HD-Zip gene (a mutation in an endogenous HD-Zip gene) in a plant, the method comprising detecting in the genome of the plant an HD-Zip gene having at least one mutation in a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 72 - 85 or 91 - 105.

53. The method according to claim 52, wherein the detected mutant HD-Zip gene comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO: 113, and / or encodes a mutant HD-Zip polypeptide having at least 90% sequence identity to SEQ ID NO:

115.

54. A method for editing a specific site in the genome of a plant cell, the method comprising: cutting a target site within an endogenous HD-Zip gene in the plant cell in a site-specific manner, the endogenous HD-Zip gene: (a) comprising a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO:69, 70, 88 or 89; (b) comprising a region consisting of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO:72 - 85 or 91 - 105; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO:71 or SEQ ID NO:90; and / or (d) encoding a polypeptide comprising a region consisting of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO:86, 87, 106, 107 or 108.

55. The method according to claim 54, further comprising regenerating a plant from the plant cell comprising the edit in the endogenous HD - Zip gene to produce a plant comprising the edit in the endogenous HD - Zip gene thereof.

56. The method according to claim 54 or claim 55, wherein the plant comprising the edit in the endogenous HD - Zip gene thereof exhibits a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes and / or reduced branching.

57. The method according to any one of claims 54 to 56, wherein the edit is located in the ethylene - responsive element - binding factor - related amphiphilic repression (EAR) motif of the HD - Zip gene.

58. The method according to any one of claims 54 to 57, wherein the edit results in a deleted HD - Zip gene.

59. The method according to any one of claims 54 to 58, wherein the edit results in a dominant - negative mutant HD - Zip gene.

60. The method according to any one of claims 54 to 59, wherein the edit causes a mutation in the endogenous HD - Zip gene, and the mutation produces a functionally - altered HD - Zip polypeptide as a gene expression regulator.

61. The method according to any one of claims 54 to 60, wherein the edit produces a non - natural mutation.

62. The method according to any one of claims 54 to 61, wherein the edit produces a mutated HD - Zip gene having at least 90% sequence identity to SEQ ID NO:113 and / or encoding a mutated HD - Zip polypeptide having at least 90% sequence identity to SEQ ID NO:

115.

63. A method for preparing a plant, the method comprising: (a) Contacting a population of plant cells comprising an endogenous HD-Zip gene with a nuclease linked to a nucleic acid binding domain (e.g., an editing system), wherein the nucleic acid binding domain binds to a target site within the endogenous HD-Zip gene, and wherein the endogenous gene: (i) comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 69, 70, 88, or 89; (ii) comprises a region of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO: 72 - 85 or 91 - 105; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; and / or (iv) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107, or 108; (b) Selecting plant cells from the population of plant cells in which the endogenous HD-Zip gene has been mutated, thereby generating plant cells comprising a mutation in the endogenous HD-Zip gene; and (c) Growing the selected plant cells into a plant comprising the mutation in the endogenous HD-Zip gene.

64. A method for increasing the number of seeds (e.g., the number of grains), increasing the seed weight (e.g., the grain weight), increasing the number of pods per node, increasing the number of pods per plant, altering the flowering time (e.g., earlier flowering time), shortening the plant height, reducing the number of nodes, and / or reducing the number of branches in a plant, the method comprising (a) Contacting a plant cell comprising an endogenous HD-Zip gene with a nuclease linked to a nucleic acid binding domain (e.g., an editing system), wherein the nucleic acid binding domain binds to a target site within the endogenous HD-Zip gene, and wherein the endogenous gene: (i) comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 69, 70, 88, or 89; (ii) comprises a region of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO: 72 - 85 or 91 - 105; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; and / or (iv) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107, or 108; and (b) Growing the plant cell comprising the mutation in the endogenous HD-Zip gene into a plant, thereby producing a plant having a mutated HD-Zip gene and exhibiting a phenotype of increased seed number (e.g., number of grains), increased seed weight (e.g., weight of grains), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching.

65. A method for producing a plant or a part thereof, the plant or the part thereof comprising at least one cell having a mutated endogenous HD-Zip gene, the method comprising: Contacting a target site within the endogenous HD-Zip gene in the plant or plant part with a nuclease, the nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site within the endogenous HD-Zip gene, wherein the endogenous HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 69, 70, 88, or 89; (b) comprises a region of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO: 72 - 85 or 91 - 105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107, or 108, thereby producing a plant or a part thereof, the plant or the part thereof comprising at least one cell having a mutation in the endogenous HD-Zip gene.

