Method of controlling meristem size to improve crops

Abstract

The present invention relates to compositions and methods for modifying the CLAVATA3 / EMBRYO SURROUNING REGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene in a plant to increase the row number of ears, optionally without a substantial reduction in ear length. The invention also relates to plants having increased row number of ears, optionally without substantial reduction of ear length, produced using the methods and compositions of the invention.

Description

[0001] This application is a divisional application of Chinese invention patent application No. 202180042838.5, with the application date of April 16, 2021 and the invention name of “Method for controlling meristem size to improve crops”.

[0002] Statement Regarding Electronic Submission of Sequence Listings

[0003] Pursuant to 37 C.FR §1.821, a sequence listing in ASCII text format with the file name 1499.28.WO_ST25.txt is submitted via EFS-Web in lieu of a paper copy thereof. The sequence listing is 395,444 bytes in size and was generated on April 14, 2021. The disclosure of this sequence listing is hereby incorporated by reference into the specification.

[0004] Priority declaration

[0005] This application claims the benefit of U.S. Provisional Application No. 63 / 010,887, filed on April 16, 2021, under 35 U.S.C. §119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0006] The present invention relates to compositions and methods for modifying the CLAVATA3 / EMBRYO SURROUNDING REGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene in plants, optionally for increasing kernel row number. The present invention also relates to plants with increased kernel row number produced using the methods and compositions of the present invention. Background Art

[0007] New plant organs are initiated at the growing tips of the plant 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 for continuous maintenance of 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.

[0008] Due to the modular nature of corn ear development, larger meristems tend to initiate more flowers, so meristem size has a direct impact on ear row number and yield. The number of flowers initiated during the development of the corn ear directly limits grain yield. The increased number of flowers initiated around the ear (ear row number or KRN) is a major trait selected during corn domestication. Significant progress through breeding has led to a sharp increase in ear row number, from 2 rows in teosinte, the ancestor of corn, to ~8-20 rows in modern elite corn varieties. In various corn strains, the ear row number can be as high as 36.

[0009] In the classical regulatory pathway described in the model plant Arabidopsis model, cells in the meristem apex secrete CLAVATA3 (CLV3) peptides, which move through the apoplast into the central stem cell domain, where it interacts with several leucine-rich receptors (Leucine Rich Receptors, LRR) including CLAVATA1 (CLV1) and CLAVATA2 (CLV2). This receptor-ligand interaction stimulates signal transduction, which ultimately plays a role in reducing WUS expression and limiting stem cell population expansion. One of the targets of WUS is the CLV3 gene itself, in which way WUS plays a role in limiting its own expression and maintaining stem cell homeostasis (Fletcher, JC, Plants 7: 87 (2018)).

[0010] Loss of function mutations in CLV1, CLV2 or CLV3 lead to amplification of the WUS domain and increased meristem size (Schoof et al., Cell 100: 635-644 (2000)). This increase in meristem size often leads to abnormal plant growth because the meristem expands uncontrollably and becomes disordered, a phenomenon known as fasciation (Je et al., Nat Genet 48: ng.3567 (2016a)). Importantly, larger meristems not only make larger organs, but also increase the number of organs around larger areas. Due to this relationship between meristem size and organ number, mutations in corn CLV-WUS signaling genes may lead to increased flower number and yield.

[0011] New strategies for regulating meristem size are needed to improve crop performance. Summary of the invention

[0012] One aspect of the present invention provides a plant or plant part thereof comprising at least one non-natural mutation in an endogenous CLAVATA3 / EMBRYO SURROUNDING REGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene encoding a CLE protein.

[0013] A second aspect of the present invention provides a plant cell comprising an editing system, wherein the editing system comprises: (a) a CRISPR-Cas effector protein; and (b) a guide nucleic acid (e.g., gRNA, gDNA, crRNA, crDNA, sgRNA, sgDNA) comprising a spacer sequence complementary to an endogenous target gene encoding a CLE protein.

[0014] A third aspect of the invention provides a corn plant cell comprising at least one non-naturally occurring mutation within a CLE gene, wherein the mutation is a substitution, insertion, deletion or inversion introduced using an editing system comprising a nucleic acid binding domain that binds to a target site in the CLE gene.

[0015] The fourth aspect of the present invention provides a method for producing / cultivating a transgenic-free edited corn plant, comprising: hybridizing the corn plant of the present invention with a transgenic-free corn plant, thereby introducing the at least one non-natural mutation into the transgenic-free corn plant; and selecting offspring corn plants that contain the at least one non-natural mutation and are transgenic-free, thereby producing a transgenic-free edited corn plant.

[0016] A fifth aspect of the invention provides a method for providing multiple corn plants with an increased number of ear 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 ear rows compared to multiple control corn plants not comprising the mutation.

[0017] The sixth aspect of the present invention provides a method for producing a variation in a mature corn CLE peptide, comprising: introducing an editing system into a corn plant cell, wherein the editing system targets a region of a corn CLE gene encoding a mature corn CLE peptide protein, wherein the region comprises a sequence having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 77, or the region is encoded by a sequence having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NO: 81-83; and contacting the region of the corn CLE gene with the editing system, thereby introducing a mutation in the mature corn CLE peptide of the corn plant cell; and producing a variation in the mature corn CLE peptide.

[0018] The seventh aspect of the present invention 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 CLE gene in a corn plant cell in a site-specific manner, the endogenous CLE gene (a) comprising (i) a sequence having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NOs: 78-80, and / or (ii) a region having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NOs: 81-83; and / or (b) a domain encoding (i) a sequence having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (ii) a sequence having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 75-77, thereby producing an edit in a portion of the endogenous gene of the corn plant cell encoding a mature CLE peptide and producing a plant cell comprising the edit in the portion of the endogenous gene.

[0019] The eighth aspect provides a method for making a corn plant, comprising: (a) contacting a population of corn plant cells comprising an endogenous CLE gene encoding a precursor CLE polypeptide with a nuclease linked to a nucleic acid binding domain (e.g., a DNA binding domain; e.g., an editing system), wherein the nucleic acid binding domain binds to a sequence that (i) has at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NOs: 78-80, (ii) has at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NOs: 81-83, (iii) encodes a sequence that has at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 72-74, or (ii) encodes a domain that has at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 75-77; (b) selecting plant cells in which the endogenous CLE gene has been mutated from the cell population, thereby producing plant cells comprising a mutation in the endogenous CLE gene; and (c) growing the selected plant cells into plants.

[0020] The ninth aspect provides a method for increasing the number of ear rows in a corn plant, comprising (a) contacting a corn plant cell comprising an endogenous CLE gene with a nuclease targeted to the endogenous CLE gene, wherein the nuclease is linked to a nucleic acid binding domain (e.g., a DNA binding domain; e.g., an editing system) that binds to a target site in the endogenous CLE gene, wherein the endogenous CLE gene: (i) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (ii) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (iii) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (iv) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 73-75. NO:75-77 has at least 95% sequence identity to any one of the amino acid sequences of NO:75-77, to produce a corn plant cell comprising a mutation in the endogenous CLE gene; and (b) growing the corn plant cell into a corn plant comprising the mutation in the endogenous CLE gene, thereby producing a corn plant with a mutated endogenous CLE gene and an increased number of ear rows.

[0021] The tenth aspect provides a method for producing a corn plant or part thereof comprising at least one cell having a mutated endogenous CLE gene, the method comprising contacting a target site in an endogenous CLE 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 CLE gene, wherein the endogenous CLE gene (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, thereby producing a corn plant or part thereof comprising at least one cell having a mutation in the endogenous CLE gene.

[0022] The eleventh aspect of the present invention provides a method for producing a corn plant or part thereof comprising a mutated endogenous CLE gene and showing an increased number of ear rows, the method comprising contacting a target site in an endogenous CLE 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 CLE gene, wherein the endogenous CLE gene: (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, thereby producing a corn plant or part thereof comprising a mutated endogenous CLE gene and showing an increased number of ear rows.

[0023] The twelfth aspect provides a guide nucleic acid that binds to a portion of a target site in a CLE gene encoding a precursor CLE peptide, wherein the target site comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83 or encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77.

[0024] In a thirteenth aspect, a system is provided, comprising a guide nucleic acid of the invention and a CRISPR-Cas effector protein bound to the guide nucleic acid.

[0025] A fourteenth aspect provides a gene editing system comprising a CRISPR-Cas effector protein bound to a guide nucleic acid, wherein the guide nucleic acid comprises a spacer sequence bound to an endogenous CLE gene.

[0026] In a fifteenth aspect, a complex comprising a CRISPR-Cas effector protein is provided, the complex comprising a cleavage domain and a guide nucleic acid, wherein the guide nucleic acid binds to a target site in an endogenous CLE gene encoding a precursor CLE polypeptide, wherein the endogenous CLE gene: (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, wherein the cleavage domain cleaves the target strand in the CLE gene.

[0027] In a sixteenth aspect, an expression cassette is provided, the expression cassette comprising (a) a polynucleotide encoding a CRISPR-Cas effector protein, the CRISPR-Cas effector protein comprising a cleavage domain and (b) a guide nucleic acid that binds to a target site in an endogenous CLE gene encoding a precursor CLE peptide, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (a) a sequence comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 78-80; (b) a region comprising a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 81-83; (c) encoding a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 72-74 amino acid sequences; and / or (d) encoding a domain having a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 75-77 amino acid sequences.

[0028] In another aspect, a method for generating a mutation in an endogenous CLE gene in a plant is provided, comprising: (a) targeting a gene editing system to a portion of the CLE gene encoding an amino acid residue at position 3, 5, 6, 8 or 9, wherein the positions are numbered with reference to the amino acid positions of SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 77, and (b) selecting a plant comprising an alternative amino acid at one of positions 3, 5, 6, 8 or 9, wherein the positions are numbered with reference to the amino acid positions of SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 77.

[0029] Another aspect of the present invention provides nucleic acids encoding a dominant negative mutation, a semi-dominant mutation, a weak loss-of-function mutation, a null allele or a hypomorphic mutation of a maize CLE protein.

[0030] In another aspect, a corn plant or part thereof comprising a nucleic acid of the invention is provided.

[0031] In another aspect, a corn plant or part thereof is provided that exhibits increased ear row number.

[0032] In some aspects, a corn plant is provided that also exhibits increased yield, and improved nematode resistance, and exhibits larger meristems.

[0033] Also provided are plants comprising in their genome one or more mutated CLE genes produced by the methods of the present invention, as well as polypeptides, polynucleotides, nucleic acid constructs, expression cassettes and vectors for making the plants of the present invention.

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

[0035] A brief description of the sequence

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

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

[0038] SEQ ID NOs: 21-22 are exemplary regulatory sequences encoding promoters and introns.

[0039] SEQ ID NOs: 23-29 are exemplary cytosine deaminase sequences that can be used in the present invention.

[0040] SEQ ID NOs: 30-40 are exemplary adenine deaminase amino acid sequences that can be used in the present invention.

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

[0042] SEQ ID NOs: 42-44 provide examples of protospacer-adjacent motif locations for type V CRISPR-Cas12a nucleases.

[0043] SEQ ID NOs: 45-47 provide exemplary peptide tags and affinity polypeptides useful in the present invention.

[0044] SEQ ID NOs: 48-58 provide examples of RNA recruitment motifs and corresponding affinity polypeptides that can be used in the present invention.

