Methods and compositions for altering plant shading

Abstract

The present invention relates to compositions and methods for modifying a phytochrome interaction factor (PIF) transcription factor gene in a plant to inhibit shade-avoiding response. The invention further relates to plants and plant parts produced using the methods and compositions of the invention.

Description

[0001] Declaration concerning the electronic file of the sequence listing

[0002] The XML-formatted sequence listing entitled 1499-122_ST26.xml, 405,907 bytes in size, generated on January 30, 2024, and submitted herewith is hereby incorporated by reference into this specification for its disclosure. Technical Field

[0003] The present invention relates to compositions and methods for modifying the phytochrome interacting factor (PIF) transcription factor gene in plants to suppress the shade avoidance response. The present invention further relates to plants and plant parts produced using the methods and compositions of the present invention.

[0004] Priority Declaration

[0005] This application claims the benefit under 35 U.S.C. §119(e) of U.S. Provisional Application No. 63 / 485,263, filed on February 16, 2023, the entire contents of which are incorporated herein by reference. 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 Exp Bot 58:3079-3089 (2007)), in which plants try to outgrow neighboring plants by growing toward resources (primarily light). Overcrowding of plants can produce shade avoidance syndrome (SAS), in which plants lack vigor and yield decreases. Shade avoidance involves the relative proportions of red and far-red light present in a plant's environment (Ballare et al., Science, 247:329-332 (1990)). Plants absorb most of the available red light 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 can include reduced branching, increased plant height, reduced leaf area, auxin redistribution, increased ethylene production, and accelerated flowering. SAS is characterized by an increase in the root / crown ratio, increased plant height, and reduced yield per plant. In typical monoculture crop environments, interplant 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 a phytochrome interacting factor (PIF) transcription factor, wherein the mutation disrupts binding of the PIF transcription factor to DNA in the plant or part thereof, 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, wherein the editing system comprises: (a) a CRISPR-Cas-associated effector protein; and (b) a guide nucleic acid (gRNA, gDNA, crRNA, crDNA), wherein the guide nucleic acid has a spacer sequence, and the spacer sequence is complementary to an endogenous target gene encoding a phytochrome interacting factor (PIF) transcription factor.

[0010] A third aspect of the present invention provides a plant cell comprising a mutation in the basic helix-loop-helix (bHLH) domain of a phytochrome-interacting factor (PIF) transcription factor, wherein the mutation is a substitution, insertion and / or deletion introduced into the endogenous PIF gene encoding the PIF transcription factor using an editing system comprising a nucleic acid binding domain that binds to a target site within an endogenous PIF gene encoding the PIF transcription factor, wherein the endogenous PIF gene: (a) comprises a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; (c) encodes a region having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: NO:71, 74, 77, 80 and / or 83 has an amino acid sequence with at least 80% sequence identity; and / or (d) encodes a polypeptide having a sequence with at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO:113, optionally wherein the mutation may be a non-natural mutation.

[0011] A fourth aspect of the present invention provides a method for providing a plurality of plants having increased yield when each of the plurality of plants are planted adjacent to each other in a planting area, the method comprising planting two or more plants of the present invention adjacent to each other, thereby providing a plurality of plants having increased yield compared to a plurality of control plants planted adjacent 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 progeny 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 phytochrome interacting factor (PIF) gene in a plant is provided, the method comprising: (a) targeting a gene editing system to a portion of the PIF gene, the portion comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; and (b) selecting a plant comprising a modification located in a region of the PIF gene, the region having at least 80% sequence identity to any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112, optionally wherein the mutation may be a non-natural mutation.

[0014] In a seventh aspect, a method for producing a change in a phytochrome interacting factor (PIF) polypeptide is provided, the method comprising introducing an editing system into a plant cell, wherein the editing system is targeted to a region of an endogenous phytochrome interacting factor (PIF) gene encoding the PIF polypeptide, and contacting the region of the endogenous PIF gene with the editing system, thereby introducing a mutation into the endogenous PIF gene and producing a change in the PIF polypeptide of the plant cell.

[0015] An eighth aspect provides a method for detecting a mutant PIF gene in a plant (a mutation in an endogenous PIF gene), the method comprising detecting a PIF gene in the genome of the plant, the PIF gene having at least one mutation in a region having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-112.

[0016] A ninth aspect provides a method for editing a specific site in the genome of a plant cell, the method comprising cleaving a target site within an endogenous phytochrome interacting factor (PIF) gene in the plant cell in a site-specific manner, the endogenous PIF gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 71, 74, 77, 80 and / or 83; and / or (d) encoding a polypeptide having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: The amino acid sequence of NO:113 has a region of at least 80% sequence identity, thereby producing editing in the endogenous PIF gene of the plant cell.

[0017] In a tenth aspect, a method for making a plant is provided, the method comprising: (a) contacting a population of plant cells comprising an endogenous gene encoding a phytochrome interacting factor (PIF) 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 any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; (iii) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 71, 74, 77, 80 and / or 83; and / or (iv) encoding a polypeptide having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: (b) selecting a plant cell from the population, the plant cell comprising a mutation in the endogenous gene encoding the PIF transcription factor, wherein the mutation is a substitution of at least one amino acid residue in the polypeptide of (iii) or (iv) or in the polypeptide encoded by any one of the nucleotide sequences of (i) or (ii), wherein the mutation modifies the bHLH domain of the PIF transcription factor; and (c) growing the selected plant cell into a plant comprising the mutation in the endogenous gene encoding the PIF transcription factor.

[0018] In an 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 phytochrome interacting factor (PIF) gene encoding a PIF 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 PIF gene, the endogenous PIF gene: (i) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; (iii) encoding a region having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: NO:71, 74, 77, 80 and / or 83 has a polypeptide with a sequence having at least 80% sequence identity to the amino acid sequence of any one of NO:71, 74, 77, 80 and / or 83; and / or (iv) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQID NO:113, thereby producing a plant cell containing a mutation in the endogenous PIF gene encoding the PIF polypeptide; and (b) growing the plant cell into a plant, thereby reducing / inhibiting the 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 phytochrome interacting factor (PIF) gene, the method comprising contacting a target site within the endogenous PIF 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 PIF gene, wherein the endogenous PIF gene: (a) comprises a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; (c) encodes a region having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: NO:71, 74, 77, 80 and / or 83 has a polypeptide having a sequence with at least 80% sequence identity to the amino acid sequence of any one of NO:71, 74, 77, 80 and / or 83; and / or (d) encoding a region with at least 80% sequence identity to the amino acid sequence of SEQ ID NO:113, thereby producing a plant or part thereof, said plant or part thereof comprising at least one cell having a mutation in said endogenous PIF gene.

[0020] In a thirteenth aspect, a method of producing a plant or part thereof comprising a mutation in a basic helix-loop-helix (bHLH) domain of a phytochrome interacting factor (PIF) transcription factor is provided, the method comprising contacting a target site within an endogenous phytochrome interacting factor (PIF) 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 PIF gene, wherein the endogenous PIF gene: (a) comprises a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; (c) encodes a region having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: NO:71, 74, 77, 80 and / or 83; and / or (d) encoding a polypeptide having a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO:113; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:113, thereby producing a plant or part thereof having a mutated phytochrome interacting factor (PIF) transcription factor containing a modified bHLH domain.

[0021] In a fourteenth aspect, a method for modifying an endogenous phytochrome interacting factor (PIF) in a plant or a part thereof to reduce / inhibit a shade avoidance response in the plant or a part thereof is provided, the method comprising modifying a target site within an endogenous PIF gene in the plant or a part thereof, wherein the endogenous PIF gene (a) comprises a nucleotide sequence having at least 80% sequence identity to any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; (b) comprises a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 71, 74, 77, 80 and / or 83; and / or (d) encodes a polypeptide having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: The invention relates to a method for modifying an endogenous PIF gene and reducing / suppressing the shade avoidance response in the 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 a phytochrome interacting factor (PIF) transcription factor, the endogenous gene comprising a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; or encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 71, 74, 77, 80 and / or 83; and / or the target site comprises a nucleotide sequence having at least 80% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; or encodes an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 113.

[0023] A further aspect provides a system comprising a guide nucleic acid of the 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 a phytochrome interacting factor (PIF) gene.

[0025] A further aspect provides 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 a phytochrome interacting factor (PIF) gene, the PIF gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 71, 74, 77, 80 and / or 83; and / or (d) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: The amino acid sequence of NO:113 has a region with at least 80% sequence identity, wherein the cleavage domain cleaves the target chain in the PIF 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 a phytochrome interacting factor (PIF) gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to the target site within the PIF gene, the PIF gene: (a) comprising a sequence having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81 and / or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112; (c) encoding a region having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: NO:71, 74, 77, 80 and / or 83; and / or (d) encoding a polypeptide having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO:113.

[0027] Additional aspects provide a nucleic acid encoding a phytochrome interacting factor (PIF) transcription factor comprising a mutated bHLH domain as described herein, and / or a nucleic acid comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134 and / or 135, and / or encoding a mutated PIF polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 121, 123, 125, 127 and / or 131.

[0028] Also provided herein is a PIF polypeptide modified as described herein. In some embodiments, the modified PIF polypeptide comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 71, 74, 77, 80, and / or 83.

[0029] Further provided are plants, plant cells and plant parts produced by the methods of the invention and comprising one or more than one mutated PIF 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 invention.

[0030] These and other aspects of the invention are set forth in more detail in the description of the invention which follows.

[0031] A brief description of the sequence

[0032] SEQ ID NOs: 1-17 are exemplary Cas12a amino acid sequences that can be used in the present invention.

[0033] SEQ ID NOs: 18-20 are exemplary Cas12a nucleotide sequences that can be used in the present invention.

[0034] SEQ ID NOs: 21-22 are exemplary regulatory sequences encoding promoters and introns.

[0035] SEQ ID NOs: 23-29 are exemplary cytosine deaminase sequences that can be used in the present invention.

[0036] SEQ ID NOs: 30-40 are exemplary adenine deaminase amino acid sequences that can be used in the present invention.

[0037] SEQ ID NO: 41 is an exemplary uracil-DNA glycosylase inhibitor (UGI) sequence that can be used in the present invention.

[0038] SEQ ID NOs: 42-44 provide exemplary peptide tags and affinity polypeptides useful in the present invention.

[0039] SEQ ID NOs: 45-55 provide exemplary RNA recruitment motifs and corresponding affinity polypeptides that can be used in the present invention.

[0040] SEQ ID NOs: 56-57 are exemplary Cas9 polypeptide sequences that can be used in the present invention.

[0041] SEQ ID NOs: 58-68 are exemplary Cas9 polynucleotide sequences that can be used in the present invention.

[0042] SEQ ID NO:69 and SEQ ID NO:72 are exemplary PIF3 genomic sequences.

[0043] SEQ ID NO:75 and SEQ ID NO:78 are exemplary PIF4 genomic sequences.

[0044] SEQ ID NO:81 is an exemplary PIF5 genomic sequence.

[0045] SEQ ID NO: 70 and SEQ ID NO: 73 are exemplary PIF3 cDNA sequences.

[0046] SEQ ID NO: 76 and SEQ ID NO: 79 are exemplary PIF4 cDNA sequences.

[0047] SEQ ID NO:82 is an exemplary PIF5 cDNA sequence.

[0048] SEQ ID NO:71 is an exemplary PIF3 polypeptide sequence encoded by SEQ ID NO:69 and SEQ ID NO:70.

[0049] SEQ ID NO:74 is an exemplary PIF3 polypeptide sequence encoded by SEQ ID NO:72 and SEQ ID NO:73.

[0050] SEQ ID NO:77 is an exemplary PIF4 polypeptide sequence encoded by SEQ ID NO:75 and SEQ ID NO:76.

[0051] SEQ ID NO:80 is an exemplary PIF4 polypeptide sequence encoded by SEQ ID NO:78 and SEQ ID NO:79.

[0052] SEQ ID NO:83 is an exemplary PIF5 polypeptide sequence encoded by SEQ ID NO:81 and SEQ ID NO:82.

[0053] SEQ ID NOs:84-112 are exemplary nucleic acid sequences (regions) from PIF polynucleotides (SEQ ID NOs:84-87 (PIF3), SEQ ID NOs:88-91 (PIF3), SEQ ID NOs:92-95 (PIF4), SEQ ID NOs:96-108 (PIF4), SEQ ID NOs:109-112 (PIF5)).

[0054] SEQ ID NO: 113 is an exemplary region from the PIF transcription factor.

[0055] SEQ ID NOs: 114-119 are exemplary spacer sequences for nucleic acid guides that can be used in the present invention.

[0056] SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and 135 are exemplary mutated PIF genomic sequences edited as described herein.

[0057] SEQ ID NOs: 121, 123, 125, 127, and 131 are exemplary mutated PIF polypeptides.

[0058] SEQ ID NO:136 is a portion of 11 consecutive nucleotides deleted from SEQ ID NO:78 to generate the mutated nucleic acid sequence SEQ ID NO:132.

[0059] SEQ ID NO:137 is a portion of 10 consecutive nucleotides deleted from SEQ ID NO:78 to generate the mutated nucleic acid sequence SEQ ID NO:134. DETAILED DESCRIPTION

[0060] The present invention will now be described hereinafter with reference to the following examples, in which embodiments of the present invention are shown. This description is not intended to be an exhaustive list of all different ways in which the present invention may be implemented or all features that may be added to the present invention. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular 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 that many variations and additions to the various embodiments set forth herein will be apparent in light of this disclosure, and these variations and additions do not deviate from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaustively describe all permutations, combinations, and variations thereof.

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

[0062] 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.

[0063] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein may be used in any combination. Furthermore, the present invention contemplates that in some embodiments of the 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 any combination thereof, may be omitted or disclaimed, individually or in any combination.

[0064] 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.

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

[0066] 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 encompass 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.

[0067] 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."

[0068] 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.

[0069] As used herein, the terms “comprise,” “comprises,” and “comprising” 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.

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

[0071] As used herein, the terms "increase," "increasing," "increased," "enhance," "enhanced," "enhancing," and "enhancement" (and 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.

[0072] 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% as 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.

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

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

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

[0076] A "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.

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

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

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

[0080] 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.

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

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

[0083] 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 mutation.

[0084] A "dominant negative mutation" is a mutation that produces an altered gene product (e.g., one that has abnormal function relative to the wild-type gene) 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 can also be referred to as a "reversal allele mutation."

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

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

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

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

[0089] A "gain of function" allele or mutation is a mutation that confers a new function to the encoded gene product and / or confers a new gene expression pattern. In some embodiments, a gain of function mutation can be dominant or semi-dominant.

[0090] As used herein, a "non-natural mutation" refers to a mutation that arises through human intervention and is distinct from a naturally occurring mutation present in the same gene (eg, occurs in nature and is not the result of modification by humans).

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

[0092] As used herein, the terms "desired allele," "target allele," and / or "allele of interest" are used interchangeably to refer to an allele associated with a desired trait. In some embodiments, a desired allele can 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 present invention, the phrases "desired allele," "target allele," or "allele of interest" refer to an allele that is 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.

[0093] A marker is "associated" with a trait when it is linked to the marker and when the presence of the marker is an indicator 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 associated with the allele or chromosomal interval and when the presence of the marker is an indicator of whether the allele or chromosomal interval is present in the plant / germplasm containing the marker.

[0094] As used herein, the terms "backcross" and "backcrossing" refer to the process of crossing a progeny plant back to one of its parents one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8, etc.). In a backcross protocol, the "donor" parent refers to the parent plant that has the desired gene or locus to be introgressed. The "recipient" parent (used one or more times) 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, in TECHNIQUES ET UTILISATIONS DESMARQUEURS 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.

[0095] As used herein, the term "cross" or "crossed" refers to the fusion of gametes by pollination to produce offspring (e.g., cells, seeds, or plants). The term encompasses sexual crossing (pollination of one plant by another) and selfing (self-pollination, e.g., when pollen and ovules are from the same plant). The term "crossing" refers to the act of fusing gametes by pollination to produce offspring.

[0096] As used herein, the terms "introgression," "introgressing," and "introgressed" refer 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. For example, the desired allele at a given locus can be passed to at least one progeny 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 progeny comprising the desired allele can be backcrossed one or more times (e.g., 1 time, 2 times, 3 times, 4 times, or more) with a line having the desired genetic background to select 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 infiltrated 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 progeny can then be backcrossed one or more times and selected until the progeny possess the genetic marker associated with increased yield under non-water stress conditions in the recurrent parent background.

[0097] A "genetic map" is a description of the genetic linkage relationships between loci on one or more chromosomes within a given species, typically depicted in a 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 polymorphism potential of each marker between different populations. The order and genetic distance between loci can vary from one genetic map to another.

[0098] As used herein, the term "genotype" refers to the genetic makeup of an individual (or individual population) at one or more genetic loci, contrasted with the proterties (phenotype) that can be observed and / or detected and / or performed. Genotype is defined by the alleles of one or more known loci inherited by an individual from its parent. The term genotype can be used to refer to the genetic makeup 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 makeup of all genes in an individual's genome. Genotype can, for example, be characterized indirectly using a mark and / or directly characterized by nucleic acid sequencing.

[0099] As used herein, the term "germplasm" refers to the genetic material of an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety, or section), or a clone derived from a line, variety, species, or culture, or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety, or section), or a clone derived from a line, variety, species, or culture. 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 makeup, 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 from which new plants can be grown, and plant parts (e.g., leaves, stems, buds, roots, pollen, cells, etc.) that can be cultured into complete plants.

[0100] 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.

[0101] As used herein, the terms "exotic," "exotic line," and "exotic germplasm" refer to any plant, line, or germplasm that is not superior. Generally, an 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., to introduce new alleles into a breeding program).

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

[0103] As used herein, the term "inbred line" 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.

[0104] A "haplotype" is the genotype of an individual at multiple genetic loci, i.e., the 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" can refer to a polymorphism at a specific locus (e.g., a single marker locus), or a polymorphism at multiple loci along a chromosome segment.

[0105] As used herein, "shade avoidance response" is defined as a certain growth habit that a plant experiences in response to a low red light: far-red light (R:FR) ratio. Inhibition of the shade avoidance response refers to the inhibition or reduction of the growth changes exhibited by a plant 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 traits of the present invention (e.g., a mutated PIF transcription factor as described herein) and isogenic plants that do not contain the traits in a controlled environment of low R:FR light ratios. 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% (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 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, 110, 120, 130, 140, 150% or lower or any range or value thereof;45,46,47,48,49,50,51,52,53,54,55 ,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,110,120,130,140,1 50% or less) (e.g., about 5% to about 10% less, about 5% to about 15% less, about 5% to about 20% less, about 5% to about 25% less, about 5% to about 30% less, about 5% to about 40% less, about 5% to about 50% less, about 10% to about 20% less, about 10% to about 30% less, about 10% to about 50% less, about 10% to about 70% less, about 15% to about 20% less, about 15% to about 30% less, about 15% to about 50% less, about 10% to about 70% less, about 15% to about 20% less, about 15% to about 30% less, about 15% to about 50% less 0%, about 20% to about 30%, about 20% to about 50%, about 20% to about 70%, about 40% to about 50%, about 40% to about 60%, about 40% to about 80%, about 40% to about 100%, about 50% to about 70%, about 50% to about 100%, about 50% to about 125%, about 75% to about 100%, about 75% to about 120%, about 75% to about 140%, etc.);

[0106] Plants exhibiting SAR exhibit hypocotyl and internode hyperelongation, longer leaves, narrow leaves, impaired root growth, early flowering and reduced seed set, low photosynthesis efficiency, enhanced green alignment, high lodging rate, accelerated senescence, reduced grain filling; and active inhibition of disease and herbaceous response mechanisms. Plants wherein SAR is reduced as described herein may have increased yield compared to plants that do not comprise SAR reduction. As used herein, "increased yield" refers to any plant trait related to growth, such as biomass, yield, nitrogen use efficiency (NUE), inflorescence size / weight, fruit yield, fruit quality, fruit size, seed size, seed number, leaf tissue weight, nodule number, nodule mass, nodulation activity, number of spikes, number of tillers, number of flowers, number of tubers, tuber mass, bulb mass, number of seeds, total seed mass, leaf emergence rate, tiller emergence rate, seedling emergence rate, root length, number of roots, size and / or weight of the root mass, or any combination thereof. Thus, in some aspects, "increased yield" can include, but is not limited to, increased inflorescence production, increased fruit yield (e.g., the number, weight, and / or size of the fruits increased; e.g., the number, weight, and / or size of the ears of, for example, corn) compared to a control plant or part thereof (e.g., 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) that does not comprise a mutated endogenous nucleic acid encoding a PIF transcription factor as described herein, including when grown in close proximity with other plants). In some aspects, the increased yield can be expressed as the number of grains produced per unit land area (e.g., bushels per acre of land).

[0107] "Seed weight" is determined by a combination of kernel morphological traits such as seed length, seed width, and seed thickness, as well as kernel filling, and all of these traits are controlled by quantitative genetics.

[0108] As used herein, " height reduction " means and suppresses stem elongation in response to abundant far-red light.Therefore, for example, as compared with the control plant that does not comprise sudden change and also grows under the shade condition, the plant with the sudden change in PIF gene as described herein shows the height that reduces when growing under the shade condition.

[0109] As used herein, " the crown of reduction: root ratio " means the reduction of above-ground biomass relative to the ratio of underground biomass.Therefore, for example, as compared with the control plant that does not comprise sudden change and also grows under the shade condition, the plant with the sudden change in the PIF gene as described herein shows the crown of reduction when growing under the shade condition: root ratio.

[0110] As used herein, "increased upright growth" means that plants with mutations in the PIF gene as described herein continue to grow upright when experiencing shade avoidance (as when planted at high density), rather than "leaning" or growing in the direction of light, compared to control plants without mutations that also experience shade avoidance. Plants with increased upright growth grow more vertically than control plants.

[0111] As used herein, "no substantial change in flowering time" means that plants having a mutation in the PIF gene as described herein maintain their normal flowering time when subjected to shade avoidance, such as when planted at high density, as compared to control plants (not comprising the mutation) subjected to shade avoidance.

