Methods and compositions for altering plant shading

Abstract

The present invention relates to compositions and methods for modifying an elongated hypocotyl 5 (HY5) transcription factor in a plant to inhibit shading responses. The invention also relates to plants and plant parts produced using the methods and compositions of the invention.

Description

[0001] Statement Regarding Electronic Submission of Sequence Listings

[0002] A sequence listing in XML format named 1499-102_ST26.xml and 314,170 bytes in size was generated and submitted on June 3, 2023, and the public content of the sequence listing is hereby incorporated into this specification by reference.

[0003] Priority declaration

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 355,730, filed on June 27, 2022, under 35 U.S.C. §119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0005] The present invention relates to compositions and methods for modifying the elongated hypocotyl 5 (HY5) transcription factor in plants to inhibit the shade avoidance response. The present invention also relates to plants and plant parts produced using the methods and compositions of the present invention. Background Art

[0006] The shade avoidance response (SAR) is a response to a decrease in the quality or quantity of available light (Kebrom and Brutnell, J ExpBot 58:3079-3089 (2007)), in which plants attempt to outcompete neighboring plants by growing toward resources (primarily light). Plant overcrowding can lead to shade avoidance syndrome (SAS), in which plants lack vigor and yield decreases. Shade avoidance involves the relative proportion of red light to far-red light present in the plant's environment (Ballare et al. Science, 247:329-332 (1990)). Plants absorb most of the red light available to them, but reflect far-red light, including reflecting this light onto nearby plants. When plants detect sustained far-red light in their environment, they will undergo morphological and physiological responses. These responses may include reduced branching, increased plant height, reduced leaf area, auxin redistribution, increased ethylene production, and accelerated flowering. SAS is characterized by an increase in root-to-crown ratio, increased plant height, and reduced yield per plant. In a typical monoculture crop environment, plant competition through shade avoidance is considered a wasteful survival mechanism.

[0007] New strategies for modulating shade avoidance responses in plants are needed to improve crop performance. Summary of the invention

[0008] One aspect of the present invention provides a plant or part thereof comprising at least one mutation in an endogenous gene encoding an elongated hypocotyl 5 (HY5) transcription factor, optionally wherein the mutation disrupts the binding of a constitutive photomorphogenesis 1 (COP1) polypeptide to the HY5 transcription factor in the plant or part thereof (i.e., disrupts the binding of COP1 to HY5 transcription), optionally wherein the mutation may be a non-natural mutation.

[0009] A second aspect of the present invention provides a plant cell comprising an editing system, which editing system comprises: (a) a CRISPR-Cas-associated effector protein; and (b) a guide nucleic acid (gRNA, gDNA, crRNA, crDNA) having a spacer sequence complementary to an endogenous target gene encoding an endogenous HY5 transcription factor.

[0010] A third aspect of the invention provides a plant cell comprising a mutation in an endogenous Elongating Hypocotyl 5 (HY5) gene, wherein the mutation is a substitution, insertion and / or deletion introduced into an endogenous HY5 gene encoding a HY5 polypeptide using an editing system, the editing system comprising a nucleic acid binding domain that binds to a target site within the endogenous HY5 gene, wherein the endogenous HY5 gene: (a) comprises a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, optionally wherein the mutation may be a non-natural mutation.

[0011] A fourth aspect of the invention provides a method for providing a plurality of plants having increased yield when each plant in the plurality of plants is planted in close proximity to each other, the method comprising planting two or more plants of the invention in close proximity to each other, thereby providing a plurality of plants having increased yield compared to a plurality of control plants lacking at least one mutation planted in close proximity to each other.

[0012] In a fifth aspect, a method for producing / cultivating a genome-edited plant that does not contain a transgene is provided, the method comprising: (a) crossing a plant of the present invention with a plant that does not contain a transgene, thereby introducing a mutation into the plant that does not contain a transgene; and (b) selecting offspring plants that contain the mutation but do not contain the transgene, thereby producing a genome-edited plant that does not contain a transgene.

[0013] In a sixth aspect, a method for generating a mutation in an endogenous elongating hypocotyl 5 (HY5) gene of a plant is provided, the method comprising: (a) targeting a gene editing system to a portion of the HY5 gene comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 75-105; and (b) selecting a plant comprising a modification located in a region of the HY5 gene having at least 80% sequence identity to any one of SEQ ID NOs: 75-105, optionally wherein the mutation may be a non-natural mutation.

[0014] In a seventh aspect, a method for producing a variation in an elongating hypocotyl 5 (HY5) polypeptide is provided, the method comprising: introducing an editing system into a plant cell, wherein the editing system targets a region of an endogenous HY5 gene encoding the HY5 polypeptide, and contacting the region of the endogenous HY5 gene with the editing system, thereby introducing a mutation into the endogenous HY5 gene and producing a variation in the HY5 polypeptide in the plant cell.

[0015] The eighth aspect provides a method for detecting a mutated elongated hypocotyl 5 (HY5) gene (mutation in an endogenous HY5 gene) in a plant, the method comprising detecting a HY5 gene in a plant genome, the gene having at least one mutation in a region having at least 80% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-105.

[0016] The 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 elongated hypocotyl 5 (HY5) gene in a plant cell in a site-specific manner, the endogenous HY5 gene: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, thereby producing an edit in the endogenous HY5 gene of the plant cell.

[0017] In a tenth aspect, a method for preparing a plant is provided, the method comprising: (a) contacting a plant cell population comprising an endogenous gene encoding an elongated hypocotyl 5 (HY5) polypeptide with a nuclease targeted to the endogenous gene, wherein the nuclease is linked to a nucleic acid binding domain that binds to a target site within the endogenous gene, the endogenous gene (i) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 72 or SEQ ID NO: 73; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 75-105; (iii) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (iv) encoding a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 75-105. NO:106 has a region with at least 80% sequence identity; (b) selecting plant cells from a population containing a mutation in an endogenous gene encoding a HY5 transcription factor, wherein the mutation is a base insertion or base deletion in the region of the endogenous gene of (ii), optionally wherein the mutation reduces or eliminates the ability of the HY5 transcription factor to bind to the constitutive photomorphogenesis 1 (COP1) polypeptide; and (c) growing the selected plant cells into plants containing a mutation in the endogenous gene encoding the HY5 transcription factor.

[0018] In the eleventh aspect, a method for reducing / inhibiting a shade avoidance response in a plant is provided, the method comprising: (a) contacting a plant cell comprising an endogenous Elongated Hypocotyl 5 (HY5) gene with a nuclease targeted to the endogenous gene, wherein the nuclease is linked to a nucleic acid binding domain that binds to a target site within the endogenous HY5 gene, the endogenous HY5 gene: (i) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; (iii) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (iv) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, thereby producing a plant cell comprising a mutation in the endogenous HY5 gene; and (b) growing the plant cell into a plant, thereby reducing / inhibiting a shade avoidance response in the plant.

[0019] In a twelfth aspect, a method for producing a plant or part thereof comprising at least one cell (e.g., one or more cells) having a mutation in an endogenous Elongating Hypocotyl 5 (HY5) gene, the method comprising contacting a target site within an endogenous HY5 gene in the plant or plant part with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain of the nuclease binds to the target site within the endogenous HY5 gene, wherein the endogenous HY5 gene (a) comprises a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, thereby producing a plant or part thereof comprising at least one cell having a mutation in an endogenous HY5 gene.

[0020] In a thirteenth aspect, a method for producing a plant or part thereof comprising a mutation in an elongating hypocotyl 5 (HY5) gene, the mutation producing a mutated HY5 transcription factor having reduced binding to a constitutive photomorphogenesis 1 (COP1) polypeptide, the method comprising contacting a target site within an endogenous HY5 gene in the plant or part thereof with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain of the nuclease binds to the target site within the endogenous HY5 gene, wherein the endogenous HY5 gene: (a) comprises a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encodes a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:75. The amino acid sequence of NO:106 has a region of at least 80% sequence identity, thereby producing a plant or part thereof comprising a mutation in the HY5 gene, which mutation produces a mutant HY5 transcription factor with reduced binding to COPl.

[0021] In a fourteenth aspect, a method is provided for modifying an endogenous elongated hypocotyl 5 (HY5) gene in a corn plant or part thereof to reduce / inhibit a shade avoidance response in the corn plant or part thereof, the method comprising altering a target site within an endogenous HY5 gene in a corn plant or part thereof, wherein the endogenous HY5 gene: (a) comprises a sequence having at least 80% sequence identity to a nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:74; and / or (d) encodes a region having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:106, thereby altering the endogenous HY5 gene and reducing / inhibiting a shade avoidance response in the corn plant or part thereof.

[0022] In a fifteenth aspect, a guide nucleic acid is provided that binds to a target site within an endogenous gene encoding an elongated hypocotyl 5 (HY5) polypeptide, the endogenous gene comprising a nucleotide sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106.

[0023] Another aspect provides a system comprising a guide nucleic acid of the present invention and a CRISPR-Cas effector protein associated with the guide nucleic acid.

[0024] In another aspect, a gene editing system is provided, 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 elongated hypocotyl 5 (HY5) gene.

[0025] Another aspect provides a complex comprising a CRISPR-Cas effector protein containing a cleavage domain and a guide nucleic acid, wherein the guide nucleic acid binds to a target site within an elongating hypocotyl 5 (HY5) gene, wherein the endogenous HY5 gene: (a) comprises a sequence having at least 80% sequence identity to a nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:74; and / or (d) encodes a region having at least 80% sequence identity to an amino acid sequence of SEQ ID NO:106, wherein the cleavage domain cleaves the target strand in the HY5 gene.

[0026] In another aspect, an expression cassette is provided, comprising (a) a polynucleotide encoding a CRISPR-Cas effector protein comprising a cleavage domain and (b) a guide nucleic acid that binds to a target site within an endogenous elongating hypocotyl 5 (HY5) gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (i) a portion of a nucleic acid having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (ii) a portion of a nucleic acid having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; (iii) 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:74; and / or (iv) a portion of a nucleic acid sequence encoding an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106.

[0027] Another aspect provides a mutant endogenous elongated hypocotyl 5 (HY5) gene encoding a HY5 transcription factor having a mutant COPl binding domain, optionally wherein the one or more mutant HY5 genes comprise a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139, optionally wherein the sequence identity may be at least 95% or the sequence identity may be 100%, and / or encodes a mutant HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138, optionally wherein the sequence identity may be at least 95% or the sequence identity may be 100%.

[0028] In another aspect, a corn plant or plant part thereof comprises at least one mutation in an endogenous elongated hypocotyl 5 (HY5) gene having a gene identification number (Gene ID) of Zm00001d015743 (SEQ ID NO: 72), optionally wherein the mutation is a non-natural mutation.

[0029] Yet another aspect of the present invention provides a guide nucleic acid that binds to a target nucleic acid within the endogenous Elongated Hypocotyl 5 (HY5) gene having a gene identification number (Gene ID) of Zm00001d015743 (Maize Genetics and Genome Database (Maize GDB)).

[0030] Also provided are plants, plant cells and plant parts produced by the methods of the present invention and comprising one or more than one mutated elongated hypocotyl 5 (HY5) gene in their genome; and polypeptides, polynucleotides, nucleic acid constructs, expression cassettes and vectors for preparing the plants, plant cells and / or plant parts of the present invention.

[0031] These and other aspects of the invention will be described in more detail in the following description of the invention.

[0032] A brief description of the sequence

[0033] SEQ ID NOs: 1-17 are exemplary Cas12a amino acid sequences that can be used in the present invention.

[0034] SEQ ID NOs: 18-20 are exemplary Cas12a nucleotide sequences that can be used in the present invention.

[0035] SEQ ID NOs: 21-22 are exemplary regulatory sequences encoding promoters and introns.

[0036] SEQ ID NOs: 23-29 are exemplary cytosine deaminase sequences that can be used in the present invention.

[0037] SEQ ID NOs: 30-40 are exemplary adenine deaminase amino acid sequences that can be used in the present invention.

[0038] SEQ ID NO:41 is an exemplary uracil-DNA glycosylase inhibitor (UGI) sequence that can be used in the present invention.

[0039] SEQ ID NOs:42-44 provide examples of the locations of the protospacer-adjacent motifs of type V CRISPR-Cas12a nucleases.

[0040] SEQ ID NOs: 45-47 provide example peptide tags and affinity polypeptides that can be used in the present invention.

[0041] SEQ ID NOs: 48-58 provide example RNA recruitment motifs and corresponding affinity polypeptides that can be used in the present invention.

[0042] SEQ ID NOs:59-60 are exemplary Cas9 polypeptide sequences that can be used in the present invention.

[0043] SEQ ID NOs:61-71 are exemplary Cas9 polynucleotide sequences that can be used in the present invention.

[0044] SEQ ID NO:72 is an exemplary HY5 genomic sequence.

[0045] SEQ ID NO:73 is an exemplary HY5 coding (cds) sequence.

[0046] SEQ ID NO:74 is an exemplary HY5 transcription factor polypeptide sequence.

[0047] SEQ ID NOs:75-105 are exemplary nucleic acid sequences (regions) from HY5 polynucleotides.

[0048] SEQ ID NO: 106 is an exemplary region of a HY5 transcription factor polypeptide.

[0049] SEQ ID NOs: 107-113 are exemplary spacer sequences that can be used in nucleic acid guide sequences of the present invention.

[0050] SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and 139 are exemplary edited HY5 nucleic acid sequences generated as described herein.

[0051] SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 and 138 are exemplary mutant HY5 polypeptides produced from the edited nucleic acid sequences of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 and 139.

[0052] SEQ ID NOs: 141, 143, 144, 146, 147 and 149 are consecutive nucleotide portions deleted from SEQ ID NOs: 123, 125, 129, 133, 135 and 137, respectively.

[0053] SEQ ID NOs: 142, 145, 148 and 150 are a portion of the consecutive amino acid residues deleted from the mutant HY5 polypeptides of SEQ ID NOs: 124, 130, 136 and 138, respectively. DETAILED DESCRIPTION

[0054] The present invention will now be described hereinafter with reference to the following examples, in which embodiments of the present invention are shown. This specific embodiment is not intended to be a detailed catalog of all different ways in which the present invention can be implemented or all features that can be added to the present invention. For example, the features shown in one embodiment may be incorporated into other embodiments, and the features shown in a specific embodiment may be deleted from the embodiment. Therefore, the present invention contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted. In addition, it will be apparent to those skilled in the art, according to the present disclosure, that many changes and additions to the various embodiments proposed herein will not deviate from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to describe in detail all its permutations, combinations and variations.

[0055] 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 the 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 be limiting of the present invention.

[0056] All publications, patent applications, patents, and other references cited herein are incorporated by reference in their entirety for the teachings relevant to the sentence and / or paragraph in which the reference is presented.

[0057] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein may be used in any combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted. For illustration, if the specification states that a composition comprises components A, B, and C, it is specifically intended that any one of A, B, or C or a combination thereof may be omitted and abandoned, either individually or in any combination.

[0058] As used in the description of the invention and the appended claims, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0059] 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 lack of a combination when interpreted in the alternative ("or").

[0060] As used herein, the term "about" when referring to a measurable value such as an amount or concentration, is intended to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value as well as the specified value. For example, "about X," where X is a measurable value, is intended to include X as well as variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. The ranges of measurable values ​​provided herein may include any other ranges and / or individual values ​​therein.

[0061] As used herein, phrases such as "between X and Y" and "between about X and Y" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" mean "between about X and about Y," and phrases such as "from about X to Y" mean "from about X to about Y."

[0062] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. For example, if a range of 10 to 15 is disclosed, 11, 12, 13, and 14 are also disclosed.

[0063] As used herein, the terms “comprising,” “including,” and “having” specify the presence of 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.

[0064] As used herein, the transitional phrase "consisting essentially of" means that the scope of the claim should be interpreted to encompass the specified materials or steps recited in the claim as well as those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. Therefore, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as being equivalent to "comprising".

[0065] As used herein, the terms "increase," "increasing," "increased," "enhance," "enhanced," "enhancing," and "enhancement" (and grammatical variations thereof) describe an increase of at least about 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to a control.

[0066] As used herein, the terms "reduce", "reduced", "reducing", "reduction", "decrease" and "lower" (and grammatical variations thereof) describe, for example, a decrease 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% compared to a control. In some embodiments, the reduction can result in no or substantially no (i.e., a negligible amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0067] As used herein, the term "expression" ("express", "expresses", "expressed" or "expression"), etc., with respect to nucleic acid molecules and / or nucleotide sequences (e.g., RNA or DNA) means that the nucleic acid molecules and / or nucleotide sequences are transcribed and optionally translated. Thus, nucleic acid molecules and / or nucleotide sequences can express a polypeptide of interest or, for example, a functional non-translated RNA.

[0068] As used herein, the term "heterologous" refers to a nucleotide / polypeptide that originates from a foreign species or, if from the same species, has been substantially modified from its native form in composition and / or genomic locus by deliberate human intervention. 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.

[0069] A "native" 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. Thus, for example, a "wild-type mRNA" is an mRNA that occurs naturally in or is endogenous to a reference organism.

[0070] As used herein, the term "heterozygous" refers to a genetic state in which different alleles are located at corresponding loci on homologous chromosomes.

[0071] As used herein, the term "homozygous" refers to a genetic state in which identical alleles are located at corresponding loci on homologous chromosomes.

[0072] As used herein, the term "allele" refers to one of two or more different nucleotides or nucleotide sequences occurring at a particular locus.

[0073] A "null allele" is a non-functional allele caused by a gene mutation that results in no production of the corresponding protein or production of a non-functional protein.

[0074] A "knockout mutation" is a mutation that results in a non-functional protein, but which may have a detectable transcript or protein.

[0075] A "recessive mutation" is a genetic mutation that produces a phenotype when homozygous, but the phenotype is not observable when the locus is heterozygous.

[0076] A "dominant mutation" is a gene mutation that produces a mutant phenotype in the presence of a non-mutated 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-of-function or weak gain-of-function.

[0077] A "dominant negative mutation" is a mutation that produces an altered gene product (e.g., one that has an abnormal function relative to the wild type) that adversely affects the function of the wild type allele or gene product. For example, a "dominant negative mutation" can block the function of a wild type gene product. A dominant negative mutation may also be referred to as a "reversal allele mutation."

[0078] 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.

[0079] A "weak loss-of-function mutation" is a mutation that results in a gene product that has partial function or reduced function (partial inactivation) compared to the wild-type gene product.

[0080] A "hypofunctional allele mutation" is a mutation that results in a partial loss of gene function, which may occur through reduced expression (e.g., reduced protein and / or reduced RNA) or reduced functional performance (e.g., reduced activity), but not a complete loss of function / activity. A "hypofunctional" allele is a semi-functional allele caused by a gene mutation that results in the production of a corresponding protein that functions at anywhere between 1% and 99% of normal efficiency.

[0081] A "hyperallelic mutation" is a mutation that results in increased expression of a gene product and / or increased activity of a gene product.

[0082] A "gain of function" allele or mutation is a mutation that confers a new function to an encoded gene product and / or confers a new gene expression pattern. In some embodiments, a gain of function mutation may be dominant or semi-dominant.

[0083] As used herein, "non-natural mutation" refers to a mutation that is produced by human intervention and is different from a naturally occurring mutation found in the same gene (eg, occurs naturally and is not the result of modification by humans).

[0084] A "locus" is the location on a chromosome where a gene or marker or allele is located. In some embodiments, a locus may encompass one or more nucleotides.

[0085] 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, a desired allele may be associated with an increase or decrease (relative to a control) in a given trait, depending on the nature of the desired phenotype. In some embodiments of the invention, the phrase "desired allele," "target allele," or "allele of interest" refers to an allele associated with an increase in yield in a plant under non-water stress conditions relative to a control plant that does not have one or more target alleles.

[0086] A marker is "associated with" a trait when it is linked to the marker and when the presence of the marker is an indication of whether and / or to what extent the desired trait or trait form occurs in the plant / germplasm containing the marker. Similarly, a marker is "associated with" an allele or chromosomal interval when it is linked to the allele or chromosomal interval and when the presence of the marker is an indication of whether the allele or chromosomal interval is present in the plant / germplasm containing the marker.

[0087] As used herein, the terms "backcross" and "backcrossing" refer to the process of backcrossing a progeny plant to one of its parents one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8 times, etc.). In a backcrossing scheme, the "donor" parent refers to the parent plant that has the desired gene or locus to be introgressed. The "recipient" parent (used once or more) or the "recurrent" parent (used two or more times) refers to the parent plant into which the gene or locus is introgressed. For example, see Ragot, M. et al., Marker-assisted Backcrossing: A Practical Example, published in T. ECHNI QUES ETUTILISATIONSDES M AR QUEURS MOLECULAIRES LES COLLOQUES, Vol. 72, pp. 45-56 (1995); and Openshaw et al., Marker-assisted Selection in Backcross Breeding, 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.

[0088] As used herein, the term "cross" or "crossed" refers to the production of offspring (e.g., cells, seeds, or plants) by the fusion of gametes through pollination. The term encompasses sexual crossing (pollination of one plant to another) and selfing (self-pollination, e.g., when pollen and ovules are from the same plant). The term "crossing" refers to the act of fusion of gametes through pollination to produce offspring.

[0089] As used herein, the term "introgression" ("introgression", "introgressing" and "introgressed") refers to the natural and artificial transmission of a desired allele or combination of desired alleles of one or more genetic loci from one genetic background to another genetic background. For example, the desired allele at a given locus can be passed to at least one offspring by sexual hybridization between two parents of the same species, wherein at least one parent has the desired allele in its genome. Alternatively, for example, the transmission of the allele can occur by recombination between two donor genomes, such as in fused protoplasts, wherein at least one donor protoplast has the desired allele in its genome. The desired allele can be a selected allele of a marker, QTL, transgenic, etc. The offspring comprising the desired allele can be backcrossed one or more times (e.g., 1, 2, 3, 4 or more times) with a strain having a desired genetic background, selecting the desired allele, with the result that the desired allele is fixed 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 possesses a genetic marker associated with increased yield under non-water stress conditions in the recurrent parent background.

[0090] A "genetic map" is a description of the genetic linkage relationships between loci on one or more chromosomes within a given species, usually depicted in a graphical or tabular form. For each genetic map, the distances between loci are measured by the frequencies 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 polymorphic potential of each marker between different populations. The order and genetic distance between loci can vary from genetic map to genetic map.

[0091] As used herein, the term "genotype" refers to the genetic constitution of an individual (or individual population) at one or more genetic loci, in contrast to observable and / or detectable and / or phenotype traits (phenotypes). Genotype is defined by the alleles of one or more known loci inherited by an individual from its parents. The term genotype can be used to refer to the genetic constitution of an individual at a single locus, a plurality of loci, or more generally, the term genotype can be used to refer to the genetic constitution of all genes in an individual genome. Genotype can be, for example, indirectly characterized using a mark and / or directly characterized by nucleic acid sequencing.

[0092] As used herein, the term "germplasm" refers to the genetic material of an individual (e.g., plant), a group of individuals (e.g., plant strains, varieties or families), or clones derived from strains, varieties, species or cultures, or from an individual (e.g., plant), a group of individuals (e.g., plant strains, varieties or families), or clones derived from strains, varieties, species or cultures. Germplasm can be a part of an organism or cell or can be separated from an organism or cell. In general, germplasm provides genetic material with a specific genetic composition, providing a basis for 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 new plants, and plant parts (e.g., leaves, stems, buds, roots, pollen, cells, etc.) that can be cultured into complete plants.

[0093] As used herein, the terms "cultivar" and "variety" refer to a group of similar plants distinguishable from other varieties within the same species by structural or genetic characteristics and / or performance.

[0094] As used herein, the terms "exotic", "exotic strains" and "exotic germplasm" refer to any plant, strain or germplasm that is not superior. Generally speaking, the exotic plant / germplasm is not derived from any known superior plant or germplasm, but is selected to introduce one or more desired genetic elements into a breeding program (e.g., introducing new alleles into a breeding program).

[0095] 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 or varieties or species, including but not limited to a cross between two inbred lines.

[0096] As used herein, the term "inbred" refers to a substantially homozygous plant or variety. 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 for a portion of the genome of particular interest.

[0097] A "haplotype" is the genotype of an individual at multiple genetic loci, i.e., a combination of alleles. Typically, the genetic loci that define a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term "haplotype" may refer to a polymorphism at a specific locus, such as a single marker locus, or a polymorphism at multiple loci along a chromosome segment.