[0045] SEQ ID NOs: 59-60 are exemplary Cas9 polypeptide sequences that can be used in the present invention.

[0046] SEQ ID NOs: 61-71 are exemplary Cas9 polynucleotide sequences that can be used in the present invention.

[0047] SEQ ID NO: 72 is an example of an unprocessed FCP1 polypeptide sequence.

[0048] SEQ ID NO: 73 is an example of an unprocessed CLE14 polypeptide sequence.

[0049] SEQ ID NO: 74 is an example of an unprocessed CLE7 polypeptide sequence.

[0050] SEQ ID NO: 75 is an example of a processed FCP1 polypeptide sequence.

[0051] SEQ ID NO: 76 is an example of a processed CLE14 polypeptide sequence.

[0052] SEQ ID NO: 77 is an example of a processed CLE7 polypeptide sequence.

[0053] SEQ ID NO: 78 is an example of an FCP1 coding (cds) sequence.

[0054] SEQ ID NO: 79 is an example of a CLE14 coding (cds) sequence.

[0055] SEQ ID NO: 80 is an example of a CLE7 coding (cds) sequence.

[0056] SEQ ID NO: 81 is an exemplary target region of the FCP1 coding (cds) sequence.

[0057] SEQ ID NO: 82 is an exemplary target region of the CLE14 coding (cds) sequence.

[0058] SEQ ID NO: 83 is an exemplary target region of the CLE7 coding (cds) sequence.

[0059] SEQ ID NOs: 84-90 are examples of mutated FCP1 polypeptide sequences.

[0060] SEQ ID NOs: 91-93 are examples of mutated CLE14 polypeptide sequences.

[0061] SEQ ID NOs: 94-100 are examples of mutated CLE7 polypeptide sequences.

[0062] SEQ ID NOs: 101-104 are exemplary spacer sequences that can be used in the nucleic acid guides of the present invention.

[0063] SEQ ID NOs: 105-109 are exemplary mutant CLE polynucleotides encoding processed CLE polypeptides. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 A map of the FCP1 coding sequence is provided showing FCP1 peptide domains and exemplary target regions. DETAILED DESCRIPTION

[0065] The present invention will now be described hereinafter with reference to the accompanying drawings and examples, in which some embodiments of the present invention are shown. This description is not intended to list in detail all the different ways in which the present invention can be performed or all the features that can be added to the present invention. For example, the features shown in relation to one embodiment can be incorporated into other embodiments, and the features shown in relation to a particular embodiment can be deleted from the embodiment. Therefore, the present invention includes that in some embodiments of the present invention, any feature or feature combination shown herein can be excluded or omitted. In addition, based on the disclosure of the present invention, many changes and additions to the various embodiments proposed herein will be apparent to those skilled in the art, and they do not depart from the present invention. Therefore, the following description is intended to illustrate some specific embodiments of the present invention, rather than to exhaustively specify all its permutations, combinations and variations.

[0066] 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 invention herein are only for the purpose of describing specific embodiments and are not intended to limit the invention.

[0067] 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.

[0068] Unless the context indicates otherwise, 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 example, if the specification states that the composition comprises components A, B, and C, it specifically indicates that any one or combination of A, B, or C may be omitted, either individually or in any combination.

[0069] 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.

[0070] Also as used herein, "and / or" is indicative of and encompasses any and all possible combinations of one or more of the associated listed items, as well as when interpreted in the alternative ("or") referring to no combinations.

[0071] When referring to a measurable value such as an amount or concentration, the term "about" as used herein is intended to encompass the specified value as well as variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the specified value. For example, "about X" (where X is a measurable value) is meant to include X as well as variations of ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. Ranges provided herein for measurable values ​​may include any other ranges and / or specific values ​​therein.

[0072] As used herein, phrases such as "between X and Y" / "XY" and "between about X and Y" / "about XY" should be interpreted to include X and Y. As used herein, phrases such as "between about X and Y" / "about XY" mean "between about X and about Y", and phrases such as "from about X to Y" mean "from about X to about Y".

[0073] 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.

[0074] As used herein, the terms "includes," "has," and "comprising," etc., refer to 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.

[0075] As used herein, the transitional phrase "consisting essentially of" means that the scope of a claim should be interpreted as covering the specified materials or steps described 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".

[0076] As used herein, the terms "increase", "increased", "enhance", "enhanced" (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 plant comprising a mutation in a CLE gene as described herein may exhibit an increased number of ear rows that is at least about 5% or more than the number of ear rows of a control plant that does not comprise the same mutation. A control plant is typically the same plant as the edited plant, but the control plant has not been similarly edited and therefore lacks the mutation. A control plant may be an isogenic plant and / or a wild-type plant. Thus, a control plant may be the same breeding line, variety or cultivar as the test plant into which the mutation described herein is introgressed, but the control breeding line, variety or cultivar does not have the mutation. In some embodiments, the comparison between the plant of the present invention and the control plant is made under the same growth conditions, such as the same environmental conditions (soil, hydration, light, heat, nutrients, etc.).

[0077] As used herein, the terms "reduce," "reduced," "lower," "reduced" (and grammatical variations thereof) describe, for example, a reduction of at least about 5%, 10%, 15%, 20%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, or 100% as compared to a control. In certain embodiments, the reduction can result in no or substantially no (i.e., an insignificant amount, e.g., less than about 10% or even 5%) detectable activity or amount.

[0078] As used herein, the terms "expression", "expressed", etc. used with respect to nucleic acid molecules and / or nucleotide sequences (e.g., RNA or DNA) indicate that the nucleic acid molecules and / or nucleotide sequences are transcribed and optionally translated. Thus, nucleic acid molecules and / or nucleotide sequences can express a polypeptide of interest or, for example, a functional non-translated RNA.

[0079] A "heterologous" or "recombinant" nucleotide sequence is a nucleotide sequence not naturally associated with a 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.

[0080] "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 cases, a "wild-type" nucleic acid is a nucleic acid that is not edited as described herein, which may be different from an "endogenous" gene (e.g., a mutated endogenous gene) that may be edited as described herein. In some cases, a "wild-type" nucleic acid (e.g., unedited) may be heterologous to an organism (e.g., a transgenic organism) in which the wild-type nucleic acid is found. As an example, a "wild-type endogenous CLAVATA3 / EMBRYOSURROUNDING REGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene" is a CLE gene, optionally a FCP1 gene, a CLE14 gene or a CLE7 gene, which is naturally present in a reference organism (e.g., a plant, such as a maize plant) or is endogenous thereto, and may be subjected to modification as described herein, after which the modified endogenous gene is no longer wild-type.

[0081] As used herein, the term "heterozygous" refers to a genetic state in which different alleles are present at corresponding loci on homologous chromosomes.

[0082] As used herein, the term "homozygous" refers to a genetic state in which identical alleles are present at corresponding loci on homologous chromosomes.

[0083] As used herein, the term "allele" refers to one of two or more different nucleotides or nucleotide sequences occurring at a particular locus.

[0084] A "null allele" is a non-functional allele caused by a gene mutation, which results in a complete lack of production of the corresponding protein or the production of a non-functional protein.

[0085] A "dominant negative mutation" is a mutation that produces an altered gene product (e.g., 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 an "anti-morphic mutation."

[0086] A "semidominant mutation" is a mutation in which the introgression of a phenotype in a heterozygous organism is less than that observed for a homozygous organism.

[0087] A "weak loss-of-function mutation" is a mutation that results in a gene product that has partial or reduced function (partial inactivation) compared to the wild-type gene product.

[0088] A "hypomorphic mutation" is a mutation that results in a partial loss of gene function, which can occur through reduced expression (e.g., reduced protein and / or reduced RNA) or reduced functional performance (e.g., reduced activity) rather than a complete loss of function / activity. A "hypomorphic" allele is a semi-functional allele caused by a genetic mutation that results in the production of a corresponding protein that functions at anywhere between 1% and 99% of normal efficiency.

[0089] A "locus" is a position on a chromosome where a gene or marker or allele is located. In some embodiments, a locus can encompass one or more nucleotides.

[0090] 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.

[0091] A marker is "associated" with a trait when it is linked to the marker and when the presence of the marker is an indicator of whether and / or to what extent the desired trait or trait form is present 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 indicator of whether the allele or chromosomal interval is present in the plant / germplasm containing the marker.

[0092] As used herein, the terms "backcrossing" and "backcrossing" refer to the process of backcrossing a progeny plant with one of its parents one or more times (e.g., 1, 2, 3, 4, 5, 6, 7, 8 times, etc.). In a backcrossing scheme, a "donor" parent refers to a parent plant with a desired gene or locus to be introgressed. A "recipient" parent (used once or more) or a "backcross" parent (used twice or more) refers to a parent plant into which a 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.

[0093] As used herein, the term "crossing" or "hybridized" refers to the fusion of gametes through pollination to produce offspring (e.g., cells, seeds, or plants). The term includes sexual crossing (one plant is pollinated by another plant) and selfing (self-pollination, e.g., when the pollen and ovules are from the same plant). The term "crossing" refers to the act of fusing gametes through pollination to produce offspring.

[0094] As used herein, the terms "introgression", "introgressing" and "introgressed" refer to the natural and artificial transmission of a desired allele or combination of desired alleles of one or more loci from one genetic background to another genetic background. For example, the desired allele at a given locus can be passed to at least one (e.g., one or more) offspring by sexual hybridization between two parents of the same species, wherein at least one parent has the desired allele in its genome. Alternatively, for example, the transmission of the allele can occur, for example, by recombination between two donor genomes in a fused protoplast, wherein at least one donor protoplast has the desired allele in its genome. The desired allele can be a selected allele of a marker, QTL, transgenic, etc. The offspring comprising the desired allele can be backcrossed once or more (e.g., 1 time, 2 times, 3 times, 4 times or more) with a strain having a desired genetic background, selecting the desired allele, with the result that the desired allele becomes fixed in the desired genetic background. For example, the mark that is associated with output increase under non-water stress condition can be gradually infiltrated from the donor into the backcross parent that does not comprise this mark and does not show output increase under non-water stress condition.Then the offspring that obtains can be backcrossed once or many times, and select, until offspring has the genetic marker that is associated with output increase under non-water stress condition under backcross parent background.

[0095] A "genetic map" is a description of the genetic linkage relationships between loci on one or more chromosomes in a given species, usually described in the form of a graph or table. 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 the 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 one genetic map to another.

[0096] As used herein, the term "genotype" refers to the genetic composition of an individual (or individual group) at one or more loci, in contrast to the proterties (phenotype) that can be observed and / or detected and / or performed. Genotype is defined by the alleles of one or more known loci of the inheritance of an individual from its parents. The term genotype can be used to refer to the genetic composition of an individual at a single locus, a plurality of loci, or more generally, the term genotype can be used to refer to the individual genetic composition of all genes in an individual genome. Genotype can for example be characterized indirectly using a mark and / or directly characterized by nucleic acid sequencing.

[0097] As used herein, the term "germplasm" refers to the genetic material of an individual (e.g., plant), a population of individuals (e.g., plant strains, varieties, or families), or a clone derived from a strain, variety, species, or culture, or the genetic material therefrom. 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 that provides a basis for some or all of the genetic properties of an organism or cell culture. As used herein, germplasm includes cells, seeds, or tissues from which new plants can be grown, and plant parts (e.g., leaves, stems, buds, roots, pollen, cells, etc.) that can be cultured into complete plants.