[0112] Compared with the plant that does not comprise the modification in (lack) at least one endogenous PIF gene, wherein at least one (for example, one or more, for example, 1,2,3 or 4 or more) endogenous PIF gene is modified as described herein (for example, comprising modification as described herein) plant can have the yield traits through improvement.As used herein, " yield traits through improvement " refers to any plant traits relevant to growth, for example biomass, output, nitrogen use efficiency (NUE), inflorescence size / weight, fruit yield, fruit quality, fruit size, seed size (for example, seed area, seed size), seed quantity, leaf tissue weight, raw node quantity, raw node quality, raw node activity, seed head quantity, tiller quantity, branch quantity, flower quantity, stem tuber quantity, stem tuber quality, corm quality, seed quantity, seed total mass, leafing rate, tiller / branch appearance rate, emergence rate, root length, root quantity, size and / or weight of root group or its any combination. In some respects, " yield traits through improvement " can include but not limited to and compare with control plant or its part (for example, do not comprise the plant of the endogenous PIF nucleic acid through sudden change as described herein), the inflorescence of increase produces, the fruit yield of increase (for example, quantity, weight and / or the size of the fruit of increase; For example, the quantity, weight and / or the length of the ear of corn for example of increase), the fruit quality of increase, the quantity of the root of increase, size and / or weight, the meristem size of increase, the seed size of increase (for example, seed area and / or seed weight), the biomass of increase, the leaf size of increase, the nitrogen use efficiency of raising, the height of increase, the internode number of increase and / or the internode length of increase. In some respects, the yield traits through improvement can be expressed as the quantity (for example, the bushel number of every acre of land) of the grain / seed produced per unit land area. In certain embodiments, one or more yield traits through improvement can be the grain row number of increase, optionally do not reduce ear length.

[0113] As used herein, " control plant " means the plant not containing the PIF gene through editing as described herein.Control plant is used for identifying and selecting the plant of editing as described herein, and it has the proterties or phenotype of enhancement or change compared with control plant.Suitable control plant can be the parental line plant of the plant comprising the PIF gene through mutation for generation, for example, lacks the wild-type plant of editing in endogenous PIF gene as described herein.Suitable control plant can also be the plant containing the recombinant nucleic acid that gives other proterties, for example, the transgenic plant that herbicide tolerance enhances.In some cases, suitable control plant can be the progeny (comprising grain weight) of heterozygous or semizygous transgenic plant line lacking the PIF gene through mutation as described herein, compared with control plant or its part, have the grain row number (optionally wherein spike length does not reduce substantially), the number of the pod of increase, the seed number of every pod of increase and the spike length of increase.

[0114] As used herein, " proterties " are physiology, morphology, biochemistry or the physical characteristic of plant or specific plant material or cell.In some cases, this characteristic is visible to the human eye and can be measured mechanically, as the size, weight, shape, form, length, height, growth rate and developmental stage of seed or plant, or can be measured by biochemical technique, as the protein, starch, some metabolite or the oil content of detection seed or leaf, or by observing metabolism or physiological process, for example, by measuring the tolerance to water shortage 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 observation such as hyperosmotic stress tolerance or output.But any technology all can be used to measure the amount, comparative level or the difference of any selected chemical compound or macromolecule in transgenic plant.

[0115] As used herein, " enhanced proterties " means the feature of the plant caused by the sudden change in the PIF gene as described herein.This type of proterties includes but is not limited to the agronomic proterties of enhancing, and the agronomic proterties of described enhancing is characterised in that plant morphology, physiology, growth and development, output enhancement, nutrition enhancement, disease or pest resistance or environment or chemical tolerance.In certain embodiments, the phenotype of the proterties / change of enhancing can be, for example, the fringe size of the increase, the every strain plant fringe dry weight of the increase, the every fringe grain number of increase, the every grain weight of increase, the every strain plant grain number of increase, the fringe empty grain of minimization, the grain filling phase of prolongation, the plant height of reduction, the root branch quantity of increase, the total root length of increase, drought tolerance, the water use efficiency of raising, cold tolerance, the nitrogen use efficiency of raising and / or the output of increase from planting to ripe days, the stem size of increase, the leaf number of increase, the nutritional stage plant height growth rate of increase, the increase, the fringe size of increase, the every strain plant fringe dry weight of increase, the every fringe grain number of increase, the every grain weight of increase, the every strain plant grain number of increase, the fringe empty grain of minimization, the grain filling phase of prolongation, the plant height of reduction, the root branch quantity of increase, the total root length of increase, drought tolerance, the water use efficiency of raising, cold tolerance, the nitrogen use efficiency of raising and / or the output of increase.In certain embodiments, proterties is the output that increases under non-stress condition or the output that increases under environmental stress condition. Stress conditions can comprise biotic stress and abiotic stress, for example, arid, shade-avoiding, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, low temperature exposure, heat exposure, osmotic stress, nitrogen nutrient availability reduction, phosphorus nutrient availability reduction and high plant density." output " can be subject to the impact of many characteristics, includes but not limited to plant height, plant biomass, pod number, pod position on plant, internode number, pod broken incidence rate, 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 ratio, seedling vigor and childhood character. Output can also be subject to the impact of the following factors: germination efficiency (comprising the germination under stress conditions), growth rate (comprising the growth rate under stress conditions), flowering time and duration, ear number, ear size, ear weight, the seed quantity of each ear or each pod, seed size, the composition (starch, oil, protein) of seed and the characteristic of seed filling.

[0116] As used herein, term " proterties modification " is contained and changes naturally occurring proterties by producing detectable characteristic difference with respect to the plant (as wild-type plant or negative segregant) that does not comprise described sudden change in the plant that comprises the sudden change in endogenous PIF gene as described herein.In some cases, proterties change can be evaluated quantitatively.For example, compared with control plant, proterties change can make observed proterties characteristic or phenotype increase or reduce.As everyone knows, the proterties of change may have natural variant.Therefore, compared with control plant, observed proterties modification can cause the normal distribution of proterties characteristic or phenotype and the variation of magnitude in plant.

[0117] The present disclosure relates to a plant with an economically relevant characteristic (more specifically the shade-avoiding of minimizing) of improvement. More specifically, the present disclosure relates to a plant comprising the sudden change as described herein in the PIF gene, wherein as compared with the control plant lacking the sudden change, the plant has the shade-avoiding response of minimizing (for example, the plant comprising sudden change as described herein produces a shorter plant than the plant of the sudden change of growing under the same conditions when growing under shade-avoiding conditions). In certain embodiments, the plant of the present disclosure further shows the proterties of the improvement relevant to output, includes but is not limited to the nitrogen use efficiency of increase, the nitrogen stress tolerance of increase, the water use efficiency of increase and / or the drought tolerance of increase, as defined below and discussed.

[0118] Yield can be defined as the measurable product with economic value from crops. Yield can be defined in terms of quantity and / or quality. Yield can directly depend on several factors, for example, the number and size of organs (for example, 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 phenotype can be the factors that determine yield. Therefore, optimizing the above factors can help to increase crop yield.

[0119] The increase / improvement of the yield correlated traits mentioned herein also can be considered to refer to the biomass (weight) increase of one or more parts of plant, and described one or more parts can comprise above-ground and / or underground (can gather in the crops) plant part.Particularly, this type of part that can gather in the crops is seed, and the execution of method of the present disclosure produces the plant that yield improves and particularly seed yield improves with respect to the seed yield of suitable control plant.The " yield " of term plant can relate to the nutrient biomass (root and / or seedling biomass), reproductive organ and / or propagule (such as seed) of described plant.In certain embodiments, implementing method of the present disclosure causes plant to have the shade-avoiding response that reduces with respect to suitable control plant.

[0120] 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. Increased yield can be attributed to increased utilization of key biochemical compounds (such as nitrogen, phosphorus, and carbohydrates), or to improved responses to environmental stresses (such as cold, heat, drought, salt, shade avoidance, high plant density, and pest or pathogen attack).

[0121] "Increased yield" can be manifested as one or more of the following: (i) an increase in plant biomass (weight), an increase in root biomass (increased number of roots, increased root thickness, increased root length) or an increase in the biomass of any other harvestable part of one or more parts of the plant, particularly the aboveground (harvestable) parts of the plant; or (ii) an increase in early vigour, defined herein as an increase in aboveground area of ​​the seedlings about three weeks after germination.

[0122] " early vigor " refers to the plant growth of active health, especially in the early stage of plant growth, and can be owing to for example plant better adapting to its environment (for example, optimizing the utilization of energy, absorbing nutrient and distributing carbon between seedling and root) the plant fitness that causes increases and produces.For example, early vigor can be the combination of the ability of seed to germinate and emerge after planting and the ability of young plant to grow and develop after emerging.Plants with early vigor also show that seedling survival rate improves and crop planting is better, and this can make field height uniform conventionally, and wherein most plants reach each developmental stage simultaneously basically, thereby usually make output increase.Therefore, early vigor can be determined by measuring various factors, as grain weight, germination percentage, emergence percentage, seedling growth, seedling height, root length, root and seedling biomass, canopy size and color etc.

[0123] Additionally, 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, e.g., 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 fill rate; an increased number of filled seeds; an increased seed size (length, width, area, girth, and / or weight), which can also affect seed composition; and / or an increased seed volume, which can also affect seed composition. In one embodiment, increased yield can be increased seed yield, e.g., increased seed weight; an increased number of filled seeds; and / or an increased harvest index.

[0124] Yield enhancement may also result from, or occur as a result of, changes in plant architecture.

[0125] Yield improvement may also be expressed as an increase in harvest index, which is expressed as the ratio of yield of harvestable parts (such as seeds) to total biomass.

[0126] The present disclosure also extends to harvestable parts of plants, such as, but not limited to, seeds, leaves, fruits, flowers, bolls, 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, starches, or proteins.

[0127] 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.

[0128] As used herein, "nitrogen use efficiency" refers to the processes that increase plant yield, biomass, vigor, and growth rate per unit of nitrogen applied. These processes can include plant absorption, assimilation, accumulation, signaling, sensing, retransfer (within the plant), and utilization of nitrogen.

[0129] 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.

[0130] 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.

[0131] 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 optimally available / applied amounts of nitrogen or under nitrogen limiting conditions.

[0132] Improved nitrogen use efficiency in plants can be translated into harvesting a similar amount of yield in the field while supplying less nitrogen, or obtaining an increase in yield by supplying an optimal / sufficient amount of nitrogen. Improved nitrogen use efficiency can improve plant nitrogen stress tolerance and can also improve crop quality and seed biochemical composition, such as protein yield and oil yield. The terms "improved nitrogen use efficiency," "enhanced nitrogen use efficiency," and "nitrogen stress tolerance" are used interchangeably in this disclosure to refer to plants with increased productivity under nitrogen-limiting conditions.

[0133] As used herein, "water use efficiency" refers to the amount of carbon dioxide assimilated by leaves per unit of transpiration. It constitutes one of the most important traits controlling plant productivity in dry environments. "Drought tolerance" refers to the degree to which a plant adapts to dry or drought conditions. The physiological response of a plant 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. Typically, plants are more susceptible to drought during flowering and seed development (reproduction stage) because the plant's resources are biased towards supporting root growth. In addition, abscisic acid (ABA) is a plant stress hormone that induces leaf stomata (microscopic pores involved in gas exchange) to close, thereby reducing water loss caused by transpiration and reducing photosynthesis rate. These responses improve the water use efficiency of plants in the short term. The terms "improved water use efficiency," "enhanced water use efficiency," and "improved drought tolerance" are used interchangeably in this disclosure to refer to plants whose productivity increases under water-restricted conditions.

[0134] 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 to grow, develop or produce faster or better when subjected to reduced available / applied amount of water (water input) or under conditions of water stress or water deficiency.

[0135] 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 short-term or long-term drought.

[0136] 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 negatively impacts grain / crop yield; a dry period (short or long / extended) that results in a lack of water and / or elevated temperatures and stresses the plant and / or causes damage to plant tissue and / or negatively impacts grain / crop yield.

[0137] As used herein, "water deficit" refers to conditions or environments that provide less than the optimal amount of water required for adequate / successful growth and development of plants.

[0138] As used herein, "water stress" refers to a condition or environment in which an inappropriate amount (less / inadequate or more / excessive) of water is provided relative to the amount 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 impacting grain / crop yield.

[0139] As used herein, "water stress" refers to conditions or circumstances that provide less / inadequate 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 impacting grain yield.

[0140] As used herein, the terms "nucleic acid," "nucleic acid molecule," "nucleotide sequence," and "polynucleotide" refer to linear or branched, single-stranded or double-stranded RNA or DNA, or hybrids thereof. The terms also encompass RNA / DNA hybrids. When dsRNA is produced synthetically, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, and the like may also be used for antisense, dsRNA, and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications may also be made, such as modifications to the 2'-hydroxyl group in the phosphodiester backbone or the RNA ribose group.

[0141] As used herein, the term "nucleotide sequence" refers to a heteropolymer of nucleotides or the sequence of these nucleotides from the 5' to 3' ends of a nucleic acid molecule, and includes DNA or RNA molecules, including cDNA, DNA fragments or portions, 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 in this article, and are represented by the standard code for representing nucleotide characters specified in U.S. sequence rules 37 CFR §§ 1.821-1.825 and World Intellectual Property Organization (WIPO) standard ST.25. As used herein, "5' district" can represent the polynucleotide district closest to the 5' end of a polynucleotide. Therefore, for example, the element in the 5' district of a polynucleotide can be located at any position of the nucleotide from the first nucleotide at the 5' end of the polynucleotide to the nucleotide in the middle of the polynucleotide. As used herein, "3' region" can refer to the region of a polynucleotide closest to the 3' end of a polynucleotide. Thus, for example, elements in the 3' region of a polynucleotide can be located anywhere from the first nucleotide at the 3' end of the polynucleotide to a nucleotide in the middle of the polynucleotide.

[0142] As used herein with respect to nucleic acids, the term "fragment" or "portion" refers 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, 560, 570, 580, 590, 600, 610, 620, 630, 640, 650, 660, 670, 680, 690, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 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 can be included in a larger polynucleotide of which it is a component. As an example, the repeat sequence of a guide nucleic acid of the present invention can comprise a portion of a wild-type CRISPR-Cas repeat sequence (e.g., a wild-type CRISPR-Cas repeat sequence; for example, a repeat 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.).

[0143] In some embodiments, the nucleic acid fragment or portion can comprise, consist essentially of, and / or consist of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 70, 75 , 80, 81, 82, 83, 84, 85, 90, 95, 100, 101, 102, 103, 104, 105, 110, 111, 112, 113, 114, 115, 120, 121, 122, 123, 124, 125, 130, 135, 140, 141, 142, 143, 144, 145, 150, 151, 152, 153, 154, 155, 160, 165, 170, 175, 176, 177, 178, 179, 180, 185, 190, 191, 192, 193, 194, 195, 200, 205, 210, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 230, 235, 240, 245, 250, 255, 256, 257, 258, 259, 260, 265, 270, 271, 272, 273, 274, 275, 280, 285, 290, 295, 300, 305, 310, 320, 330, 335, 336, 337, 338, 339, 340, 350, 360, 370, 380, 390, 395, 400, 410, 415, 420, 425, 430, 435, 440, 445, 450, 500, 501 , 2250, 2300, 2350, 2400, 2450, 2460, 2470, 2480, 2490, 2500, 2550 or 2600 or more consecutive nucleotides, or any range or value therein,Optionally, a fragment of a PIF 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 350 nucleotides, from about 100 nucleotides to about 250 nucleotides, from about 100 nucleotides to about 350 nucleotides, from about 150 nucleotides to about 400 nucleotides, e.g., from about 60, 80, 100, 120, 140, 160, 180, or 200 nucleotides to about 210, 220, 240, 260, 280, 300, or 350 or more consecutive nucleotides (e.g., any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82, e.g., SEQ ID NOs: 84-112, optionally with SEQ ID NOs: 85-86). NO: 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 or 112).

[0144] 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 that is 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 polypeptide fragments may be included in the larger polypeptide of which they are a component. In some embodiments, the 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, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 300, 350, 400, or more contiguous amino acids of a reference polypeptide. In some embodiments, fragments of a PIF transcription factor comprise, consist essentially of, or consist 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, or 30 or more contiguous amino acids (e.g., any one of SEQ ID NOs: 71, 74, 77, 80, 83, e.g., a fragment or portion of SEQ ID NO: 113).

[0145] In some embodiments, a "portion" can relate to the number of amino acids deleted from a polypeptide. Thus, for example, a deleted "portion" of a PIF transcription factor polypeptide can comprise at least one amino acid residue (e.g., at least 1 or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 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, In some embodiments, the percent identity may be at least 85%. In some embodiments, the percent identity may be at least 90%. In some embodiments, the percent identity may be at least 95%. In some embodiments, the percent identity may be 100%.

[0146] " district " of polynucleotide or polypeptide refers to the part of continuous nucleotide or continuous amino acid residue of described polynucleotide or polypeptide respectively.For example, the district of PIF polynucleotide sequence can include but not limited to any one in the nucleotide sequence of SEQ ID NO:84-87,88-91,92-95,96-108 or 109-112.In certain embodiments, the district of PIF peptide sequence can include but not limited to the aminoacid sequence of SEQ ID NO:113.In certain embodiments, the district can be the target region or the target site for modifying in PIF polynucleotide or PIF transcription factor.

[0147] In some embodiments, a "sequence-specific nucleic acid binding domain" (e.g., a sequence-specific DNA binding domain) can bind to a PIF gene (e.g., SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82) and / or to one or more fragments, portions, or regions of a PIF nucleic acid (e.g., SEQ ID NO: 84-87, 88-91, 92-95, 96-108, or 109-112).

[0148] 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 polypeptide fragment that retains one or more activities of a native reference polypeptide.

[0149] 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 can include both coding and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, terminator sequences and / or 5' and 3' untranslated regions). A gene can be "isolated," which means that nucleic acid is substantially or substantially free of the components that are typically present with nucleic acid under natural conditions. Such components include other cell materials, culture medium from recombinant production and / or various chemicals for chemically synthesized nucleic acids.

[0150] The term "mutation" refers to a point mutation (e.g., missense or nonsense, or the insertion or deletion of a single base pair that causes a frameshift), insertion, deletion, and / or truncation. When a mutation is a residue in an amino acid sequence that is replaced by another residue, or a deletion or insertion of one or more residues in the sequence, the mutation is typically described by identifying the original residue, then identifying the position of the residue in the sequence, and the identity of the newly replaced residue. In certain embodiments, the deletion or insertion is an in-frame deletion or an in-frame insertion. In certain embodiments, the deletion or insertion can be a frameshift deletion or a frameshift insertion. In certain embodiments, the deletion can result in a frameshift mutation that produces a premature stop codon, thereby truncating the protein. Truncation can include truncation at the C-terminal end of a polypeptide or at the N-terminal end of a polypeptide. The truncation of a polypeptide can be the result of a corresponding 5' end or 3' end deletion of a gene encoding a polypeptide.

[0151] As used herein, the terms "complementary" or "complementarity" refer to the natural binding of polynucleotides through base pairing under permissive salt and temperature conditions. For example, the sequence "AGT" (5' to 3') binds to the complementary sequence "TCA" (3' to 5'). Complementarity between two single-stranded molecules can be "partial," where only some of the nucleotides bind, or complete, where perfect complementarity exists between the single-stranded molecules. The degree of complementarity between nucleic acid strands has a significant impact on the efficiency and strength of hybridization between nucleic acid strands.

[0152] As used herein, "complementary" 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%, 98%, 99% etc. complementarity, e.g., substantial complementarity).

[0153] Different nucleic acids or proteins with homology are referred to as "homologs" in this article. 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 acids and / or amino acid sequences, expressed as a percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Therefore, the compositions and methods of the present invention further comprise homologs of the nucleotide sequences and polypeptide sequences of the present invention. As used herein, "orthologs" refer to homologous nucleotide sequences and / or amino acid sequences in different species, which are derived from common ancestral genes during the speciation process. 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.

[0154] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are invariant over the entire component (eg, nucleotide or amino acid) alignment 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, eds.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primers. Primer (Gribskov, M. and Devereux, J., eds.) Stockton Press, New York (1991).

[0155] As used herein, term " sequence identity percentage " or " identity percentage " refer to when two sequences are optimally aligned, compared with test (" subject ") polynucleotide molecule (or its complementary strand), the percentage ratio of identical nucleotides in the linear polynucleotide sequence of reference (" query ") polynucleotide molecule (or its complementary strand).In certain embodiments, " sequence identity percentage " can refer to compared with reference polypeptide, the percentage ratio of identical amino acids in amino acid sequence.About PIF gene, sequence can have at least about 80% sequence identity with the nucleotide sequence of any one in SEQ ID NO:69,70,72,73,75,76,78,79,81 and / or 82.In certain embodiments, PIF gene can have at least about 85% sequence identity with the nucleotide sequence of any one in SEQ ID NO:69,70,72,73,75,76,78,79,81 and / or 82. In some embodiments, the PIF gene may have at least about 90% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81, and / or 82. In some embodiments, the PIF gene may have at least about 95% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81, and / or 82, optionally wherein the PIF gene may have about 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81, and / or 82. A PIF polypeptide as described herein may have at least about 80% sequence identity with the polypeptide sequence of any one of SEQ ID NOs: 71, 74, 77, 80, and / or 83. In some embodiments, the PIF polypeptide may have at least about 85% sequence identity to the polypeptide sequence of any one of SEQ ID NOs: 71, 74, 77, 80, and / or 83. In some embodiments, the PIF polypeptide may have at least about 90% sequence identity to the polypeptide sequence of any one of SEQ ID NOs: 71, 74, 77, 80, and / or 83. In some embodiments, the PIF polypeptide may have at least about 95% sequence identity to the polypeptide sequence of any one of SEQ ID NOs: 71, 74, 77, 80, and / or 83, optionally wherein the PIF polypeptide may have about 100% sequence identity to the polypeptide sequence of any one of SEQ ID NOs: 71, 74, 77, 80, and / or 83.With respect to a region or portion of a PIF gene, the region or portion can have at least about 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112, optionally at least about 80% sequence identity to any one of SEQ ID NOs: 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 and / or 112. In some embodiments, a region or portion of a PIF gene may have at least about 85% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112, optionally at least about 85% sequence identity to any one of SEQ ID NOs: 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, and / or 112. In some embodiments, a region or portion of a PIF gene can have at least about 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112, optionally having at least about 90% sequence identity to any one of SEQ ID NOs: 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, and / or 112. In some embodiments, a region or portion of a PIF gene can have at least about 95% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 and / or 109-112, optionally having at least about 95% sequence identity to any one of SEQ ID NOs: 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, and / or 112.In some embodiments, a region or portion of a PIF gene can have about 100% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, and / or 109-112, optionally about 100% sequence identity to any one of SEQ ID NOs: 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, and / or 112. With respect to a region or portion of a PIF polypeptide, the region or portion can have at least about 80% sequence identity to the polypeptide sequence of SEQ ID NO: 113. In some embodiments, a region or portion of a PIF polypeptide can have at least about 85% sequence identity with the polypeptide sequence of SEQ ID NO: 113. In some embodiments, a region or portion of a PIF polypeptide can have at least about 90% sequence identity with the polypeptide sequence of SEQ ID NO: 113. In some embodiments, a region or portion of a PIF polypeptide can have at least about 95% sequence identity with the polypeptide sequence of SEQ ID NO: 113, optionally wherein a region or portion of a PIF polypeptide can have about 100% sequence identity with the polypeptide sequence of SEQ ID NO: 113. In some embodiments, a mutated PIF gene can have at least about 90% sequence identity with a mutated PIF gene having the nucleotide sequence of any one of SEQ ID NO: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135. In some embodiments, the mutated PIF gene may have at least about 95% sequence identity to a mutated PIF gene having the nucleotide sequence of any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135. In some embodiments, the mutated PIF gene may have about 100% sequence identity to a mutated PIF gene having the nucleotide sequence of any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135. In some embodiments, the mutated PIF polypeptide may have at least about 90% sequence identity to a mutated PIF polypeptide having the amino acid sequence of any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131. In some embodiments, the mutated PIF polypeptide may have at least about 95% sequence identity to a mutated PIF polypeptide having the amino acid sequence of any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131.In some embodiments, the mutated PIF polypeptide may have about 100% sequence identity to a mutated PIF polypeptide having the amino acid sequence of any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131.

