Methods and compositions for controlling meristem size for crop improvement

Abstract

The present invention relates to compositions and methods for modifying the FASCIATED EAR2 (FEA2) gene (optionally to alter meristem size) in a plant. The invention also relates to plants having increased grain row number produced using the methods and compositions of the invention.

Description

[0001] Statement Regarding Electronic Submission of Sequence Listings

[0002] A sequence listing in XML format named 1499-104_ST26.xml and with a size of 222,389 bytes was generated and submitted on June 8, 2023, and the public content of the sequence listing is hereby incorporated into this specification by reference.

[0003] Priority declaration

[0004] This application claims the benefit of U.S. Provisional Application No. 63 / 356,527, filed on June 29, 2022, under 35 USC § 119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0005] The present invention relates to compositions and methods for modifying the FASCIATED EAR2 (FEA2) gene in plants, optionally to alter meristem size. The present invention also relates to plants with increased kernel row number produced using the methods and compositions of the present invention. Background Art

[0006] New plant organs begin at the growing tips of plants called meristems. In the meristems, a population of undifferentiated stem cells is maintained. During growth, the meristems distribute stem cells to newly formed organs, including seeds, while retaining some stem cells to continue to maintain the meristem. Several conserved molecular mechanisms have been described that control the size of the stem cell population to ensure organized growth and appropriate meristem size.

[0007] Due to the modular nature of corn ear development, larger meristems tend to cause more flowers, and therefore, meristem size has a direct impact on the number of kernel rows and yield. The number of flowers caused during corn ear development directly limits grain yield. A major trait selected during corn domestication is the increase in the number of flowers that begin around the ear (kernel row number or KRN). The significant progress achieved through breeding has led to a sharp increase in the number of kernel rows, from 2 rows in the corn ancestor Teosinte to about 8 to 20 rows in modern improved corn varieties. In different corn strains, the number of kernel rows can reach up to 36.

[0008] In the typical regulatory pathway described in the model plant Arabidopsis, the CLAVATA3 (CLV3) peptide is secreted from cells at the top of the meristem and moves through the apoplast to the central stem cell domain, where it interacts with several leucine-rich receptors (LRRs), including CLAVATA1 (CLV1) and CLAVATA2 (CLV2). This receptor-ligand interaction stimulates signal transduction, which ultimately serves to reduce WUS expression and limit the expansion of stem cell populations. One of the targets of WUS is the CLV3 gene itself, and WUS acts in this way to limit its own expression and maintain stem cell homeostasis (Fletcher, JC, Plants 7: 87 (2018)).

[0009] Loss-of-function mutations in CLV1, CLV2, or CLV3 result in amplification of the WUS domain and increased meristem size (Schoof et al., Cell 100: 635–644 (2000)). Typically, this increase in meristem size results in abnormal plant growth because the meristem expands uncontrollably and becomes disorganized, a phenomenon known as fasciation (Je et al., Nat Genet 48: ng.3567 (2016a)). Importantly, larger meristems do not simply create larger organs, but increase the number of organs around a larger area. Due to this relationship between meristem size and organ number, mutations in maize CLV-WUS signaling genes can result in increased flower number and yield. However, although a strong loss-of-function mutation in the maize CLV2 ortholog FASCIATED EAR2 (FEA2) resulted in meristem enlargement and increased KRN, the ear was disorganized and thus yield was not increased (Taguchi-Shiobara et al., Gene Dev 15:2755–2766 (2001)).

[0010] New strategies for regulating meristem size are needed to improve crop performance. Summary of the invention

[0011] One aspect of the present invention provides a corn plant or plant part thereof comprising at least one mutation in an endogenous FASCIATED EAR2 (FEA2) gene encoding a FEA2 protein, optionally wherein the at least one mutation may be a non-natural mutation.

[0012] A second aspect of the present invention provides a corn plant cell comprising an editing system, which comprises: (a) a CRISPR-Cas effector protein; and (b) a guide nucleic acid (gRNA, gDNA, crRNA, crDNA, sgRNA, sgDNA) comprising a spacer sequence complementary to an endogenous target gene encoding a FEA2 protein.

[0013] A third aspect of the invention provides a corn plant cell comprising at least one mutation within the FEA2 gene, wherein the mutation is a replacement, insertion or deletion introduced using an editing system comprising a nucleic acid binding domain that binds to a target site in the FEA2 gene, optionally wherein the at least one mutation may be a non-natural mutation.

[0014] A fourth aspect of the present invention provides a method for producing / cultivating a transgenic-free edited corn plant, the method comprising: crossing the corn plant of the present invention with a transgenic-free corn plant, thereby introducing the at least one mutation into the transgenic-free corn plant; and selecting offspring corn plants that contain the at least one mutation and are free of transgenes, thereby producing a transgenic-free edited corn plant, optionally wherein the at least one mutation may be a non-natural mutation.

[0015] A fifth aspect of the invention provides a method for providing multiple corn plants with an increased number of kernel rows, the method comprising planting two or more plants of the invention in a growing area, thereby providing multiple corn plants with an increased number of kernel rows compared to multiple control corn plants not comprising the mutation.

[0016] A sixth aspect of the invention provides a method for producing a variation in a region of a corn FEA2 protein, the method comprising: introducing an editing system into a corn plant cell, wherein the editing system targets a region of a FEA2 gene encoding a FEA2 polypeptide, and contacting the region of the FEA2 gene with the editing system, thereby introducing a mutation into the FEA2 gene and producing a variation in the region of the FEA2 protein of the corn plant cell.

[0017] The seventh aspect of the present invention provides a method for detecting a mutated FEA2 gene (mutation in an endogenous FEA2 gene) in a corn plant, the method comprising detecting in the genome of the corn plant a FEA2 gene having at least one mutation in a region having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NOs: 75-77.

[0018] In an eighth aspect, a method for generating a mutation in an endogenous FEA2 gene of a corn plant is provided, the method comprising: (a) targeting a gene editing system to a portion of the FEA2 gene comprising a sequence having at least 80% sequence identity with any one of SEQ ID NOs:75-77; and (b) selecting a corn plant comprising a modification located in a region of the FEA2 gene having at least 80% sequence identity with any one of SEQ ID NOs:75-77.

[0019] The ninth aspect provides a method for editing a specific site in the genome of a corn plant cell, the method comprising: cutting a target site within an endogenous FEA2 gene in a corn plant cell in a site-specific manner, the endogenous FEA2 gene: (a) comprising a nucleotide sequence having at least 90% sequence identity with SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NOs:75-77; (c) encoding an amino acid sequence having at least 90% sequence identity with SEQ ID NO:74; and / or (d) encoding a polypeptide comprising a region having at least 90% sequence identity with SEQ ID NOs:78-80, thereby producing an edit in the endogenous FEA2 gene of the corn plant cell, and producing a corn plant cell comprising the edit in the endogenous FEA2 gene.

[0020] The tenth aspect provides a method for preparing a corn plant, the method comprising: (a) contacting a population of corn plant cells comprising a wild-type endogenous FEA2 gene with a nuclease linked to a nucleic acid binding domain (e.g., an editing system), the nucleic acid binding domain binding to a sequence having at least 90% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-77 or binding to a sequence encoding an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NOs: 78-80; (b) selecting corn plant cells from a population in which the endogenous FEA2 gene has been mutated, thereby producing corn plant cells comprising at least one mutation in the endogenous FEA2 gene; and (c) growing the selected corn plant cells into corn plants.

[0021] The eleventh aspect provides a method for increasing the number of kernel rows in a corn plant, the method comprising (a) contacting a corn plant cell comprising an endogenous FEA2 gene with a nuclease targeted to the endogenous FEA2 gene, wherein the nuclease is linked to a nucleic acid binding domain (e.g., an editing system) that binds to a target site within the endogenous FEA2 gene, wherein the endogenous FEA2 gene: (i) comprises a nucleotide sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (ii) encodes an amino acid sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 74; (iii) comprises a region having at least 90% sequence identity to a nucleotide sequence of any one of SEQ ID NOs: 75-77; and / or (iv) encodes a region having at least 90% sequence identity to a nucleotide sequence of SEQ ID NOs: NO:78-80 has a polypeptide region with at least 90% sequence identity to any of the amino acid sequences, to produce a corn plant cell comprising a mutation in a wild-type endogenous FEA2 gene; and (b) growing the corn plant cell into a corn plant comprising a mutation in the endogenous FEA2 gene, thereby producing a corn plant having a mutated endogenous FEA2 gene and exhibiting increased yield and / or increased number of kernel rows, optionally exhibiting increased number of kernel rows without substantially reducing ear length.

[0022] The twelfth aspect provides a method for producing a corn plant or part thereof comprising at least one cell having a mutation in an endogenous FEA2 gene, the method comprising contacting a target site within an endogenous FEA2 gene in a corn plant or plant part with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site in the endogenous FEA2 gene, wherein the endogenous FEA2 gene (a) comprises a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:74; (c) comprises a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:75-77; and / or (d) encodes a polypeptide region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs:78-80, thereby producing a corn plant or part thereof comprising at least one cell having a mutation in an endogenous FEA2 gene.

[0023] The thirteenth aspect of the present invention provides a method for producing a corn plant or part thereof comprising a mutated endogenous FEA2 gene and exhibiting an increased number of kernel rows (e.g., producing an ear with an increased number of kernel rows, optionally without substantially reducing ear length), the method comprising contacting a target site within an endogenous FEA2 gene in a corn plant or plant part with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site within the endogenous FEA2 gene, wherein the endogenous FEA2 gene: (a) comprises a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:74; (c) comprises a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:75-77; and / or (d) encodes a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:75-77; and / or A polypeptide region of a region having at least 90% sequence identity to the amino acid sequence of any one of NO:78-80, thereby producing a corn plant or part thereof comprising an endogenous FEA2 gene with a mutation and exhibiting increased yield and / or increased number of kernel rows, optionally wherein when the number of kernel rows is increased, the ear length is not substantially reduced.

[0024] In the fourteenth aspect, a method for modifying an endogenous FEA2 gene in a corn plant or part thereof to improve the yield traits of the corn plant or part thereof is provided, the method comprising altering a target site within an endogenous FEA2 gene in a corn plant or part thereof, wherein the endogenous FEA2 gene: (a) comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-77; (c) encodes a polypeptide comprising a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NOs:74; and / or (d) encodes a region having at least 90% sequence identity to the amino acid sequence of SEQ ID NOs:78-80, thereby modifying the endogenous FEA2 gene and improving the yield traits of the corn plant or part thereof.

[0025] In a fifteenth aspect, a guide nucleic acid is provided that binds to a target site in a FEA2 gene, the target site comprising a nucleotide sequence of any one of SEQ ID NOs: 75-77 or a nucleotide sequence encoding an amino acid sequence of any one of SEQ ID NOs: 78-80.

[0026] A sixteenth aspect provides a system comprising a guide nucleic acid of the present invention and a CRISPR-Cas effector protein associated with the guide nucleic acid.

[0027] In a seventeenth aspect, a gene editing system is provided, comprising a CRISPR-Cas effector protein associated with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to an endogenous FEA2 gene.

[0028] In the eighteenth aspect, a complex is provided, the complex comprising a guide nucleic acid and a CRISPR-Cas effector protein containing a cleavage domain, wherein the guide nucleic acid binds to a target site within an endogenous FEA2 gene, wherein the endogenous FEA2 gene, wherein the endogenous FEA2 gene: (a) comprises a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (b) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 74; (c) comprises a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-77; and / or (d) encodes a polypeptide region comprising a region having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 78-80, wherein the cleavage domain cleaves the target strand in the FEA2 gene.

[0029] In the nineteenth aspect, an expression cassette is provided, comprising (a) a polynucleotide encoding a CRISPR-Cas effector protein comprising a cleavage domain and (b) a guide nucleic acid that binds to a target site within an endogenous FEA2 gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (i) a portion of a nucleic acid encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 74; (ii) a portion of a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (iii) a portion of a sequence having at least 90% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 75-77; and / or (iv) a portion of a nucleic acid encoding a polypeptide region having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 78-80.

[0030] In a twentieth aspect, a nucleic acid encoding a dominant negative mutation, a semi-dominant mutation, or a weak loss-of-function mutation of a maize FEA2 protein is provided.

[0031] Also provided herein are FEA2 polypeptides having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97 or 99, wherein the modified FEA2 polypeptide comprises a mutation in at least one non-natural mutation located in the 10th LRR repeat sequence of the encoded FEA2 polypeptide SEQ ID NO: 74.

[0032] In another aspect, a corn plant or part thereof is provided comprising one or more mutated FEA2 genes, wherein the one or more mutated FEA2 genes comprise a sequence having at least 90% sequence identity to any one of SEQ ID NOs:82, 84, 86, 88, 90, 92, 94, 96 or 98, and / or encode a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs:83, 85, 87, 89, 91, 93, 95, 97 or 99.

[0033] In another aspect, a corn plant or part thereof is provided comprising at least one mutation in an endogenous FEA2 gene, the corn plant or part thereof exhibiting increased yield and / or increased number of kernel rows (e.g., producing ears with increased number of kernel rows, optionally without substantially reducing ear length), optionally wherein the at least one mutation may be a non-natural mutation. In some aspects, a corn plant is provided that also exhibits increased yield and larger floral meristems.

[0034] Also provided are corn plants comprising in their genome one or more mutated FASCIATED EAR2 (FEA2) genes produced by the methods of the invention, as well as polypeptides, polynucleotides, nucleic acid constructs, expression cassettes and vectors for making the corn plants of the invention.

[0035] These and other aspects of the invention will be described in more detail in the following description of the invention.

[0036] A brief description of the sequence

[0037] SEQ ID NOs: 1-17 are exemplary Cas12a amino acid sequences that can be used in the present invention.

[0038] SEQ ID NOs: 18-20 are exemplary Cas12a nucleotide sequences that can be used in the present invention.

[0039] SEQ ID NOs: 21-22 are exemplary regulatory sequences encoding promoters and introns.

[0040] SEQ ID NOs: 23-29 are exemplary cytosine deaminase sequences that can be used in the present invention.

[0041] SEQ ID NOs: 30-40 are exemplary adenine deaminase amino acid sequences that can be used in the present invention.

[0042] SEQ ID NO: 41 is an exemplary uracil-DNA glycosylase inhibitor (UGI) sequence that can be used in the present invention.

[0043] SEQ ID NOs: 42-44 provide examples of the locations of the protospacer-adjacent motifs of type V CRISPR-Cas12a nucleases.

[0044] SEQ ID NOs:45-47 provide exemplary peptide tags and affinity polypeptides useful in the present invention.

[0045] SEQ ID NOs:48-58 provide exemplary RNA recruitment motifs and corresponding affinity polypeptides that can be used in the present invention.

[0046] SEQ ID NOs:59-60 are exemplary Cas9 polypeptide sequences that can be used in the present invention.

[0047] SEQ ID NOs:61-71 are exemplary Cas9 polynucleotide sequences that can be used in the present invention.

[0048] SEQ ID NO:72 is an exemplary FEA2 genomic sequence.

[0049] SEQ ID NO:73 is an exemplary FEA2 coding (cds) sequence.

[0050] SEQ ID NO:74 is an exemplary FEA2 polypeptide sequence.

[0051] SEQ ID NOs:75-77 are exemplary target regions of the FEA2 genomic sequence that can be used in the present invention.

[0052] SEQ ID NOs:77-80 are exemplary target regions of FEA2 polypeptides useful in the present invention.

[0053] SEQ ID NO: 81 is an exemplary spacer sequence of a nucleic acid guide sequence that can be used in the present invention.

[0054] SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 and 98 are exemplary edited FEA2 nucleic acid sequences generated as described herein.

[0055] SEQ ID NOs:83, 85, 87, 89, 91, 93, 95, 97, and 99 are exemplary mutated FEA2 polypeptides generated from the edited nucleic acid sequences of SEQ ID NOs:82, 84, 86, 88, 90, 92, 94, 96, and 98, respectively. DETAILED DESCRIPTION

[0056] The present invention will now be described hereinafter with reference to the following examples, in which embodiments of the present invention are shown. This specific embodiment is not intended to be a detailed catalog of all different ways in which the present invention can be implemented or all features that can be added to the present invention. For example, the features shown in one embodiment may be incorporated into other embodiments, and the features shown in a specific embodiment may be deleted from the embodiment. Therefore, the present invention contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted. In addition, it will be apparent to those skilled in the art, according to the present disclosure, that many changes and additions to the various embodiments proposed herein will not deviate from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to describe in detail all its permutations, combinations and variations.

[0057] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. The terms used in the description of the present invention are for the purpose of describing particular embodiments only and are not intended to be limiting of the present invention.

[0058] 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.

[0059] Unless the context indicates otherwise, it is specifically intended that the various features of the invention described herein may be used in any combination. In addition, the present invention also contemplates that in some embodiments of the present invention, any feature or combination of features set forth herein may be excluded or omitted. For illustration, if the specification states that a composition comprises components A, B, and C, it is specifically intended that any one of A, B, or C or a combination thereof may be omitted and abandoned, either individually or in any combination.

[0060] 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.

[0061] Also as used herein, "and / or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of a combination when interpreted in the alternative ("or").

[0062] As used herein, the term "about" when referring to a measurable value such as an amount or concentration, is intended to encompass variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value as well as the specified value. For example, "about X," where X is a measurable value, is intended to include X as well as variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. The ranges of measurable values ​​provided herein may include any other ranges and / or single values ​​therein.

[0063] 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."

[0064] 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.

[0065] As used herein, the terms “comprising,” “including,” and “having” specify the presence of stated features, integers, steps, operations, elements, and / or components, but do not preclude the presence or addition of one or more other features, integers, steps, operations, elements, components, and / or groups thereof.

[0066] As used herein, the transitional phrase "consisting essentially of" means that the scope of the claim should be interpreted to encompass the specified materials or steps recited in the claim as well as those materials or steps that do not materially affect the basic and novel characteristics of the claimed invention. Therefore, when used in the claims of the present invention, the term "consisting essentially of" is not intended to be interpreted as being equivalent to "comprising".

[0067] As used herein, the terms "increase", "increasing", "increased", "enhance", "enhanced", "enhancing", and "enhancement" (and grammatical variations thereof) describe an increase of at least about 5%, 10%, 15%, 20%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more compared to a control. For example, a corn plant comprising a mutation in a FEA2 gene as described herein can exhibit an increased number of kernel rows (e.g., produce ears having an increased number of kernel rows) that is at least about 5% or more greater than the number of kernel rows of a control plant that does not comprise the same mutation, optionally wherein the length of the ear comprising the increased number of kernel rows is not substantially reduced (e.g., less than 30% reduction in length as compared to an ear of a corn plant that does not comprise the same FEA2 mutation). A control plant is typically the same plant as the edited plant, but the control plant has not been similarly edited and therefore does not comprise 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 infiltrated, but the control breeding line, variety or cultivar does not have a mutation. In some embodiments, the comparison between the plant of the present invention and the control plant is performed under the same growth conditions, such as the same environmental conditions (soil, hydration, light, heat, nutrients, etc.).

[0068] As used herein, the terms "reduce", "reduced", "reducing", "reduction", "reduction" and "lowering" (and grammatical variations thereof) describe, e.g., a reduction of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% compared to a control. In certain 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.

[0069] As used herein, the term "expression" ("express", "expresses", "expressed" or "expression"), etc., with respect to nucleic acid molecules and / or nucleotide sequences (e.g., RNA or DNA) means that the nucleic acid molecules and / or nucleotide sequences are transcribed and optionally translated. Thus, nucleic acid molecules and / or nucleotide sequences can express a polypeptide of interest or, for example, a functional non-translated RNA.

[0070] 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. A "heterologous" nucleotide / polypeptide may originate from a foreign species or, if from the same species, has been substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.

[0071] A corn plant in which at least one (e.g., one or more, e.g., 1, 2, 3, or 4 or more) endogenous FEA2 gene is modified as described herein (e.g., comprises a modification as described herein) can have an improved yield trait compared to a corn plant that does not comprise (lack) the modification in at least one endogenous FEA2 gene. As used herein, "improved yield trait" refers to any plant trait associated with growth, such as biomass, yield, nitrogen use efficiency (NUE), inflorescence size / weight, fruit yield, fruit quality, fruit size, seed size (e.g., seed area, seed size), seed number, leaf tissue weight, nodule number, nodule quality, nodulation activity, seed ear number, tiller number, branch number, flower number, tuber number, tuber quality, bulb quality, seed number, total seed mass, leaf emergence rate, tiller / branch emergence rate, seedling emergence rate, root length, root number, root mass size and / or weight, or any combination thereof. In some aspects, "improved yield traits" may include, but are not limited to, increased inflorescence yield, increased fruit yield (e.g., increased number, weight, and / or size of fruits; e.g., increased number, weight, and / or length of ears, e.g., for corn), increased fruit quality, increased number, size, and / or weight of roots, increased meristem size, increased seed size (e.g., seed area and / or seed weight), increased biomass, increased leaf size, improved nitrogen use efficiency, increased height, increased number of internodes, and / or increased internode length, compared to a control plant or part thereof (e.g., a corn plant that does not contain a mutated endogenous FEA2 nucleic acid as described herein). In some aspects, the improved yield trait may be expressed as the number of grains / seeds produced per unit area of ​​land (e.g., bushels per acre of land). In some embodiments, a corn plant comprising a mutated endogenous FEA2 nucleic acid as described herein exhibits increased yield, optionally wherein the corn plant exhibits an increased kernel row number (KRN) phenotype, optionally wherein ear length is not substantially reduced, compared to a control plant lacking the at least one mutation.

[0072] As used herein, "control plant" means a plant that does not contain one or more edited FEA2 genes as described herein that impart enhanced / improved traits (e.g., yield traits) or altered phenotypes (e.g., increased yield, increased number of grain rows, optionally substantially unreduced ear length). Control plants are used to identify and select plants edited as described herein, which have enhanced traits or altered phenotypes compared to control plants. Suitable control plants can be parental line plants for producing plants containing mutated FEA2 genes, for example, wild-type plants lacking editing in endogenous FEA2 genes as described herein. Suitable control plants can also be plants containing recombinant nucleic acids that impart other traits, for example, transgenic plants with enhanced herbicide tolerance. In some cases, suitable control plants can be progeny of heterozygous or hemizygous transgenic plant lines lacking mutated FEA2 genes as described herein, referred to as negative segregants or negative isogenic lines. Comparison with control plants can be carried out under the same environmental conditions.

[0073] Enhanced traits (e.g., improved yield traits) may include, for example, reduced days from planting to maturity, increased stem size, increased leaf number, increased vegetative stage plant height growth rate, increased ear size, increased dry weight per plant ear, increased number of grains per ear, increased weight per grain, increased number of grains per plant, reduced empty ears, extended grain filling period, reduced plant height, increased number of root branches, increased total root length, increased yield, increased nitrogen use efficiency, and / or increased water use efficiency compared to control plants. The altered phenotype may be, for example, plant height, biomass, canopy area, anthocyanin content, chlorophyll content, water application, water content, and water use efficiency.

[0074] In some embodiments, plants of the present invention may comprise one or more improved yield traits, including but not limited to, in some embodiments, one or more improved yield traits include higher yield (bushels per acre), increased biomass, increased plant height, increased stem diameter, increased leaf area, increased number of flowers, increased number of kernel rows (optionally wherein ear length is not substantially reduced), increased number of kernels, increased kernel size, increased ear length, reduced number of tillers, reduced number of tassel branches, increased number of pods (including increased number of pods per node and / or increased number of pods per plant), increased number of seeds per pod, increased number of seeds, increased seed size, and / or increased seed weight (e.g., increased 100-seed weight) compared to a control plant lacking at least one mutation. In some embodiments, plants of the present invention may comprise one or more improved yield traits including, but not limited to, optionally increased yield (bushels per acre), seed size (including kernel size), seed weight (including kernel weight), increased number of kernel rows (optionally wherein ear length is not substantially reduced), increased number of pods, increased number of seeds per pod, and increased ear length, as compared to control plants or parts thereof.

[0075] As used herein, "properties" are physiology, morphology, biochemistry or physical characteristics of plants or specific plant materials or cells. In some cases, this feature is visible to the human eye and can be measured mechanically, such as the size, weight, shape, form, length, height, growth rate and developmental stage of seeds or plants, or can be measured by biochemical techniques, such as protein, starch, some metabolites or the oil content of detection seeds or leaves, or by observing metabolism or physiological processes, for example, by measuring the tolerance to lack of water or specific salt or sugar concentration, or by measuring the expression level of one or more genes, for example, by adopting Northern analysis, RT-PCR, microarray gene expression array or reporter gene expression system, or by agricultural observations such as high osmotic stress tolerance or yield. However, any technology can be used to measure the amount, comparative level or difference of any selected chemical compound or macromolecule in transgenic plants.

[0076] As used herein, "enhanced proterties" refers to plant characteristics caused by mutations in FEA2 genes as described herein. Such proterties include, but are not limited to, enhanced agronomic traits, characterized in that enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environment or chemical tolerance. In some embodiments, the phenotype of enhanced proterties / changes can be, for example, the number of days from planting to maturity, the stem size of increase, the number of leaves increased, the growth rate of the plant height in the nutrient stage increased, the ear size of increase, the dry weight of each plant ear of increase, the number of grains per ear of increase, the weight of each grain of increase, the number of grains per plant of increase, the empty grains of the ear of reduction, the filling period of extension, the plant height of reduction, the number of root branches increased, the total root length of increase, drought tolerance, the water use efficiency of improvement, cold tolerance, the nitrogen use efficiency of improvement and / or the yield of increase. In some embodiments, proterties are the yield increased under non-stress conditions or the yield increased under environmental stress conditions. Stress conditions can include biotic and abiotic stresses, such as drought, shading, fungal diseases, viral diseases, bacterial diseases, insect infestation, nematode infestation, low temperature exposure, heat exposure, osmotic stress, nitrogen nutrient availability reduction, phosphorus nutrient availability reduction and high plant density. "Yield" can be affected by many characteristics, including but not limited to plant height, plant biomass, pod number, pod position on plant, internode number, pod cracking incidence, grain size, ear size, ear tip filling degree, grain abortion, nodulation and nitrogen fixation efficiency, nutrient assimilation efficiency, biotic and abiotic stress resistance, carbon assimilation, plant configuration, lodging resistance, seed germination percentage, seedling vigor and juvenile traits. Yield can also be affected by the following factors: germination efficiency (including germination under stress conditions), growth rate (including growth rate under stress conditions), flowering time and duration, ear number, ear size, ear weight, number of seeds per ear or per pod, seed size, composition (starch, oil, protein) of seed and the characteristics of seed filling.