[0098] As used herein, the terms "cultivar" and "variety" refer to a group of similar plants that are distinguishable from other varieties of the same species by structural or genetic characteristics and / or performance.

[0099] As used herein, the terms "exotic," "exotic lines," and "exotic germplasm" refer to any non-elite plant, line, or germplasm. In general, an exotic plant / germplasm is not derived from any known superior plant or germplasm, but is selected to introduce one or more desired genetic elements into a breeding program (e.g., to introduce novel alleles into a breeding program).

[0100] As used herein, the term "hybrid" in the context of plant breeding refers to 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.

[0101] 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 particular portion of the genome of interest.

[0102] A "haplotype" is the genotype, i.e., the combination of alleles, of an individual at multiple loci. Typically, the genetic loci that define a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term "haplotype" can refer to a polymorphism at a specific locus, such as a single marker locus, or a polymorphism at multiple loci along a chromosome segment.

[0103] As used herein, the term "heterologous" refers to a nucleotide / polypeptide that originates from a foreign species or, if from the same species, is substantially modified from its native form in composition and / or genomic locus by deliberate human intervention.

[0104] Plants in which at least one (e.g., one or more, such as 1, 2, 3 or 4 or more) CLE genes (e.g., one or more CLE genes) are modified as described herein (e.g., comprise modifications as described herein) may have improved yield traits compared to plants that do not comprise said modifications in said at least one CLE 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, seed number, leaf tissue weight, nodule number, nodule quality, nodulation activity, number of seed heads, number of tillers, number of branches, number of flowers, number of tubers, tuber quality, bulb quality, number of seeds, total seed mass, leaf emergence rate, rate of tiller / branch emergence, emergence rate, root length, number of roots, size and / or weight of root balls, or any combination thereof. Thus, in some aspects, "improved yield traits" may include, but are not limited to, increased inflorescence production, increased fruit production (e.g., increased number, weight and / or size of fruits; e.g., increased number, weight and / or size of ears of, e.g., corn), increased fruit quality, increased number, size and / or weight of roots, increased meristem size, increased seed size, increased biomass, increased leaf size, increased nitrogen use efficiency, increased height, increased number of internodes and / or increased internode length compared to a control plant or part thereof (e.g., a plant that does not contain a mutated endogenous CLE nucleic acid (e.g., a mutated CLE gene)). Improved yield traits may also result from increased planting density of the plants of the invention. Thus, in some aspects, the plants of the invention can be planted at increased density (due to changes in plant architecture caused by the endogenous mutations), which results in improved yield traits compared to control plants planted at the same density. In some aspects, the improved yield trait may be expressed as the amount of grain produced per land area (e.g., bushels per acre of land).

[0105] As used herein, "control plants" means plants that do not contain one or more edited CLE genes as described herein, which confer enhanced / improved traits (e.g., yield traits) or altered phenotypes. Control plants are used to identify and select plants that are edited as described herein and have enhanced traits or altered phenotypes compared to control plants. Suitable control plants can be plants of the parental line used to produce plants containing mutated CLE genes, for example, wild-type plants without editing in endogenous CLE genes as described herein. Suitable control plants can also be plants containing recombinant nucleic acids that confer other traits, such as transgenic plants with enhanced herbicide tolerance. In some cases, suitable control plants can be progeny of heterozygous or hemizygous transgenic plant lines that do not contain mutated CLE genes as described herein, referred to as negative segregants or negative isogenic lines.

[0106] The enhanced traits can be, for example, compared to control plants, a reduction in the number of days from planting to maturity, an increase in stalk size, an increase in the number of leaves, an increase in plant height growth rate in the vegetative stage, an increase in ear size, an increase in ear dry weight per plant, an increase in the number of grains per ear, an increase in the weight of each grain, an increase in the number of grains per plant, a reduction in ear spacing, an extension of the grain filling phase, a reduction in plant height, an increase in the number of root branches, an increase in total root length, an increase in yield, an increase in nitrogen use efficiency, and an increase in water use efficiency. The altered phenotype can be, for example, plant height, biomass, canopy area, anthocyanin content, chlorophyll content, water applied, water content, and water use efficiency.

[0107] As used herein, "properties" are the 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 seed or plant size, weight, shape, form, length, height, growth rate and development stage, or can be measured by biochemical techniques, such as detecting the oil content of protein, starch, some metabolites or seeds or leaves, or by observing metabolism or physiological processes, such as 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, such as by adopting Northern analysis, RT-PCR, microarray gene expression determination or reporter gene expression system, or by agricultural observation, such as high osmotic stress tolerance or output. However, any technology can be used to measure the amount, comparative level or difference of any selected compound or macromolecule in transgenic plants.

[0108] As used herein, "enhanced traits" means plant characteristics resulting from mutations in CLE genes as described herein. Such traits include, but are not limited to, enhanced agronomic traits characterized by enhanced plant morphology, physiology, growth and development, yield, nutritional enhancement, disease or pest resistance, or environmental or chemical tolerance. In some embodiments, the enhanced trait / altered phenotype can be, for example, a reduction in the number of days from planting to maturity, an increase in stalk size, an increase in the number of leaves, an increase in plant height growth rate during the growth stage, an increase in ear size, an increase in ear dry weight per plant, an increase in the number of grains per ear, an increase in the weight of each grain, an increase in the number of grains per plant, a reduction in ear gaps, an extension of the grain filling period, a reduction in plant height, an increase in the number of root branches, an increase in total root length, drought tolerance, an increase in water use efficiency, cold tolerance, an increase in nitrogen use efficiency, and an increase in yield. In some embodiments, the trait is an increase in yield under non-stress conditions or an increase in yield under environmental stress conditions. Stress conditions may include biotic and abiotic stresses, such as drought, shading, fungal diseases, viral diseases, bacterial diseases, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability, and high plant density. "Yield" may be affected by many properties, including but not limited to plant height, plant biomass, pod number, pod position on plant, internode number, incidence of pod breakage, grain size, ear size, ear tip filling, kernel abortion, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stresses, carbon assimilation, plant architecture, resistance to lodging, seed germination percentage, seedling vigor, and early traits. Yield may also be affected by 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, seed number per ear or pod, seed size, seed composition (starch, oil, protein), and seed filling characteristics.

[0109] The term "trait modification" as also used herein includes altering the trait by causing a detectable difference in a characteristic in a plant comprising a mutation in an endogenous CLE gene as described herein, relative to a plant not comprising the mutation (e.g., a wild-type plant or a negative isolate). In some cases, the trait modification can be quantitatively assessed. For example, a trait modification may be an increase or decrease in an observed trait characteristic or phenotype compared to a control plant. It is known that there can be natural variation in the modified trait. Therefore, an observed trait modification requires a normal distribution and a change in the magnitude of the trait characteristic or phenotype in the plant compared to a control plant.

[0110] The present disclosure relates to plants with improved economically important properties, more specifically increased yield. More specifically, the present disclosure relates to plants comprising a mutation in a CLE gene as described herein, wherein the plant has increased yield compared to a control plant without the mutation. In some embodiments, the plants produced as described herein exhibit increased yield or improved yield trait components compared to control plants. In some embodiments, the plants of the present disclosure exhibit improved traits associated with yield, including but not limited to increased nitrogen use efficiency, increased nitrogen stress tolerance, increased water use efficiency, and increased drought tolerance, as defined and discussed below.

[0111] Output can be defined as the measurable output of the economic value of crop. Productivity can be defined in the scope of quantity and / or quality. Output can directly depend on several factors, such as the number and size of organs, plant structure (such as the number of branches, plant biomass, such as increased root biomass, steeper root angle and / or longer root etc.), flowering time and duration, grain filling time. Root structure and development, photosynthetic efficiency, nutrient uptake, stress tolerance, early vigor, delayed senescence and function keep green phenotype can be the factor of determining output. Therefore, optimizing the above-mentioned factors can contribute to increase crop yield.

[0112] Mention the increase / improvement of yield-related traits herein and can also represent the increase of the biomass (weight) of one or more parts of plant, it can comprise above-ground and / or underground (harvesting) plant parts.Especially, this harvestable part is seed, and the execution of the disclosed method causes the plant that yield, particularly seed yield increase relative to suitable control plant.Term " yield " of plant can relate to the nutrient biomass (root and / or bud biomass), reproductive organ and / or propagule (for example seed) of this plant.

[0113] The increased yield of 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., seeds or seed weight per acre), bushels per acre, tons per acre, or thousands per hectare. The increased yield can be caused by improved utilization of key biochemical compounds (e.g., nitrogen, phosphorus, and carbohydrates), or by improved response to environmental stresses (e.g., cold, heat, drought, salt, shade, high plant density, and attack by pests or pathogens).

[0114] "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, particularly 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 improved aboveground area of ​​seedlings at about three weeks after germination.

[0115] "Early vigor" refers to active healthy plant growth, particularly during the early stages of plant growth, which can be caused by an increase in plant adaptability due to, for example, a plant better adapting to its environment (e.g., optimizing the use of energy, the uptake of nutrients, and the carbon allocation between buds and roots). For example, early vigor can be a combination of the ability of seeds to germinate and emerge after planting and the ability of young plants to grow and develop after development. Plants with early vigor also show increased seedling survival and better crop growth, which usually results in highly uniform fields, in which most plants substantially reach various developmental stages at the same time, which usually results in increased yield. 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 bud biomass, canopy size and color, etc.

[0116] Additionally, increased yield may also be manifested as increased total seed yield, which may result from one or more of: an increase in seed biomass (seed weight) due to increased seed weight per plant and / or on an individual seed basis, such as an increase in the number of flowers / panicles per plant; an increase in the number of pods; an increase in the number of nodes; an increase in the number of flowers ("florets") per inflorescence / plant; an increase in seed filling rate; an increase in the number of filled seeds; an increase in seed size (length, width, area, girth), which may also affect seed composition; and / or an increase in seed volume, which may also affect seed composition. In one embodiment, increased yield may be increased seed yield, such as increased seed weight; an increased number of filled seeds; and an increased harvest index.

[0117] Increased yield may also result from modified architecture, or may occur as a result of improved plant architecture.

[0118] Increased yield may also be manifested as an increased harvest index, expressed as the ratio of the yield of harvestable parts, such as seeds, to the total biomass.

[0119] The present disclosure also extends to the harvestable parts of plants, such as but not limited to seeds, leaves, fruits, flowers, bolls, pods, siliques, nuts, stems, rhizomes, tubers and bulbs. The present disclosure also relates to products derived from the harvestable parts of such plants, such as dry particles, powders, oils, fats and fatty acids, starch or proteins.

[0120] The present disclosure provides a method for increasing the "yield" of a plant or the "broad acre yield" of a plant or plant part, which is defined as the harvestable plant part per unit area, such as seeds or seed weight per acre, pounds per acre, bushels per acre, tons per acre, tons per acre, kilograms per hectare.

[0121] As used herein, "nitrogen use efficiency" refers to processes that result in increased plant yield, biomass, vigor, and growth rate per unit of nitrogen applied. The methods may include plant uptake, assimilation, accumulation, signaling, sensing, relocation (within the plant), and use of nitrogen.