66. A method for producing a plant or a part thereof, the plant or the part thereof comprising a mutated endogenous HD-Zip gene and exhibiting a phenotype of increased seed number (e.g., number of grains), increased seed weight (e.g., weight of grains), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching, the method comprising contacting a target site within the endogenous HD-Zip gene in the plant or plant part with a nuclease, the nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site within the HD-Zip gene, wherein the HD-Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO: 69, 70, 88, or 89; (b) comprises a region of contiguous nucleotides having at least 80% identity to any one of SEQ ID NO: 72 - 85 or 91 - 105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) A polypeptide encoding a region consisting of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NO: 86, 87, 106, 107, or 108, thereby generating a plant or a part thereof, wherein the plant or the part thereof contains an endogenous HD-Zip gene with a mutation and exhibits a phenotype of increased seed number (e.g., number of grains), increased seed weight (e.g., weight of grains), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching.

67. The method according to any one of claims 63 to 66, wherein the nuclease cleaves the endogenous HD-Zip gene and introduces the mutation into the ethylene response element binding factor-associated amphiphilic repression (EAR) motif of the endogenous HD-Zip gene.

68. The method according to any one of claims 63 to 67, wherein the mutation generates a dominant negative allele.

69. The method according to any one of claims 63 to 68, wherein the mutation is a base deletion, optionally an in-frame deletion.

70. The method according to any one of claims 63 to 69, wherein the mutation comprises a base substitution of A, T, G, or C.

71. The method according to any one of claims 63 to 70, wherein the mutation is a substitution of at least one base pair.

72. The method according to any one of claims 63 to 71, wherein the mutation generates a mutated HD-Zip polypeptide with an altered function as a gene expression regulator.

73. The method according to any one of claims 63 to 69 or 72, wherein the mutation is a base deletion, comprising the deletion of one or more contiguous nucleotides from a region of the HD-Zip gene, optionally wherein the region contains a nucleic acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 72 - 85 or 91 - 105, optionally wherein the deletion is within the region of the HD-Zip gene at nucleotide position numbers 2206 to 2220 of reference SEQ ID NO: 69 and / or at nucleotide position numbers 2179 to 2193 of reference SEQ ID NO:

88.

74. The method according to claim 73, wherein the base deletion is the deletion of three or more contiguous amino acid residues from a region of the HD-Zip polypeptide.

75. The method according to claim 73 or claim 74, wherein the base deletion causes one or more amino acids to be deleted from a region having at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NO: 86, 87, 106, 107 or 108, optionally one or more amino acid residues at positions 11 to 15 of the HD-Zip polypeptide numbered according to the amino acid positions of reference SEQ ID NO: 71 or SEQ ID NO: 90 are deleted, optionally at least 1, 2, 3, 4 or 5 amino acid residues of SEQ ID NO: 71 or SEQ ID NO: 90 are deleted.

76. The method according to any one of claims 63 to 75, wherein the plant or a part thereof comprising the at least one mutation exhibits a phenotype of increased seed number (e.g., number of grains), increased seed weight (e.g., weight of grains), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes and / or reduced branching compared to a control plant or a plant part thereof that does not comprise the mutation.

77. The method according to any one of claims 63 to 76, wherein the nuclease is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an endonuclease (e.g., Fok1) or a CRISPR-Cas effector protein.

78. The method according to any one of claims 63 to 77, wherein the nucleic acid binding domain is a zinc finger, a transcription activator-like DNA binding domain (TAL), an Argonaute or a CRISPR-Cas effector DNA binding domain.

79. The method according to any one of claims 63 to 78, wherein the mutation generates a mutated HD-Zip gene having at least 90% sequence identity with SEQ ID NO: 113 and / or encoding a mutated HD-Zip polypeptide having at least 90% sequence identity with SEQ ID NO:

115.