[0077] As also used herein, the term "trait modification" encompasses changing naturally occurring traits by producing detectable characteristic differences in plants comprising a mutation in an endogenous FEA2 gene as described herein relative to a plant that does not comprise the mutation (such as a wild-type plant, or a negative segregant). In some cases, trait modifications can be quantitatively evaluated. For example, compared to control plants, trait modifications can result in an increase or decrease in observed trait characteristics or phenotypes. It is well known that modified traits may have natural variations. Therefore, compared to control plants, observed trait modifications can result in a normal distribution and magnitude change in trait characteristics or phenotypes in plants.

[0078] The present disclosure relates to a corn plant with improved economically relevant characteristics (more specifically, increased yield and / or increased KRN), optionally wherein the increase in KRN does not result in a significant decrease in ear length (e.g., a decrease of 30% or less) compared to a control plant. More specifically, the present disclosure relates to a corn plant comprising a mutation in a FEA2 gene as described herein, wherein the corn plant exhibits increased yield and / or increased kernel row number (KRN) as compared to a control plant lacking the mutation, optionally wherein ear length is not substantially reduced. In some embodiments, the plants of the present disclosure exhibit further improved traits associated with yield, including but not limited to increased nitrogen use efficiency, increased nitrogen stress tolerance, increased water use efficiency, and / or increased drought tolerance, as defined and discussed below.

[0079] Yield can be defined as a measurable product with economic value from a crop. Yield can be defined in terms of quantity and / or quality. Yield can depend directly on several factors, such as the number and size of organs (e.g., number of flowers), plant configuration (such as the number of branches, plant biomass, such as increased root biomass, steeper root angles and / or longer roots, etc.), flowering time and duration, grain filling period. Root configuration and development, photosynthetic efficiency, nutrient absorption, stress tolerance, early vigor, delayed senescence and functional stay-green phenotypes may be factors that determine yield. Therefore, optimizing the above factors can help increase crop yield.

[0080] The increase / improvement of the yield correlated traits mentioned herein also can be considered to refer to the increase of the biomass (weight) of one or more parts of plant, and these one or more parts can comprise above-ground and / or underground (can gather in the crops) plant parts.Particularly, this type of part that can gather in the crops is seed, and the execution of the disclosed method produces the plant of the seed yield with the yield of increase, particularly 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 this plant.

[0081] The increased yield of the plants of the present disclosure can be measured in a variety of ways, including test weight, number of seeds per plant, seed weight, number of seeds per unit area (e.g., number of seeds per acre or seed weight), bushels per acre, tons per acre, or kilograms per hectare. The increased yield can be attributed to improved utilization of key biochemical compounds (such as nitrogen, phosphorus, and carbohydrates), or to improved responses to environmental stresses (such as cold, heat, drought, salt, shade, high plant density, and pest or pathogen attack).

[0082] "Increased yield" can be manifested as one or more of the following: (i) increased plant biomass (weight) of one or more parts of the plant, in particular the aboveground (harvestable) parts of the plant, increased root biomass (increased number of roots, increased root thickness, increased root length) or increased biomass of any other harvestable part; or (ii) increased early vigour, defined herein as increased aboveground area of ​​seedlings about three weeks after germination.

[0083] "Early vigor" refers to active and healthy plant growth, especially in the early stages of plant growth, and can be due to, for example, plant better adapting to its environment (for example, optimizing the utilization of energy, nutrient absorption and carbon distribution between seedling and root) caused by the increase in plant fitness. For example, early vigor can be a combination of the ability of seeds to germinate and emerge after planting and the ability of seedlings to grow and develop after emergence. Plants with early vigor also show increased seedling survival rate and better crop planting, which can usually make the field highly uniform, wherein most plants basically reach each development stage at the same time, thereby usually increasing output. Therefore, early vigor can be determined by measuring various factors, such as grain weight, germination percentage, emergence percentage, seedling growth, seedling height, root length, root and seedling biomass, canopy size and color, etc.

[0084] In addition, increased yield can also be manifested as increased total seed yield, which can be attributed to one or more of the following: an increase in seed biomass (seed weight) due to an increase in seed weight per plant and / or individual seed, for example, an increased number of flowers / panicles per plant; an increased number of pods; an increased number of nodes; an increased number of flowers ("florets") per panicle / plant; an increased seed filling rate; an increased number of filled seeds; an increased seed size (length, width, area, circumference and / or weight), which can also affect seed composition; and / or an increased seed volume, which can also affect seed composition. In one embodiment, the increased yield can be an increased seed yield, for example, an increased seed weight; an increased number of filled seeds; and / or an increased harvest index.

[0085] Increased production may also result in configurational changes or may be due to

[0086] Occurs due to changes in plant architecture.

[0087] Increased yield can also be expressed as an increased harvest index, which is expressed as

[0088] The ratio of the yield of harvestable parts (such as seeds) to the total biomass

[0089] The present disclosure also extends to harvestable parts of plants, such as, but not limited to, seeds, leaves, fruits, flowers, pods, pods, siliques, nuts, stems, rhizomes, tubers and bulbs. The present disclosure also relates to products derived from harvestable parts of such plants, such as dry pellets, powders, oils, fats and fatty acids, starch or proteins.

[0090] 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.

[0091] As used herein, "nitrogen use efficiency" refers to the processes that lead to increased plant yield, biomass, vigor, and growth rate per unit of nitrogen applied. These processes may include plant absorption, assimilation, accumulation, signal transduction, sensing, retransfer (within the plant), and utilization of nitrogen.

[0092] 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.

[0093] 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.

[0094] As used herein, "increased nitrogen stress tolerance" refers to the ability of a plant to grow, develop or produce normally, or to grow, develop or produce faster or better, when subjected to less than optimal available / applied amounts of nitrogen or under nitrogen limiting conditions.

[0095] The improved plant nitrogen utilization efficiency can be converted into a similar amount of yield in the field while supplying less nitrogen, or an increased yield can be obtained by supplying an optimum / sufficient amount of nitrogen. The improved nitrogen utilization efficiency can improve plant nitrogen stress tolerance, and can also improve the biochemical composition of crop quality and seeds, such as protein yield and oil yield. The terms "improved nitrogen utilization efficiency", "enhanced nitrogen utilization efficiency" and "nitrogen stress tolerance" are used interchangeably in this disclosure and refer to plants with improved productivity under nitrogen limiting conditions.

[0096] As used herein, "water use efficiency" refers to the amount of carbon dioxide assimilated by leaves per unit of transpiration water vapor. It constitutes one of the most important traits controlling plant productivity in dry environments. "Drought tolerance" refers to the degree to which plants adapt to dry or drought conditions. The physiological response of plants to water shortage includes leaf wilting, reduced leaf area, leaf shedding, and stimulating root growth by directing nutrients to the underground parts of the plant. Generally, plants are more susceptible to drought during flowering and seed development (reproduction stage) because the resources of the plant are biased to support root growth. In addition, abscisic acid (ABA) is a plant stress hormone that induces the closure of leaf stomata (microscopic pores involved in gas exchange), thereby reducing water loss caused by transpiration and reducing photosynthesis rate. These reactions improve the water use efficiency of plants in the short term. The terms "improved water use efficiency", "enhanced water use efficiency" and "increased drought tolerance" are used interchangeably in this disclosure and refer to plants with improved productivity under water-limited conditions.

[0097] As used herein, "increased water use efficiency" refers to the ability of a plant to grow, develop or produce faster or better than normal when subjected to the same available / applied amount of water as under normal or standard conditions; the ability of a plant to grow, develop or produce normally, or the ability to grow, develop or produce faster or better when subjected to reduced available / applied amounts of water (water input) or under conditions of water stress or water deficiency stress.

[0098] As used herein, "increased drought tolerance" refers to the ability of a plant to grow, develop or produce normally, or to grow, develop or produce faster or better than normal, when subjected to reduced available / applied amounts of water and / or under conditions of short-term or long-term drought; the ability of a plant to grow, develop or produce normally when subjected to reduced available / applied amounts of water (water input) or under conditions of water stress or under conditions of short-term or long-term drought.

[0099] As used herein, "drought stress" refers to a dry period (short or long / extended) that results in a lack of water and stresses the plant and / or causes damage to plant tissue and / or has a negative impact on grain / crop yield; a dry period (short or long / extended) that results in a lack of water and / or an increase in temperature and stresses the plant and / or causes damage to plant tissue and / or has a negative impact on grain / crop yield.

[0100] As used herein, "water deficit" refers to conditions or circumstances that provide less than the optimal amount of water required for adequate / successful growth and development of plants.

[0101] As used herein, "water stress" refers to a condition or environment that provides an inappropriate amount of water (less / insufficient or more / excessive) relative to the amount of water required for adequate / successful growth and development of the plant / crop, thereby stressing the plant and / or causing damage to plant tissue and / or negatively affecting grain / crop yield.

[0102] As used herein, "water stress" refers to a condition or environment that provides less / insufficient water than required for adequate / successful growth and development of a plant / crop, thereby stressing the plant and / or causing damage to plant tissue and / or negatively affecting grain yield.

[0103] "Natural" or "wild-type" nucleic acid, nucleotide sequence, polypeptide or amino acid sequence refers to a naturally occurring or endogenous nucleic acid, nucleotide sequence, polypeptide or amino acid sequence. In some contexts, a "wild-type" nucleic acid is an unedited nucleic acid as described herein, and may be different from an "endogenous" gene (e.g., a mutated endogenous gene) that can be edited as described herein. In some contexts, a "wild-type" nucleic acid (e.g., unedited) may be heterologous to an organism (e.g., a transgenic organism) in which a wild-type nucleic acid is found. As an example, a "wild-type endogenous FASCIATED EAR2 (FEA2) gene" is a FEA2 gene that is naturally present in a reference organism such as a plant (e.g., a corn plant) or endogenous to it, and may be subjected to modification as described herein, after which this modified endogenous gene is no longer wild-type.

[0104] As used herein, the term "heterozygous" refers to a genetic state in which different alleles are located at corresponding loci on homologous chromosomes.

[0105] As used herein, the term "homozygous" refers to a genetic state in which identical alleles are located at corresponding loci on homologous chromosomes.

[0106] As used herein, the term "allele" refers to one of two or more different nucleotides or nucleotide sequences occurring at a particular locus.

[0107] 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.

[0108] A "recessive mutation" is a genetic mutation that produces a phenotype when homozygous, but the phenotype is not observable when the locus is heterozygous.

[0109] 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.

[0110] A "dominant negative mutation" is a mutation that produces an altered gene product (e.g., one that has an abnormal function relative to the wild type) that adversely affects the function of the wild type allele or gene product. For example, a "dominant negative mutation" can block the function of a wild type gene product. A dominant negative mutation may also be referred to as a "reversal allele mutation."

[0111] A "semidominant mutation" is a mutation in which the penetrance of the phenotype in a heterozygous organism is less than the penetrance of the phenotype observed in a homozygous organism.

[0112] 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.

[0113] A "hypofunctional allele mutation" is a mutation that results in a partial loss of gene function, which may occur through reduced expression (e.g., reduced protein and / or reduced RNA) or reduced functional performance (e.g., reduced activity), but not a complete loss of function / activity. A "hypofunctional" allele is a semi-functional allele caused by a gene mutation that results in the production of a corresponding protein that functions at anywhere between 1% and 99% of normal efficiency.

[0114] A "hyperallelic mutation" is a mutation that results in increased expression of a gene product and / or increased activity of a gene product.

[0115] A "gain of function" allele or mutation is a mutation that confers a new function to an encoded gene product and / or confers a new gene expression pattern. In some embodiments, a gain of function mutation may be dominant or semi-dominant.

[0116] As used herein, "non-natural mutation" refers to a mutation that is produced by human intervention and is different from a naturally occurring mutation found in the same gene (eg, occurs naturally and is not the result of modification by humans).

[0117] A "locus" is the location on a chromosome where a gene or marker or allele is located. In some embodiments, a locus may encompass one or more nucleotides.

[0118] As used herein, the terms "desired allele," "target allele," and / or "allele of interest" are used interchangeably to refer to an allele associated with a desired trait. In some embodiments, a desired allele may be associated with an increase or decrease (relative to a control) in a given trait, depending on the nature of the desired phenotype.

[0119] A marker is "associated with" a trait when it is linked to the marker and when the presence of the marker is an indication of whether and / or to what extent the desired trait or trait form occurs in the plant / germplasm containing the marker. Similarly, a marker is "associated with" an allele or chromosomal interval when it is linked to the allele or chromosomal interval and when the presence of the marker is an indication of whether the allele or chromosomal interval is present in the plant / germplasm containing the marker.

[0120] 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 times, etc.). In a backcrossing 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 des Marqueurs 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.

[0121] As used herein, the term "cross" or "crossed" refers to the production of offspring (e.g., cells, seeds, or plants) by the fusion of gametes through pollination. The term encompasses sexual crossing (pollination of one plant to another) and selfing (self-pollination, e.g., when pollen and ovules are from the same plant). The term "crossing" refers to the act of fusion of gametes through pollination to produce offspring.

[0122] As used herein, the term "introgression" ("introgression", "introgressing" and "introgressed") refers to the natural and artificial transmission of a desired allele or combination of desired alleles of one or more genetic loci from one genetic background to another genetic background. For example, the desired allele at a given locus can be transmitted 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 offspring containing the desired allele can be backcrossed one or more times (e.g., 1, 2, 3, 4 or more times) with a strain having a desired genetic background, selecting the desired allele, with the result that the desired allele is fixed in the desired genetic background. For example, a marker associated with increased yield under non-water stress conditions can be introgressed from a donor into a recurrent parent that does not contain the marker and does not exhibit increased yield under non-water stress conditions. The resulting offspring can then be backcrossed one or more times and selected until the progeny possess the genetic marker associated with increased yield under non-water stress conditions in the recurrent parent background.

[0123] A "genetic map" is a description of the genetic linkage relationships between loci on one or more chromosomes within a given species, usually depicted in a graphical or tabular form. For each genetic map, the distances between loci are measured by the frequencies of recombination between them. A variety of markers can be used to detect recombination between loci. A genetic map is a product of the mapping population, the type of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distance between loci can vary from genetic map to genetic map.

[0124] As used herein, the term "genotype" refers to the genetic constitution of an individual (or individual population) at one or more genetic loci, in contrast to observable and / or detectable and / or phenotype traits (phenotypes). Genotype is defined by the alleles of one or more known loci inherited by an individual from its parents. The term genotype can be used to refer to the genetic constitution of an individual at a single locus, a plurality of loci, or more generally, the term genotype can be used to refer to the genetic constitution of all genes in an individual genome. Genotype can be, for example, indirectly characterized using a mark and / or directly characterized by nucleic acid sequencing.

[0125] As used herein, the term "germplasm" refers to the genetic material of an individual (e.g., plant), a group of individuals (e.g., plant strains, varieties or families), or clones derived from strains, varieties, species or cultures, or from an individual (e.g., plant), a group of individuals (e.g., plant strains, varieties or families), or clones derived from strains, varieties, species or cultures. Germplasm can be a part of an organism or cell or can be separated from an organism or cell. In general, germplasm provides genetic material with a specific genetic composition, providing a basis for some or all of the genetic qualities of an organism or cell culture. As used herein, germplasm includes cells, seeds or tissues that can grow new plants, and plant parts (e.g., leaves, stems, buds, roots, pollen, cells, etc.) that can be cultured into complete plants.

[0126] 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.

[0127] As used herein, the terms "exotic", "exotic strains" and "exotic germplasm" refer to any plant, strain or germplasm that is not superior. Generally speaking, the exotic plant / germplasm is not derived from any known superior plant or germplasm, but is selected to introduce one or more desired genetic elements into a breeding program (e.g., introducing new alleles into a breeding program).

[0128] As used herein, the term "hybrid" in the context of plant breeding refers to a plant that is the offspring of genetically distinct parents produced by crossing plants of different lines or varieties or species, including but not limited to a cross between two inbred lines.

[0129] As used herein, the term "inbred" refers to a substantially homozygous plant or variety. The term can refer to a plant or plant variety that is substantially homozygous throughout the genome, or a plant or plant variety that is substantially homozygous for a portion of the genome of particular interest.

[0130] A "haplotype" is the genotype of an individual at multiple genetic loci, i.e., a combination of alleles. Typically, the genetic loci that define a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term "haplotype" may refer to a polymorphism at a specific locus, such as a single marker locus, or a polymorphism at multiple loci along a chromosome segment.

[0131] As used herein, the terms "nucleic acid", "nucleic acid molecule", "nucleotide sequence" and "polynucleotide" refer to linear or branched, single-stranded or double-stranded RNA or DNA, or a hybrid thereof. The term also encompasses RNA / DNA hybrids. When synthesizing dsRNA, less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine, etc. can also be used for antisense, dsRNA and ribozyme pairing. For example, polynucleotides containing C-5 propyne analogs of uridine and cytidine have been shown to bind RNA with high affinity and are potent antisense inhibitors of gene expression. Other modifications may also be made, such as modifications to the 2'-hydroxyl in the phosphodiester backbone or the RNA ribose group.

[0132] As used herein, the term "nucleotide sequence" refers to a heteropolymer of nucleotides or a sequence of these nucleotides from the 5' to 3' end of a nucleic acid molecule, and includes DNA or RNA molecules, including cDNA, DNA fragments or parts, genomic DNA, synthetic (e.g., chemically synthesized) DNA, plasmid DNA, mRNA and antisense RNA, any of which can be single-stranded or double-stranded. The terms "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid construct", "oligonucleotide" and "polynucleotide" are also used interchangeably herein to refer to a heteropolymer of nucleotides. Nucleic acid molecules and / or nucleotide sequences provided herein are presented in 5' to 3' directions from left to right herein, and are represented by the standard code for representing nucleotide characters specified in the World Intellectual Property Organization (WIPO) standard ST.26. As used herein, "5' region" can represent the polynucleotide region closest to the 5' end of a polynucleotide. Therefore, for example, the elements in the 5' region of a polynucleotide can be located at any position from the first nucleotide at the 5' end of a polynucleotide to the nucleotide located in the middle of a polynucleotide. As used herein, "3' region" can represent the polynucleotide region closest to the 3' end of a polynucleotide. Thus, for example, elements in the 3' region of a polynucleotide can be located anywhere from the first nucleotide at the 3' end of the polynucleotide to a nucleotide located in the middle of the polynucleotide.

[0133] As used herein with respect to nucleic acids, the terms "fragment" or "portion" refer to a portion of a nucleic acid that is reduced in length (e.g., by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 400, 450, 500, 550, 660, 670, 680, 690, 710, 720, 730, 740, 750, 760, 770, 780, 790, 800 The nucleic acid fragment may be a nucleic acid having a length of 100, 200, 300, 400, 500, 600, 700, 750, 800, 850 or 900 nucleotides or any range or value therein) and comprising, consisting essentially of and / or consisting of a nucleotide sequence of contiguous nucleotides that are identical or nearly identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the corresponding portion of the reference nucleic acid. Where appropriate, such a nucleic acid fragment may be contained in a larger polynucleotide of which the nucleic acid fragment is a component. As an example, the repetitive sequence of the guide nucleic acid of the present invention may include a "portion" of a wild-type CRISPR-Cas repetitive sequence (e.g., a wild-type CRISPR-Cas repetitive sequence; for example, a repetitive sequence from a CRISPR Cas system, such as Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or Cas14c, etc.).

[0134] In some embodiments, the nucleic acid fragment may comprise, consist essentially of, or consist of the following consecutive nucleotides: about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45 of the nucleic acid encoding the FEA2 polypeptide. ,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,70,75,80,85,90,95,100,105,110,115,120,125,130,135,140,145,150,155,160,165,170,175,180,185,190,195,200,205,21 0, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 320, 330, 340, 350, 360, 370, 380, 390, 395, 400, 410, 415, 420, 425, 430, 435, 440, 445, 450, 500, 550, 600 , 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, 1500, 1550, 1600, 1650, 1700, 1750, 1800, 1850, 1900, 1950, 2000, 2500, 3000, 3500 or 4000 or more consecutive nucleotides, or any range or value therein,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,5 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 3, 104, 105, 106, 107, 108, 109, 110, 115, 120, 125, 130, 135, 140, 145, 150 consecutive nucleotides to about 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 221, 222, 223, 224, 225, 230, 240 , 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395 or 400 or more consecutive nucleotides, or any range or value therein (e.g., a fragment or portion of SEQ ID NO:72 or SEQ ID NO:73 (e.g., SEQ ID NOs:75-77)),

[0135] In some embodiments, a "sequence-specific nucleic acid binding domain" (eg, a sequence-specific DNA binding domain) can bind to one or more fragments or portions of a nucleotide sequence encoding a FEA2 polypeptide as described herein.

[0136] As used herein with respect to polypeptides, the term "fragment" or "portion" may refer to a polypeptide that is reduced in length relative to a reference polypeptide and comprises, consists essentially of, and / or consists of an amino acid sequence of contiguous amino acids that are identical or nearly identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the corresponding portion of the reference polypeptide. Where appropriate, such a polypeptide fragment may be contained in a larger polypeptide of which it is a component. In some embodiments, a polypeptide fragment comprises, consists essentially of, or consists of at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 225, 250, 260, 270, 280, 290 or more contiguous amino acids of a reference polypeptide. In some embodiments, the FEA2 polypeptide fragment may comprise, consist essentially of, 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, 35, 40, 45, 50, 60, 70, 71, 72, 73, 74, 75, 80, 90, 100, 125, 150, 175, 200, 225, 250, 275, 300, 350, 400, 450, 500, 550, or 600 consecutive amino acid residues, or any range or value therein (e.g., a fragment or portion of SEQ ID NO: 74 (e.g., SEQ ID NOs: 78-80)). In some embodiments, a fragment of a FEA2 polypeptide or FEA2 polynucleotide may be the result of a deletion produced in the FEA2 gene, or may be the result of an amino acid substitution resulting in a truncated polypeptide. A deletion may result in an in-frame deletion allele or an out-of-frame deletion allele. The FEA2 gene may be edited at more than one position, thereby providing a FEA2 gene comprising more than one mutation.

[0137] In some embodiments, reference to a "portion" or "region" of a nucleic acid refers to at least 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 115, 120, 121 5. 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 210, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, 230, 240, 250, 260, 270, 280, 285, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 or more consecutive nucleotides from a gene (e.g., consecutive nucleotides from the FEA2 gene),Optionally, a “portion” or “region” of a FEA2 gene can be about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 6, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 80, 90, 100, 101, 102, 103, 104, 105, 110, 115, 120, 125, 130, 135, 140, 141, 142, 143, 144, 145, or 150 consecutive nucleotides to about 155, 160, 165, 170、175、180、185、190、195、200、205、210、215、220、221、222、223、224、225、230、235、240、245、250、255、255、260、265、270、275、280、285、290、295、300、305、310、315、320、 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 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, 330, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445 or 450 or more consecutive nucleotides in length, or any range or value therein (e.g., a portion or region of consecutive nucleotides from SEQ ID NO: 72; e.g., SEQ ID NOs: 75-77). In some embodiments, reference to a "portion" or "region" of a polypeptide refers to at least 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, or 35 contiguous amino acid residues to about 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 , 43, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 75, 76, 77, 78, 79 or 80 or more consecutive amino acid residues from a polypeptide (e.g., a FEA2 polypeptide, e.g., SEQ ID NO: 74; see, e.g., SEQ ID NOs: 78-80). ,

[0138] In some embodiments, a "sequence-specific nucleic acid binding domain" can bind to one or more fragments or portions of a nucleotide sequence encoding a FEA2 polypeptide as described herein.

[0139] As used herein with respect to nucleic acids, the term "functional fragment" refers to a nucleic acid encoding a functional fragment of a polypeptide. A "functional fragment" with respect to a polypeptide is a fragment of a polypeptide that retains one or more activities of a native reference polypeptide.

[0140] As used herein, the term "gene" refers to a nucleic acid molecule that can be used to produce mRNA, antisense RNA, miRNA, anti-microRNA antisense oligodeoxyribonucleotides (AMOs), etc. A gene may or may not be used to produce a functional protein or gene product. A gene may include coding regions and non-coding regions (e.g., introns, regulatory elements, promoters, enhancers, termination sequences, and / or 5' and 3' untranslated regions). A gene can be "isolated," which means that the nucleic acid is substantially or substantially free of components that are usually present with the nucleic acid under natural conditions. Such components include other cell materials, culture media from recombinant production, and / or various chemicals for chemically synthesizing nucleic acids.

[0141] The term "mutation" refers to a point mutation (e.g., missense or nonsense, or insertion or deletion of a single base pair resulting in a frameshift), insertion, deletion and / or truncation. When a mutation is a replacement of one residue within an amino acid sequence by another residue, or a deletion or insertion of one or more residues within a sequence, the mutation is typically described by identifying the original residue, followed by the position of the residue within the sequence, and the identity of the newly replaced residue. Truncation may include truncation at the C-terminus of a polypeptide or the N-terminus of a polypeptide. Truncation of a polypeptide may be the result of a deletion of the corresponding 5' or 3' end of a gene encoding the polypeptide. Frameshift mutations occur when a deletion or insertion of one or more base pairs is introduced into a gene. Frameshift mutations in a gene may result in the production of a polypeptide that is longer, shorter, or the same length as a wild-type polypeptide, depending on when the first stop codon appears after the mutated region of the gene.

[0142] As used herein, the terms "complementary" or "complementarity" refer to the natural binding of polynucleotides by base pairing under permissive salt and temperature conditions. For example, the sequence "AGT" (5' to 3') binds to the complementary sequence "TCA" (3' to 5'). The complementarity between two single-stranded molecules can be "partial", in which only some of the nucleotides bind, or it can be complete when there is complete complementarity between the single-stranded molecules. The degree of complementarity between nucleic acid chains has a significant effect on the efficiency and strength of hybridization between nucleic acid chains.

[0143] As used herein, "complement" can mean 100% complementarity to a comparison nucleotide sequence, or it can mean less than 100% complementarity to a comparison nucleotide sequence (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, etc. complementarity).

[0144] Different nucleic acids or proteins with homology are referred to herein as "homologs". The term homolog includes homologous sequences from the same species and other species and orthologous sequences from the same species and other species." Homology " refers to the level of similarity between two or more nucleic acid and / or amino acid sequences, expressed as a percentage of positional identity (i.e., sequence similarity or identity). Homology also refers to the concept of having similar functional properties between different nucleic acids or proteins. Therefore, the compositions and methods of the present invention also include homologs of the nucleotide sequences and polypeptide sequences of the present invention. As used herein, "orthologs" refer to homologous nucleotide sequences and / or amino acid sequences in different species, which are derived from common ancestral genes during the process of speciation. Homologs of the nucleotide sequences of the invention have substantial sequence identity (e.g., at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to the nucleotide sequences of the invention.