[0122] 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 amount of available / applied nitrogen as under normal or standard conditions; the ability of a plant to grow, develop or produce faster or better than normal when subjected to less than an optimal amount of available / applied nitrogen, or under nitrogen limiting conditions.

[0123] 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 plant metabolism, growth, reproductive success and / or vigor.

[0124] As used herein, "increased nitrogen stress tolerance" refers to the ability of a plant to grow, develop or yield normally, or to grow, develop or yield faster or better, when subjected to less than optimal amounts of available / applied nitrogen, or under nitrogen limiting conditions.

[0125] Increased plant nitrogen use efficiency can be understood in the art as a similar amount of yield harvested with less nitrogen supply, or increased yield obtained by supplying an optimal / sufficient amount of nitrogen. Increased nitrogen use efficiency can improve plant nitrogen stress tolerance, and can also improve crop quality and biochemical components of seeds, such as protein yield and oil yield. The terms "improved nitrogen use efficiency", "increased nitrogen use efficiency" and "nitrogen stress tolerance" are used interchangeably in the present disclosure to refer to plants with increased productivity under nitrogen limiting conditions.

[0126] As used herein, "water use efficiency" refers to the amount of carbon dioxide assimilated by leaves per unit of water vapor of transpiration. It constitutes one of the most important characteristics controlling plant productivity in dry environments. "Drought tolerance" refers to the degree to which plants adapt to drought or drought conditions. The physiological response of plants to water deficiency includes leaf atrophy, reduction of leaf area, leaf dehiscence, and stimulating root growth by directing nutrients to the underground parts of plants. Generally, plants are more susceptible to drought during flowering and seed development (reproductive stage) because the resources of plants are biased to support root growth. In addition, abscisic acid (ABA), a plant stress hormone, induces leaf stomata (microscopic pores involving gas exchange) to close, thereby reducing water loss through transpiration and reducing the rate of photosynthesis. These responses improve the water use efficiency of plants in the short term. The terms "increased water use efficiency", "improved water use efficiency" and "increased drought tolerance" are used interchangeably in the present disclosure to refer to plants with improved productivity under water-restrictive conditions.

[0127] As used herein, "increased water use efficiency" refers to the ability of a plant to grow, develop or gain faster or better than normal when subjected to the same amount of available / applied water as under normal or standard conditions; the ability of a plant to grow, develop or gain normally or to grow, develop or gain faster or better when subjected to reduced amounts of available / applied water (water input) or under conditions of water stress or water deficiency stress.

[0128] As used herein, "increased drought tolerance" refers to the ability of a plant to grow, develop or gain normally, or to grow, develop or gain faster or better than normal when subjected to reduced amounts of available / applied water and / or under conditions of acute or chronic drought; or the ability of a plant to grow, develop or gain normally when subjected to reduced amounts of available / applied water (water input) or under conditions of water stress or under conditions of acute or chronic drought.

[0129] As used herein, "drought stress" refers to a dry period (acute or chronic / long term) that results in a lack of water and subjects plants to stress and / or damage to plant tissues and / or negatively affects grain / crop yield; a dry period (acute or chronic / long term) that results in a lack of water and / or higher temperatures and subjects plants to stress and / or damage to plant tissues and / or negatively affects grain / crop yield.

[0130] 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.

[0131] As used herein, "water stress" refers to conditions or circumstances in which an inappropriate amount of water (less / insufficient or more / excessive) is provided than required for adequate / successful growth and development of a plant / crop, thereby causing the plant to experience stress and / or damage to plant tissues and / or adversely affecting grain / crop yield.

[0132] As used herein, "water deficit stress" refers to conditions or circumstances in which less / inadequate water is provided than required for adequate / successful growth and development of a plant / crop, thereby causing the plant to experience stress and / or damage to plant tissues and / or adversely affecting grain yield.

[0133] 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 includes 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 ribose group of the RNA.

[0134] As used herein, the term "nucleotide sequence" refers to a heteropolymer of nucleotides or a sequence of these nucleotides from the 5' end to the 3' end of a nucleic acid molecule, including 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 a 5' to 3' direction from left to right herein, and are represented using the standard code for representing nucleotide features set forth in the U.S. sequencing rules 37CFR§§1.821-1.825 and the World Intellectual Property Organization (WIPO) standard ST.25. As used herein, "5' district" may refer to the region closest to the 5' end of a polynucleotide. Thus, for example, an element in the 5' region of a polynucleotide may be located anywhere from the first nucleotide at the 5' end of the polynucleotide to a nucleotide in the middle of the polynucleotide. As used herein, a "3' region" may refer to a region of a polynucleotide that is closest to the 3' end of the polynucleotide. Thus, for example, an element in the 3' region of a polynucleotide may be located anywhere from the first nucleotide at the 3' end of the polynucleotide to a nucleotide in the middle of the polynucleotide.

[0135] As used herein with respect to nucleic acids, the terms "fragment" or "portion" refer to a portion of a nucleic acid that is shorter in length (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 400, 450, 500, 550, 600, 701, 711, 721, 731, 743, 751, 764, 775 The invention relates to a nucleic acid having a length of 50, 650, 700, 750, 800, 850 or 900 nucleotides or more, or any range or value therebetween, and which comprises, consists essentially of and / or consists of a nucleotide sequence of consecutive 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. If appropriate, such a nucleic acid fragment can be contained in a larger polynucleotide as a component thereof. For example, the repeat sequence of the guide nucleic acid of the invention can comprise a "portion" of a wild-type CRISPR-Cas repeat sequence (e.g., a wild-type CRISPR-Cas repeat sequence; e.g., repeats 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.).

[0136] In some embodiments, the nucleic acid fragment may comprise 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、50、55、60、65、70、75、80、85、90、95、100、110、120、130、140、150、160、170、180、190、200、210、220、230、240、250、260、270、280、285、290、300、310、3 350, 375, 3839, or 40 consecutive nucleotides in length, about 85, 90, 95, 100 consecutive nucleotides to about 300, 320, 350, 375, 400, or more consecutive nucleotides in length, or any range or value therein. In some embodiments, a fragment of a nucleic acid encoding a CLE polypeptide can be about 36 contiguous nucleotides in length.

[0137] In some embodiments, a "sequence-specific nucleic acid binding domain" can bind to one or more fragments or portions of a nucleotide sequence (eg, DNA, RNA) encoding a CLE polypeptide, such as described herein.

[0138] As used herein with respect to polypeptides, the term "fragment" or "portion" may refer to a polypeptide of reduced length relative to a reference polypeptide, which comprises or consists essentially of an amino acid sequence of consecutive amino acids identical or nearly identical (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical) to the corresponding portion of the reference polypeptide. Where appropriate, such a polypeptide fragment may be included in a larger polypeptide as a constituent part thereof. In some embodiments, the 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, or 290 or more contiguous amino acids of a reference polypeptide. In some embodiments, the polypeptide fragment can comprise about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 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 0, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135 or 136 or more consecutive amino acid residues, or any range or value therebetween (e.g., SEQ ID NO: 1

[00136] In some embodiments, the invention relates to fragments or portions of SEQ ID NOs: 66-68 (e.g., SEQ ID NOs: 69-71)), consisting essentially of, or consisting of.

[0139] In some embodiments, such a deletion may be a dominant negative allele, a semi-dominant allele, a weak loss-of-function allele, a null allele, or a hypomorphic mutation, which when included in a plant may result in the plant exhibiting an increased number of ear rows compared to a plant not comprising the deletion. In some embodiments, such plants may also exhibit increased yield and increased nematode resistance, as well as larger meristems. A CLE gene may be edited at more than one position, thereby providing a CLE gene comprising more than one mutation. In some embodiments, a CLE polypeptide mutated as described herein may comprise more than one edit, which may result in a peptide having more than one amino acid substitution.

[0140] In some embodiments, a “portion” with respect to 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 ,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,8 2, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 285, 2 In some embodiments, the portion of the genomic sequence encoding a CLE polypeptide may be about 285, 399, or 411 consecutive nucleotides in length (e.g., SEQ ID NOs: 78-80; e.g., a CLE coding sequence). In some embodiments, the portion of a CLE polypeptide gene may be about 36 consecutive nucleotides in length (e.g., SEQ ID NOs: 81-83). In some embodiments, by “portion” with respect to a polypeptide is meant at least 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 or more contiguous amino acid residues from a polypeptide (e.g., a CLE polypeptide).In some embodiments, a "portion" of a CLE polypeptide can have about 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32 or more contiguous amino acid residues (e.g., amino acid residues 53-104, 63-94, 73-84 of SEQ ID NO:72 or SEQ ID NO:73; or amino acid residues 78-129, 88-119, 98-109 of SEQ ID NO:74).

[0141] As used herein with respect to nucleic acids, the term "functional fragment" refers to a nucleic acid that encodes a functional fragment of a polypeptide.

[0142] 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 be used or not 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 may be "isolated", which refers to nucleic acid being substantial or substantially free of components that are usually found to be associated with the nucleic acid present in its natural state. Such components include other cell materials, culture media, and / or various chemicals for chemically synthesizing the nucleic acid from recombinant production.

[0143] The term "mutation" refers to a point mutation (e.g., missense or nonsense, or insertion or deletion of a single base pair that results in a frameshift), insertion, deletion, inversion, and / or truncation. When a mutation is a replacement of a residue in an amino acid sequence by another residue, or a deletion or insertion of one or more residues in a sequence, the mutation is typically described by listing the original residue, followed by the position of the residue in the sequence, and the identity of the newly replaced residue. Truncation may include truncation at the C-terminus of a polypeptide or at the N-terminus of a polypeptide. Truncation of a polypeptide may be the result of a deletion of the corresponding 5' end or 3' end of a gene encoding the polypeptide. Frameshift mutations may occur when deletions or insertions of one or more base pairs are introduced into a gene. Frameshift mutations in a gene may result in the production of a polypeptide that is longer, shorter, or of the same length as a wild-type polypeptide, depending on when the first stop codon appears after the mutation region of the gene. In some embodiments, a mutation may be a DNA transversion of about 10 to about 2000 consecutive base pairs.

[0144] 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.

[0145] As used herein, "complementary" can mean 100% complementarity to a comparison nucleotide sequence, or it can mean less than 100% complementarity to a comparison nucleotide sequence (e.g., 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., complementary).

[0146] 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 expressed as a percentage of positional identity (i.e., sequence similarity or identity) between two or more nucleic acids and / or amino acid sequences. Homology also refers to the concept of similar functional properties between different nucleic acids or proteins. Therefore, the compositions and methods of the present invention also include homologs to the nucleotide sequences and polypeptide sequences of the present invention. As used herein, "orthologous" refers to homologous nucleotide sequences and / or amino acid sequences in different species produced from a common ancestral gene during species formation. Homologs of the nucleotide sequences of the invention may 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.

[0147] As used herein, "sequence identity" refers to the degree to which two optimally aligned polynucleotide or polypeptide sequences are unchanged over a component (e.g., nucleotide or amino acid) comparison window. "Identity" can be readily calculated by known methods, including, but not limited to, those described in Computational Molecular Biology (Lesk, AM, ed.) Oxford University Press, New York (1988); Biocomputing: Informatics and Genome Projects (Smith, DW, ed.) Academic Press, New York (1993); Computer Analysis of Sequence Data, Part I (Griffin, AM and Griffin, HG, ed.) Humana Press, New Jersey (1994); Sequence Analysis in Molecular Biology (von Heinje, G., ed.) Academic Press (1987); and Sequence Analysis Primer (Gribskov, M. and Devereux, J., ed.) Stockton Press, New York (1991).