80. The method according to any one of claims 63 to 79, wherein the mutation is a non-natural mutation.

81. A plant produced by any one of the methods according to claims 63 to 80.

82. A method for modifying an endogenous HD-Zip gene in a plant or a part thereof to increase seed number, increase seed weight, increase the number of pods per stem node, increase the number of pods per plant, alter flowering time, shorten plant height, reduce the number of stem nodes and / or reduce branching in the plant or a part thereof, the method comprising modifying a target site within the endogenous HD-Zip gene in the plant or a part thereof, wherein the endogenous HD-Zip gene: (a) comprises a nucleotide sequence having at least 80% sequence identity with any one of SEQ ID NO: 69, 70, 88 or 89; (b) comprises a region consisting of contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 72 - 85 or 91 - 105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to SEQ ID NO: 71 or SEQ ID NO: 90; and / or (d) encodes a polypeptide comprising a region consisting of contiguous amino acid residues having at least 90% sequence identity to any one of SEQ ID NOs: 86, 87, 106, 107 or 108, whereby the endogenous HD - Zip gene is modified and the number of seeds, the seed weight, the number of pods per internode, the number of pods per plant, the flowering time is changed, the plant height is reduced, the number of internodes is reduced and / or the number of branches is reduced in the plant or a part thereof.

83. The method according to claim 82, wherein the target site is a region of the HD - Zip gene having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 72 - 85 and / or 91 - 105, optionally the target site is in a region comprising at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 72 - 85 and / or 91 - 105.

84. A guide nucleic acid that binds to a target site within an endogenous HD - Zip gene, the target site comprising a sequence having at least 80% identity to any one or more nucleotide sequences of the nucleotide sequence of SEQ ID NO: 72 - 85 or 91 - 105.

85. The guide nucleic acid according to claim 84, wherein the guide nucleic acid comprises a spacer having the nucleotide sequence of any one of SEQ ID NOs: 109 - 112.

86. A system comprising the guide nucleic acid according to claim 84 or claim 85 and a CRISPR - Cas effector protein, the CRISPR - Cas effector protein being associated with the guide nucleic acid.

87. The system according to claim 86, further comprising a tracr nucleic acid, the tracr nucleic acid being associated with the guide nucleic acid and the CRISPR - Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

88. A gene editing system comprising a CRISPR - Cas effector protein associated with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to an endogenous HD - Zip gene.

89. The gene editing system according to claim 88, wherein the HD - Zip gene (a) comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 69, 70, 88 or 89; (b) comprises a region consisting of contiguous nucleotides having at least 80% identity to any one of SEQ ID NOs: 72 - 85 or 91 - 105; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO:71 or SEQ ID NO:90; and / or (d) encoding a polypeptide comprising a region consisting of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO:86, 87, 106, 107 or 108.

90. The gene editing system according to claim 88 or claim 89, wherein the guide nucleic acid comprises a spacer sequence having the nucleotide sequence of any one of SEQ ID NO:109 - 112.

91. The gene editing system according to any one of claims 88 to 90, further comprising a tracr nucleic acid that associates with the guide nucleic acid and the CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

92. A complex comprising a CRISPR-Cas effector protein and a guide nucleic acid, the CRISPR-Cas effector protein comprising a cleavage domain, wherein the guide nucleic acid binds to a target site within an endogenous HD-Zip gene, (a) comprising a nucleotide sequence having at least 80% sequence identity with any one of SEQ ID NO:69, 70, 88 or 89; (b) comprising a region consisting of contiguous nucleotides having at least 80% identity with any one of SEQ ID NO:72 - 85 or 91 - 105; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity with SEQ ID NO:71 or SEQ ID NO:90; and / or (d) encoding a polypeptide comprising a region consisting of contiguous amino acid residues having at least 90% sequence identity with any one of SEQ ID NO:86, 87, 106, 107 or 108, and the cleavage domain cleaves the target strand in the HD-Zip gene.

93. An expression cassette comprising (a) a polynucleotide encoding a CRISPR-Cas effector protein, the CRISPR-Cas effector protein comprising a cleavage domain, and (b) a guide nucleic acid that binds to a target site within an endogenous HD-Zip gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (i) a portion of a nucleic acid encoding an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:71 or SEQ ID NO:90; (ii) a portion of a nucleic acid encoding an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NO:86, 87, 106, 107 or 108; (iii) a portion of a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:69, 70, 88 or 89; and / or (iii) a portion of a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:72-85 or 91-105.

94. A nucleic acid encoding an HD-Zip polypeptide having a mutated ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif, wherein the mutated EAR motif comprises a mutation that alters its function as a gene expression regulator, optionally wherein the nucleic acid encoding the HD-Zip polypeptide has the gene identification number (SoyBase database) Glyma.20g014400 (HD-Zip17-1) or Glyma.07g218000 (HD-Zip17-2).