[0145] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are unchanged over the entire component (e.g., nucleotide or amino acid) comparison window."Identity" can be easily calculated by known methods, including but not limited to the methods described in the following documents: Computational Molecular Biology (Lesk, AM edited) Oxford University Press, New York (1988); Biocomputing: Informatics andGenome Projects (Smith, DW edited) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG edited) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G. edited) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J. edited) Stockton Press, New York (1991).

[0146] As used herein, the term "percent sequence identity" or "percent identity" refers to the percentage of identical nucleotides in a linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) compared to a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percent sequence identity" may refer to the percentage of identical amino acids in an amino acid sequence compared to a reference polypeptide. With respect to the FEA2 gene, the sequence may have at least 90% sequence identity with the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73. In some embodiments, the FEA2 gene may have at least 95% sequence identity with the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73, optionally wherein the FEA2 gene may have 100% sequence identity with the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73. The FEA2 polypeptide as described herein may have at least 90% sequence identity with the polypeptide sequence of SEQ ID NO:74. In some embodiments, the FEA2 polypeptide may have at least 95% sequence identity with the polypeptide sequence of SEQ ID NO: 74, optionally wherein the FEA2 polypeptide may have 100% sequence identity with the polypeptide sequence of SEQ ID NO: 74. With respect to a region or portion of the FEA2 gene, the region or portion may have at least 90% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-77, optionally at least 90% sequence identity with any one of SEQ ID NOs: 75, 76, and / or 77. In some embodiments, a region or portion of the FEA2 gene may have at least 95% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-77, optionally at least 90% sequence identity with any one of SEQ ID NOs: 75, 76, and / or 77. In some embodiments, a region or portion of a FEA2 gene may have 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-77, optionally 100% sequence identity with any one of SEQ ID NOs: 75, 76, and / or 77. In some embodiments, a region or portion of a FEA2 polypeptide may have at least 90% sequence identity with the amino acid sequence of SEQ ID NOs: 78-80, optionally at least 90% sequence identity with any one of SEQ ID NOs: 78, 79, and / or 80. In some embodiments, a region or portion of a FEA2 polypeptide may have at least 95% sequence identity with the amino acid sequence of SEQ ID NOs: 78-80, optionally at least 95% sequence identity with any one of SEQ ID NOs: 78, 79, and / or 80.In some embodiments, a region or portion of a FEA2 polypeptide may have 100% sequence identity with an amino acid sequence of SEQ ID NOs: 78-80, optionally 100% sequence identity with any one of SEQ ID NOs: 78, 79, and / or 80. With respect to a mutated or edited FEA2 gene, the mutated or edited FEA2 gene may have at least 90% sequence identity with a nucleotide sequence of any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, and / or 98. In some embodiments, the mutated or edited FEA2 gene may have at least 95% sequence identity with a nucleotide sequence of any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, and / or 98. In some embodiments, the mutated or edited FEA2 gene may have at least 100% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 and / or 98. In some embodiments, the mutated FEA2 polypeptide may have at least 90% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, or 99. In some embodiments, the mutated FEA2 polypeptide may have at least 95% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, or 99. In some embodiments, the mutated FEA2 polypeptide may have 100% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, or 99.

[0147] As used herein, the phrases "substantially identical" or "substantial identity" in the context of two nucleic acid molecules, nucleotide sequences, or polypeptide sequences, means that two or more sequences or subsequences have at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms, or by visual inspection. In some embodiments of the invention, substantial identity exists over a region of contiguous nucleotides of a nucleotide sequence of the invention, the region being from about 10 nucleotides to about 20 nucleotides, from about 10 nucleotides to about 25 nucleotides, from about 10 nucleotides to about 30 nucleotides, from about 15 nucleotides to about 25 nucleotides, from about 30 nucleotides to about 40 nucleotides, from about 50 nucleotides to about 60 nucleotides, from about 70 nucleotides to about 80 nucleotides, from about 90 nucleotides to about 100 nucleotides, from about 100 nucleotides to about 200 nucleotides, from about 100 nucleotides to about 300 nucleotides, from about 100 nucleotides to about 400 nucleotides, from about 100 nucleotides to about 500 nucleotides, from about 100 nucleotides to about 600 nucleotides, from about 100 nucleotides to about 800 nucleotides, from about 100 nucleotides to about 900 nucleotides, or longer, or any range therein, up to the full length of the sequence. In some embodiments, the nucleotide sequences may be substantially identical over at least about 20 nucleotides (e.g., about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, or 80 nucleotides or more).

[0148] 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 25 amino acid residues, from about 7 amino acid residues to about 30 amino acid residues, from about 10 amino acid residues to about 25 amino acid residues, from about 15 amino acid residues to about 30 amino acid residues, from about 20 amino acid residues to about 40 amino acid residues, from about 25 amino acid residues to about 40 amino acid residues, from about 25 amino acid residues to about 50 amino acid residues, from about 30 amino acid residues to about 50 amino acid residues, from about 40 amino acid residues to about 50 amino acid residues, from about 40 amino acid residues to about 70 amino acid residues, from about 50 amino acid residues to about 70 amino acid residues, from about 60 amino acid residues to about 80 amino acid residues, from about 70 amino acid residues to about 80 amino acid residues, from about 90 amino acid residues to about 100 amino acid residues, or more, and any ranges therein, up to the full length of the sequence. In some embodiments, the polypeptide sequence can be within at least about 8 to about 350 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, 75 150, 175, 200, 225, 250, 300, 350 or more amino acids or more consecutive amino acid residues) are substantially identical to each other.In some embodiments, two or more FEA2 polypeptides can be identical or substantially identical (e.g., at least 70% to 99.9% identical, e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% identical, or any range or value therein) over about 8, 9, 10, 11, 12, 13, 14, or 15 consecutive amino acids to about 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 consecutive amino acids.

[0149] For sequence comparison, usually one sequence acts as a reference sequence compared to a test sequence. When using a sequence comparison algorithm, the test sequence and the reference sequence are input into a computer, subsequence coordinates are specified if necessary, and sequence algorithm program parameters are specified. Then, the sequence comparison algorithm calculates the sequence identity percentage of the test sequence relative to the reference sequence based on the specified program parameters.

[0150] The sequence optimal comparison for comparing the comparison window is well known to those skilled in the art, and can be carried out 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 carried out by the computerized realization of these algorithms, such as GAP, BESTFIT, FASTA and TFASTA, they can be used as GCG Wisconsin Package (Accelrys Inc., San Diego, CA) a part obtains. " Identity score " for the comparison fragment of test sequence and reference sequence is the quantity of the same components shared by two comparison sequences divided by the total number of components in the reference sequence fragment (for example, the smaller definition part of whole reference sequence or reference sequence). The sequence identity percentage is expressed as identity score multiplied by 100. The comparison of one or more polynucleotide sequences can be a comparison with full-length polynucleotide sequence or a part thereof, or a comparison with a longer polynucleotide sequence. For purposes of the present invention, "percent identity" may also be determined using BLASTX version 2.0 for translated nucleotide sequences and BLASTN version 2.0 for polynucleotide sequences.

[0151] 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.

[0152] In the context of nucleic acid hybridization experiments (such as Southern and Northern hybridizations), "stringent hybridization conditions" and "stringent hybridization wash conditions" are sequence-dependent and are different under different environmental parameters. An extensive guide to nucleic acid hybridization can be found in Tijssen Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays" Elsevier, New York (1993). In general, highly stringent hybridization and wash conditions are selected to be higher than the thermal melting point (Tf) for the specific sequence at a defined ionic strength and pH. m ) is about 5ºC lower.

[0153] T m It is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe. Very stringent conditions are chosen to be equal to the T for a specific probe. m. An example of stringent hybridization conditions for hybridization of complementary nucleotide sequences with more than 100 complementary residues on a filter in a Southern or Northern blot is 50% formamide with 1 mg of heparin at 42ºC overnight. An example of highly stringent wash conditions is 0.15 M NaCl at 72ºC for about 15 minutes. An example of stringent wash conditions is 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. An example of a medium stringency wash for a duplex of, for example, more than 100 nucleotides is 1x SSC at 45ºC for 15 minutes. An example of a low stringency wash for a duplex of, for example, more than 100 nucleotides is 4-6x SSC at 40ºC for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature of typically at least about 30°C. Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. In general, a signal-to-noise ratio that is 2 times (or higher) that observed for an unrelated probe in a particular hybridization assay indicates that specific hybridization has been detected. Nucleotide sequences that do not hybridize to each other under stringent conditions are still substantially identical if the proteins encoded by the nucleotide sequences are substantially identical. This occurs, for example, when a copy of a nucleotide sequence is produced using the maximum codon degeneracy permitted by the genetic code.

[0154] The polynucleotides and / or recombinant nucleic acid constructs (e.g., expression cassettes and / or vectors) of the invention may be codon optimized for expression. In some embodiments, the polynucleotides, nucleic acid constructs, expression cassettes and / or vectors of the editing systems of the invention (e.g., comprising / encoding sequence-specific nucleic acid binding domains (e.g., sequence-specific nucleic acid binding domains from: polynucleotide-guided endonucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), Argonaute proteins and / or CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins) The 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, type V CRISPR-Cas effector proteins, or type VI CRISPR-Cas effector proteins)), nucleases (e.g., endonucleases (e.g., Fok1), polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, and / or transcription activator-like effector nucleases (TALENs)), deaminase proteins / domains (e.g., adenine deaminase, cytosine deaminase), polynucleotides encoding reverse transcriptase proteins or domains, polynucleotides encoding 5'-3' exonuclease polypeptides, and / or affinity polypeptides, peptide tags, etc.) can be codon-optimized for expression in plants. In some embodiments, a codon-optimized nucleic acid, polynucleotide, expression cassette, and / or vector of the invention has about 70% to about 99.9% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100%) or more identity to a reference nucleic acid, polynucleotide, expression cassette, and / or vector that has not been codon-optimized.

[0155] In the embodiments described herein, the polynucleotides or nucleic acid constructs of the present invention may be operably associated with a variety of promoters and / or other regulatory elements for expression in plants and / or plant cells. Therefore, in some embodiments, the polynucleotides or nucleic acid constructs of the present invention may also include one or more promoters, introns, enhancers and / or terminators operably connected to one or more nucleotide sequences. In some embodiments, a promoter may be operably associated with an intron (e.g., an Ubil promoter and an intron). In some embodiments, a promoter associated with an intron may be referred to as a "promoter region" (e.g., an Ubil promoter and an intron).

[0156] As used herein, "operably linked" or "operably associated" in relation to a polynucleotide means that the indicated elements are functionally related to each other, and usually also physically related. Thus, as used herein, the term "operably linked" or "operably associated" refers to functionally associated nucleotide sequences on a single nucleic acid molecule. Thus, a first nucleotide sequence that is operably linked to a second nucleotide sequence refers to a situation where the first nucleotide sequence is in a functional relationship with the second nucleotide sequence. For example, if a promoter affects the transcription or expression of a nucleotide sequence, then the promoter is operably associated with the nucleotide sequence. Those skilled in the art will understand that a control sequence (e.g., a promoter) does not have to be adjacent to the nucleotide sequence to which it is operably associated, as long as the function of the control sequence is to direct its expression. Thus, for example, there may be an intervening untranslated but transcribed nucleic acid sequence between a promoter and a nucleotide sequence, and the promoter may still be considered to be "operably linked" to the nucleotide sequence.

[0157] As used herein, the term "linked" in relation to a polypeptide refers to the connection of one polypeptide to another polypeptide. A polypeptide can be linked to another polypeptide (at the N-terminus or C-terminus) directly (eg, via a peptide bond) or via a linker.

[0158] The term "joint" is recognized in the art and refers to a chemical group or molecule that connects two molecules or parts, such as two domains of a fusion protein, such as, for example, a nucleic acid binding polypeptide or domain and a peptide tag and / or a reverse transcriptase and an affinity polypeptide bound to the peptide tag; or a DNA endonuclease polypeptide or domain and a peptide tag and / or a reverse transcriptase and an affinity polypeptide bound to the peptide tag. The joint may consist of a single connecting molecule, or may contain more than one connecting molecule. In some embodiments, the joint may be an organic molecule, a group, a polymer, or a chemical moiety, such as a divalent organic moiety. In some embodiments, the joint may be an amino acid, or may be a peptide. In some embodiments, the joint is a peptide.

[0159] In some embodiments, the length of a peptide linker useful in the invention can be from about 2 to about 100 or more amino acids, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more amino acids (e.g., about 2 to about 40, about 2 to about 50, about 2 to about 60, about 4 to about 40, about 4 to about 50, about 4 to about 60, about 5 to about 40, about 5 to about 50, about 5 to about 60 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50,51,52,53,54,55,56,57,58,59,50 In some embodiments, the peptide linker can be a GS linker.

[0160] As used herein, the term "connection" or "fusion" related to polynucleotides refers to the connection of one polynucleotide to another polynucleotide. In some embodiments, two or more polynucleotide molecules can be connected by a joint, which can be an organic molecule, a group, a polymer or a chemical part, such as a divalent organic part. Polynucleotides can be connected or fused to another polynucleotide (at the 5' end or 3' end) by covalent or non-covalent bonds or in conjunction (including, for example, by Watson-Crick base pairing or by one or more connecting nucleotides). In some embodiments, a polynucleotide motif of a certain structure can be inserted into another polynucleotide sequence (for example, an extension of a hairpin structure in a guide RNA). In some embodiments, the connecting nucleotides can be naturally occurring nucleotides. In some embodiments, the connecting nucleotides can be non-naturally occurring nucleotides.

[0161] "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 a functional RNA. Generally, "promoter" refers to a nucleotide sequence that contains a binding site for RNA polymerase II and guides transcription initiation. In general, the promoter is located at 5' or upstream of the start of the coding region of the corresponding coding sequence. The promoter may include other elements that act as gene expression regulatory factors; for example, a promoter region. These include a TATA box consensus sequence, and generally also include a CAAT box consensus sequence (Breathnach and Chambon, (1981) Annu. Rev. Biochem. 50: 349). In plants, the CAAT box can be replaced by an AGGA box (Messing et al., (1983) in Genetic Engineering of Plants, T. Kosuge, C. Meredith and A. Hollaender (eds.), Plenum Press, pp. 211-227).

[0162] Promoters useful in the present invention may include, for example, constitutive, inducible, temporally regulated, developmentally regulated, chemically regulated, tissue-preferred and / or tissue-specific promoters for use in preparing recombinant nucleic acid molecules, for example, "synthetic nucleic acid constructs" or "protein-RNA complexes." These different types of promoters are known in the art.

[0163] The selection of promoters may vary depending on the temporal and spatial requirements of expression, and may also vary depending on the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge available in the art, appropriate promoters may be selected for a particular host organism of interest. Thus, for example, promoters upstream of genes that are highly constitutively expressed in model organisms are well known, and this knowledge may be easily obtained and implemented in other systems where appropriate.

[0164] In some embodiments, a promoter functional in plants can be used 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 the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr), and the promoter of the repeat carbonic anhydrase gene 1 (Pdca1) (see Walker et al., 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 7SL promoter from corn (Zea mays) can be used in the constructs of the present invention. In some embodiments, the U6c promoter and / or the 7SL promoter from corn can be used to drive expression of the guide nucleic acid. In some embodiments, the U6c promoter, U6i promoter and / or 7SL promoter from soybean (Glycine max) can be used in the constructs of the present invention. In some embodiments, the U6c promoter, U6i promoter and / or 7SL promoter from soybean can be used to drive expression of the guide nucleic acid.

[0165] Examples of constitutive promoters useful in plants include, but are not limited to, the cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), the rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; and U.S. Pat. No. 5,641,876), the CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), the CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), the nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA 84:5745-5749), the Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 85:5745-5749), the 84:6624-6629), sucrose synthase promoter (Yang and Russell (1990) Proc. Natl. Acad. Sci. USA 87:4144-4148) and ubiquitin promoter. Constitutive promoters derived from ubiquitin accumulate in many cell types. Ubiquitin promoters have been cloned from several plant species for use in transgenic plants, for example, sunflower (Binet et al., 1991. Plant Science 79: 87-94), corn (Christensen et al., 1989. Plant Molec. Biol. 12: 619-632) and Arabidopsis thaliana (Norris et al., 1993. Plant Molec. Biol. 21:895-906). The 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 sequence of the present invention in transgenic plants, especially monocots. In addition, the promoter expression cassette described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified for expression of the nucleotide sequence of the present invention, and is particularly suitable for monocot hosts.

[0166] In some 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 tissues include many promoters of genes involved in photosynthesis, many of which are cloned from monocots and dicots. In one embodiment, promoters useful for the present invention are corn PEPC promoters (Hudspeth and Grula, Plant Molec. Biol. 12:579-589 (1989)) from phosphoenol carboxylase genes. Non-limiting examples of tissue-specific promoters include those associated with genes encoding seed storage proteins (such as β-conglycinin, cruciferin, napin, and phaseolin), zein or oil body proteins (such as oleosin), or proteins involved in fatty acid biosynthesis (including acyl carrier protein, stearoyl-ACP desaturase, and fatty acid desaturase (fad 2-1), as well as other nucleic acids expressed during embryo development (such as Bce4, see, e.g., Kridl et al. (1991) Seed Sci. Res. 1:209-219; and EP Patent No. 255378). Tissue-specific or tissue-preferred promoters useful for expressing the nucleotide sequences of the present invention in plants, particularly corn, include, but are not limited to, those that direct expression in roots, pith, leaves, or pollen. Such promoters are disclosed, for example, in WO 93 / 07278 (incorporated herein by reference in its entirety).Other non-limiting examples of tissue-specific or tissue-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) that drives expression of the maize trpA gene; the tuberose yellow leaf curl virus promoter disclosed 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. Pat. No. 10,421,972), PLA from Arabidopsis thaliana. 2 -δ promoter (U.S. Pat. No. 7,141,424) and / or the ZmC5 promoter from maize (International PCT Publication No. WO 1999 / 042587).

[0167] 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 corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), the S-adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant and Cell Physiology, 37(8):1108-1115), the maize light harvesting complex promoter (Bansal et al. (1992) Proc. Natl. Acad. Sci. USA 89:3654-3658), the maize heat shock protein promoter (O'Dell et al. (1985) EMBO J. 5:451-458; and Rochester et al. (1986) EMBO J. 5:451-458), the pea small subunit RuBP carboxylase promoter (Cashmore, "Nuclear genes encoding the small subunit of ribulose-1,5-bisphosphate carboxylase" pp. 29-39, in: Genetic Engineering of Plants (Hollaender, ed., Plenum Press 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), Ti plasmid mannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), Ti plasmid nopaline synthase promoter (Langridge et al. (1989), supra), petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), leguminous glycine-rich protein 1 promoter (Keller et al. (1989) Genes Dev.3:1639-1646), a truncated CaMV 35S promoter (O'Dell et al. (1985) Nature 313:810-812), a potato glycoprotein promoter (Wenzler et al. (1989) Plant Mol. Biol. 13:347-354), a root cell promoter (Yamamoto et al. (1990) Nucleic Acids Res. 18:7449), a zein promoter (Kriz et al. (1987) Mol. Gen. Genet. 207:90-98; Langridge et al. (1983) Cell 34:1015-1022; Reina et al. (1990) Nucleic Acids Res. 18:6425; Reina et al. (1990) Nucleic Acids Res. 18:7449; and Wandelt et al. (1989) Nucleic Acids Res. 17:2354), globulin-1 promoter (Belanger et al. (1991) Genetics 129:863-872), α-tubulin cab promoter (Sullivan et al. (1989) Mol. Gen. Genet. 215:431-440), PEPCase promoter (Hudspeth and Grula (1989) Plant Mol. Biol. 12:579-589), R gene complex-associated promoter (Chandler et al. (1989) Plant Cell 1:1175-1183), and chalcone synthase promoter (Franken et al. (1991) EMBO J. 10:2605-2612).

[0168] Useful for seed-specific expression is the vicilin promoter (Czako et al. (1992) Mol. Gen. Genet. 235:33-40; and seed-specific promoters disclosed in U.S. Pat. No. 5,625,136). Useful promoters for expression in mature leaves are those that switch at the onset of senescence, such as the SAG promoter from Arabidopsis thaliana (Gan et al. (1995) Science 270:1986-1988).

[0169] In addition, promoters that are functional in chloroplasts can be used. Non-limiting examples of such promoters include phage T3 gene 9 5'UTR and other promoters disclosed in U.S. Patent No. 7,579,516. Other promoters that can be used in the present invention include, but are not limited to, the S-E9 small subunit RuBP carboxylase promoter and the Kunitz trypsin inhibitor gene promoter (Kti3).

[0170] 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.

[0171] Introns that can be used in the present invention can be identified in plants and separated from plants, and then inserted into introns in expression cassettes for plant transformation. As will be appreciated by those skilled in the art, introns can include sequences required for self-excision and are incorporated into nucleic acid constructs / expression cassettes in frame. Introns can be used as spacers to separate multiple protein coding sequences in a nucleic acid construct, or introns can be used in a protein coding sequence, for example, to stabilize mRNA. If they are used in a protein coding sequence, they are inserted "in frame" and include excision sites. Introns can also be associated with promoters to improve or modify expression. As an example, promoter / intron combinations that can be used in the present invention include but are not limited to promoter / intron combinations of corn Ubil promoters and introns (see, for example, SEQ ID NO:21 and SEQ ID NO:22).

[0172] 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.

[0173] In some embodiments, the polynucleotides and / or nucleic acid constructs of the present invention may be "expression cassettes", or may be contained within expression cassettes. As used herein, "expression cassettes" refer to recombinant nucleic acid molecules comprising, for example, one or more polynucleotides of the present invention (e.g., polynucleotides encoding sequence-specific nucleic acid (e.g., DNA) binding domains, polynucleotides encoding deaminase proteins or domains, polynucleotides encoding reverse transcriptase proteins or domains, polynucleotides encoding 5'-3' exonuclease polypeptides or domains, guide nucleic acids and / or reverse transcriptase (RT) templates), wherein the polynucleotides are operably associated with one or more control sequences (e.g., promoters, terminators, etc.). Thus, in some embodiments, one or more expression cassettes may be provided, which are designed to express, for example, a nucleic acid construct of the invention (e.g., a polynucleotide encoding a sequence-specific nucleic acid binding domain, a polynucleotide encoding a nuclease polypeptide / domain, a polynucleotide encoding a deaminase protein / domain, a polynucleotide encoding a reverse transcriptase protein / domain, a polynucleotide encoding a 5'-3' exonuclease polypeptide / domain, a polynucleotide encoding a peptide tag, and / or a polynucleotide encoding an affinity polypeptide, etc., or comprising a guide nucleic acid, an extended guide nucleic acid, and / or an RT template, etc.). When the expression cassette of the invention comprises more than one polynucleotide, the polynucleotide may be operably linked to a single promoter that drives expression of all polynucleotides, or the polynucleotide may be operably linked to one or more separate promoters (e.g., three polynucleotides may be driven by one, two, or three promoters in any combination). When two or more separate promoters are used, the promoters may be the same promoter, or they may be different promoters. Thus, when contained in a single expression cassette, a polynucleotide encoding a sequence-specific nucleic acid binding domain, a polynucleotide encoding a nuclease protein / domain, a polynucleotide encoding a CRISPR-Cas effector protein / domain, a polynucleotide encoding a deaminase protein / domain, a polynucleotide encoding a reverse transcriptase polypeptide / domain (e.g., an RNA-dependent DNA polymerase) and / or a polynucleotide encoding a 5'-3' exonuclease polypeptide / domain, a guide nucleic acid, an extended guide nucleic acid and / or an RT template can each be operably linked to a single promoter or separate promoters in any combination.

[0174] The expression cassette comprising the nucleic acid construct of the invention may be chimeric, meaning that at least one of its components is heterologous relative to at least one of its other components (e.g., a promoter from a host organism is operably linked to a polynucleotide of interest to be expressed in the host organism, wherein the polynucleotide of interest is from an organism different from the host, or is not normally associated with the promoter). The expression cassette may also be naturally occurring, but has been obtained in a recombinant form that can be used for heterologous expression.

[0175] The expression cassette may optionally include a transcription and / or translation termination region (i.e., termination region) and / or an enhancer region that is functional in a selected host cell. A variety of transcription terminators and enhancers are known in the art and can be used in expression cassettes. Transcription terminators are responsible for terminating transcription and correcting mRNA polyadenylation. Termination regions and / or enhancer regions may be natural to the transcription initiation region, may be natural to the following: for example, a gene encoding a sequence-specific nucleic acid binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase, etc., or may be natural to a host cell, or may be natural to another source (e.g., to a promoter, a gene encoding a sequence-specific nucleic acid binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase, etc., or to a host cell, or any combination thereof, exogenous or heterologous).

[0176] The expression cassette of the present invention may also include a polynucleotide encoding a selectable marker that can be used to select transformed host cells. As used herein, a "selectable marker" refers to a polynucleotide sequence that, when expressed, imparts a unique phenotype to the host cell expressing the marker, thereby allowing such transformed cells to be distinguished from cells without the marker. This polynucleotide sequence can encode a selectable or screenable marker, depending on whether the marker imparts a trait that can be selected by chemical means, such as by using a selection agent (e.g., antibiotics, etc.), or whether the marker is simply a trait that can be identified by observation or testing, such as by screening (e.g., fluorescence). Many examples of suitable selectable markers are known in the art and can be used in expression cassettes described herein.

[0177] In addition to expression cassettes, nucleic acid molecules / constructs and polynucleotide sequences described herein can also be used in combination with vectors. The term "vector" refers to a composition for transferring, delivering or introducing nucleic acids (or multiple nucleic acids) into a cell. Vectors include nucleic acid constructs (e.g., expression cassettes) comprising nucleotide sequences to be transferred, delivered or introduced. Vectors for transforming host organisms are well known in the art. Non-limiting examples of general class vectors include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, phages, artificial chromosomes, minicircles or double-stranded or single-stranded linear or circular forms of Agrobacterium binary vectors, which may or may not be self-transferable or removable. In some embodiments, viral vectors may include, but are not limited to, retroviruses, slow viruses, adenoviruses, adeno-associated viruses or herpes simplex virus vectors. Vectors as defined herein can be transformed prokaryotic or eukaryotic hosts by being integrated into the cell genome or being present outside the chromosome (e.g., autonomously replicating plasmids with a replication origin). In addition, shuttle vectors are also included, and shuttle vectors refer to DNA vectors that can be naturally or intentionally replicated in two different host organisms, and host organisms can be selected from actinomycetes and related species, bacteria and eukaryotic organisms (for example, higher plants, mammals, yeast or fungal cells). In some embodiments, the nucleic acid in the vector is controlled by a suitable promoter or other regulatory elements, and is operably connected to a suitable promoter or other regulatory elements so as to be transcribed in a host cell. The vector can be a bifunctional expression vector that works in a variety of hosts. In the case of genomic DNA, this can include its own promoter and / or other regulatory elements, and in the case of cDNA, this can be controlled by a suitable promoter and / or other regulatory elements so as to be expressed in a host cell. Therefore, the nucleic acid or polynucleotide of the present invention and / or the expression cassette comprising the nucleic acid or polynucleotide can be included in a vector as described herein and known in the art.