[0148] As used herein, the term "percentage of sequence identity" or "percentage of identity" refers to the percentage of identical nucleotides in the linear polynucleotide sequence of a reference ("query") polynucleotide molecule (or its complementary strand) and the linear polynucleotide sequence of a test ("subject") polynucleotide molecule (or its complementary strand) when the two sequences are optimally aligned. In some embodiments, "percentage of sequence identity" may refer to the percentage of identical amino acids in an amino acid sequence compared to a reference polypeptide.

[0149] As used herein, the phrases "substantially identical" / "substantially identical" or "substantial identity" in the context of two nucleic acid molecules, nucleotide sequences, or polypeptide sequences refer to two or more sequences or subsequences having at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, or 100% nucleotide or amino acid residue identity when compared and aligned for maximum correspondence as measured using one of the following sequence comparison algorithms or by visual inspection. In some embodiments of the invention, substantial identity exists over a contiguous region of nucleotides of a nucleotide sequence of the invention, the contiguous region of nucleotides being about 10 nucleotides to about 20 nucleotides, about 10 nucleotides to about 25 nucleotides, about 10 nucleotides to about 30 nucleotides, about 15 nucleotides to about 25 nucleotides, about 30 nucleotides to about 40 nucleotides, about 50 nucleotides to about 60 nucleotides, about 70 nucleotides to about 80 nucleotides, about 90 nucleotides to about 100 nucleotides, about 100 nucleotides to about 200 nucleotides, about 100 nucleotides to about 300 nucleotides, about 100 nucleotides to about 400 nucleotides, about 100 nucleotides to about 500 nucleotides, about 100 nucleotides to about 600 nucleotides, about 100 nucleotides to about 800 nucleotides, about 100 nucleotides to about 900 nucleotides, or more nucleotides in length, or any range therebetween, 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).

[0150] 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 about 3 amino acid residues to about 20 amino acid residues, about 5 amino acid residues to about 25 amino acid residues, about 7 amino acid residues to about 30 amino acid residues, about 10 amino acid residues to about 25 amino acid residues, about 15 amino acid residues to about 30 amino acid residues, about 20 amino acid residues to about 40 amino acid residues, about 25 amino acid residues to about 40 amino acid residues, about 25 amino acid residues to about 50 amino acid residues, about 30 amino acid residues to about 50 amino acid residues, about 40 amino acid residues to about 50 amino acid residues, about 40 amino acid residues to about 70 amino acid residues, about 50 amino acid residues to about 70 amino acid residues, about 60 amino acid residues to about 80 amino acid residues, about 70 amino acid residues to about 80 amino acid residues, about 90 amino acid residues to about 100 amino acid residues, or more amino acid residues in length, and any ranges therein, up to the full length of the sequence. 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, 111 150, 175, 200, 225, 250, 300, 350 or more amino acids, or more consecutive amino acid residues).In some embodiments, two or more CLE polypeptides may be identical or substantially identical (e.g., at least 70% to 99.9% identical); for example, 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 therebetween.

[0151] For sequence comparison, usually one sequence is used as a reference sequence to which a test sequence is compared. 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 one or more test sequences relative to the reference sequence based on the specified program parameters.

[0152] Optimal alignment of sequences for aligning a comparison window is well known to those skilled in the art and can be performed by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the similarity search method of Pearson and Lipman, and optionally by computerized implementations of these algorithms (such as GAP, BESTFIT, FASTA and TFASTA (available as Wisconsin (Accelrys Inc., San Diego, Ca)). The "identity score" of the aligned segments of a test sequence and a reference sequence is the number of identical components shared by the two aligned sequences divided by the total number of components in the segment of the reference sequence (e.g., the entire reference sequence or a smaller defined portion of the reference sequence). The percent sequence identity is expressed as the identity score multiplied by 100. The comparison of one or more polynucleotide sequences can be a comparison to the full-length polynucleotide sequence or a portion thereof, or a comparison to a longer polynucleotide sequence. For the purposes of the present invention, BLASTX version 2.0 may also be used for translated nucleotide sequences, and BLASTN version 2.0 may be used for polynucleotide sequences to determine the "percent identity".

[0153] 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 considered to be substantially complementary hybridize to each other under highly stringent conditions.

[0154] "Stringent hybridization conditions" and "stringent hybridization wash conditions" in the context of nucleic acid hybridization experiments such as Southern and Northern hybridizations are sequence-dependent and are different under different environmental parameters. A detailed guide to nucleic acid hybridization can be found in Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Part I, Chapter 2 "Overview of principles of hybridization and the strategy of nucleic acid probe assays", Elsevier, New York (1993). Generally, highly stringent hybridization and wash conditions are selected to be higher than the thermal melting point (Tk) for the specific sequence at a defined ionic strength and pH. m ) is about 5℃ lower.

[0155] 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 selected to be equal to the T for a specific probe. m. In Southern or Northern blotting, an example of stringent hybridization conditions for hybridization of complementary nucleotide sequences having more than 100 complementary residues on a filter membrane is 50% formamide and 1 mg heparin at 42° C., where hybridization is performed overnight. An example of highly stringent washing conditions is washing with 0.15 M NaCl at 72° C. for about 15 minutes. An example of stringent washing conditions is washing with 0.2x SSC at 65° C. for 15 minutes (for a description of SSC buffer, see Sambrook, supra). Typically, a high stringency wash is preceded by a low stringency wash to remove background probe signals. For example, for duplexes of more than 100 nucleotides, an example of a moderate stringency wash is washing with 1x SSC at 45° C. for 15 minutes. For example, for duplexes of more than 100 nucleotides, an example of a low stringency wash is washing with 4-6x SSC at 40° C. for 15 minutes. For short probes (e.g., about 10 to 50 nucleotides), stringent conditions typically involve a salt concentration of less than about 1.0 M Na ions, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3, and a temperature of typically at least about 30° C. Stringent conditions can also be achieved by adding destabilizing agents such as formamide. In general, a signal-to-noise ratio of 2 times (or higher) that observed for an unrelated probe in a particular hybridization assay indicates that specific hybridization has been detected. If the proteins encoded by nucleotide sequences that do not hybridize to each other under stringent conditions are substantially identical, the nucleotide sequences are still substantially identical. This can occur, for example, when a copy of a nucleotide sequence is produced using the maximum codon degeneracy allowed by the genetic code.

[0156] 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., DNA binding domains) (e.g., from polynucleotide-guided endonucleases, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), Argonaute proteins and / or CRISPR-Cas endonucleases (e.g., CRISPR-Cas effector proteins) (e.g., type I CRISPR-Cas effector proteins, type II CRISPR-Cas effector proteins, type III CRISPR-Cas effector proteins, type IV CRISPR-Cas effector proteins, 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 In some embodiments, the codon-optimized nucleic acids, polynucleotides, expression cassettes and / or vectors of the invention are codon-optimized for expression in plants. In some embodiments, the codon-optimized nucleic acids, polynucleotides, expression cassettes and / or vectors of the invention are codon-optimized with reference nucleic acids that are not codon-optimized. The nucleic acids, polynucleotides, expression cassettes and / or vectors may have about 70% to about 99.9% (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or 100%) identity or more.

[0157] In any of the embodiments described herein, the polynucleotides or nucleic acid constructs of the present invention can be operably connected to 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, the promoter may be operably associated with an intron (e.g., an Ubil promoter and an intron). In some embodiments, the promoter associated with an intron may be referred to as a "promoter region" (e.g., an Ubil promoter and an intron).

[0158] As used herein, "operably connected" or "operably associated" when referring to a polynucleotide means that the elements shown are functionally related to each other, and usually also physically related. Therefore, as used herein, the term "operably connected" or "operably associated" refers to a nucleotide sequence that is functionally associated on a single nucleic acid molecule. Therefore, a first nucleotide sequence that is operably connected to a second nucleotide sequence means a situation when the first nucleotide sequence is placed in a functional relationship with the second nucleotide sequence. For example, if a promoter achieves transcription or expression of a nucleotide sequence, the promoter is operably associated with the nucleotide sequence. It will be understood by those skilled in the art that a control sequence (e.g., a promoter) does not need to be adjacent to a nucleotide sequence that is operably associated with it, as long as the control sequence is able to direct its expression. Therefore, for example, an intervening, untranslated, but still transcribed nucleic acid sequence may be present between a promoter and the nucleotide sequence, and the promoter may still be considered to be "operably connected" to the nucleotide sequence.

[0159] As used herein, the term "linked" when referring to polypeptides refers to the attachment of one polypeptide to another polypeptide. A polypeptide can be linked to another polypeptide (at the N-terminus or C-terminus) directly (e.g., by a peptide bond) or through a linker.

[0160] The term "joint" is recognized in the art and refers to a chemical group or molecule that connects two molecules or parts (e.g., two domains of a fusion protein, such as a nucleic acid binding polypeptide or domain and a peptide tag and / or a reverse transcriptase and an affinity polypeptide bound to a peptide tag; or a DNA endonuclease polypeptide or domain and a peptide tag and / or a reverse transcriptase and an affinity polypeptide bound to a peptide tag). The joint can consist of a single connecting molecule, or can contain more than one connecting molecule. In some embodiments, the joint can be an organic molecule, a group, a polymer, or a chemical part, such as a divalent organic part. In some embodiments, the joint can be an amino acid or it can be a peptide. In some embodiments, the joint is a peptide.

[0161] In some embodiments, the length of a peptide linker useful in the present invention can be from about 2 to about 100 or more amino acids, for example, about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 7, 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 50, about 4 to about 60, about 5 to about 50, about 5 ...60, about 5 to about 50, about 5 to about 60, about 5 to about 60, about 5 to about 70, about 5 to about 70, about 5 to about 70, about 5 to about 70, about 5 to about 70, about 5 35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110 100, 110, 115, 120, 130, 140, 150 or more amino acids in length). In some embodiments, the peptide linker can be a GS linker.

[0162] As used herein, the term "connected" or "fused" with respect to polynucleotides refers to the attachment 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. A polynucleotide can be connected or fused to another polynucleotide (at the 5' end or the 3' end) by covalent or non-covalent bonding or binding (including, for example, 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 (e.g., 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.

[0163] "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 directs the initiation of transcription. Generally, the promoter is located 5' or upstream relative to the start of the coding region of the corresponding coding sequence. The promoter may contain other elements that act as regulators of gene expression; for example, a promoter region. These include a TATA box consensus sequence, and are typically 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).

[0164] 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, such as "synthetic nucleic acid constructs" or "protein-RNA complexes." These different types of promoters are known in the art.

[0165] The selection of promoter can vary according to the time and space requirements of expression, and also can vary according to the host cell to be transformed. Promoters for many different organisms are well known in the art. Based on the extensive knowledge in this area, suitable promoters can be selected for specific target host organisms. Therefore, for example, promoters upstream of genes highly constitutively expressed in model organisms are well understood, and this type of knowledge can be easily accessed and applied to other systems in appropriate situations.