[0098] As used herein, "shade avoidance response" is defined as the growth of a plant in response to a low red light: far-red light (R:FR light) ratio. Inhibition of the shade avoidance response refers to the inhibition of growth changes in response to a low R:FR light ratio. In one aspect, inhibition of the shade avoidance response can be shown by measuring the height of plants comprising the trait of the invention (e.g., a mutant HY5 transcription factor as described herein) and isogenic plants that do not contain the trait in a controlled environment of a low R:FR light ratio. When grown under the same conditions in the presence of an R:FR ratio of 0.16, plants comprising the trait of the invention will be at least 5% shorter (e.g., height measured at the coleoptile, V1 sheath or V2 sheath) than isogenic plants not comprising the trait (e.g., about 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%, 111%, 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%, 110%, 120%, 130%, 140%, 150% or more, or any range or value thereof,For example, from about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25% 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%, 9%, 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%, 110%, 120%, 130%, 140%, 150% or more) (e.g., less than about 5% to about 10%, less than about 5% to about 15%, less than about 5% to about 20%, less than about 5% to about 25%, less than about 5% to about 30%, less than about 5% to about 40%, less than about 5% to about 50%, less than about 10% to about 20%, less than about 10% to about 30%, less than about 10% to about 50%, less than about 10% to about 70%, less than about 15% to about 80%, less than about 15% to about 90%, less than about 10% to about 20%, less than about 10% to about 30%, less than about 10% to about 50%, less than about 10% to about 70%, less than about 15% to about 100%, less than about 15% to about 20%, less than about 15% to about 25%, less than about 10% to about 30%, less than about 15% to about % to about 20%, less than about 15% to about 30%, less than about 15% to about 50%, less than about 20% to about 30%, less than about 20% to about 50%, less than about 20% to about 70%, less than about 40% to about 50%, less than about 40% to about 60%, less than about 40% to about 80%, less than about 40% to about 100%, less than about 50% to about 70%, less than about 50% to about 100%, less than about 50% to about 125%, less than about 75% to about 100%, less than about 75% to about 120%, less than about 75% to about 140%, etc.),

[0099] Plants exhibiting reduced SAR when planted in close proximity to one or more plants exhibit increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, essentially no change in flowering time, increased photosynthetic efficiency and grain filling, and / or enhanced defense responses to pathogens and herbivores, compared to control plants planted in close proximity.

[0100] Plants with reduced SAR as described herein may have increased yield compared to plants without reduced SAR. As used herein, "increased yield" refers to any plant trait associated with growth, such as biomass, yield, nitrogen use efficiency (NUE), inflorescence size / weight, fruit yield, fruit quality, fruit size, seed size, seed number, leaf tissue weight, nodule number, nodule quality, nodulation activity, ear number, tiller number, flower number, tuber number, tuber quality, bulb quality, seed number, total seed mass, leaf emergence rate, tiller emergence rate, seedling emergence rate, root length, root number, root mass size and / or weight, or any combination thereof. Thus, in some aspects, "increased yield" may include, but is not limited to, increased inflorescence yield, increased fruit yield (e.g., increased fruit number, weight, and / or size; e.g., increased number, weight, and / or size of ears of, e.g., corn), increased fruit quality, increased root number, size, and / or weight, increased meristem size, increased seed size, increased biomass, increased nitrogen use efficiency, as compared to a control plant or part thereof (e.g., a plant that does not contain a mutant endogenous nucleic acid encoding a HY5 transcription factor as described herein, grown in an environment with a low R:FR light ratio (e.g., a shaded environment; e.g., an R:FR ratio of about 0.16), including when grown in close proximity to other plants). In some aspects, increased yield may be expressed as the number of grains produced per unit area of ​​land (e.g., bushels per acre of land).

[0101] As used herein, "reduced height" means inhibition of stem elongation in response to abundant far-red light. Thus, for example, plants having a mutation in the HY5 gene as described herein exhibit reduced height when grown under shade conditions, as compared to control plants that do not contain the mutation and are also grown under shade conditions. The reduction in height can be about 15% to about 80% (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%, 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%, 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%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 200%,

[0102] As used herein, "reduced stem:root ratio" means a reduction in the ratio of above-ground biomass to below-ground biomass. Thus, for example, plants having a mutation in the HY5 gene as described herein exhibit a reduced stem:root ratio when grown under shade conditions, as compared to control plants that do not contain the mutation and are also grown under shade conditions. The reduction in the shoot:root ratio can be a reduction of about 5% to about 50% (e.g., about 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%, or any range or value therein) compared to a control.

[0103] As used herein, "increased upright growth" means that plants having a mutation in the HY5 gene as described herein continue to grow upright when experiencing shade (such as when planted at high density), rather than "leaning" or growing in the direction of light, compared to control plants without the mutation that are also experiencing shade. Plants with increased upright growth grow more vertically than control plants. In some embodiments, the increase in upright growth can be an increase of about 5% to about 50% (e.g., about 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%, or any range or value therein) compared to a control.

[0104] As used herein, "reduced leaf length" means that the distance from the top of the leaf to the bottom of the leaf of a plant having a mutation in the HY5 gene as described herein is reduced as compared to a control plant not comprising the mutation. In some embodiments, the reduction in leaf length can be a reduction of about 5% to about 50% (e.g., about 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%, or any range or value therein) compared to a control.

[0105] As used herein, "increased mechanical strength of stems" means that plants having a mutation in the HY5 gene as described herein have an improvement in one or more of bending strength, elastic modulus, tensile strength, critical buckling load, compressive strength, shear strength, and / or shear modulus as compared to a control plant that does not contain the mutation. In some embodiments, the reduced mechanical strength of the stem can be about 5% to about 65% (e.g., about 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%, 100%, 101%, 102%, 63%, 64%, or 65%, or any range or value therein).

[0106] As used herein, "lodging rate" means the ratio of the number of plants that are tilted >45° relative to the vertical axis to the total number of plants in the evaluated population. Thus, for example, plants having a mutation in the HY5 gene as described herein exhibit "reduced lodging rate" as compared to control plants that do not contain the mutation. In some embodiments, the "reduction in lodging rate" can be from about 20% to about 100% (e.g., about 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%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or any range or value therein).

[0107] As used herein, "senescence" means the process of plant aging. For example, as compared to control plants that do not contain mutations, plants with mutations in the HY5 gene as described herein have a delay in one or both of stress-induced or age-related developmental aging (senescence). In some embodiments, the delay in senescence can be about 10% to about 80% (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 , 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%, or 80% or any range or value therein).

[0108] As used herein, "increased photosynthesis efficiency" means that a plant having a mutation in the HY5 gene as described herein exhibits an increase in the efficiency of energy transfer from harvested light to the electron transport chain as compared to a control plant that does not contain the mutation. In some embodiments, the increase in photosynthesis efficiency can be from about 10% to about 100%% compared to a control (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%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% or any range or value therein).

[0109] As used herein, "grain filling" refers to the time between flowering and physiological maturity. For example, as compared to control plants that do not contain the mutation, plants having a mutation in the HY5 gene as described herein have increased grain filling (e.g., a longer grain filling period), resulting in greater grain weight, higher harvest index and / or higher grain yield per plant. In some embodiments, the increase in grain filling can be an increase in the length of the grain filling period compared to a control by 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%, %, 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% or any range or value thereof).

[0110] As used herein, "enhanced defense response against pathogens and herbivores" means that plants having a mutation in the HY5 gene as described herein have an improvement in basal resistance (e.g., innate immunity) and / or systemic acquired resistance in response to pathogens (e.g., viruses, bacteria, nematodes and / or fungal pathogens) and herbivores as compared to control plants that do not contain the mutation. In some embodiments, the enhanced defense response can be an enhancement / improvement or increase in defense response by about 30% to about 100%% (e.g., about 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%, %, 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% or any range or value thereof).

[0111] As used herein, "no substantial change in flowering time" means that a plant having a mutation in the HY5 gene as described herein, when subjected to shade, such as when planted at high density, maintains substantially no change in its normal flowering time, as compared to a control plant (not comprising the mutation) subjected to shade. In some embodiments, a plant having a mutation in HY5 as described herein may have substantially no change in flowering time, wherein "substantially no change" means that the change in flowering time is less than 15% (e.g., no change or a change of less than 1%, 2%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14% or 15% or any range or value therein) as compared to a control. Plants that do not have substantially a change in flowering time can also be described as having a flowering time that is at least 85% (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or any range or value therein) of the flowering time of control plants.

[0112] Thus, a plant in which at least one (e.g., one or more, e.g., 1, 2, 3, or 4 or more) endogenous HY5 gene is modified as described herein (e.g., comprises a modification as described herein) can have improved yield traits compared to a plant that does not comprise (lack) the modification in at least one endogenous HY5 gene. As used herein, "improved yield trait" refers to any plant trait associated with 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 quality, nodulation activity, seed ear number, tiller number, branch number, flower number, tuber number, tuber quality, bulb quality, seed number, total seed mass, leaf emergence rate, tiller / branch emergence rate, seedling emergence rate, root length, root number, root mass size and / or weight, or any combination thereof. In some aspects, "improved yield traits" may 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 ears, e.g., for corn), increased 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, improved nitrogen use efficiency, increased height, increased number of internodes and / or increased internode length, compared to a control plant or part thereof (e.g., a plant not comprising a mutant endogenous HY5 nucleic acid as described herein), increased biomass, increased leaf size, increased nitrogen use efficiency, increased height, increased number of internodes and / or increased internode length. In some aspects, the improved yield trait may 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 may be an increased number of rows of grains, optionally without reducing ear length.

[0113] As used herein, "control plant" means a plant that does not contain an edited HY5 transcription factor gene as described herein. Control plants are used to identify and select plants edited as described herein, which have enhanced traits or altered phenotypes compared to control plants. Suitable control plants can be parental line plants used to produce plants containing mutated HY5 transcription factor genes, for example, wild-type plants lacking editing in endogenous HY5 transcription factor genes as described herein. Suitable control plants can also be plants containing recombinant nucleic acids that confer other traits, for example, transgenic plants with enhanced herbicide tolerance. In some cases, suitable control plants can be offspring of heterozygous or hemizygous transgenic plant lines lacking mutated HY5 transcription factor genes as described herein, referred to as negative segregants or negative isogenic lines.

[0114] Enhanced traits (e.g., improved yield traits) may include, for example, reduced days from planting to maturity, increased stem size, increased leaf number, increased vegetative stage plant height growth rate, increased ear size, increased dry weight per plant ear, increased number of grains per ear, increased weight per grain, increased number of grains per plant, reduced empty ears, extended grain filling period, reduced plant height, increased number of root branches, increased total root length, increased yield (e.g., increase in harvestable seeds), increased nitrogen use efficiency, and / or increased water use efficiency, compared to control plants. The altered phenotype may be, for example, plant height, biomass, canopy area, anthocyanin content, chlorophyll content, water application, water content, and water use efficiency.

[0115] In some embodiments, the plants of the present invention may comprise one or more improved yield traits. In some embodiments, the one or more improved yield traits include higher yield (bushels per acre), increased biomass, increased plant height, increased stem diameter, increased leaf area, increased number of flowers, increased number of rows of seeds (optionally wherein ear length is not substantially reduced), increased number of seeds, increased seed size, increased ear length, reduced number of tillers, reduced number of tassel branches, increased number of pods (including increased number of pods per node and / or increased number of pods per plant), increased number of seeds per pod, increased number of seeds, increased seed size, and / or increased seed weight (e.g., increased 100-seed weight) compared to a control plant lacking at least one non-natural mutation. In some embodiments, plants of the present invention may comprise one or more improved yield traits 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 wherein ear length is not substantially reduced), increased number of pods, increased number of seeds per pod, and increased ear length, as compared to control plants or parts thereof.

[0116] As used herein, "properties" are physiology, morphology, biochemistry or physical characteristics of plants or specific plant materials or cells. In some cases, this feature is visible to the human eye and can be measured mechanically, such as the size, weight, shape, form, length, height, growth rate and developmental stage of seeds or plants, or can be measured by biochemical techniques, such as protein, starch, some metabolites or the oil content of detection seeds or leaves, or by observing metabolism or physiological processes, for example, by measuring the tolerance to lack of water or specific salt or sugar concentration, or by measuring the expression level of one or more genes, for example, by adopting Northern analysis, RT-PCR, microarray gene expression array or reporter gene expression system, or by agricultural observations such as high osmotic stress tolerance or yield. However, any technology can be used to measure the amount, comparative level or difference of any selected chemical compound or macromolecule in transgenic plants.

[0117] As used herein, "enhanced proterties" refers to plant characteristics caused by mutations in HY5 transcription factor genes as described herein. Such proterties include, but are not limited to, enhanced agronomic traits, characterized in that enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In some embodiments, the phenotype of enhanced proterties / changes can be, for example, reduced days from planting to maturity, increased stem size, increased leaf number, increased nutrient stage plant height growth rate, increased ear size, increased per plant ear dry weight, increased per ear grain number, increased per grain weight, increased per plant grain number, reduced ear empty grains, extended filling period, reduced plant height, increased root branch number, increased total root length, drought tolerance, improved water use efficiency, cold tolerance, improved nitrogen use efficiency and / or increased yield. In some embodiments, proterties are the yield increased under non-stress conditions or the yield increased under environmental stress conditions. Stress conditions can include biotic and abiotic stresses, such as drought, shading, fungal diseases, viral diseases, bacterial diseases, insect infestation, nematode infestation, low temperature exposure, heat exposure, osmotic stress, nitrogen nutrient availability reduction, phosphorus nutrient availability reduction and high plant density. "Yield" can be affected by many characteristics, including but not limited to plant height, plant biomass, pod number, pod position on plant, internode number, pod cracking incidence, grain size, ear size, ear tip filling degree, grain abortion, nodulation and nitrogen fixation efficiency, nutrient assimilation efficiency, biotic and abiotic stress resistance, carbon assimilation, plant configuration, lodging resistance, seed germination percentage, 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, ear number, ear size, ear weight, number of seeds per ear or per pod, seed size, composition (starch, oil, protein) of seed and the characteristics of seed filling.

[0118] As also used herein, the term "trait modification" encompasses changing a naturally occurring trait by producing a detectable characteristic difference in a plant comprising a mutation in an endogenous HY5 transcription factor gene as described herein relative to a plant that does not comprise the mutation (such as a wild-type plant, or a negative segregant). In some cases, trait modification can be quantitatively evaluated. For example, a trait modification can result in an increase or decrease in an observed trait characteristic or phenotype compared to a control plant. It is well known that altered traits may have natural variation. Therefore, the observed trait modification can result in a normal distribution and magnitude change in trait characteristics or phenotypes in plants compared to control plants.

[0119] The present disclosure relates to a plant with improved economically relevant characteristics (more specifically reduced shade avoidance). More specifically, the present disclosure relates to a plant comprising a mutation in a HY5 transcription factor gene as described herein, wherein the plant has a reduced shade avoidance response as compared to a control plant lacking the mutation (e.g., a plant comprising a mutation as described herein when grown under shade conditions results in a shorter plant than a plant lacking the mutation grown under the same conditions). In some embodiments, the plants of the present disclosure further exhibit improved traits associated with 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.

[0120] Yield can be defined as a measurable product with economic value from a crop. Yield can be defined in terms of quantity and / or quality. Yield can depend directly on several factors, such as the number and size of organs (e.g., number of flowers), plant configuration (such as the number of branches, plant biomass, such as increased root biomass, steeper root angles and / or longer roots, etc.), flowering time and duration, grain filling period. Root configuration and development, photosynthetic efficiency, nutrient absorption, stress tolerance, early vigor, delayed senescence and functional stay-green phenotypes may be factors that determine yield. Therefore, optimizing the above factors can help increase crop yield.

[0121] The increase / improvement of the yield-related traits mentioned herein can also be considered to refer to the increase of the biomass (weight) of one or more parts of a plant, which can include above-ground and / or underground (harvesting) plant parts. Specifically, such harvestable parts are seeds, and the execution of the disclosed method produces a plant with an output of increase, particularly with respect to the seed yield of a suitable control plant, with an increase in seed yield. The term "output" of a plant can relate to the vegetative biomass (root and / or seedling biomass), reproductive organs and / or propagules (such as seeds) of the plant. In some embodiments, the disclosed method causes a plant to have a shade-avoiding reaction that is reduced with respect to a suitable control plant.

[0122] The increased yield of the plants of the present disclosure can be measured in a variety of ways, including test weight, number of seeds per plant, seed weight, number of seeds per unit area (e.g., number of seeds per acre or seed weight), bushels per acre, tons per acre, or kilograms per hectare. The increased yield can be attributed to improved 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).

[0123] "Increased yield" can be manifested as one or more of the following: (i) increased plant biomass (weight) of one or more parts of the plant, in particular the aboveground (harvestable) parts of the plant, increased root biomass (increased number of roots, increased root thickness, increased root length) or increased biomass of any other harvestable part; or (ii) increased early vigour, defined herein as increased aboveground area of ​​seedlings about three weeks after germination.

[0124] "Early vigor" refers to active and healthy plant growth, especially in the early stages of plant growth, and can be due to, for example, plant better adapting to its environment (for example, optimizing the utilization of energy, nutrient absorption and carbon distribution between seedling and root) caused by the increase in plant fitness. For example, early vigor can be a combination of the ability of seeds to germinate and emerge after planting and the ability of seedlings to grow and develop after emergence. Plants with early vigor also show increased seedling survival rate and better crop planting, which can usually make the field highly uniform, wherein most plants basically reach each development stage at the same time, thereby usually increasing output. Therefore, early vigor can be determined by measuring various factors, such as grain weight, germination percentage, emergence percentage, seedling growth, seedling height, root length, root and seedling biomass, canopy size and color, etc.

[0125] In addition, increased yield can also be manifested as increased 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 individual seed, for example, an increased number of flowers / panicles per plant; an increased number of pods; an increased number of nodes; an increased number of flowers ("florets") per panicle / plant; an increased seed filling rate; an increased number of filled seeds; an increased seed size (length, width, area, circumference and / or weight), which can also affect seed composition; and / or an increased seed volume, which can also affect seed composition. In one embodiment, the increased yield can be an increased seed yield, for example, an increased seed weight; an increased number of filled seeds; and / or an increased harvest index.

[0126] Increased production may also result in configurational changes or may be due to

[0127] Occurs due to changes in plant architecture.

[0128] Increased yield can also be expressed as an increased harvest index, which is expressed as

[0129] The ratio of the yield of harvestable parts (such as seeds) to the total biomass

[0130] The present disclosure also extends to harvestable parts of plants, such as, but not limited to, seeds, leaves, fruits, flowers, pods, pods, siliques, nuts, stems, rhizomes, tubers and bulbs. The present disclosure also relates to products derived from harvestable parts of such plants, such as dry pellets, powders, oils, fats and fatty acids, starch or proteins.

[0131] The present disclosure provides a method for increasing the "yield" of a plant or the "broadacre yield" of a plant or plant part, which is defined as the harvestable plant part per unit area, such as number of seeds per acre or seed weight, pounds per acre, bushels per acre, tons per acre, tons per acre, kilograms per hectare.

[0132] As used herein, "nitrogen use efficiency" refers to the processes that lead to increased plant yield, biomass, vigor, and growth rate per unit of nitrogen applied. These processes may include plant absorption, assimilation, accumulation, signal transduction, sensing, retransfer (within the plant), and utilization of nitrogen.

[0133] As used herein, "increased 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 the ability to grow, develop or produce faster or better when subjected to less than optimal available / applied amounts of nitrogen or under nitrogen limiting conditions.

[0134] As used herein, "nitrogen limiting conditions" refers to growing conditions or environments that provide less than the optimal amount of nitrogen required for adequate or successful metabolism, growth, reproductive success, and / or survival of a plant.

[0135] As used herein, "increased nitrogen stress tolerance" refers to the ability of a plant to grow, develop or produce normally, or to grow, develop or produce faster or better, when subjected to less than optimal available / applied amounts of nitrogen or under nitrogen limiting conditions.

[0136] The improved plant nitrogen utilization efficiency can be converted into a similar amount of yield in the field while supplying less nitrogen, or an increased yield can be obtained by supplying an optimum / sufficient amount of nitrogen. The improved nitrogen utilization efficiency can improve plant nitrogen stress tolerance, and can also improve the biochemical composition of crop quality and seeds, such as protein yield and oil yield. The terms "improved nitrogen utilization efficiency", "enhanced nitrogen utilization efficiency" and "nitrogen stress tolerance" are used interchangeably in this disclosure and refer to plants with improved productivity under nitrogen limiting conditions.

[0137] As used herein, "water use efficiency" refers to the amount of carbon dioxide assimilated by leaves per unit of transpiration water vapor. It constitutes one of the most important traits controlling plant productivity in dry environments. "Drought tolerance" refers to the degree to which plants adapt to dry or drought conditions. The physiological response of plants to water shortage includes leaf wilting, reduced leaf area, leaf shedding, and stimulating root growth by directing nutrients to the underground parts of the plant. Generally, plants are more susceptible to drought during flowering and seed development (reproduction stage) because the resources of the plant are biased to support root growth. In addition, 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 caused by transpiration and reducing photosynthesis rate. These reactions improve the water use efficiency of plants in the short term. The terms "improved water use efficiency", "enhanced water use efficiency" and "increased drought tolerance" are used interchangeably in this disclosure and refer to plants with improved productivity under water-limited conditions.

[0138] 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 the ability to grow, develop or produce faster or better when subjected to reduced available / applied amounts of water (water input) or under conditions of water stress or water deficiency stress.

[0139] As used herein, "increased drought tolerance" refers to the ability of a plant to grow, develop or produce normally, or to grow, develop or produce faster or better than normal, when subjected to reduced available / applied amounts 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 reduced available / applied amounts of water (water input) or under conditions of water stress or under conditions of short-term or long-term drought.

[0140] As used herein, "drought stress" refers to a dry period (short or long / extended) that results in a lack of water and stresses the plant and / or causes damage to plant tissue and / or has a negative impact on grain / crop yield; a dry period (short or long / extended) that results in a lack of water and / or an increase in temperature and stresses the plant and / or causes damage to plant tissue and / or has a negative impact on grain / crop yield.

[0141] As used herein, "water deficit" refers to conditions or circumstances that provide less than the optimal amount of water required for adequate / successful growth and development of plants.

[0142] 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.

[0143] 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.

[0144] 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 a hybrid thereof. The term also encompasses RNA / DNA hybrids. When synthesizing dsRNA, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. can 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.

[0145] 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 5' to 3' directions from left to right herein, and are represented by the standard code for representing nucleotide characters set forth in World Intellectual Property Organization (WIPO) standard ST.26. As used herein, "5' region" can represent the polynucleotide region closest to the 5' end of a polynucleotide. Therefore, for example, the element in the 5' region of a polynucleotide can be located at any position from the first nucleotide at the 5' end of a polynucleotide to the nucleotide at the middle of the polynucleotide. As used herein, "3' region" can represent the polynucleotide region closest to the 3' end of a polynucleotide. Thus, for example, elements in the 3' region of a polynucleotide can be located anywhere from the first nucleotide at the 3' end of the polynucleotide to a nucleotide located in the middle of the polynucleotide.

[0146] As used herein with respect to nucleic acids, the terms "fragment" or "portion" refer to a portion of a nucleic acid that is reduced in length (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 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, 610, 620, 630, 640, 650, 660, 670, 680, 690, 711, 720, 730, 740, 750, 760, 770, 780, 790, 800 The nucleic acid fragment may be a nucleic acid having a length of 50, 700, 750, 800, 850, 900, 950 or 1000 or more nucleotides or any range or value therein) and comprising or consisting essentially of and / or consisting of a nucleotide sequence of contiguous nucleotides that are identical or nearly identical (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% identical) to the corresponding portion of the reference nucleic acid. Where appropriate, such a nucleic acid fragment may be contained in a larger polynucleotide of which the nucleic acid fragment is a component. As an example, the repetitive sequence of the guide nucleic acid of the present invention may include a portion of a wild-type CRISPR-Cas repetitive sequence (e.g., a wild-type CRISR-Cas repetitive sequence; for example, a repetitive sequence from a CRISPR Cas system, such as Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or Cas14c, etc.).In some embodiments, the nucleic acid fragment may comprise, consist essentially of, and / or consist of the following consecutive nucleotides: about 1, 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, 175, 200, 225, 250, 300, 350, 400, 450, 50 0, 550, 600, 660, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000 or more consecutive nucleotides. A decrease in the activity of the HY5 gene (e.g., a decrease in the binding of COPl to HY5) can result in a reduced shade avoidance response in a plant.

[0147] In some embodiments, a nucleic acid fragment or portion may comprise, consist essentially of, and / or consist of the following consecutive nucleotides: 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, 6, 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, 141, 142, 143, 145, 146, 147, 148, 149, 150, 175, 200, 210, 220, 225, 2 3, 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, 241, 242, 243, 245, 245, 246, 247, 248, 249, 250, 260, 270, 280, 290, 300, 350, 360, 361, 362, 363, 364 , 365, 366, 367, 368, 369, 370, 380, 390, 400, 410, 411, 412, 413, 414, 415, 416, 417, 418, 419, 420, 430, 440, 450, 460, 470, 480, 490, 491, 492, 493, 494, 618, 619, or 620 or more consecutive nucleotides, or any range or value therein, and optionally, a fragment of the HY5 polynucleotide can be from about 20 nucleotides to about 120 nucleotides, from about 20 nucleotides to about 250 nucleotides, from about 20 nucleotides to about 620 nucleotides, from about 100 nucleotides to about 250 nucleotides, from about 150 nucleotides to about 200 nucleotides, from about 100 nucleotides to about 250 nucleotides, from about 150 nucleotides to about 200 nucleotides, from about 100 nucleotides to about 250 nucleotides, from about 150 nucleotides to about 200 nucleotides, from about 100 nucleotides to about 250 nucleotides, from about 150 nucleotides to about 200 nucleotides, from about 100 nucleotides to about 250 nucleotides, from about 150 nucleotides to about 200 nucleotides,about 100 nucleotides to about 400 nucleotides, about 150 nucleotides to about 400 nucleotides, about 200 nucleotides to about 620 nucleotides, such as about 60, 80, 100, 120, 140, 160, 180 or 200 nucleotides to about 210, 220, 240, 260, 280, 300, 350, 400, 450, 500, 550, 600, 615 or 620 or more consecutive nucleotides (e.g., consecutive nucleotides of SEQ ID NO: 72, such as SEQ ID NO: 75-105, optionally with SEQ ID NO: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105 having at least 80% sequence identity).