[0178] As used herein, "contact," "contacting," "contacted," and grammatical variations thereof refer to bringing together the components of a desired reaction under conditions suitable for carrying out the desired reaction (e.g., transformation, transcriptional control, genome editing, nicking and / or cleavage). As an example, a target nucleic acid can be contacted with a sequence-specific nucleic acid binding protein (e.g., a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., a CRISPR-Cas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN) and / or an Argonaute protein)) and a deaminase or a nucleic acid construct encoding these under the following conditions: the sequence-specific nucleic acid binding protein, the reverse transcriptase and / or the deaminase are expressed, the sequence-specific nucleic acid binding protein binds to the target nucleic acid, and the reverse transcriptase and / or the deaminase can be fused to the sequence-specific nucleic acid binding protein or recruited to the sequence-specific nucleic acid binding protein (e.g., by a peptide tag fused to the sequence-specific nucleic acid binding protein and an affinity tag fused to the reverse transcriptase and / or the deaminase), so that the deaminase and / or the reverse transcriptase are located near the target nucleic acid, thereby modifying the target nucleic acid. Other methods of recruiting reverse transcriptases and / or deaminases using other protein-protein interactions can be used, and RNA-protein interactions and chemical interactions can also be used for protein-protein and protein-nucleic acid recruitment.

[0179] As used herein, "modification" ("modifying" or "modification") of a target nucleic acid includes editing (e.g., mutation), covalent modification, exchanging / replacing nucleic acid / nucleotide bases, deletion, cutting, nicking, and / or altering transcriptional control of a target nucleic acid. In some embodiments, the modification may include any type of one or more single base changes (SNPs).

[0180] "Introducing", "introduce", "introduced" (and grammatical variations thereof) in the context of a polynucleotide of interest refers to presenting a nucleotide sequence of interest (e.g., a polynucleotide, RT template, nucleic acid construct and / or guide nucleic acid) to a plant, a plant part thereof, or a cell thereof in a manner that enables the nucleotide sequence to enter the interior of the cell.

[0181] The terms "transformation" or "transfection" are used interchangeably and as used herein refer to the introduction of a heterologous nucleic acid into a cell. The transformation of a cell can be stable or transient. Thus, in some embodiments, a host cell or host organism (e.g., a plant) can be stably transformed with a polynucleotide / nucleic acid molecule of the present invention. In some embodiments, a host cell or host organism can be transiently transformed with a polynucleotide / nucleic acid molecule of the present invention.

[0182] "Transient transformation" in the context of a polynucleotide means that the polynucleotide is introduced into a cell but is not integrated into the genome of the cell.

[0183] "Stably introduced" or "stably introduced" in the context of a polynucleotide being introduced into a cell means that the introduced polynucleotide is stably incorporated into the genome of the cell, so that the cell is stably transformed with the polynucleotide.

[0184] As used herein, "stable transformation" or "stably transformed" refers to a nucleic acid molecule that is introduced into a cell and integrated into the genome of the cell. Thus, the integrated nucleic acid molecule is capable of being inherited by its progeny, more specifically, by successive generations of progeny. As used herein, "genome" includes nuclear and plastid genomes, and thus includes integration of nucleic acids into, for example, chloroplast or mitochondrial genomes. As used herein, stable transformation may also refer to a transgene that is maintained extrachromosomally, for example, as a minichromosome or plasmid.

[0185] 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 of the genomic DNA of the cells with nucleic acid sequences that specifically hybridize with nucleotide sequences of transgenics introduced into an organism (e.g., a plant). Stable transformation of cells can be detected by, for example, Northern blot hybridization assays of the RNA of the cells with nucleic acid sequences that specifically hybridize with nucleotide sequences of transgenics introduced into a host organism. Stable transformation of cells can also be detected by, for example, polymerase chain reaction (PCR) or other amplification reactions well known in the art, which employ specific primer sequences that hybridize with target sequences of transgenics, resulting in amplification of transgenic sequences, which can be detected according to standard methods. Transformation can also be detected by direct sequencing and / or hybridization protocols well known in the art.

[0186] Thus, in some embodiments, the nucleotide sequences, polynucleotides, nucleic acid constructs and / or expression cassettes of the invention can be transiently expressed and / or they can be stably incorporated into the genome of a host organism. Thus, in some embodiments, the nucleic acid constructs of the invention (e.g., one or more expression cassettes comprising polynucleotides for editing as described herein) can be transiently introduced into a cell along with a guide nucleic acid, and therefore, DNA is not maintained in the cell.

[0187] The nucleic acid construct of the present invention can be introduced into corn plant cells by any method known to those skilled in the art. The non-limiting examples of transformation methods include conversion by bacterial-mediated nucleic acid delivery (for example, by Agrobacterium), virus-mediated nucleic acid delivery, silicon carbide or nucleic acid whisker-mediated nucleic acid delivery, liposome-mediated nucleic acid delivery, microinjection, microparticle bombardment, calcium phosphate-mediated conversion, cyclodextrin-mediated conversion, electroporation, nanoparticle-mediated conversion, ultrasonic treatment, infiltration, PEG-mediated nucleic acid uptake, and any other electrical, chemical, physical (mechanical) and / or biological mechanism that nucleic acid is introduced into 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. Nat. Biotechnol. 31: 233-239; Ran et al., 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)).

[0188] In some embodiments of the present invention, the transformation of cells may include nuclear transformation. In other embodiments, the transformation of cells may include plastid transformation (e.g., chloroplast transformation). In further embodiments, nucleic acids of the present invention may be introduced into cells by conventional breeding techniques. In some embodiments, one or more of polynucleotides, expression cassettes and / or vectors may be introduced into plant cells by Agrobacterium transformation.

[0189] Therefore, polynucleotide can be introduced into plant, plant part, plant cell in any number of ways known in the art.Method of the present invention does not rely on the ad hoc method for introducing one or more nucleotide sequences into plant, as long as they can enter the interior of the cell.If more than one polynucleotide is to be introduced, they can be assembled as a part of a single nucleic acid construct or assembled as an independent nucleic acid construct, and can be located on the same or different nucleic acid constructs.Therefore, polynucleotide can be introduced into interested cell in a single transformation event, or introduced into interested cell in an independent transformation event, or alternatively, polynucleotide can be incorporated into plant as a part of a breeding scheme.

[0190] The present invention provides methods and compositions for reducing the effects of genes that normally act to limit meristem size in order to produce plants with larger floral meristems, thereby increasing kernel rows (optionally without substantially reducing ear length (e.g., ear length is reduced by no more than 30% compared to ears of corn plants that do not contain the same FEA2 mutation)) and yield (e.g., increasing seed number).

[0191] CLV1, CLV2 and CLV3 genes are part of several overlapping signal transduction pathways that utilize similar mechanisms to regulate meristem size. Plants express many CLV3-like (CLE) peptides that are sensed by many leucine-rich repeat (LRR) domains, and downstream signal transduction regulates numerous growth processes in plants (Fletcher, JC, Plants 7:87 (2018)). The CLV2 ortholog in corn is FASCIATED EAR2 (FEA2). Similar to FEA2, other CLV orthologs can be edited using the strategies described herein to improve yield traits in a variety of plant species.

[0192] Thus, the plants of the present invention may comprise one or more improved yield traits, which may include, but are not limited to, increased floral meristem size and / or increased kernel row number (KRN), optionally wherein the increase in KRN does not result in a significant reduction in ear length. Plants with increased kernel row number may also exhibit increased yield.

[0193] As used herein, "increased yield" means an increase in yield of about 15% to about 100% (e.g., about 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 97%, 98%, 99%, or 100%, or any range or value therein). The increase in yield can be measured, for example, by the number, size, or weight of seeds or grains.

[0194] As used herein, "larger floral meristem" or "floral meristem of increased size" refers to an increase in size by about 1% to about 20% (e.g., about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, or any range or value therein).

[0195] As used herein, an "increase in KRN" refers to an increase in KRN by about 5% to about 35% (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%, or 35% or any range or value therein) as compared to control plants.

[0196] As used herein, "an increase in kernel row number (KRN)" means an increase in the number of rows in an ear by at least about 1 to about 10 (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 or more), optionally wherein ear length is not substantially decreased, as compared to a control plant lacking a mutation as described herein. By “increase in KRN” is meant an increase in KRN by about 5% to about 50% (e.g., about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%, or any range or value therein; for example, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, and any range or value therein) as compared to a control plant lacking a mutated endogenous FEA2 gene.

[0197] As used herein, the phrase “ear length is not substantially reduced” may mean that the ear length is reduced by no more than 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% or 30%, or any range or value therein, as compared to the ear of a plant lacking the same mutation. In some embodiments, the ear with increased KRN can have an ear length that can be at least about 70% or more (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%, or 100% or any range or value therein) of the ear length of a plant lacking the same mutation.

[0198] Therefore, as described herein, editing techniques are used to target the FEA2 gene in plants to produce plants with larger floral meristems, increased seed rows and / or increased yields. Mutations that can be used to produce plants showing increased seed rows include, for example, substitutions, deletions, and insertions. In some aspects, the mutations produced by the editing techniques can be point mutations, dominant negative mutations, semi-dominant mutations, or weak loss-of-function mutations.

[0199] In some embodiments, the present invention provides a corn plant or plant part thereof, comprising at least one mutation (e.g., 1, 2, 3, 4, or 5 or more mutations) in an endogenous FASCIATED EAR2 (FEA2) gene encoding a FEA2 protein. In some embodiments, the endogenous FASCIATED EAR2 (FEA2) gene has a gene identification number (gene ID) of Zm00001d051012. In some embodiments, the at least one mutation may result in a dominant negative mutation and / or a weak loss-of-function mutation. In some embodiments, the at least one mutation may be a non-natural mutation.

[0200] The mutation in the FEA2 gene of corn plant, its plant part or corn plant cell can be any type of mutation, including displacement, deletion and / or insertion. In some embodiments, the mutation can be a non-natural mutation. In some embodiments, the mutation can include base substitutions to A, T, G or C. In some embodiments, the mutation can be a deletion of at least one base pair (e.g., 1 base pair to about 50 base pairs) or an insertion of at least one base pair (e.g., 1 base pair to about 50 base pairs). In some embodiments, the deletion can include 1 base pair to about 10 consecutive base pairs (e.g., 1 bp, 2bp to about 3bp, 4bp, 5bp, 6bp, 7bp, 8bp, 9bp, or 10bp; for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 consecutive base pairs), 1 base pair to about 20 consecutive base pairs (e.g., 1, 2, 3, 4, 56, 7, 8, 9, 101, 12, 13, 14, 15, 16, 17, 18, 19, or 20 consecutive base pairs; for example, 1 bp, 2bp, 3bp, 4bp, 5bp, 6bp to about 7, 8, 9, 10, 11, 12, 13, 14, 15, 47, 48, 49, 50 consecutive base pairs or more, or any value or range therein.

[0201] In some embodiments, the at least one mutation may be an out-of-frame deletion or insertion or an in-frame deletion or insertion, optionally resulting in a dominant negative mutation and / or a weak loss-of-function mutation. In some embodiments, the at least one mutation may be an out-of-frame deletion or an out-of-frame insertion resulting in a dominant negative mutation and / or a weak loss-of-function mutation. In some embodiments, the at least one mutation may be an in-frame deletion or an in-frame insertion resulting in a dominant negative mutation and / or a weak loss-of-function mutation. In some embodiments, the at least one mutation may be located in the 10th LRR repeat sequence of the encoded FEA2 polypeptide. In some embodiments, the at least one mutation may result in a modified, truncated FEA2 polypeptide. In some embodiments, the at least one mutation may be a base substitution resulting in one or more amino acid substitutions. In some embodiments, the at least one mutation may result in a substituted amino acid residue at or near position 332 (with reference to the amino acid position numbering of SEQ ID NO: 74) of the encoded FEA2 polypeptide, optionally a substitution of G332. As used herein, "around position 332" means that the at least one mutation (e.g., substituted amino acid residue) can occur within about 15 consecutive amino acids of position 332 (towards the N-terminus or toward the C-terminus) with reference to the amino acid position numbering of SEQ ID NO: 74, for example, from about residue position 318 to about residue position 348; for example, with reference to SEQ ID NO: 74. 317, 318, 319, 320, 321, 322, 323, 324, 325, 326, 327, 328, 329, 330, 331, 332, 333, 334, 335, 336, 337, 338, 339, 340, 341, 342, 343, 345, 346, 347 and / or 348 of amino acid position numbering of NO:74.

[0202] The endogenous FEA2 gene that can be used in the present invention may (a) comprise a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:72 or SEQ ID NO:73, (b) comprise a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-77, (c) encode an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74, and / or (d) encode a polypeptide comprising a region having at least 90% sequence identity to any one of SEQ ID NOs:78-80, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 95%, optionally the sequence identity may be 100%. Mutations in the endogenous FEA2 gene can result in a mutated FEA2 polypeptide, optionally wherein the mutated FEA2 protein can exhibit an altered ability to complex with other polypeptides, optionally wherein the mutated FEA2 protein can exhibit an altered ability to cross the plasma membrane (e.g., when complexed with a protein such as CRN, the modified FEA2 as described herein has a reduced ability to localize to the plasma membrane, thereby trapping FEA2 in the ER). In some embodiments, the mutated FEA2 gene can comprise a nucleotide sequence having at least about 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, or 98, optionally wherein the sequence identity can be at least 95%, optionally the sequence identity can be 100%.

[0203] In some embodiments, corn plants comprising at least one mutation (e.g., a non-natural mutation) in an endogenous FEA2 gene may exhibit increased maintenance of meristems and / or increased number of kernel rows, optionally with substantially no reduction in ear length (e.g., a reduction of less than 30%, optionally with an ear length of at least 70% of the control ear), as compared to a control corn plant without the at least one mutation (e.g., a non-natural mutation). In some embodiments, corn plants comprising at least one mutation in an endogenous FEA2 gene exhibit increased number of kernel rows. In some embodiments, corn plants comprising at least one mutation in an endogenous FEA2 gene may exhibit increased yield. In some embodiments, a corn plant can be regenerated from a corn plant part and / or corn plant cell of the present invention, wherein the regenerated corn plant comprises a mutated endogenous FEA2 gene and exhibits a phenotype of increased number of kernel rows as compared to a control corn plant that does not comprise the mutation, optionally wherein the length of the ear with increased number of kernel rows is not substantially reduced (e.g., as compared to an ear of a corn plant that does not comprise the same FEA2 mutation, exhibiting no more than a 30% reduction in ear length, optionally an ear length that is at least 70% of the ear of a control plant), optionally the regenerated corn plant also exhibits increased yield.

[0204] In some embodiments, a corn plant cell is provided, the corn plant cell comprising an editing system, the editing system comprising: (a) CRISPR-Cas effector protein; and (b) a guide nucleic acid (gRNA, gDNA, crRNA, crDNA, sgRNA, sgDNA) comprising a spacer sequence complementary to an endogenous target gene encoding a FEA2 protein. The editing system can be used to generate mutations in an endogenous target gene encoding a FEA2 protein. In some embodiments, the mutation is a non-natural mutation. In some embodiments, the guide nucleic acid of the editing system may comprise the nucleotide sequence (spacer sequence) of SEQ ID NO: 81.

[0205] In some embodiments, a corn plant cell is provided, the corn plant cell comprising at least one mutation in the FEA2 gene, wherein the mutation is a displacement, insertion or deletion introduced using an editing system, the editing system comprising a nucleic acid binding domain that binds to a target site in the FEA2 gene. In some embodiments, the displacement, insertion or deletion in the FEA2 gene results in a dominant negative allele or a weak loss of function allele. In some embodiments, the mutation can be a non-natural mutation. In some embodiments, the mutation can be a point mutation. In some embodiments, the target site is within a region of the FEA2 gene that comprises a sequence having at least 90% sequence identity (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity) to the nucleotide sequence of any one of SEQ ID NOs:75-77, optionally wherein the sequence identity may be at least 95% or it may be 100%, and / or encodes a sequence having at least 90% sequence identity (e.g., about 95%, 96%, 97%, 98%, 99%, 99.5%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% sequence identity) to the amino acid sequence of any one of SEQ ID NOs:78-80, optionally wherein the sequence identity may be at least 95% or it may be 100%. In some embodiments, the editing system further comprises a nuclease, the nucleic acid binding domain binds to a target site within a sequence having at least 90% sequence identity with the nucleotide sequence of any one of SEQ ID NOs: 75-77, optionally wherein the sequence identity can be at least 95% or it can be 100%, and generates at least one mutation within the FEA2 gene after being cleaved by the nuclease. In some embodiments, the endogenous FEA2 gene comprises a sequence having at least 90% sequence identity with the nucleotide sequence of SEQ ID NO: 72 or SEQ ID NO: 73, optionally wherein the sequence identity can be at least 95% or it can be 100%, or encodes a sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 74, optionally wherein the sequence identity can be at least 95% or it can be 100%. In some embodiments, the mutation results in an amino acid substitution in the encoded polypeptide. In some embodiments, the mutation is an out-of-frame insertion or out-of-frame deletion resulting in a mutated FEA2 polypeptide, optionally wherein the mutation in the FEA2 polypeptide is located in the 10th LRR.

[0206] In some embodiments, at least one mutation within the FEA2 gene in a corn plant cell can result in an altered ability of the encoded FEA2 polypeptide to complex with other polypeptides and / or to translocate from the endoplasmic reticulum to the plasma membrane after forming a protein complex with other proteins such as a pseudokinase (e.g., CORYNE (CRN)) and / or the heterotrimeric G protein COMPACT PLANT2 (e.g., a reduced ability of the FEA2 complex to interact with the export machinery to transfer from the ER to the plasma membrane).

[0207] In some embodiments, the mutated FEA2 gene contained in the corn plant cell may have at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%.

[0208] In some embodiments, a corn plant cell or corn plant part comprising a mutated FEA2 gene as described herein can be regenerated into a corn plant comprising a mutated FEA2 gene (optionally comprising a mutated FEA2 polypeptide). In some embodiments, a corn plant part or corn plant cell comprising a mutated FEA2 gene as described herein is not regenerated into a corn plant.

[0209] In some embodiments, a method for producing / cultivating an edited corn plant that is free of transgenes is provided, the method comprising: crossing a corn plant of the present invention (e.g., a corn plant comprising a mutation in the FEA2 gene and having an increased number of kernel rows, optionally without substantially reduced ear length (e.g., a reduction of less than 30%)) with a corn plant that is free of transgenes, thereby introducing at least one mutation into the corn plant that is free of transgenes; and selecting offspring corn plants that comprise at least one mutation and are free of transgenes, thereby producing an edited corn plant that is free of transgenes.

[0210] In some embodiments, a method of providing a plurality of corn plants having an increased number of kernel rows is provided, the method comprising planting two or more corn 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 corn plants comprising a mutation in the FEA2 polypeptide and having an increased number of kernel rows, optionally with substantially no reduction in ear length (e.g., a reduction of less than 30%)) in a growing area (e.g., a field (e.g., a cultivated field, an agricultural field), a growth chamber, a greenhouse, a recreational area, a lawn, and / or a roadside, etc.), thereby providing a plurality of corn plants having an increased number of kernel rows compared to a plurality of control corn plants that do not comprise the mutation. In some embodiments, the plurality of corn plants may also exhibit larger floral meristems and / or increased yield.

[0211] In some embodiments, a method for generating a variation in a region of a corn FEA2 polypeptide is provided, the method comprising introducing an editing system into a corn plant cell, wherein the editing system targets a region of a FEA2 gene encoding a FEA2 polypeptide, and contacting a region of the FEA2 gene with the editing system, thereby introducing a mutation into the FEA2 gene and generating a variation in the region of the corn FEA2 polypeptide in the corn plant cell. In some embodiments, the FEA2 gene: (a) comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:72 or SEQ ID NO:73, (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-77, (c) encodes an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74, and / or (d) encodes a polypeptide comprising a region having at least 90% sequence identity to any one of SEQ ID NOs:78-80, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, the region of the FEA2 gene that is targeted has at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-77, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, a region of an endogenous FEA2 gene in a corn plant cell is contacted with an editing system to produce a corn plant cell comprising an edited endogenous FEA2 gene in its genome, and the method further comprises (a) regenerating a corn plant from the corn plant cell; (b) selfing the corn plant to produce a corn offspring plant (E1); (c) assaying the offspring plant of (b) for increased yield and / or increased number of kernel rows (optionally wherein ear length does not substantially decrease when kernel rows increase); and (d) selecting offspring corn plants that exhibit increased yield and / or increased number of kernel rows (optionally wherein ear length does not substantially decrease when kernel rows increase) to produce selected offspring corn plants that exhibit increased yield and / or increased number of kernel rows (optionally wherein ear length does not substantially decrease when kernel rows increase) compared to a control corn plant.In some embodiments, the method may also include (e) selfing the selected offspring corn plant in (d) to produce an offspring corn plant (E2); (f) determining the offspring corn plant of (e) for increased yield and / or increased number of rows of kernels (optionally wherein ear length does not substantially decrease when the number of rows of kernels increases); and (g) selecting offspring corn plants that exhibit increased yield and / or increased number of rows of kernels (optionally wherein ear length does not substantially decrease when the number of rows of kernels increases) compared to control corn plants to produce selected offspring corn plants that exhibit increased yield and / or increased number of rows of kernels (optionally wherein ear length does not substantially decrease when the number of rows of kernels increases), optionally repeating (e) to (g) one or more times.

[0212] In some embodiments, the present invention provides a method for detecting a mutant FEA2 gene in a corn plant (detecting a mutation in an endogenous FEA2 gene), the method comprising detecting in the genome of a corn plant a FEA2 gene having at least one mutation in a region having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NO: 75-77, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, the detected mutant FEA2 gene may comprise a mutant nucleotide sequence as described herein.

[0213] In some embodiments, a method for editing a specific site in the genome of a corn plant cell is provided, the method comprising: cleaving a target site within an endogenous FEA2 gene in a corn plant cell in a site-specific manner, the endogenous FEA2 gene: (a) comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-77; (c) encoding an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74; and / or (d) encoding a polypeptide comprising a region having at least 90% sequence identity to any one of SEQ ID NOs:78-80, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 95%, optionally the sequence identity can be 100%, thereby generating an edit in the endogenous FEA2 gene of the corn plant cell, and generating a corn plant cell comprising an edit in the endogenous FEA2 gene. Endogenous FEA2 gene encodes FEA2 protein, and editing causes amino acid variation in the coding region of FEA2 protein, for example, editing causes amino acid replacement in the encoded FEA2 polypeptide. In some embodiments, editing causes deletion, replacement or insertion, optionally wherein the deletion, replacement or insertion can be a dominant negative allele or a weak loss-of-function allele. In some embodiments, editing causes mutation, which is an out-of-frame deletion or out-of-frame insertion. In some embodiments, the target site comprises a sequence having at least 90% sequence identity with the nucleotide sequence of any one of SEQ ID NO:75-77, optionally wherein the sequence identity can be at least 95%, optionally the sequence identity can be 100%, or comprises a sequence encoding an amino acid sequence having at least 90% sequence identity with any one of SEQ ID NO:78-80, optionally wherein the sequence identity can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, with reference to the amino acid position numbering of SEQ ID NO:74, editing causes amino acid replacement at residue 332, optionally wherein the editing causes replacement of G332. In some embodiments, the edit is at or near residue 332, optionally wherein the edit results in an out-of-frame deletion or out-of-frame insertion, with reference to the amino acid position numbering of SEQ ID NO: 74. In some embodiments, the mutation alters the ability of the FEA2 polypeptide to complex with other proteins and / or alters the ability of the FEA2 polypeptide to translocate from the endoplasmic reticulum (ER) to the plasma membrane when the FEA2 polypeptide is complexed with another protein such as the pseudokinase CORYNE (CRN) (e.g., FEA2 is trapped in the ER when complexed with a polypeptide such as CRN).In some embodiments, the editing results in a non-natural mutation, optionally a mutated FEA2 gene, comprising a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, optionally wherein the sequence identity can be at least 95%, optionally the sequence identity can be 100%.

[0214] In some embodiments, the editing method may further include regenerating a corn plant from a corn plant cell comprising an edit in an endogenous FEA2 gene, thereby producing a corn plant comprising an edit in its endogenous FEA2 gene and having a phenotype of increased number of kernel rows (e.g., producing one or more ears with increased number of kernel rows) when compared to a control corn plant that does not comprise the edit, optionally wherein the length of the one or more ears with increased number of kernel rows is not substantially reduced, and optionally wherein the corn plant exhibits increased yield when compared to a control corn plant that does not comprise the edit.

[0215] In some embodiments, a method for preparing a corn plant is provided, the method comprising: (a) contacting a population of corn plant cells comprising an endogenous FEA2 gene with a nuclease linked to a nucleic acid binding domain (e.g., a DNA binding domain, such as an editing system) that binds to a sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-77 (optionally, wherein the sequence identity can be at least 95%, or it can be 100%) or to a sequence region encoding an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 78-80 (optionally, wherein the sequence identity can be at least 95%, or it can be 100%); (b) selecting corn plant cells from the population in which the endogenous FEA2 gene has been mutated, thereby producing corn plant cells comprising at least one mutation in the endogenous FEA2 gene; and (c) growing the selected corn plant cells into corn plants.

[0216] In some embodiments, a method of increasing the number of kernel rows in a corn plant, optionally without reducing ear length, is provided, the method comprising (a) contacting a corn plant cell comprising an endogenous FEA2 gene with a nuclease targeted to the endogenous FEA2 gene, wherein the nuclease is linked to a nucleic acid binding domain (e.g., an editing system) that binds to a target site in the endogenous FEA2 gene, wherein the endogenous FEA2 gene: (i) comprises a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (ii) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO: 74; (iii) comprises a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-11770; and / or (iv) encodes a region having at least 90% sequence identity to the nucleotide sequence of SEQ ID NOs: NO:78-80 has a polypeptide region with a region having at least 90% sequence identity, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) can be at least 95%, optionally the sequence identity can be 100%, to produce a corn plant cell comprising a mutation in an endogenous FEA2 gene; and (b) growing the corn plant cell into a corn plant comprising a mutation in an endogenous FEA2 gene, thereby producing a corn plant having a mutated endogenous FEA2 gene and exhibiting increased yield and / or increased number of kernel rows, optionally exhibiting increased number of kernel rows while significantly reducing ear length, optionally wherein the ear length is not substantially reduced (e.g., reduced by less than 30%).