[0156] As used herein, the phrases "substantially identical" or "substantial identity" in the context of two nucleic acid molecules, nucleotide sequences, or polypeptide sequences refers to two or more sequences or subsequences having 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 measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the invention, substantial identity exists over a contiguous region of nucleotides of a nucleotide sequence of the invention, said region being about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 15 nucleotides to about 25 nucleotides, about 30 nucleotides to about 40 nucleotides, about 50 nucleotides to about 60 nucleotides, about 70 nucleotides to about 80 nucleotides, about 90 nucleotides to about 100 nucleotides, about 100 nucleotides to about 200 nucleotides, about 100 nucleotides to about 300 nucleotides, about 100 nucleotides to about 400 nucleotides, about 100 nucleotides to about 500 nucleotides, about 100 nucleotides to about 600 nucleotides, about 100 nucleotides to about 800 nucleotides, about 100 nucleotides to about 900 nucleotides, or more, and any ranges therein, up to the full length of the sequence. In some embodiments, the nucleotide sequences may be substantially identical over at least about 20 contiguous nucleotides (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2100, 2200, 2300 or more nucleotides).In some embodiments, two or more PIF genes can be present in 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, 71, 72, 73, 75, 76, 78, 79, 81, or 82 (see, e.g., SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112). 500, 520, 540, 560, 580, 600 or more consecutive nucleotides).

[0157] 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 , 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 130, 140, 150, 175, 200, 225, 250, 275, 300, 325, 350, 400, 450, 500 or more amino acids or more consecutive amino acid residues).75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, or 150 residues or more) of SEQ ID NO: 71, 74, 77, 80, or 83, or any range or value therein, see, e.g., SEQ ID NO: 113. In some embodiments, a substantially identical nucleotide or protein sequence can perform substantially the same function as the substantially identical nucleotide (or encoded protein sequence) to which it is substantially identical.

[0158] For sequence comparison, typically one sequence serves as a reference sequence to which a test sequence is compared. When using a sequence comparison algorithm, the test and reference sequences are input into a computer, subsequence coordinates are specified, if necessary, and sequence algorithm program parameters are specified. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the specified program parameters.

[0159] 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 an aligned segment of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the reference sequence segment (e.g., the entire reference sequence or a smaller defined portion of a reference sequence). The percent sequence identity is expressed as the identity score multiplied by 100. Comparison of one or more polynucleotide sequences can be to a full-length polynucleotide sequence or a portion thereof, or to a longer polynucleotide sequence. For the purposes of the present invention, "percent identity" can also be determined using BLASTX version 2.0 for transcribed nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0160] 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.

[0161] 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 temperature (Tf) for the specific sequence at a defined ionic strength and pH. m ) is about 5℃ lower.

[0162] 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 having more than 100 complementary residues on a filter membrane in a Southern or Northern blot is hybridization with 50% formamide and 1 mg of heparin at 42°C overnight. An example of highly stringent wash conditions is washing with 0.15M NaCl at 72°C for about 15 minutes. An example of stringent wash 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 signal. 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 can also be achieved by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2 times (or greater) 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 copies of a nucleotide sequence are created using the maximum codon degeneracy permitted by the genetic code.

[0163] The polynucleotides and / or recombinant nucleic acid constructs (e.g., expression cassettes and / or vectors) of the present 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 present 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 effector proteins) are codon-optimized for expression. 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 protein), 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, the codon-optimized nucleic acids, polynucleotides, expression cassettes and / or vectors of the invention are 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%) identical or greater to a reference nucleic acid, polynucleotide, expression cassette and / or vector that is not codon-optimized.

[0164] The polynucleotides or nucleic acid constructs of the present invention can 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 further comprise one or more promoters, introns, enhancers and / or terminators operably linked to one or more nucleotide sequences. In some embodiments, a promoter can be operably associated with an intron (e.g., an Ubil promoter and an intron). In some embodiments, a promoter associated with an intron can be referred to as a "promoter region" (e.g., an Ubil promoter and an intron) (see, e.g., SEQ ID NO: 21 and SEQ ID NO: 22).

[0165] As used herein, "operably linked" or "operably associated" in reference to a polynucleotide means that the indicated elements are functionally related to each other, and usually also physically related. Thus, as used herein, the terms "operably linked" or "operably associated" refer to functionally related 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. It will be understood by those skilled in the art that a control sequence (e.g., a promoter) does not have to be adjacent to the nucleotide sequence with 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.

[0166] 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 (e.g., via a peptide bond) or via a linker.

[0167] The term "linker" is recognized in the art and refers to a chemical group or molecule that connects two molecules or moieties, 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. A linker can consist of a single linking molecule or can contain more than one linking molecule. In some embodiments, a linker can be an organic molecule, group, polymer, or chemical moiety, such as a divalent organic moiety. In some embodiments, a linker can be an amino acid, or can be a peptide. In some embodiments, a linker is a peptide.

[0168] In some embodiments, the length of the peptide linkers useful in the present 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, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 , 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 in length (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 47,48,49,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,59,50,51,52,53,54,55,56,57,59,50,5 , 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (e.g., about 105, 110, 115, 120, 130, 140, 150 or more amino acids in length). In some embodiments, the peptide linker can be a GS linker.

[0169] In some embodiments, two or more polynucleotide molecules can be connected by a joint, and the joint 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, the 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, the connecting nucleotides can be naturally occurring nucleotides. In some embodiments, the connecting nucleotides can be non-naturally occurring nucleotides.

[0170] "Promoter" is a nucleotide sequence that controls or regulates the transcription of a nucleotide sequence (e.g., a coding sequence) that is operably associated with the promoter. The coding sequence controlled or regulated by the promoter can encode a polypeptide and / or functional RNA. Generally, "promoter" refers to a nucleotide sequence that comprises a binding site for RNA polymerase II and guides transcription initiation. In general, the promoter is located 5' or upstream of the starting point of the coding region relative to the corresponding coding sequence. The promoter may include other elements that act as gene expression regulators; 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) "Annuals of Biochemistry (Annu. Rev. Biochem.)" 50:349). In plants, the CAAT box may 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).

[0171] 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, e.g., "synthetic nucleic acid constructs" or "protein-RNA complexes." These different types of promoters are known in the art.

[0172] The selection of promotor can be different because of the time and space requirement of expression, also can be different because of the host cell that will transform.The promotor of many different organisms is well-known in the art.Based on the extensive knowledge that exists in this area, can be selected suitable promotor for the specific host organism of paying close attention to.Therefore, for example, the promotor of the gene upstream of height compositional expression in the model organism is known very much, and this knowledge can be obtained at easy speed, and in appropriate time, implement in other system.

[0173] In certain embodiments, a promoter functional in plants can be used together with the construct 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 actin gene (Pactin), the promoter of nitrate reductase gene (Pnr) and the promoter of repeat carbonic anhydrase gene 1 (Pdca1) (see Walker et al., Plant Cell Reports (Plant Cell Rep) .23:727-735 (2005); Li et al., Gene 403:132-142 (2007); Li et al., Mol Biol. Rep .37:1143-1154 (2010)). PrbcS1 and Pactin are constitutive promoters, and Pnr and Pdca1 are inducible promoters. Pnr is induced by nitrate and repressed by ammonium (Li et al., Gene 403:132-142 (2007)), and Pdca1 is induced by salt (Li et al., Mol Biol Rep 37:1143-1154 (2010)). In some embodiments, the promoter useful in the present invention is an RNA polymerase II (Pol II) promoter. In some embodiments, the U6 promoter or the 7SL promoter from maize (Zea mays) can be used in the constructs of the present invention. In some embodiments, the U6c promoter and / or the 7SL promoter from maize can be used to drive expression of the guide nucleic acid. In some embodiments, the U6c promoter, U6i promoter, and / or the 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 the 7SL promoter from soybean can be used to drive expression of the guide nucleic acid.

[0174] 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) PNAS 84:6624-6629), the sucrose synthase promoter (Yang and Russell (1990) PNAS 87:4144-4148), and the ubiquitin promoter. Constitutive promoters derived from ubiquitin accumulate in many cell types. The ubiquitin promoter has 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 maize ubiquitin promoter (UbiP) has been developed in transgenic monocot systems, and its sequence and vectors constructed for monocot transformation are disclosed in patent publication EP 0 342 926. The ubiquitin promoter is suitable for expressing the nucleotide sequences of the present invention in transgenic plants, particularly monocots. In addition, the promoter expression cassette described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be readily modified for expression of the nucleotide sequences of the present invention and is particularly suitable for monocot hosts.

[0175] In certain embodiments, tissue-specific / tissue-preferred promoters can be used for expressing 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 expressing in green tissue include many promoters of genes involved in photosynthesis, many of which are cloned from monocots and dicots. In one embodiment, the promoter that can be used for the present invention is the corn PEPC promoter (Hudspeth and Grula, Plant Molecular Biology .12:579-589 (1989)) 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, for example, Kridl et al. (1991) Seed Sci. Res. 1: 209-219; and EP Patent No. 255378). Tissue-specific or tissue-preferred promoters that can be used to express 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-preferred 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 (FEBS 290:103-106 (1991); 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 stalk-specific promoter described in WO 01 / 73087; and pollen-specific or -preferred promoters, including but not limited to ProOsLPS10 and ProOsLPS11 from rice (Nguyen et al., Plant Biotechnol. Reports 9(5):297-306 (2015)), ZmSTK2_USP from maize (Wang et al., Genome 60( 6):485-495 (2017)), LAT52 and LAT59 from tomato (Twell et al., Development 109(3):705-713 (1990)), Zm13 (U.S. Patent No. 10,421,972), PLA2-δ promoter from Arabidopsis thaliana (U.S. Patent No. 7,141,424) and / or ZmC5 promoter from maize (International PCT Publication No. WO1999 / 042587).

[0176] Additional examples of plant tissue-specific / tissue-preferred promoters include, but are not limited to, the root hair-specific cis-element (RHE) (Kim et al., The Plant Cell 18:2958-2970 (2006)), the root-specific promoters RCc3 (Jeong et al., Plant Physiol. 153:185-197 (2010)) 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. Res. 12:3983-4000), S-adenyl-L-methionine synthase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115), maize light-harvesting complex promoter (Bansal et al. (1992) Proceedings of the National Academy of Sciences of the United States of America 89:3654-3658), maize heat shock protein promoter (O'Dell et al. (1985) Journal of the European Molecular Biology Association (EMBO J)).5:451-458; and Rochester et al. (1986) EMBO J 5:451-458), pea small subunit RuBP carboxylase promoter (Cashmore, "Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase," pp. 29-39, In: Genetic Engineering of Plants (Hollaender, ed., Plum Press, 1983); and Poulsen et al. (1986) Mol. Genet. Genomics 205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al. (1989) PNAS 86:3219-3223), Ti plasmid nopaline synthase promoter (Langridge et al. (1989), supra), petunia chalcone isomerase promoter (van Tunen et al. (1988) Journal of the European Molecular Biology Association 7:1257-1263), 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. Genet. Genomics 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), globin-1 promoter (Belanger et al. (1991) Nucleic Acids Res. 18:106-110), globin-2 promoter (Belanger et al. (1991) Nucleic Acids Res. 18:106-110), globin-3 promoter (Belanger et al. (1991) Nucleic Acids Res. 18:106-110), globin-4 promoter (Belanger et al. (1991) Nucleic Acids Res. 18:106-110), globin-5 promoter (Belanger et al. (1991) Nucleic Acids Res. 18:106-110), globin-6 promoter (Belanger et al. (1991) Nucleic Acids Res. 18:106-110), globin-7 promoter (Belanger et al. (1991) Nucleic Acids Res. 18:106-110), globin-8 promoter (Belanger et al. (1991) Nucleic Acids Res. (1991) Genetics 129:863-872), the α-tubulin cab promoter (Sullivan et al. (1989) Mol. Genet. Genomics 215:431-440), the PEPCase promoter (Hudspeth and Grula (1989) Plant Mol. Biol. 12:579-589), the R gene complex-associated promoter (Chandler et al. (1989) Plant Cell 1:1175-1183), and the chalcone synthase promoter (Franken et al. (1991) EMBO J 10:2605-2612).

[0177] Useful promoters for seed-specific expression include the vicilin promoter (Czako et al. (1992) Mol. Genet. Genomics 235:33-40) and the 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).

[0178] In addition, promoters that are functional in chloroplasts can be used. Non-limiting examples of such promoters include the 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).

[0179] 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.

[0180] Introns that can be used for the present invention can be identified in plants and separated from plants, and then inserted into the introns in the expression cassette for plant transformation. As understood by those skilled in the art, introns can include the sequence required for self-excision and be incorporated into the nucleic acid construct / expression cassette 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 it is used in a protein coding sequence, it is inserted "in frame" and includes an excision site. Introns can also be associated with promoters to improve or change expression. As an example, promoter / intron combinations that can be used for the present invention include but are not limited to the promoter / intron combination of corn Ubil promoter and intron (see, for example, SEQ ID NO:21 and SEQ ID NO:22).

[0181] 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.

[0182] In some embodiments, the polynucleotides and / or nucleic acid constructs of the present invention can be, or can be contained within, an "expression cassette." As used herein, an "expression cassette" refers to a recombinant nucleic acid molecule comprising, for example, one or more polynucleotides of the present invention (e.g., a polynucleotide encoding a sequence-specific nucleic acid binding domain, a polynucleotide encoding a deaminase protein or domain, a polynucleotide encoding a reverse transcriptase protein or domain, a polynucleotide encoding a 5'-3' exonuclease polypeptide or domain, a guide nucleic acid, and / or a reverse transcriptase (RT) template), wherein the polynucleotide is operably associated with one or more control sequences (e.g., a promoter, a terminator, etc.). Thus, in some embodiments, one or more expression cassettes can be provided that are designed to express, for example, 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 an expression cassette of the invention comprises more than one polynucleotide, the polynucleotides can be operably linked to a single promoter that drives expression of all polynucleotides, or the polynucleotides can be operably linked to one or more separate promoters (e.g., three polynucleotides can be driven by one, two, or three promoters in any combination). When two or more separate promoters are used, the promoters can be the same promoter, or they can be different promoters. Thus, when contained 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 to separate promoters in any combination.

[0183] The expression cassette comprising the nucleic acid construct of the present invention may be chimeric, meaning that at least one of its components is heterologous with respect 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 useful for heterologous expression.

[0184] The expression cassette can optionally include a transcription and / or translation termination region (i.e., termination region) and / or an enhancer region that functions in the selected host cell. A variety of transcription terminators and enhancers are known in the art and can be used in expression cassettes. The transcription terminator is responsible for terminating transcription and correcting mRNA polyadenylation. The termination region and / or enhancer region can be natural to the transcription initiation region, can 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 can be natural to the host cell, or can be natural to another source (for example, 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 the host cell, or exogenous or heterologous to any combination thereof).

[0185] 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, confers a unique phenotype to the host cell expressing the marker, thereby allowing such transformed cells to be distinguished from cells without the marker. Such a polynucleotide sequence can encode a selectable or screenable marker, depending on whether the marker confers a trait that can be selected by chemical means, such as by using a selection agent (e.g., 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 the expression cassettes described herein.

[0186] In addition to expression cassettes, nucleic acid molecules / constructs and polynucleotide sequences as described herein can also be used in combination with vectors.Term " vector " refers to the composition for nucleic acid (or multiple nucleic acids) transfer, delivery or introduction into cell.Carrier comprises nucleic acid construct (for example, expression cassette), and described nucleic acid construct comprises the nucleotide sequence to be transferred, delivered or introduced.Carrier for transforming host organism is well known in the art.Non-limiting examples of general category carriers include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, Fosmid (fosmid) vectors, phage, artificial chromosomes, mini-circles or double-stranded or single-stranded linear or circular form of Agrobacterium (Agrobacterium) binary vectors, and these carriers can be or can not be self-transferable or removable.In certain embodiments, viral vectors can include but are not limited to retrovirus, slow virus, adenovirus, adeno-associated virus or herpes simplex virus vectors.Carrier as defined herein can be by being integrated into cell genome or being present in extrachromosomal (for example, autonomously replicating plasmid with replication origin) and transforming protokaryon or eukaryotic host. 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 eukaryotes (for example, higher plants, mammals, yeast or fungal cells). In certain embodiments, the nucleic acid in the vector is controlled by a suitable promoter or other regulatory elements, and is operably connected with a suitable promoter or other regulatory elements, so that it is transcribed in the 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 that it is expressed in the host cell. Therefore, nucleic acid of the present invention or polynucleotides and / or expression cassettes comprising the nucleic acid or polynucleotides can be included in vectors as described herein and known in the art.

[0187] As used herein, "contact," "contacting," "conacted," and grammatical variations thereof, refer to bringing together the components of a desired reaction under conditions suitable for the desired reaction (e.g., transformation, transcriptional control, genome editing, nicking, and / or cleavage). As an example, a target nucleic acid can be contacted with a sequence-specific 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 nucleic acid constructs 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 deaminase can be fused to 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 deaminase), so that the deaminase and / or reverse transcriptase are located near the target nucleic acid, thereby modifying the target nucleic acid. Other methods of recruiting reverse transcriptases and / or deaminases that utilize 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.

[0188] As used herein, "modification" or "modification" of a target nucleic acid includes editing (e.g., mutation), covalently altering, exchanging / replacing nucleic acid / nucleotide bases, deleting, cutting, creating a nick, and / or altering transcriptional control of the target nucleic acid. In some embodiments, the modification may include one or more single base changes (SNPs) of any type.

[0189] 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 influence the expression of one or more genes, thereby changing the phenotype, e.g., the response to light.

[0190] "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 such that the nucleotide sequence enters the interior of the cell.

[0191] The terms "transformation" and "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.

[0192] "Transient transformation" in the context of a polynucleotide refers to the introduction of a polynucleotide into a cell but without its integration into the cell's genome.

[0193] "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, and thus the cell is stably transformed with the polynucleotide.

[0194] As used herein, "stable transformation" or "stably transformed" refers to a nucleic acid molecule that is introduced into a cell and integrated into the cell's genome. Thus, the integrated nucleic acid molecule is capable of being inherited by its progeny, more specifically, by successive generations. As used herein, "genome" includes both nuclear and plastid genomes, and thus includes integration of a 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.

[0195] 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 transgenics introduced into an organism. Stable transformation of cells can be detected by, for example, Southern blot hybridization assays with nucleic acid sequences to the genomic DNA of the cells, which specifically hybridize to the nucleotide sequences of the transgenics introduced into an organism (e.g., a plant). Stable transformation of cells can be detected by, for example, Northern blot hybridization assays with nucleic acid sequences to the RNA of the cells, which specifically hybridize to the nucleotide sequences of the transgenics introduced into the 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 to the target sequences of the transgenics, resulting in the amplification of the transgenic sequences, thereby enabling the transgenic sequences to be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0196] Thus, in some embodiments, the nucleotide sequences, polynucleotides, nucleic acid constructs and / or expression cassettes of the invention can be transiently expressed and / or they can be stably incorporated into the genome of a host organism. Thus, in some embodiments, the nucleic acid constructs of the invention (e.g., one or more expression cassettes comprising a polynucleotide for editing as described herein) can be transiently introduced into a cell along with a guide nucleic acid, and thus, the DNA is not maintained in the cell.

[0197] The nucleic acid construct of the present invention can be introduced into plant cells by any method known to persons skilled in the art.The limiting examples of transformation method include by bacterial-mediated nucleic acid delivery (for example, by agrobacterium) conversion, 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 conversion, cyclodextrin-mediated conversion, electroporation, nanoparticle-mediated conversion, ultrasonic treatment, infiltration, PEG-mediated nucleic acid uptake, and nucleic acid is introduced into any other electricity, chemistry, physics (mechanics) and / or biological mechanism in plant cells, including any combination thereof.The program for transforming eukaryotic organisms and prokaryotic organisms is well known in the art and conventional, and is described in the literature (see, for example, Jiang et al., 2013. " Natural Biotechnology (Nat.Biotechnol.) " 31:233-239; Ran et al., " Natural Scheme (Nature Protocols) " 8:2281–2308 (2013)). 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 (Cell. Mol. Biol. Lett. 7:849-858 (2002)).

[0198] In some embodiments of the present invention, the transformation of the cell can comprise nuclear transformation. In other embodiments, the transformation of the cell can comprise plastid transformation (e.g., chloroplast transformation). In further embodiments, nucleic acid of the present invention can be introduced into the cell by conventional cultivation techniques. In certain embodiments, one or more of polynucleotides, expression cassettes and / or vectors can be introduced into plant cells by Agrobacterium transformation.

[0199] Therefore, polynucleotide can be introduced into plant, plant part, plant cell in the mode of any amount well known in the art.Method of the present invention does not rely on the ad hoc approach that is used for one or more nucleotide sequences are introduced into plant, as long as it can enter cell interior.If introduce and exceed a polynucleotide, it can be assembled into the part of single nucleic acid construct or be assembled into independent nucleic acid construct, and can be positioned on identical or different nucleic acid construct.Therefore, polynucleotide can be introduced into the cell of being paid close attention to in single transformation event, or in independent transformation event, introduce into the cell of being paid close attention to, or alternatively, polynucleotide can be mixed in the plant as the part of cultivation scheme.

[0200] Plants respond to their neighbors to better compete with them for resources, particularly light. While this is an adaptive advantage in natural or wild environments, in monoculture, plants compete with plants of the same species that contribute to yield, 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 causes poor plant vigor and reduced plant yield (shade avoidance syndrome; SAS). For example, corn yield (bushels per acre) has steadily increased through intensive cultivation. However, yield increases have recently begun to plateau, and significant investments in field evaluation and cultivation are required to demonstrate genetic gain. New genetic modification methods are needed to significantly increase yield, which is not possible using traditional methods. Crop yield can be increased in two fundamentally different ways: 1) by increasing yield itself, where engineered plants gain advantages such as improved photosynthesis or optimized carbohydrate partitioning, or 2) by removing degenerate survival mechanisms that are inconsistent with high-yield agriculture. The shade avoidance response (SAR) or shade avoidance syndrome (SAS) is one such 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.

[0201] 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 these changes. The components of the phytochrome system include phytochromes, phytochrome-interacting factors (PIFs), and a signal transduction cascade that includes transcription factors such as HB53 and ultimately culminates in hormone production (particularly auxins) and cell elongation.