[0148] As used herein with respect to polypeptides, the term "fragment" or "portion" may refer to a polypeptide that is reduced in length relative to a reference polypeptide and comprises, consists essentially of, and / or consists of an amino acid sequence of contiguous amino acids that are identical or nearly identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the corresponding portion of the reference polypeptide. Where appropriate, such a polypeptide fragment may be included in a larger polypeptide of which it is a component. In some embodiments, a polypeptide fragment comprises, consists essentially of, or consists of at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, or more consecutive amino acids of a reference polypeptide. In some embodiments, the HY5 transcription factor fragment comprises, consists essentially of, or consists of at least about 15, 20, 25, 30, 35, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, or 160 or more consecutive amino acids (e.g., a fragment or portion of SEQ ID NO: 74, e.g., SEQ ID NO: 106).

[0149] In some embodiments, a "portion" may relate to the number of amino acids deleted from a polypeptide. Thus, for example, a "portion" of a HY5 transcription factor polypeptide may include a deletion from the amino acid sequence of SEQ ID NO: 74 (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 the amino acid sequence of SEQ ID NO: 74; optionally wherein the sequence identity may be at least 85%, it may be at least 90% or it may be at least 95%, or the sequence identity may be 100%)). 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 50 or more consecutive amino acid residues) (e.g., a deletion of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 to about 30, 31, 32, 33, 34, 35, 36, 37, 38, 37 or 40 residues, for example, SEQ ID NO: 106 or a portion thereof), optionally a deletion in a region adjacent to or in the first and / or second exon of the HY5 gene can result in one or more than one amino acid residue being deleted from the encoded HY5 polypeptide. In some embodiments, the deleted portion of the HY5 transcription factor gene can be, for example, an in-frame deletion or an out-of-frame deletion in which at least one amino acid (e.g., one or more than one amino acid) is deleted from the N-terminal portion of the encoded HY5 transcription factor, optionally, a deletion of at least a portion of the consecutive amino acid residues (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more consecutive amino acid residues) of SEQ ID NO: 74. In some embodiments, when the mutation is an out-of-frame deletion, a premature stop codon may be generated, which results in a truncated HY5 protein.

[0150] In some embodiments, the deletion of one or more amino acid residues from the HY5 transcription factor polypeptide as described herein can result in a dominant mutation and / or a dominant negative mutation, which when included in a plant can result in the plant exhibiting a reduced shade avoidance response (SAR) as compared to a plant not comprising the deletion. In some embodiments, the deletion of one or more amino acid residues from the HY5 transcription factor polypeptide as described herein can result in a plant exhibiting increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, substantially no change in flowering time, increased photosynthetic efficiency and grain filling, and / or enhanced defense responses to pathogens and herbivores when planted in close proximity to one or more plants compared to control plants planted in close proximity. In some embodiments, such a mutation results in reduced binding of the HY5 transcription factor to a constitutive photomorphogenesis 1 (COP1) polypeptide. In some embodiments, the mutation is a dominant negative mutation.

[0151] A "region" of a polynucleotide or polypeptide refers to a portion of consecutive nucleotides or consecutive amino acid residues of the polynucleotide or polypeptide, respectively. For example, a region of a HY5 polynucleotide sequence may include, but is not limited to, any one of the nucleic acid sequences of SEQ ID NOs: 75-105, optionally with 80% sequence identity to any one of SEQ ID NOs: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, or 103-105, optionally wherein 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 at least 100%. In some embodiments, a region of a HY5 polypeptide sequence may include, but is not limited to, the amino acid sequence of SEQ ID NO: 106.

[0152] In some embodiments, a "sequence-specific nucleic acid binding domain" (e.g., a sequence-specific DNA binding domain) can bind to a HY5 transcription factor gene (e.g., SEQ ID NO:72 or SEQ ID NO:73) and / or one or more fragments, portions or regions of a HY5 transcription factor nucleic acid (e.g., SEQ ID NOs:75-105).

[0153] 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" with respect to a polypeptide is a fragment of a polypeptide that retains one or more activities of a native reference polypeptide.

[0154] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotides (AMOs), etc. A gene may or may not be used to produce a functional protein or gene product. A gene may include coding regions 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," which means that the nucleic acid is substantially or substantially free of components that are usually present with the nucleic acid under natural conditions. Such components include other cell materials, culture media from recombinant production, and / or various chemicals for chemically synthesizing nucleic acids.

[0155] The term "mutation" refers to a point mutation (e.g., missense or nonsense, or insertion or deletion of a single base pair that results in a frameshift), insertion, deletion, and / or truncation. When a mutation is a replacement of a residue within an amino acid sequence by another residue, or a deletion or insertion of one or more residues within a sequence, the mutation is typically described by identifying the original residue, then identifying the position of the residue within the sequence, and the identity of the newly replaced residue. Truncation may include truncation at the C-terminus of a polypeptide or the N-terminus of a polypeptide. Truncation of a polypeptide may be the result of a corresponding 5' or 3' end deletion of a gene encoding a polypeptide. When a deletion or insertion of one or more base pairs is introduced into a gene, a frameshift mutation may occur, optionally resulting in an out-of-frame mutation or an in-frame mutation. A frameshift mutation in a gene may result in a polypeptide that is longer, shorter, or the same length as a wild-type polypeptide, depending on when the first stop codon appears after the mutation region of the gene. As an example, an out-of-frame mutation that results in an early stop codon may result in a polypeptide that is shorter than a wild-type polypeptide, or in some embodiments, the polypeptide may not be present / detectable. DNA inversion is the result of rotation of a genetic fragment within a chromosomal region.

[0156] As used herein, the terms "complementary" or "complementarity" refer to the natural binding of polynucleotides by base pairing under permissive salt and temperature conditions. For example, the sequence "AGT" (5' to 3') binds to the complementary sequence "TCA" (3' to 5'). The complementarity between two single-stranded molecules can be "partial", in which only some of the nucleotides bind, or it can be complete when there is complete complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid chains has a significant effect on the efficiency and strength of hybridization between nucleic acid chains.

[0157] As used herein, "complement" can mean 100% complementarity to a compared nucleotide sequence, or it can mean less than 100% complementarity to a compared nucleotide sequence (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%, etc. complementarity; e.g., substantial complementarity).

[0158] Different nucleic acids or proteins with homology are referred to herein as "homologs". The term homolog includes homologous sequences from the same species and other species and 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 having similar functional properties between different nucleic acids or proteins. Therefore, the compositions and methods of the present invention also include homologs of the nucleotide sequences and polypeptide sequences of the present invention. As used herein, "orthologs" refer to homologous nucleotide sequences and / or amino acid sequences in different species, which are derived from common ancestral genes during the process of speciation. Homologs of the nucleotide sequences of the invention have substantial sequence identity (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%) to the nucleotide sequences of the invention.

[0159] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are unchanged over the entire component (e.g., nucleotide or amino acid) comparison window. "Identity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, ed.) 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., ed.) Stockton Press, New York (1991).

[0160] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in a 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 HY5 gene, the sequence may have at least 80% sequence identity with the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73. In some embodiments, the HY5 gene may have at least 85% sequence identity with the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73. In some embodiments, the HY5 gene may have at least 90% sequence identity with the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73. In some embodiments, the HY5 gene may have at least 95% sequence identity with the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73, optionally wherein the HY5 gene may have at least 100% sequence identity with the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73. A HY5 polypeptide as described herein may have at least 80% sequence identity with the polypeptide sequence of SEQ ID NO:74. In some embodiments, the HY5 polypeptide may have at least 85% sequence identity with the polypeptide sequence of SEQ ID NO:74. In some embodiments, the HY5 polypeptide may have at least 90% sequence identity with the polypeptide sequence of SEQ ID NO:74. In some embodiments, the HY5 polypeptide may have at least 95% sequence identity with the polypeptide sequence of SEQ ID NO:74, optionally wherein the HY5 polypeptide may have at least 100% sequence identity with the polypeptide sequence of SEQ ID NO:74. Regarding the region or portion of the HY5 gene, the region or portion may have at least 80% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-105, optionally at least 80% sequence identity with any one of SEQ ID NOs: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, or 103-105. In some embodiments, the region or portion of the HY5 gene may have at least 85% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-105, optionally at least 85% sequence identity with any one of SEQ ID NOs: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, or 103-105.In some embodiments, a region or portion of the HY5 gene may have at least 90% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-105, optionally at least 90% sequence identity with any one of SEQ ID NOs: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, or 103-105. In some embodiments, a region or portion of the HY5 gene may have at least 95% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-105, optionally at least 95% sequence identity with any one of SEQ ID NOs: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, or 103-105. In some embodiments, a region or portion of the HY5 gene may have at least 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-105, optionally at least 100% sequence identity with any one of SEQ ID NOs: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, or 103-105. With respect to a region or portion of a HY5 polypeptide, the region or portion may have at least at least 80% sequence identity with the polypeptide sequence of SEQ ID NO: 106. In some embodiments, a region or portion of a HY5 polypeptide may have at least 85% sequence identity with the polypeptide sequence of SEQ ID NO: 106. In some embodiments, a region or portion of a HY5 polypeptide may have at least 90% sequence identity with the polypeptide sequence of SEQ ID NO: 106. In some embodiments, a region or portion of a HY5 polypeptide may have at least 95% sequence identity with the polypeptide sequence of SEQ ID NO: 106. In some embodiments, a region or portion of a HY5 polypeptide may have at least 100% sequence identity with the polypeptide sequence of SEQ ID NO: 106. With respect to the edited HY5 nucleic acid sequence, the edited HY5 nucleic acid sequence may have at least 90% sequence identity with any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, or 139. In some embodiments, the edited HY5 nucleic acid sequence may have at least 95% sequence identity with any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, or 139.In some embodiments, the edited HY5 nucleic acid sequence may have 100% sequence identity with any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, or 139. With respect to the mutated HY5 amino acid sequence, the mutated HY5 polypeptide may have at least 90% sequence identity with any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136, and 138. In some embodiments, the mutated HY5 polypeptide may have at least 95% sequence identity with any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136, and 138. In some embodiments, the mutant HY5 polypeptide may have 100% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136, and 138.

[0161] As used herein, the phrases "substantially identical" or "substantial identity" in the context of two nucleic acid molecules, nucleotide sequences, or polypeptide sequences, means that two or more sequences or subsequences 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 as determined using one of the following sequence comparison algorithms, or by visual inspection. In some embodiments of the invention, substantial identity exists over a region of contiguous nucleotides of a nucleotide sequence of the invention, the region 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, and any ranges therein, up to the full length of the sequence. 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300, or more nucleotides).In some embodiments, two or more HY5 transcription factor genes may be present within at least about 30 or more consecutive nucleotides (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 consecutive nucleotides).

[0162] SEQ ID NO:75-105).

[0163] In some embodiments of the invention, substantial identity exists over a region of contiguous amino acid residues of a polypeptide of the invention that is from about 3 amino acid residues to about 20 amino acid residues, from about 5 amino acid residues to about 10 amino acid residues, from about 5 amino acid residues to about 55 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, about 25 amino acid residues to about 50 amino acid residues, about 30 amino acid residues to about 50 amino acid residues, about 40 amino acid residues to about 50 amino acid residues, about 40 amino acid residues to about 70 amino acid residues, about 50 amino acid residues to about 70 amino acid residues, about 60 amino acid residues to about 80 amino acid residues, about 70 amino acid residues to about 80 amino acid residues, about 90 amino acid residues to about 100 amino acid residues or more, and any ranges therein, up to the full length of the sequence. In some embodiments, the polypeptide sequence may be at least about 8, 9, 10, 11, 12, 13, 14, or more consecutive amino acid residues (e.g., 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, 75 175, 200, 225, 250, 275, 300, 325, 350, 400, 450, 500 or more amino acids or more consecutive amino acid residues) are substantially identical to each other.In some embodiments, two or more HY5 transcription factor polypeptides may be substantially identical to each other over at least about 10 to about 150 consecutive amino acid residues of SEQ ID NO:74 (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, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 residues or more) of SEQ ID NO: 106, or any range or value therein). In some embodiments, a substantially identical nucleotide or protein sequence can perform substantially the same function as its substantially identical nucleotide (or encoded protein sequence).

[0164] For sequence comparison, usually one sequence acts as a reference sequence compared to a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.

[0165] 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 can be used as Wisconsin (Accelrys Inc., San Diego, CA). The "identity score" for the alignment fragment of the test sequence and the 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 fragment (e.g., the entire reference sequence or a smaller defined portion of the reference sequence). The percentage of sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more polynucleotide sequences can be a comparison with a full-length polynucleotide sequence or a portion thereof, or a comparison with a longer polynucleotide sequence. For purposes of the present invention, BLASTX version 2.0 can also be used to determine the "identity percentage" for the translated nucleotide sequence, and BLASTN version 2.0 can be used to determine the "identity percentage" for the polynucleotide sequence.

[0166] When two nucleotide sequences hybridize to each other under stringent conditions, the two sequences may also be considered to be substantially complementary. In some embodiments, two nucleotide sequences that are considered to be substantially complementary hybridize to each other under highly stringent conditions.

[0167] 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. An extensive guide to nucleic acid hybridization can be found 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). In general, highly stringent hybridization and wash conditions are selected to be higher than the thermal melting point (Tf) for the specific sequence at a defined ionic strength and pH. m ) is about 5℃ lower.

[0168] T m It is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are chosen to be equal to the T for a specific probe. m. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues on a filter membrane in a Southern or Northern blot is hybridization overnight with 50% formamide and 1 mg of heparin at 42°C. An example of highly stringent washing conditions is washing with 0.15M NaCl at 72°C for about 15 minutes. An example of stringent washing conditions is washing with 0.2x SSC at 65°C for 15 minutes (for a description of SSC buffer, see Sambrook below). Typically, a low stringency wash is performed before a high stringency wash to remove background probe signals. For example, an example of a medium stringency wash for a duplex of more than 100 nucleotides is washing with 1x SSC at 45°C for 15 minutes. For example, an example of a low stringency wash for a duplex of more than 100 nucleotides is washing with 4-6x SSC at 40°C for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature of typically at least about 30° C. Stringent conditions may also be achieved by adding a destabilizing agent such as formamide. In general, a signal-to-noise ratio of 2 times (or higher) that observed for an unrelated probe in a particular hybridization assay indicates that specific hybridization has been detected. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins encoded by the nucleotide sequences are substantially identical. This occurs, for example, when a copy of a nucleotide sequence is produced using the maximum codon degeneracy allowed by the genetic code.

[0169] The polynucleotides and / or recombinant nucleic acid constructs (e.g., expression cassettes and / or vectors) of the invention may be codon optimized for expression. In some embodiments, the polynucleotides, nucleic acid constructs, expression cassettes and / or vectors of the editing systems of the invention (e.g., comprising / encoding sequence-specific nucleic acid binding domains (e.g., sequence-specific nucleic acid binding domains from: polynucleotide-guided endonucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), Argonaute proteins and / or CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins) (e.g., type I CRISPR-Cas effector proteins, type II CRISPR-Cas effector proteins, type III CRISPR-Cas effector proteins, type IV CRISPR-Cas s effector protein, type V CRISPR-Cas effector protein or type VI CRISPR-Cas effector protein)), nucleases (e.g., 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)), deaminase proteins / domains (e.g., adenine deaminase, cytosine deaminase), polynucleotides encoding reverse transcriptase proteins or domains, polynucleotides encoding 5'-3' exonuclease polypeptides and / or affinity polypeptides, peptide tags, etc.) can be codon-optimized for expression in plants. In some embodiments, a codon-optimized nucleic acid, polynucleotide, expression cassette, and / or vector of the invention has 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%) or more identity to a reference nucleic acid, polynucleotide, expression cassette, and / or vector that has not been codon-optimized.

[0170] The polynucleotides or nucleic acid constructs of the present invention may be operably associated with a variety of promoters and / or other regulatory elements for expression in plants and / or plant cells. Therefore, in some embodiments, the polynucleotides or nucleic acid constructs of the present invention may also include one or more promoters, introns, enhancers and / or terminators operably connected to one or more nucleotide sequences. In some embodiments, a promoter may be operably associated with an intron (e.g., Ubil promoter and intron). In some embodiments, a promoter associated with an intron may be referred to as a "promoter region" (e.g., Ubil promoter and intron) (see, e.g., SEQ ID NO: 21 and SEQ ID NO: 22).

[0171] As used herein, "operably linked" or "operably associated" in relation to a polynucleotide means that the indicated elements are functionally related to each other, and usually also physically related. Thus, as used herein, the term "operably linked" or "operably associated" refers to functionally associated nucleotide sequences on a single nucleic acid molecule. Thus, a first nucleotide sequence that is 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, then the promoter is operably associated with the nucleotide sequence. Those skilled in the art will understand that a control sequence (e.g., a promoter) does not have to be adjacent to the nucleotide sequence to which it is operably associated, as long as the function of the control sequence is to direct its expression. Thus, for example, there may be an intervening untranslated but transcribed nucleic acid sequence between a promoter and a nucleotide sequence, and the promoter may still be considered to be "operably linked" to the nucleotide sequence.

[0172] As used herein, the term "linked" in relation to a polypeptide refers to the connection of one polypeptide to another polypeptide. A polypeptide can be linked to another polypeptide (at the N-terminus or C-terminus) directly (eg, via a peptide bond) or via a linker.

[0173] The term "joint" is recognized in the art and refers to a chemical group or molecule that connects two molecules or parts, such as two domains of a fusion protein, such as, for example, a DNA binding polypeptide or domain and a peptide tag and / or a reverse transcriptase and an affinity polypeptide bound to the peptide tag; or a DNA endonuclease polypeptide or domain and a peptide tag and / or a reverse transcriptase and an affinity polypeptide bound to the peptide tag. The joint may consist of a single connecting molecule, or may contain more than one connecting molecule. In some embodiments, the joint may be an organic molecule, a group, a polymer, or a chemical moiety, such as a divalent organic moiety. In some embodiments, the joint may be an amino acid, or may be a peptide.

[0174] In some embodiments, the linker is a peptide.

[0175] In some embodiments, the length of a peptide linker useful in the invention can be from about 2 to about 100 or more amino acids, for example, 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 , 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., about 2 to about 40, about 2 to about 50, about 2 to about 60, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 5 to about 40, about 5 to about 50, about 5 to about 60 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50 In some embodiments, the peptide linker can be a GS linker.

[0176] In some embodiments, two or more polynucleotide molecules can be connected by a joint, which can be an organic molecule, a group, a polymer or a chemical part, such as a divalent organic part. Polynucleotides can be connected or fused to another polynucleotide (at the 5' end or 3' end) by covalent or non-covalent bonds or in combination (including, for example, by Watson-Crick base pairing or by one or more connecting nucleotides). In some embodiments, a polynucleotide motif of a certain structure can be inserted into another polynucleotide sequence (for example, an extension of a hairpin structure in a guide RNA). In some embodiments, connecting nucleotides can be naturally occurring nucleotides. In some embodiments, connecting nucleotides can be non-naturally occurring nucleotides.

[0177] "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, "promoter" refers to a nucleotide sequence that contains a binding site for RNA polymerase II and guides transcription initiation. In general, the promoter is located at 5' or upstream of the start of the coding region of the corresponding coding sequence. The promoter may include other elements that act as gene expression regulatory factors; for example, a promoter region. These include a TATA box consensus sequence, and generally also include a CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50: 349). In plants, the CAAT box can be replaced by an AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).

[0178] Promoters useful in the present invention may include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and / or tissue-specific promoters for use in preparing recombinant nucleic acid molecules, for example, "synthetic nucleic acid constructs" or "protein-RNA complexes." These different types of promoters are known in the art.

[0179] The selection of promoters may vary depending on the temporal and spatial requirements of expression, and may also vary depending on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge available in the art, appropriate promoters may be selected for a particular host organism of interest. Thus, for example, promoters upstream of genes that are highly constitutively expressed in model organisms are well known, and this knowledge may be easily obtained and implemented in other systems where appropriate.

[0180] 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 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 repeat carbonic anhydrase gene 1 (Pdca1) (see Walker et al. (2005) Plant Cell Rep. 23: 727-735; Li et al. (2007) Gene 403: 132-142; Li et al. (2010) Mol Biol. Rep. 37: 1143-1154). PrbcS1 and Pactin are constitutive promoters, and Pnr and Pdca1 are inducible promoters. Pnr is induced by nitrate and repressed by ammonium (Li et al. (2007) Gene 403: 132-142), and Pdca1 is induced by salt (Li et al. (2010) Mol Biol. Rep. 37: 1143-1154). In some embodiments, the promoter useful in the present invention is an RNA polymerase II (Pol II) promoter. In some embodiments, the U6 promoter or 7SL promoter from corn (Zea mays) can be used in the constructs of the present invention. In some embodiments, the U6c promoter and / or the 7SL promoter from corn 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 soybean (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.

[0181] Examples of constitutive promoters useful in plants include, but are not limited to, the cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; and U.S. Pat. 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 84:5745-5749), the 84:6624-6629), sucrose synthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148) and ubiquitin promoter. Constitutive promoters derived from ubiquitin accumulate in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al. (1991) Plant Science 79:87-94), corn (Christensen et al. (1989) Plant Molec. Biol. 12:619-632) and Arabidopsis thaliana (Norris et al. (1993) Plant Molec. Biol. 21:895-906). The corn ubiquitin promoter (UbiP) has been developed in a transgenic monocot system, 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 sequence of the present invention in transgenic plants, especially monocotyledons. In addition, the promoter expression cassette described by McElroy et al. ((1991) Mol. Gen. Genet. 231: 150-160) can be easily modified for expression of the nucleotide sequence of the present invention, and is particularly suitable for monocotyledon hosts.

[0182] 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 tissues include many promoters of genes involved in photosynthesis, many of which are cloned from monocots and dicots. In one embodiment, a promoter useful in the present invention is the corn PEPC promoter (Hudspeth and Grula (1989) Plant Molec.Biol.12:579-589) from the phosphoenol carboxylase gene. 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 oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase, and fatty acid desaturase (fad 2-1), as well as other nucleic acids expressed during embryo development (such as Bce4, see, e.g., 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 corn, 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 (incorporated herein by reference in its entirety).Other non-limiting examples of tissue-specific or tissue-preferential promoters that can be used in the present invention are the cotton rubisco promoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Pat. No. 5,604,121; the root-specific promoter described by de Framond ((1991) FEBS 290:103-106; EP 0 452 269 to Ciba-Geigy); the stem-specific promoter described in U.S. Pat. No. 5,625,136 (to Ciba-Geigy), which drives expression of the maize trpA gene; the tuberose yellow leaf curl virus promoter disclosed in WO 01 / 73087; and pollen-specific or -preferential promoters, including but not limited to ProOsLPS10 and ProOsLPS11 from rice (Nguyen et al. (2015) Plant Biotechnol. Reports 9(5):297-306), ZmSTK2_USP from maize (Wang et al. (2017) Genome 60(6):485-495), LAT52 and LAT59 from tomato (Twell et al. (1990) Development 109(3):705-713), Zm13 (U.S. Pat. No. 10,421,972), PLA2-δ promoter from Arabidopsis thaliana (U.S. Pat. No. 7,141,424) and / or ZmC5 promoter from maize (International PCT Publication No. WO1999 / 042587).

[0183] 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. (2006) The Plant Cell 18:2958-2970), the root-specific promoters RCc3 (Jeong et al. (2010) Plant Physiol. 153:185-197) and RB7 (U.S. Pat. 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 synthetase (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, 1996), 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), the Ti plasmid mannopine 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), truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810-812), potato glycoprotein promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354), root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), zein promoter (Kriz et al. (1987) Mol. Gen. Genet. 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18: 7449; and Wandelt et al. (1989) Nucleic Acids Res. 17: 2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129: 863-872), α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet. 215: 431-440), PEPCase promoter (Hudspeth and Grula (1989) Plant Mol. Biol. 12: 579-589), R gene complex-associated promoter (Chandler et al. (1989) Plant Cell 1: 1175-1183) and chalcone synthase promoter (Franken et al. (1991) EMBO J. 10: 2605-2612).