[0166] In some embodiments, promoters functional in plants can be used with the constructs of the present invention. Non-limiting examples of promoters that can be used to drive expression in plants include the promoter of RubisCo small subunit gene 1 (PrbcS1), the promoter of the actin gene (Pactin), the promoter of the nitrate reductase gene (Pnr), and the promoter of the repeat carbonic anhydrase gene 1 (Pdca1) (see Walker et al., 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 can be used in the constructs of the present invention. In some embodiments, the U6c promoter and / or the 7SL promoter from corn can be used to drive the expression of the guide nucleic acid. In some embodiments, the U6c promoter, U6i promoter and / or the 7SL promoter from soybean can be used in the constructs of the present invention. In some embodiments, the U6c promoter, U6i promoter and / or the 7SL promoter from soybean can be used to drive the expression of the guide nucleic acid.

[0167] Examples of constitutive promoters that can be used in plants include, but are not limited to, cestrum virus promoter (cmp) (U.S. Pat. No. 7,166,770), rice actin 1 promoter (Wang et al. (1992) Mol. Cell. Biol. 12:3399-3406; and U.S. Pat. No. 5,641,876), CaMV 35S promoter (Odell et al. (1985) Nature 313:810-812), CaMV 19S promoter (Lawton et al. (1987) Plant Mol. Biol. 9:315-324), nos promoter (Ebert et al. (1987) Proc. Natl. Acad. Sci. USA 84:5745-5749), Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:5745-5749), and Adh promoter (Walker et al. (1987) Proc. Natl. Acad. Sci. USA 84:5745-5749). 84:6624-6629), sucrose synthase promoter (Yang & 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 transgenic plants, such as sunflower (Binet et al., 1991. Plant Science 79:87-94), corn (Christensen et al., 1989, Plant Molec. Biol. 12:619-632) and Arabidopsis thaliana (Norris et al. 1993. Plant Molec. Biol. 21:895-906). The corn ubiquitin promoter (UbiP) has been developed in a transgenic monocot system, and its sequence and construction of vectors for monocot transformation are disclosed in patent publication EP 0 342 926. The ubiquitin promoter is suitable for expressing the nucleotide sequence of the present invention in transgenic plants, especially monocotyledons. In addition, the promoter expression cassette described by McElroy et al. (Mol. Gen. Genet. 231: 150-160 (1991)) can be easily modified to express the nucleotide sequence of the present invention, and is particularly suitable for monocotyledon hosts.

[0168] In some embodiments, tissue-specific / tissue-preferred promoters can be used to express heterologous polynucleotides in plant cells. Tissue-specific or preferred expression patterns include, but are not limited to, green tissue-specific or preferred, root-specific or preferred, stem-specific or preferred, flower-specific or preferred, or pollen-specific or preferred expression patterns. Promoters suitable for expression in green tissues include many promoters that regulate the genes involved in photosynthesis, and many of these promoters have been cloned from monocots and dicots. In one embodiment, the promoter that can be used for the present invention is the corn PEPC promoter (Hudspeth & Grula, Plant Molec. Biol. 12: 579-589 (1989)) from the phosphoenol carboxylase gene. Non-limiting examples of tissue-specific promoters include those associated with genes encoding seed storage proteins such as β-conglycinin, cruciferin, napin, and phaseolin, zein or oil body proteins such as oleosin, or proteins involved in fatty acid biosynthesis, including acyl carrier protein, stearoyl-ACP desaturase, and fatty acid desaturase (fad 2-1), as well as other nucleic acids expressed during embryonic development (such as Bce4, see, e.g., Kridl et al. (1991) Seed Sci. Res. 1:209-219; and European 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, promoters that direct expression in roots, pith, leaves, or pollen. Such promoters are disclosed, e.g., in WO 93 / 07278 (incorporated herein by reference in its entirety).Other non-limiting examples of tissue-specific or tissue-preferential promoters that can be used in the present invention include the cotton rubisco promoter disclosed in U.S. Pat. No. 6,040,504; the rice sucrose synthase promoter disclosed in U.S. Pat. No. 5,604,121; the root-specific promoter described by de Framond (FEBS 290: 103-106 (1991); EP 0 452 269 to Ciba-Geigy); the stem-specific promoter described in U.S. Pat. No. 5,625,136 (to Ciba-Geigy), which drives expression of the maize trpA gene; the tuberose yellow leaf curl virus promoter disclosed in WO 01 / 73087; and pollen-specific or -preferential promoters, including but not limited to ProOsLPS10 and ProOsLPS11 from rice (Nguyen et al., Plant Biotechnol. Reports 2004). 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), PLA2-δ promoter from Arabidopsis thaliana (U.S. Pat. No. 7,141,424) and / or ZmC5 promoter from maize (International PCT Publication No. WO1999 / 042587).

[0169] Other examples of plant tissue-specific / tissue-preferred promoters include, but are not limited to, root hair-specific cis-element (RHE) (Kim et al., The Plant Cell 18:2958-2970 (2006)), root-specific promoters RCc3 (Jeong et al., Plant Physiol. 153:185-197 (2010)) and RB7 (U.S. Pat. No. 5,459,252), plant lectin promoter (Lindstrom et al. (1990) Der. Genet. 11:160-167; and Vodkin (1983) Prog. Clin. Biol. Res. 138:87-98), corn alcohol dehydrogenase 1 promoter (Dennis et al. (1984) Nucleic Acids Res. 12:3983-4000), S-adenosyl-L-methionine synthetase (SAMS) (Vander Mijnsbrugge et al. (1996) Plant Physiol. 154:185-197 (2010)), and RB7 (U.S. Pat. No. 5,459,252). 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: Genetic Engineering of Plants (Hollaender, ed., Plenum Press, 1996). 1983; and Poulsen et al. (1986) Mol. Gen. Genet. 205:193-200), the Ti plasmid mannopine synthase promoter (Langridge et al. (1989) Proc. Natl. Acad. Sci. USA 86:3219-3223), the Ti plasmid nopaline synthase promoter (Langridge et al. (1989), supra), the petunia chalcone isomerase promoter (van Tunen et al. (1988) EMBO J. 7:1257-1263), the soybean 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 tuber storage protein (patatin) 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 & 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).

[0170] Useful for seed-specific expression is the vicilin promoter of pea (Czako et al. (1992) Mol. Gen. Genet. 235:33-40); and the seed-specific promoters disclosed in U.S. Pat. No. 5,625,136. Useful promoters for expression in mature leaves are those that are switched at the onset of senescence, such as the SAG promoter from Arabidopsis thaliana (Gan et al. (1995) Science 270:1986-1988).

[0171] In addition, promoters that are functional in chloroplasts can be used. Non-limiting examples of such promoters include the 5'UTR of bacteriophage T3 gene 9 and other promoters disclosed in U.S. Pat. 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).

[0172] Other regulatory elements useful in the present invention include, but are not limited to, introns, enhancers, termination sequences, and / or 5' and 3' untranslated regions.

[0173] Introns useful in the present invention can be introns identified and isolated from plants, and then inserted into expression cassettes for plant transformation. As will be appreciated by those skilled in the art, introns can contain sequences required for self-excision and be 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 inside a protein coding sequence, for example, to stabilize mRNA. If they are used in protein coding sequences, they are inserted "in-frame," including excision sites. Introns can also be combined with promoters to improve or modify expression. For example, promoter / intron combinations useful in the present invention include, but are not limited to, combinations of maize Ubil promoters and introns (see, for example, SEQ ID NO: 21 and SEQ ID NO: 22).

[0174] Non-limiting examples of introns that can be used in the present invention include introns from the ADHI gene (e.g., Adh1-S introns 1, 2, and 6), the ubiquitin gene (Ubil), the RuBisCO small subunit (rbcS) gene, the RuBisCO large subunit (rbcL) gene, the actin gene (e.g., the actin-1 intron), the pyruvate dehydrogenase kinase gene (pdk), the nitrate reductase gene (nr), the repetitive carbonic anhydrase gene 1 (Tdca1), the psbA gene, the atpA gene, or any combination thereof.

[0175] In some embodiments, the polynucleotides and / or nucleic acid constructs of the present invention may be "expression cassettes" or may be contained in expression cassettes. As used herein, "expression cassettes" means recombinant nucleic acid molecules comprising, for example, one or more polynucleotides of the present invention (e.g., polynucleotides encoding sequence-specific nucleic acid binding domains, polynucleotides encoding deaminase proteins or domains, polynucleotides encoding reverse transcriptase proteins or domains, polynucleotides encoding 5'-3' exonuclease polypeptides or domains, guide nucleic acids and / or reverse transcriptase (RT) templates), wherein one or more polynucleotides are operably associated with one or more control sequences (e.g., promoters, terminators, etc.). Therefore, in some embodiments, one or more expression cassettes may be provided, which are designed to express, for example, nucleic acid constructs of the present invention (e.g., polynucleotides encoding sequence-specific nucleic acid binding domains, polynucleotides encoding nuclease polypeptides / domains, polynucleotides encoding deaminase proteins / domains, polynucleotides encoding reverse transcriptase proteins / domains, polynucleotides encoding 5'-3' exonuclease polypeptides / domains, polynucleotides encoding peptide tags and / or polynucleotides encoding affinity polypeptides, etc., or guide nucleic acids, extended guide nucleic acids and / or RT templates, etc.). When the expression cassette of the present invention comprises more than one polynucleotide, the polynucleotide can be operably linked to a single promoter that drives the expression of all polynucleotides, or the polynucleotide can be operably linked to one or more separate promoters (e.g., three polynucleotides can be driven by one, two, or three promoters in any combination). When two or more different promoters are used, the promoters can be the same promoter or different promoters. Thus, when contained in a single expression cassette, polynucleotides encoding sequence-specific nucleic acid binding domains, polynucleotides encoding nuclease proteins / domains, polynucleotides encoding CRISPR-Cas effector proteins / domains, polynucleotides encoding deaminase proteins / domains, polynucleotides encoding reverse transcriptase polypeptides / domains (e.g., RNA-dependent DNA polymerases), and / or polynucleotides encoding 5'-3' exonuclease polypeptides / domains, guide nucleic acids, extended guide nucleic acids, and / or RT templates can each be operably linked to a single promoter or independent promoters in any combination.

[0176] The expression cassette comprising the nucleic acid construct of the invention may be chimeric, meaning that at least one (e.g., one or more) 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 found associated with the promoter). The expression cassette may also be one that occurs naturally but has been obtained in a recombinant form for heterologous expression.

[0177] The expression cassette may optionally include a transcription and / or translation termination region (i.e., termination region) and / or an enhancer region that is functional in the selected host cell. Various transcription terminators and enhancers are known in the art and are available for use in expression cassettes. The transcription terminator is responsible for the termination of transcription and correct mRNA polyadenylation. The termination region and / or enhancer region may be natural for the transcription initiation region, may be natural for, 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 the host cell, or may be natural to another source (e.g., for example, a promoter, a gene encoding a sequence-specific nucleic acid binding protein, a gene encoding a nuclease, a gene encoding a reverse transcriptase, a gene encoding a deaminase, etc., or may be foreign or heterologous to the host cell or any combination thereof).