[0217] In some embodiments, a method for producing a corn plant or part thereof comprising at least one cell having a mutated endogenous FEA2 gene is provided, the method comprising contacting a target site within an endogenous FEA2 gene in a corn plant or plant part with a nuclease comprising a cleavage domain and a nucleic acid binding domain (e.g., a DNA binding domain), wherein the nucleic acid binding domain binds to the target site in the endogenous FEA2 gene, wherein the endogenous FEA2 gene (a) comprises a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:74; (c) comprises a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:75-77; and / or (d) encodes a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:75-77; and / or A polypeptide region having an amino acid sequence of any one of NO:78-80 having a region with at least 90% sequence identity, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 95%, optionally the sequence identity can be 100%, thereby producing a corn plant or part thereof comprising at least one cell having a mutation in an endogenous FEA2 gene.

[0218] Also provided herein is a method for producing a corn plant or part thereof comprising a mutated endogenous FEA2 gene and exhibiting increased kernel row number, increased yield, and larger floral meristem, the method comprising contacting a target site in an endogenous FEA2 gene in a corn plant or plant part with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to the target site in the endogenous FEA2 gene, wherein the endogenous FEA2 gene: (a) comprises a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:74; (c) comprises a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:75-77; and / or (d) encodes a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs:75-77; NO:78-80 any one of the amino acid sequences has a polypeptide region of the region of at least 90% sequence identity, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 95%, optionally the sequence identity can be 100%, thereby producing an endogenous FEA2 gene comprising a mutation and showing an increased number of kernel rows (e.g., producing one or more ears with increased kernel rows) compared to a control corn plant not comprising the mutation, or a part thereof, optionally wherein the length of the one or more ears with increased kernel rows is not substantially reduced (e.g., reduced by less than 30%). In some embodiments, the corn plant may also show increased yield compared to a control corn plant not comprising the mutation.

[0219] In some embodiments, the nuclease contacting the corn plant cell, the corn plant cell population and / or the target site cuts the endogenous FEA2 gene, thereby introducing the mutation into the endogenous FEA2 gene. In some embodiments, the nuclease that can be used for the present invention can cut the endogenous FEA2 gene, thereby introducing the mutation into the endogenous FEA2 gene. In some embodiments, the nuclease can include but is not limited to zinc finger nucleases, transcription activator-like effector nucleases (TALEN), endonucleases (e.g., Fok1) and / or CRISPR-Cas effector proteins. Similarly, the nucleic acid binding domains (e.g., DNA binding domains, RNA binding domains) that can be used for the present invention can be any nucleic acid binding domain that can be used to edit / modify the target nucleic acid. Such nucleic acid binding domains include but are not limited to zinc fingers, transcription activator-like DNA binding domains (TAL), argonaute and / or CRISPR-Cas effector DNA binding domains.

[0220] In some embodiments, a corn plant or part thereof comprising at least one cell having a mutation in an endogenous FEA2 gene as described herein comprises a sequence having at least 90% identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, and / or encodes a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97 or 99, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%.

[0221] In some embodiments, a method for modifying an endogenous FEA2 gene in a corn plant or part thereof to improve the yield trait of the corn plant or part thereof is provided, the method comprising altering a target site within an endogenous FEA2 gene in a corn plant or part thereof, wherein the endogenous FEA2 gene: (a) comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-77; (c) encodes a polypeptide comprising a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NOs:74; and / or (d) encodes a region having at least 90% sequence identity to the amino acid sequence of SEQ ID NOs:78-80, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 95%, optionally the sequence identity can be 100%, thereby modifying the endogenous FEA2 gene and improving the yield trait of the corn plant or part thereof.

[0222] In some embodiments, a method for editing an endogenous FEA2 gene in a corn plant or a plant part is provided, the method comprising contacting a target site in a FEA2 gene in a corn plant or a plant part with a cytosine base editing system, the cytosine base editing system comprising a cytosine deaminase and a nucleic acid binding domain that binds to a target site in the FEA2 gene, wherein the FEA2 gene (a) comprises a nucleotide sequence having at least 90% sequence identity to a sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) encodes an amino acid sequence having at least 90% sequence identity to a sequence of SEQ ID NO:74; (c) comprises a region having at least 90% sequence identity to a nucleotide sequence of any one of SEQ ID NOs:75-77; and / or (d) encodes a polypeptide region having a region having at least 90% sequence identity to an amino acid sequence of any one of SEQ ID NOs:78-80, thereby editing an endogenous FEA2 gene in a corn plant or a plant part thereof and producing a corn plant or a plant part thereof comprising at least one cell having a mutation in the endogenous FEA2 gene.

[0223] In some embodiments, a method for editing an endogenous FEA2 gene in a corn plant or plant part is provided, the method comprising contacting a target site in a FEA2 gene in a corn plant or plant part with an adenosine base editing system, the adenosine base editing system comprising an adenosine deaminase and a nucleic acid binding domain that binds to a target site in the FEA2 gene, wherein the FEA2 gene (a) comprises a nucleotide sequence having at least 90% sequence identity to a sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) encodes an amino acid sequence having at least 90% sequence identity to a sequence of SEQ ID NO:74; (c) comprises a region having at least 90% sequence identity to a nucleotide sequence of any one of SEQ ID NOs:75-77; and / or (d) encodes a polypeptide region comprising a region having at least 90% sequence identity to an amino acid sequence of any one of SEQ ID NOs:78-80, thereby editing an endogenous FEA2 gene in a corn plant or plant part and producing a corn plant or plant part comprising at least one cell having a mutation in the endogenous FEA2 gene.

[0224] In some embodiments, a method for generating a mutation in an endogenous FEA2 gene of a corn plant is provided, the method comprising: (a) targeting a gene editing system to a portion of the FEA2 gene comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs:75-77; and (b) selecting a corn plant comprising a modification located in a region of the FEA2 gene having at least 90% sequence identity to any one of SEQ ID NOs:75-77, optionally wherein the sequence identity of (a) and / or (b) can be at least 95%, optionally the sequence identity can be 100%.

[0225] In some embodiments, a mutation in an endogenous FEA2 gene edited as described herein results in a sequence having at least 90% identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, or 98, optionally wherein the sequence identity may be at least 95%, optionally wherein the sequence identity may be 100%, and / or encodes a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, or 99, optionally wherein the sequence identity may be at least 95%, optionally wherein the sequence identity may be 100%.

[0226] In some embodiments, a method for detecting a mutated FEA2 gene (mutation in an endogenous FEA2 gene) is provided, the method comprising detecting in the genome of a corn plant a FEA2 gene having at least one mutation in a region having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NOs: 75-77, optionally wherein the sequence identity can be at least 95%, optionally the sequence identity can be 100%.

[0227] In some embodiments, a method for detecting a mutated FEA2 gene (a mutation in an endogenous FEA2 gene) is provided, the method comprising detecting a mutation in a nucleic acid encoding the amino acid sequence of SEQ ID NO: 74 in the genome of a corn plant, wherein the mutation is located in the 10th LRR of the encoded polypeptide.

[0228] In some embodiments, a method for detecting a mutated FEA2 gene (a mutation in an endogenous FEA2 gene) is provided, the method comprising detecting a mutation in a nucleic acid encoding an amino acid sequence of SEQ ID NO:74 in the genome of a corn plant, wherein the mutation is located at or near amino acid residue 332 with reference to the amino acid position numbering of SEQ ID NO:74.

[0229] In some embodiments, a method for detecting a mutation in an endogenous FEA2 gene is provided, the method comprising detecting a mutant FEA2 gene in the genome of a corn plant. In some embodiments, the mutant FEA2 gene comprises a sequence having at least 90% sequence identity to any one of SEQ ID NO: 82, 84, 86, 88, 90, 92, 94, 96 or 98, optionally wherein the sequence identity may be at least 95%, optionally wherein the sequence identity may be 100%, and / or encodes a mutant FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NO: 83, 85, 87, 89, 91, 93, 95, 97 or 99, optionally wherein the sequence identity may be at least 95%, optionally wherein the sequence identity may be 100%.

[0230] In some embodiments, a method for producing a corn plant comprising a mutation in an endogenous FEA2 gene and at least one polynucleotide of interest is provided, the method comprising crossing a corn plant of the present invention (a first plant) comprising at least one mutation in an endogenous FEA2 gene with a second plant comprising at least one polynucleotide of interest to produce progeny plants; and selecting progeny plants comprising at least one mutation in the FEA2 gene and at least one polynucleotide of interest, thereby producing a corn plant comprising a mutation in the endogenous FEA2 gene and at least one polynucleotide of interest.

[0231] In some embodiments, the present invention provides a method for producing a corn plant comprising a mutation in an endogenous FEA2 gene and at least one polynucleotide of interest, the method comprising introducing at least one polynucleotide of interest into a corn plant of the present invention comprising at least one mutation in an endogenous FEA2 gene, thereby producing a corn plant comprising at least one mutation in the FEA2 gene and at least one polynucleotide of interest.

[0232] The polynucleotide of interest can be any polynucleotide that can confer a desired phenotype on a plant or otherwise alter the phenotype or genotype of a plant. In some embodiments, the polynucleotide of interest can be a polynucleotide that confer herbicide tolerance, insect resistance, disease resistance, increased yield, increased nutrient use efficiency, or abiotic stress resistance.

[0233] The polynucleotide of interest may be any polynucleotide that can confer a desired phenotype on a plant or otherwise alter the phenotype or genotype of a plant. In some embodiments, the polynucleotide of interest may be a polynucleotide that confer herbicide tolerance, insect resistance, nematode resistance, disease resistance, increased yield, increased nutrient use efficiency, or abiotic stress resistance.

[0234] Therefore, plants or plant cultivars to be preferentially treated according to the invention include all plants which, by genetic modification, have acquired genetic material which imparts particularly advantageous useful properties ("traits") to these plants. Examples of such properties are better plant growth, vigor, stress tolerance, uprightness, resistance to lodging, nutrient uptake, plant nutrition and / or yield, in particular improved growth, increased tolerance to high or low temperatures, increased tolerance to drought or water or soil salinity levels, enhanced flowering performance, easier harvesting, accelerated maturation, higher yield, higher quality and / or higher nutritional value of the harvested product, better storage life and / or processability of the harvested product.

[0235] A further example of such a property is an increased resistance to animal and microbial pests, such as insects, arachnids, nematodes, mites, slugs and snails, due, for example, to toxins formed in the plant. Among the DNA sequences encoding proteins conferring tolerance properties to such animal and microbial pests, in particular insects, mention will be made in particular of the genetic material encoding the Bt protein from Bacillus thuringiensis, which is widely described in the literature and is well known to the person skilled in the art. Mention will also be made of proteins extracted from bacteria such as Photorhabdus (WO 97 / 17432 and WO 98 / 08932). Specifically, mention will be made of 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 Cry1F protein or hybrids derived from Cry1F protein (e.g., hybrid Cry1A-CrylF protein or toxic fragments thereof), Cry1A-type proteins or toxic fragments thereof, preferably Cry1Ac protein or hybrids derived from Cry1Ac protein (e.g., hybrid Cry1Ab-CrylAc protein) or Cry1Ab or Bt2 protein or toxic fragments thereof, Cry2Ae, Cry2Af or Cry2Ag protein or toxic fragments thereof, Cry1A.105 protein or toxic fragments thereof, VIP3Aa19 protein, VIP3Aa20 protein, VIP3A protein produced in COT202 or COT203 cotton events, as described in Estruch et al. (1996), Proc Natl Acad Sci US A. 28; 93 (11): 5389-94 or a toxic fragment thereof, such as the Cry protein described in WO2001 / 47952, an insecticidal protein from a strain of Xenorhabdus (such as described in WO98 / 50427), Serratia (especially from S. entomophila) or Photorhabdus, such as the Tc protein from Photorhabdus described in WO98 / 08932. In addition, any variant or mutant of any of these proteins that differs from any of the above-named sequences in some amino acids (1-10, preferably 1-5) is also included herein, and these sequences are particularly the sequences of their toxic fragments, or are fused to a transit peptide, such as a plastid transit peptide, or another protein or peptide.

[0236] Another particularly emphasized example of such properties is imparting tolerance to one or more herbicides, such as imidazolinone, sulfonylurea, glyphosate or glufosinate. In the DNA sequences (i.e., polynucleotides of interest) encoding proteins that impart the properties of tolerance to certain herbicides in transformed plant cells and plants, bar or PAT genes or Streptomyces coelicolor genes described in WO2009 / 152359 will be particularly mentioned, which impart tolerance to glufosinate herbicides; genes encoding suitable EPSPS (5-enolpyruvylshikimate-3-phosphate-synthetase), which impart tolerance to herbicides (especially herbicides such as glyphosate and its salts) targeting EPSPS; genes encoding glyphosate-n-acetyltransferase, or genes encoding glyphosate oxidoreductase. Additional suitable herbicide tolerance traits include at least one ALS (acetolactate synthase) inhibitor (e.g., WO2007 / 024782); a mutated Arabidopsis ALS / AHAS gene (e.g., U.S. Pat. No. 6,855,533); a gene encoding 2,4-D-monooxygenase, which confers tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid); and a gene encoding dicamba monooxygenase, which confers tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid).

[0237] 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.

[0238] Particularly useful transgenic events in transgenic plants or plant cultivars that can be preferentially treated according to the present invention include: event 531 / PV-GHBK04 (cotton, insect control, described in WO2002 / 040677), event 1143-14A (cotton, insect control, not deposited, described in WO2006 / 128569); event 1143-51B (cotton, insect control, not deposited, described in WO2006 / 128570); event 1445 (cotton, herbicide tolerance, not deposited, described in US-A 2002-120964 or WO2002 / 034946); event 17053 (rice, herbicide tolerance, deposited as PTA-9843, described in WO2010 / 117737); event 17314 (rice, herbicide tolerance, deposited as PTA-9844, described in WO2010 / 117735); Event 281-24-236 (cotton, insect control-herbicide tolerance, deposited as PTA-6233, described in WO2005 / 103266 or US-A 2005-216969); Event 3006-210-23 (cotton, insect control-herbicide tolerance, deposited as PTA-6233, described in US-A 2007-143876 or WO2005 / 103266); Event 3272 (maize, quality traits, deposited as PTA-9972, described in WO2006 / 098952 or US-A 2006-230473); Event 33391 (wheat, herbicide tolerance, deposited as PTA-2347, described in WO2002 / 027004), event 40416 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-11508, described in WO 11 / 075593); event 43A47 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-11509, described in WO2011 / 075595); event 5307 (corn, insect control, deposited as ATCC PTA-9561, described in WO2010 / 077816); event ASR-368 (benthgrass, herbicide tolerance, deposited as ATCC PTA-4816, described in US-A 2006-162007 or WO2004 / 053062); event B16 (maize, herbicide tolerance, not deposited, described in US-A 2003-126634); event BPS-CV127-9 (soybean, herbicide tolerance, deposited as NCIMB No. 41603, described in WO2010 / 080829); event BLR1 (canola, male sterility restoration, deposited as NCIMB 41193, described in WO2005 / 074671),Event CE43-67B (cotton, insect control, deposited as DSM ACC2724, described in US-A 2009-217423 or WO2006 / 128573); Event CE44-69D (cotton, insect control, not deposited, described in US-A 2010-0024077); Event CE44-69D (cotton, insect control, not deposited, described in WO2006 / 128571); Event CE46-02A (cotton, insect control, not deposited, described in WO2006 / 128572); Event COT102 (cotton, insect control, not deposited, described in US-A 2006-130175 or WO2004 / 039986); Event COT202 (cotton, insect control, not deposited, described in US-A 2007-067868 or WO2005 / 054479); event COT203 (cotton, insect control, not deposited, described in WO2005 / 054480);); event DAS21606-3 / 1606 (soybean, herbicide tolerance, deposited as PTA-11028, described in WO2012 / 033794), event DAS40278 (corn, herbicide tolerance, deposited as ATCC PTA-10244, described in WO2011 / 022469); event DAS-44406-6 / pDAB8264.44.06.1 (soybean, herbicide 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-A 2009-137395 or WO 08 / 112019); event DP-305423-1 (soybean, quality traits, not deposited, described in US-A 2008-312082 or WO2008 / 054747); event DP-32138-1 (corn, hybrid system,deposited as ATCC PTA-9158, described in US-A 2009-0210970 or WO2009 / 103049); event DP-356043-5 (soybean, herbicide tolerance, deposited as ATCC PTA-8287, described in US-A 2010-0184079 or WO2008 / 002872); event EE-I (eggplant, insect control, not deposited, described in WO 07 / 091277); event Fil 17 (corn, herbicide tolerance, deposited as ATCC 209031, described in US-A 2006-059581 or WO 98 / 044140); event FG72 (soybean, herbicide tolerant, deposited as PTA-11041, described in WO2011 / 063413), event GA21 (corn, herbicide tolerant, deposited as ATCC 209033, described in US-A2005-086719 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 tolerance, deposited as ATCC 209030, described in US-A 2005-188434 or WO98 / 044140); event GM RZ13 (sugar beet, virus resistance, deposited as NCIMB-41601, described in WO2010 / 076212); event H7-1 (sugar beet, herbicide tolerance, deposited as NCIMB 41158 or NCIMB 41159, described in US-A 2004-172669 or WO2004 / 074492); event JOPLIN1 (wheat, disease resistance, 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-A 2003-097687); event LLRICE06 (rice, herbicide tolerance, deposited as ATCC 203353, described in US 6,468,747 or WO2000 / 026345); event LLRICE62 (rice, herbicide tolerance, deposited as ATCC 203352, described in WO2000 / 026345), event LLRICE601 (rice, herbicide tolerance, deposited as ATCC PTA-2600, described in US-A 2008-2289060 or WO2000 / 026356); event LY038 (maize, quality traits, deposited as ATCC PTA-5623, described in US-A 2007-028322 or WO2005 / 061720); event MIR162 (corn, insect control, deposited as PTA-8166, described in US-A 2009-300784 or WO2007 / 142840); event MIR604 (corn, insect control, not deposited, described in US-A 2008-167456 or WO2005 / 103301); event MON15985 (cotton, insect control, deposited as ATCC PTA-2516, described in US-A 2004-250317 or WO2002 / 100163); event MON810 (corn, insect control, not deposited, described in US-A 2002-102582); event MON863 (corn, insect control, deposited as ATCC PTA-2605, described in WO2004 / 011601 or US-A 2006-095986); event MON87427 (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,MON87754 (soybean, quality traits, deposited as ATCC PTA-9385, described in WO2010 / 024976); event MON87769 (soybean, quality traits, deposited as ATCC PTA-8911, described in US-A 2011-0067141 or WO2009 / 102873); event MON88017 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-5582, described in US-A 2008-028482 or WO2005 / 059103); event MON88913 (cotton, herbicide tolerance, deposited as ATCC PTA-4854, described in WO2004 / 072235 or US-A 2006-059590); event MON88302 (canola, herbicide tolerance, deposited as PTA-10955, described in WO2011 / 153186), event MON88701 (cotton, herbicide tolerance, deposited as PTA-11754, described in WO2012 / 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 tolerance, deposited as ATCC PTA-6708, described in US-A 2006-282915 or WO2006 / 130436); event MS1 1 (canola, pollination control-herbicide tolerance, deposited as ATCC PTA-850 or PTA-2485, described in WO2001 / 031042); event MS8 (canola, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO2001 / 041558 or US-A 2003-188347); event NK603 (maize, herbicide tolerance, deposited as ATCC PTA-2478, described in US-A 2007-292854); event PE-7 (rice, insect control, not deposited, described in WO2008 / 114282); event RF3 (canola, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO2001 / 041558 or US-A 2003-188347); event RT73 (canola, herbicide tolerant, not deposited, described in WO2002 / 036831 or US-A 2008-070260); event SYHT0H2 / SYN-000H2-5 (soybean, herbicide tolerant, deposited as PTA-11226, described in WO2012 / 082548),Event T227-1 (sugar beet, herbicide tolerance, not deposited, described in WO2002 / 44407 or US-A 2009-265817); Event T25 (corn, herbicide tolerance, not deposited, described in US-A 2001-029014 or WO2001 / 051654); Event T304-40 (cotton, insect control-herbicide tolerance, deposited as ATCC PTA-8171, described in US-A 2010-077501 or WO2008 / 122406); Event T342-142 (cotton, insect control, not deposited, described in WO2006 / 128568); Event TC1507 (corn, insect control-herbicide tolerance, not deposited, described in US-A 2005-039226 or WO2004 / 099447); event VIP1034 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-3925, described in WO2003 / 052073), event 32316 (corn, insect control-herbicide tolerance, deposited as PTA-11507, described in WO2011 / 084632), event 4114 (corn, insect control-herbicide tolerance, deposited as PTA-11506, described in WO2011 / 084621), event EE-GM3 / FG72 (soybean, herbicide tolerance, ATCC accession number PTA-11041), optionally stacked with event EE-GM1 / LL27 or event EE-GM2 / LL55 (WO2011 / 063413A2), Event DAS-68416-4 (soybean, herbicide tolerance, ATCC Accession No. PTA-10442, WO2011 / 066360A1), Event DAS-68416-4 (soybean, herbicide tolerance, ATCC Accession No. PTA-10442, WO2011 / 066384A1), Event DP-040416-8 (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 (soybean, herbicide tolerance, ATCC Accession No. PTA-11509, WO2011 / 075595A1), Event DP-004114-3 (soybean, herbicide tolerance, 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, (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 (canola, herbicide tolerance, ATCC Accession No. PTA-10955, WO2011 / 153186A1), event DAS-21606-3 (soybean, herbicide tolerance,ATCC Accession No. PTA-11028, WO2012 / 033794A2), event MON-87712-4 (soybean, quality traits, ATCC Accession 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 (canola, herbicide tolerance, no deposit number available, WO2012071039A1), event DP-073496-4 (canola, herbicide tolerance, no deposit number available, US2012131692), event 8264.44.06.1 (soybean, herbicide tolerance stacked, accession number PTA-11336, WO2012075426A2), event 8291.45.36.2 (soybean, herbicide tolerance stacked, accession number 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, with superimposed herbicide tolerance, ATCC accession number PTA-11993, WO2013 / 010094A1), event MZDT09Y (corn, ATCC accession number PTA-13025, WO2013 / 012775A1). ,

[0239] The genes / events that confer the desired traits in question (e.g., polynucleotides of interest) may also be present in combination with one another in transgenic plants. Examples of transgenic plants that may be mentioned are important crop plants such as cereals (wheat, rice, triticale, barley, rye, oats), corn, soybeans, potatoes, sugar beets, sugar cane, tomatoes, peas and other types of vegetables, cotton, tobacco, rapeseed and fruit plants (fruits are apples, pears, citrus fruits and grapes), with particular emphasis on corn, soybeans, wheat, rice, potatoes, cotton, sugar cane, tobacco and rapeseed. Traits that are particularly emphasized are the increased resistance of the plants to insects, arachnids, nematodes, slugs and snails, and the increased resistance of the plants to one or more herbicides.

[0240] Commercially available examples of such plants, plant parts or plant seeds that may be preferentially treated according to the present invention include commercial products, such as plant seeds sold or distributed under the trade names GENUITY®, DROUGHTGARD®, SMARTSTAX®, RIB COMPLETE®, ROUNDUP READY®, VT DOUBLE PRO®, VT TRIPLE PRO®, BOLLGARD II®, ROUNDUP READY 2 YIELD®, YIELDGARD®, ROUNDUP READY® 2 XTEND™, INTACTA RR2 PRO®, VISTIVE GOLD® and / or XTENDFLEX™.

[0241] FEA2 genes useful in the present invention include any FEA2 gene, wherein a mutation as described herein can confer increased kernel row number and optionally increased yield in a corn plant or part thereof comprising the mutation. In some embodiments, the FEA2 polypeptide comprises an amino acid sequence having at least 90% identity (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% sequence identity) to SEQ ID NO: 74, optionally wherein the sequence identity can be at least 95% sequence identity or 100% sequence identity) or a region within the FEA2 polypeptide having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 78-80. In some embodiments, the FEA2 gene may comprise a sequence having at least about 90% sequence identity (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% sequence identity) to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73, or the FEA2 gene comprises therein a sequence (region or portion) having at least 90% identity (e.g., about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 100% sequence identity, optionally wherein the sequence identity may be at least 95% sequence identity or 100% sequence identity) to any one of the nucleotide sequences of SEQ ID NOs:75-77.

[0242] In some embodiments, at least one mutation generated in an endogenous FEA2 gene in a corn plant can be a substitution, deletion, and / or insertion. In some embodiments, at least one mutation in an endogenous FEA2 gene in a corn plant can be a substitution, deletion, and / or insertion that results in a dominant negative mutation or a weak loss-of-function mutation, optionally wherein a corn plant comprising the mutation can exhibit a phenotype of increased number of rows of kernels (e.g., a phenotype of producing an ear with increased number of rows of kernels) and / or increased yield compared to a control corn plant that does not comprise the edit / mutation, optionally wherein the ear with increased number of rows of kernels does not have a significantly reduced length (e.g., a reduction of less than 30% compared to a corn plant that does not comprise the same FEA2 mutation). For example, the mutation can be a substitution, deletion and / or insertion of one or more amino acid residues (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acids of the FEA2 polypeptide), or the mutation can be a substitution, deletion and / or insertion of at least 1 nucleotide to about 50 consecutive nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 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 In some embodiments, the at least one mutation may be a base substitution of A, T, G, or C. In some embodiments, the mutation or edit may be an out-of-frame insertion or deletion and / or may result in one or more amino acid substitutions. In some embodiments, the mutation or edit in the FEA2 gene can alter the ability of the mutated FEA2 polypeptide to complex with other proteins and / or the mutation alters the ability of the FEA2 polypeptide to translocate from the ER to the plasma membrane when the FEA2 polypeptide is complexed with another protein such as the pseudokinase CORYNE (CRN) (e.g., FEA2 is trapped in the ER when complexed with a polypeptide such as CRN).

[0243] In some embodiments, the mutations produced by the methods of the invention result in a mutated FEA2 gene comprising an edited nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, or 98, optionally wherein the sequence identity may be at least 95%, optionally wherein the sequence identity may be 100%, and / or encodes a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, or 99, optionally wherein the sequence identity may be at least 95% sequence identity or 100% sequence identity.