[0184] Useful for seed-specific expression is the vicilin promoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; and seed-specific promoters disclosed in U.S. Pat. No. 5,625,136). Useful promoters for expression in mature leaves are those that switch at the onset of senescence, such as the SAG promoter from Arabidopsis thaliana (Gan et al. (1995) Science 270:1986-1988).

[0185] In addition, promoters that are functional in chloroplasts can be used. Non-limiting examples of such promoters include phage 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).

[0186] 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.

[0187] Introns that can be used in the present invention can be identified in plants and separated from plants, and then inserted into introns in expression cassettes for plant transformation. As will be appreciated by those skilled in the art, introns can include sequences required for self-excision and are incorporated into nucleic acid constructs / expression cassettes in frame. Introns can be used as spacers to separate multiple protein coding sequences in a nucleic acid construct, or introns can be used in a protein coding sequence, for example, to stabilize mRNA. If they are used in a protein coding sequence, they are inserted "in frame" and include excision sites. Introns can also be associated with promoters to improve or modify expression. As an example, promoter / intron combinations that can be used in the present invention include but are not limited to promoter / intron combinations of corn Ubil promoters and introns (see, for example, SEQ ID NO:21 and SEQ ID NO:22).

[0188] Non-limiting examples of introns that can be used in the present invention include introns from the following genes: ADHI gene (e.g., Adh1-S intron 1, 2 and 6), ubiquitin gene (Ubil), 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), repeated carbonic anhydrase gene 1 (Tdca1), psbA gene, atpA gene, or any combination thereof.

[0189] In some embodiments, the polynucleotides and / or nucleic acid constructs of the present invention may be "expression cassettes", or may be contained within expression cassettes. As used herein, "expression cassettes" refer to recombinant nucleic acid molecules comprising, for example, one or more polynucleotides of the present invention (e.g., polynucleotides encoding sequence-specific nucleic acid binding domains, polynucleotides encoding deaminase proteins or domains, polynucleotides encoding reverse transcriptase proteins or domains, polynucleotides encoding 5'-3' exonuclease polypeptides or domains, guide nucleic acids and / or reverse transcriptase (RT) templates), wherein the polynucleotides are operably associated with one or more control sequences (e.g., promoters, terminators, etc.). Thus, in some embodiments, one or more expression cassettes may be provided, which are designed to express, for example, a nucleic acid construct of the invention (e.g., a polynucleotide encoding a sequence-specific nucleic acid binding domain (e.g., a sequence-specific DNA 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 comprising a guide nucleic acid, an extended guide nucleic acid, and / or an RT template, etc.). When the expression cassette of the invention comprises more than one polynucleotide, the polynucleotide may be operably linked to a single promoter that drives expression of all the polynucleotides, or the polynucleotide may be operably linked to one or more separate promoters (e.g., three polynucleotides may be driven by one, two, or three promoters in any combination). When two or more separate promoters are used, the promoters may be the same promoter, or they may be different promoters. Thus, when contained in a single expression cassette, a polynucleotide encoding a sequence-specific nucleic acid binding domain, a polynucleotide encoding a nuclease protein / domain, a polynucleotide encoding a CRISPR-Cas effector protein / domain, a polynucleotide encoding a deaminase protein / domain, a polynucleotide encoding a reverse transcriptase polypeptide / domain (e.g., an RNA-dependent DNA polymerase) and / or a 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 separate promoters in any combination.

[0190] The expression cassette comprising the nucleic acid construct of the invention may 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, wherein the polynucleotide of interest is from an organism different from the host, or is not normally associated with the promoter). The expression cassette may also be naturally occurring, but has been obtained in a recombinant form that can be used for heterologous expression.

[0191] The expression cassette may optionally include a transcription and / or translation termination region (i.e., termination region) and / or an enhancer region that is functional in the selected host cell. Various transcription terminators and enhancers are known in the art and can be used in expression cassettes. Transcription terminators are responsible for terminating transcription and correcting mRNA polyadenylation. Termination regions and / or enhancer regions may be natural to the transcription initiation region, may be natural to the following: for example, a gene encoding a sequence-specific DNA binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase, etc., or may be natural to a host cell, or may be natural to another source (e.g., to a promoter, a gene encoding a sequence-specific DNA binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase, etc., or to a host cell, or any combination thereof, exogenous or heterologous).

[0192] The expression cassette of the present invention may also include a polynucleotide encoding a selectable marker that can be used to select transformed host cells. As used herein, a "selectable marker" refers to a polynucleotide sequence that, when expressed, imparts a unique phenotype to the host cell expressing the marker, thereby allowing such transformed cells to be distinguished from cells without the marker. This polynucleotide sequence can encode a selectable or screenable marker, depending on whether the marker imparts a trait that can be selected by chemical means, such as by using a selection agent (e.g., antibiotics, etc.), or whether the marker is simply a trait that can be identified 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 expression cassettes described herein.

[0193] In addition to expression cassettes, nucleic acid molecules / constructs and polynucleotide sequences described herein can also be used in combination with vectors. The term "vector" refers to a composition for transferring, delivering or introducing nucleic acids (or multiple nucleic acids) into a cell. Vectors include nucleic acid constructs (e.g., expression cassettes) comprising nucleotide sequences to be transferred, delivered or introduced. Vectors for transforming host organisms are well known in the art. Non-limiting examples of general class vectors include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, phages, artificial chromosomes, minicircles or double-stranded or single-stranded linear or circular forms of Agrobacterium binary vectors, which may or may not be self-transferable or removable. In some embodiments, viral vectors may include, but are not limited to, retroviruses, slow viruses, adenoviruses, adeno-associated viruses or herpes simplex virus vectors. Vectors as defined herein can be transformed prokaryotic or eukaryotic hosts by being integrated into the cell genome or being present outside the chromosome (e.g., autonomously replicating plasmids with a replication origin). In addition, shuttle vectors are also included, and shuttle vectors refer to DNA vectors that can be naturally or intentionally replicated in two different host organisms, and host organisms can be selected from actinomycetes and related species, bacteria and eukaryotic organisms (for example, higher plants, mammals, yeast or fungal cells). In some embodiments, the nucleic acid in the vector is controlled by a suitable promoter or other regulatory elements, and is operably connected to a suitable promoter or other regulatory elements so as to be transcribed in a host cell. The vector can be a bifunctional expression vector that works in a variety of hosts. In the case of genomic DNA, this can include its own promoter and / or other regulatory elements, and in the case of cDNA, this can be controlled by a suitable promoter and / or other regulatory elements so as to be expressed in a host cell. Therefore, the nucleic acid or polynucleotide of the present invention and / or the expression cassette comprising the nucleic acid or polynucleotide can be included in a vector as described herein and known in the art.

[0194] As used herein, "contact," "contacting," "contacted," and grammatical variations thereof refer to bringing together the components of a desired reaction under conditions suitable for carrying out 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 DNA 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 these under the following conditions: the sequence-specific DNA binding protein, the reverse transcriptase and the deaminase are expressed and the sequence-specific DNA binding protein binds to the target nucleic acid, and the reverse transcriptase and / or the deaminase can be fused to the sequence-specific DNA binding protein or recruited to the sequence-specific DNA binding protein (e.g., by a peptide tag fused to the sequence-specific DNA binding protein and an affinity tag fused to the reverse transcriptase and / or the deaminase), so that the deaminase and / or the reverse transcriptase are located near the target nucleic acid, thereby modifying the target nucleic acid. Other methods of recruiting reverse transcriptases and / or deaminases 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.

[0195] As used herein, "modification" ("modifying" or "modification") of a target nucleic acid includes editing (e.g., mutation), covalently changing, exchanging / replacing nucleic acid / nucleotide bases, deleting, cutting, creating nicks, and / or changing the transcriptional control of the target nucleic acid. In some embodiments, the modification may include one or more single base changes (SNPs) of any type.

[0196] The term "regulate," as used in the context of a transcription factor "regulating" a phenotype, e.g., a response to light (e.g., a light response, e.g., a shade avoidance response), refers to the ability of the transcription factor to affect the expression of one or more genes, thereby changing the phenotype, e.g., the response to light.

[0197] "Introducing", "introduce", "introduced" (and grammatical variations thereof) in the context of a polynucleotide of interest refers to presenting a nucleotide sequence of interest (e.g., a polynucleotide, RT template, nucleic acid construct and / or guide nucleic acid) to a plant, a plant part thereof, or a cell thereof in a manner that enables the nucleotide sequence to enter the interior of the cell.

[0198] The terms "transformation" or "transfection" are used interchangeably and as used herein refer to the introduction of a heterologous nucleic acid into a cell. The transformation of a cell can be stable or transient. Thus, in some embodiments, a host cell or host organism (e.g., a plant) can be stably transformed with a polynucleotide / nucleic acid molecule of the present invention. In some embodiments, a host cell or host organism can be transiently transformed with a polynucleotide / nucleic acid molecule of the present invention.

[0199] "Transient transformation" in the context of a polynucleotide means that the polynucleotide is introduced into a cell but is not integrated into the genome of the cell.

[0200] "Stably introduced" or "stably introduced" in the context of a polynucleotide being introduced into a cell means that the introduced polynucleotide is stably incorporated into the genome of the cell, so that the cell is stably transformed with the polynucleotide.

[0201] As used herein, "stable transformation" or "stably transformed" refers to a nucleic acid molecule that is introduced into a cell and integrated into the genome of the cell. Thus, the integrated nucleic acid molecule is capable of being inherited by its progeny, more specifically, by successive generations of progeny. As used herein, "genome" includes nuclear and plastid genomes, and thus includes integrating the nucleic acid into, for example, a chloroplast or mitochondrial genome. As used herein, stable transformation may also refer to a transgene that is maintained extrachromosomally, for example, as a minichromosome or plasmid.

[0202] Transient transformation can be detected by, for example, enzyme-linked immunosorbent assay (ELISA) or Western blotting, which can detect the presence of peptides or polypeptides encoded by one or more transgenes introduced into an organism. Stable transformation of cells can be detected by, for example, Southern blot hybridization assays of the genomic DNA of the cells with nucleic acid sequences that specifically hybridize with nucleotide sequences of transgenes introduced into an organism (e.g., a plant). Stable transformation of cells can be detected by, for example, Northern blot hybridization assays of the RNA of the cells with nucleic acid sequences that specifically hybridize with nucleotide sequences of transgenes introduced into a host organism. Stable transformation of cells can also be detected by, for example, polymerase chain reaction (PCR) or other amplification reactions well known in the art, which employ specific primer sequences that hybridize with target sequences of transgenes, resulting in amplification of transgenic sequences, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0203] 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 polynucleotides for editing as described herein) can be transiently introduced into a cell along with a guide nucleic acid, and therefore, DNA is not maintained in the cell.

[0204] The nucleic acid construct 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 bacterial-mediated nucleic acid delivery (e.g., by Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated transformation, cyclodextrin-mediated transformation, electroporation, nanoparticle-mediated transformation, ultrasonic treatment, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical) and / or biological mechanism for introducing nucleic acid into plant cells, including any combination thereof. The procedures for transforming eukaryotic organisms 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. (2013) Nature Protocols 8: 2281–2308). General guides to the 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, BR and Thompson, JE, eds. (CRC Press, Inc., Boca Raton, 1993), pp. 67-88) and Rakowoczy-Trojanowska ((2002) Cell. Mol. Biol. Lett. 7:849-858).

[0205] In some embodiments of the present invention, the transformation of cells may include nuclear transformation. In other embodiments, the transformation of cells may include plastid transformation (e.g., chloroplast transformation). In further embodiments, nucleic acids of the present invention may be introduced into cells by conventional breeding techniques. In some embodiments, one or more of polynucleotides, expression cassettes and / or vectors may be introduced into plant cells by Agrobacterium transformation.

[0206] Therefore, polynucleotide can be introduced into plant, plant part, plant cell in any number of ways known in the art.Method of the present invention does not rely on the ad hoc method for introducing one or more nucleotide sequences into plant, as long as they can enter the interior of the cell.If more than one polynucleotide is to be introduced, they can be assembled as a part of a single nucleic acid construct or assembled as an independent nucleic acid construct, and can be located on the same or different nucleic acid constructs.Therefore, polynucleotide can be introduced into interested cell in a single transformation event, or introduced into interested cell in an independent transformation event, or alternatively, polynucleotide can be incorporated into plant as a part of a breeding scheme.

[0207] Plants respond to neighboring plants in order to better compete with them for resources, particularly access to light. While this is an adaptive advantage in natural or wild environments, in monocrop agriculture, plants compete with the same species of crops that contribute to yield together, so when averaged across a farm, the net gain of individual plants is lost. This response to neighboring plants is called the shade avoidance response (SAR), which results in poor plant vigor and reduced plant yield (shade avoidance syndrome; SAS). For example, corn yield (bushels per acre) has steadily increased through intensive breeding. However, increases in yield have recently begun to level off, and significant investments in field evaluation and breeding are required to demonstrate genetic gain. New genetic modification methods are needed to significantly increase yield, which is not possible through traditional methods. Crop yield can be increased in two fundamentally different ways: 1) an increase in yield itself, in which engineered plants gain advantages, such as improved photosynthesis or optimized carbohydrate partitioning, or 2) the removal of degenerate survival mechanisms that are inconsistent with high-yield agriculture. The shade avoidance response (SAR) or shade avoidance syndrome (SAS) is such a survival mechanism. SAS / SAR is characterized by an increased root-to-shoot ratio, increased plant height, and reduced yield per plant, and in typical monoculture crop environments, this response to competition is a wasteful survival mechanism.

[0208] The environmental signal that plants use to detect neighboring plants is a change in the R:FR light ratio, where light reflected from leaf tissue is shifted toward FR wavelengths relative to light reaching the plant directly. Plants use a system of phytochromes to detect wavelengths of light and signal changes in the plant.

[0209] The present invention solves the problems associated with increased planting density and reduced yield losses (on an acre basis) by using gene editing to modify key regulatory factors that trigger crop shade avoidance. Plants with such edited genomes will have reduced shade avoidance capabilities. HY5 encodes a basic leucine zipper (bZIP) transcription factor and plays a role in promoting photomorphogenesis. The HY5 gene regulates the shade avoidance response through its interaction with the constitutive photomorphogenesis 1 (COP1) polypeptide. The activity of HY5 is negatively regulated by COP1. COP1 contains three conserved domains important for protein-protein interactions: an N-terminal Ring finger domain, a coiled coil for dimerization, and a WD-40 repeat domain for substrate recognition. In the dark, the COP1 protein is mainly located in the nucleus and becomes abundant in the cytoplasm when exposed to light. COP1 acts as an E3 ligase and degrades transcription factors (such as HY5) in the dark by the 26S proteasome, thereby reducing sensitivity to light signaling. COP1 is degraded in the absence of shade. The abundance of HY5 protein is negatively correlated with light intensity and is regulated by darkness-dependent degradation mediated by the 26S proteasome through interaction with COP1.

[0210] The methods of the present invention relate to editing the HY5 gene, thereby reducing or eliminating the effects of the shade response in a plant or part thereof comprising the edited HY5 gene, for example, so that the activity of the encoded HY5 polypeptide is no longer regulated by COP1, for example, thereby reducing the regulation of the HY5 transcription factor by COP1. In some embodiments, the HY5 gene can be edited so as to modify the COP1 binding site in the encoded HY5 transcription factor, thereby reducing or eliminating the binding of COP1 to the altered HY5 transcription factor. When the plant is under light (e.g., not experiencing far-red light), COP1 is located in the cytoplasm, where it does not interact with other proteins. When the plant experiences far-red light, COP1 is in the nucleus, where it can interact with the HY5 transcription factor, resulting in the degradation of the HY5 transcription factor. Without wishing to be limited by any particular theory, when the plant experiences far-red light, the HY5 transcription factor modified as described herein will not degrade, and therefore, the plant will not show morphological changes associated with shade avoidance. A single copy of the HY5 gene modified as described herein may be sufficient to reduce or eliminate SAR / SAS, since production of the HY5 transcription factor polypeptide modified so as not to be regulated by COPl should accumulate when the plant is subjected to far-red light.

[0211] The types of mutations that can be used to generate plants with mutations in endogenous HY5 genes that result in plants having reduced / inhibited shade avoidance responses, e.g., substitutions, deletions, and / or insertions. In some aspects, mutations in endogenous HY5 genes can result in mutant HY5 polypeptides having enhanced activity. In some aspects, the mutations generated by the editing technique can be point mutations. In some embodiments, the mutations can be non-natural mutations.

[0212] Thus, the present invention provides a plant or part thereof comprising at least one mutation (e.g., one or more mutations) in an endogenous elongated hypocotyl 5 (HY5) gene encoding a HY5 transcription factor, wherein the HY5 transcription factor encoded by the endogenous gene is capable of regulating a response to light (e.g., a shade avoidance response (SAR)) in a plant. The HY5 transcription factor useful in the present invention comprises a constitutive photomorphogenesis 1 (COP1) binding site. COP1 is a ubiquitin ligase (e.g., an E3 ubiquitin ligase, RING refers to a ubiquitin ligase). In some embodiments, the at least one mutation may be located in the COP1 binding site of the HY5 transcription factor encoded by the HY5 gene. In some embodiments, the at least one mutation may disrupt binding of a COP1 polypeptide to the HY5 transcription factor in the plant or part thereof, optionally wherein the at least one mutation results in the HY5 transcription factor having no or reduced COP1 binding. In some embodiments, the binding of COPl to a HY5 transcription factor mutated as described herein can be reduced by about 30% to about 100% (e.g., about 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%, 121%, 122%, 123%, 124%, 125 %, 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%, or any range or value thereof).

[0213] In some embodiments, the endogenous gene encoding the HY5 transcription factor useful in the present invention: (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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106. In some embodiments, the at least one mutation may be in a region of the endogenous HY5 transcription factor that has at least 80% sequence identity with SEQ ID NO:106 (SSSERSSSSGHHVDMEVKEGMESDDEIRRVPELGLEL), 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 at least 100%.

[0214] In some embodiments, a plant or plant part of the invention comprises at least one mutation (e.g., one or more mutations) in an endogenous elongated hypocotyl 5 (HY5) gene encoding a HY5 transcription factor, optionally wherein the mutation disrupts binding of the HY5 transcription factor by COPl in the plant or part thereof, wherein the endogenous HY5 transcription factor 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; (c) encodes a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; NO:74; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, 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 at least 100%. In some embodiments, the HY5 transcription factor comprises a COPl binding domain in a region of the HY5 transcription factor polypeptide that has at least about 80% identity to SEQ ID NO:106, optionally wherein 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 at least 100%.

[0215] The mutation in the endogenous elongated hypocotyl 5 (HY5) transcription factor gene in the plant can be any type of mutation, including but not limited to base substitution, base deletion and / or base insertion. In some embodiments, the mutation in the endogenous HY5 gene causes the HY5 transcription factor to disrupt the binding of the constitutive photomorphogenesis 1 (COP1) ubiquitin ligase to the HY5 transcription factor. For example, the mutation can be a substitution, deletion and / or insertion of one or more bases of the transcription factor gene or one or more amino acid residues of the HY5 transcription factor. In some embodiments, the at least one mutation is an out-of-frame deletion or an in-frame deletion. In some embodiments, the at least one mutation is an in-frame deletion. In some embodiments, the at least one mutation may include a base substitution for A, T, G or C, which results in an amino acid substitution, thereby disrupting the binding of the COPl polypeptide to the HY5 transcription factor. In some embodiments, the mutation that can be used in the present invention is a non-natural mutation.

[0216] In some embodiments, at least one mutation (e.g., one or more mutations) in the endogenous HY5 gene may comprise a deletion. In some embodiments, the endogenous HY5 gene encodes a HY5 transcription factor polypeptide comprising a COPl binding domain, and the deletion in the HY5 gene is an in-frame deletion comprising the HY5 transcription factor gene (e.g., SEQ ID 72), a deletion of at least three consecutive base pairs (e.g., at least 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 114, 117, 120 or more consecutive base pairs. In some embodiments, the deletion in the endogenous HY5 transcription factor gene can be a deletion of part or all of the COPl binding region, optionally wherein the deletion is an in-frame deletion.

[0217] In some embodiments, at least one mutation (e.g., one or more mutations) may produce a dominant negative mutation or a dominant mutation. In some embodiments, the mutation in the endogenous HY5 gene useful in the present invention is a dominant negative mutation. In some embodiments, the dominant negative mutation results in the loss of binding of COPl to the HY5 transcription factor, thereby reducing the function of the encoded HY5 transcription factor.

[0218] In some embodiments, plants comprising a mutation in the HY5 gene as described herein can be planted at an increased density without reducing plant yield on a per plant basis as compared to a control, optionally wherein the planting density is increased by about 5% to about 75% (e.g., about 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%, 111%, 112%, 113%, 94%, 95%, 96%, 97%, 98%, 99% or 100% or any range or value therein) without reducing plant yield on a per plant basis. For comparison purposes, for example, for corn, the standard planting density can be about 25,000 plants per acre to about 35,000 plants, while for example, for soybeans, the standard planting density can be in the range of about 100,000 plants per acre to about 125,000 plants. As understood in the art, the standard planting density will vary at least according to the crop species. In some embodiments, plants comprising a mutation in the HY5 gene when planted in close proximity to one or more plants may exhibit increased yield, increased upright growth, reduced height, reduced root-to-crown ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, essentially no change in flowering time, increased photosynthetic efficiency and grain filling, and / or enhanced defense responses to pathogens and herbivores compared to control plants planted in close proximity.

[0219] "Close proximity" refers to a high planting density of any particular plant species that can result in SAR. For example, in some embodiments, "close proximity" includes seeds of plants spaced about 6.1 inches or less apart (e.g., about 6.1, 6, 5.9, 5.8, 5.7, 5.6, 5.5, 5.4, 5.3, 5.2, 5.2, 5.1, 5, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4, 3.9, 3.8, 3.7 , 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1, 0.9, 0.8, 0.7, 0.6, 0.5 inches, etc., or any range or value therein). As will be appreciated by those skilled in the art, to achieve high density planting, the number of seeds planted per acre will vary depending on the plant species. For example, high density planting of corn includes more than 35,000 seeds per acre with a row spacing of 30 inches or greater.

[0220] In some embodiments, the mutation can result in a mutated HY5 gene having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, or 139 and / or encoding a mutated HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138.

[0221] In some embodiments, a plant cell comprising an editing system is provided, the editing system comprising: (a) a CRISPR-Cas-associated effector protein; and (c) a guide nucleic acid (gRNA, gDNA, crRNA, crDNA) having a spacer sequence complementary to an endogenous target gene encoding an elongated hypocotyl 5 (HY5) transcription factor. The endogenous HY5 transcription factor can be any HY5 transcription factor involved in the shade avoidance response. In some embodiments, the endogenous HY5 gene encodes a basic leucine zipper (bZIP) transcription factor (e.g., a HY5 transcription factor). In some embodiments, the HY5 transcription factor gene that shares complementarity with the spacer sequence of the guide 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:75. NO:106 has a region of at least 80% sequence identity, 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 at least 100%. In some embodiments, the spacer sequence useful in the present invention may include, but is not limited to, the nucleotide sequence of any one of SEQ ID NO:107-113, or its reverse complement, or a combination thereof.

[0222] In some embodiments, a plant cell is provided that comprises at least one mutation in an endogenous elongating hypocotyl 5 (HY5) gene, wherein the mutation is a substitution, insertion and / or deletion introduced into the endogenous HY5 gene using an editing system comprising a nucleic acid binding domain that binds to a target site within the endogenous HY5 gene, wherein the endogenous HY5 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; (c) encodes a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; NO:74 amino acid sequence having at least 80% sequence identity; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, 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 at least 100%. In some embodiments, the nucleic acid binding domain of the editing system may be 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.

[0223] In some embodiments, the deletion or insertion produced in the endogenous HY5 gene can be from 1 base pair to about 100 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 98, 99, or 100 base pairs, or any range or value therein) or more. In some embodiments, the deletion can be an in-frame deletion or an out-of-frame deletion. In some embodiments, the mutation may be a deletion in a region of the HY5 gene encoding a COPl binding site, optionally wherein the region may encode a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 106 (optionally, the sequence identity may be at least 90% or it may be at least 95%, optionally the sequence identity may be at least 100%). In some embodiments, the mutation may be a deletion of all or part of the COPl binding domain of the HY5 transcription factor. In some embodiments, the mutation may be a base substitution of A, T, G or C, optionally wherein the base substitution results in an amino acid substitution. In some embodiments, the mutation is a non-natural mutation.

[0224] Plants or plant parts useful in the present invention may be dicots or monocots. Non-limiting examples of plants useful in the present invention or parts thereof include, but are not limited to, corn, soybean, rape, 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 species (Brassica spp.). In some embodiments, plant part can be a cell from a plant, and the plant includes but is not limited to corn, soybean, rape, wheat, rice, cotton, sugarcane, beet, barley, oat, 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 species. In some embodiments, plant or its part can be corn. In some embodiments, plant can be regenerated from plant cell or plant part of the present invention. In some aspects, plant cell can be a non-reproduction plant cell that no longer generates a plant. As compared with a plant lacking at least one mutation, the plant of the present invention comprising at least one mutation in the HY5 transcription factor gene can include a weakened shade avoidance response (SAR). In some embodiments, plants comprising at least one mutation in the HY5 gene may exhibit increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, essentially no change in flowering time, increased photosynthesis efficiency and grain filling, and / or enhanced defense responses to pathogens and herbivores when planted in close proximity to one or more plants, compared to control plants planted in close proximity.