[0178] The expression cassette of the present invention may also include a polynucleotide encoding a selective marker, which can be used to select a transformed host cell. As used herein, a "selective marker" refers to a polynucleotide sequence that, when expressed, gives a unique phenotype to a host cell expressing the marker, thereby distinguishing such transformed cells from those that do not have the marker. This polynucleotide sequence can encode a selective marker or a screenable marker, depending on whether the marker gives a trait that can be selected by chemical means, such as by using a selection agent (e.g., antibiotics, etc.), or depends on whether the marker is only a trait that can be identified by observation or testing, such as by screening (e.g., fluorescence). Many examples of suitable selective markers are known in the art and can be used in expression cassettes as described herein.

[0179] In addition to the expression cassette, 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 acid (or multiple nucleic acids) into a cell. The vector comprises a nucleic acid construct (e.g., an expression cassette) containing one or more nucleotide sequences to be transferred, delivered or introduced. The vector used to transform a host organism is well known in the art. Non-limiting examples of general classes of vectors include viral vectors, plasmid vectors, phage vectors, phagemid vectors, cosmid vectors, fosmid vectors, phages, artificial chromosomes, mini-rings or Agrobacterium (Agrobacterium) binary vectors in double-stranded or single-stranded linear or circular forms, which may or may not be self-transmitted 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. The vector defined herein can be transformed into a prokaryotic or eukaryotic host by being integrated into the cell genome or being present outside the chromosome (e.g., an autonomously replicating plasmid with a replication origin). Also include shuttle vectors, which refer to DNA vectors that can be replicated in two different host organisms naturally or by design, and the host organisms can be selected from actinomycetes and related species, bacteria and eukaryotic organisms (e.g., higher plants, mammals, yeast or fungal cells). In some embodiments, the nucleic acid in the vector is under the control of a suitable promoter or other regulatory elements for transcription in the host cell and is operably connected thereto. The vector can be a bifunctional expression vector that works in a variety of hosts. In the case of genomic DNA, this may include its own promoter and / or other regulatory elements, and in the case of cDNA, this may be under the control of a suitable promoter and / or other regulatory elements, so as to express in the host cell. Therefore, nucleic acids or polynucleotides of the present invention and / or expression cassettes comprising them may be included in vectors described herein and known in the art.

[0180] 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). For example, the target nucleic acid can be contacted with a sequence-specific nucleic acid binding protein (e.g., a polynucleotide-guided endonuclease, a CRISPR-Cas endonuclease (e.g., a CRISPR-Cas effector protein), a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and / or an Argonaute protein)) and a deaminase or a nucleic acid construct encoding the same, under conditions where the sequence-specific DNA binding protein, the reverse transcriptase, and / or the deaminase are expressed and 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 for 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.

[0181] As used herein, "modification" or "modification" of a target nucleic acid includes editing (e.g., mutation), covalent modification, exchanging / replacing nucleic acid / nucleotide bases, deleting, cutting, nicking, and / or changing the transcriptional control of the target nucleic acid. In some embodiments, the modification may include any type of one or more single base changes (SNPs).

[0182] "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 gain access to the interior of the cell.

[0183] 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 invention. In some embodiments, a host cell or host organism can be transiently transformed with a polynucleotide / nucleic acid molecule of the invention.

[0184] "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.

[0185] "Stably introducing" or "stably introduced" in the context of a polynucleotide introduced into a cell is intended to mean that the introduced polynucleotide is stably integrated into the genome of the cell, such that the cell is stably transformed with the polynucleotide.

[0186] As used herein, "stable transformation" or "stably transformed" means that a nucleic acid molecule is introduced into a cell and integrated into the genome of the cell. Thus, the integrated nucleic acid molecule is capable of being inherited by its progeny, more specifically, by multiple successive generations of progeny. As used herein, "genome" includes both 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 (e.g., as a minichromosome or plasmid).

[0187] Transient transformation can be detected by, for example, enzyme-linked immunosorbent assay (ELISA) or Western blot, 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 analysis of cell genomic DNA with nucleic acid sequences, which 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 assay of cell RNA with nucleic acid sequences, which 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 use 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 schemes well known in the art.

[0188] 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 integrated into the genome of the 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 cells with guide nucleic acids, and thus, no DNA is retained in the cells.

[0189] The nucleic acid construct of the present invention can be introduced into plant cells by any method known to those skilled in the art. The non-limiting examples of transformation methods include nucleic acid delivery (for example, by agrobacterium) mediated by bacteria, nucleic acid delivery mediated by virus, nucleic acid delivery mediated by silicon carbide or nucleic acid whiskers, nucleic acid delivery mediated by liposomes, 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 (including any combination thereof) conversion that causes nucleic acid to be introduced into plant cells. The method for transforming eukaryotic organisms and prokaryotic organisms is a conventional method well known in the art, and is described in the entire document (see, for example, Jiang et al. 2013.Nat.Biotechnol.31:233-239; Ran et al. Nature Protocols 8:2281-2308 (2013)). General guides to 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)).

[0190] In some embodiments of the present invention, the transformation of cells can include nuclear transformation. In other embodiments, the transformation of cells can include plastid transformation (e.g., chloroplast transformation). In another other embodiment, nucleic acid of the present invention can be introduced into cells by conventional breeding techniques. In some embodiments, one or more of polynucleotides, expression cassettes and / or vectors can be introduced into plant cells by Agrobacterium transformation.

[0191] Therefore, polynucleotide can be introduced into plant, plant part, plant cell in many ways well known in the art.Method of the present invention does not rely on the ad hoc method that one or more nucleotide sequences are introduced into plant, only they are required to enter cell interior.When introducing more than one polynucleotide, they can be assembled as a part of single nucleic acid construct, or assembled as independent nucleic acid construct, and can be located on same or different nucleic acid construct.Therefore, described polynucleotide can be introduced into interested cell in single transformation event or in the transformation event separated, perhaps, polynucleotide can be integrated into plant as a part of breeding scheme.

[0192] The present invention provides methods and compositions for reducing the effects of genes that normally act to limit meristem size, to produce plants with larger meristems, to increase the number of ear rows, optionally without substantially reducing ear length, to provide improved yield traits, and to improve resistance to nematode infection. In some embodiments, the methods of the present invention provide a dominant negative effect, wherein gene function is reduced in a genetically dominant manner, allowing the introduced mutation to be effective in hybrid crop systems and / or when genetic redundancy may be an issue.

[0193] Plants express a variety of CLAVATA3 / EMBRYO SURROUNDING REGION-RELATED (CLV3 / ESR-RELATED) CLV3-like peptides (CLEs) that act in different parts of the plant to control stem cell function and growth, including regulating meristem size. CLE peptides are sensed by many leucine-rich repeat (LRR) proteins, and downstream signaling regulates numerous growth processes in plants (Fletcher, JC, Plants 7:87 (2018)). CLE proteins are expressed as longer pre-propeptides, which are processed into shorter active peptides that function as extracellular ligands.

[0194] Thus, as described herein, editing techniques are used to target CLE genes in plants to alter stem cell homeostasis, thereby producing plants with larger meristems, increased ear rows, increased yields, and improved nematode resistance. Mutations that can be used to produce plants that exhibit increased ear rows include, for example, substitutions, deletions, insertions, and / or transversions. In some aspects, the mutations generated by the editing techniques can be point mutations. In some embodiments, the mutations generated as described herein can be dominant negative mutations, semi-dominant mutations, null mutations, sub-effect mutations, or weak loss-of-function mutations.

[0195] CLE genes / peptides include three related genes / peptides, CLE7 (e.g., ZmCLE7 in maize), the CLV3 ortholog in Arabidopsis, and FCP1 and CLE14 (e.g., ZmFCP1 and ZmCLE14 in maize). In some embodiments, the present invention describes editing the mature peptide sequence of an endogenous CLE gene to mediate a weak loss of function or a dominant negative effect on meristem growth, thereby increasing yield. For example, a P9L mutation that results in a weak loss of function, and a G6T or D8A mutation that provides a dominant negative effect, are generated.

[0196] In some embodiments, the present invention provides a plant or plant part thereof, comprising at least one (e.g., one or more) non-natural mutations in an endogenous CLAVATA3 / EMBRYO SURROUNDING REGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene encoding a wild-type precursor CLE polypeptide. In some embodiments, the at least one non-natural mutation provides a dominant negative mutation, a semi-dominant mutation, a null mutation, a hypomorphic mutation, and / or a weak loss-of-function mutation.

[0197] In some embodiments, a plant cell is provided, the plant cell comprising an editing system, the editing system comprising: (a) a CRISPR-Cas effector protein; and (b) a guide nucleic acid (e.g., gRNA, gDNA, crRNA, crDNA, sgRNA, sgDNA), the guide nucleic acid comprising a spacer sequence complementary to an endogenous target gene encoding a CLE protein. The editing system can be used to generate a mutation in an endogenous target gene encoding a CLE protein. In some embodiments, the mutation is a non-natural mutation. In some embodiments, the guide nucleic acid of the editing system may include a nucleotide sequence (spacer sequence, such as one or more spacers) of any one of SEQ ID NOs: 101-104 (e.g., SEQ ID NO: 101 (FCP1), SEQ ID NO: 102 (CLE14), SEQ ID NO: 103 (CLE7), SEQ ID NO: 104 (CLE7)) and a nucleotide sequence (spacer sequence) of any one of SEQ ID NOs: 110-117 (e.g., SEQ ID NO: 110 (CLE7), SEQ ID NO: 111 (CLE7), SEQ ID NO: 112 (FCP1), SEQ ID NO: 113 (FCP1), SEQ ID NO: 114 (FCP1), SEQ ID NO: 115 (CLE14), SEQ ID NO: 116 (CLE14), SEQ ID NO: 117 (CLE14)).

[0198] In some embodiments, the mutation in the CLE gene is within the portion of the endogenous CLE gene that encodes a mature CLE peptide, and the mutation results in a mature CLE peptide having an amino acid substitution compared to a wild-type mature CLE peptide.

[0199] The mutation in the CLE gene of a plant, a plant part thereof, or a plant cell can be any type of mutation, including base substitution, base deletion, base insertion, and / or transversion. In some embodiments, the non-natural mutation can include a base substitution for A, T, G, or C. In some embodiments, the non-natural mutation can be a deletion of at least one base pair (e.g., 1 base pair to about 5 base pairs, such as 1, 2, 3, 4, 5 consecutive base pairs) or an insertion of at least one base pair (e.g., 1 base pair to about 5 base pairs, such as 1, 2, 3, 4, 5 consecutive base pairs). In some embodiments, the non-natural mutation can be a transversion of about 10 to about 2000 consecutive base pairs of the CLE gene.

[0200] In some embodiments, the mutation in the CLE gene is located within the portion of the endogenous CLE gene encoding the mature CLE peptide, and the mutation results in an amino acid substitution in the mature CLE peptide compared to the wild-type mature CLE peptide. In some embodiments, the mutation results in one or more substituted amino acid residues at positions 3, 5, 6, 8, or 9, relative to the amino acid position numbering of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77.