[0244] In some embodiments, mutations in the endogenous FEA2 gene can be generated after being cleaved 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 FEA2 gene), wherein the target nucleic acid: (a) comprises a nucleotide sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (b) encodes an amino acid sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 74; (c) comprises a region having at least 90% sequence identity to a nucleotide sequence of any one of SEQ ID NOs: 75-77; and / or (d) encodes a polypeptide region comprising a region having at least 90% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 78-80, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, the nuclease cleaves the endogenous FEA2 gene, and mutations are introduced into the endogenous FEA2 gene. In some embodiments, the mutations generated by the editing system may result in one or more than one amino acid substitutions. In some embodiments, the mutations may change the ability of the FEA2 polypeptide to complex with other proteins. In some embodiments, the mutations may change the ability of the FEA2 polypeptide to transport from the endoplasmic reticulum (ER) to the plasma membrane when complexed with another protein such as the pseudokinase CORYNE (CRN) (e.g., FEA2 is trapped in the ER when complexed with polypeptides such as CRN). In some embodiments, the mutations may be out-of-frame mutations (e.g., out-of-frame deletions or insertions). In some embodiments, the mutations may result in the replacement of one or more amino acid residues, optionally located at the 332nd residue numbered with reference to SEQ ID NO:74. In some embodiments, the mutation is located in the 10th LRR of the FEA2 polypeptide. In some embodiments, with reference to the amino acid position numbering of SEQ ID NO:74, the mutation is located at or near residue 332 of the FEA2 polypeptide. In some embodiments, cleavage results in a mutated endogenous FEA2 gene comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%, and / or encoding a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97 or 99, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%.

[0245] Nucleases that can be used in the present invention can cut the endogenous FEA2 gene, thereby introducing mutations into the endogenous FEA2 gene. 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, nucleic acid binding domains that can be used in the present invention (e.g., DNA binding domains, RNA binding domains) include any nucleic acid binding domains that can be used to edit / modify target nucleic acids. Such nucleic acid binding domains include, but are not limited to, zinc fingers, transcription activator-like DNA binding domains (TALs), argonautes, and / or CRISPR-Cas effector DNA binding domains.

[0246] Also provided herein are guide nucleic acids (e.g., gRNA, gDNA, crRNA, crDNA) that bind to a target site within an endogenous FEA2 gene, wherein the endogenous FEA2 gene: (a) comprises a nucleotide sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (b) encodes an amino acid sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 74; (c) comprises a region having at least 90% sequence identity to a nucleotide sequence of any one of SEQ ID NOs: 75-77; and / or (d) encodes a polypeptide region comprising a region having at least 90% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 78-80. In some embodiments, the target site can be a region in any one of the nucleotide sequences of SEQ ID NOs: 75-77, or a region of a nucleotide sequence encoding a region having an amino acid sequence of any one of SEQ ID NOs: 78-80, optionally wherein the sequence identity of (a), (b), (c) and / or (d) can be at least 95%, optionally the sequence identity can be 100%. In some embodiments, a guide nucleic acid is provided that binds to a target nucleic acid in a FASCIATED EAR2 (FEA2) gene in a corn plant, wherein the FEA2 gene has a gene identification number (gene ID) of Zm00001d051012. In some embodiments, the guide nucleic acid comprises a spacer having a nucleotide sequence of SEQ ID NO: 81.

[0247] 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, a system is provided comprising a guide nucleic acid and a CRISPR-Cas effector protein associated with the guide nucleic acid, the guide nucleic acid comprising a spacer having a nucleotide sequence of SEQ ID NO: 81. In some embodiments, the system may further comprise a tracr nucleic acid associated with the guide nucleic acid and the CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

[0248] In some embodiments, a gene editing system is also provided, the gene editing system comprising a CRISPR-Cas effector protein associated with a guide nucleic acid, and the guide nucleic acid comprises a spacer sequence that binds to a FEA2 gene, wherein the FEA2 gene (a) comprises a nucleotide sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (b) encodes an amino acid sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 74; (c) comprises a region having at least 90% sequence identity to a nucleotide sequence of any one of SEQ ID NOs: 75-77; and / or (d) encodes a polypeptide region comprising a region having at least 90% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 78-80, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 95%, optionally the sequence identity may be 100%. In some embodiments, the spacer sequence of the guide nucleic acid may comprise the nucleotide sequence of SEQ ID NO: 81. In some embodiments, the gene editing system may further comprise a tracr nucleic acid associated with a guide nucleic acid and a CRISPR-Cas effector protein, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

[0249] 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 in a gene.

[0250] In some embodiments, a complex comprising a guide nucleic acid and a CRISPR-Cas effector protein containing a cleavage domain is provided, wherein the guide nucleic acid binds to a target site in an endogenous FEA2 gene, wherein the endogenous FEA2 gene: (a) comprises a nucleotide sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (b) encodes an amino acid sequence having at least 90% sequence identity to a sequence of SEQ ID NO: 74; (c) comprises a region having at least 90% sequence identity to a nucleotide sequence of any one of SEQ ID NOs: 75-77; and / or (d) encodes a polypeptide region comprising a region having at least 90% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 78-80, optionally wherein the sequence identity of (a), (b), (c) and / or (d) may be at least 95%, optionally the sequence identity may be 100%, and the cleavage domain cleaves the target strand in the FEA2 gene. In some embodiments, the cleavage domain cleaves the target strand in the FEA2 gene, resulting in a mutation in an endogenous FEA2 gene, the endogenous FEA2 gene comprising a sequence having at least 90% identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, or 98, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%, and / or encoding a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, or 99, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%.

[0251] In some embodiments, an expression cassette is provided, the 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 in an endogenous FEA2 gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (i) a portion of a nucleic acid encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 74; (ii) a portion of a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 72 or SEQ ID NO: 73; (iii) a portion of a sequence having at least 90% sequence identity to any one of the nucleotide sequences of any one of SEQ ID NOs: 75-77; and / or (iv) a portion of a nucleic acid encoding a polypeptide region having at least 90% sequence identity to the amino acid sequence of any one of SEQ ID NOs: 78-80, optionally wherein the sequence identity of (i), (ii), (iii) and / or (iv) may be at least 95%, optionally the sequence identity may be 100%.

[0252] Also provided herein are nucleic acids encoding a mutated FEA2 gene which, when present in a corn plant or plant part, results in a corn plant comprising a phenotype of increased number of kernel rows (e.g., a phenotype of producing an ear with an increased number of kernel rows), and optionally increased yield, compared to a control corn plant not comprising the FEA2 mutation, optionally wherein the ear with an increased number of kernel rows does not have a significantly reduced length (e.g., a reduction of less than 30% in length compared to an ear of a corn plant not comprising the same FEA2 mutation). In some embodiments, the mutated FEA2 gene may comprise a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%, or encodes a polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97 or 99, optionally wherein the sequence identity may be at least 95%, optionally the sequence identity may be 100%.

[0253] The nucleic acid constructs of the present invention (e.g., constructs comprising a sequence-specific nucleic acid binding domain, a CRISPR-Cas effector domain, a deaminase domain, a reverse transcriptase (RT), an RT template and / or a guide nucleic acid, etc.) and expression cassettes / vectors comprising the same can be used as the editing system of the present invention for modifying target nucleic acids (e.g., endogenous FEA2 genes) and / or their expression.

[0254] Any corn plant containing an endogenous FEA2 gene capable of conferring increased kernel row number can be modified (e.g., mutated, such as base editing, cleavage, incision, etc.) as described herein (e.g., using the polypeptides, polynucleotides, RNPs, nucleic acid constructs, expression cassettes and / or vectors of the invention) to increase kernel row number in the corn plant. A corn plant exhibiting increased kernel row number can have an increase in kernel row number from about 5% to about 50% (e.g., about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, or 50%, or any range or value therein; for example, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 10% to about 50%, about 20% to about 50%, about 30% to about 50%, and any range or value therein) as compared to a corn plant, or part thereof, that does not comprise a mutated endogenous FEA2 gene. In some embodiments, corn plants that exhibit increased kernel rows as described herein (e.g., plants that produce ears with increased kernel rows) produce ears that are also not substantially reduced in length. As used herein, ears that are "not substantially reduced in length" of corn plants comprising mutations as described herein have a length that is reduced by less than 30% (e.g., 0% or about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, or any range or value therein), as compared to corn plants that do not comprise the same FEA2 mutation.

[0255] In some embodiments, a corn plant or plant part thereof is provided, comprising at least one mutation in at least one endogenous FASCIATED EAR2 (FEA2) gene having a gene identification number (Gene ID) of Zm00001d051012, wherein the mutated endogenous FEA2 gene comprises a nucleic acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, optionally wherein the sequence identity may be at least 95%, optionally wherein the sequence identity may be 100%, and / or encodes a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97 or 99, optionally wherein the sequence identity may be at least 95%, optionally wherein the sequence identity may be 100%, optionally wherein the at least one mutation is a non-natural mutation.

[0256] As used herein, the term "plant part" includes, but is not limited to, reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, buds, ovules, seeds, and embryos); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, shoots, branches, bark, apical meristems, axillary buds, cotyledons, hypocotyls, and leaves); vascular tissues (e.g., phloem and xylem); specialized cells, such as epidermal cells, parenchyma cells, collenchyma cells, stomata, guard cells, cuticles, mesophyll cells; callus; and cuttings. The term "plant part" also includes plant cells, including intact plant cells in plants and / or plant parts, plant protoplasts, plant tissues, plant organs, plant cell tissue cultures, plant callus, plant clumps, and the like. As used herein, "shoot" refers to the above-ground parts, including leaves and stems. As used herein, the term "tissue culture" encompasses cultures of tissues, cells, protoplasts, and callus.

[0257] As used herein, "plant cell" refers to the structural and physiological unit of a plant, which generally includes a cell wall, but also includes a protoplast. The plant cell of the present invention can be in the form of an isolated unicellular cell, or can be a cultured cell, or can be a part of a higher organizational unit such as, for example, a plant tissue (including callus) or a plant organ. "Protoplast" is a plant cell that does not have a cell wall or only has a partial cell wall. Therefore, in some embodiments of the present invention, the transgenic cell comprising the nucleic acid molecule of the present invention and / or the nucleotide sequence is a cell of any plant or plant part, including but not limited to root cells, leaf cells, tissue culture cells, seed cells, flower cells, fruit cells, pollen cells, etc. In some aspects of the present invention, a plant part can be a plant germplasm. In some aspects, a plant cell can be a non-breeding plant cell that no longer generates a plant.

[0258] "Plant cell culture" refers to cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0259] As used herein, a "plant organ" is a distinct and clearly structured and differentiated part of a plant, such as a root, stem, leaf, flower bud, or embryo.

[0260] As used herein, "plant tissue" refers to a group of plant cells organized into a structural and functional unit. Included is any plant tissue in situ or in culture. The term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue cultures, and any group of plant cells organized into a structural and / or functional unit. The term, when used with or without any specific type of plant tissue listed above or included in this definition, is not intended to exclude any other type of plant tissue.

[0261] In some embodiments of the invention, transgenic tissue cultures or transgenic plant cell cultures are provided, wherein the transgenic tissue or cell culture comprises the nucleic acid molecules / nucleotide sequences of the invention. In some embodiments, the transgenic tissue or cell culture is provided, wherein the transgenic tissue or cell culture comprises the nucleic acid molecules / nucleotide sequences of the invention. In some embodiments, the transgenic tissue or cell is provided, wherein the transgenic tissue or cell culture ...

[0262] The editing system that can be used for the present invention can be any site-specific (sequence-specific) genome editing system that is now known or developed later, which can introduce mutations in a target-specific manner. For example, the editing system (e.g., a site-specific or sequence-specific editing system) may include, but is not limited to, a CRISPR-Cas editing system, a meganuclease editing system, a zinc finger nuclease (ZFN) editing system, a transcription activator-like effector nuclease (TALEN) editing system, a base editing system, and / or a lead editing system, each of which may include one or more polypeptides and / or one or more polynucleotides that can modify (mutate) a target nucleic acid in a sequence-specific manner when expressed as a system in a cell. In some embodiments, the editing system (e.g., a site-specific or sequence-specific editing system) may include one or more polynucleotides and / or one or more polypeptides, including but not limited to a nucleic acid binding domain (DNA binding domain), a nuclease, and / or other polypeptides and / or polynucleotides.

[0263] In some embodiments, the editing system may include one or more sequence-specific nucleic acid binding domains (DNA binding domains), which may be derived from, for example, polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, transcription activator-like effector nucleases (TALENs), and / or Argonaute proteins. In some embodiments, the editing system may include one or more cleavage domains (e.g., nucleases), including but not limited to endonucleases (e.g., Fok1), polynucleotide-guided endonucleases, CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins), zinc finger nucleases, and / or transcription activator-like effector nucleases (TALENs). In some embodiments, the editing system may include one or more polypeptides, including but not limited to deaminases (e.g., cytosine deaminases, adenine deaminases), reverse transcriptases, Dna2 polypeptides, and / or 5' flap endonucleases (FENs). In some embodiments, the editing system may comprise one or more polynucleotides, including but not limited to a CRISPR array (CRISPR guide sequence) nucleic acid, an extended guide nucleic acid, and / or a reverse transcriptase template.

[0264] In some embodiments, a method of modifying or editing a FEA2 gene may comprise contacting a target nucleic acid (e.g., a nucleic acid encoding FEA2) with a base editing fusion protein (e.g., a sequence-specific nucleic acid binding protein, a sequence-specific DNA binding protein (e.g., a CRISPR-Cas effector protein or domain) fused to a deaminase domain (e.g., an adenine deaminase and / or a cytosine deaminase) and a guide nucleic acid, wherein the guide nucleic acid is capable of directing / targeting the base editing fusion protein to the target nucleic acid, thereby editing a locus within the target nucleic acid. In some embodiments, the base editing fusion protein and the guide nucleic acid may be contained in one or more expression cassettes. In some embodiments, the target nucleic acid may be contacted with the base editing fusion protein and an expression cassette containing the guide nucleic acid. In some embodiments, the sequence-specific nucleic acid binding fusion protein and the guide sequence may be provided as a ribonucleoprotein (RNP). In some embodiments, a cell may be contacted with more than one base editing fusion protein and / or one or more guide nucleic acids, which may target one or more target nucleic acids in the cell.

[0265] In some embodiments, a method of modifying or editing a FEA2 gene may include contacting a target nucleic acid (e.g., a nucleic acid encoding FEA2) with a sequence-specific nucleic acid binding fusion protein (e.g., a sequence-specific DNA binding protein (e.g., a CRISPR-Cas effector protein or domain) fused to a peptide tag, a deaminase fusion protein comprising a deaminase domain (e.g., adenine deaminase and / or cytosine deaminase) fused to an affinity polypeptide capable of binding to the peptide tag, and a guide nucleic acid, wherein the guide nucleic acid is capable of guiding / targeting the sequence-specific nucleic acid binding fusion protein to the target nucleic acid, and the sequence-specific nucleic acid binding fusion protein is capable of recruiting the deaminase fusion protein to the target nucleic acid through a peptide tag-affinity polypeptide interaction. To the target nucleic acid, thereby editing the locus within the target nucleic acid. In some embodiments, the sequence-specific nucleic acid binding fusion protein can be fused to an affinity polypeptide that binds a peptide tag, and the deaminase can be fused to the peptide tag, thereby recruiting the deaminase to the sequence-specific nucleic acid binding fusion protein and the target nucleic acid. In some embodiments, the sequence-specific binding fusion protein, the deaminase fusion protein, and the guide nucleic acid can be included in one or more expression cassettes. In some embodiments, the target nucleic acid can be contacted with the sequence-specific binding fusion protein, the deaminase fusion protein, and the expression cassette containing the guide nucleic acid. In some embodiments, the sequence-specific nucleic acid binding fusion protein, the deaminase fusion protein, and the guide sequence can be provided as a ribonucleoprotein (RNP).

[0266] In some embodiments, methods such as lead editing can be used to generate mutations in the endogenous FEA2 gene. 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 confer the ability to recognize and bind to targets in a sequence-specific manner, and can also cause nicks containing PAM chains within the target. The nucleic acid binding domain can be a CRISPR-Cas effector protein, and in this case, the CRISPR array or guide RNA can be an extended guide sequence comprising an extension portion that includes a primer binding site (PSB) and an edit to be incorporated into the genome (template). Similar to base editing, lead editing can utilize various methods to recruit proteins for target site editing, including non-covalent and covalent interactions between proteins and nucleic acids used in selected processes of genome editing.

[0267] As used herein, a "CRISPR-Cas effector protein" is a protein or polypeptide or a domain thereof that cleaves or cuts a nucleic acid, binds a nucleic acid (e.g., a target nucleic acid and / or a guide nucleic acid), and / or identifies, recognizes, or binds a guide nucleic acid as defined herein. In some embodiments, a CRISPR-Cas effector protein may be an enzyme (e.g., a nuclease, an endonuclease, a nickase, etc.) or a portion thereof and / or may act as an enzyme. In some embodiments, a CRISPR-Cas effector protein refers to a CRISPR-Cas nuclease polypeptide or a domain thereof that comprises nuclease activity or wherein the nuclease activity has been reduced or eliminated, and / or comprises nickase activity or wherein the nickase activity has been reduced or eliminated, and / or comprises single-stranded DNA cleavage activity (ss DNA enzyme activity) or wherein the ss DNA enzyme activity has been reduced or eliminated, and / or comprises self-processing RNA enzyme activity or wherein the self-processing RNA enzyme activity has been reduced or eliminated. A CRISPR-Cas effector protein may bind to a target nucleic acid.

[0268] In some embodiments, the sequence-specific nucleic acid binding domain (e.g., a sequence-specific DNA binding domain) can be a CRISPR-Cas effector protein. In some embodiments, 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.

[0269] In some embodiments, the CRISPR-Cas effector protein may include, but is not limited to, Cas9, C2c1, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5 nuclease, optionally wherein the CRISPR-Cas effector protein can be Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (Cas12f), (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or Cas14c effector proteins.

[0270] 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 a 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", e.g., dCas. In some embodiments, a CRISPR-Cas effector protein domain or polypeptide having a mutation in its nuclease active site may have impaired activity or reduced activity compared to the same CRISPR-Cas effector protein (e.g., a nickase, e.g., a Cas9 nickase, a Cas12a nickase) without the mutation.

[0271] CRISPR Cas9 effector proteins or CRISPR Cas9 effector domains useful in the present invention can be any known or later identified Cas9 nuclease. In some embodiments, the CRISPR Cas9 polypeptide can be a Cas9 polypeptide from, for example, Streptococcus spp. (e.g., S. pyogenes, S. thermophilus), Lactobacillus spp., Bifidobacterium spp., Kandleria spp., Leuconostoc spp., Oenococcus spp., Pediococcus spp., Weissella spp., and / or Olsenella spp. Example Cas9 sequences include, but are not limited to, the amino acid sequences of SEQ ID NO:59 and SEQ ID NO:60 or the nucleotide sequences of SEQ ID NO:61-71.

[0272] In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus pyogenes and recognizes the PAM sequence motifs NGG, NAG, NGA (Mali et al., Science 2013; 339(6121): 823-826). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus thermophiles and recognizes the PAM sequence motifs NGGNG and / or NNAGAAW (W=A or T) (see, e.g., Horvath et al., Science, 2010; 327(5962): 167-170, and Deveau et al., J Bacteriol 2008; 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus mutans and recognizes the PAM sequence motifs NGG and / or NAAR (R=A or G) (see, e.g., Deveau et al., J BACTERIOL 2008; 190(4): 1390-1400). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus aureus and recognizes the PAM sequence motifs NNGRR (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus aureus and recognizes the PAM sequence motifs N GRRT (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus aureus that recognizes the PAM sequence motifs N GRRV (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Neisseria meningitidis and recognizes the PAM sequence motif N GATT or N GCTT (R = A or G, V = A, G or C) (see, e.g., Hou et al., PNAS 2013, 1-6). In the above embodiments, N can be any nucleotide residue, such as any one of A, G, C or T.In some embodiments, the CRISPR-Cas effector protein can be a Cas13a protein derived from Leptotrichia shahii, 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.

[0273] In some embodiments, the CRISPR-Cas effector protein may be derived from Cas12a, which is a V-type regularly spaced clustered short palindromic repeats (CRISPR)-Cas nuclease (see, e.g., SEQ ID NO: 1-20). Cas12a is different from the more well-known type II CRISPR Cas9 nuclease in several aspects. For example, Cas9 recognizes the G-rich protospacer adjacent motif (PAM) (3'-NGG) located 3' of its guide RNA (gRNA, sgRNA, crRNA, crDNA, CRISPR array) binding site (protospacer, target nucleic acid, target DNA), while Cas12a recognizes the T-rich PAM (5'-TTN, 5'-TTTN) located 5' of the target nucleic acid. In fact, the orientation of Cas9 and Cas12a binding to their guide RNAs is almost opposite relative to their N and C termini. In addition, the Cas12a enzyme uses a single guide RNA (gRNA, CRISPR array, crRNA), rather than the dual guide RNA (sgRNA (e.g., crRNA and tracrRNA)) found in the natural Cas9 system, and Cas12a processes its own gRNA. In addition, the Cas12a nuclease activity produces staggered DNA double-strand breaks, rather than the blunt ends produced by the Cas9 nuclease activity, and Cas12a relies on a single RuvC domain to cleave two DNA chains, while Cas9 utilizes the HNH domain and the RuvC domain to cleave.

[0274] CRISPR Cas12a effector proteins / domains useful in the present invention may be any known or later identified Cas12a polypeptide (formerly known as Cpf1) (see, e.g., U.S. Pat. No. 9,790,490, which is incorporated by reference for its disclosure of Cpf1 (Cas12a) sequences). The term "Cas12a", "Cas12a polypeptide" or "Cas12a domain" refers to an RNA-guided nuclease comprising a Cas12a polypeptide or a fragment thereof, the fragment comprising a guide nucleic acid binding domain of Cas12a and / or an active, inactive or partially active DNA cleavage domain of Cas12a. In some embodiments, Cas12a useful in the present invention may comprise a mutation in a nuclease active site (e.g., a RuvC site of a Cas12a domain). A Cas12a domain or Cas12a polypeptide having a mutation in its nuclease active site and thus no longer comprising nuclease activity is generally referred to as a dead Cas12a (e.g., dCas12a). In some embodiments, a Cas12a domain or a Cas12a polypeptide having a mutation in its nuclease active site may have impaired activity, for example, may have nickase activity.

[0275] Any deaminase domain / polypeptide that can be used for base editing can be used in the present invention. In some embodiments, the deaminase domain can be a cytosine deaminase domain or an adenine deaminase domain. The cytosine deaminase (or cytidine deaminase) that can be used in the present invention can be any known or later identified cytosine deaminase from any organism (see, e.g., U.S. Patent No. 10,167,457 and Thuronyi et al., Nat. Biotechnol. 37:1070–1079 (2019), each of which is incorporated herein by reference for its disclosure of cytosine deaminase). Cytosine deaminase can catalyze the hydrolysis and deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. Therefore, in some embodiments, the deaminase or deaminase domain that can be used in the present invention can be a cytidine deaminase domain that catalyzes the hydrolysis and deamination of cytosine to uracil. In some embodiments, the cytosine deaminase can be a variant of a naturally occurring cytosine deaminase, including but not limited to primates (e.g., humans, monkeys, chimpanzees, gorillas), dogs, cows, rats, or mice. Thus, in some embodiments, the cytosine deaminase useful in the present invention can be about 70% to about 100% identical to a wild-type cytosine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a naturally occurring cytosine deaminase, and any ranges or values ​​therein).

[0276] In some embodiments, the cytosine deaminase that can be used in the present invention can be an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase can be an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, an APOBEC4 deaminase, a human activation-induced deaminase (hAID), rAPOBEC1, FERNY and / or CDA1, optionally pmCDA1, atCDA1 (e.g., At2g19570), and their evolved forms (e.g., SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29). In some embodiments, the cytosine deaminase can be an APOBEC1 deaminase having an amino acid sequence of SEQ ID NO: 23. In some embodiments, the cytosine deaminase may be an APOBEC3A deaminase having the amino acid sequence of SEQ ID NO: 24. In some embodiments, the cytosine deaminase may be a CDA1 deaminase, optionally a CDA1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the cytosine deaminase may be a FERNY deaminase, optionally a FERNY having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a cytosine deaminase useful in the present invention may be about 70% to about 100% identical (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% identical) to the amino acid sequence of a naturally occurring cytosine deaminase (e.g., an evolved deaminase).In some embodiments, a cytosine deaminase useful in the present invention may be about 70% to about 99.5% identical (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identical) to the amino acid sequence of any one 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 any one 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 is at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of any one of NO:29. In some embodiments, the polynucleotide encoding the cytosine deaminase can be codon-optimized for expression in plants, and the codon-optimized polypeptide can be about 70% to 99.5% identical to the reference polynucleotide.

[0277] In some embodiments, the nucleic acid constructs of the present invention may also encode uracil glycosylase inhibitor (UGI) (e.g., uracil-DNA glycosylase inhibitor) polypeptides / domains. Thus, in some embodiments, nucleic acid constructs encoding CRISPR-Cas effector proteins and cytosine deaminase domains (e.g., encoding a fusion protein comprising a CRISPR-Cas effector protein domain fused to a cytosine deaminase domain, and / or a CRISPR-Cas effector protein domain fused to a peptide tag or an affinity polypeptide capable of binding a peptide tag, and / or a deaminase protein domain fused to a peptide tag or an affinity polypeptide capable of binding a peptide tag) may also encode uracil-DNA glycosylase inhibitor (UGI), optionally wherein UGI may be codon-optimized for expression in plants. In some embodiments, the present invention provides fusion proteins comprising CRISPR-Cas effector polypeptides, deaminase domains, and UGI and / or one or more polynucleotides encoding them, optionally wherein one or more polynucleotides may be codon-optimized for expression in plants. In some embodiments, the present invention provides fusion proteins, wherein the CRISPR-Cas effector polypeptide, the deaminase domain and the UGI can be fused to any combination of peptide tags and affinity polypeptides as described herein, thereby recruiting the deaminase domain and the UGI to the CRISPR-Cas effector polypeptide and the target nucleic acid. In some embodiments, the guide nucleic acid can be linked to a recruitment RNA motif, and one or more of the deaminase domain and / or the UGI can be fused to an affinity polypeptide capable of interacting with the recruitment RNA motif, thereby recruiting the deaminase domain and the UGI to the target nucleic acid.

[0278] "Uracil glycosylase inhibitors" useful in the present invention may be any protein capable of inhibiting uracil-DNA glycosylase base excision repair enzymes. In some embodiments, the UGI domain comprises a wild-type UGI or a fragment thereof. In some embodiments, the UGI domain useful in the present invention may be about 70% to about 100% identical to the amino acid sequence of a naturally occurring UGI domain (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or 100% identical and any range or value therein). In some embodiments, the UGI domain may comprise the amino acid sequence of SEQ ID NO:41, or a polypeptide having about 70% to about 99.5% sequence identity to the amino acid sequence of SEQ ID NO:41 (e.g., at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the amino acid sequence of SEQ ID NO:41). For example, in some embodiments, the UGI domain may comprise a fragment of the amino acid sequence of SEQ ID NO:41 that is 100% identical to a portion of consecutive nucleotides (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides; e.g., about 10, 15, 20, 25, 30, 35, 40, 45 to about 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides) of the amino acid sequence of SEQ ID NO:41. In some embodiments, the UGI domain can be a variant of a known UGI (e.g., SEQ ID NO: 41) having about 70% to about 99.5% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity, and any ranges or values ​​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.