95. The nucleic acid according to claim 94, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to SEQ ID NO:

113.

96. The nucleic acid according to claim 94 or claim 95, wherein the nucleic acid encodes an amino acid sequence having at least 90% sequence identity to SEQ ID NO:

115.

97. A modified HD-Zip polypeptide having at least 90% sequence identity to SEQ ID NO:

115.

98. A plant or a part thereof comprising the nucleic acid according to any one of claims 94 to 96 and / or the polypeptide according to claim 97, optionally wherein the plant is a soybean plant.

99. The plant or a part thereof according to claim 98, wherein the plant exhibits a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching compared to a control plant that does not comprise the HD-Zip polypeptide having a mutated ethylene-responsive element binding factor-associated amphiphilic repression (EAR) motif.

100. A soybean plant or a plant part thereof, wherein the soybean plant or the plant part thereof comprises a mutation in at least one endogenous HD-Zip gene having Gene Identification Number (SoyBase database) Glyma.20g014400 (HD-Zip17-1) or Glyma.07g218000 (HD-Zip17-2).

101. The soybean plant or a part thereof according to claim 100, wherein the soybean plant or the plant part thereof exhibits a phenotype of increased seed number (e.g., grain number), increased seed weight (e.g., grain weight), increased number of pods per plant, altered flowering time (e.g., earlier flowering time), shorter plant height, reduced number of stem nodes, and / or reduced branching as compared to a control plant that does not contain the HD-Zip polypeptide having a mutated ethylene response element binding factor-associated amphiphilic repression (EAR) motif.

102. A method of producing a plant that comprises a mutation in an endogenous HD-Zip gene and at least one polynucleotide of interest, the method comprising crossing a first plant with a second plant to produce a progeny plant, the first plant being a plant according to any one of claims 1 to 23, 29, 30, 43, 81, or 98 to 101, and the second plant comprising the at least one polynucleotide of interest; and selecting a progeny plant that comprises the mutation in the HD-Zip gene and the at least one polynucleotide of interest, thereby producing the plant that comprises a mutation in an endogenous HD-Zip gene and at least one polynucleotide of interest.

103. A method of producing a plant that comprises a mutation in an endogenous HD-Zip gene and at least one polynucleotide of interest, the method comprising introducing the at least one polynucleotide of interest into a plant according to any one of claims 1 to 23, 29, 30, 43, 81, or 98 to 101, thereby producing a plant that comprises a mutation in the HD-Zip gene and at least one polynucleotide of interest.

104. The method according to claim 102 or claim 103, wherein the polynucleotide of interest is a polynucleotide that confers herbicide tolerance, insect resistance, disease resistance, increased yield, increased nutrient use efficiency, or abiotic stress resistance.

105. A method of producing a plant that comprises a mutation in an endogenous HD-Zip gene and exhibits a phenotype of improved yield traits and / or improved plant architecture, the method comprising crossing a first plant with a second plant, the first plant being a plant according to any one of claims 1 to 23, 29, 30, 43, 81, or 98 to 101, and the second plant exhibiting a phenotype of improved yield traits and / or improved plant architecture; and Select progeny plants comprising the mutation in the HD-Zip gene and a phenotype of improved yield traits and / or improved plant architecture, thereby generating the plant, which comprises a mutation in an endogenous HD-Zip gene and exhibits a phenotype of improved yield traits and / or improved plant architecture compared to a control plant.

106. A method of controlling weeds in a container (e.g., a pot or a seed tray, etc.), a growth chamber, a greenhouse, a field, a recreational area, a lawn, or along a roadside, the method comprising applying a herbicide to one or more (multiple) plants according to any one of claims 1 to 23, 29, 30, 43, 81, or 98 to 101 growing in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn, or along a roadside, thereby controlling the weeds in the container, the growth chamber, the greenhouse, the field, the recreational area, the lawn, or along the roadside in which the one or more plants grow.

107. A method of reducing insect predation on a plant, the method comprising applying an insecticide to one or more plants according to any one of claims 1 to 23, 29, 30, 43, 81, or 98 to 101, thereby reducing insect predation on the one or more plants.

108. A method of reducing fungal diseases on a plant, the method comprising applying a fungicide to one or more plants according to any one of claims 1 to 23, 29, 30, 43, 81, or 98 to 101, thereby reducing fungal diseases on the one or more plants.

109. The method according to claim 107 or claim 108, wherein the one or more plants grow in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn, or along a roadside.

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