[0202] PIF is a positive regulator of the shade avoidance response (Levar et al. Plant Cell 21(11):3535-3553 (2009); Quail, PH Annual Plant Reviews. 81–105 (2018); Shi et al. Biochem Bioph Res Co 516:112–119 (2019); Hornitschek et al. EMBO J 28:3893–3902 (2009)). Under high density (HD) planting, when there is a low R:FR ratio, PIF forms a transcription factor complex to promote cell elongation (Oh et al. Plant Cell 21(2):403-419 (2009)). Under normal light conditions, photoactive phyB interacts with PIF, resulting in its phosphorylation and degradation via the 26S proteasome or inactivation by less understood mechanisms (Pham et al., Plant Physiology 176(2), 1025-1038(2018)). PIF transcription factors are structurally defined by belonging to the basic helix-loop-helix (bHLH) class, which functions by dimerization and acts as a positive regulator of SAR. The bHLH domain located at the C-terminus plays a role in DNA binding and dimer formation. In addition, many bHLH proteins have been identified by mutants or functional characterization. For example, antagonistic pairs of bHLH proteins, positive regulator of grain length 1 (PGL1) and antagonist of PGL1 (APG) are involved in controlling grain yield components in rice (Heang et al., Breeding Science 62(2): 133-141(2012)). APG is a bHLH transcription factor that restricts grain size by heterodimerizing with PGL1.

[0203] The present invention uses gene editing to modify regulatory factors that trigger shade avoidance in crops (e.g., dominant negative mutants) to address the problems associated with increased tolerance to planting density and reduced yield losses due to planting variability (on an acre basis). Plants with such edited genomes will have reduced shade avoidance.

[0204] Therefore, the invention provides a kind of plant or its part, described plant or its part are included in at least one sudden change (for example, 1,2,3,4 or 5 or more sudden change) in the endogenous gene of coding phytochrome interacting factor (PIF) transcription factor, wherein said sudden change destroys the combination of the DNA in described PIF transcription factor and described plant or its part.In certain embodiments, PIF transcription factor is basic helix-loop-helix (bHLH) transcription factor.In certain embodiments, at least one sudden change can be in the district of the endogenous gene of basic helix-loop-helix (bHLH) domain of coding PIF transcription factor.In certain embodiments, PIF transcription factor can regulate the response (for example, shade avoidance response (SAR)) to illumination in described plant.In certain embodiments, PIF transcription factor can be basic helix-loop-helix (bHLH) transcription factor, optionally phytochrome interacting factor 3 (PIF3) transcription factor, phytochrome interacting factor 4 (PIF4) transcription factor or phytochrome interacting factor 5 (PIF5) transcription factor. In some embodiments, the endogenous gene encoding the PIF transcription factor can be a phytochrome interacting factor 3 (PIF3) gene, a phytochrome interacting factor 4 (PIF4) gene, or a phytochrome interacting factor 5 (PIF5) gene. In some embodiments, the plant or part thereof comprising a mutated PIF gene comprises a mutated PIF gene comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135, and / or the mutated PIF gene encodes a mutated PIF polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131.

[0205] In some embodiments, the endogenous gene encoding the PIF 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 any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; or (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 any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally NO: 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 and / or 112 having at least 80% sequence identity; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 85% or at least 90%, or the sequence identity may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, at least one mutation may be in a region of the encoded PIF transcription factor having at least 80% sequence identity to SEQ ID NO:113.Thus, the plants or plant parts of the present invention can comprise at least one mutation (e.g., one or more mutations) in an endogenous gene encoding a phytochrome interacting factor (PIF) transcription factor, optionally wherein the mutation disrupts binding of the PIF transcription factor to DNA in the plant or part thereof, wherein the endogenous gene encoding a phytochrome interacting factor (PIF) transcription factor (e.g., an endogenous PIF 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 any one of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (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 any one of SEQ ID NO: any one of the nucleotide sequences of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with a region having at least 80% sequence identity to any one of SEQ ID NOs: 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, and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 71, 74, 77, 80, or 83; and / or (d) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NOs: The amino acid sequence of NO:113 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 the sequence identity can be at least 95%, optionally the sequence identity can be 100%.

[0206] The mutation in the endogenous phytochrome interacting factor (PIF) 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 that can be used in the present invention is a non-natural mutation.

[0207] In certain embodiments, the sudden change in endogenous PIF gene can make PIF transcription factor have basic helix-loop-helix (bHLH) structural domain (for example, basic domain) through destruction, and optionally produce the combination through destruction that the DNA that is combined by the PIF transcription factor that mutates is carried out.For example, described sudden change can be the replacement, disappearance and / or insertion of one or more bases of PIF transcription factor gene.In certain embodiments, described at least one sudden change can produce the modification (for example, replacement, insertion, disappearance) of the amino-acid residue in PIF transcription factor.In certain embodiments, described at least one sudden change can be the replacement of the amino-acid residue that produces PIF transcription factor, and optionally the base replacement of the replacement of the amino-acid residue in the basic domain of PIF transcription factor replaces.In certain embodiments, at least one non-natural mutation can comprise the base replacement that becomes A, T, G or C, and this produces aminoacid replacement, destroys basic domain thus, and optionally destroys the combination of PIF transcription factor to DNA.

[0208] In certain embodiments, the at least one sudden change (for example, one or more sudden changes) in endogenous PIF gene can include base replacement, and optionally wherein said base replacement causes the replacement of amino acid residue.In certain embodiments, base replacement causes the replacement of amino acid residue in the basic domain of PIF transcription factor, and optionally wherein said amino acid replacement destroys the bHLH domain of PIF transcription factor, thus destroys the combination of PIF transcription factor and DNA.In certain embodiments, the at least one sudden change is at the residue E361 place with reference to SEQ ID NO:71 residue position numbering, at the residue E430 place with reference to SEQ ID NO:74 residue position numbering, at the residue E260 place with reference to SEQ ID NO:77 residue position numbering, at the residue E341 place with reference to SEQ ID NO:80 residue position numbering, or at the residue E232 place with reference to SEQ ID NO:83 residue position numbering producing the replacement of amino acid replacement.In certain embodiments, the at least one sudden change can be at the residue E6 position with reference to SEQ ID NO:113 residue position numbering producing the replacement of amino acid replacement. In some embodiments, the amino acid substitution can be a substitution that changes glutamic acid (E) to lysine (K) (E>K), optionally with reference to E6K of SEQ ID NO: 113.

[0209] In some embodiments, at least one mutation (eg, one or more mutations) in the endogenous gene encoding the PIF transcription factor can produce a dominant negative allele, a recessive allele, a null allele, a weak loss-of-function allele, or a hypomorphic allele.

[0210] In some embodiments, plants comprising a mutation in a PIF gene as described herein can be planted at an increased density without reducing plant yield on a per plant basis 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 , 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, or 75%, or any range or value therein), without reducing plant yield on a per plant basis. For comparative purposes, for example, for corn, a standard planting density can be from about 25,000 plants per acre to about 35,000 plants per acre, while for example, for soybeans, a standard planting density can be in the range of about 100,000 plants per acre to about 125,000 plants per acre. As is understood in the art, standard planting density will vary depending at least on the crop species. As used herein, "planting at increased density without reducing plant yield" means a yield reduction of 10% or less compared to a null / wild-type line grown under normal light. That is, when planted at increased density, the yield of plants containing mutations of the present invention that reduce SAR will be approximately 90%-100% of the yield of a null / wild-type line grown under normal light.

[0211] In some embodiments, a plant comprising a mutation in a PIF gene as described herein can exhibit a reduced shade avoidance response when planted adjacent to one or more plants, compared to a plant that does not comprise a reduced shade avoidance response when planted adjacent to one or more plants, optionally exhibiting at least one of the following phenotypes when planted adjacent to one or more plants: increased yield, reduced height, reduced crown:root ratio, reduced leaf length; increased stem mechanical strength; reduced lodging rate; delayed senescence; increased photosynthetic efficiency and grain filling; and / or enhanced defense response to pathogens and herbivores.

[0212] In some embodiments, the at least one mutation in the endogenous phytochrome interacting factor (PIF) gene results in a mutated PIF gene having at least 90% sequence identity (e.g., at least 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5%, optionally the sequence identity can be 100%) to a mutated PIF gene comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135, and / or the mutated PIF gene encodes a mutated PIF polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131.

[0213] In some embodiments, a plant cell is provided, comprising an editing system comprising: (a) a CRISPR-Cas-associated effector protein; and (b) a guide nucleic acid (gRNA, gDNA, crRNA, crDNA), wherein the guide nucleic acid has a spacer sequence that is complementary to an endogenous target gene encoding a phytochrome interacting factor (PIF) transcription factor. The endogenous PIF transcription factor can be any PIF transcription factor that participates in the shade avoidance response. In some embodiments, the endogenous PIF gene encodes a basic helix-loop-helix (bHLH) transcription factor (e.g., a PIF transcription factor). In some embodiments, the PIF gene with which the spacer sequence of the guide nucleic acid shares complementarity (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 any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; or (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 any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with SEQ ID NOs: NO: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 and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO:71, 74, 77, 80 or 83; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:113 ， Optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or the sequence identity can be at least 95%, and optionally the sequence identity can be 100%. In certain embodiments, the spacer sequence that can be used for the present invention can include but is not limited to the nucleotide sequence of any one in SEQ ID NO:114-119 or its reverse complementary sequence or its combination. The editing system can be used for producing sudden change in the endogenous target gene of coding PIF protein. In certain embodiments, sudden change is non-natural sudden change.

[0214] In some embodiments, a plant cell is provided that comprises a mutation in the basic helix-loop-helix (bHLH) domain of a phytochrome-interacting factor (PIF) transcription factor, wherein the mutation is a substitution, insertion, and / or deletion introduced into the endogenous PIF gene using an editing system comprising a nucleic acid binding domain that binds to a target site within an endogenous PIF gene encoding the PIF transcription factor, wherein the endogenous PIF 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; (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 any one of the nucleotide sequences of SEQ ID NO: NO: 84-87, 88-91, 92-95, 96-108 or 109-112, optionally with SEQ ID NO: 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 and / or 112 having at least 80% sequence identity; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO: NO:113 amino acid sequence 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 the sequence identity can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, the nucleic acid binding domain of the editing system can be derived from a polynucleotide-guided nuclease, a CRISPR-Cas nuclease (e.g., a CRISPR-Cas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) and / or an Argonaute protein.

[0215] In some embodiments, the deletion or insertion produced in the endogenous PIF 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, In certain embodiments, the mutation may be a replacement of one or more base pairs in the PIF gene. In certain embodiments, the mutation may be a replacement of one or more base pairs in the PIF gene. In certain embodiments, the replacement may be in the district of the coding basic helix-loop-helix (bHLH) domain of the PIF gene. In some embodiments, the mutation in the PIF gene may be located in a region of the PIF gene that has at least 80% sequence identity with the nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:113.

[0216] In some embodiments, the mutation may be a base substitution to A, T, G, or C, optionally wherein the base substitution produces an amino acid substitution. In some embodiments, at least one mutation may be a base substitution to produce an amino acid substitution at residue E361 with reference to the residue position numbering of SEQ ID NO: 71, at residue E430 with reference to the residue position numbering of SEQ ID NO: 74, at residue E260 with reference to the residue position numbering of SEQ ID NO: 77, at residue E341 with reference to the residue position numbering of SEQ ID NO: 80, or at residue E232 with reference to the residue position numbering of SEQ ID NO: 83. In some embodiments, the at least one mutation may be a substitution to produce an amino acid substitution at residue E6 with reference to the residue position numbering of SEQ ID NO: 113. In some embodiments, the amino acid substitution may be a substitution to produce an amino acid substitution at residue E6 with reference to the residue position numbering of SEQ ID NO: 113 (E>K), optionally with reference to E6K of SEQ ID NO: 113. In certain embodiments, at least one sudden change (for example, one or more sudden changes) in the endogenous gene of coding PIF transcription factor can produce dominant negative allele, recessive allele, invalid allele, weak function loss type allele or hypomorphic allele.In certain embodiments, sudden change can be non-natural sudden change.

[0217] The plant or plant part that can be used in the present invention can be dicotyledonous plant or monocotyledonous plant. Non-limiting examples of the plant or plant part that can be used in the present invention include but are not limited to 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, raspberry or Brassica spp. In certain embodiments, plant part can be the cell from plant, and described plant includes but not limited to monocotyledon or dicotyledon, optionally corn, soybean, Semen Brassicae Campestris, 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, raspberry or Brassica.In certain embodiments, plant can be by plant cell of the present invention or plant part regeneration.In some aspects, plant cell can be the non-breeding plant cell that no longer generates plant.The plant of the present invention that comprises at least one sudden change in the PIF gene can comprise the shade-avoiding response (SAR) of weakening. In certain embodiments, compared with the plant that does not comprise the shade avoidance response of reduction planted in the close proximity of one or more strain plants, the plant regenerated by the plant cell of the present invention that comprises at least one mutation in the PIF gene can show the shade avoidance response of reduction when being planted in the close proximity of one or more strain plants, optionally can show at least one of following phenotypes when being planted in the close proximity of one or more strain plants: the yield of increase, the height of reduction, the crown: root ratio of reduction, the leaf length of reduction; The mechanical strength of the stem that increases; The lodging rate that reduces; The senescence of delay; The photosynthetic efficiency and the grain filling of increase; And / or the defense response to pathogen and herbivore that enhance.In certain embodiments, regenerated plant can be planted with the density that increases, and does not reduce the plant yield based on every strain plant, optionally wherein planting density has increased by about 5% to about 75%, and does not reduce the plant yield based on every strain plant.

[0218] In some embodiments, the mutated PIF gene contained in the plant cell can have at least 90% sequence identity to any one of SEQ ID NO: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134 and / or 135 (optionally the sequence identity can be at least 85% or at least 90%, or the sequence identity can be at least 95%, optionally the sequence identity can be 100%), and / or can encode a mutated PIF polypeptide having at least 90% sequence identity to any one of SEQ ID NO: 121, 123, 125, 127 and / or 131.

[0219] Also provided herein is a method of providing a plurality of plants having increased yield (e.g., increased floret fecundity, increased seed number, and / or increased seed weight) when each of the plurality of plants are planted adjacent to each other in a planting area, 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., comprising a mutation in a PIF gene and having a reduced shade avoidance response) in a planting area, thereby providing a plurality of plants having increased yield compared to a plurality of control plants not comprising the mutation (e.g., compared to isogenic wild-type plants not comprising the mutation). The growing area can be any area where multiple plants can be grown together, including but not limited to fields (e.g., cultivated land, agricultural fields), growth chambers, greenhouses, recreational areas, lawns, and / or roadsides, etc.

[0220] "In close proximity" refers to a high planting density of any particular plant species that can produce SAR. For example, in some embodiments, "in close proximity" includes plants with seeds 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, inch 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 greater than 35,000 seeds per acre at 30 inches or greater row spacing.

[0221] In some embodiments, a method for producing / cultivating a plant that is not edited by a transgenic plant is provided, the method comprising: crossing a plant of the present invention (e.g., a plant comprising a mutation in an endogenous PIF gene as described herein and having a weakened shade avoidance response) with a plant that is not transgenic, thereby introducing at least one mutation (e.g., one or more mutations) into the plant that is not transgenic (e.g., introduced into a progeny plant); and selecting a progeny plant that comprises the at least one mutation and is not transgenic, thereby producing a plant that is not edited by a transgenic plant (e.g., through base editing). In some embodiments, at least one mutation can be a non-natural mutation.

[0222] In some embodiments, the present invention provides a method for generating a mutation in an endogenous phytochrome interacting factor (PIF) gene in a plant, the method comprising: (a) targeting a gene editing system to a portion of the PIF gene, the portion (i) comprising a sequence having at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity) to any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (ii) 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 any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally NO: 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 and / or 112; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (iv) encodes a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO: (b) selecting a plant comprising a modification in a region of the PIF gene that is identical to any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108 or 109-112, optionally ... NO: 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, and / or 112. In some embodiments, the mutations generated result in a nucleic acid having at least 90% sequence identity to any one of SEQ ID NO: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135 and / or a polypeptide having at least 90% sequence identity to any one of SEQ ID NO: 121, 123, 125, 127, and / or 131.

[0223] In some embodiments, a method for producing a change in a PIF gene is provided, the method comprising: introducing an editing system into a plant cell, wherein the editing system is targeted to a region of an endogenous phytochrome interacting factor (PIF) gene encoding the PIF polypeptide, and contacting the region of the endogenous PIF gene with the editing system, thereby introducing a mutation into the endogenous PIF gene and producing a change in the PIF polypeptide of the plant cell. In some embodiments, the altered PIF 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 any one of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82 and / or encodes an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NO: 71, 74, 77, 80, or 83. In some embodiments, the region of the PIF gene that can be targeted comprises at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally to any one of SEQ ID NOs: 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, and / or 112, optionally wherein the region of the PIF gene that can be targeted encodes an amino acid sequence that has at least 80% sequence identity to SEQ ID NO: 113. In some embodiments, contacting a region of an endogenous PIF gene in a plant cell with an editing system produces a plant cell comprising an edited endogenous PIF 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 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 the progeny 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 comprise (e) selfing the selected progeny plant of (d) to produce a progeny plant (E2); (f) determining the reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) of the progeny plant of (e); and (g) selecting the progeny plant that exhibits a reduced shade avoidance response (SAR) / shade avoidance syndrome (SAS) compared to the control plant, optionally repeating (e) to (g) one or more additional times.

[0224] In some embodiments, the mutated PIF gene produced by the methods of the present invention can comprise a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135, and / or encode a mutated PIF polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131.

[0225] In certain embodiments, plant can comprise one or more (for example, at least one, for example 1, 2, 3, 4, 5, 6 or more) PIF genes through mutation as described herein, optionally wherein for one or more mutations at any given allele, plant through editing can be heterozygous or isozygous or its combination.In certain embodiments, plant can be heterozygous, and the specific locus place in its genome comprises the mutation in an allele of PIF gene, and the same locus place in the second copy of same gene is wild type.In certain embodiments, in specific PIF locus, plant can comprise the different mutations at each allele of specific PIF gene, or can be included in the identical mutation at each allele.

[0226] In some embodiments, a method of detecting a mutant PIF gene (a mutation in an endogenous PIF gene) in a plant or plant part (e.g., a plant cell) is provided, the method comprising detecting a PIF gene in the genome of the plant having a nucleotide sequence identical to any one of SEQ ID NOs: 84-112, optionally 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, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, and / or 112. 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, 126, 128, 129, 130, 132, 133, 134, and / or 135. In some embodiments, the mutant PIF gene detected comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131, and / or encodes a mutated PIF polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131.

[0227] 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 in an endogenous PIF gene in the plant cell in a site-specific manner, the endogenous PIF 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 any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with SEQ ID NOs: NO: 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 and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: NO:113 amino acid sequence has the district of at least 80% sequence identity, optionally wherein (a), (b), (c) and / or (d) sequence identity can be at least 85% or at least 90%, or described sequence identity can be at least 95%, optionally described sequence identity can be 100%, thus produces editing in the described endogenous PIF gene of described plant cell.In certain embodiments, PIF gene encodes the PIF transcription factor that comprises basic helix-loop-helix (bHLH) domain, and described editing produces mutation in basic helix-loop-helix (bHLH) domain encoded by endogenous PIF gene.In certain embodiments, editing produces mutation in endogenous PIF transcription factor gene, optionally non-natural mutation, and described mutation produces the PIF transcription factor with reduced DNA binding. In some embodiments, the edits can be in a region of the PIF transcription factor that has at least 80% sequence identity to SEQ ID NO: 113 (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity, optionally at least 90% or 95%, optionally 100%).In some embodiments, the editing in the endogenous PIF gene can produce a mutated PIF gene having at least 90% sequence identity (e.g., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) with a mutated PIF gene as described herein, and / or a mutated PIF transcription factor having at least 90% sequence identity (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%) with a mutated PIF transcription factor polypeptide having the amino acid sequence of any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%). In some embodiments, a plant can be regenerated from a plant cell comprising an editing in an endogenous PIF gene to produce a plant comprising the editing in its endogenous PIF gene. In some embodiments, the plant is not regenerated from a plant cell.In some embodiments, a plant comprising an edit in its endogenous PIF gene exhibits a reduced shade avoidance response compared to a control plant that does not comprise the edit.

[0228] Plants comprising an endogenous PIF gene edited as described herein to provide a PIF transcription factor, optionally with reduced DNA binding, can exhibit a reduced shade avoidance response when compared to a control plant lacking the endogenous PIF gene edited as described herein. Plants comprising an edited endogenous PIF 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)).

[0229] In some embodiments, a method for making a plant is provided, the method comprising: (a) contacting a population of plant cells comprising an endogenous gene encoding a phytochrome interacting factor (PIF) 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; (ii) 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 any one of the nucleotide sequences of SEQ ID NO: any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with a region having at least 80% sequence identity to any one of SEQ ID NOs: 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, and / or 112; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 71, 74, 77, 80, or 83; and / or (iv) encodes a region having at least 80% sequence identity to any one of SEQ ID NOs: NO:113 has a region of at least 80% sequence identity, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) can be at least 85% or at least 90%, or the sequence identity can be at least 95%, optionally the sequence identity can be 100%; (b) selecting plant cells from the population, the plant cells comprising a mutation in the endogenous gene encoding the PIF transcription factor, wherein the mutation is a substitution of at least one amino acid residue in the polypeptide of (iii) or (iv) or in the polypeptide encoded by any one of the nucleotide sequences of (i) or (ii), wherein the mutation modifies the bHLH domain of the PIF transcription factor; and (c) growing the selected plant cells into plants comprising the mutation in the endogenous gene encoding the PIF transcription factor, optionally wherein the mutation reduces or eliminates the ability of the PIF transcription factor to bind to DNA.In some embodiments, the mutation in the endogenous PIF gene can produce a mutated PIF gene having at least 90% sequence identity (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%) to any of the nucleic acids of SEQ ID NO: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135 and / or can encode an amino acid sequence having at least 90% sequence identity to any of SEQ ID NO: 121, 123, 125, 127, and / or 131.

[0230] 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 phytochrome interacting factor (PIF) gene encoding a PIF 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 PIF gene, the endogenous PIF 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; (ii) 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 any one of the nucleotide sequences of SEQ ID NO: any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with a region having at least 80% sequence identity to any one of SEQ ID NOs: 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, and / or 112; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 71, 74, 77, 80, or 83; and / or (iv) encodes a region having at least 80% sequence identity to any one of SEQ ID NOs: (b) growing the plant cells into plants, thereby reducing / inhibiting the shade avoidance response in the plants.In some embodiments, the regenerated plant comprises a mutated PIF gene having at least 90% sequence identity (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, optionally the sequence identity may be at least 95%, optionally the sequence identity may be 100%) to any of the nucleic acids of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135 and / or encodes a mutated PIF polypeptide having at least 90% sequence identity to any of SEQ ID NOs: 121, 123, 125, 127, and / or 131.