[0225] Also provided herein is a method of providing a plurality of plants having an increase in yield (e.g., increased floret fertility, increased seed number, and / or increased seed weight) when each plant in the plurality of plants is planted in close proximity to one another, the method comprising planting two or more plants of the invention (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, 200, 300, 400, 500, 1000, 2000, 3000, 400, 5000, or 10,000 or more plants of the invention) (e.g., in the HY5 transcription factor gene Comprising a mutation and having a reduced shade avoidance response, optionally as compared to a control plant planted closely adjacent, when planted closely adjacent to one or more plants, exhibiting increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, substantially no change in flowering time, increased photosynthetic efficiency and grain filling, and / or enhanced defense response to pathogens and herbivores), thereby providing a plurality of plants with increased yield compared to a plurality of control plants not comprising the mutation (e.g., as compared to an isogenic wild-type plant not comprising the mutation). The growing area can be any area where a plurality of plants can be planted together, including but not limited to fields (e.g., cultivated land, farmland), growth chambers, greenhouses, recreational areas, lawns, and / or roadsides, etc.

[0226] In some embodiments, a method for producing / cultivating an edited plant that does not contain a transgene is provided, the method comprising: crossing a plant of the present invention (e.g., a plant comprising a mutation in a HY5 transcription factor gene as described herein (and having a reduced shade avoidance response when planted in close proximity to one or more plants as compared to a control plant planted in close proximity, and optionally exhibiting increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, essentially no change in flowering time, increased photosynthetic efficiency and grain filling, and / or enhanced defense response to pathogens and herbivores) with a plant that does not contain a transgene, thereby introducing at least one mutation (e.g., one or more mutations) into the plant that does not contain a transgene (e.g., into a progeny plant); and selecting a progeny plant that comprises the at least one mutation and does not contain a transgene, thereby producing an edited (e.g., base-edited) plant that does not contain a transgene.

[0227] In some embodiments, the present invention provides a method for generating a mutation in an endogenous Elongated Hypocotyl 5 (HY5) gene of a plant, the method comprising: (a) targeting a gene editing system to a portion of the HY5 gene, the portion 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 to SEQ ID NOs: 75-105, optionally the sequence identity can be at least 90% or it can be at least 95%, optionally the sequence identity can be at least 100%); and (b) selecting a plant comprising a modification located in a region of the HY5 gene having at least 80% sequence identity to any one of SEQ ID NOs: 75-105. In some embodiments, plants comprising a mutation in an endogenous HY5 gene may exhibit increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, essentially no change in flowering time, increased photosynthetic efficiency and grain filling, and / or enhanced defense responses to pathogens and herbivores when planted in close proximity to one or more plants, compared to control plants planted in close proximity.

[0228] In some embodiments, a method for generating a variation in an Elongating Hypocotyl 5 (HY5) gene is provided, the method comprising: introducing an editing system into a plant cell, wherein the editing system targets a region of the HY5 gene encoding a HY5 transcription factor; and contacting the region of the HY5 gene with the editing system, thereby introducing a mutation into the HY5 gene and generating a variation in the HY5 gene in the plant cell. In some embodiments, the variant HY5 gene 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 either SEQ ID NO:72 or SEQ ID NO:73, and / or encodes an amino acid sequence having at least 80% sequence identity to SEQ ID NO:74, optionally wherein 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 at least 100%. In some embodiments, the region of the HY5 gene that may be targeted comprises at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-105, optionally wherein the region of the HY5 gene that may be targeted encodes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 106, optionally wherein 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 at least 100%. In some embodiments, contacting a region of an endogenous HY5 gene in a plant cell with an editing system produces a plant cell comprising an edited endogenous HY5 gene in its genome. In some embodiments, the method may further comprise (a) regenerating a plant from the plant cell; (b) selfing the plant to produce an offspring plant (E1); (c) determining the reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) of the offspring plant of (b); and (d) selecting an offspring plant that exhibits a reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) compared to a control plant lacking the mutation. In some embodiments, the method may further include (e) selfing the selected offspring plant of (d) to produce an offspring plant (E2); (f) determining the reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) of the offspring plant of (e); and (g) selecting offspring plants that exhibit a reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) compared to control plants, optionally repeating (e) to (g) one or more times.

[0229] In some embodiments, a method for detecting a mutated HY5 transcription factor gene (a mutation in an endogenous HY5 transcription factor gene) is provided, the method comprising detecting in a plant genome a mutation in any one of the nucleotide sequences, e.g., SEQ ID NO: 72 or SEQ ID NO: 73, optionally wherein the mutation is a deletion of at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides), optionally an in-frame deletion. In some embodiments, a method for detecting a mutated HY5 gene (mutation in an endogenous HY5 gene) in a plant or plant part (e.g., a plant cell) is provided, the method comprising detecting in the plant genome a HY5 gene having at least one mutation within a region 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: 75-105, optionally the sequence identity may be at least 90% or it may be at least 95%, optionally the sequence identity may be at least 100%), optionally wherein the mutation is a deletion or insertion of at least one nucleotide (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or more nucleotides).

[0230] In some embodiments, a method for detecting a mutation in an endogenous HY5 gene is provided, the method comprising detecting a mutant HY5 gene in a plant genome. In some embodiments, the mutant HY5 gene comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137, or 139, and / or encodes a mutant HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138.

[0231] In some embodiments, a method for editing a specific site in the genome of a plant cell is provided, the method comprising cleaving a target site within an endogenous elongated hypocotyl 5 (HY5) gene in a plant cell in a site-specific manner, the endogenous HY5 gene: (a) 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encoding a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:75. The amino acid sequence of NO:106 has a region of at least 80% sequence identity, 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 at least 100%, thereby producing an edit in the endogenous HY5 gene of the plant cell. In some embodiments, the HY5 gene encodes a HY5 transcription factor comprising a COP1 binding region, and the editing results in a mutation in the COP1 binding region of the HY5 transcription factor. In some embodiments, the editing can result in a HY5 transcription factor having no or reduced COPl binding, optionally wherein the binding of the COPl polypeptide to the HY5 transcription factor can be reduced by about 30% to about 100% (e.g., about 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%, 121%, 122%, 123%, 124%, 125 %, 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%).In some embodiments, the edit may be located in a region of the HY5 transcription factor 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 at least 95%, optionally or at least 100%) to SEQ ID NO: 106.

[0232] In some embodiments, the editing in the endogenous HY5 gene can result in an endogenous HY5 gene that has at least 90% (e.g., 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 at least 100%) with any of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139 and / or encodes a mutated HY5 polypeptide that has at least 90% sequence identity with any of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138.

[0233] In some embodiments, plants can be regenerated from plant cells comprising an edit in an endogenous HY5 transcription factor gene to produce plants comprising an edit in its endogenous HY5 transcription factor gene. In some embodiments, the plant is not regenerated from a plant cell. In some embodiments, a plant comprising an edit in its endogenous HY5 transcription factor gene exhibits a weakened shade avoidance response compared to a control plant that does not comprise an edit. In some embodiments, a plant comprising an edit in an endogenous HY5 gene may exhibit increased yield, increased upright growth, reduced height, reduced root-to-crown ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, substantially no change in flowering time, increased photosynthesis efficiency and grain filling, and / or enhanced defense responses to pathogens and herbivores when planted in close proximity to one or more plants compared to a control plant planted in close proximity.

[0234] Plants comprising an endogenous HY5 transcription factor gene edited as described herein to provide a HY5 transcription factor with reduced COP1 binding can exhibit a reduced shade avoidance response (optionally also exhibiting increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, essentially no change in flowering time, increased photosynthesis efficiency and grain filling, and / or enhanced defense responses to pathogens and herbivores) when compared to control plants that do not comprise the edited endogenous HY5 transcription factor gene. Plants comprising an edited endogenous HY5 transcription factor gene as described herein can be compared to plants that have not been so edited when grown under the same environmental conditions, e.g., an environment with a low R:FR light ratio, such as shade conditions (e.g., an R:FR ratio of about 0.16; or an R:FR ratio ranging from about 0.09 to about 0.7 (e.g., about 0.09, 0.10, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 018, 0.19, 0.2, 0.21, 0.23, 0.24, 0.25 to about 0.26, 0.27, 0.28, 0.29, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, or any range or value therein)).

[0235] In some embodiments, a method of making a plant is provided, the method comprising: (a) contacting a population of plant cells comprising an endogenous gene encoding an elongated hypocotyl 5 (HY5) transcription factor with a nuclease targeted to the endogenous gene, wherein the nuclease is linked to a nucleic acid binding domain that binds to a target site within the endogenous gene, the endogenous gene (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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105, optionally with SEQ ID NO: NO:79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105 have 80% sequence identity; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (iv) encodes a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: The amino acid sequence of NO:106 has a region of at least 80% sequence identity, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) may be at least 85% or at least 90% or it may be at least 95%, optionally the sequence identity may be at least 100%; (b) selecting plant cells from a plant cell population in which an endogenous gene encoding a HY5 transcription factor has been mutated, thereby producing plant cells comprising a mutation in the endogenous gene; and (c) growing the selected plant cells into plants comprising a mutation in the endogenous gene encoding the HY5 transcription factor, optionally wherein the mutation reduces or eliminates the ability of the HY5 transcription factor to bind to a constitutive photomorphogenesis 1 (COP1) polypeptide.

[0236] In some embodiments, a method for reducing / inhibiting a shade avoidance response in a plant is provided, the method comprising: (a) contacting a plant cell comprising an endogenous elongated hypocotyl 5 (HY5) gene with a nuclease targeted to the endogenous gene, wherein the nuclease is linked to a nucleic acid binding domain that binds to a target site within the endogenous gene, the endogenous gene (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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105, optionally with SEQ ID NO: NO:79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105 have 80% sequence identity; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (iv) encodes a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: NO:106 has a region of at least 80% sequence identity, 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 at least 100%, thereby producing a plant cell comprising a mutation in the endogenous HY5 gene; and (b) growing the plant cell into a plant, thereby reducing / inhibiting the shade avoidance response in the plant, optionally increasing yield, increasing upright growth, reducing height, reducing root-to-crown ratio, reducing leaf length, increasing stem mechanical strength, reducing lodging rate, delaying senescence, substantially no change in flowering time, increasing photosynthesis efficiency and grain filling, and / or enhancing defense responses to pathogens and herbivores when planted in close proximity to one or more plants compared to control plants planted in close proximity.

[0237] In some embodiments, a method for producing a plant or part thereof comprising at least one cell (e.g., one or more cells) having a mutation in an endogenous elongated hypocotyl 5 (HY5) gene, the method comprising contacting a target site within the endogenous HY5 gene in the plant or plant part with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain of the nuclease binds to the target site within the endogenous HY5 gene, wherein the endogenous HY5 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; NO:75-105; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, 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 at least 100%, thereby producing a plant or part thereof comprising at least one cell having a mutation in an endogenous HY5 gene. In some embodiments, at least one cell in a plant or part thereof having a mutated endogenous HY5 transcription factor gene produces a HY5 transcription factor with reduced binding to COPl.

[0238] In some embodiments, a method for producing a plant or part thereof comprising a mutation in an endogenous elongated hypocotyl 5 (HY5) gene that produces a mutant HY5 transcription factor that has reduced binding to a constitutive photomorphogen 1 polypeptide is provided, the method comprising contacting a target site within the endogenous HY5 gene in the plant or part thereof with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain of the nuclease binds to the target site within the endogenous HY5 gene, wherein the endogenous HY5 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; NO:75-105 has a region with at least 80% sequence identity, optionally with any one of SEQ ID NO:79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105 has a region with at least 80% sequence identity; (c) encodes a polypeptide comprising a sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encodes a region with at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, 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 at least 100%, thereby producing a plant or part thereof having a mutation in an endogenous HY5 gene, which mutation produces a mutant HY5 transcription factor with reduced binding to COPl.

[0239] In some embodiments, the target site can be a region of the HY5 gene or within a region of the 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 to the nucleotide sequence of any one of SEQ ID NOs: 75-105, optionally the sequence identity can be at least 90% or it can be at least 95%, optionally the sequence identity can be at least 100%), optionally with any one of SEQ ID NOs: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, or 103-105.

[0240] In some embodiments, the nuclease can be a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an endonuclease (e.g., Fok1), or a CRISPR-Cas effector protein, wherein the nuclease cleaves the endogenous HY5 gene and introduces a mutation into the HY5 gene.

[0241] In some embodiments, the mutation may be a base substitution, a base deletion and / or a base insertion, optionally a non-natural mutation. In some embodiments, the mutation is a base substitution for A, T, G or C. In some embodiments, the mutation in the HY5 gene may be a deletion or insertion. In some embodiments, the mutation may be located in the region of the HY5 gene encoding the COPl binding site, optionally wherein the region may encode a sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 106 (optionally, the sequence identity may be at least 90% or it may be at least 95%, optionally the sequence identity may be at least 100%). In some embodiments, the mutation results in the mutated HY5 transcription factor having altered activity. In some embodiments, the mutation may reduce or eliminate the ability of the HY5 transcription factor to bind to the constitutive photomorphogenesis 1 (COP1) polypeptide. In some embodiments, the mutation in the endogenous HY5 gene may be a dominant mutation and / or or a dominant negative mutation.

[0242] In some embodiments, the plant or part thereof produced by the methods of the present invention comprises a mutated endogenous HY5 transcription factor gene as described herein, and exhibits a weakened / reduced shade avoidance response compared to a control plant that does not comprise a mutation in the endogenous HY5 transcription factor gene, e.g., the plant or plant part does not have a target site within its endogenous HY5 transcription factor gene that is contacted with an editing system or a nuclease comprising a cleavage domain and a nucleic acid binding domain (e.g., a DNA binding domain). In some embodiments, a comparison with a control plant can be made between the edited plant and the control plant when grown under the same environmental conditions (e.g., a shaded environment, e.g., a low R:FR ratio environment).

[0243] Plants comprising a mutated endogenous HY5 transcription factor as described herein and exhibiting a weakened / reduced shade avoidance response when planted in close proximity to one or more other plants also exhibit one or more phenotypes, including but not limited to increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length; increased stem mechanical strength; reduced lodging rate; delayed senescence; increased photosynthetic efficiency and grain filling; essentially no change in flowering time, and / or enhanced defense responses to pathogens and herbivores, as compared to plants that do not comprise a mutated endogenous HY5 transcription factor as described herein and are planted in close proximity to one or more other plants. In some embodiments, plants with reduced SAR are at least about 5% shorter than control plants grown under the same environmental conditions (e.g., a shaded environment, e.g., a low R:FR ratio environment) (e.g., about 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 %, 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%, 110%, 120%, 130%, 140%, 150% or less, or any range or value therein).

[0244] In some embodiments, the methods of the present invention produce a plant or part thereof having a mutated HY5 gene having at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity) to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139, optionally the sequence identity may be at least 95%, optionally the sequence identity may be at least 100%) and / or encoding a mutated HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138.

[0245] In some embodiments, the nuclease contacting the plant cell, the plant cell population and / or the target site cuts the endogenous HY5 transcription factor gene, thereby introducing the mutation into the DNA binding site of the endogenous HY5 transcription factor gene. The nuclease that can be used in the present invention can be any nuclease that can be used to edit / modify the target nucleic acid. 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, any nucleic acid binding domain (e.g., DNA binding domain) that can be used for the nuclease of the present invention 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), argonautes and / or CRISPR-Cas effector DNA binding domains.

[0246] In some embodiments, a method for modifying an endogenous elongated hypocotyl 5 (HY5) gene in a corn plant or part thereof to attenuate or reduce a shade avoidance response in a corn plant or part thereof is provided, the method comprising altering a target site within an endogenous HY5 gene in a corn plant or part thereof, wherein the endogenous HY5 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; (c) encodes a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; NO:74 amino acid sequence having at least 80% sequence identity; and / or (d) encoding a region having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:106, 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 at least 100%, thereby altering the endogenous HY5 gene and attenuating or reducing the shade avoidance response in corn plants or parts thereof. In some embodiments, the target site is a region of the HY5 gene having at least 80% sequence identity with the nucleotide sequence of any one of SEQ ID NOs:75-105.

[0247] In some embodiments, a method for editing an endogenous HY5 transcription factor gene in a plant or a plant part is provided, the method comprising contacting a target site within a HY5 transcription factor gene in the plant or a plant part with a cytosine base editing system, the cytosine base editing system comprising a cytosine deaminase and a nucleic acid binding domain that binds to a target site within the HY5 transcription factor gene, wherein the HY5 transcription factor 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; (c) encodes a region having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: NO:74 has at least 80% sequence identity; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, 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 at least 100%, thereby producing a plant or part thereof comprising an endogenous HY5 transcription factor gene having a mutation. In some embodiments, the mutation reduces the binding of COPl to the HY5 transcription factor. In some embodiments, plants comprising an endogenous HY5 transcription factor gene having a mutation exhibit reduced shade avoidance response (SAR) (e.g., increased yield when planted in close proximity to other plants).

[0248] In some embodiments, a method for editing an endogenous HY5 transcription factor gene in a plant or a plant part is provided, the method comprising contacting a target site within a HY5 transcription factor gene in the plant or a plant part with an adenosine base editing system, the adenosine base editing system comprising an adenosine deaminase and a nucleic acid binding domain that binds to a target site within the HY5 transcription factor gene, wherein the HY5 transcription factor 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; (c) encodes a region having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO: NO:74 has at least 80% sequence identity; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, 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 at least 100%, thereby producing a plant or part thereof comprising an endogenous HY5 transcription factor gene having a mutation. In some embodiments, the mutation reduces the binding of COPl to the HY5 transcription factor. In some embodiments, plants comprising an endogenous HY5 transcription factor gene having a mutation exhibit reduced shade avoidance response (SAR) (e.g., increased yield when planted in close proximity to other plants).

[0249] In some embodiments, the present invention provides a method for producing a plant comprising a mutation in an endogenous HY5 transcription factor gene and at least one polynucleotide of interest, the method comprising crossing a plant of the present invention (a first plant) comprising at least one mutation in an endogenous HY5 transcription factor gene with a second plant comprising at least one polynucleotide of interest to produce offspring plants; and selecting offspring plants comprising at least one mutation in the HY5 transcription factor gene and at least one polynucleotide of interest, thereby producing a plant comprising a mutation in the endogenous HY5 transcription factor gene and at least one polynucleotide of interest.

[0250] Also provided is a method for producing a plant comprising a mutation in an endogenous HY5 transcription factor gene and at least one polynucleotide of interest, the method comprising introducing at least one polynucleotide of interest into the plant of the present invention comprising at least one mutation in the HY5 transcription factor gene, thereby producing a plant comprising at least one mutation in the HY5 transcription factor gene and at least one polynucleotide of interest.

[0251] Also provided is a method for producing a plant comprising a mutation in an endogenous HY5 transcription factor gene and exhibiting a phenotype of improved yield traits, improved plant architecture and / or improved defense traits, the method comprising crossing a first plant (i.e., a plant of the present invention (e.g., comprising at least one mutation in a HY5 transcription factor gene)) with a second plant exhibiting a phenotype of improved yield traits, improved plant architecture and / or improved defense traits; and selecting offspring plants comprising a mutation in the HY5 transcription factor gene and having a phenotype of improved yield traits, improved plant architecture and / or improved defense traits, thereby producing a plant comprising a mutation in an endogenous HY5 transcription factor gene and exhibiting a phenotype of improved yield traits, improved plant architecture and / or improved defense traits compared to a control plant.

[0252] In some embodiments, the present invention provides a method for producing a plant comprising a mutation in an endogenous Elongated Hypocotyl 5 (HY5) gene and at least one polynucleotide of interest, the method comprising crossing a first plant (i.e., a plant of the present invention (e.g., comprising at least one mutation in an endogenous HY5 gene)) with a second plant comprising at least one polynucleotide of interest to produce offspring plants; and selecting offspring plants comprising a mutation in a HY5 transcription factor gene and at least one polynucleotide of interest, thereby producing a plant comprising a mutation in an endogenous HY5 gene and at least one polynucleotide of interest.

[0253] Also provided is a method for controlling weeds in a container (e.g., a pot or seed tray, etc.), a growth chamber, a greenhouse, a field, a recreational area, a lawn, or a roadside, the method comprising applying a herbicide to one or more (several) plants of the present invention (e.g., comprising at least one mutation in an endogenous HY5 transcription factor gene) growing in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn, or a roadside, thereby controlling the weeds in the container, growth chamber, greenhouse, field, recreational area, lawn, or roadside where the one or more plants grow.

[0254] In some embodiments, methods of reducing insect predation on plants are provided, the methods comprising applying an insecticide to one or more plants of the present invention (e.g., comprising at least one mutation in an endogenous HY5 transcription factor gene), thereby reducing insect predation on the one or more plants.

[0255] In some embodiments, a method of reducing fungal diseases on plants is provided, 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 HY5 transcription factor gene), thereby reducing fungal diseases on the one or more plants, optionally wherein the one or more plants are grown in containers, growth chambers, greenhouses, fields, recreational areas, lawns, or grown on roadsides.

[0256] In some embodiments, a method of reducing bacterial diseases on plants is provided, 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 HY5 transcription factor gene), thereby reducing bacterial diseases on the one or more plants, optionally wherein the one or more plants are grown in containers, growth chambers, greenhouses, fields, recreational areas, lawns, or grown on roadsides.

[0257] The polynucleotide of interest may be any polynucleotide that can confer a desired phenotype on a plant or otherwise alter the phenotype or genotype of a plant. In some embodiments, the polynucleotide of interest may include, but is not limited to, polynucleotides that confer herbicide tolerance, insect resistance, nematode resistance, disease resistance, increased yield, increased nutrient use efficiency, and / or abiotic stress resistance.

[0258] Therefore, plants or plant cultivars to be preferentially treated according to the invention include all plants which, by genetic modification, have acquired genetic material which imparts particularly advantageous useful properties ("traits") to these plants. Examples of such properties are better plant growth, vigor, stress tolerance, uprightness, resistance to lodging, 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 maturation, higher yield, higher quality and / or higher nutritional value of the harvested product, better storage life and / or processability of the harvested product.

[0259] A further particularly emphasized example of such properties is increased resistance to animal and microbial pests, such as insects, arachnids, nematodes, mites, slugs and snails, due, for example, to toxins formed in the plant. Among the DNA sequences encoding proteins which confer tolerance properties to such animal and microbial pests, in particular insects, mention will be made in particular of the genetic material encoding the Bt protein from Bacillus thuringiensis, which is widely described in the literature and is well known to the person skilled in the art. Mention will also be made of proteins extracted from bacteria such as Photorhabdus (WO 97 / 17432 and WO 98 / 08932). In particular, BtCry or VIP proteins will be mentioned, which include CrylA, CryIAb, CryIAc, CryIIA, CryIIIA, CryIIIB2, Cry9cCry2Ab, Cry3Bb and CryIF proteins or toxic fragments thereof, and hybrids or combinations thereof, in particular CrylF protein or hybrids derived from CrylF protein (e.g., hybrid CrylA-CrylF protein or toxic fragments thereof), CrylA-type proteins or toxic fragments thereof, preferably CrylAc protein or hybrids derived from CrylAc protein (e.g., hybrid CrylAb-CrylAc protein) or CrylAb or Bt2 protein or toxic fragments thereof, Cry2Ae, Cry2Af or Cry2Ag protein or toxic fragments thereof, CrylA.105 protein or toxic fragments thereof, VIP3Aa19 protein, VIP3Aa20 protein, VIP3A protein produced in COT202 or COT203 cotton events, as described in Estruch et al. (1996), Proc Natl Acad Sci US A.28; 93 (11): 5389-94 or a toxic fragment thereof, such as the Cry protein described in WO2001 / 47952, an insecticidal protein from a strain of Xenorhabdus (such as described in WO98 / 50427), Serratia (especially from S. entomophila) or Photorhabdus, such as the Tc protein from Photorhabdus described in WO98 / 08932. In addition, any variant or mutant of any of these proteins that differs from any of the above-named sequences in some amino acids (1-10, preferably 1-5) is also included herein, and these sequences are particularly the sequences of their toxic fragments, or are fused to a transit peptide, such as a plastid transit peptide, or another protein or peptide.

[0260] Another particularly emphasized example of such properties is the conferring of tolerance to one or more herbicides, such as imidazolinone, sulfonylurea, glyphosate or glufosinate. Among the DNA sequences (i.e., polynucleotides of interest) encoding proteins that confer the properties of tolerance to certain herbicides on transformed plant cells and plants, particular mention will be made of the bar or PAT gene or the Streptomyces coelicolor gene described in WO2009 / 152359, which confer tolerance to glufosinate herbicides; genes encoding suitable EPSPS (5-enolpyruvylshikimate-3-phosphate-synthetase), which confer tolerance to herbicides targeting EPSPS, in particular herbicides such as glyphosate and its salts; genes encoding glyphosate-n-acetyltransferase, or genes encoding glyphosate oxidoreductase. Additional suitable herbicide tolerance traits include at least one ALS (acetolactate synthase) inhibitor (e.g., WO2007 / 024782); a mutated Arabidopsis ALS / AHAS gene (e.g., U.S. Pat. No. 6,855,533); a gene encoding 2,4-D-monooxygenase, which confers tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid); and a gene encoding dicamba monooxygenase, which confers tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid).