[0279] Adenine deaminase (or adenosine deaminase) useful in the present invention can be any known or later identified adenine deaminase from any organism (see, e.g., U.S. Pat. No. 10,113,163, the disclosure of which is incorporated herein by reference for adenine deaminase). Adenine deaminase can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, adenine deaminase can catalyze the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, adenosine deaminase can catalyze the hydrolytic deamination of adenine or adenosine in DNA. In some embodiments, adenine deaminase encoded by a nucleic acid construct of the present invention can produce an A→G transition in the sense (e.g., "+"; template) strand of a target nucleic acid or a T→C transition in the antisense (e.g., "-", complementary) strand of a target nucleic acid.

[0280] In some embodiments, the adenosine deaminase may be a variant of a naturally occurring adenine deaminase. Thus, in some embodiments, the adenosine deaminase may be about 70% to 100% identical to a wild-type adenine deaminase (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identical to a naturally occurring adenine deaminase, and any range or value therein). In some embodiments, the deaminase or deaminase is not naturally occurring and may be referred to as an engineered, mutated or evolved adenosine deaminase. Thus, for example, an engineered, mutated or evolved adenine deaminase polypeptide or adenine deaminase domain may 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%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to a naturally occurring adenine deaminase polypeptide or adenine deaminase domain, and any range or value therein). In some embodiments, the adenosine deaminase may 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 may be codon-optimized for expression in plants.

[0281] In some embodiments, the adenine deaminase domain can be a wild-type tRNA-specific adenosine deaminase domain, such as a tRNA-specific adenosine deaminase (TadA), and / or a mutant / evolved adenosine deaminase domain, such as a mutant / evolved tRNA-specific adenosine deaminase domain (TadA*). In some embodiments, the TadA domain can be from Escherichia coli (E. coli). In some embodiments, TadA can be modified, such as truncated, with one or more N-terminal and / or C-terminal amino acids missing relative to full-length TadA (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 6, 17, 18, 19 or 20 N-terminal and / or C-terminal amino acid residues may be missing relative to full-length TadA). In some embodiments, the TadA polypeptide or TadA domain does not include an N-terminal methionine. In some embodiments, the wild-type E. coli TadA comprises the amino acid sequence of SEQ ID NO: 30. In some embodiments, the mutant / evolved E. coli TadA* comprises the amino acid sequence of SEQ ID NO: 31-40 (e.g., SEQ ID NO: 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40). In some embodiments, the polynucleotide encoding TadA / TadA* can be codon-optimized for expression in plants.

[0282] Cytosine deaminase catalyzes the deamination of cytosine and produces thymidine (via a uracil intermediate), causing a C to T transition or a G to A transition in the complementary strand of the genome. Thus, in some embodiments, a cytosine deaminase encoded by a polynucleotide of the invention produces a C→T transition in the sense (e.g., "+"; template) strand of a target nucleic acid or a G→A transition in the antisense (e.g., "-", complementary) strand of a target nucleic acid.

[0283] In some embodiments, an adenine deaminase encoded by a nucleic acid construct of the invention produces an A→G transition in the sense (e.g., "+"; template) strand of a target nucleic acid or a T→C transition in the antisense (e.g., "-", complementary) strand of a target nucleic acid.

[0284] The nucleic acid construct encoding the base editor comprising a sequence-specific nucleic acid binding protein and a cytosine deaminase polypeptide of the present invention, and the nucleic acid construct / expression cassette / vector encoding the same, can be used in combination with a guide nucleic acid for modifying a target nucleic acid, including but not limited to generating a C→T or G→A mutation in a target nucleic acid (including but not limited to a plasmid sequence); generating a C→T or G→A mutation in a coding sequence to change the amino acid identity; generating a C→T or G→A mutation in a coding sequence to generate a stop codon; generating a C→T or G→A mutation in a coding sequence to destroy a start codon; generating a point mutation in genomic DNA to destroy function; and / or generating a point mutation in genomic DNA to destroy a splice junction.

[0285] The nucleic acid constructs of the present invention encoding base editors comprising sequence-specific nucleic acid binding proteins and adenine deaminase polypeptides, as well as expression cassettes and / or vectors encoding the same, can be used in combination with guide nucleic acids to modify target nucleic acids, including but not limited to generating A→G or T→C mutations in target nucleic acids (including but not limited to plasmid sequences); generating A→G or T→C mutations in coding sequences to change amino acid identity; generating A→G or T→C mutations in coding sequences to generate stop codons; generating A→G or T→C mutations in coding sequences to destroy start codons; generating point mutations in genomic DNA to destroy function; and / or generating point mutations in genomic DNA to destroy splice junctions.

[0286] The nucleic acid constructs of the present invention comprising a CRISPR-Cas effector protein or a fusion protein thereof can be used in combination with a guide RNA (gRNA, CRISPR array, CRISPR RNA, crRNA) to modify a target nucleic acid, which is designed to function with the encoded CRISPR-Cas effector protein or domain. The guide nucleic acid that can be used in the present invention comprises at least one spacer sequence and at least one repeat sequence. The guide nucleic acid is capable of forming a complex with the CRISPR-Cas nuclease domain encoded and expressed by the nucleic acid construct of the present invention, and the spacer sequence is capable of hybridizing with the target nucleic acid, thereby guiding the complex (e.g., a CRISPR-Cas effector fusion protein (e.g., a CRISPR-Cas effector domain fused to a deaminase domain and / or a CRISPR-Cas effector domain fused to a peptide tag or affinity polypeptide to recruit a deaminase domain and optionally a UGI) to a target nucleic acid, wherein the target nucleic acid can be modified (e.g., cleaved or edited) or regulated (e.g., regulated transcription) by the deaminase domain.

[0287] As an example, a nucleic acid construct encoding a Cas9 domain (e.g., a fusion protein) connected to a cytosine deaminase domain can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the cytosine deaminase domain of the fusion protein deaminates cytosine bases in the target nucleic acid, thereby editing the target nucleic acid. In another example, a nucleic acid construct encoding a Cas9 domain (e.g., a fusion protein) connected to an adenine deaminase domain can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the adenine deaminase domain of the fusion protein deaminates adenosine bases in the target nucleic acid, thereby editing the target nucleic acid.

[0288] Similarly, encoding a Cas12a domain (or other selected CRISPR-Cas nucleases, such as C2c1, C2c3, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9) linked to a cytosine deaminase domain or an adenine deaminase domain (also known as Csnl and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, Csx10, Csx16, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 Nucleic acid constructs containing (dinG) and / or Csf5) (e.g., fusion proteins) can be used in combination with a Cas12a guide nucleic acid (or a guide nucleic acid of another selected CRISPR-Cas nuclease) to modify a target nucleic acid, wherein the cytosine deaminase domain or the adenine deaminase domain of the fusion protein deaminates cytosine bases in the target nucleic acid, thereby editing the target nucleic acid.

[0289] 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 to) 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 repeat sequence of a type V C2c1 CRISPR Cas system, or a fragment thereof; e.g., 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, Cs The gRNA of the present invention can be designed based on a type I, type II, type III, type IV, type V or type VI CRISPR-Cas system.

[0290] 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'.

[0291] In some embodiments, the guide nucleic acid may comprise more than one repeat-spacer sequence (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more repeat-spacer sequences) (e.g., repeat-spacer-repeat, e.g., repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer-repeat-spacer, etc.). The guide nucleic acids of the invention are synthetic, artificial, and do not exist in nature. The gRNA can be very long and can be used as an aptamer (e.g., in the MS2 recruitment strategy) or other RNA structures with hanging spacers.

[0292] As used herein, "repetitive sequence" refers to any repetitive sequence of, for example, a wild-type CRISPR Cas locus (e.g., Cas9 locus, Cas12a locus, C2c1 locus, etc.) or a repetitive sequence of a synthetic crRNA that works together with a CRISPR-Cas effector protein encoded by a nucleic acid construct of the present invention. The repetitive sequence that can be used in the present invention can be any known or later identified repetitive sequence (e.g., type I, type II, type III, type IV, type V, or type VI) of the CRISPR-Cas locus, or it can be a synthetic repetitive sequence designed to work in a type I, II, III, IV, V, or VI CRISPR-Cas system. The repetitive sequence may include a hairpin structure and / or a stem-loop structure. In some embodiments, the repetitive sequence may form a pseudoknot-like structure (i.e., "handle") at its 5' end. Thus, in some embodiments, the repetitive sequence may be identical or substantially identical to a repetitive sequence from a wild-type type I CRISPR-Cas locus, a type II CRISPR-Cas locus, a type III CRISPR-Cas locus, a type IV CRISPR-Cas locus, a type V CRISPR-Cas locus, and / or a type VI CRISPR-Cas locus. Repetitive sequences from wild-type CRISPR-Cas loci can be determined by established algorithms, such as using CRISPRfinder provided by CRISPRdb (see, Grissa et al., Nucleic Acids Res. 35(web server album):W52-7). In some embodiments, the repetitive sequence or a portion thereof is linked to the 5' end of the spacer sequence at its 3' end, thereby forming a repeat-spacer sequence (e.g., a guide nucleic acid, a guide RNA / DNA, a crRNA, a crDNA).

[0293] In some embodiments, the repetitive sequence comprises, consists essentially of, or consists of at least 10 nucleotides, depending on the particular repetitive sequence and whether the guide nucleic acid comprising the repetitive sequence is processed or unprocessed (e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 to 100 or more nucleotides, or any range or value therein). In some embodiments, the repetitive sequence comprises, consists essentially of, or consists of about 10 to about 20, about 10 to about 30, about 10 to about 45, about 10 to about 50, about 15 to about 30, about 15 to about 40, about 15 to about 45, about 15 to about 50, about 20 to about 30, about 20 to about 40, about 20 to about 50, about 30 to about 40, about 40 to about 80, about 50 to about 100, or more nucleotides.

[0294] The repeat sequence linked to the 5' end of the spacer sequence may comprise a portion of the repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive nucleotides of the wild-type repeat sequence). In some embodiments, the portion of the repeat sequence linked to the 5' end of the spacer sequence can be about five to about ten contiguous nucleotides in length (e.g., about 5, 6, 7, 8, 9, 10 nucleotides) and have at least 90% sequence identity (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%) to the same region (e.g., 5' end) of a wild-type CRISPR Cas repeat nucleotide sequence. In some embodiments, a portion of the repeat sequence can include a pseudoknot-like structure (e.g., a "handle") at its 5' end.

[0295] As used herein, a "spacer sequence" is a nucleotide sequence that is substantially complementary to a target nucleic acid (e.g., target DNA) (e.g., a pre-spacer) (e.g., substantially complementary to contiguous nucleotides of a portion / region of a nucleotide sequence that (a) has at least 90% sequence identity with any one of SEQ ID NOs: 72, 73, and / or 75-77 or (b) encodes an amino acid sequence that has at least 90% sequence identity with SEQ ID NO: 74 or SEQ ID NOs: 78-80, optionally wherein the sequence identity of (a) or (b) may be at least 95%, optionally the sequence identity may be 100%). In some embodiments, the spacer sequence may include, but is not limited to, the nucleotide sequence of SEQ ID NO: 81. The spacer sequence can be completely complementary or substantially complementary (e.g., at least about 70% complementary (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more (e.g., 99.1%, 99.2%, 99.3%, 99.4%, 99.6%, 99.7%, 99.8%, 99.9%, 99.1%, 99.2%, 99.3%, 99.9%, 99.1%, 99.2%, 99.3%, 99.9%, 99.1%, 99.2%, 99.3%, 99.9%, 99.8%, 99.9%, 99.9%, 99.1%, 99.2%, 99.3 ... .4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100%)). Thus, in some embodiments, the spacer sequence may have one, two, three, four or five mismatches as compared to the target nucleic acid, which mismatches may be continuous or discontinuous. In some embodiments, the spacer sequence may be 70% complementary to the target nucleic acid. In other embodiments, the spacer nucleotide sequence may be 80% complementary to the target nucleic acid. In still other embodiments, the spacer nucleotide sequence may be 100% complementary to the target nucleic acid (pre-spacer). In some embodiments, the spacer sequence is 100% complementary to the target nucleic acid. The spacer sequence can have a length of about 15 nucleotides to about 30 nucleotides (e.g., 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 nucleotides or any range or value therein). Therefore, in some embodiments, the spacer sequence can have complete complementarity or basic complementarity (e.g., at least 70% complementarity) in a region (e.g., pre-spacer) at least about 15 nucleotides to about 30 nucleotides long of the target nucleic acid. In some embodiments, the length of the spacer is about 20 nucleotides. In some embodiments, the length of the spacer is about 21, 22 or 23 nucleotides.

[0296] In some embodiments, the 5' region of the spacer sequence of the guide nucleic acid may be identical to the target DNA, while the 3' region of the spacer may be substantially complementary to the target DNA (see, e.g., the spacer sequence of the type V CRISPR-Cas system), or the 3' region of the spacer sequence of the guide nucleic acid may be identical to the target DNA, while the 5' region of the spacer may be substantially complementary to the target DNA (see, e.g., the spacer sequence of the type II CRISPR-Cas system), and thus, the overall complementarity of the spacer sequence to the target DNA may be less than 100%. Thus, for example, in the guide sequence of the type V CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 5' region (i.e., the seed region) of the 20-nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary to the target DNA (e.g., at least about 70% complementary). In some embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8 nucleotides, and any ranges therein) of the 5' end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary to the target DNA (e.g., at least about 50% complementary (e.g., 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)).

[0297] As another example, in a guide sequence of a type II CRISPR-Cas system, the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides in the 3' region (i.e., seed region) of, for example, a 20-nucleotide spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary to the target DNA (e.g., at least about 70% complementary). In some embodiments, the first 1 to 10 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 nucleotides, and any ranges therein) of the 3' end of the spacer sequence may be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary to the target DNA (e.g., at least about 50% complementary (e.g., at least about 50%, 55%, 60%, 65%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more or any range or value therein)).

[0298] In some embodiments, the seed region of the spacer can be about 8 to about 10 nucleotides long, about 5 to about 6 nucleotides long, or about 6 nucleotides long.

[0299] As used herein, "target nucleic acid," "target DNA," "target nucleotide sequence," "target region," or "target region in a genome" refers to a region of a plant genome that is fully complementary (100% complementary) or substantially complementary (e.g., at least 70% complementary (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more)) to a spacer sequence in a guide nucleic acid of the invention. The target region that can be used for a CRISPR-Cas system can be positioned immediately 3' (e.g., a type V CRISPR-Cas system) or immediately 5' (e.g., a type II CRISPR-Cas system) to a PAM sequence in the genome of an organism (e.g., a plant genome). The target region can be selected from any region of at least 15 consecutive nucleotides (e.g., 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 nucleotides, etc.) located immediately adjacent to the PAM sequence.

[0300] "Pre-spacer sequence" refers to the target double-stranded DNA, specifically to the portion of the target DNA (e.g., or a target region in the genome) that is completely or substantially complementary (and hybridizes) to the spacer sequence of a CRISPR repeat-spacer sequence (e.g., a guide nucleic acid, a CRISPR array, a crRNA).

[0301] In the case of type V CRISPR-Cas (e.g., Cas12a) systems and type II CRISPR-Cas (Cas9) systems, the protospacer sequence is flanked by (e.g., immediately adjacent to) a protospacer adjacent motif (PAM). For type IV CRISPR-Cas systems, the PAM is located at the 5' end of the non-target strand and the 3' end of the target strand (see below, as an example).

[0302]

[0303] In the case of type II CRISPR-Cas (e.g., Cas9) systems, the PAM is positioned immediately 3' of the target region. The PAM of type I CRISPR-Cas systems is located 5' of the target strand. There is no known PAM for type III CRISPR-Cas systems. Makarova et al. describe the nomenclature of all classes, types, and subtypes of CRISPR systems (Nature Reviews Microbiology 13:722–736 (2015)). R. Barrangou (Genome Biol. 16:247 (2015)) describes guide structures and PAMs.

[0304] Typical Cas12a PAMs are T-rich. In some embodiments, typical Cas12a PAM sequences can be 5'-TTN, 5'-TTTN, or 5'-TTTV. In some embodiments, typical Cas9 (e.g., Streptococcus pyogenes) PAMs can be 5'-NGG-3'. In some embodiments, atypical PAMs can be used, but the efficiency may be lower.

[0305] Those skilled in the art can determine additional PAM sequences by established experiments and calculation methods. Therefore, for example, experimental methods include targeting sequences of all possible nucleotide sequences flanking and identifying sequence members that do not undergo targeting, such as by transformation of target plasmid DNA (Esvelt et al., 2013. Nat. Methods 10: 1116-1121; Jiang et al., 2013. Nat. Biotechnol. 31: 233-239). In some aspects, calculation methods may include performing BLAST searches on natural spacers to identify the original target DNA sequence in phage or plasmid, and aligning these sequences to determine conserved sequences adjacent to the target sequence (Briner and Barrangou, 2014. Appl. Environ. Microbiol. 80: 994-1001; Mojica et al., 2009. Microbiology 155: 733-740).

[0306] In some embodiments, the present invention provides expression cassettes and / or vectors comprising nucleic acid constructs of the present invention (e.g., one or more components of the editing system of the present invention). In some embodiments, expression cassettes and / or vectors comprising nucleic acid constructs of the present invention and / or one or more guide nucleic acids may be provided. In some embodiments, nucleic acid constructs of the present invention encoding base editors (e.g., constructs (e.g., fusion proteins) comprising CRISPR-Cas effector proteins and deaminase domains) or components for base editing (e.g., CRISPR-Cas effector proteins fused to peptide tags or affinity polypeptides, deaminase domains fused to peptide tags or affinity polypeptides, and / or UGI fused to peptide tags or affinity polypeptides) may be included in an expression cassette or vector that is the same as or separate from an expression cassette or vector comprising one or more guide nucleic acids. When the nucleic acid construct encoding a base editor or the components for base editing are contained on an expression cassette or vector separate from the expression cassette or vector comprising a guide nucleic acid, the target nucleic acid can be contacted with (e.g., provided together with) the expression cassette or vector encoding the base editor or the components for base editing in any order with each other and the guide nucleic acid, for example, before, simultaneously with, or after providing the expression cassette comprising the guide nucleic acid (e.g., contacting with the target nucleic acid).

[0307] The fusion protein of the present invention may comprise a sequence-specific nucleic acid binding domain, a CRISPR-Cas polypeptide and / or a deaminase domain fused to a peptide tag as known in the art or an affinity polypeptide that interacts with a peptide tag, for recruiting the deaminase to the target nucleic acid. The recruitment method may also include a guide nucleic acid connected to an RNA recruitment motif and a deaminase fused to an affinity polypeptide that can interact with the RNA recruitment motif, thereby recruiting the deaminase to the target nucleic acid. Alternatively, chemical interactions can be used to recruit polypeptides (e.g., deaminase) to target nucleic acids.

[0308] Peptide tags (e.g., epitopes) that can be used in the present invention may include, but are not limited to, GCN4 peptide tags (e.g., Sun-Tag), c-Myc affinity tags, HA affinity tags, His affinity tags, S affinity tags, methionine-His affinity tags, RGD-His affinity tags, octapeptides sold under the trade name FLAG®, strep tags or strep tags II, V5 tags, and / or VSV-G epitopes. Any epitope that can be linked to a polypeptide and has a corresponding affinity polypeptide that can be linked to another polypeptide can be used as a peptide tag in the present invention. In some embodiments, the peptide tag may include 1 or 2 or more copies of a peptide tag (e.g., repeating units, multimeric epitopes (e.g., tandem repeats)) (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more repeating units). In some embodiments, the affinity polypeptide that interacts / binds with the peptide tag can be an antibody. In some embodiments, the antibody can be a scFv antibody. In some embodiments, the affinity polypeptide that binds to the peptide tag can be synthetic (e.g., evolved for affinity interaction), including but not limited to affibodies, anticalins, monobodies, and / or DARPins (see, e.g., Sha et al., Protein Sci. 26(5):910-924 (2017)); Gilbreth (Curr OpinStruc Biol 22(4):413-420 (2013)), U.S. Patent No. 9,982,053, each of which is incorporated by reference for its teachings on affinity bodies, anticalins, monobodies, and / or DARPins. Example peptide tag sequences and affinity polypeptides thereof include but are not limited to the amino acid sequences of SEQ ID NOs: 45-47.

[0309] In some embodiments, the guide nucleic acid can be linked to an RNA recruitment motif, and the polypeptide to be recruited (e.g., a deaminase) can be fused to an affinity polypeptide that binds to the RNA recruitment motif, wherein the guide sequence binds to the target nucleic acid and the RNA recruitment motif binds to the affinity polypeptide, thereby recruiting the polypeptide to the guide sequence and contacting the target nucleic acid with the polypeptide (e.g., a deaminase). In some embodiments, two or more polypeptides can be recruited to the guide nucleic acid, thereby contacting the target nucleic acid with two or more polypeptides (e.g., a deaminase). Example RNA recruitment motifs and affinity polypeptides thereof include, but are not limited to, sequences of SEQ ID NOs: 48-58.

[0310] In some embodiments, the polypeptide fused to the affinity polypeptide can be a reverse transcriptase, and the guide nucleic acid can be an extended guide nucleic acid connected to an RNA recruitment motif. In some embodiments, the RNA recruitment motif can be located at the 3' end of the extended portion of the extended guide nucleic acid (e.g., 5'-3', repeat-spacer-extension portion (RT template-primer binding site)-RNA recruitment motif). In some embodiments, the RNA recruitment motif can be embedded in the extension portion.

[0311] In some embodiments of the invention, the extended guide RNA and / or guide RNA can be linked to one or two or more RNA recruitment motifs (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more motifs; e.g., at least 10 to about 25 motifs), optionally wherein the two or more RNA recruitment motifs can be the same RNA recruitment motif or different RNA recruitment motifs. In some embodiments, the RNA recruitment motif and the corresponding affinity polypeptide may include, but are not limited to, the telomerase Ku binding motif (e.g., Ku binding hairpin) and the corresponding affinity polypeptide Ku (e.g., Ku heterodimer), the telomerase Sm7 binding motif and the corresponding affinity polypeptide Sm7, the MS2 phage operator stem loop and the corresponding affinity polypeptide MS2 coat protein (MCP), the PP7 phage operator stem loop and the corresponding affinity polypeptide PP7 coat protein (PCP), the SfMu phage Com stem loop and the corresponding affinity polypeptide Com RNA binding protein, the PUF binding site (PBS) and the affinity polypeptide Pumilio / fem-3 mRNA binding factor (PUF), and / or a synthetic RNA aptamer and an aptamer ligand as the corresponding affinity polypeptide. In some embodiments, the RNA recruitment motif and the corresponding affinity polypeptide may be the MS2 phage operator stem loop and the affinity polypeptide MS2 coat protein (MCP). In some embodiments, the RNA recruitment motif and the corresponding affinity polypeptide can be a PUF binding site (PBS) and an affinity polypeptide Pumilio / fem-3m RNA binding factor (PUF).

[0312] In some embodiments, the components used to recruit polypeptides and nucleic acids can be those that function through chemical interactions, which can include, but are not limited to, rapamycin-induced FRB-FKBP dimerization; biotin-streptavidin; SNAP tag; Halo tag; CLIP tag; compound-induced DmrA-DmrC heterodimer; bifunctional ligands (e.g., two protein-binding chemicals fused together; such as dihydrofolate reductase (DHFR).

[0313] In some embodiments, a nucleic acid construct, expression cassette, or vector of the invention optimized for expression in a plant may be about 70% to 100% identical (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100%) to a nucleic acid construct, expression cassette, or vector comprising the same polynucleotide but that has not been codon-optimized for expression in a plant.

[0314] Also provided herein are cells comprising one or more polynucleotides, guide nucleic acids, nucleic acid constructs, expression cassettes or vectors of the invention.

[0315] The present invention will now be described with reference to the following examples. It should be understood that these examples are not intended to limit the scope of the claims of the present invention, but are intended to be examples of certain embodiments. Any changes to the exemplary methods that occur to the technician are intended to fall within the scope of the present invention. Example

[0316] Example 1: Design of editing constructs for FEA2 editing

[0317] The genomic sequence of the FEA2 gene was identified in a proprietary corn line. Based on the reference sequence, a spacer sequence (e.g., SEQ ID NO: 81) is designed for editing constructs. The editing construct comprises a CRISPR-Cas effector and a spacer sequence designed to target the 10th LRR repeat sequence of the FEA2 protein encoded by the FEA2 gene (e.g., SEQ ID NO: 72). The spacer is deployed with a Cas effector cleavage enzyme or a Cas effector base editing complex.

[0318] Example 2: Transformation and selection of edited E0 plants

[0319] Agrobacterium was used to introduce a vector encoding a spacer region (SEQ ID NO: 81) and a selected CRISPR-Cas effector into dried ex vivo maize embryos. The transformed tissue was maintained in vitro with antibiotic selection to regenerate positive transformants. Healthy non-chimeric plants (E0) were selected and planted in growth trays. Tissues were collected from regenerated plants (E0 generation) for DNA extraction, and molecular screening was subsequently used to identify the edits in FEA2. Plants identified as (1) healthy, non-chimeric and fertile, with (2) low transgenic copies and (3) edits in the 10th LRR repeat sequence of the FEA2 polypeptide encoded by the FEA2 gene were cultivated to the next generation. E0 plants that met all of the above criteria were self-pollinated to produce the E1 generation. The selected E1 was self-pollinated to produce the E2 generation. We identified several families with deletions and substitutions in or around the 10th LRR.

[0320] Example 3: Phenotypic Assessment of Trait Activity

[0321] The seeds of E1 and E2 materials are sown on the flat ground, and then transferred to pots after seedling formation. All materials are cultivated under standard greenhouse conditions and grown to reproductive maturity. According to standard practice, the newly grown ear is covered with a small paper bag before silking, and the male tassel is covered by each plant during flowering to capture pollen. In some cases, flowering and silking are not synchronized, and the ear is not pollinated. We refer to these ears as "unpollinated" ears, and once all ears are taken out from the corn plant after drying, they are evaluated to determine the number of grain rows (as described below).

[0322] After the ears were harvested and dried, the number of kernel rows was manually counted for all ears. The data represent the average of three row counts per ear, taken from the middle of the ear, where the row pedigree is most clear. To prevent duplicate counting of rows, a marker (e.g., a paper clip) was inserted between the rows where counting began to specify where the row count should stop.