[0231] 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 phytochrome interacting factor (PIF) gene is provided, the method comprising contacting a target site within the endogenous PIF 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 PIF gene, wherein the endogenous PIF 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; (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 any one of the nucleotide sequences of SEQ ID NO: any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with a region having at least 80% sequence identity to any one of SEQ ID NOs: 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, and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 71, 74, 77, 80, or 83; and / or (d) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: The aminoacid sequence of NO:113 has the district of at least 80% sequence identity, optionally wherein (a), (b), (c) and / or the sequence identity of (d) can be at least 85% or at least 90%, or described sequence identity can be at least 95%, optionally described sequence identity can be 100%, produce plant or its part thus, described plant or its part comprise at least one cell with the sudden change in described endogenous PIF gene.In certain embodiments, plant with endogenous PIF gene through sudden change or at least one cell in its part produces and is combined with DNA the PIF transcription factor that reduces.In some embodiments, the plant produced comprises a mutated PIF gene having at least 90% sequence identity (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, optionally the sequence identity may be at least 95%, optionally the sequence identity may be 100%) to any of the nucleic acids of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135 and / or encodes a mutated PIF polypeptide having at least 90% sequence identity to any of SEQ ID NOs: 121, 123, 125, 127, and / or 131.

[0232] In some embodiments, a method of producing a plant or part thereof comprising a mutation in a basic helix-loop-helix (bHLH) domain of a phytochrome interacting factor (PIF) transcription factor, the method comprising contacting a target site within an endogenous phytochrome interacting factor (PIF) 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 PIF gene, wherein the endogenous PIF 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; (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 any one of the nucleotide sequences of SEQ ID NO: any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with a region having at least 80% sequence identity to any one of SEQ ID NOs: 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, and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 71, 74, 77, 80, or 83; and / or (d) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: The amino acid sequence of NO:113 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 the sequence identity can be at least 95%, optionally the sequence identity can be 100%, thereby producing a plant or part thereof, which has a mutated phytochrome interacting factor (PIF) transcription factor containing a modified bHLH domain.In some embodiments, the methods can produce a plant or part thereof comprising a mutated PIF gene having at least 90% sequence identity (e.g., at least 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100%, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%) to any of the nucleic acids of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135 and / or encoding a mutated PIF polypeptide having at least 90% sequence identity to any of SEQ ID NOs: 121, 123, 125, 127, and / or 131.

[0233] In some embodiments, the endogenous PIF gene may be a PIF3 gene, a PIF4 gene, or a PIF5 gene, which encodes a PIF transcription factor capable of regulating the response to light (eg, shade avoidance response (SAR)) in the plant.

[0234] In some embodiments, the target site can be a region of the PIF gene or within the region that has a nucleotide sequence identical to any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally identical to any one of SEQ ID NOs: NO: 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 and / or 112 have at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity, optionally the sequence identity can be at least 85% or can be at least 90%, or the sequence identity can be at least 95%, optionally the sequence identity can be 100%), or have 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 SEQ ID NO: 84, 85, 86, 87, 88, 89, The nucleotide sequence of the amino acid sequence of NO:113 has at least 80% sequence identity.

[0235] In certain embodiments, sudden change can be base substitution, base deletion and / or base insertion, optionally non-natural mutation.In certain embodiments, sudden change is the base substitution that becomes A, T, G or C.In certain embodiments, sudden change in the PIF gene causes the amino acid replacement in the encoded PIF transcription factor, optionally wherein said amino acid replacement destroys the bHLH domain of the PIF transcription factor. In certain embodiments, the mutation can be at the residue E361 with reference to the residue position number of SEQ ID NO:71, at the residue E430 with reference to the residue position number of SEQ ID NO:74, at the residue E260 with reference to the residue position number of SEQ ID NO:77, at the residue E341 with reference to the residue position number of SEQ ID NO:80, or at the residue E232 with reference to the residue position number of SEQ ID NO:83, producing a replacement of an amino acid replacement, optionally wherein said at least one mutation is at the residue E6 position with reference to the residue position number of SEQ ID NO:113, producing a replacement of an amino acid replacement, optionally glutamic acid (E) is changed into the replacement (e.g., with reference to the E6K of SEQ ID NO:113) of lysine (K) (E>K). In certain embodiments, the mutation in endogenous PIF gene produces the PIF transcription factor with the DNA combination of reduction. In certain embodiments, the mutation in endogenous PIF gene can be dominant negative mutation, recessive mutation, null mutation, weak loss-of-function mutation or hypoactive mutation.

[0236] In certain embodiments, the plant produced by the method of the present invention or its part comprises the endogenous PIF gene through mutation and / or the PIF transcription factor through mutation as described herein, and with the control plant for the mutation lacking endogenous PIF gene, for example, described plant or plant part do not have in its endogenous PIF gene the target site that the nuclease that comprises cleavage domain and nucleic acid binding domain (for example, DNA binding domain) contacts and compares, show the shade avoidance response of weakening / reduction.In certain embodiments, when grown under identical environmental conditions (for example shade avoidance environment, for example low R:FR ratio environment), can carry out comparison with control plant between edited plant and control plant.

[0237] Plants comprising a mutated endogenous PIF gene as described herein and exhibiting an attenuated / reduced shade avoidance response further exhibit one or more phenotypes when planted in close proximity to one or more other plants, compared to plants lacking a mutated endogenous PIF transcription factor as described herein that are planted in close proximity to one or more other plants, the one or more phenotypes may include, but are not limited to: increased yield, increased upright growth, decreased height, decreased crown:root ratio, decreased leaf length; increased stem mechanical strength; decreased lodging rate; delayed senescence; increased photosynthetic efficiency and grain filling; no change in flowering time and / or enhanced defense responses to pathogens and herbivores. In some embodiments, plants with reduced SAR are at least about 5% shorter (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 97, 98, 99, 100, 110, 120, 130, 140, 150% or lower or any range or value thereof). Plants comprising a mutated endogenous PIF transcription factor as described herein and exhibiting an attenuated / reduced shade avoidance response can be planted at increased density without reducing plant yield on a per plant basis, optionally wherein the planting density can be 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 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, 126, 127, 128, 129, 200, 201

[0238] In certain embodiments, the nuclease contacted with plant cells, plant cell colonies and / or target sites cuts endogenous PIF genes, thereby introducing mutations into basic helix-loop-helix (bHLH) domains encoded by endogenous PIF genes. The nuclease that can be used for the present invention can be any nuclease that can be used for editing / modifying target nucleic acids. 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 domains (e.g., DNA binding domains) that can be used for the nuclease of the present invention can be any nucleic acid binding domains that can be used for editing / modifying target nucleic acids. Such nucleic acid binding domains include but are not limited to zinc finger, transcription activator-like DNA binding domains (TAL), argonaute and / or CRISPR-Cas effector DNA binding domains. In certain embodiments, mutations are non-natural mutations.

[0239] In some embodiments, a method of editing an endogenous PIF gene in a plant or plant part is provided, the method comprising contacting a target site within the PIF gene in the plant or plant part with a cytosine base editing system comprising a cytosine deaminase and a nucleic acid binding domain that binds to the target site within the PIF gene, wherein the PIF 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 any one of the nucleotide sequences of SEQ ID NOs: 84-112, optionally with any one of SEQ ID NOs: 84-113. NO: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 and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO:71, 74, 77, 80 or 83; and / or (d) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO:71, 74, 77, 80 or 83; In certain embodiments, the amino acid sequence of IDNO:113 has the district of at least 80% sequence identity, optionally wherein (a), (b), (c) and / or the sequence identity of (d) can be at least 85% or at least 90%, or described sequence identity can be at least 95%, optionally described sequence identity can be 100%, edits the described endogenous PIF gene in described plant or plant part thus.In certain embodiments, sudden change has reduced the DNA combination that is carried out by the PIF transcription factor.In certain embodiments, the plant that comprises the endogenous PIF gene with sudden change as described herein shows the shade-avoiding response (SAR) (for example, the output that increases when planting with other plant next-door neighbour).

[0240] In some embodiments, a method of editing an endogenous PIF gene in a plant or plant part is provided, the method comprising contacting a target site within the PIF gene in the plant or plant part with a cytosine base editing system comprising an adenine deaminase and a nucleic acid binding domain that binds to the target site within the PIF gene, wherein the PIF 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 any one of the nucleotide sequences of SEQ ID NOs: 84-112, optionally with any one of SEQ ID NOs: 84-113. NO: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 and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO:71, 74, 77, 80 or 83; and / or (d) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO:71, 74, 77, 80 or 83; In certain embodiments, the amino acid sequence of IDNO:113 has the district of at least 80% sequence identity, optionally wherein (a), (b), (c) and / or the sequence identity of (d) can be at least 85% or at least 90%, or described sequence identity can be at least 95%, optionally described sequence identity can be 100%, edits the described endogenous PIF gene in described plant or plant part thus.In certain embodiments, sudden change has reduced the DNA combination that is carried out by the PIF transcription factor.In certain embodiments, the plant that comprises the endogenous PIF gene with sudden change as described herein shows the shade-avoiding response (SAR) (for example, the output that increases when planting with other plant next-door neighbour).

[0241] In some embodiments, a method for modifying an endogenous PIF gene in a plant or a part thereof to reduce / suppress shade avoidance response (SAR) in the plant or part thereof is provided, the method comprising modifying a target site within the endogenous PIF gene in the plant or part thereof, wherein the endogenous PIF 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 any one of the nucleotide sequences of SEQ ID NOs: 84-112, optionally with any one of SEQ ID NOs: NO: 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 and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: In some embodiments, the amino acid sequence of NO:113 has the district 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 said sequence identity can be at least 95%, or said sequence identity can be 100%, modifies said endogenous PIF gene thus and reduces / suppresses SAR plant or its part.In certain embodiments, the target site is the district of PIF gene, any one nucleotide sequence in said district and SEQ ID NO:84-112 has at least 80% sequence identity, optionally wherein the sequence identity can be at least 85% or at least 90%, or said sequence identity can be at least 95%, or said sequence identity can be 100%.

[0242] In some embodiments, the present invention provides a method for producing a plant comprising a mutation in an endogenous PIF 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 PIF gene with a second plant comprising at least one polynucleotide of interest to produce a progeny plant; and selecting the progeny plant comprising at least one mutation in the PIF gene and the at least one polynucleotide of interest, thereby producing the plant comprising the mutation in the endogenous PIF gene and at least one polynucleotide of interest.

[0243] Also provided is a method for producing a plant comprising a mutation in an endogenous PIF gene and at least one polynucleotide of interest, the method comprising introducing at least one polynucleotide of interest into a plant of the present invention comprising at least one mutation in the PIF gene, thereby producing a plant comprising at least one mutation in the PIF gene and at least one polynucleotide of interest.

[0244] Also provided is a method for producing a plant comprising a mutation in an endogenous PIF 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 of the present invention (e.g., comprising at least one mutation in an endogenous PIF gene) with a second plant that exhibits a phenotype of improved yield traits, improved plant architecture and / or improved defense traits; and selecting progeny plants comprising the mutation in the PIF gene and a phenotype of improved yield traits, improved plant architecture and / or improved defense traits, thereby producing the plant comprising the mutation in the endogenous PIF gene and exhibiting a phenotype of improved yield traits, improved plant architecture and / or improved defense traits compared to a control plant.

[0245] In some embodiments, the present invention provides a method for producing a plant comprising a mutation in an endogenous PIF gene and at least one polynucleotide of interest, the method comprising crossing a first plant that is a plant of the present invention (e.g., comprising at least one mutation in an endogenous PIF gene) with a second plant comprising at least one polynucleotide of interest to produce a progeny plant; and selecting the progeny plant comprising the mutation in the PIF gene and the at least one polynucleotide of interest, thereby producing the plant comprising the mutation in the endogenous PIF gene and the at least one polynucleotide of interest.

[0246] Also provided is a method for producing a plant comprising a mutation in an endogenous PIF gene and at least one polynucleotide of interest, the method comprising introducing at least one polynucleotide of interest into a plant of the present invention (e.g., comprising at least one mutation in an endogenous PIF gene), thereby producing a plant comprising a mutation in a PIF gene and at least one polynucleotide of interest.

[0247] In some embodiments, a method of producing a plant is provided, the plant comprising a mutation in an endogenous PIF 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 of the present invention (e.g., comprising at least one mutation in an endogenous PIF gene) with a second plant that exhibits a phenotype of improved yield traits, improved plant architecture and / or improved defense traits; and selecting progeny plants that comprise the mutation in the PIF gene and the phenotype of improved yield traits, improved plant architecture and / or improved defense traits, thereby producing the plant comprising the mutation in the endogenous PIF gene and exhibiting a phenotype of improved yield traits, improved plant architecture and / or improved defense traits compared to a control plant.

[0248] 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 on a roadside, the method comprising applying a herbicide to one or more (plural) plants of the present invention (e.g., comprising at least one mutation in an endogenous PIF gene) growing in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn, or on a roadside, thereby controlling the weeds in the container, the growth chamber, the greenhouse, the field, the recreational area, the lawn, or on the roadside in which the one or more plants are growing.

[0249] In some embodiments, a method of reducing insect predation on a plant is provided, comprising applying an insecticide to one or more plants of the present invention (e.g., comprising at least one mutation in an endogenous PIF gene), thereby reducing insect predation on the one or more plants.

[0250] In some embodiments, a method of reducing fungal diseases on a plant is provided, comprising applying a fungicide to one or more plants of the present invention (e.g., comprising at least one mutation in an endogenous PIF gene), thereby reducing fungal diseases on the one or more plants, optionally 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.

[0251] In some embodiments, a method of reducing bacterial disease on a plant is provided, comprising applying a bactericide to one or more plants of the invention (e.g., comprising at least one mutation in an endogenous PIF gene), thereby reducing bacterial disease on the one or more plants, optionally wherein the one or more plants are grown in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn, or on a roadside.

[0252] The polynucleotides of interest can be any polynucleotides that can confer a desired phenotype on a plant or otherwise alter the phenotype or genotype of a plant. In some embodiments, the polynucleotides of interest can include, but are 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.

[0253] Thus, plants or plant cultivars to be treated preferentially according to the present 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.

[0254] Another example of such properties is increased resistance to animal and microbial pests, such as insects, arachnids, nematodes, mites, slugs and snails, which is caused, for example, by toxins formed in the plant. Among the DNA sequences encoding proteins that 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 proteins from Bacillus thuringiensis, which are widely described in the literature and are well known to those 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, mention will be made of the Bt Cry or VIP proteins, which include Cry1A, Cry1Ab, Cry1Ac, CryIIA, CryIIIA, CryIIIB2, Cry9c Cry2Ab, Cry3Bb and CryIF proteins or toxic fragments thereof, and hybrids or combinations thereof, in particular CrylF protein or a hybrid derived from CrylF protein (e.g., a hybrid CrylA-CrylF protein or a toxic fragment thereof), a CrylA-type protein or a toxic fragment thereof, preferably CrylAc protein or a hybrid derived from CrylAc protein (e.g., a hybrid CrylAb-CrylAc protein) or CrylAb or Bt2 protein or a toxic fragment thereof, Cry2Ae, Cry2Af or Cry2Ag protein or a toxic fragment thereof, CrylA.105 protein or a toxic fragment thereof, VIP3Aa19 protein, VIP3Aa20 protein In particular, the VIP3A protein produced in the COT202 or COT203 cotton event, such as the VIP3Aa protein described in Estruch et al. (1996), Proc. Natl. Acad. Sci. USA 28;93(11):5389-94, or a toxic fragment thereof, such as the Cry protein described in WO 2001 / 47952, an insecticidal protein from a strain of Xenorhabdus (such as described in WO 98 / 50427), Serratia (particularly from S. entomophila), or Photorhabdus, such as the Tc protein from Photorhabdus described in WO 98 / 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), particularly the sequence of a toxic fragment thereof, or fused to a transit peptide, such as a plastid transit peptide, or another protein or peptide is also included herein.

[0255] 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 tolerance to certain herbicides on transformed plant cells and plants, the bar or PAT genes or Streptomyces coelicolor genes described in WO 2009 / 152359 are particularly mentioned, which confer tolerance to glufosinate-ammonium herbicides; genes encoding suitable EPSPS (5-enolpyruvylshikimate-3-phosphate-synthetase), which confer tolerance to herbicides targeting EPSPS, particularly 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., WO 2007 / 024782); a mutated Arabidopsis ALS / AHAS gene (e.g., U.S. Patent 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).

[0256] 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.

[0257] 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 tolerant, deposited as PTA-9843, described in WO2010 / 117737); Event 17314 (rice, herbicide tolerant, deposited as PTA-9844, described in WO2010 / 117735); Event 281-24-236 (cotton, insect control-herbicide tolerant, deposited as PTA-6233, described in WO2005 / 103266 or US-A 2005-216969); Event 3006-210-23 (cotton, insect control-herbicide tolerant, 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 (evergreen grass, herbicide tolerant, deposited as ATCC PTA-4816, described in US-A2006-162007 or WO2004 / 053062); Event B16 (corn, herbicide tolerant, not deposited, described in US-A2003-126634); Event BPS-CV127-9 (soybean, herbicide tolerant, deposited as NCIMB No. 41603, described in WO2010 / 080829); Event BLR1 (rapeseed, restoration of male sterility, deposited as NCIMB 41193, described in WO2005 / 074671), Event CE43-67B (cotton, insect control, deposited as DSMACC2724, described in WO2005 / 074671).2006-130175 or WO2004 / 039986); Event COT202 (cotton, insect control, not deposited, described in US-A 2006-130175 or WO2004 / 039986); 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 2006-130175 or WO2004 / 039986); 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 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 event COT203 (cotton, insect control, not deposited, described in WO2005 / 054480); event DAS21606-3 / 1606 (soybean, herbicide tolerant, deposited as PTA-11028, described in WO2012 / 033794); event DAS40278 (corn, herbicide tolerant, deposited as ATCC PTA-10244, described in WO2011 / 022469); event DAS-44406-6 / pDAB8264.44.06.1 (soybean, herbicide tolerant, deposited as PTA-11336, described in WO2012 / 075426); event DAS-14536-7 / pDAB8291.45.36.2 (soybean, herbicide tolerant, deposited as PTA-11335, described in WO2012 / 075429), event DAS-59122-7 (corn, insect control-herbicide tolerant, deposited as ATCC PTA 11384, described in US-A 2006-070139); event DAS-59132 (corn, insect control-herbicide tolerant, not deposited, described in WO2009 / 100188); event DAS68416 (soybean, herbicide tolerant, 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-A2009-137395 or WO 08 / 112019); event DP-305423-1 (soybean, quality traits, not deposited, described in US-A2008-312082 or WO2008 / 054747); event DP-32138-1 (corn, hybrid system, deposited as ATCC PTA-9158, described in US-A2009-0210970 or WO2009 / 103049); event DP-356043-5 (soybean, herbicide tolerance,deposited as ATCC PTA-8287, described in US-A 2010-0184079 or WO 2008 / 002872); event EE-1 (eggplant, insect control, not deposited, described in WO 07 / 091277); event Fil 17 (corn, herbicide tolerant, deposited as ATCC 209031, described in US-A 2006-059581 or WO 98 / 044140); event FG72 (soybean, herbicide tolerant, deposited as PTA-11041, described in WO 2011 / 063413), event GA21 (corn, herbicide tolerant, deposited as ATCC 209033, described in US-A 2005-086719 or WO 98 / 044140); event GG25 (corn, herbicide tolerant, deposited as ATCC 209032, described in US-A 2005-188434 or WO98 / 044140); Event GHB119 (cotton, insect control-herbicide tolerant, 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 (corn, herbicide tolerant, deposited as ATCC 209030, described in US-A 2005-188434 or WO98 / 044140); Event GM RZ13 (sugar beet, virus resistant, deposited as NCIMB-41601, described in WO 2010 / 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 tolerant, not deposited, described in US-A 2008-064032); event LL27 (soybean, herbicide tolerant, deposited as NCIMB 41658, described in WO 2006 / 108674 or US-A 2008-320616); event LL55 (soybean, herbicide tolerant, deposited as NCIMB 41660, described in WO 2006 / 108675 or US-A 2008-196127); event LLcotton25 (cotton, herbicide tolerant, deposited as ATCC PTA-3343, described in WO2003 / 013224 or US A2003-097687); event LLRICE06 (rice, herbicide tolerant, deposited as ATCC 203353, described in US 6,468,747 or WO2000 / 026345); event LLRice62 (rice, herbicide tolerant,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 (maize, insect control, deposited as PTA-8166, described in US-A 2009-300784 or WO2007 / 142840); event MIR604 (maize, 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 (maize, insect control, not deposited, described in US-A 2002-102582); event MON863 (maize, insect control, deposited as ATCC PTA-2605, described in WO2004 / 011601 or US-A2006-095986); event MON87427 (maize, pollination control, deposited as ATCC PTA-7899, described in WO2011 / 062904); event MON87460 (maize, 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 tolerant, deposited as ATCC PTA-5582, described in US-A 2008-028482 or WO 2005 / 059103); event MON88913 (cotton, herbicide tolerant, deposited as ATCC PTA-4854, described in WO 2004 / 072235 or US-A 2006-059590); event MON88302 (rapeseed, herbicide tolerant, deposited as PTA-10955, described in WO 2011 / 153186), event MON88701 (cotton, herbicide tolerant, deposited as PTA-11754, described in WO 2012 / 134808), event MON89034 (corn, 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-A2006-282915 or WO2006 / 130436); event MS11 (rapeseed, pollination control-herbicide tolerant, deposited as ATCC PTA-850 or PTA-2485, described in WO2001 / 031042); event MS8 (rapeseed, pollination control-herbicide tolerant, deposited as ATCC PTA-730, described in WO2001 / 041558 or US-A2003-188347); event NK603 (corn, herbicide tolerant, deposited as ATCC PTA-2478, described in US-A2007-292854); Event PE-7 (rice, insect control, not deposited, described in WO2008 / 114282); Event RF3 (rapeseed, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO 2001 / 041558 or US-A 2003-188347); Event RT73 (rapeseed, herbicide tolerant, not deposited, described in WO 2002 / 036831 or US-A 2008-070260); Event SYHT0H2 / SYN-000H2-5 (soybean, herbicide tolerant, deposited as PTA-11226, described in WO 2012 / 082548), Event T227-1 (sugar beet, herbicide tolerant, not deposited, described in WO 2002 / 44407 or US-A 2009-265817); Event T25 (corn, herbicide tolerant, not deposited, described in US-A 2001-029014 or WO2001 / 051654); Event T304-40 (cotton, insect control-herbicide tolerance, deposited as ATCC PTA-8171,described in US-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-A2005-039226 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 tolerant, deposited as PTA-11507, described in WO2011 / 084632), event 4114 (corn, insect control-herbicide tolerant, deposited as PTA-11506, described in WO2011 / 084621), event EE-GM3 / FG72 (soybean, herbicide tolerant, ATCC accession number PTA-11041), optionally stacked with event EE-GM1 / LL27 or event EE-G M2 / 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 / 066384A1), Event DP-040416-8 (corn, insect control, ATCC Accession No. PTA-11508, WO2011 / 075593A1), Event DP-043A47 (soybean, herbicide tolerant, ATCC Accession No. PTA-10442, WO2011 / 066384A1), Event DP-040416-8 (corn, insect control, ATCC Accession No. PTA-11508, WO2011 / 075593A1), Event DP-043A47 -3 (corn, insect control, ATCC Accession No. PTA-11509, WO2011 / 075595A1), event DP-004114-3 (corn, insect control, ATCC Accession No. PTA-11506, WO2011 / 084621A1), event DP-032316-8 (corn, insect control, ATCC Accession No. PTA-11507, WO2011 / 084632A1), event MON-88302-9 (rapeseed, herbicide tolerance, ATCC Accession No. PTA-10955, WO2011 / 15 3186A1), event DAS-21606-3 (soybean, herbicide tolerance, ATCC Accession No. PTA-11028, WO2012 / 033794A2), event MON-87712-4 (soybean, quality traits, ATCC Accession No. PTA-10296, WO2012 / 051199A2), event DAS-44406-6 (soybean, stacked herbicide tolerance, ATCC Accession No. PTA-11336, WO2012 / 075426A1), event DAS-14536-7 (soybean, stacked herbicide tolerance,ATCC Accession No. PTA-11335, WO2012 / 075429A1), event SYN-000H2-5 (soybean, herbicide tolerance, ATCC Accession No. PTA-11226, WO2012 / 082548A2), event DP-061061-7 (rapeseed, herbicide tolerance, not deposited and unavailable, WO2012071039A1), event DP-073496-4 (rapeseed, herbicide tolerance, not deposited and unavailable, US2012131692), event 8264.44.06.1 (soybean, stacked herbicide tolerance, Accession No. PTA-11336, WO2012075426A2), event 8291.45.36.2 (soybean, stacked herbicide tolerance, Accession No. PTA -11335, WO2012075429A2), event SYHT0H2 (soybean, ATCC accession number PTA-11226, WO2012 / 082548A2), event MON88701 (cotton, ATCC accession number PTA-11754, WO2012 / 134808A1), event KK179-2 (alfalfa ATCC accession number PTA-11833, WO2013 / 003558A1), event pDAB8264.42.32.1 (soybean, stacked herbicide tolerance, ATCC accession number PTA-11993, WO2013 / 010094A1), event MZDT09Y (corn, ATCC accession number PTA-13025, WO2013 / 012775A1).