[0261] Further examples of such traits are increased resistance to phytopathogenic fungi, bacteria and / or viruses due to, for example, systemic acquired resistance (SAR), systemins, phytoalexins, elicitors and resistance genes and correspondingly expressed proteins and toxins.

[0262] Particularly useful transgenic events in transgenic plants or plant cultivars that can be preferentially treated 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-A 2005-216969); Event 3006-210-23 (cotton, insect control-herbicide tolerance, deposited as PTA-6233, described in US-A 2007-143876 or WO2005 / 103266); event 3272 (corn, quality traits, deposited as PTA-9972, described in WO2006 / 098952 or US-A 2006-230473); event 33391 (wheat, herbicide tolerance, deposited as PTA-2347, described in WO2002 / 027004), event 40416 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-11508, described in WO 11 / 075593); event 43A47 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-11509, described in WO2011 / 075595); event 5307 (corn, insect control, deposited as ATCC PTA-9561, described in WO2010 / 077816); event ASR-368 (benthgrass, herbicide tolerant, deposited as ATCC PTA-4816, described in US-A 2006-162007 or WO2004 / 053062); event B16 (maize, herbicide tolerant, not deposited, described in US-A 2003-126634); event BPS-CV127-9 (soybean, herbicide tolerant, deposited as NCIMB No. 41603, described in WO2010 / 080829); event BLR1 (canola, male sterility restoration, deposited as NCIMB 41193, described in WO2005 / 074671), event CE43-67B (cotton,insect control, deposited as DSM ACC2724, described in US-A 2009-217423 or WO2006 / 128573); event CE44-69D (cotton, insect control, not deposited, described in US-A 2010-0024077); 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 (corn, 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 (corn, insect control - herbicide tolerance, deposited as ATCC PTA11384, described in US-A 2006-070139); event DAS-59132 (corn, 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 (corn, herbicide tolerance, deposited as ATCC PTA-8296, described in US-A 2009-137395 or WO 08 / 112019); event DP-305423-1 (soybean, quality traits, not deposited, described in US-A 2008-312082 or WO2008 / 054747); event DP-32138-1 (maize, hybrid system, deposited as ATCC PTA-9158,described in US-A 2009-0210970 or WO2009 / 103049); event DP-356043-5 (soybean, herbicide tolerance, deposited as 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 Fil 17 (corn, herbicide tolerance, deposited as ATCC 209031, described in US-A 2006-059581 or WO98 / 044140); event FG72 (soybean, herbicide tolerance, deposited as PTA-11041, described in WO2011 / 063413), event GA21 (corn, herbicide tolerance, deposited as ATCC 209033, described in US-A 2005-086719 or WO98 / 044140); event GG25 (corn, herbicide tolerance, deposited as ATCC 209032, described in US-A 2005-188434 or WO98 / 044140); event GHB119 (cotton, insect control-herbicide tolerance, deposited as ATCC PTA-8398, described in WO2008 / 151780); event GHB614 (cotton, herbicide tolerant, deposited as ATCC PTA-6878, described in US-A 2010-050282 or WO2007 / 017186); event GJ11 (maize, herbicide tolerant, deposited as ATCC 209030, described in US-A2005-188434 or WO98 / 044140); event GM RZ13 (sugar beet, virus resistant, deposited as NCIMB-41601, described in WO2010 / 076212); event H7-1 (sugar beet, herbicide tolerant, deposited as NCIMB 41158 or NCIMB 41159, described in US-A 2004-172669 or WO 2004 / 074492); event JOPLIN1 (wheat, disease tolerance, not deposited, described in US-A 2008-064032); event LL27 (soybean, herbicide tolerance, deposited as NCIMB 41658, described in WO 2006 / 108674 or US-A 2008-320616); event LL55 (soybean, herbicide tolerance, deposited as NCIMB 41660, described in WO 2006 / 108675 or US-A 2008-196127); event LLcotton25 (cotton, herbicide tolerance, deposited as ATCC PTA-3343,WO2003 / 013224 or US-A2003-097687); event LLRICE06 (rice, herbicide tolerance, deposited as ATCC 203353, described in US 6,468,747 or WO2000 / 026345); event LLRICE62 (rice, herbicide tolerance, deposited as ATCC 203352, described in WO2000 / 026345), event LLRICE601 (rice, herbicide tolerance, deposited as ATCC PTA-2600, described in US-A 2008-2289060 or WO2000 / 026356); event LY038 (maize, quality traits, deposited as ATCC PTA-5623, described in US-A 2007-028322 or WO2005 / 061720); event MIR162 (corn, insect control, deposited as PTA-8166, described in US-A 2009-300784 or WO2007 / 142840); event MIR604 (corn, insect control, not deposited, described in US-A 2008-167456 or WO2005 / 103301); event MON15985 (cotton, insect control, deposited as ATCC PTA-2516, described in US-A 2004-250317 or WO2002 / 100163); event MON810 (corn, insect control, not deposited, described in US-A 2002-102582); event MON863 (corn, insect control, deposited as ATCC PTA-2605, described in WO2004 / 011601 or US-A 2006-095986); event MON87427 (corn, pollination control, deposited as ATCC PTA-7899, described in WO2011 / 062904); event MON87460 (corn, stress tolerance, deposited as ATCC PTA-8910, described in WO2009 / 111263 or US-A 2011-0138504); event MON87701 (soybean, insect control, deposited as ATCC PTA-8194, described in US-A 2009-130071 or WO2009 / 064652); event MON87705 (soybean, quality trait - herbicide tolerance, deposited as ATCC PTA-9241, described in US-A 2010-0080887 or WO2010 / 037016); event MON87708 (soybean, herbicide tolerance, deposited as ATCC PTA-9670, described in WO2011 / 034704); event MON87712 (soybean, yield, deposited as PTA-10296, described in WO2012 / 051199),Event MON87754 (soybean, quality traits, deposited as ATCC PTA-9385, described in WO2010 / 024976); Event MON87769 (soybean, quality traits, deposited as ATCC PTA-8911, described in US-A 2011-0067141 or WO2009 / 102873); Event MON88017 (corn, 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 (canola, herbicide tolerant, deposited as PTA-10955, described in WO2011 / 153186), event MON88701 (cotton, herbicide tolerant, 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-A 2008-260932); event MON89788 (soybean, herbicide tolerant, deposited as ATCC PTA-6708, described in US-A 2006-282915 or WO2006 / 130436); event MS11 (canola, pollination control-herbicide tolerance, deposited as ATCC PTA-850 or PTA-2485, described in WO2001 / 031042); event MS8 (canola, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO2001 / 041558 or US-A 2003-188347); event NK603 (maize, herbicide tolerance, deposited as ATCC PTA-2478, described in US-A 2007-292854); event PE-7 (rice, insect control, not deposited, described in WO2008 / 114282); event RF3 (canola, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO2001 / 041558 or US-A 2003-188347); event RT73 (rapeseed, herbicide tolerant, not deposited, described in WO2002 / 036831 or US-A 2008-070260); event SYHT0H2 / SYN-000H2-5 (soybean, herbicide tolerant, deposited as PTA-11226, described in WO2012 / 082548), event T227-1 (sugar beet, herbicide tolerant, not deposited,described in WO2002 / 44407 or US-A 2009-265817); Event T25 (corn, herbicide tolerance, not deposited, described in US-A2001-029014 or WO2001 / 051654); Event T304-40 (cotton, insect control-herbicide tolerance, deposited as ATCC PTA-8171, described in US-A2010-077501 or WO2008 / 122406); Event T342-142 (cotton, insect control, not deposited, described in WO2006 / 128568); Event TC1507 (corn, insect control-herbicide tolerance, not deposited, described in US-A 2005-039226 or WO2004 / 099447); event VIP1034 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-3925, described in WO2003 / 052073), event 32316 (corn, insect control-herbicide tolerance, deposited as PTA-11507, described in WO2011 / 084632), event 4114 (corn, insect control-herbicide tolerance, deposited as PTA-11506, described in WO2011 / 084621), event EE-GM3 / FG72 (soybean, herbicide tolerance, ATCC accession number PTA -11041), optionally stacked with event EE-GM1 / LL27 or event EE-GM2 / LL55 (WO2011 / 063413A2), event DAS-68416-4 (soybean, herbicide tolerant, ATCC Accession No. PTA-10442, WO2011 / 066360A1), event DAS-68416-4 (soybean, herbicide tolerant, ATCC Accession No. PTA-10442, WO2011 / 066384A 1), event DP-040416-8 (corn, insect control, ATCC accession number PTA-11508, WO2011 / 075593A1), event DP-043A47-3 (corn, insect control, ATCC accession number PTA-11509, WO2011 / 075595A1), event DP-004114-3 (corn, insect control, ATCC accession number PTA-11506, WO2011 / 084621A1), Event DP-032316-8 (corn, insect control, ATCC Accession No. PTA-11507, WO2011 / 084632A1), Event MON-88302-9 (canola, herbicide tolerance, ATCC Accession No. PTA-10955, WO2011 / 153186A1), Event DAS-21606-3 (soybean, herbicide tolerance, ATCC Accession No. PTA-11028, WO2012 / 033794A2),Event MON-87712-4 (soybean, quality traits, ATCC accession number PTA-10296, WO2012 / 051199A2), event DAS-44406-6 (soybean, stacked herbicide tolerance, ATCC accession number PTA-11336, WO2012 / 075426A1), event DAS-14536-7 (soybean, stacked herbicide tolerance, ATCC accession number PTA-11335, WO2012 / 075429 A1), event SYN-000H2-5 (soybean, herbicide tolerance, ATCC accession number PTA-11226, WO2012 / 082548A2), event DP-061061-7 (canola, herbicide tolerance, no deposit number available, WO2012071039A1), event DP-073496-4 (canola, herbicide tolerance, no deposit number available, US2012131692), event 8264.44.06.1 (soybean, added herbicide tolerance, accession number PTA-11336, WO2012075426A2), event 8291.45.36.2 (soybean, superimposed herbicide tolerance, accession number PTA-11335, WO2012075429A2), event SYHT0H2 (soybean, ATCC accession number PTA-11226, WO2012 / 082548A2), event MON88701 (cotton, ATCC accession number PTA-11754, WO 2012 / 134808A1), event KK179-2 (alfalfa, ATCC accession number PTA-11833, WO2013 / 003558A1), event pDAB8264.42.32.1 (soybean, superimposed with herbicide tolerance, ATCC accession number PTA-11993, WO2013 / 010094A1), event MZDT09Y (corn, ATCC accession number PTA-13025, WO2013 / 012775A1). ,

[0263] The genes / events that confer the desired traits in question (e.g., polynucleotides of interest) may also be present in combination with one another in transgenic plants. Examples of transgenic plants that may be mentioned are important crop plants such as cereals (wheat, rice, triticale, barley, rye, oats), corn, soybeans, potatoes, sugar beets, sugar cane, tomatoes, peas and other types of vegetables, cotton, tobacco, rapeseed and fruit plants (fruits are apples, pears, citrus fruits and grapes), with particular emphasis on corn, soybeans, wheat, rice, potatoes, cotton, sugar cane, tobacco and rapeseed. Traits that are particularly emphasized are the increased resistance of the plants to insects, arachnids, nematodes, slugs and snails, and the increased resistance of the plants to one or more herbicides.

[0264] Commercially available examples of such plants, plant parts or plant seeds that may be preferentially treated according to the invention include commercial products such as ROUNDUP VT DOUBLE VT TRIPLE BOLLGARD ROUNDUP READY 2 ROUNDUP 2XTEN DTM 、INTACTA RR2 VISTIVE and / or XTENDFLEX TM Plant seeds sold or distributed under a trade name.

[0265] HY5 transcription factor genes useful in the present invention include any HY5 transcription factor gene that is capable of regulating responses to light in plants (e.g., shade avoidance response (SAR)), and wherein a mutation as described herein can confer reduced SAR / SAS in a plant or part thereof comprising the mutation. In some embodiments, the HY5 gene encodes a basic leucine zipper (bZIP) transcription factor and plays a role in promoting photomorphogenesis, such as a HY5 transcription factor. In some embodiments, the HY5 transcription factor 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or (d) encodes a polypeptide having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:75. The amino acid sequence of NO:106 has a region of at least 80% sequence identity, 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 at least 100%.

[0266] In some embodiments, the mutation in the endogenous HY5 gene may be a non-natural mutation. In some embodiments, the mutation may be any mutation in the endogenous HY5 gene that results in a HY5 transcription factor that can confer a reduced SAR response in a plant comprising a mutated HY5 gene and / or an increased yield when planted in close proximity to other plants. In some embodiments, at least one mutation (e.g., one or more mutations) in the endogenous HY5 transcription factor gene is a point mutation, optionally a base substitution, base insertion, and / or base deletion. In some embodiments, at least one mutation in the endogenous HY5 transcription factor gene is a dominant negative mutation. In some embodiments, at least one mutation in the endogenous HY5 transcription factor gene in a plant may be a base substitution, base deletion, and / or base insertion that results in a plant having a reduced SAR response and / or an increased yield when planted in close proximity to other plants. In some embodiments, at least one mutation in the endogenous HY5 transcription factor gene in a plant may be a substitution, deletion, and / or insertion that results in a dominant negative mutation or a dominant mutation and the plant having a reduced SAR response and / or an increased yield when planted in close proximity to other plants. For example, the mutation can be a substitution, deletion and / or insertion (e.g., in-frame insertion or in-frame deletion) of 1 nucleotide or 2, 3, 4 or 5 consecutive nucleotides to about 100 consecutive nucleotides, optionally 3, 6, 9, 12, 18, 21, 24, 27, 30 or more consecutive nucleotides. In some embodiments, the at least one mutation can be a base substitution of A, T, G or C. In some embodiments, the at least one mutation can be a partial or complete deletion of the encoding COPl binding domain of the HY5 transcription factor. In some embodiments, the at least one mutation can be an in-frame deletion of 3 or more consecutive nucleotides.

[0267] In some embodiments, a guide nucleic acid (e.g., gRNA, gDNA, crRNA, crDNA) is provided that binds to a target site within an endogenous elongated hypocotyl 5 (HY5) gene, the endogenous HY5 gene 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) to SEQ ID NO: 72 or SEQ ID NO: 73; comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105, optionally to SEQ ID NO: 76. NO:79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105 have a region with at least 80% sequence identity; encodes a polypeptide comprising a sequence with at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or encodes a region with at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, optionally wherein 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 at least 100%.

[0268] In some embodiments, the guide nucleic acid binds to a target nucleic acid within the endogenous elongated hypocotyl 5 (HY5) gene having a gene identification number (Gene ID) of Zm00001d015743.

[0269] In some embodiments, the target site to which the guide nucleic acid of the invention can bind can comprise a nucleotide sequence or a portion thereof 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 any one of the nucleotide sequences of SEQ ID NOs: 75-105, optionally the sequence identity can be at least 90% or it can be at least 95%, optionally the sequence identity can be at least 100%), optionally a nucleotide sequence or a portion thereof having at least 80% sequence identity to any one of SEQ ID NOs: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105, and / or can encode a nucleotide sequence or a portion thereof having at least 80% sequence identity to any one of SEQ ID NOs: The amino acid sequence of NO:106 has at least 80% sequence identity (or a portion thereof).

[0270] Exemplary spacer sequences that can be used as guide sequences of the present invention may comprise complementarity with the following sequences: a fragment or portion 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 to any of the nucleotide sequences of SEQ ID NO: 72 or SEQ ID NO: 73, optionally the sequence identity may be at least 90% or it may be at least 95%, optionally the sequence identity may be at least 100%); optionally with SEQ ID NO: 74 or SEQ ID NO: 75; any one of the nucleotide sequences of SEQ ID NOs:75-105 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 90% sequence identity or at least 95% sequence identity, or at least 100% sequence identity), optionally a fragment or portion of a nucleotide sequence having 80% sequence identity to any one of SEQ ID NOs:79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105; and / or a fragment or portion of a nucleotide sequence encoding a polypeptide comprising the polypeptide identical to SEQ ID NOs:79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105; NO:74 has an amino acid 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 90% or it can be at least 95%, optionally the sequence identity can be at least 100%) sequence identity; optionally with SEQ ID The amino acid sequence of NO:106 has a sequence with 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% sequence identity or at least 95% sequence identity, or at least 100% sequence identity).

[0271] In some embodiments, the target nucleic acid is an endogenous HY5 transcription factor gene capable of regulating the plant's response to light. In some embodiments, the target site within the target nucleic acid may 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 a region, portion, or fragment of SEQ ID NO: 72 or SEQ ID NO: 73 (e.g., SEQ ID NO: 75-105, optionally having at least 80% sequence identity to any one of SEQ ID NO: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101, or 103-105), or may encode 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, optionally the sequence identity may be at least 90% or it may be at least 95%, optionally the sequence identity may be at least 100%) to SEQ ID NO: 72 or SEQ ID NO: 73. The amino acid sequence of NO:74 has a region of amino acid sequence with at least 80% sequence identity (eg, SEQ ID NO:106).

[0272] In some embodiments, the guide nucleic acid may comprise a spacer having a nucleotide sequence of any one of SEQ ID NOs: 107-113, or a reverse complement thereof, or any combination thereof.

[0273] In some embodiments, a system is provided, comprising a guide nucleic acid of the invention and a CRISPR-Cas effector protein associated with the guide nucleic acid. In some embodiments, the system may further comprise a tracr nucleic acid 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.

[0274] As used herein, “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.

[0275] In some embodiments, a gene editing system is provided, the 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 a HY5 gene. In some embodiments, a HY5 transcription factor gene that can be used in a gene editing system: (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) to a nucleotide sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 74; and / or (d) encodes a polypeptide having at least 80% sequence identity to an amino acid sequence of SEQ ID NO: 75. The amino acid sequence of NO:106 has a region of at least 80% sequence identity, optionally wherein 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 at least 100%.

[0276] In some embodiments, the guide nucleic acid of the gene editing system may comprise a region, portion or fragment 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 to any one of the nucleotide sequences of SEQ ID NO: 72 or SEQ ID NO: 73, optionally the sequence identity may be at least 90% or it may be at least 95%, optionally the sequence identity may be at least 100%) (e.g., SEQ ID NO: 75-105, optionally with SEQ ID NO: 76-110). NO:79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105 have at least 80% sequence identity) has a spacer sequence that is complementary, or can encode a region, portion or fragment of a sequence that has at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74 (e.g., SEQ ID NO:106). In some embodiments, the gene editing system can also include a tracr nucleic acid associated with a guide nucleic acid and a CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked. In some embodiments, a guide nucleic acid is provided that binds to a target nucleic acid in an endogenous HY5 gene having a gene identification number (gene ID) of Zm00001d015743 (SEQ ID NO: 72) (Maize Genetics and Genome Database (Maize GDB)), optionally wherein (a) the target region within Zm00001d015743 (SEQ ID NO: 72) may comprise a portion of contiguous nucleotides of any one or more of the nucleotide sequences of SEQ ID NOs: 75-105, optionally having at least 80% sequence identity with any one of SEQ ID NOs: 79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105.

[0277] The present invention also provides a complex comprising a CRISPR-Cas effector protein containing a cleavage domain and a guide nucleic acid, wherein the guide nucleic acid binds to a target site within an elongated hypocotyl 5 (HY5) transcription factor gene, wherein the HY5 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 SEQ ID NO: 72 or SEQ ID NO: 73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 75-105; (c) encodes an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 75; The amino acid sequence of NO:106 has an amino acid sequence with at least 80% sequence identity, optionally wherein 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 at least 100%, wherein the cleavage domain cleaves the target chain in the HY5 transcription factor gene.

[0278] Also provided herein is an expression cassette comprising (a) a polynucleotide encoding a CRISPR-Cas effector protein comprising a cleavage domain and (b) a guide nucleic acid that binds to a target site within an elongated hypocotyl 5 (HY5) gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to a target site within the HY5 gene, wherein the HY5 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 SEQ ID NO: 72 or SEQ ID NO: 73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: NO:74 has at least 80% sequence identity; and / or (d) encodes an amino acid sequence having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:106, optionally wherein 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 at least 100%.

[0279] In some embodiments, a nucleic acid encoding an elongated hypocotyl 5 (HY5) transcription factor having a mutated COPl binding site is provided, wherein the mutated COPl binding site comprises a mutation that disrupts binding of the HY5 transcription factor to a COPl polypeptide. In some embodiments, the mutation eliminates binding of the HY5 transcription factor to COPl, or reduces the ability of the HY5 transcription factor to bind to a COPl polypeptide by at least 75% (e.g., at least about 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more). In some embodiments, a nucleic acid is provided comprising a mutant HY5 gene having at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 99% or 100% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139, optionally the sequence identity may be at least 95%, optionally the sequence identity may be at least 100%) and / or encoding a mutant HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136, or 138.

[0280] Also provided is a plant or part thereof comprising a HY5 nucleic acid of a mutation as described herein. In some embodiments, the plant may be a corn (maize) plant. In some embodiments, the plant is a wheat plant. In some embodiments, as compared to one or more plants planted closely adjacent to each other but lacking a mutation and not showing a reduced shade-avoiding response, when planted closely adjacent to one or more plants comprising a HY5 of a mutation as described herein and having a reduced SAR, a plant, corn plant and / or wheat plant comprising a HY5 of a mutation as described herein and having a reduced SAR may show increased yield, increased upright growth, reduced height, reduced root-crown ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, increased photosynthetic efficiency and grain filling, flowering time are substantially unchanged, and / or enhanced defense responses to pathogens and herbivores. In some embodiments, when planted closely adjacent to each other, the plant of the present invention comprising a reduced SAR is at least about 5% shorter than a control plant grown under the same conditions (e.g., a shaded environment, e.g., a low R:FR ratio environment). In some embodiments, a corn plant or part thereof is provided, comprising a mutation in an endogenous elongated hypocotyl 5 (HY5) gene having a gene identification number (Gene ID) of Zm00001d015743 (SEQ ID NO: 72), optionally wherein the mutation is a non-natural mutation.

[0281] In some embodiments, the methods of the present invention may further comprise regenerating a plant from a plant cell or plant part comprising at least one mutation (e.g., one or more mutations) in an endogenous elongated hypocotyl 5 (HY5) gene, optionally wherein the mutation disrupts the binding of a COPl polypeptide to an encoded HY5 polypeptide. In some embodiments, plants comprising at least one mutation in an endogenous HY5 gene may exhibit increased yield, increased upright growth, reduced height, reduced root-to-crown ratio, reduced leaf length, increased mechanical stem strength, reduced lodging rate, delayed senescence, increased photosynthetic efficiency and grain filling, substantially no change in flowering time, and / or enhanced defense responses to pathogens and herbivores when they are planted in close proximity to one or more other plants, compared to control plants that do not comprise at least one mutation in the endogenous HY5 gene and therefore do not comprise a reduced shade avoidance response when planted in close proximity to one or more other plants. In some embodiments, the mutation is a non-naturally occurring mutation. In some embodiments, the mutation is a deletion, optionally an in-frame deletion. In some embodiments, the deletion results in a dominant negative mutation or a dominant mutation.

[0282] The editing system that can be used for the present invention can be any site-specific (sequence-specific) genome editing system that is now known or developed later, which can introduce mutations in a target-specific manner. For example, the editing system (e.g., a site-specific or sequence-specific editing system) may include, but is not limited to, a CRISPR-Cas editing system, a meganuclease editing system, a zinc finger nuclease (ZFN) editing system, a transcription activator-like effector nuclease (TALEN) editing system, a base editing system, and / or a lead editing system, each of which may include 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., a site-specific or sequence-specific editing system) may include 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.

[0283] In some embodiments, the editing system may include one or more sequence-specific nucleic acid binding domains (DNA binding domains), which may 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. In some embodiments, the editing system may include 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, the editing system may include one or more polypeptides, including but not limited to deaminases (e.g., cytosine deaminases, adenine deaminases), reverse transcriptases, Dna2 polypeptides, and / or 5' flap endonucleases (FENs). In some embodiments, the editing system may comprise one or more polynucleotides, including but not limited to a CRISPR array (CRISPR guide sequence) nucleic acid, an extended guide nucleic acid, and / or a reverse transcriptase template.

[0284] In some embodiments, a method of modifying or editing a HY5 gene may include contacting a target nucleic acid (e.g., a target region of a HY5 gene) with a base editing fusion protein (e.g., 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 the locus within the target nucleic acid. In some embodiments, the base editing fusion protein and the guide nucleic acid may be contained in one or more expression cassettes. In some embodiments, the target nucleic acid may be contacted with a base editing fusion protein and an expression cassette comprising a guide nucleic acid. In some embodiments, the sequence-specific DNA binding fusion protein and the guide sequence may be provided as a ribonucleoprotein (RNP). In some embodiments, a cell may be contacted with more than one base editing fusion protein and / or one or more guide nucleic acids, which may target one or more target nucleic acids in a cell.