[0323] All ears were photographed and recorded with a Canon digital camera and EOS application. The images were subsequently imported into ImageJ, and all ears were measured using the line trace function. The ear length was determined in centimeters by the set scale in the image analysis program, and the distance was output in centimeters after tracking the ear from the ear top to the ear base with a line along the ear length. Unedited germplasm and strains transformed with Gus plasmids were used as wild-type controls for phenotypic analysis.

[0324] Example 4: Edited Alleles of FEA2

[0325] The edited allele of the FEA2 gene Zm00001d051012 was generated as described in Example 1 and Example 2 and is further described in Table 1.

[0326] Table 1. Edited alleles

[0327] Example 5: Phenotypic analysis of edited alleles

[0328] Plants containing the edited allele of FEA2 described in Example 4 were evaluated for phenotypic changes in ears as described in Example 3. The observations are summarized in Table 2 and demonstrate that the edited allele of FEA2 has altered ear formation and the number of kernels, and can increase overall plant yield.

[0329] The entry "complex het in frame" represents a group of mixed plants containing a combination of alleles D, E, G or H without the presence of any wild-type sequence.

[0330] Table 2. Analysis of kernel row numbers of edited alleles

[0331] In some jurisdictions, products obtained specifically by essentially biological processes are excluded from patent protection. Therefore, the claimed plants, plant parts and cells and their offspring can be defined as only those plants, plant parts and cells and their offspring obtained by technical intervention (regardless of any further reproduction by hybridization and selection). One embodiment of the present invention relates to plants or plant parts or offspring produced or obtained by introducing RNA-specific CRISPR / Cas systems or one or more polynucleotide sequences encoding the RNA-specific CRISPR / Cas systems into plants or plant parts using gene editing technology by stable or transient transformation for or targeting nucleotide sequences encoding FEA2 proteins. Alternatively, the subject matter excluded from patentability can be abandoned. One embodiment of the present invention relates to plants, plant parts or their offspring comprising changes in FEA2 genes as described elsewhere herein, provided that the plant, plant part or plant or offspring is not specifically obtained by essentially biological processes, wherein essentially biological processes are processes for producing plants or animals, if they are completely composed of natural phenomena such as hybridization or selection.

[0332] The foregoing is illustrative of the present invention and should not be construed as limiting the present invention. The present invention is defined by the following claims, with equivalents of the claims to be included therein.

Claims

1. A corn plant or plant part thereof comprising at least one mutation in an endogenous FASCIATED EAR2 (FEA2) gene encoding a FEA2 polypeptide.

2. The corn plant or part thereof of claim 1, wherein the endogenous FASCIATED EAR2 (FEA2) gene has a gene identification number (gene ID) of Zm00001d051012.

3. The corn plant or part thereof of claim 1 or claim 2, wherein the at least one mutation is a dominant negative mutation or a weak loss-of-function mutation.

4. The corn plant or part thereof according to any one of claims 1 to 3, wherein the at least one mutation is a base substitution, deletion and / or insertion.

5. The corn plant or part thereof of claim 4, wherein the at least one mutation is a deletion, optionally an in-frame deletion or an out-of-frame deletion.

6. The corn plant or part thereof as claimed in any one of the preceding claims, wherein the at least one mutation comprises a base substitution to A, T, G or C.

7. The corn plant or part thereof as claimed in any one of the preceding claims, wherein the at least one mutation is located in the 10th LRR repeat sequence of the reference encoded FEA2 polypeptide SEQ ID NO:

74.

8. The corn plant or part thereof as claimed in any one of the preceding claims, wherein the at least one mutation results in a modified, truncated FEA2 polypeptide.

9. The corn plant or part thereof as claimed in any one of the preceding claims, wherein the at least one mutation results in a substituted amino acid residue at position 332 numbered with reference to the amino acid position of SEQ ID NO:

74.

10. The corn plant or part thereof as claimed in any one of the preceding claims, wherein the at least one mutation results in a replacement of G332.

11. A corn plant or part thereof as described in any of the preceding claims, wherein the endogenous FASCIATEDEAR2 (FEA2) gene (a) comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-77; (c) encodes an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74; and / or (d) encodes a region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs:78-80.

12. The corn plant or part thereof of any of the preceding claims, wherein the at least one mutation results in a mutated FEA2 polypeptide having an altered ability to complex with other polypeptides, and / or an altered ability to cross the plasma membrane and / or to localize from the endoplasmic reticulum to the plasma membrane.

13. A corn plant or part thereof as described in any of the preceding claims, wherein the corn plant comprising the at least one mutation exhibits increased yield compared to a control plant lacking the at least one mutation, optionally wherein the corn plant exhibits an increased kernel row number (KRN) phenotype, optionally wherein ear length is not substantially reduced.

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

15. A corn plant or part thereof as described in any of the preceding claims, wherein the at least one mutation results in a mutated FEA2 gene, the mutated FEA2 gene comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NO:82, 84, 86, 88, 90, 92, 94, 96 or 98, and / or encoding a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NO:83, 85, 87, 89, 91, 93, 95, 97 or 99.

16. A corn plant cell, comprising an editing system, wherein the editing system comprises: (a) CRISPR-Cas effector proteins; and (b) A guide nucleic acid comprising a spacer sequence complementary to an endogenous target gene encoding a FEA2 protein.

17. The corn plant cell of claim 16, wherein the endogenous target gene (a) comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:72 or SEQ ID NO:73, (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-77, (c) encodes an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74, and / or (d) encodes a polypeptide comprising a region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs:78-80.

18. The corn plant cell of claim 16 or claim 17, wherein the guide nucleic acid comprises the nucleotide sequence of SEQ ID NO:

81.

19. A corn plant regenerated from the corn plant part of any one of claims 1 to 15 or the corn plant cell of any one of claims 16 to 18.

20. The corn plant of claim 19, wherein the corn plant exhibits increased yield and / or exhibits increased kernel row number, optionally wherein ear length does not substantially decrease when kernel row number increases.

21. The corn plant of claim 19 or claim 20, wherein the corn plant comprises a mutant FEA2 gene comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, or 98, and / or encodes a mutant FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, or 99.

22. A corn plant cell comprising at least one mutation within a FEA2 gene, wherein the mutation is a substitution, insertion, or deletion introduced using an editing system comprising a nucleic acid binding domain that binds to a target site in the FEA2 gene.

23. The corn plant cell of claim 22, wherein the at least one mutation is a dominant negative allele or a weak loss-of-function allele.

24. The corn plant cell of claim 22 or claim 23, wherein the target site is located within a region of the FEA2 gene that comprises a sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-77.

25. The corn plant cell of any one of claims 22 to 24, wherein the editing system further comprises a nuclease, wherein the nucleic acid binding domain binds to a target site within a sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-77, and upon cleavage by the nuclease, generates at least one mutation within the FEA2 gene.

26. The corn plant cell of claim 25, 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.

27. The corn plant cell of any one of claims 22 to 26, wherein the nucleic acid binding domain is a zinc finger, a transcription activator-like DNA binding domain (TAL), an argonaute, or a CRISPR-Cas effector nucleic acid binding domain.

28. The corn plant cell of any one of claims 22 to 27, wherein the at least one mutation within the FEA2 gene is a substitution or a deletion, optionally an in-frame deletion or an out-of-frame deletion.

29. The corn plant cell of any one of claims 22 to 28, wherein the at least one mutation within the FEA2 gene comprises a point mutation.

30. The corn plant cell of any one of claims 22 to 29, wherein the at least one mutation is located in the 10th LRR repeat sequence of the reference encoded FEA2 polypeptide.

31. A corn plant cell as described in any one of claims 22 to 30, wherein the at least one mutation results in a substituted amino acid residue located at position 322 with reference to the amino acid position numbering of SEQ ID NO:74, optionally wherein the at least one mutation results in a displacement of G332.

32. The corn plant cell of any one of claims 22 to 31, wherein the at least one mutation results in a mutated FEA2 polypeptide having an altered ability to complex with other polypeptides, and / or an altered ability to cross the plasma membrane and / or to localize from the endoplasmic reticulum to the plasma membrane.

33. The corn plant cell of any one of claims 22 to 32, wherein the at least one mutation is a non-natural mutation.

34. The corn plant cell of any one of claims 22 to 33, wherein the at least one mutation results in a mutated FEA2 gene having at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, and / or encodes a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97 or 99.

35. A corn plant regenerated from the corn plant cell of any one of claims 22 to 34, comprising the at least one mutation in the FEA2 gene.

36. The corn plant of claim 35, wherein the corn plant exhibits an increased yield and / or increased kernel row number phenotype when compared to a control plant not comprising the FEA2 gene comprising the at least one mutation, optionally wherein ear length is not substantially reduced when kernel row number is increased.

37. A method of producing / growing a transgene-free edited corn plant, the method comprising: Crossing the corn plant of any one of claims 1 to 15, 19 to 21, 35 or 36 with a corn plant without the transgene, thereby introducing the at least one mutation into the corn plant without the transgene; and Progeny corn plants that contain the at least one mutation and are free of the transgene are selected, thereby producing transgene-free edited corn plants.

38. A method for providing a plurality of corn plants having increased yield and / or increased number of rows of kernels, the method comprising planting two or more plants of any one of claims 1 to 15, 19 to 21, 35 or 36 in a growing area, thereby providing a plurality of corn plants having increased yield and / or increased number of rows of kernels compared to a plurality of control corn plants lacking the at least one mutation.

39. A method for generating a mutation in an endogenous FEA2 gene of a corn plant, the method comprising: (a) targeting a gene editing system to a portion of the FEA2 gene, the portion comprising a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 75-77; and (b) selecting a corn plant comprising a modification in a region of the FEA2 gene having at least 90% sequence identity to any one of SEQ ID NOs: 75-77.

40. A method of generating a variation in a region of a maize FEA2 protein, the method comprising: introducing an editing system into a corn plant cell, wherein the editing system targets a region of the FEA2 gene encoding the FEA2 polypeptide, and The region of the FEA2 gene is contacted with the editing system, thereby introducing mutations into the FEA2 gene and generating variations in the region of the maize FEA2 polypeptide in the maize plant cell.

41. The method of claim 40, wherein the FEA2 gene: (a) comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:72 or SEQ ID NO:73, (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-77, (c) encodes an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74, and / or (d) encodes a polypeptide comprising a region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs:78-80.

42. The method of claim 40 or claim 41, wherein the region of the FEA2 gene that is targeted comprises at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-77.

43. The method of any one of claims 40 to 42, wherein contacting the region of the endogenous FEA2 gene in the corn plant cell with the editing system produces a corn plant cell comprising the edited endogenous FEA2 gene in its genome, the method further comprising (a) regenerating a corn plant from the corn plant cell; (b) selfing the corn plant to produce a corn offspring plant (E1); (c) assaying the offspring plant of (b) for increased yield and / or increased number of kernel rows (optionally wherein ear length does not substantially decrease when kernel rows increase); and (d) selecting offspring corn plants that exhibit increased yield and / or increased number of kernel rows (optionally wherein ear length does not substantially decrease when kernel rows increase) to produce selected offspring corn plants that exhibit increased yield and / or increased number of kernel rows (optionally wherein ear length does not substantially decrease when kernel rows increase) compared to a control corn plant.

44. The method of claim 43, further comprising (e) selfing the selected offspring corn plant of (d) to produce an offspring corn plant (E2); (f) determining the offspring corn plant of (e) for increased yield and / or increased number of kernel rows (optionally wherein ear length does not substantially decrease when kernel rows increase); and (g) selecting offspring corn plants that exhibit increased yield and / or increased number of kernel rows (optionally wherein ear length does not substantially decrease when kernel rows increase) compared to control corn plants to produce selected offspring corn plants that exhibit increased yield and / or increased number of kernel rows (optionally wherein ear length does not substantially decrease when kernel rows increase), optionally repeating (e) to (g) one or more times.

45. A method for detecting a mutated FEA2 gene (mutation in an endogenous FEA2 gene) in a corn plant, the method comprising detecting in the genome of the corn plant a FEA2 gene having at least one mutation in a region having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NOs: 75-77.

46. ​​The method of claim 45, wherein the mutant FEA2 gene being detected comprises a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, and / or encodes a mutant FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97 or 99.

47. A method for editing a specific site in the genome of a corn plant cell, the method comprising: The target site within the endogenous FEA2 gene in the corn plant cell is cut in a site-specific manner, wherein the endogenous FEA2 gene: (a) comprises a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:72 or SEQ ID NO:73; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:75-77; (c) encodes an amino acid sequence having at least 90% sequence identity to SEQ ID NO:74; and / or (d) encodes a polypeptide comprising a region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs:78-80, thereby generating an edit in the endogenous FEA2 gene of the corn plant cell, and generating a corn plant cell comprising the edit in the endogenous FEA2 gene.

48. The method of claim 46, wherein the target site comprises a sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-77, or a sequence encoding a polypeptide comprising a region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 78-80.

49. The method of claim 47 or claim 48, further comprising regenerating a corn plant from the corn plant cell comprising the edit in the endogenous FEA2 gene to produce a corn plant comprising the edit in its endogenous FEA2 gene.

50. The method of any one of claims 47 to 49, wherein the editing results in a non-natural mutation.

51. The method of claim 47 or claim 48, wherein the corn plant comprising the edit in its endogenous FEA2 gene has a phenotype of increased yield and / or increased number of kernel rows, optionally wherein the increase in the number of kernel rows does not significantly reduce ear length.

52. The method of any one of claims 47 to 51, wherein the editing results in a deletion and / or an amino acid substitution, optionally wherein the mutation is an in-frame deletion or an out-of-frame deletion.

53. The method of any one of claims 47 to 52, wherein the edit is located in the 10th LRR repeat sequence of the reference encoded FEA2 polypeptide.

54. The method of any one of claims 47 to 53, wherein the editing results in a substituted amino acid residue at position 322 numbered with reference to the amino acid position of SEQ ID NO: 74, optionally wherein the at least one mutation results in a substitution of G332.

55. The method of any one of claims 47 to 54, wherein the editing results in a mutant FEA2 polypeptide having an altered ability to complex with other polypeptides, and / or an altered ability to cross the plasma membrane and / or to localize from the endoplasmic reticulum to the plasma membrane.

56. The method of any one of claims 47 to 55, wherein editing in the endogenous FEA2 gene results in a non-natural mutation.

57. The method of any one of claims 47 to 56, wherein the editing results in a mutated FEA2 gene comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, and / or encoding a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97 or 99.

58. A method for preparing a corn plant, the method comprising: (a) contacting a population of corn plant cells comprising a wild-type endogenous FEA2 gene with a nuclease linked to a nucleic acid binding domain (e.g., an editing system) that binds to a sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-77, or to a sequence encoding an amino acid sequence having at least 90% sequence identity to any one of SEQ ID NOs: 78-80; (b) selecting a corn plant cell from the population in which an endogenous FEA2 gene has been mutated, thereby producing a corn plant cell comprising at least one mutation in the endogenous FEA2 gene; and (c) growing the selected corn plant cells into corn plants.

59. A method of increasing the number of kernel rows in a corn plant, the method comprising (a) contacting a corn plant cell comprising an endogenous FEA2 gene with a nuclease targeted to the endogenous FEA2 gene, wherein the nuclease is linked to a nucleic acid binding domain (e.g., an editing system) that binds to a target site in the endogenous FEA2 gene, wherein the endogenous FEA2 gene: (i) comprising a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:72 or SEQ ID NO:73; (ii) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:74; (iii) comprising a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-77; and / or (iv) encoding a polypeptide region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 78-80, to produce a corn plant cell comprising a mutation in said wild-type endogenous FEA2 gene; and (b) growing the corn plant cell into a corn plant comprising a mutation in the endogenous FEA2 gene, thereby producing a corn plant having a mutated endogenous FEA2 gene and exhibiting increased yield and / or increased number of kernel rows, optionally exhibiting increased number of kernel rows without substantially reducing ear length.

60. A method for producing a corn plant or part thereof comprising at least one cell having a mutated endogenous FEA2 gene, the method comprising: contacting a target site in an endogenous FEA2 gene in the corn plant or plant part with a nuclease comprising a cleavage domain and a DNA binding domain, wherein the nucleic acid binding domain binds to the target site in the endogenous FEA2 gene, The endogenous FEA2 gene (a) comprising a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:74; (c) comprises a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-77; and / or (d) encoding a polypeptide region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 78-80, thereby producing a corn plant or part thereof comprising at least one cell having a mutation in the endogenous FEA2 gene.

61. A method for producing a corn plant or part thereof comprising a mutated endogenous FEA2 gene and exhibiting increased kernel row number, the method comprising: contacting a target site in an endogenous FEA2 gene in the corn plant or plant part with a nuclease comprising a cleavage domain and a DNA binding domain, wherein the nucleic acid binding domain binds to the target site in the endogenous FEA2 gene, Wherein the endogenous FEA2 gene: (i) comprising a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:72 or SEQ ID NO:73; (ii) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:74; (iii) comprising a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-77; and / or (iv) encoding a polypeptide region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 78-80, thereby producing a corn plant or part thereof comprising an endogenous FEA2 gene having a mutation and exhibiting increased yield and / or increased number of kernel rows, optionally wherein when the number of kernel rows is increased, ear length is not substantially reduced.

62. The method of any one of claims 58 to 61, wherein the nuclease cleaves the endogenous FEA2 gene, thereby introducing the mutation into the endogenous FEA2 gene.

63. The method of any one of claims 58 to 62, wherein the mutation is a non-natural mutation.

64. The method of any one of claims 58 to 63, wherein the mutation is a dominant negative mutation or a weak loss-of-function mutation.

65. The method of any one of claims 58 to 64, wherein the mutation is a substitution, insertion and / or deletion, optionally wherein the mutation is an in-frame deletion or an out-of-frame deletion.

66. The method of any one of claims 58 to 65, wherein the mutation comprises a point mutation.

67. The method of any one of claims 58 to 66, wherein the mutation is in the 10th LRR repeat of the reference encoded FEA2 polypeptide.

68. The method of any one of claims 58 to 67, wherein the mutation results in a substituted amino acid residue at position 322 numbered with reference to the amino acid position of SEQ ID NO: 74, optionally wherein the at least one mutation results in a substitution of G332.

69. The method of any one of claims 58 to 68, wherein the mutation results in a mutated FEA2 polypeptide having an altered ability to complex with other polypeptides, and / or an altered ability to cross the plasma membrane and / or to localize to the plasma membrane from the endoplasmic reticulum.

70. The method of any one of claims 58 to 69, wherein the mutation results in a mutated FEA2 gene comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96 or 98, and / or encoding a mutated FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97 or 99.

71. The method of any one of claims 58 to 70, wherein the nuclease is a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), an endonuclease, or a CRISPR-Cas effector protein.

72. The method of any one of claims 58 to 71, wherein the nucleic acid binding domain is a zinc finger, a transcription activator-like DNA binding domain (TAL), an argonaute, or a CRISPR-Cas effector DNA binding domain.

73. A method for modifying an endogenous FASCIATED EAR2 (FEA2) gene in a corn plant or part thereof to improve yield traits in said corn plant or part thereof, said method comprising modifying a target site in said endogenous FEA2 gene in said corn plant or part thereof, wherein said endogenous FEA2 gene: (a) comprising a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) comprising a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-77; (c) encodes a polypeptide comprising a sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 74, and / or (d) encoding a polypeptide region comprising a region having at least 90% sequence identity to the amino acid sequence of SEQ ID NOs: 78-80, Thereby the endogenous IPT7 gene is modified and the yield trait of the corn plant or part thereof is improved.

74. The method of claim 73, wherein the target site is a region of the IPT7 gene that has at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 72-107.

75. The method of claim 73 or claim 74, wherein the improved yield trait is increased yield and / or increased number of rows of kernels, optionally wherein when the number of rows of kernels is increased, ear length is not substantially reduced compared to a corn plant lacking the at least one mutation.

76. A corn plant produced by any one of the methods of claims 58 to 75.

77. A guide nucleic acid that binds to a target site in the FEA2 gene, wherein the target site comprises a nucleotide sequence of SEQ ID NOs: 75-77 or a nucleotide sequence encoding any one of the amino acid sequences of SEQ ID NOs: 78-80.

78. The guide nucleic acid of claim 77, wherein the guide nucleic acid comprises a spacer comprising the nucleotide sequence of SEQ ID NO:

81.

79. A system comprising a guide nucleic acid as claimed in claim 77 or claim 78 and a CRISPR-Cas effector protein associated with the guide nucleic acid.

80. The system of claim 79, 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.

81. A gene editing system comprising a CRISPR-Cas effector protein associated with a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence that binds to an endogenous FEA2 gene.

82. The gene editing system of claim 81, wherein the FEA2 gene: (a) comprising a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:74; (c) comprises a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-77; and / or (d) encodes a polypeptide region comprising a region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 78-80.

83. The gene editing system of claim 81 or 82, wherein the guide nucleic acid comprises a spacer sequence comprising a nucleotide sequence of SEQ ID NO:

81.

84. The gene editing system of any one of claims 81 to 83, 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.

85. A complex comprising a guide nucleic acid and a CRISPR-Cas effector protein comprising a cleavage domain, wherein the guide nucleic acid binds to a target site in an endogenous FEA2 gene, wherein the endogenous FEA2 gene: (a) comprising a nucleotide sequence having at least 90% sequence identity to the sequence of SEQ ID NO:72 or SEQ ID NO:73; (b) encodes an amino acid sequence having at least 90% sequence identity to the sequence of SEQ ID NO:74; (c) comprises a region having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 75-77; and / or (d) encoding a polypeptide region comprising a region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 78-80, wherein the cleavage domain cleaves the target strand in the FEA2 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 in an endogenous FEA2 gene, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (i) a portion of a nucleic acid encoding an amino acid sequence having at least 90% sequence identity to the amino acid sequence of SEQ ID NO: 74; (ii) a portion of a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:72 or SEQ ID NO:73; (iii) a portion of a sequence having at least 90% sequence identity to any of the nucleotide sequences of any of SEQ ID NOs: 75-77; and / or (iv) a portion of a nucleic acid encoding a polypeptide region having at least 90% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 78-80.

87. A nucleic acid encoding a dominant negative mutation or a weak loss-of-function mutation of a corn FEA2 protein, optionally wherein the corn FEA2 protein has a gene identification number (gene ID) of Zm00001d051012, optionally wherein the dominant negative mutation or the weak loss-of-function mutation is a non-natural mutation.

88. The nucleic acid of claim 87, wherein the nucleic acid comprises a sequence having at least 90% sequence identity to any one of SEQ ID NOs: 82, 84, 86, 88, 90, 92, 94, 96, or 98, and / or encodes a mutant FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NOs: 83, 85, 87, 89, 91, 93, 95, 97, or 99.

89. A modified maize FEA2 polypeptide comprising a mutation in the 10th LRR repeat sequence with reference to the endogenous FEA2 polypeptide SEQ ID NO:

74.

90. The modified corn FEA2 polypeptide of claim 89, wherein the modified FEA2 polypeptide comprises a replacement of the amino acid residue at G332.

91. The modified corn FEA2 polypeptide of claim 89 or claim 90, wherein the ability of the modified FEA2 polypeptide to complex with other polypeptides is altered and / or its ability to cross the plasma membrane and / or to localize to the plasma membrane from the endoplasmic reticulum is altered.

92. A modified corn FEA2 polypeptide having at least 90% sequence identity to any one of SEQ ID NO:83, 85, 87, 89, 91, 93, 95, 97 or 99.

93. A corn plant or part thereof comprising the nucleic acid of any one of claims 87 to 88 or the modified corn FEA2 polypeptide of claims 89 to 92.

94. A corn plant or plant part thereof, comprising at least one mutation in at least one endogenous FASCIATED EAR2 (FEA2) gene having a gene identification number (Gene ID) of Zm00001d051012, optionally wherein the at least one mutation is a non-natural mutation.

95. A guide nucleic acid that binds to a target nucleic acid in the FASCIATED EAR2 (FEA2) gene having a gene identification number (gene ID) of Zm00001d051012.

96. A method of producing a corn plant comprising a mutation in an endogenous FEA2 gene and at least one polynucleotide of interest, the method comprising: Crossing a first corn plant that is a corn plant as described in any one of claims 1 to 15, 19 to 21, 35, 36, 76, 93 or 94 with a second corn plant comprising the at least one polynucleotide of interest to produce a progeny corn plant; and Progeny corn plants are selected that contain the mutation in the FEA2 gene and the at least one polynucleotide of interest, thereby producing the corn plant that contains the mutation in the endogenous FEA2 gene and the at least one polynucleotide of interest.

97. A method of producing a corn plant comprising a mutation in an endogenous FEA2 gene and at least one polynucleotide of interest, the method comprising: Introducing at least one polynucleotide of interest into a corn plant as described in any one of claims 1 to 15, 19 to 21, 35, 36, 76, 93 or 94, thereby producing a corn plant comprising a mutation in the FEA2 gene and at least one polynucleotide of interest.

98. The method of claim 96 or claim 97, 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.

99. A method of producing a corn plant comprising a mutation in an endogenous FEA2 gene and exhibiting a phenotype of an improved yield trait, improved plant architecture, and / or improved defense trait, the method comprising: Crossing a first corn plant that is a corn plant as set forth in any one of claims 1 to 15, 19 to 21, 35, 36, 76, 93 or 94 with a second corn plant that exhibits a phenotype of an improved yield trait, improved plant architecture and / or improved defense trait; as well as Selecting progeny corn plants that contain the mutation in the FEA2 gene and have a phenotype of improved yield traits, improved plant architecture and / or improved defense traits, thereby producing the corn plant that contains the mutation in the endogenous FEA2 gene and exhibits a phenotype of improved yield traits, improved plant architecture and / or improved defense traits compared to control corn plants.

100. A method of controlling weeds in a container (e.g., a pot or seed tray, etc.), a growth chamber, a greenhouse, a field, a recreational area, a lawn, or a roadside, the method comprising: Applying a herbicide to one or more (a large number of) plants as described in any one of claims 1 to 15, 19 to 21, 35, 36, 76, 93 or 94 growing in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn or a roadside, thereby controlling the weeds in the container, the growth chamber, the greenhouse, the field, the recreational area, the lawn or the roadside where the one or more plants are growing.

101. A method of reducing insect predation on corn plants, the method comprising applying an insecticide to one or more plants of any one of claims 1 to 15, 19 to 21, 35, 36, 76, 93 or 94, thereby reducing insect predation on the one or more corn plants.

102. A method of reducing fungal diseases on corn plants, the method comprising applying a fungicide to one or more plants of any one of claims 1 to 15, 19 to 21, 35, 36, 76, 93 or 94, thereby reducing fungal diseases on the one or more corn plants.

103. The method of claim 101 or claim 102, wherein the one or more corn plants are grown in a container, a growth chamber, a greenhouse, a field, a recreational area, a lawn, or a roadside.

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