[0258] The genes / events that confer the desired trait in question (e.g., the polynucleotides of interest) can 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 such as 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 increased resistance of the plants to insects, arachnids, nematodes, slugs and snails, as well as increased resistance of the plants to one or more herbicides.

[0259] 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 RIB ROUNDUP VT DOUBLE VT TRIPLE BOLLGARD ROUNDUP READY 2 ROUNDUP 2XTENDTM, INTACTA RR2 VISTIVE and / or XTENDFLEX TM Plant seeds sold or distributed under a trade name.

[0260] The PIF gene that can be used for the present invention includes any endogenous PIF gene that can regulate the response to illumination (for example, shade avoidance response (SAR)) in the plant, and wherein mutation as described herein can give the SAR / SAS of reduction in the plant comprising mutation or its part.In certain embodiments, PIF gene encoding basic leucine zipper (bZIP) transcription factor and works in promoting photomorphogenesis, for example PIF transcription factor. In some embodiments, the PIF 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; (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 any one of the nucleotide sequences of SEQ ID NO: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally to any one of the nucleotide sequences of SEQ ID NO: NO: 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 and / or 112 having at least 80% sequence identity; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 85% or at least 90%, or the sequence identity may be at least 95%, optionally the sequence identity may be 100%.

[0261] In certain embodiments, the sudden change in the endogenous PIF gene can be any sudden change that produces the PIF transcription factor, and described any sudden change can give the SAR response of minimizing in the plant comprising the PIF gene through sudden change, and optionally can give the output of increase when planting with other plants next door.In certain embodiments, the sudden change in the endogenous PIF gene can be a non-natural mutation.In certain embodiments, at least one sudden change (for example, one or more sudden changes) in the endogenous PIF gene is a point mutation, optionally base substitution, base insertion and / or base deletion.In certain embodiments, at least one sudden change in the endogenous PIF gene is a dominant negative mutation, recessive mutation, null mutation, weak function loss type sudden change or hypoactive mutation.In certain embodiments, the sudden change in the endogenous PIF gene in the plant can be base substitution, base deletion and / or base insertion, and described sudden change produces the plant with the SAR response of minimizing and / or the output of increase when planting with other plants next door. In certain embodiments, the sudden change in the endogenous PIF gene in the plant can be to replace, lack and / or insert, and described sudden change causes dominant negative mutation, recessive mutation, null mutation, weak function loss type sudden change or hypoeffective mutation and when planting with other plant next door, has the SAR response of minimizing and / or the plant of the output of increase.For example, sudden change can be that 1 Nucleotide or 2,3,4 or 5 continuous nucleotides replace, lack and / or be inserted into about 100 continuous nucleotides.In certain embodiments, sudden change can be the base that becomes A, T, G or C and replace.In certain embodiments, the sudden change in the PIF gene produces the aminoacid replacement in the coded PIF transcription factor, and optionally wherein said aminoacid replacement destroys the bHLH structural domain of the PIF transcription factor. In some embodiments, the mutation can be a substitution producing an amino acid substitution at residue E361 with reference to the residue position numbering of SEQ ID NO: 71, at residue E430 with reference to the residue position numbering of SEQ ID NO: 74, at residue E260 with reference to the residue position numbering of SEQ ID NO: 77, at residue E341 with reference to the residue position numbering of SEQ ID NO: 80, or at residue E232 with reference to the residue position numbering of SEQ ID NO: 83, optionally wherein the at least one mutation is a substitution producing an amino acid substitution at residue E6 position with reference to the residue position numbering of SEQ ID NO: 113, optionally a substitution (e.g., with reference to E6K of SEQ ID NO: 113) of glutamic acid (E) to lysine (K) (E>K). In some embodiments, the mutation in the endogenous PIF gene produces a PIF transcription factor with reduced DNA binding.

[0262] In some embodiments, the mutation generated by the methods of the present invention results in a mutated PIF gene comprising an edited nucleotide sequence having at least 90% sequence identity (e.g., at least 95%, optionally the sequence identity can be 100%) to any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135, and / or encoding a mutated PIF polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131, optionally wherein the mutation in the mutated PIF gene is a non-natural mutation.

[0263] In some embodiments, mutations in the endogenous PIF gene can be made following cleavage by an editing system comprising a nuclease and a nucleic acid binding domain (e.g., a DNA binding domain) that binds to a target site within a target nucleic acid (e.g., a PIF gene). Thus, in some embodiments, the present invention provides a method for modifying an endogenous phytochrome interacting factor (PIF) in a plant or part thereof to reduce / inhibit a shade avoidance response in the plant or part thereof, the method comprising modifying a target site within an endogenous PIF gene in the plant or part thereof, wherein the endogenous PIF 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 any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with any one of SEQ ID NOs: NO: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 and / or 112 having at least 80% sequence identity; (c) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO:71, 74, 77, 80 or 83; and / or (d) encoding a polypeptide comprising a sequence having at least 80% sequence identity to the amino acid sequence of any one of SEQ ID NO: The amino acid sequence of NO:113 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 the sequence identity can be at least 95%, optionally the sequence identity can be 100%, thereby modifying the endogenous PIF gene and reducing / suppressing the shade avoidance response in the plant or part thereof. In some embodiments, the modification of the endogenous PIF gene produces a mutated endogenous PIF gene comprising a sequence that is at least 90% identical to any one of SEQ ID NO: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134 and / or 135, optionally wherein the percent identity to SEQ ID NO: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134 and / or 135 is at least 95%, or the percent identity is 100%.

[0264] Nucleases that can be used for the present invention can cut endogenous PIF gene, thus mutation is introduced into endogenous PIF gene.Such nucleases include but are not limited to zinc finger nucleases, transcription activator-like effector nucleases (TALEN), endonucleases (for example, Fok1) and / or CRISPR-Cas effector proteins.Similarly, nucleic acid binding domains that can be used for the present invention (for example, DNA binding domain, RNA binding domain) include any nucleic acid binding domain that can be used for editing / modifying target nucleic acid.Such nucleic acid binding domains include but are not limited to zinc finger, transcription activator-like DNA binding domain (TAL), argonaute and / or CRISPR-Cas effector DNA binding domain.

[0265] In some embodiments, a guide nucleic acid (e.g., gRNA, gDNA, crRNA, crDNA) is provided that binds to a target site within an endogenous phytochrome interacting factor (PIF) 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; or encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80, or 83; wherein the target site comprises a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80, or 83; Any one of the nucleotide sequences of NO: 84-87, 88-91, 92-95, 96-108, or 109-112 ，SEQ ID NO: 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 and / or 112 has a nucleotide sequence with at least 80% sequence identity, optionally wherein the sequence identity may be at least 85% or at least 90%, or the sequence identity may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, the guide nucleic acid binds to a target nucleic acid within an endogenous PIF gene having a gene identification number (Gene ID) of Zm00001d040536 (SEQ ID NO: 69), Zm00001d008205 (SEQ ID NO: 72), Zm00001d031044 (SEQ ID NO: 75), Zm00001d033267 (SEQ ID NO: 78), and / or Zm00001d034298 (SEQ ID NO: 81) (Maize Genetics and Genomics Database (Maize GDB)), optionally wherein the target region within Zm00001d040536 (SEQ ID NO: 69) can comprise a portion of any one or more contiguous nucleotides of the nucleotide sequence of SEQ ID NOs: 84-87, the target region within Zm00001d008205 (SEQ ID NO: 72) can comprise SEQ ID NO: 83. In some embodiments, the target region may include a portion of consecutive nucleotides of any one or more of the nucleotide sequences of SEQ ID NOs: 88-91, the target region within Zm00001d031044 (SEQ ID NO: 75) may include a portion of consecutive nucleotides of any one or more of the nucleotide sequences of SEQ ID NOs: 92-95, the target region within Zm00001d033267 (SEQ ID NO: 78) may include a portion of consecutive nucleotides of any one or more of the nucleotide sequences of SEQ ID NOs: 96-108, and the target region within Zm00001d034298 (SEQ ID NO: 81) may include a portion of consecutive nucleotides of any one or more of the nucleotide sequences of SEQ ID NOs: 109-112. In some embodiments, the target region may include a portion of consecutive nucleotides of a nucleic acid encoding the amino acid sequence of SEQ ID NO: 113.

[0266] In some embodiments, a target site to which a 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, optionally the sequence identity can be at least 85% or at least 90%, or the sequence identity can be at least 95%, optionally the sequence identity can be 100%) to any of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, or having at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity, optionally the sequence identity can be at least 85% or at least 90%, or the sequence identity can be at least 95%, optionally the sequence identity can be 100%) to any of the nucleotide sequences of SEQ ID NOs: Any of NO: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 and / or 112 has at least 80% sequence identity, and / or can encode a sequence identical to SEQ ID The amino acid sequence of NO:113 has a sequence with at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity, optionally the sequence identity can be at least 85% or at least 90%, or the sequence identity can be at least 95%, optionally the sequence identity can be 100%).

[0267] Exemplary spacer sequences useful in guides of the present invention may comprise complementarity to a fragment or portion of a nucleotide sequence to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82, optionally SEQ ID NO: 69, 72, 75, 78 or 81 and / or SEQ ID NO: 84-112 (optionally, to any one of SEQ ID NO: 84-112). NO: 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 and / or 112) having at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity, optionally the sequence identity can be at least 85% or at least 90%, or the sequence identity can be at least 95%, optionally the sequence identity can be 100%); or a fragment or portion of a nucleotide sequence encoding a nucleotide sequence comprising a residue identical to SEQ ID NO: 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 NO:71, 74, 77, 80 or 83 and / or a polypeptide having a sequence with at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity, optionally the sequence identity can be at least 85% or at least 90%, or the sequence identity can be at least 95%, optionally the sequence identity can be 100%).

[0268] In certain embodiments, target nucleic acid is the endogenous PIF gene that can regulate the response to illumination in plant.In certain embodiments, the target site in target nucleic acid can comprise and SEQ ID NO:69,70,72,73,75,76,78,79,81 or 82, referring to for example the district, part or fragment of any one in the nucleotide sequence of SEQ ID NO:84-112 have at least 80% sequence identity (for example, 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), or can encode and SEQ ID NO:71,74,77,80 or 83 (for example, SEQ ID NO:113) amino acid sequence any one has the district of the aminoacid sequence of at least 80% sequence identity.

[0269] In some embodiments, the guide nucleic acid can comprise a spacer having a nucleotide sequence of any one of SEQ ID NOs: 114-119, or a reverse complement thereof, or any combination thereof.

[0270] 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 can further comprise a tracr nucleic acid associated with the guide nucleic acid and the CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid is covalently linked to the guide nucleic acid.

[0271] 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.

[0272] In some embodiments, 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 a phytochrome interacting factor (PIF) gene. In some embodiments, the PIF gene that can be used in the 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; (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 any one of the nucleotide sequences of SEQ ID NO: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with any one of the nucleotide sequences of SEQ ID NO: 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 and / or 112 having at least 80% sequence identity; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 85% or at least 90%, or the sequence identity may be at least 95%, optionally the sequence identity may be 100%.

[0273] In some embodiments, the guide nucleic acid of a gene editing system can comprise a spacer sequence that has complementarity with 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 NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82 (e.g., SEQ ID NOs: 84-112), or can encode a region, portion, or fragment of a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 71, 74, 77, 80, or 83 (e.g., SEQ ID NO: 113), optionally wherein the region, portion, or fragment is complementary to a region, portion, or fragment of a nucleotide sequence of SEQ ID NOs: The sequence identity of any one of NO: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, and / or 112 may be at least 85% or at least 90%, or the sequence identity may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, the gene editing 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 is covalently linked to the guide nucleic acid. In some embodiments, the PIF gene is a PIF3 gene, a PIF4 gene, or a PIF5 gene.In some embodiments, a guide nucleic acid is provided that binds to a target nucleic acid in an endogenous PIF gene having a gene identification number (gene ID) of Zm00001d040536 (SEQ ID NO: 69), Zm00001d008205 (SEQ ID NO: 72), Zm00001d031044 (SEQ ID NO: 75), Zm00001d033267 (SEQ ID NO: 78), and / or Zm00001d034298 (SEQ ID NO: 81) (Maize Genetics and Genomic Database (Maize GDB)), optionally wherein the target region within Zm00001d040536 (SEQ ID NO: 69) can comprise any one or more of the nucleotide sequences of SEQ ID NOs: 84-87, optionally SEQ ID NOs: 85-88. the target region within Zm00001d008205 (SEQ ID NO: 72) may comprise any one or more of the nucleotide sequences of SEQ ID NOs: 88-91, optionally a portion of the consecutive nucleotides of any one or more of SEQ ID NOs: 88, 89, 90 and / or 91, the target region within Zm00001d031044 (SEQ ID NO: 75) may comprise any one or more of the nucleotide sequences of SEQ ID NOs: 92-95, optionally a portion of the consecutive nucleotides of any one or more of SEQ ID NOs: 92, 93, 94 and / or 95, the target region within Zm00001d033267 (SEQ ID NO: 78) may comprise any one or more of the nucleotide sequences of SEQ ID NOs: 96-108, optionally a portion of the consecutive nucleotides of SEQ ID NOs: NO: 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107 and / or 108, and the target region within Zm00001d034298 (SEQ ID NO: 81) may include any one or more of the nucleotide sequences of SEQ ID NOs: 109-112, optionally a portion of consecutive nucleotides of any one or more of SEQ ID NOs: 109, 110, 111 and / or 112. In some embodiments, the target region may include a portion of consecutive nucleotides of a nucleic acid encoding the amino acid sequence of SEQ ID NO: 113.

[0274] The present invention further provides 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 a phytochrome interacting factor (PIF) gene, the PIF 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 any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with any one of the nucleotide sequences of SEQ ID NOs: NO: 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 and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: In some embodiments, the amino acid sequence of NO:113 has a district 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 said sequence identity can be at least 95%, optionally said sequence identity can be 100%, wherein said cleavage domain cuts the target strand in the PIF gene.In certain embodiments, the PIF gene is PIF3 gene, PIF4 gene or PIF5 gene.In certain embodiments, the cleavage domain cuts the target strand in the PIF gene, thereby producing a sudden change in the endogenous PIF gene comprising a sequence of at least 90% identity with any one of SEQ ID NO:120,122,124,126,128,129,130,132,133,134 and / or 135. In some embodiments, the sequence identity to any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135 can be at least 95%. In some embodiments, the sequence identity to any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135 can be 100%. In some embodiments, the mutation in the endogenous PIF gene is a non-natural mutation.

[0275] Also provided herein are expression cassettes 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 a phytochrome interacting factor (PIF) gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to the target site within the PIF gene, the PIF 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; (b) 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 any one of the nucleotide sequences of SEQ ID NO: any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112, optionally with a region having at least 80% sequence identity to any one of SEQ ID NOs: 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, and / or 112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 71, 74, 77, 80, or 83; and / or (d) encodes a region having at least 80% sequence identity to any one of SEQ ID NOs: In certain embodiments, the amino acid sequence of NO:113 has the district of at least 80% sequence identity, and optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 85% or at least 90%, or said sequence identity can be at least 95%, and optionally said sequence identity can be 100%.In certain embodiments, the PIF gene is PIF3 gene, PIF4 gene or PIF5 gene.

[0276] In some embodiments, nucleic acids are provided that encode a phytochrome interacting factor (PIF) transcription factor comprising a mutated bHLH domain, optionally wherein the mutation disrupts DNA binding by the PIF transcription factor. In some embodiments, the mutation can be at residue E361 with reference to SEQ ID NO: 71, at residue E430 with reference to SEQ ID NO: 74, at residue E260 with reference to SEQ ID NO: 77, at residue E341 with reference to SEQ ID NO: 80, or at residue E232 with reference to SEQ ID NO: 83, producing an amino acid substitution. In some embodiments, the mutation can be at residue E6 with reference to SEQ ID NO: 113, producing an amino acid substitution, optionally E6K. In some embodiments, the nucleic acid comprises a mutated PIF gene, wherein the mutated PIF gene can comprise a sequence having at least 90% sequence identity (e.g., at least about 90, 91, 92, 93, 94, 95, 96, 97, 99, or 100% sequence identity, optionally the sequence identity can be at least 95%, optionally the sequence identity can be 100%) to any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, and / or 135, and / or encodes a mutated PIF polypeptide comprising an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 121, 123, 125, 127, and / or 131. Also provided are modified PIF polypeptides comprising any of the modified amino acid sequences of SEQ ID NO: 121, 123, 125, 127 and / or 131.

[0277] Further provide and comprise the PIF nucleic acid through mutation as described herein and / or the plant or its part through the PIF transcription factor polypeptide through mutation.In certain embodiments, described plant can be corn plant.In certain embodiments, described plant can be wheat plant.In certain embodiments, with next-door neighboring plantation but lack sudden change and do not show the strain or multiple strain plants of the shade-avoiding response of reduction, compare, comprise the PIF through sudden change as described herein and have the SAR of reduction, corn plant and / or wheat plant when next-door neighboring plantation with the PIF through sudden change as described herein and have the SAR of reduction, can show the output of increase, the upright growth of increase, the height of reduction, the crown of reduction: root ratio, the leaf length of reduction, the stem mechanical strength of increase, the lodging rate of reduction; Delayed senescence; The photosynthetic efficiency that increases and grain filling, the flowering time do not change and / or the defense response to pathogen and herbivore that enhance. In some embodiments, when planted in close proximity to each other, plants of the invention comprising reduced SAR can be at least about 5% shorter (e.g., 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, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105 , 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, 110, 120, 130, 140, 150% or less). In some embodiments, corn plants comprising a mutant PIF nucleic acid and / or a mutant PIF transcription factor polypeptide as described herein exhibit a short stature / semi-dwarf phenotype.In some embodiments, a corn plant or part thereof is provided, comprising at least one mutation in an endogenous phytochrome interacting factor (PIF) gene having a gene identification number (Gene ID) of Zm00001d040536, Zm00001d008205, Zm00001d031044, Zm00001d033267, or Zm00001d034298 (e.g., SEQ ID NO: 69, SEQ ID NO: 72, SEQ ID NO: 75, SEQ ID NO: 78, and / or SEQ ID NO: 81, respectively) (Maize Genetics and Genomics Database (Maize GDB)), optionally wherein the mutation is a non-natural mutation.

[0278] In certain embodiments, method of the present invention can further comprise by comprising at least one sudden change (for example, one or more sudden changes) in endogenous phytochrome interacting factor (PIF) gene plant cell or plant part regeneration plant, optionally wherein said sudden change destroys the combination of coded PIF polypeptide and DNA.In certain embodiments, with not comprising at least one sudden change in endogenous PIF gene and therefore do not comprise the control plant of the shade-avoiding response of reduction when planting in the next-door neighbourhood of one or more strains other plant, the plant of at least one sudden change in endogenous PIF gene can show the SAR response of reduction, the output of increase, the upright growth of increase, the height of reduction, the crown of reduction: root ratio, the leaf length of reduction, the stem mechanical strength of increase, the lodging rate of reduction when planting in the next-door neighbourhood of one or more strains other plant; Delayed senescence; The photosynthetic efficiency and the grain filling, flowering time of increase do not have the defense response to pathogen and herbivore of change and / or enhancement.In certain embodiments, sudden change can be non-natural sudden change.In certain embodiments, sudden change is base substitution, optionally produces the replacement of the amino acid residue in coded PIF polypeptide. In some embodiments, the substitution causes a dominant negative mutation, a recessive mutation, a null mutation, a weak loss-of-function mutation, or a hypomorphic mutation.

[0279] 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, and the system can introduce mutations in a target-specific manner. For example, an editing system (e.g., a site-specific or sequence-specific editing system) can 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 can 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, an editing system (e.g., a site-specific or sequence-specific editing system) can 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.

[0280] In some embodiments, the editing system may comprise one or more sequence-specific nucleic acid binding domains (DNA binding domains), which may be derived from, for example, 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. In some embodiments, the editing system may comprise one or more cleavage domains (e.g., nucleases), including but not limited to endonucleases (e.g., Fok1), polynucleotide-guided endonucleases, a CRISPR-Cas endonuclease (e.g., a CRISPR-Cas effector protein), a zinc finger nuclease, and / or a transcription activator-like effector nuclease (TALEN). In some embodiments, the editing system may comprise 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) nucleic acid, an extended guide nucleic acid, and / or a reverse transcriptase template.