[0285] In some embodiments, a method of modifying or editing a HY5 gene may include contacting a target nucleic acid (e.g., a target region of a HY5 gene) with a sequence-specific DNA binding fusion protein (e.g., a sequence-specific DNA binding protein (e.g., a CRISPR-Cas effector protein or domain) fused to a peptide tag, a deaminase fusion protein comprising a deaminase domain (e.g., adenine deaminase and / or 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 DNA binding fusion protein to the target nucleic acid, and the sequence-specific DNA binding fusion protein is capable of recruiting the deaminase fusion protein to the target nucleic acid through a peptide tag-affinity polypeptide interaction. Target nucleic acid, thereby editing the locus within the target nucleic acid. In some embodiments, the sequence-specific DNA binding fusion protein can be fused to an affinity polypeptide that binds a peptide tag, and the deaminase can be fused to the peptide tag, thereby recruiting the deaminase to the sequence-specific DNA 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 included 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 the expression cassette containing the guide nucleic acid. In some embodiments, the sequence-specific DNA binding fusion protein, the deaminase fusion protein, and the guide sequence can be provided as a ribonucleoprotein (RNP).

[0286] In some embodiments, methods such as lead editing can be used to generate mutations in the endogenous HY5 transcription factor gene. In lead 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 the target in a sequence-specific manner, and can also cause a nick containing a PAM chain 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 includes an extension that includes a primer binding site (PSB) and an edit to be incorporated into the genome (template). Similar to base editing, lead editing can utilize various methods to recruit proteins for target site editing, including non-covalent and covalent interactions between proteins and nucleic acids used in selected processes of genome editing.

[0287] In some embodiments, the mutation or modification of the endogenous HY5 transcription factor gene can be an insertion, a deletion and / or a point mutation that produces a mutated HY5 transcription factor (e.g., a mutated HY5 transcription factor) that has reduced binding to a constitutive photomorphogenesis 1 (COP1) polypeptide. In some embodiments, the plant part can be a cell. In some embodiments, the plant or its plant part can be any plant or part thereof as described herein. In some embodiments, the plant that can be used in the present invention can be corn, soybean, rapeseed, wheat, rice, cotton, sugarcane, sugar beet, barley, oat, 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 species. In some embodiments, plants comprising a mutated endogenous HY5 transcription factor with reduced COP1 binding may comprise increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, increased photosynthetic efficiency and grain filling, essentially no change in flowering time, and / or enhanced defense responses to pathogens and herbivores when they are planted in close proximity to one or more other plants, compared to control plants that do not comprise at least one non-natural mutation in the endogenous HY5 gene and therefore do not comprise a reduced shade avoidance response (the plants are planted in close proximity to one or more other plants). In some embodiments, the plant can be a corn plant comprising a mutated endogenous HY5 transcription factor with reduced COP1 binding and optionally exhibiting increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, increased photosynthetic efficiency and grain filling, essentially no change in flowering time, and / or enhanced defense responses to pathogens and herbivores.

[0288] In some embodiments, the mutation introduced into the endogenous HY5 gene can be a non-natural mutation, which optionally results in a reduced binding of COPl to the encoded HY5 polypeptide. In some embodiments, the mutation introduced into the endogenous HY5 gene can optionally result in a reduced binding of COPl to the encoded HY5 polypeptide. In some embodiments, the mutation introduced into the endogenous HY5 gene can be a substitution, insertion and / or deletion of one nucleotide or at least two consecutive nucleotides (e.g., 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 or 30 or more), wherein the mutation optionally results in a reduced binding of COPl to the encoded HY5 polypeptide. In some embodiments, the mutation introduced into the endogenous HY5 gene can be a deletion, optionally an in-frame deletion. In some embodiments, the deletion may result in the complete or partial deletion of the COPl binding region in the HY5 transcription factor polypeptide, for example, deleting one, two or three or more nucleotides (e.g., consecutive nucleotides) from the region of the HY5 gene encoding the amino acid sequence of SEQ ID NO:106.

[0289] In some embodiments, sequence-specific nucleic acid binding domains (sequence-specific DNA binding domains) useful in the editing systems of the present invention may 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.

[0290] In some embodiments, the sequence-specific nucleic acid binding domain may be a CRISPR-Cas effector protein, optionally wherein the CRISPR-Cas effector protein may be 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 may be from a type II CRISPR-Cas system or a type V CRISPR-Cas system. In some embodiments, the CRISPR-Cas effector protein may be a type II CRISPR-Cas effector protein, such as a Cas9 effector protein. In some embodiments, the CRISPR-Cas effector protein may be a type V CRISPR-Cas effector protein, such as a Cas12 effector protein.

[0291] As used herein, a "CRISPR-Cas effector protein" is a protein or polypeptide or a domain thereof that cuts or cleaves a nucleic acid, binds a nucleic acid (e.g., a target nucleic acid and / or a guide nucleic acid), and / or identifies, recognizes, or binds a guide nucleic acid as defined herein. In some embodiments, a CRISPR-Cas effector protein may be an enzyme (e.g., a nuclease, an endonuclease, a nickase, etc.) or a portion thereof and / or may act as an enzyme. In some embodiments, a CRISPR-Cas effector protein refers to a CRISPR-Cas nuclease polypeptide or a domain thereof that comprises nuclease activity or wherein the nuclease activity has been reduced or eliminated, and / or comprises nickase activity or wherein the nickase activity has been reduced or eliminated, and / or comprises single-stranded DNA cleavage activity (ss DNA enzyme activity) or wherein the ss DNA enzyme activity has been reduced or eliminated, and / or comprises self-processing RNA enzyme activity or wherein the self-processing RNA enzyme activity has been reduced or eliminated. A CRISPR-Cas effector protein may bind to a target nucleic acid.

[0292] In some embodiments, the CRISPR-Cas effector protein may include, but is not limited to, Cas9, C2c1, 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, CsxlO, Csxl6, 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.

[0293] In some embodiments, the CRISPR-Cas effector protein useful in the present invention may comprise a mutation in its nuclease active site (e.g., RuvC, HNH, e.g., a RuvC site of a Cas12a nuclease domain; e.g., a RuvC site and / or an HNH site of a Cas9 nuclease domain). The CRISPR-Cas effector protein has a mutation in its nuclease active site and therefore no longer comprises nuclease activity, and is often referred to as "dead", e.g., dCas. In some embodiments, a CRISPR-Cas effector protein domain or polypeptide having a mutation in its nuclease active site may have impaired activity or reduced activity compared to the same CRISPR-Cas effector protein (e.g., a nickase, e.g., a Cas9 nickase, a Cas12a nickase) without the mutation.

[0294] CRISPR Cas9 effector proteins or CRISPR Cas9 effector domains 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 NOs: 59-60 or the polynucleotide sequences of SEQ ID NOs: 61-71.

[0295] In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus pyogenes and recognizes 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 thermophiles and recognizes 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 recognizes 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 recognizes the PAM sequence motifs NNGRR (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus aureus and recognizes the PAM sequence motifs 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 motifs N GRRV (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Neisseria meningitidis and recognizes the PAM sequence motif NGATT or NGCTT (R = A or G, V = A, G or C) (see, e.g., Hou et al. (2013) Proc. Natl. Acad. Sci. USA 110(39): 15644-15649). 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, which recognizes a single 3' A, U or C protospacer flanking sequence (PFS) (or RNA PAM (rPAM)) sequence motif that can be located within the target nucleic acid.

[0296] In some embodiments, the CRISPR-Cas effector protein may be derived from Cas12a, which is a V-type regularly interspaced clustered short palindromic repeat (CRISPR)-Cas nuclease (see, e.g., SEQ ID NO: 1-17 amino acid sequence, SEQ ID NO: 18-20 nucleic acid sequence). Cas12a differs from the more well-known type II CRISPR Cas9 nuclease in several aspects. For example, Cas9 recognizes a G-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 T-rich PAM (5'-TTN, 5'-TTTN) located 5' of the target nucleic acid. In fact, the orientations of Cas9 and Cas12a binding to their guide RNAs are almost opposite relative 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 natural Cas9 system, and Cas12a processes its own gRNA. In addition, the Cas12a nuclease activity produces staggered DNA double-strand breaks, rather than the flat ends produced by the Cas9 nuclease activity, and Cas12a relies on a single RuvC domain to cut two DNA chains, while Cas9 uses the HNH domain and the RuvC domain to cut.

[0297] CRISPR Cas12a effector proteins / domains useful in the present invention may be any known or later identified Cas12a polypeptide (formerly known as Cpf1) (see, e.g., U.S. Pat. No. 9,790,490, which is incorporated by reference for its disclosure of Cpf1 (Cas12a) sequences). The term "Cas12a", "Cas12a polypeptide" or "Cas12a domain" refers to an RNA-guided nuclease comprising a Cas12a polypeptide or a fragment thereof, the fragment comprising a guide nucleic acid binding domain of Cas12a and / or an active, inactive or partially active DNA cleavage domain of Cas12a. In some embodiments, Cas12a useful in the present invention may comprise a mutation in a nuclease active site (e.g., a RuvC site of a 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 a dead Cas12a (e.g., dCas12a). In some embodiments, a Cas12a domain or Cas12a polypeptide having a mutation in its nuclease active site may have impaired activity, for example, may have nickase activity.

[0298] 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, e.g., U.S. Patent No. 10,167,457 and Thuronyi et al., Nat. Biotechnol. 37: 1070–1079 (2019), each of which is incorporated herein by reference for its disclosure of cytosine deaminase). Cytosine deaminase can catalyze the hydrolysis and deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. Therefore, 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 hydrolysis and 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., humans, monkeys, chimpanzees, gorillas), dogs, cows, rats, or mice. Thus, in some embodiments, the cytosine deaminase useful in the present invention can be about 70% to about 100% identical to a 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 a naturally occurring cytosine deaminase, and any range or value therein).

[0299] In some embodiments, the cytosine deaminase that can be used in the present invention can be an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase can be an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, an APOBEC4 deaminase, a human activation-induced deaminase (hAID), rAPOBEC1, FERNY and / or CDA1, optionally pmCDA1, atCDA1 (e.g., At2g19570), and their evolutionary forms (e.g., SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29). In some embodiments, the cytosine deaminase can be an APOBEC1 deaminase having an amino acid sequence of SEQ ID NO: 23. In some embodiments, the cytosine deaminase may be an APOBEC3A deaminase having the amino acid sequence of SEQ ID NO: 24. In some embodiments, the cytosine deaminase may be a CDA1 deaminase, optionally a CDA1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the cytosine deaminase may be a FERNY deaminase, optionally a FERNY having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a cytosine deaminase useful in the present invention can be about 70% to about 100% identical (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) to the amino acid sequence of a naturally occurring cytosine deaminase (e.g., an evolved deaminase).In some embodiments, a cytosine deaminase useful in the present invention may be about 70% to about 99.5% 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%, or 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) 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: NO:29 is 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). In some embodiments, the polynucleotide encoding the cytosine deaminase can be codon-optimized for expression in plants, and the codon-optimized polypeptide can be about 70% to 99.5% identical to the reference polynucleotide.

[0300] In some embodiments, the nucleic acid constructs of the present invention may also encode uracil glycosylase inhibitor (UGI) (e.g., uracil-DNA glycosylase inhibitor) polypeptides / domains. Thus, in some embodiments, nucleic acid constructs encoding CRISPR-Cas effector proteins and cytosine deaminase domains (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 also encode uracil-DNA glycosylase inhibitor (UGI), optionally wherein UGI may be codon-optimized for expression in plants. In some embodiments, the present invention provides fusion proteins comprising CRISPR-Cas effector polypeptides, deaminase domains, and UGI and / or one or more polynucleotides encoding them, optionally wherein one or more polynucleotides may be codon-optimized for expression in plants. In some embodiments, the present invention provides fusion proteins, wherein the CRISPR-Cas effector polypeptide, the deaminase domain and the UGI can 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 can be linked to a recruitment RNA motif, and one or more of the deaminase domain and / or the UGI can 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.

[0301] The "uracil glycosylase inhibitor" useful in the present invention may be any protein capable of inhibiting uracil-DNA glycosylase base excision repair enzymes. In some embodiments, the UGI domain comprises a 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 to 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 of the amino acid sequence of SEQ ID NO:41 that is 100% identical to a portion 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 can be a variant of a known UGI (e.g., SEQ ID NO: 41) having about 70% to about 99.5% sequence identity (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) to the known UGI. In some embodiments, the polynucleotide encoding the UGI can be codon-optimized for expression in a plant (e.g., a plant), and the codon-optimized polypeptide can be about 70% to about 99.5% identical to the reference polynucleotide.

[0302] 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. Pat. No. 10,113,163, the disclosure of which is incorporated herein by reference for adenine deaminase). 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, adenine deaminase encoded by a nucleic acid construct of the present invention can produce an A→G transition in the sense (e.g., "+"; template) strand of a target nucleic acid or a T→C transition in the antisense (e.g., "-", complementary) strand of a target nucleic acid.

[0303] In some embodiments, the adenosine deaminase may be a variant of a naturally occurring adenine deaminase. Thus, in some embodiments, the adenosine deaminase may be about 70% to 100% identical to a wild-type adenine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a naturally occurring adenine deaminase, and any range or value therein). In some embodiments, the deaminase or deaminase is not naturally occurring and may 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 a naturally occurring adenine deaminase polypeptide / domain (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 11 In some embodiments, the adenine deaminase polypeptide / domain may be codon-optimized for expression in plants.

[0304] In some embodiments, the adenine deaminase domain can be a wild-type tRNA-specific adenosine deaminase domain, such as a tRNA-specific adenosine deaminase (TadA), and / or a mutant / evolved adenosine deaminase domain, such as a mutant / 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, with one or more N-terminal and / or C-terminal amino acids missing relative to full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or 20 N-terminal and / or C-terminal amino acid residues may be missing relative to full-length TadA). In some embodiments, the TadA polypeptide or TadA domain does not include an N-terminal methionine. In some embodiments, the wild-type E. coli TadA comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the mutant / evolved E. coli TadA* comprises the amino acid sequence of SEQ ID NO: 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.

[0305] Cytosine deaminase catalyzes the deamination of cytosine and produces thymidine (via a uracil intermediate), causing a C to T transition or a G to A transition in the complementary strand of the genome. Thus, in some embodiments, a cytosine deaminase encoded by a polynucleotide of the invention produces a C→T transition in the sense (e.g., "+"; template) strand of a target nucleic acid or a G→A transition in the antisense (e.g., "-", complementary) strand of a target nucleic acid.

[0306] In some embodiments, an adenine deaminase encoded by a nucleic acid construct of the invention produces an A→G transition in the sense (e.g., "+"; template) strand of a target nucleic acid or a T→C transition in the antisense (e.g., "-", complementary) strand of a target nucleic acid.

[0307] The nucleic acid construct encoding the base editor comprising a sequence-specific DNA binding protein and a cytosine deaminase polypeptide of the present invention, and the nucleic acid construct / expression cassette / vector 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 a C→T or G→A mutation in a target nucleic acid (including but not limited to a plasmid sequence); generating a C→T or G→A mutation in a coding sequence to change the amino acid identity; generating a C→T or G→A mutation in a coding sequence to generate a stop codon; generating a C→T or G→A mutation in a coding sequence to destroy a start codon; generating a point mutation in genomic DNA to destroy transcription factor binding; and / or generating a point mutation in genomic DNA to destroy a splice junction.

[0308] The nucleic acid constructs of the present invention encoding base editors comprising sequence-specific DNA binding proteins and adenine deaminase polypeptides, as well as expression cassettes and / or vectors encoding the same, can be used in combination with guide nucleic acids to modify target nucleic acids, including but not limited to generating A→G or T→C mutations in target nucleic acids (including but not limited to plasmid sequences); generating A→G or T→C mutations in coding sequences to change amino acid identity; generating A→G or T→C mutations in coding sequences to generate stop codons; generating A→G or T→C mutations in coding sequences to destroy start codons; generating point mutations in genomic DNA to destroy function; and / or generating point mutations in genomic DNA to destroy splice junctions.

[0309] 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) to modify a target nucleic acid, which is designed to function 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 construct of the present invention, and the spacer sequence is capable of hybridizing with 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 affinity polypeptide to recruit a deaminase domain and optionally a UGI) to a target nucleic acid, wherein the target nucleic acid can be modified (e.g., cut or edited) or regulated (e.g., regulated transcription) by the deaminase domain.

[0310] As an example, a nucleic acid construct encoding a Cas9 domain (e.g., a fusion protein) connected 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 cytosine bases 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) connected 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 adenosine bases in the target nucleic acid, thereby editing the target nucleic acid.

[0311] Similarly, encoding a Cas12a domain (or other selected CRISPR-Cas nucleases, such as 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, Nucleic acid constructs of Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4(dinG) and / or Csf5) (e.g., fusion proteins) can be used in combination with a Cas12a guide nucleic acid (or a guide nucleic acid of another selected CRISPR-Cas nuclease) to modify a target nucleic acid, wherein the cytosine deaminase domain or adenine deaminase domain of the fusion protein deaminates cytosine bases in the target nucleic acid, thereby editing the target nucleic acid.

[0312] As used herein, “guide nucleic acid,” “guide RNA,” “gRNA,” “CRISPR RNA / DNA,” “crRNA,” or “crDNA” refers to a sequence comprising at least one spacer sequence complementary to (and hybridizing with) a target DNA (e.g., a protospacer) 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 type V C2c1 CRISPR Repeat sequences of the Cas system, or fragments thereof; for example, 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, CsxlO, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5 CRISPR-Cas system repetitive sequence, or its fragment) nucleic acid, wherein the repetitive sequence can be connected 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 system.

[0313] In some embodiments, the Cas12a gRNA may comprise a repeat sequence (full length or a portion thereof ("handle"); e.g., a pseudoknot-like structure) and a spacer sequence from 5' to 3'.

[0314] In some embodiments, the guide nucleic acid may comprise more than one repeat-spacer sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeat-spacer sequences) (e.g., repeat-spacer-repeat, e.g., repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer, etc.). The guide nucleic acids of the invention are synthetic, artificial, and do not exist in nature. The gRNA can be very long and can be used as an aptamer (e.g., in the MS2 recruitment strategy) or other RNA structures with hanging spacers.

[0315] As used herein, "repetitive sequence" refers to any repetitive sequence of, for example, a wild-type CRISPR Cas locus (e.g., Cas9 locus, Cas12a locus, C2c1 locus, etc.) or a repetitive sequence of a synthetic crRNA that works together with a CRISPR-Cas effector protein encoded by a nucleic acid construct of the present invention. The repetitive sequence that can be used in the present invention can be any known or later identified repetitive sequence (e.g., type I, type II, type III, type IV, type V, or type VI) of the CRISPR-Cas locus, or it can be a synthetic repetitive sequence designed to work in a type I, II, III, IV, V, or VI CRISPR-Cas system. The repetitive sequence may include a hairpin structure and / or a stem-loop structure. In some embodiments, the repetitive sequence may form a pseudoknot-like structure (i.e., "handle") at its 5' end. Thus, in some embodiments, the repetitive sequence may be identical or substantially identical to a repetitive sequence from a wild-type type I CRISPR-Cas locus, a type II CRISPR-Cas locus, a type III CRISPR-Cas locus, a type IV CRISPR-Cas locus, a type V CRISPR-Cas locus, and / or a type VI CRISPR-Cas locus. Repetitive 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 album): W52-7). In some embodiments, the repetitive sequence or a portion thereof is linked to the 5' end of the spacer sequence at its 3' end, thereby forming a repeat-spacer sequence (e.g., a guide nucleic acid, a guide RNA / DNA, a crRNA, a crDNA).

[0316] In some embodiments, the repetitive sequence comprises, consists essentially of, or consists of at least 10 nucleotides, depending on the particular repetitive sequence and whether the guide nucleic acid comprising the repetitive 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 repetitive sequence comprises, consists essentially of, or consists of about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100, or more nucleotides.

[0317] The repeat sequence linked to the 5' end of the spacer sequence can include 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 a wild-type repeat sequence). In some embodiments, the 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) in length and has at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) to the same region (e.g., 5' end) of the wild-type CRISPR Cas repeat nucleotide sequence. In some embodiments, a portion of a repeat sequence may comprise a pseudoknot-like structure (eg, a "handle") at its 5' end.

[0318] As used herein, a "spacer sequence" is a nucleotide sequence that is complementary to a portion of a target nucleic acid (eg, target DNA) (eg, a protospacer). In some embodiments, the spacer sequence is complementary to a portion of consecutive nucleotides of a HY5 gene, wherein the HY5 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) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105, optionally a region having at least 80% sequence identity to any one of SEQ ID NO:79-81, 83-85, 87-89, 91-93, 95-97, 99-101 or 103-105; (c) encodes a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: NO:74 amino acid sequence having at least 80% sequence identity, and / or (d) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:106, optionally wherein 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 at least 100%. The spacer sequence may be fully 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 100%)). In some embodiments, as compared with the target nucleic acid, the spacer sequence may have one, two, three, four or five mispairings, which may be continuous or discontinuous. In some embodiments, the spacer sequence may have 70% complementarity with the target nucleic acid. In other embodiments, the spacer nucleotide sequence may have 80% complementarity with the target nucleic acid. In still other embodiments, the spacer nucleotide sequence may have 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementarity with the target nucleic acid (pre-spacer), etc. In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid. The spacer sequence may have a length of 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 sequence may have full complementarity or substantial complementarity over a region of the target nucleic acid (e.g., pre-spacer) that is at least about 15 nucleotides to about 30 nucleotides in length. In some embodiments, the spacer is about 20 nucleotides in length. In some embodiments, the spacer is about 21, 22, or 23 nucleotides in length. In some embodiments, the spacer sequence may comprise any one of SEQ ID NOs: 107-113, or any combination thereof.

[0319] In some embodiments, the 5' region of the spacer sequence of the guide nucleic acid may be identical to the target DNA, while the 3' region of the spacer may be substantially complementary to the target DNA (such as the spacer of the type V CRISPR-Cas system), or the 3' region of the spacer sequence of the guide nucleic acid may be identical to the target DNA, while the 5' region of the spacer may be substantially complementary to the target DNA (such as the spacer of the type II CRISPR-Cas system), and thus, the overall complementarity of the spacer sequence to the target DNA may be less than 100%. Thus, for example, in the guide sequence of the type V CRISPR-Cas system, for example, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 5' region (i.e., the seed region) of the 20-nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary 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 ranges therein) of the 5' end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary 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)).

[0320] As another example, in the guide sequence of a type II CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 3' region (i.e., seed region) of, for example, a 20-nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary 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 ranges therein) of the 3' end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary 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)).

[0321] In some embodiments, the seed region of the spacer can be about 8 to about 10 nucleotides long, about 5 to about 6 nucleotides long, or about 6 nucleotides long.

[0322] As used herein, "target nucleic acid," "target DNA," "target nucleotide sequence," "target region," or "target region in a genome" refers to a region of a plant genome that is fully 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 a spacer sequence in a guide nucleic acid of the invention. The target region that can be used for a CRISPR-Cas system can be positioned immediately 3' (e.g., a type V CRISPR-Cas system) or immediately 5' (e.g., a type II CRISPR-Cas system) to a PAM sequence in the genome of an organism (e.g., a plant genome). The target region can be selected from any region 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.

[0323] "Pre-spacer sequence" refers to the target double-stranded DNA, specifically to the portion of the target DNA (e.g., or a target region in the genome) that is completely 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).

[0324] In the case of type V CRISPR-Cas (e.g., Cas12a) systems and type II CRISPR-Cas (Cas9) systems, the protospacer sequence is flanked by (e.g., immediately adjacent to) a protospacer adjacent motif (PAM). For type IV CRISPR-Cas systems, the PAM is located at the 5' end of the non-target strand and the 3' end of the target strand (see below, as an example).

[0325]

[0326] In the case of type II CRISPR-Cas (e.g., Cas9) systems, the PAM is positioned immediately 3' to the target region. The PAM of type I CRISPR-Cas systems is located 5' to the target strand. There is no known PAM for type III CRISPR-Cas systems. Makarova et al. describe the nomenclature for all classes, types, and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722–736 (2015)). R. Barrangou (Genome Biol. 16:247 (2015)) describes guide structures and PAMs.

[0327] Typical Cas12a PAMs are T-rich. In some embodiments, typical Cas12a PAM sequences can be 5'-TTN, 5'-TTTN, or 5'-TTTV. In some embodiments, typical Cas9 (e.g., Streptococcus pyogenes) PAMs can be 5'-NGG-3'. In some embodiments, atypical PAMs can be used, but the efficiency may be lower.