[0281] In some embodiments, the method for modifying or editing the PIF gene can include contacting a target nucleic acid (e.g., a nucleic acid encoding a PIF transcription factor) with a base editing fusion protein (e.g., a sequence-specific DNA binding protein (e.g., 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 can guide / target the base editing fusion protein to the target nucleic acid, thereby editing the locus in the target nucleic acid. In some embodiments, the base editing fusion protein and the guide nucleic acid can be contained in one or more expression cassettes. In some embodiments, the target nucleic acid can 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 can be provided as a ribonucleoprotein (RNP). In some embodiments, the cell can be contacted with a guide nucleic acid that is greater than a base editing fusion protein and / or one or more target nucleic acids that can target the cell.

[0282] In some embodiments, a method for modifying or editing a PIF gene may comprise contacting a target nucleic acid (e.g., a nucleic acid encoding a PIF transcription factor) with a sequence-specific DNA binding fusion protein fused to a peptide tag (e.g., a sequence-specific DNA binding protein (e.g., a CRISPR-Cas effector protein or domain)), a deaminase fusion protein comprising a deaminase domain fused to an affinity polypeptide capable of binding to a peptide tag (e.g., adenine deaminase and / or cytosine deaminase), and a guide nucleic acid, wherein the guide nucleic acid is capable of directing / 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 via a peptide tag-affinity polypeptide interaction, thereby editing a 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 to 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 contained in one or more expression cassettes. In some embodiments, a target nucleic acid can be contacted with a sequence-specific binding fusion protein, a deaminase fusion protein, and an expression cassette comprising a guide nucleic acid. In some embodiments, the sequence-specific DNA binding fusion protein, the deaminase fusion protein, and the guide can be provided as a ribonucleoprotein (RNP).

[0283] In some embodiments, methods such as lead editing can be used to produce mutations in endogenous PIF genes. In lead editing, RNA-dependent DNA polymerase (reverse transcriptase, RT) and reverse transcriptase template (RT template) are used in combination with sequence-specific nucleic acid binding domains, which give the ability to identify and bind targets in a sequence-specific manner, and can also cause nicks containing PAM chains in targets. 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 comprising an extension portion comprising a primer binding site (PSB) and an editor to be incorporated into the genome (template). Similar to base editing, lead editing can utilize various methods to recruit proteins for target site editing, and such methods include non-covalent and covalent interactions between proteins and nucleic acids used in the selected genome editing process.

[0284] In certain embodiments, the sudden change of PIF gene or modification can be base substitution, base insertion, base deletion and / or point mutation, and described sudden change or modification produce the PIF transcription factor (for example, the PIF transcription factor) through sudden change that has the DNA combination of reduction and / or give the SAR of reduction on the plant or its part of comprising sudden change / modified PFI gene.In certain embodiments, plant part can be cell.In certain embodiments, plant or its plant part can be any plant as described herein or its part.In certain embodiments, plant that can be used for 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 or Brassica. In some embodiments, a plant comprising a mutated endogenous PIF transcription factor comprising a mutation in its basic helix-loop-helix (bHLH) domain (e.g., comprising a mutated PIF gene comprising a mutation in an encoded basic helix-loop-helix (bHLH) domain) can comprise reduced SAR, increased yield, increased upright growth, reduced height, reduced crown:root ratio, reduced leaf length, increased stem mechanical strength, reduced lodging rate, delayed senescence; increased photosynthetic efficiency and grain filling, no change in flowering time, and / or enhanced defense responses to pathogens and herbivores when planted in close proximity to one or more other plants, compared to a control plant lacking at least one mutation in the endogenous PIF gene and thus lacking a reduced shade avoidance response when the plant is planted in close proximity to one or more other plants. In some embodiments, the plant can be a corn plant comprising a mutated endogenous PIF transcription factor with a mutated basic domain (optionally, reduced DNA binding), and optionally exhibiting reduced SAR, increased yield, increased upright growth, reduced height, reduced crown:root ratio, reduced leaf length; increased stem mechanical strength, reduced lodging rate; delayed senescence; increased photosynthetic efficiency and grain filling, no change in flowering time and / or enhanced defense responses to pathogens and herbivores.

[0285] In certain embodiments, the mutation introduced into the endogenous PIF gene can be a non-natural mutation, and the mutation optionally causes the DNA binding carried out by the encoded PIF polypeptide to be reduced. In certain embodiments, the mutation introduced into the endogenous PIF gene can be the replacement, insertion and / or deletion of at least one nucleotide, at least two consecutive nucleotides or at least three consecutive nucleotides, wherein the mutation can be in the basic domain, and optionally causes the DNA binding carried out by the encoded PIF polypeptide to be reduced. In certain embodiments, the mutation introduced into the endogenous PIF gene can be the replacement of at least one nucleotide (e.g., one or more nucleotides, e.g., 1, 2, 3, 4, 5, 6, 7, 8 or 9 or more nucleotides), and optionally the replacement causes the replacement of the amino acid residue in the encoded PIF transcription factor. In certain embodiments, the replacement of the amino acid residue in the PIF transcription factor can be in the basic domain of the PIF transcription factor, optionally wherein the replacement is in the region of an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 113 in the coding of the PIF gene.

[0286] In some embodiments, sequence-specific nucleic acid binding domains (sequence-specific DNA binding domains) useful in the editing systems of the present invention can be derived from, for example, polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or Argonaute proteins.

[0287] In some embodiments, the sequence-specific nucleic acid binding domain can be a CRISPR-Cas effector protein, optionally wherein the CRISPR-Cas effector protein can 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 can be from a type II CRISPR-Cas system or a type V CRISPR-Cas system. In some embodiments, the CRISPR-Cas effector protein can be a type II CRISPR-Cas effector protein, such as a Cas9 effector protein. In some embodiments, the CRISPR-Cas effector protein can be a type V CRISPR-Cas effector protein, such as a Cas12 effector protein.

[0288] As used herein, a "CRISPR-Cas effector protein" is a protein or polypeptide or domain thereof that cuts or cleaves nucleic acids, binds nucleic acids (e.g., target nucleic acids and / or guide nucleic acids), and / or identifies, recognizes, or binds to guide nucleic acids as defined herein. In some embodiments, a CRISPR-Cas effector protein can be an enzyme (e.g., a nuclease, an endonuclease, a nickase, etc.) or a portion thereof and / or can 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 DNase activity) or wherein the ss DNase activity has been reduced or eliminated, and / or comprises self-processing RNase activity or wherein the self-processing RNase activity has been reduced or eliminated. A CRISPR-Cas effector protein can bind to a target nucleic acid.

[0289] 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, Csy1, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl,

[00135] 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.

[0290] 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). A 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," such as 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.

[0291] The CRISPR Cas9 effector protein or CRISPR Cas9 effector domain that can be used in the present invention can be any known or later identified Cas9 nuclease. In some embodiments, the CRISPR Cas9 polypeptide can be a Cas9 polypeptide from, for example, Streptococcus spp. (e.g., S. pyogenes, S. thermophilus), Lactobacillus spp., Bifidobacterium spp., Kandleria spp., Leuconostoc spp., Oenococcus spp., Pediococcus spp., Weissella spp., and / or Olsenella spp. Exemplary Cas9 sequences include, but are not limited to, the amino acid sequences of SEQ ID NO: 56 and SEQ ID NO: 57 or the nucleotide sequences of SEQ ID NO: 58-68.

[0292] 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 motif 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 motif NNGRR (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 protein derived from Streptococcus aureus that recognizes the PAM sequence motif NGRRT (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus aureus that recognizes the PAM sequence motif NGRRV (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Neisseria meningitidis and recognizes the PAM sequence motif NGATT or NGCTT (R = A or G, V = A, G or C) (see, for example, Hou et al., Proceedings of the National Academy of Sciences (PNAS) 2013, 1-6). In the above embodiments, N can be any nucleotide residue, such as any one of A, G, C or T. In some embodiments, the CRISPR-Cas effector protein can be a Cas13a protein derived from Leptotrichia shahii, 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.

[0293] In some embodiments, the CRISPR-Cas effector protein can be derived from Cas12a, which is a V-type regularly interspaced clustered short palindromic repeats (CRISPR)-Cas nuclease, see, for example, SEQ ID NO: 1-17 amino acid sequences, SEQ ID NO: 18-20 nucleic acid sequences. Cas12a differs from the more well-known type II CRISPR Cas9 nuclease in several aspects. For example, Cas9 recognizes a G-rich pre-spacer adjacent motif (PAM) (3'-NGG) located 3' to its guide RNA (gRNA, sgRNA, crRNA, crDNA, CRISPR array) binding site (pre-spacer, target nucleic acid, target DNA), while Cas12a recognizes a T-rich PAM (5'-TTN, 5'-TTTN) located 5' to 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, Cas12a nuclease activity produces staggered DNA double-strand breaks, rather than the blunt ends produced by 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.

[0294] The CRISPR Cas12a effector protein / domain that can be used in the present invention can be any known or later identified Cas12a polypeptide (formerly known as Cpf1) (see, e.g., U.S. Patent No. 9,790,490, 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, the Cas12a that can be used in the present invention can comprise a mutation in a nuclease active site (e.g., a RuvC site of a Cas12a domain). A Cas12a domain or Cas12a polypeptide that has a mutation in its nuclease active site and therefore no longer comprises 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.

[0295] 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., Nature Biotechnology 37: 1070–1079 (2019), each of which is incorporated herein by reference for disclosures about its 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).

[0296] In some embodiments, the cytosine deaminase useful in the present invention may be an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase may 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 an evolved form thereof (e.g., SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: 29). In some embodiments, the cytosine deaminase may be an APOBEC1 deaminase having the 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 can 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: 29. In some embodiments, the polynucleotide encoding the cytosine deaminase may be codon-optimized for expression in plants, and the codon-optimized polypeptide may be about 70% to 99.5% identical to the reference polynucleotide.

[0297] In some embodiments, the nucleic acid constructs of the present invention may further encode a uracil glycosylase inhibitor (UGI) (e.g., a uracil-DNA glycosylase inhibitor) polypeptide / domain. Thus, in some embodiments, a nucleic acid construct encoding a CRISPR-Cas effector protein and a cytosine deaminase domain (e.g., encoding a fusion protein comprising a CRISPR-Cas effector protein domain fused to a cytosine deaminase domain, and / or a CRISPR-Cas effector protein domain fused to a peptide tag or an affinity polypeptide capable of binding a peptide tag, and / or a deaminase protein domain fused to a peptide tag or an affinity polypeptide capable of binding a peptide tag) may further encode a uracil-DNA glycosylase inhibitor (UGI), optionally wherein the UGI may be codon-optimized for expression in plants. In some embodiments, the present invention provides a fusion protein comprising a CRISPR-Cas effector polypeptide, a deaminase domain, and a UGI and / or one or more polynucleotides encoding the same, optionally wherein the one or more polynucleotides may be codon-optimized for expression in plants. In some embodiments, the present invention provides fusion proteins in which the CRISPR-Cas effector polypeptide, deaminase domain, and UGI can be fused to any combination of peptide tags and affinity polypeptides as described herein, thereby recruiting the deaminase domain and UGI to the CRISPR-Cas effector polypeptide and 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 UGI can be fused to an affinity polypeptide capable of interacting with the recruitment RNA motif, thereby recruiting the deaminase domain and UGI to the target nucleic acid.

[0298] "Uracil glycosylase inhibitors" useful in the present invention can be any protein capable of inhibiting uracil-DNA glycosylase base excision repair enzymes. In some embodiments, the UGI domain comprises wild-type UGI or a fragment thereof. In some embodiments, the UGI domain useful in the present invention can 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 can 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 can 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.

[0299] The adenine deaminase (or adenine deaminase) that can be used in the present invention can be any known or later identified adenine deaminase from any organism (see, e.g., U.S. Patent No. 10,113,163, the disclosure of which is incorporated herein by reference for its adenine deaminase). The adenine deaminase can catalyze the hydrolytic deamination of adenine or adenine. In some embodiments, the adenine deaminase can catalyze the hydrolytic deamination of adenine or deoxyadenine to inosine or deoxyinosine, respectively. In some embodiments, the adenine deaminase can catalyze the hydrolytic deamination of adenine or adenine in DNA. In some embodiments, the adenine deaminase encoded by the 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.

[0300] In some embodiments, the adenine deaminase can be a variant of a naturally occurring adenine deaminase. Thus, in some embodiments, the adenine deaminase can 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, one or more deaminases are not naturally occurring and can be referred to as engineered, mutated, or evolved adenine deaminases. 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%, 1 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identical and any range or value therein. In some embodiments, the adenine deaminase can be from bacteria (e.g., Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, Caulobacter crescentus, etc.). In some embodiments, the polynucleotide encoding the adenine deaminase polypeptide / domain can be codon-optimized for expression in plants.

[0301] In some embodiments, the adenine deaminase domain can be a wild-type tRNA-specific adenine deaminase domain, such as a tRNA-specific adenine deaminase (TadA), and / or a mutated / evolved adenine deaminase domain, such as a mutated / evolved tRNA-specific adenine 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, to lack one or more N-terminal and / or C-terminal amino acids 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 relative to full-length TadA). In some embodiments, the TadA polypeptide or TadA domain does not comprise an N-terminal methionine. In some embodiments, 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.

[0302] Cytosine deaminases catalyze the deamination of cytosine and produce thymidine (via a uracil intermediate), resulting in a C to T transition or a G to A transition in the complementary strand of the genome. Thus, in some embodiments, the cytosine deaminases encoded by the polynucleotides of the invention produce 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.

[0303] In some embodiments, the adenine deaminase encoded by the nucleic acid constructs 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.

[0304] The nucleic acid constructs encoding base editors comprising sequence-specific DNA binding proteins and cytosine deaminase polypeptides of the present invention, as well as nucleic acid constructs / expression cassettes / vectors encoding the same, can be used in combination with guide nucleic acids for modifying target nucleic acids, including but not limited to generating C→T or G→A mutations in target nucleic acids (including but not limited to plasmid sequences); generating C→T or G→A mutations in coding sequences to change amino acid identity; generating C→T or G→A mutations in coding sequences to generate stop codons; generating C→T or G→A mutations in coding sequences to destroy start codons; generating point mutations in genomic DNA to disrupt transcription factor binding; and / or generating point mutations in genomic DNA to disrupt splice junctions.

[0305] The nucleic acid constructs of the present invention encoding base editors comprising a sequence-specific DNA binding protein and an adenine deaminase polypeptide, and 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.

[0306] 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, wherein the guide RNA 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 the deaminase domain and, optionally, a UGI) to the target nucleic acid, wherein the target nucleic acid can be modified (e.g., cleaved or edited) or regulated (e.g., transcription is regulated) by the deaminase domain.

[0307] As an example, a nucleic acid construct encoding a Cas9 domain (e.g., a fusion protein) linked to a cytosine deaminase domain can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the cytosine deaminase domain of the fusion protein deaminates 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) linked to an adenine deaminase domain can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the adenine deaminase domain of the fusion protein deaminates adenine bases in the target nucleic acid, thereby editing the target nucleic acid.

[0308] Similarly, encoding a Cas12a domain (or other selected CRISPR-Cas nuclease, 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, Csy1, Csy2, Csy3, Cse1, 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 other selected CRISPR-Cas nucleases) to modify a target nucleic acid, wherein the cytosine deaminase domain or the adenine deaminase domain of the fusion protein deaminates cytosine bases in the target nucleic acid, thereby editing the target nucleic acid.

[0309] 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, Csy1, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, The gRNA of the present invention can be based on a type I, type II, type III, type IV, type V or type VI CRISPR-Cas system.

[0310] 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'.

[0311] In some embodiments, the guide nucleic acid can 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 present invention are synthetic, artificial, and do not exist in nature. gRNAs can be very long and can be used as aptamers (e.g., in MS2 recruitment strategies) or other RNA structures with hanging spacers.

[0312] 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 plays a role together with the CRISPR-Cas effector protein encoded by the nucleic acid construct of the present invention. The repetitive sequence that can be used for 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 play a role in an I, II, III, IV, V, or VI type CRISPR-Cas system. The repetitive sequence may include a hairpin structure and / or a stem-loop structure. In certain 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. The repetitive sequence from the wild-type CRISPR-Cas locus can be determined by established algorithms, such as using CRISPRfinder provided by CRISPRdb (see, Grissa et al. Nucleic Acids Research, 35 (Web Server Special): W52-7). In some embodiments, the repetitive sequence or portion thereof is linked at its 3' end to the 5' end of the spacer sequence to form a repeat-spacer sequence (e.g., a guide nucleic acid, a guide RNA / DNA, a crRNA, a crDNA).

[0313] In some embodiments, the repeat sequence comprises, consists essentially of, or consists of at least 10 nucleotides, depending on the particular repeat sequence and whether the guide nucleic acid comprising the repeat sequence is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides, or any range or value therein). In some embodiments, the 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.

[0314] The repeat sequence linked to the 5' end of the spacer sequence can comprise a portion of the repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive nucleotides of 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.

[0315] As used herein, " spacer sequence " is a nucleotide sequence complementary to a portion of a target nucleic acid (e.g., target DNA) (e.g., pre-spacer). In certain embodiments, the spacer sequence is complementary to a portion of a continuous nucleotide of a PIF gene, wherein the PIF gene (a) comprises a sequence having at least 80% sequence identity (e.g., at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 99 or 100% sequence identity) with any one of the nucleotide sequences of SEQ ID NO: 84-87, 88-91, 92-95, 96-108 or 109-112, optionally with any one of the nucleotide sequences of SEQ ID NO: 84-87, 88-91, 92-95, 96-108 or 109-112. NO: 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 and / or 112 having at least 80% sequence identity; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encodes a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 85% or at least 90%, or the sequence identity may be at least 95%, optionally the sequence identity may be 100%. The spacer sequence can 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 more)). In some embodiments, the spacer sequence can have one, two, three, four, or five mismatches, which can be contiguous or discontinuous, as compared to the target nucleic acid. In some embodiments, the spacer sequence can have 70% complementarity to the target nucleic acid. In other embodiments, the spacer nucleotide sequence can have 80% complementarity to the target nucleic acid. In still other embodiments, the spacer nucleotide sequence can be 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementary to the target nucleic acid (pre-spacer), etc. In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid.The length of the spacer sequence can be about 15 nucleotides to about 30 nucleotides (for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides or any range or value therein). Therefore, in some embodiments, the spacer sequence can have complete complementarity or substantial complementarity in a region where the target nucleic acid (for example, a preceding spacer) has a length of at least about 15 nucleotides to about 30 nucleotides. In certain embodiments, the length of the spacer is about 20 nucleotides. In certain embodiments, the length of the spacer is about 21, 22 or 23 nucleotides. In certain embodiments, the spacer sequence can include any one of the sequences of SEQ ID NO:114-119 or its reverse complementary sequence or any combination.

[0316] In some embodiments, the 5' region of the spacer sequence of the guide nucleic acid can be identical to the target DNA, while the 3' region of the spacer can be substantially complementary to the target DNA (such as the spacer of the type V CRISPR-Cas system), or the 3' region of the spacer sequence of the guide nucleic acid can be identical to the target DNA, while the 5' region of the spacer can be substantially complementary to the target DNA (such as the spacer of the type II CRISPR-Cas system), and thus, the overall complementarity of the spacer sequence to the target DNA can be less than 100%. Thus, for example, in a guide 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 a 20-nucleotide spacer sequence can 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 range therein) of the 5' end of the spacer sequence can 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)).

[0317] As another example, in a guide 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 can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary (e.g., at least about 7...

Claims

1. A plant or part thereof comprising at least one mutation in an endogenous gene encoding a phytochrome interacting factor (PIF) transcription factor, wherein the mutation disrupts binding of the PIF transcription factor to DNA in the plant or part thereof.

2. The plant or part thereof according to claim 1, wherein the PIF transcription factor is a basic helix-loop-helix (bHLH) transcription factor.

3. The plant or part thereof of claim 1 or claim 2, wherein the at least one mutation is in a region of the endogenous gene encoding the basic helix-loop-helix (bHLH) domain of the PIF transcription factor.

4. The plant or part thereof according to any one of the preceding claims, wherein the PIF transcription factor is capable of regulating the response to light (e.g., shade avoidance response (SAR)) in the plant.

5. according to each described plant or its part in the preceding claim, wherein described at least one sudden change in the endogenous gene of coding PIF transcription factor produces dominant negative allelotrope, recessive allelotrope, null allelotrope, weak function loss type allelotrope or hypomorphic allelotrope.

6. according to each described plant or its part in the aforementioned claim 1, the endogenous gene of wherein said coding PIF transcription factor is phytochrome interaction factor 3 (PIF3) gene, phytochrome interaction factor 4 (PIF4) gene or phytochrome interaction factor 5 (PIF5) gene.

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

8. The plant or part thereof of any one of the preceding claims, wherein the plant is 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, or Brassica spp.

9. A plant or part thereof according to any one of the preceding claims, wherein the endogenous gene encoding the PIF transcription factor: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

113.

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

11. The plant or part thereof according to any one of the preceding claims, wherein the at least one mutation comprises a base substitution to A, T, G or C, which results in an amino acid substitution.

12. The plant or part thereof of claim 11, wherein the amino acid substitution disrupts the bHLH domain of the PIF transcription factor.

13. The plant or part thereof according to any one of the preceding claims, wherein the at least one mutation is a substitution resulting in an amino acid substitution at residue E361 as numbered with reference to residue position of SEQ ID NO: 71, at residue E430 as numbered with reference to residue position of SEQ ID NO: 74, at residue E260 as numbered with reference to residue position of SEQ ID NO: 77, at residue E341 as numbered with reference to residue position of SEQ ID NO: 80, or at residue E232 as numbered with reference to residue position of SEQ ID NO:

83.

14. The plant or part thereof according to any one of the preceding claims, wherein the at least one mutation is a substitution resulting in an amino acid substitution at residue E6 as numbered with reference to the residue positions of SEQ ID NO:

113.

15. The plant or part thereof according to any one of claims 11 to 14, wherein the amino acid substitution is from glutamic acid (E) to lysine (K).

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

17. The plant or plant part of any preceding claim, wherein the plant exhibits a reduced shade avoidance response when planted adjacent to one or more plants, compared to a plant that does not comprise a reduced shade avoidance response when planted adjacent to the one or more plants, optionally exhibiting at least one of the following phenotypes when planted adjacent to the one or more plants: increased yield, reduced height, reduced crown:root ratio, reduced leaf length; increased stem mechanical strength; reduced lodging rate; delayed senescence; increased photosynthetic efficiency and grain filling; and / or enhanced defense response to pathogens and herbivores.