[0328] Those skilled in the art can determine additional PAM sequences by established experimental and computational methods. Therefore, 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 may include performing BLAST searches on natural spacers to identify the original target DNA sequence in phage or plasmids, and aligning these sequences to determine conserved sequences adjacent to the target sequence (Briner and Barrangou, 2014. Appl. Environ. Microbiol. 80: 994-1001; Mojica et al., 2009. Microbiology 155: 733-740).

[0329] In some embodiments, the present invention provides expression cassettes and / or vectors comprising nucleic acid constructs of the present invention (e.g., one or more components of the editing system of the present invention). In some embodiments, expression cassettes and / or vectors comprising nucleic acid constructs of the present invention and / or one or more guide nucleic acids may be provided. In some embodiments, nucleic acid constructs of the present invention encoding base editors (e.g., constructs (e.g., fusion proteins) comprising CRISPR-Cas effector proteins and deaminase domains) or components for base editing (e.g., CRISPR-Cas effector proteins fused to peptide tags or affinity polypeptides, deaminase domains fused to peptide tags or affinity polypeptides, and / or UGI fused to peptide tags or affinity polypeptides) may be included in an expression cassette or vector that is the same as or separate from an expression cassette or vector comprising one or more guide nucleic acids. When the nucleic acid construct encoding a base editor or the components for base editing are contained on an expression cassette or vector separate from the expression cassette or vector comprising a guide nucleic acid, the target nucleic acid can be contacted with (e.g., provided together with) the expression cassette or vector encoding the base editor or the components for base editing in any order with each other and the guide nucleic acid, for example, before, simultaneously with, or after providing the expression cassette comprising the guide nucleic acid (e.g., contacting with the target nucleic acid).

[0330] The fusion protein of the present invention may comprise a sequence-specific nucleic acid binding domain, a CRISPR-Cas polypeptide and / or a deaminase domain fused to a peptide tag as 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 may also include a guide nucleic acid connected to an RNA recruitment motif and a deaminase fused to an affinity polypeptide that can interact with the RNA recruitment motif, thereby recruiting the deaminase to the target nucleic acid. Alternatively, chemical interactions can be used to recruit polypeptides (e.g., deaminase) to target nucleic acids.

[0331] Peptide tags (e.g., epitopes) that can be used in the present invention may 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, and peptide tags sold under the trade names Octapeptides, strep tags or strep tags II, V5 tags and / or VSV-G epitopes sold. In some embodiments, peptide tags may also include phosphorylated tyrosine in a specific sequence background recognized by an SH2 domain, a characteristic consensus sequence containing phosphoserine recognized by a 14-3-3 protein, a proline-rich peptide motif recognized by an SH3 domain, a PDZ protein interaction domain or a PDZ signal sequence, and an AGO hook motif from a plant. Peptide tags are disclosed in WO 2018 / 136783 and U.S. Patent Application Publication No. 2017 / 0219596 (which is incorporated herein by reference for disclosures regarding peptide tags). Any epitope that can be connected to a polypeptide and has a corresponding affinity polypeptide that can be connected to another polypeptide can be used as a peptide tag in the present invention. The peptide tag may comprise or be present in one copy or two or more copies of a peptide tag (e.g., a multimeric peptide tag or multimeric epitope) (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 9, 20, 21, 22, 23, 24, or 25 or more peptide tags). When multimerized, the peptide tags may be directly fused to each other, or they may be linked to each other by one or more amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more amino acids, optionally about 3 to about 10, about 4 to about 10, about 5 to about 10, about 5 to about 15, or about...

Claims

1. A plant or plant part thereof comprising at least one mutation in an endogenous elongated hypocotyl 5 (HY5) gene encoding a HY5 transcription factor.

2. The plant or plant part thereof of claim 1, wherein the HY5 transcription factor is capable of regulating a response to light (eg, shade avoidance response (SAR)) in a plant.

3. The plant or plant part thereof of claim 1 or claim 2, wherein the at least one mutation is located in the N-terminal region of the endogenous HY5 transcription factor.

4. The plant or plant part thereof of any one of claims 1 to 3, wherein the HY5 transcription factor comprises a constitutive photomorphogenesis 1 (COP1) binding site, and the at least one mutation is located in the COP1 binding site of the HY5 transcription factor encoded by the HY5 gene.

5. The plant or plant part thereof of any one of claims 1 to 4, wherein the at least one mutation results in a HY5 transcription factor having no or reduced COPl binding, optionally wherein the binding is reduced by about 30% to about 100% compared to a HY5 transcription factor lacking the at least one mutation.

6. The plant or plant part thereof according to any one of claims 1 to 5, wherein the at least one mutation is located in a region of the endogenous HY5 transcription factor having at least 80% sequence identity to SEQ ID NO:

106.

7. The plant or plant part thereof as claimed in any one of the preceding claims, wherein the endogenous HY5 gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

106.

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

9. The plant or plant part thereof as claimed in any one of the preceding claims, wherein the mutation is an in-frame deletion, an out-of-frame deletion, an in-frame insertion or an out-of-frame insertion.

10. The plant or plant part thereof of claim 8 or claim 9, wherein the deletion or insertion in the endogenous HY5 gene is a deletion or insertion of 1 to about 100 base pairs.

11. The plant or plant part thereof according to any one of claims 1 to 10, wherein the at least one mutation in the endogenous gene encoding the HY5 polypeptide is an out-of-frame deletion or an in-frame deletion, optionally wherein the at least one mutation is an in-frame deletion.

12. The plant or plant part thereof of any one of claims 1 to 8, wherein the at least one mutation comprises a base substitution to A, T, G or C.

13. The plant or plant part thereof according to any one of the preceding claims, wherein the at least one mutation in the endogenous gene encoding the HY5 polypeptide is a dominant allele or a dominant negative allele.

14. The plant or part thereof as claimed in any one of the preceding claims, wherein the at least one mutation is a non-natural mutation.

15. The plant or plant part thereof as claimed in any one of the preceding claims, wherein the plant is a monocot or a dicot.

16. The plant or plant part thereof as claimed in any one of the preceding claims, wherein the plant is planted at an increased density without reducing the plant yield on a per plant basis.

17. The plant or plant part thereof of claim 16, wherein the planting density is increased by about 5% to about 75% without decreasing plant yield on a per plant basis.

18. The plant or plant part thereof of any one of the preceding claims, wherein the plant is corn, soybean, canola, wheat, rice, cotton, sugarcane, sugar beet, barley, oat, 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 species.

19. The plant or plant part thereof as claimed in any one of the preceding claims, wherein the plant is a corn plant.

20. The plant or plant part thereof of any one of the preceding claims, wherein the at least one mutation in the endogenous Elongated Hypocotyl 5 (HY5) gene results in a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139 and / or encoding a mutated HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138.

21. A plant cell comprising an editing system, the editing system comprising: (a) CRISPR-Cas-associated effector proteins; and (b) Guide nucleic acids (gRNA, gDNA, crRNA, crDNA) comprising a spacer sequence complementary to an endogenous target gene encoding the elongated hypocotyl 5 (HY5) transcription factor.

22. The plant cell of claim 21, wherein the endogenous target gene encoding the HY5 transcription factor polypeptide (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

106.

23. The plant cell of claim 21 or claim 22, wherein the guide nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 107-113.

24. The plant cell of any one of claims 21 to 23, wherein the plant cell is a cell from corn, soybean, rapeseed, wheat, rice, cotton, sugarcane, sugar beet, barley, oat, 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 a Brassica species.

25. The plant cell of any one of claims 21 to 24, wherein the plant cell is a corn cell.

26. A plant cell comprising at least one mutation in an endogenous elongated hypocotyl 5 (HY5) gene, wherein the at least one mutation is a substitution, insertion and / or deletion introduced into the endogenous HY5 gene using an editing system comprising a nucleic acid binding domain that binds to a target site within the endogenous HY5 gene, wherein the endogenous HY5 gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

106.

27. The plant cell of claim 26, wherein the nucleic acid binding domain of the editing system is 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.

28. The plant cell of claim 26 or claim 27, wherein the at least one mutation is a substitution, deletion and / or insertion.

29. The plant cell of any one of claims 26 to 28, wherein the at least one mutation is a deletion in the region of the HY5 gene encoding the COPl binding site, optionally wherein the coding region comprises a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

106.

30. The plant cell of any one of claims 26 to 29, wherein the deletion or insertion in the endogenous HY5 gene is a deletion or insertion of 1 to about 100 base pairs.

31. The plant cell of any one of claims 26 to 30, wherein the deletion is an in-frame deletion or an out-of-frame deletion.

32. The plant cell of any one of claims 26 to 31, wherein the at least one mutation is a non-natural mutation.

33. A plant cell as described in any one of claims 26 to 32, wherein the plant cell is a cell from corn, soybean, rapeseed, wheat, rice, cotton, sugarcane, sugar beet, barley, oat, 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 species.

34. The plant cell of any one of claims 26 to 33, wherein the plant cell is a corn cell.

35. The plant cell of any one of claims 26 to 34, wherein the at least one mutation in the endogenous Elongated Hypocotyl 5 (HY5) gene results in a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139 and / or encoding a mutated HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138.

36. A plant regenerated from the plant part of any one of claims 1 to 20 or from the plant cell of any one of claims 21 to 35.

37. The plant of any one of claims 1 to 20 or 36, wherein the plant comprises a reduced shade avoidance response.

38. The plant of claim 36 or claim 37, wherein the plant is corn.

39. A method of providing a plurality of plants having increased yield when each plant in the plurality of plants is planted in close proximity to each other, the method comprising Planting two or more plants as claimed in any one of claims 1 to 20 or 36 to 38 in close proximity to each other provides a plurality of plants having increased yield compared to a plurality of control plants planted in close proximity to each other.

40. A method of producing / growing a genome-edited (e.g., base-edited) plant that is free of transgenes, the method comprising: (a) crossing the plant of any one of claims 1 to 20 or 36 to 38 with a plant that does not contain a transgene, thereby introducing the mutation into the plant that does not contain a transgene; as well as (b) selecting progeny plants that comprise the mutation but do not contain the transgene, thereby producing genome-edited (e.g., base-edited) plants that do not contain the transgene.

41. A method for generating a mutation in an endogenous elongated hypocotyl 5 (HY5) gene of a plant, the method comprising: (a) targeting a gene editing system to a portion of the HY5 gene, the portion comprising a nucleotide sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; and (b) selecting a plant comprising a modification in a region of the HY5 gene having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105.

42. A method for generating a variation in the Elongated Hypocotyl 5 (HY5) gene, the method comprising: introducing an editing system into a plant cell, wherein the editing system targets a region of the HY5 gene encoding the HY5 transcription factor, and The region of the HY5 gene is contacted with the editing system, thereby introducing mutations into the HY5 gene and generating variations in the HY5 gene in the plant cell.

43. The method of claim 42, wherein the HY5 gene comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NO:72 or SEQ ID NO:73, and / or encodes an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

74.

44. The method of claim 42 or claim 43, wherein the targeted region of the HY5 gene comprises at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-105.

45. The method of any one of claims 42 to 44, wherein contacting the region of the endogenous HY5 gene in the plant cell with the editing system produces a plant cell comprising the edited endogenous HY5 gene in its genome, the method further comprising (a) regenerating a plant from the plant cell; (b) selfing the plant to produce a progeny plant (E1); (c) determining the reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) of the progeny plant of (b); and (d) selecting progeny plants that exhibit a reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) compared to a control plant lacking the mutation.

46. ​​The method of claim 45, further comprising (e) selfing the selected offspring plant of (d) to produce an offspring plant (E2); (f) determining the reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) of the offspring plant of (e); and (g) selecting offspring plants that exhibit reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) compared to control plants, optionally repeating (e) to (g) one or more times.

47. A method for detecting a mutant elongated hypocotyl 5 (HY5) gene (mutation in an endogenous HY5 gene) in a plant, the method comprising detecting a HY5 gene in the genome of the plant having at least one mutation within a region having at least 80% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-105.

48. The method of claim 47, wherein the mutant HY5 gene detected comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139, and / or encodes a mutant HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138.

49. A method for editing a specific site in the genome of a plant cell, the method comprising: to cleave in a site-specific manner a target site within an endogenous Elongated Hypocotyl 5 (HY5) gene in the plant cell, wherein the endogenous HY5 gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 106, thereby producing editing in an endogenous HY5 gene of the plant cell.

50. The method of claim 49, wherein the HY5 gene encodes a HY5 transcription factor comprising a COPl binding region, and the editing results in a mutation in the COPl binding region of the HY5 transcription factor.

51. The method of claim 49 or claim 50, wherein the editing results in no or reduced COPl binding of the HY5 transcription factor, optionally wherein the binding is reduced by about 30% to about 100% compared to a control plant lacking the editing.

52. The method of any one of claims 49 to 51, wherein the editing is located in a region of the HY5 transcription factor having at least 80% sequence identity to SEQ ID NO:

106.

53. The method of any one of claims 49 to 52, wherein the editing in the endogenous HY5 gene results in an endogenous HY5 gene having at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139 and / or encoding a mutated HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138.

54. The method of any one of claims 49 to 53, further comprising regenerating a plant from the plant cell comprising the edit in the endogenous HY5 gene to produce a plant comprising the edit in its endogenous HY5 gene.

55. The method of claim 54, wherein a plant comprising said edit in its endogenous HY5 gene has a reduced shade avoidance response compared to a control plant lacking said edit.

56. A method for preparing a plant, the method comprising: (a) contacting a plant cell population comprising an endogenous gene encoding an elongated hypocotyl 5 (HY5) transcription factor with a nuclease targeted to the endogenous gene, wherein the nuclease is linked to a nucleic acid binding domain that binds to a target site within the endogenous gene, the endogenous gene (i) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (iv) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 106; (b) selecting, from the plant cell population, a plant cell in which the endogenous gene encoding the HY5 transcription factor has been mutated, thereby producing a plant cell comprising a mutation in the endogenous gene; and (c) growing the selected plant cell into a plant comprising a mutation in said endogenous gene encoding a HY5 transcription factor, optionally wherein said mutation reduces or eliminates the ability of said HY5 transcription factor to bind a constitutive photomorphogenesis 1 (COP1) polypeptide.

57. A method for reducing / inhibiting a shade avoidance response in a plant, the method comprising: (a) contacting a plant cell comprising an endogenous elongated hypocotyl 5 (HY5) gene with a nuclease targeted to the endogenous gene, wherein the nuclease is linked to a nucleic acid binding domain that binds to a target site within the endogenous gene, the endogenous gene (i) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (iv) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 106, thereby producing a plant cell comprising a mutation in the endogenous HY5 gene; and (b) growing the plant cell into a plant, thereby reducing / inhibiting a shade avoidance response in the plant.

58. A method for producing a plant or part thereof comprising at least one cell having a mutation in an endogenous Elongated Hypocotyl 5 (HY5) gene, the method comprising: contacting a target site within the endogenous HY5 gene in the plant or plant part with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain of the nuclease binds to the target site within the endogenous HY5 gene, wherein the endogenous HY5 gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 106, thereby producing a plant or part thereof comprising at least one cell having a mutation in the endogenous HY5 gene.

59. A method for producing a plant or part thereof comprising a mutation in an endogenous Elongated Hypocotyl 5 (HY5) gene, said mutation producing a mutated HY5 transcription factor having reduced binding to a Constitutive Photomorphogenesis 1 (COP1) polypeptide, said method comprising contacting a target site within said endogenous HY5 gene in said plant or plant part with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein said nucleic acid binding domain of said nuclease binds to the target site within said endogenous HY5 gene, wherein said endogenous HY5 gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 106, thereby producing a plant or part thereof having a mutation in the endogenous HY5 gene, said mutation resulting in a mutated HY5 transcription factor having reduced binding to the COPl polypeptide.

60. The method of any one of claims 56-59, wherein the nuclease cleaves the endogenous HY5 gene, thereby introducing the mutation into the endogenous HY5 gene, optionally wherein the mutation is introduced into the region of the HY5 gene encoding the COP1 binding region of the HY5 transcription factor, optionally wherein the COP1 binding region is located at the N-terminus of the encoded HY5 transcription factor.

61. The method of any one of claims 56 to 60, wherein the mutation is a non-natural mutation.

62. The method of any one of claims 56 to 61, wherein the mutation is a base substitution, a base insertion and / or a base deletion.

63. The method of any one of claims 56 to 62, wherein the mutation is a dominant mutation or a dominant negative mutation.

64. The method of any one of claims 56 to 63, wherein the mutation is an in-frame or out-of-frame insertion or an in-frame or out-of-frame deletion.

65. The method of any one of claims 56 to 64, wherein the mutation comprises a point mutation.

66. The method of any one of claims 56 to 65, wherein the mutation is a deletion of one base pair to about 100 base pairs.

67. The method of any one of claims 56 to 66, 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.

68. The method of any one of claims 42 to 46, 50 to 55 or 59 to 67, wherein the HY5 transcription factor is capable of regulating a response to light (eg, a shade avoidance response (SAR)) in the plant.

69. The method of any one of claims 42 to 46, 50 to 55 or 59 to 67, wherein the HY5 transcription factor comprises a COPl binding region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

106.

70. The method of any one of claims 56 to 69, wherein the plant produced comprises a mutated endogenous HY5 gene comprising at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139 and / or encodes a mutated HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138.

71. The method of any one of claims 56 to 70, wherein the resulting plant exhibits a reduced shade avoidance response compared to a control plant lacking the mutation.

72. A method as claimed in claim 71, wherein the plant with a reduced shade avoidance response comprises at least one of the following phenotypes when planted closely adjacent to one or more plants comprising a reduced shade avoidance response: increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, essentially no change in flowering time, increased photosynthetic efficiency and grain filling, and / or enhanced defense responses to pathogens and herbivores, as compared to a plant not comprising a reduced shade avoidance response planted closely adjacent to one or more plants but lacking the mutation and not exhibiting the reduced shade avoidance response.

73. A method for modifying an endogenous elongated hypocotyl 5 (HY5) gene in a corn plant or part thereof to reduce a shade avoidance response in the corn plant or part thereof, the method comprising modifying a target site within the endogenous HY5 gene in the corn plant or part thereof, wherein the endogenous HY5 gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 106, thereby modifying the endogenous HY5 gene and reducing the shade avoidance response in the corn plant or part thereof.

74. The method of claim 73, wherein the target site is located within a region of the HY5 gene that has at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-105.

75. A plant produced by any method of claims 36 or 56 to 75.

76. The plant of claim 75, wherein the plant comprises a mutant HY5 gene, wherein the mutant HY5 gene has at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139 and / or encodes a mutant HY5 polypeptide as described herein having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138.

77. The plant of claim 75 or claim 76, wherein the plant exhibits a reduced shade avoidance response.

78. A guide nucleic acid that binds to a target site within an endogenous HY5 gene, wherein the endogenous HY5 gene comprises a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO:75-105; encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:74; and / or encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

106.

79. A guide nucleic acid that binds to a target site within an endogenous HY5 gene, wherein the target site is located in a region of the endogenous HY5 gene that has at least 80% sequence identity to any one of SEQ ID NOs: 75-105.

80. The guide nucleic acid of claim 78 or claim 79, wherein the guide nucleic acid comprises a spacer having a nucleotide sequence of any one of SEQ ID NOs: 107-113.

81. A system comprising a guide nucleic acid as described in any one of claims 78 to 80 and a CRISPR-Cas effector protein associated with the guide nucleic acid.

82. The system of claim 81 , further comprising a tracr nucleic acid associated with the guide nucleic acid and a CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

83. 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 elongated hypocotyl 5 (HY5) gene.

84. The gene editing system of claim 83, wherein the HY5 gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

106.

85. The gene editing system of claim 83 or 84, wherein the guide nucleic acid comprises a spacer sequence having a nucleotide sequence of any one of SEQ ID NOs: 107-113.

86. The gene editing system of any one of claims 83 to 85, further comprising a tracr nucleic acid associated with the guide nucleic acid and a CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

87. A complex comprising a CRISPR-Cas effector protein comprising a cleavage domain and a guide nucleic acid, wherein the guide nucleic acid binds to a target site within an elongated hypocotyl 5 (HY5) gene, the HY5 gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 106, wherein the cleavage domain cleaves the target strand in the HY5 gene.

88. An expression cassette comprising (a) a polynucleotide encoding a CRISPR-Cas effector protein comprising a cleavage domain and (b) a guide nucleic acid that binds to a target site within an elongating hypocotyl 5 (HY5) gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to a target site within the HY5 gene, wherein the HY5 gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-105; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (d) encoding a region having at least 80% sequence identity with the amino acid sequence of SEQ ID NO:

106.

89. A nucleic acid encoding an elongated hypocotyl 5 (HY5) transcription factor having a mutated COPl binding region, optionally wherein the mutation is in a nucleic acid having at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139 and / or encoding a mutated HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138.

90. A nucleic acid comprising a mutated endogenous elongated hypocotyl 5 (HY5) gene, wherein the mutated endogenous HY5 gene has at least 90% sequence identity to any one of SEQ ID NOs: 114, 116, 118, 119, 121, 123, 125, 127, 129, 131, 133, 135, 137 or 139 and / or encodes a mutated HY5 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 115, 117, 120, 122, 124, 126, 128, 130, 132, 134, 136 or 138.

91. A plant or plant part thereof comprising the nucleic acid of claim 89 or claim 90.

92. A corn plant or plant part thereof comprising the nucleic acid of claim 89 or claim 90, optionally wherein the corn plant comprises a dwarf stature / semi-dwarf phenotype.

93. A corn plant or plant part thereof comprising at least one mutation in the endogenous Elongated Hypocotyl 5 (HY5) gene having the Gene Identification Number (Gene ID) of Zm00001d015743.

94. The plant of claim 91 or the corn plant of claim 92 or claim 93, wherein the plant or corn plant exhibits a reduced shade avoidance response compared to a plant lacking the mutation.

95. The plant or corn plant of claim 94, further comprising increased yield, increased upright growth, reduced height, reduced root-to-shoot ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence, essentially no change in flowering time, increased photosynthetic efficiency and grain filling, and / or enhanced defense responses to pathogens and herbivores when planted closely adjacent to one or more plants comprising a reduced shade avoidance response, as compared to a plant not comprising a reduced shade avoidance response planted closely adjacent to one or more plants but lacking the mutation and not exhibiting the reduced shade avoidance response.

96. A guide nucleic acid that binds to a target nucleic acid within the endogenous Elongated Hypocotyl 5 (HY5) gene having a gene identification number (Gene ID) of Zm00001d015743 (Maize Genetics and Genome Database (Maize GDB)).

97. A method for producing a plant comprising a mutation in an endogenous Elongated Hypocotyl 5 (HY5) gene and at least one polynucleotide of interest, the method comprising: crossing a first plant that is a plant of any one of claims 1 to 20, 36 to 38, 75 to 77, or 91 to 95 with a second plant comprising the at least one polynucleotide of interest to produce progeny plants; and Progeny plants are selected that comprise the mutation in the HY5 gene and the at least one polynucleotide of interest, thereby producing the plant comprising the mutation in the endogenous HY5 gene and the at least one polynucleotide of interest.

98. A method for producing a plant comprising a mutation in an endogenous HY5 gene and at least one polynucleotide of interest, the method comprising: Introducing at least one polynucleotide of interest into a plant as described in any one of claims 1 to 20, 36 to 38, 75 to 77 or 91 to 95, thereby producing a plant comprising a mutation in the HY5 gene and at least one polynucleotide of interest.

99. The method of any one of claim 97 or claim 98, 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.

100. A method of producing a plant comprising a mutation in an endogenous HY5 gene and exhibiting a phenotype of improved yield traits, improved plant architecture and / or improved defense traits, the method comprising: Crossing a first plant that is a plant as claimed in any one of claims 1 to 20, 36 to 38, 75 to 77 or 91 to 95 with a second plant that exhibits a phenotype of improved yield traits, improved plant architecture and / or improved defense traits; and Selecting offspring plants that contain the mutation in the HY5 gene and have a phenotype of improved yield traits, improved plant architecture and / or improved defense traits, thereby producing the plant that contains the mutation in the endogenous HY5 gene and exhibits a phenotype of improved yield traits, improved plant architecture and / or improved defense traits compared to control plants.

101. A method of controlling weeds in a container (e.g., pots or seed trays, etc.), a growth chamber, a greenhouse, a field, a recreational area, a lawn, or a roadside, the method comprising: Applying a herbicide to one or more (several) plants as described in any one of claims 1 to 20, 36 to 38, 75 to 77 or 91 to 95 growing in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn or a roadside, thereby controlling the weeds in the container, the growth chamber, the greenhouse, the field, the recreational area, the lawn or the roadside where the one or more plants grow.

102. A method of reducing insect predation on a plant, the method comprising applying an insecticide to one or more plants of any one of claims 1 to 20, 36 to 38, 75 to 77 or 91 to 95, thereby reducing insect predation on the one or more plants.

103. A method of reducing fungal diseases on plants, the method comprising applying a fungicide to one or more plants of any one of claims 1 to 20, 36 to 38, 75 to 77 or 91 to 95, thereby reducing fungal diseases on the one or more plants.

104. The method of claim 102 or claim 103, wherein the one or more plants are grown in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn, or a roadside.

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