18. Plant or part thereof according to any one of the preceding claims, wherein the plant can be grown at increased density without reducing plant yield on a per plant basis.

19. The plant or part thereof of claim 18, wherein the planting density is increased by about 5% to about 75% without reducing plant yield on a per plant basis.

20. according to plant described in any one of the preceding claims or its part, wherein said at least one sudden change produces the PIF gene through mutation, said PIF gene through mutation comprises and SEQ ID NO:120,122,124,126,128,129,130,132,133,134 or 135 any one has the nucleotide sequence of at least 90% sequence identity, and / or said PIF gene encoding through mutation has the PIF polypeptide through mutation of at least 90% sequence identity with SEQ ID NO:121,123,125,127 or 131 any one.

21. A plant cell comprising an editing system, the editing system comprising: (a) CRISPR-Cas-associated effector proteins; and (b) A guide nucleic acid (gRNA, gDNA, crRNA, crDNA) having a spacer sequence complementary to an endogenous target gene encoding a phytochrome interacting factor (PIF) transcription factor.

22. The plant cell of claim 21, wherein the endogenous gene encoding the PIF transcription factor is a phytochrome interacting factor 3 (PIF3) gene, a phytochrome interacting factor 4 (PIF4) gene, or a phytochrome interacting factor 5 (PIF5) gene.

23. The plant cell of claim 21 or claim 22, wherein the endogenous gene encoding the PIF transcription factor: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

113.

24. The plant cell of any one of claims 21 to 23, wherein the guide nucleic acid comprises the nucleotide sequence (spacer) of any one of SEQ ID NOs: 114-119 or any combination thereof.

25. A plant cell comprising a mutation in a basic helix-loop-helix (bHLH) domain of a phytochrome-interacting factor (PIF) transcription factor, wherein the mutation is a substitution, insertion and / or deletion introduced into the endogenous PIF gene encoding the PIF transcription factor using an editing system comprising a nucleic acid binding domain that binds to a target site within the endogenous PIF gene, wherein the endogenous PIF gene: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

113.

26. The plant cell of claim 25, wherein the endogenous gene PIF gene is a phytochrome interacting factor 3 (PIF3) gene, a phytochrome interacting factor 4 (PIF4) gene, or a phytochrome interacting factor 5 (PIF5) gene.

27. The plant cell of claim 25 or claim 26, wherein the nucleic acid binding domain of the editing system is derived from a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., a CRISPR-Cas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and / or an Argonaute protein.

28. The plant cell of any one of claims 25 to 27, wherein the mutation comprises a base substitution to A, T, G or C, optionally wherein the base substitution results in an amino acid substitution.

29. The plant cell of any one of claims 25 to 28, wherein the mutation is a substitution of at least one amino acid residue in a region of the PIF transcription factor that has at least 90% sequence identity to the amino acid sequence of SEQ ID NO:

113.

30. The plant cell of any one of claims 25 to 29, 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, or canola, optionally wherein the plant cell is from corn.

31. The plant cell of any one of claims 25 to 30, wherein the mutation is a non-natural mutation.

32. according to the plant cell described in any one in claim 25 to 30, wherein at least one sudden change produces the PIF gene through mutation, the described PIF gene through mutation comprises and SEQ ID NO:120,122,124,126,128,129,130,132,133,134 or 135 any one has the nucleotide sequence of at least 90% sequence identity, and / or the described PIF gene encoding through mutation and SEQ ID NO:121,123,125,127 or 131 any one has the PIF polypeptide through mutation of at least 90% sequence identity.

33. a plant, it is by the plant part according to any one in claim 1 to 20 or by the plant cell regeneration according to any one in claim 21 to 32, and comprises the described sudden change in the endogenous PIF gene.

34. The plant of claim 33, wherein the plant exhibits a reduced shade avoidance response when planted adjacent to one or more plants, compared to plants that do not comprise a reduced shade avoidance response when planted adjacent to the one or more plants, optionally exhibiting at least one of the following phenotypes when planted adjacent to the one or more plants: increased yield, reduced height, reduced crown:root ratio, reduced leaf length; increased stem mechanical strength; reduced lodging rate; delayed senescence; increased photosynthetic efficiency and grain filling; and / or enhanced defense responses to pathogens and herbivores.

35. The plant of claim 33 or claim 34, wherein the plant can be grown at increased density without reducing plant yield on a per plant basis.

36. The plant or part thereof of claim 35, wherein the planting density is increased by about 5% to about 75% without reducing plant yield on a per plant basis.

37. A method of providing a plurality of plants having increased yield when each of the plurality of plants are planted adjacent to each other in a planting area, the method comprising planting two or more plants according to any one of claims 1 to 20 or 33 to 36 adjacent to each other in a planting area, thereby providing a plurality of plants having increased yield compared to a plurality of control plants planted adjacent to each other.

38. A method of producing / growing a genome-edited (e.g., base-edited) plant that is free of transgenes, the method comprising: (a) crossing a plant according to any one of claims 1 to 20 or 33 to 36 with a plant that does not contain a transgene, thereby introducing the mutation into the plant that does not contain a transgene; and (b) selecting progeny plants that comprise the mutation but do not have the transgene, thereby producing genome-edited (e.g., base-edited) plants that do not have the transgene.

39. A method for generating a mutation in an endogenous phytochrome interacting factor (PIF) gene in a plant, the method comprising: (a) targeting a gene editing system to a portion of the PIF gene, the portion comprising a sequence having at least 80% sequence identity to any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; and (b) selecting a plant comprising a modification in a region of the PIF gene that has at least 80% sequence identity to any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112.

40. A method for producing a change in a phytochrome interacting factor (PIF) polypeptide, the method comprising: introducing an editing system into a plant cell, wherein the editing system is targeted to a region of an endogenous phytochrome interacting factor (PIF) gene encoding the PIF polypeptide, and The region of the endogenous PIF gene is contacted with the editing system, thereby introducing mutations into the endogenous PIF gene and producing changes in the PIF polypeptide in the plant cell.

41. The method of claim 40, wherein the endogenous PIF gene comprises a nucleotide sequence that has at least 80% sequence identity to any one of SEQ ID NO:69, 70, 72, 73, 75, 76, 78, 79, 81 or 82, and / or encodes an amino acid sequence that has at least 80% sequence identity to any one of SEQ ID NO:71, 74, 77, 80 or 83.

42. The method of claim 40 or claim 41, wherein the region of the endogenous PIF gene that is targeted comprises at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112.

43. according to the method described in any one of claim 40 to 42, wherein the change is produced in the district of described PIF polypeptide, and described district comprises the amino acid sequence that has at least 80% sequence identity with any one in SEQ ID NO:

113.

44. according to the method described in any one in claim 40 to 43, wherein the described district of the described endogenous PIF gene in the described plant cell is contacted with the described editing system to produce plant cell, and described plant cell comprises the PIF gene through editing in its genome, and described method further comprises (a) regenerating plant by described plant cell; (b) making described plant self-pollinate to produce progeny plant (E1); (c) measuring the shade avoidance response of the reduction of the described progeny plant of (b); and (d) selecting the described progeny plant that shows the phenotype of the shade avoidance response of reduction compared with control plant.

45. The method of claim 44, further comprising (e) selfing the selected progeny plant of (d) to produce a progeny plant (E2); (f) determining the reduced shade avoidance response of the progeny plant of (e); and (g) selecting the progeny plant that exhibits a phenotype of reduced shade avoidance response compared to the control plant, optionally repeating (e) to (g) one or more additional times.

46. ​​A method for detecting a mutant PIF gene (mutation in an endogenous PIF gene) in a plant, the method comprising detecting a PIF gene in the genome of the plant, the PIF gene having at least one mutation in a region having at least 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 84-112.

47. method according to claim 46, wherein the described mutant PIF gene of being detected comprises the nucleotide sequence that has at least 90% sequence identity with any one of SEQ ID NO:120,122,124,126,128,129,130,132,133,134 or 135, and / or the described mutant PIF gene encoding has the PIF polypeptide through mutation that has at least 90% sequence identity with any one of SEQ ID NO:121,123,125,127 or 131.

48. A method for editing a specific site in the genome of a plant cell, the method comprising cleaving a target site in an endogenous phytochrome interacting factor (PIF) gene in the plant cell in a site-specific manner, the endogenous PIF gene: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113, thereby producing an edit in the endogenous PIF gene of the plant cell.

49. The method of claim 48, wherein the editing generates a mutation in the basic helix-loop-helix (bHLH) domain of the PIF polypeptide encoded by the endogenous PIF gene.

50. The method of claim 48 or claim 49, further comprising regenerating a plant from the plant cell comprising the edit in the endogenous PIF gene to produce a plant comprising the edit in its endogenous PIF gene.

51. The method of claim 50, wherein the plant comprising the edit in its endogenous PIF transcription factor gene has a reduced shade avoidance response compared to a control plant lacking the edit.

52. The method of any one of claims 48 to 51, wherein the editing produces a mutated PIF gene comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 120, 122, 124, 126, 128, 129, 130, 132, 133, 134, or 135, and / or the mutated PIF gene encodes a mutated PIF polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 121, 123, 125, 127, or 131.

53. A method for preparing a plant, the method comprising: (a) contacting a population of plant cells comprising an endogenous gene encoding a phytochrome interacting factor (PIF) 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 any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (iv) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113; (b) selecting a plant cell from the population, the plant cell comprising a mutation in the endogenous gene encoding the PIF transcription factor, wherein the mutation is a substitution of at least one amino acid residue in the polypeptide of (iii) or (iv) or in the polypeptide encoded by any of the nucleotide sequences of (i) or (ii), wherein the mutation modifies the bHLH domain of the PIF transcription factor; and (c) growing the selected plant cell into a plant comprising the mutation in the endogenous gene encoding the PIF transcription factor.

54. A method for reducing / inhibiting a shade avoidance response in a plant, the method comprising: (a) contacting a plant cell comprising an endogenous phytochrome-interacting factor (PIF) gene encoding a PIF 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 PIF gene, wherein the endogenous PIF gene: (i) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (ii) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (iii) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (iv) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113, thereby generating a plant cell comprising a mutation in said endogenous PIF gene encoding a PIF polypeptide; and (b) growing the plant cell into a plant, thereby reducing / inhibiting the shade avoidance response in the plant.

55. A method for producing a plant or part thereof comprising at least one cell having a mutation in an endogenous phytochrome interacting factor (PIF) gene, the method comprising contacting a target site in the endogenous PIF 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 in the endogenous PIF gene, wherein the endogenous PIF gene: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113, thereby producing a plant or part thereof comprising at least one cell having a mutation in the endogenous PIF gene.

56. A method of producing a plant or part thereof comprising a mutation in a basic helix-loop-helix (bHLH) domain of a phytochrome-interacting factor (PIF) transcription factor, the method comprising contacting a target site within an endogenous phytochrome-interacting factor (PIF) 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 PIF gene, wherein the endogenous PIF gene: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 113, thereby producing a plant or part thereof having a mutated phytochrome-interacting factor (PIF) transcription factor containing a modified bHLH domain.

57. The method of any one of claims 53 to 56, 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.

58. according to the method described in any one in claim 53 to 57, wherein said nuclease cuts endogenous PIF transcription factor gene, and sudden change is introduced in the described bHLH structural domain of the described PIF transcription factor encoded by described endogenous PIF transcription factor gene.

59. according to the method described in any one in claim 53 to 58, wherein said target site is the district of said PIF gene, said district has at least 80% sequence identity with the nucleotide sequence of any one in SEQ ID NO:84-87,88-91,92-95,96-108 or 109-112 or has at least 80% sequence identity with the nucleotide sequence of encoding the amino acid sequence of SEQ ID NO:

113.

60. according to each described method in claim 53 to 59, wherein said endogenous PIF gene is PIF3 gene, PIF4 gene or PIF5 gene.

61. The method of claim 53, 54, or 56 to 60, wherein the PIF transcription factor is capable of regulating a response to light (eg, shade avoidance response (SAR)) in the plant.

62. The method according to any one of claims 53 to 61, wherein the at least one mutation is a base substitution, a base deletion and / or a base insertion.

63. The method of any one of claims 53 to 62, wherein the mutation is a base substitution to A, T, G, or C.

64. according to the method described in any one of claim 53 to 63, wherein said mutation produces amino acid substitution, optionally wherein said amino acid substitution destroys the described bHLH domain of described PIF transcription factor.

65. The method of any one of claims 53 to 64, wherein the mutation is a substitution resulting in an amino acid substitution at residue E361 as numbered with reference to residue position of SEQ ID NO: 71, at residue E430 as numbered with reference to residue position of SEQ ID NO: 74, at residue E260 as numbered with reference to residue position of SEQ ID NO: 77, at residue E341 as numbered with reference to residue position of SEQ ID NO: 80, or at residue E232 as numbered with reference to residue position of SEQ ID NO:

83.

66. The method of any one of claims 53 to 65, wherein the mutation is a substitution resulting in an amino acid substitution at residue E6, numbered with reference to residue positions of SEQ ID NO:

113.

67. The method of claim 65 or claim 66, wherein the substitution is glutamic acid (E) to lysine (K).

68. The method of any one of claims 53 to 65, wherein the mutation is a non-natural mutation.

69. according to the method described in any one in claim 53 to 68, the described mutation in wherein said endogenous PIF gene produces the PIF transcription factor with reduced DNA binding.

70. according to the method described in any one in claim 53 to 69, the described sudden change in wherein said endogenous PIF gene is dominant negative mutation, recessive mutation, null mutation, weak loss-of-function mutation or hypoactive mutation.

71. The method of any one of claims 53 to 70, wherein the resulting plants exhibit a reduced shade avoidance response compared to control plants.

72. The method of claim 71, wherein the plant exhibiting a reduced shade avoidance response exhibits at least one of the following phenotypes when planted adjacent to one or more plants, compared to a plant that does not exhibit a reduced shade avoidance response when planted adjacent to the one or more plants: increased yield, reduced height, reduced crown:root ratio, reduced leaf length; increased stem mechanical strength; reduced lodging rate; delayed senescence; increased photosynthetic efficiency and grain filling; and / or enhanced defense responses to pathogens and herbivores.

73. A method according to claim 71 or claim 72, wherein the plants are grown at an increased density without reducing plant yield on a per plant basis.

74. The method of claim 73, wherein the planting density is increased by about 5% to about 75% without decreasing plant yield on a per plant basis.

75. according to the method described in any one of claim 53 to 74, wherein said mutation produces a mutated PIF gene, said mutated PIF gene comprises a nucleotide sequence with at least 90% sequence identity to any one of SEQ ID NO:120, 122, 124, 126, 128, 129, 130, 132, 133, 134 or 135, and / or said mutated PIF gene encodes a mutated PIF polypeptide with at least 90% sequence identity to any one of SEQ ID NO:121, 123, 125, 127 or 131.

76. A guide nucleic acid that binds to a target site within an endogenous gene encoding a phytochrome-interacting factor (PIF) transcription factor, wherein the endogenous gene comprises a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 69, 70, 72, 73, 75, 76, 78, 79, 81, or 82; or encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 71, 74, 77, 80, or 83; and / or the target site comprises a nucleotide sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; or encodes an amino acid sequence having at least 80% sequence identity to SEQ ID NO:

113.

77. The guide nucleic acid of claim 76, wherein the guide nucleic acid comprises a spacer having the nucleotide sequence of any one of SEQ ID NOs: 114-119.

78. A system comprising the guide nucleic acid of claim 76 or claim 77 and a CRISPR-Cas effector protein associated with the guide nucleic acid.

79. The system of claim 78, 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.

80. 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 a phytochrome interacting factor (PIF) gene.

81. The gene editing system of claim 80, wherein the PIF gene: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

113.

82. The gene editing system of claim 80 or claim 81, wherein the guide nucleic acid comprises a spacer sequence having a nucleotide sequence of any one of SEQ ID NOs: 114-119.

83. The gene editing system of any one of claims 80 to 82, 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.

84. The gene editing system of any one of claims 80 to 83, wherein the PIF gene is a PIF3 gene, a PIF4 gene, or a PIF5 gene.

85. 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 a phytochrome interacting factor (PIF) gene, the PIF gene: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encoding a region having at least 80% sequence identity with the amino acid sequence of SEQ ID NO: 113, wherein the cleavage domain cleaves the target strand in the PIF gene.

86. 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 a phytochrome interacting factor (PIF) gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to the target site within the PIF gene, wherein the PIF gene: (a) comprising a sequence having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 69, 70, 72, 73, 75, 76, 78, 79, 81 or 82; (b) comprising a region having at least 80% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 84-87, 88-91, 92-95, 96-108, or 109-112; (c) encodes a polypeptide comprising a sequence having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NO: 71, 74, 77, 80 or 83; and / or (d) encoding a region having at least 80% sequence identity to the amino acid sequence of SEQ ID NO:

113.

87. The complex of claim 85 or the expression cassette of claim 86, wherein the PIF gene is a PIF3 gene, a PIF4 gene or a PIF5 gene.

88. A nucleic acid encoding a phytochrome-interacting factor (PIF) transcription factor comprising a mutated bHLH domain.

89. The nucleic acid of claim 88, wherein the PIF transcription factor is a PIF3 transcription factor, a PIF4 transcription factor, or a PIF5 transcription factor.

90. The nucleic acid of claim 88 or claim 89, wherein the mutation is a substitution producing an amino acid substitution at residue E361 as numbered with reference to residue position of SEQ ID NO: 71, at residue E430 as numbered with reference to residue position of SEQ ID NO: 74, at residue E260 as numbered with reference to residue position of SEQ ID NO: 77, at residue E341 as numbered with reference to residue position of SEQ ID NO: 80, or at residue E232 as numbered with reference to residue position of SEQ ID NO:

83.

91. The nucleic acid of any one of claims 88 to 90, wherein the mutation is a substitution resulting in an amino acid substitution at residue E6, numbered with reference to the residue positions of SEQ ID NO:

113.

92. according to the nucleic acid of any one of claim 88 to 91, wherein said nucleic acid comprises a mutated PIF gene, said mutated PIF gene comprises a nucleotide sequence with at least 90% sequence identity to any one of SEQ ID NO:120, 122, 124, 126, 128, 129, 130, 132, 133, 134 or 135, and / or said mutated PIF gene encodes a mutated PIF polypeptide with at least 90% sequence identity to any one of SEQ ID NO:121, 123, 125, 127 or 131.

93. A modified PIF transcription factor having at least 90% sequence identity to any one of SEQ ID NO: 121, 123, 125, 127 or 131.

94. A plant or part thereof comprising a nucleic acid according to any one of claims 88 to 92 or a modified PIF transcription factor according to claim 93.

95. A corn plant or part thereof comprising a nucleic acid according to any one of claims 88 to 92 and / or a modified PIF transcription factor according to claim 93.

96. A wheat plant or part thereof comprising a nucleic acid according to any one of claims 88 to 92 and / or a modified PIF transcription factor according to claim 93.

97. The plant of claim 94, the corn plant of claim 95, or the wheat plant of claim 96, which exhibits a phenotype of reduced shade avoidance response.

98. The plant, corn plant or wheat plant of claim 97, wherein and does not comprise a reduced shade avoidance response and said plant, corn plant or wheat plant further exhibits increased yield, reduced height, reduced crown:root ratio, reduced leaf length when planted adjacent to one or more plants, corn plants and / or wheat plants; increased stem mechanical strength; reduced lodging rate; delayed senescence; increased photosynthetic efficiency and grain filling; and / or enhanced defense responses to pathogens and herbivores compared to corn plants or wheat plants planted adjacent to one or more plants, corn plants and / or wheat plants.

99. The plant, corn plant or wheat plant of claim 97 or claim 98, wherein the plant, corn plant or wheat plant is grown at increased density without reducing plant yield on a per plant basis.

100. The plant, corn plant or wheat plant of claim 99, wherein the planting density is increased by about 5% to about 75% without decreasing plant yield on a per plant basis.

101. A corn plant or part thereof, comprising at least one mutation in an endogenous phytochrome interacting factor (PIF) gene having a gene identification number (Gene ID) of Zm00001d040536, Zm00001d008205, Zm00001d031044, Zm00001d033267 or Zm00001d034298 (Maize GDB), optionally wherein the mutation is a non-natural mutation.

102. A guide nucleic acid that binds to a target nucleic acid in an endogenous phytochrome interacting factor (PIF) gene having a gene identification number (Gene ID) of Zm00001d040536, Zm00001d008205, Zm00001d031044, Zm00001d033267, or Zm00001d034298 (Maize GDB).

103. A method for producing a plant comprising a mutation in an endogenous PIF gene and at least one polynucleotide of interest, the method comprising: crossing a first plant that is a plant according to any one of claims 1 to 20, 33 to 36, or 94 to 101 with a second plant comprising the at least one polynucleotide of interest to produce a progeny plant; and Progeny plants comprising the mutation in the PIF gene and the at least one polynucleotide of interest are selected, thereby producing the plant comprising the mutation in the endogenous PIF gene and the at least one polynucleotide of interest.

104. A method for producing a plant comprising a mutation in an endogenous PIF gene and at least one polynucleotide of interest, the method comprising: Introducing at least one polynucleotide of interest into a plant according to any one of claims 1 to 20, 33 to 36 or 94 to 101, thereby producing a plant comprising a mutation in the PIF gene and the at least one polynucleotide of interest.

105. The method of any one of claim 103 or claim 104, 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.

106. A method for producing a plant comprising a mutation in an endogenous PIF gene and exhibiting a phenotype that exhibits improved yield traits, improved plant configuration and / or improved defense traits, the method comprising Crossing a first plant that is a plant according to any one of claims 1 to 20, 33 to 36 or 94 to 101 with a second plant that exhibits a phenotype of improved yield traits, improved plant architecture and / or improved defense traits; and Progeny plants comprising the mutation in the PIF gene and the phenotype of improved yield traits, improved plant architecture and / or improved defense traits are selected to thereby produce plants comprising the mutation in the endogenous PIF gene and exhibiting the phenotype of improved yield traits, improved plant architecture and / or improved defense traits compared to control plants.

107. 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 on a roadside, the method comprising applying a herbicide to one or more plants according to any one of claims 1 to 20, 33 to 36, or 94 to 101 growing in the container, growth chamber, greenhouse, field, recreational area, lawn, or on a roadside, thereby controlling the weeds in the container, growth chamber, greenhouse, field, recreational area, lawn, or on the roadside in which the one or more plants are growing.

108. A method of reducing insect predation on a plant, the method comprising applying an insecticide to one or more plants according to any one of claims 1 to 20, 33 to 36 or 94 to 101, thereby reducing insect predation on the one or more plants.

109. A method of reducing fungal diseases on plants, the method comprising applying a fungicide to one or more plants according to any one of claims 1 to 20, 33 to 36, or 94 to 101, thereby reducing fungal diseases on the one or more plants.

110. The method of claim 108 or claim 109, wherein the one or more plants are grown in containers, growth chambers, greenhouses, fields, recreational areas, lawns or roadsides.

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