[0201] Endogenous CLE genes useful in the present invention encode FON2-LIKE CLE PROTEIN1 (FCP1) FCP1 protein, CLE7 protein or CLE14 protein. In some embodiments, the endogenous CLE gene (e.g., target gene) may comprise a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 78, SEQ ID NO: 79 or SEQ ID NO: 80, a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 83; a sequence having at least 95% sequence identity to SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74, or a domain having a sequence having at least 95% sequence identity to SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 77. In some embodiments, the wild-type mature peptide comprises an amino acid sequence having a length of 12 amino acids and R at position 1, V at position 3, P at position 4, G at position 6, P at position 9, H at position 11, and H at position 12, optionally wherein the wild-type processed peptide comprises any one of the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77. Exemplary non-natural mutations in a CLE gene can provide a mutant CLE gene. Exemplary non-natural mutations in an endogenous CLE gene can encode a mutant CLE protein.

[0202] In some embodiments, the CLE peptide comprises a mutation of one or more amino acid residues at positions 3, 5, 6, 8, or 9 relative to the amino acid position numbering of any one of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77. In some embodiments, the mutation may be a base substitution from C to T (C>T), G to A (G>A), A to G (A>G), or T to C (T>C). In some embodiments, the mutant CLE peptide comprises an amino acid substitution of valine (V) at position 3 to isoleucine (I) or methionine (M), glutamic acid (E) at position 5 to lysine (K), glycine (G) at position 6 to threonine (T), aspartic acid (D) at position 8 to alanine (A), aspartic acid (D) at position 8 to glycine (G), and / or proline (P) at position 9 to leucine (L) (e.g., comprising mutations of V3I, V3M, E5K, G6T, D8A, D8G, and / or P9L) relative to the amino acid position numbering of any one of SEQ ID NOs: 84-100. In some embodiments, the mature mutant CLE peptide of the present invention may comprise the sequence of any one of the amino acid sequences of SEQ ID NOs: 84-100.

[0203] In some embodiments, a plant (e.g., a corn plant) comprising at least one (e.g., one or more) non-natural mutation in an endogenous CLE gene exhibits an increased number of ear rows (e.g., an increase of 2, 4, 6, 8, 10, 12, 14 or more rows) compared to a plant that does not have the at least one non-natural mutation. In some embodiments, the plant comprising at least one mutation in an endogenous CLE gene is a corn plant that exhibits an increased number of ear rows. In some embodiments, the plant comprising at least one mutation in an endogenous CLE gene is a corn plant that exhibits increased yield and increased nematode resistance. In some embodiments, a plant (e.g., a corn plant) can be regenerated from a plant part and / or plant cell of the present invention, wherein the regenerated plant (e.g., a regenerated corn plant) comprises the mutation in the endogenous CLE gene, has a phenotype of increased number of ear rows, optionally without substantially reducing ear length, compared to a plant lacking the same CLE mutation.

[0204] As used herein, the term "without substantially reducing the length of the ear" refers to the length of an ear having an increased number of ear rows due to one or more mutations in one or more CLE genes as described herein, wherein the length of the ear is not reduced by more than about 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, for, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29% or 30% as compared to an ear of a plant lacking the same mutation in the same CLE gene.

[0205] In some embodiments, a corn plant cell is provided, the corn plant cell comprising at least one (e.g., one or more) non-natural mutation within an endogenous CLE gene (e.g., CLE7, CLE14, FCP1), wherein the mutation is a substitution, insertion, deletion, or transversion introduced using an editing system comprising a nucleic acid binding domain that binds to a target site in the CLE gene. In some embodiments, the substitution, insertion, deletion, or transversion produces a dominant negative allele, a semi-dominant allele, a weak loss-of-function allele, or a hypomorphic allele. In some embodiments, the at least one non-natural mutation is a point mutation. In some embodiments, the target site within a CLE gene is located within a region of the CLE 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 SEQ ID NO:81, SEQ ID NO:82, or SEQ ID NO:83 and / or encodes a sequence having at least 95% 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 SEQ ID NO:75, SEQ ID NO:76, or SEQ ID NO:77. In some embodiments, the CLE gene comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 72 or SEQ ID NO: 73 or encodes a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 74. In some embodiments, the mutation is generated after cleavage by an editing system comprising a nuclease and a nucleic acid binding domain that binds to a target site within a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 81-83. In some embodiments, the at least one non-natural mutation produces one or more substituted amino acid residues at positions 3, 5, 6, 8, or 9 (e.g., V3I, V3M, E5K, G6T, D8A, D8G, and / or P9L) relative to the amino acid position numbering of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77. In some embodiments, the corn plant cell is regenerated into a corn plant comprising the at least one non-natural mutation, and the mutation results in a dominant negative allele.In some embodiments, a corn plant regenerated from the corn plant cell exhibits a phenotype of increased ear row number compared to a wild-type plant not comprising / lacking the allele (e.g., an isogenic wild-type plant) (optionally without substantially reducing ear length; for example, without reducing ear length by 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% compared to ears of a plant not comprising the same CLE mutation).

[0206] In some embodiments, a method for producing / cultivating a transgenic-free edited corn plant is provided, the method comprising: crossing a corn plant of the present invention (e.g., a corn plant comprising one or more mutations (e.g., non-natural mutations) in one or more CLE genes and having an increased number of ear rows) with a transgenic-free corn plant, thereby introducing the mutation into the transgenic-free corn plant; and selecting offspring corn plants comprising the mutation and being transgenic-free, thereby producing a transgenic-free edited corn plant.

[0207] Also provided herein is a method of producing a plurality of corn plants having an increased number of ear rows, the method comprising planting two or more corn plants of the present invention (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10 or more corn plants comprising one or more mutations (e.g., non-natural mutations) in one or more CLE polypeptides and having an increased number of ear rows) in a growing area (e.g., a field (e.g., a cultivated field, a farmland), 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 ear rows (optionally, without substantially reducing ear length (e.g., ear length is not reduced by more than 5-30%)) compared to a plurality of control corn plants lacking the mutation. In some embodiments, the plurality of plants may also exhibit larger meristems, increased yield, and increased nematode resistance.

[0208] The present invention also provides a method for generating a variation in a mature corn CLE peptide, comprising: introducing an editing system into a corn plant cell, wherein the editing system targets a region of a corn CLE gene encoding the mature corn CLE peptide protein, wherein the region comprises a sequence having at least 95% sequence identity with any one of the amino acid sequences of SEQ ID NO: 81, SEQ ID NO: 82, or SEQ ID NO: 83, or the region is encoded by a sequence having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NO: 81-83; and contacting the region of the corn CLE gene with the editing system, thereby introducing a mutation into a mature corn CLE peptide in a corn plant cell; and generating a variation in the mature corn CLE peptide. In some embodiments, the mutation can be performed after cleavage by the editing system, wherein the editing system comprises a nuclease and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to a target site within a sequence having at least 90% sequence identity with any one of the nucleotide sequences of SEQ ID NO: 81-83.

[0209] In some embodiments, a specific site in the genome of a corn plant cell is edited, the method comprising: cleaving a target site within an endogenous CLE gene in a corn plant cell in a site-specific manner, the endogenous CLE gene (a) comprising (i) a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80, and / or (ii) a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; and / or (b) a domain encoding (i) a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (ii) a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, thereby generating an edit in a portion of the endogenous gene of the corn plant cell encoding a mature CLE peptide, and generating a plant cell comprising the edit in the portion of the endogenous gene.

[0210] In some embodiments, the editing results in a non-naturally occurring mutation, including but not limited to a deletion, substitution, insertion, or transversion, wherein the editing can result in a dominant negative mutation, a semi-dominant mutation, a hypomorphic mutation, or a weak loss-of-function mutation. In some embodiments, the editing can be a nucleotide substitution of C to T (C>T), G to A (G>A), A to G (A>G), or T to C (T>C). In some embodiments, the editing results in a change in an amino acid in the coding region of the CLE protein. For example, relative to the amino acid position numbering of any one of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77, the editing can result in a change in an amino acid residue at position 3, 5, 6, 8, or 9. In some embodiments, the editing produces the following amino acid substitutions in the CLE polypeptide relative to the amino acid position numbering of any one of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77: valine (V) to isoleucine (I) or methionine (M) at position 3, glutamic acid (E) to lysine (K) at position 5, glycine (G) to threonine (T) at position 6, aspartic acid (D) to alanine (A) at position 8, aspartic acid (D) to glycine (G) at position 8, and / or proline (P) to leucine (L) at position 9 (e.g., mutations comprising V3I, V3M, E5K, G6T, D8A, D8G, and / or P9L). In some embodiments, the edited mature CLE peptide of the invention may comprise the sequence of any one of the amino acid sequences of SEQ ID NO: 84-100.

[0211] In some embodiments, the editing method can further include regenerating a corn plant from a corn plant cell comprising the edit in the endogenous CLE gene, thereby producing a corn plant comprising the edit in its endogenous CLE gene and having a phenotype of increased ear row number compared to a control corn plant not comprising the edit.

[0212] In some embodiments, a method for making a corn plant comprises: (a) contacting a population of corn plant cells comprising an endogenous CLE gene encoding a precursor CLE polypeptide with a nuclease linked to a nucleic acid binding domain (e.g., a DNA binding domain, such as an editing system), wherein the nucleic acid binding domain binds to a sequence that (i) has at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80, (ii) has at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83, (iii) encodes a sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74, or (ii) encodes a sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77; (b) selecting plant cells from the population in which the endogenous CLE gene has been mutated, thereby producing plant cells comprising a mutation in the endogenous CLE gene; and (c) growing the selected plant cells into plants.

[0213] In some embodiments, a method of increasing the number of ear rows in a corn plant comprises: (a) contacting a corn plant cell comprising an endogenous CLE gene with a nuclease targeted to the endogenous CLE gene, wherein the nuclease is linked to a nucleic acid binding domain (e.g., a DNA binding domain, such as an editing system) that binds to a target site in the endogenous CLE gene, wherein the endogenous CLE gene: (i) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (ii) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (iii) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (iv) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 73-75. (a) producing a corn plant cell comprising a mutation in an endogenous CLE gene; and (b) growing the corn plant cell into a corn plant comprising the mutation in the endogenous CLE gene, thereby producing a corn plant having a mutated endogenous CLE gene and an increased number of ear rows.

[0214] In some embodiments, a method for producing a corn plant or part thereof comprising at least one cell having a mutation in an endogenous CLE gene, the method comprising contacting a target site in an endogenous CLE 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 CLE gene, wherein the endogenous CLE gene (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, thereby producing a corn plant or part thereof comprising at least one cell having a mutation in the endogenous CLE gene.

[0215] Also provided herein is a method for producing a corn plant or part thereof comprising a mutated endogenous CLE gene and exhibiting increased ear row number, the method comprising contacting a target site in an endogenous CLE gene in the 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 CLE gene, wherein the endogenous CLE gene: (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, thereby producing a corn plant or part thereof comprising an endogenous CLE gene having a mutation and exhibiting increased ear row number.

[0216] In some embodiments, the nuclease can cut the endogenous CLE gene, thereby introducing a mutation into the endogenous CLE gene. The nuclease that can be used in the present invention can be any nuclease that can be used to edit / modify the target nucleic acid. Such nucleases include, but are not limited to, zinc finger nucleases, transcription activator-like effector nucleases (TALENs), endonucleases (e.g., Fok1), and / or CRISPR-Cas effector proteins. Similarly, any nucleic acid binding domain that can be used in the present invention can be any DNA binding domain or RNA binding domain that can be used to edit / modify the target nucleic acid. Such nucleic acid binding domains include, but are not limited to, zinc fingers, transcription activator-like DNA binding domains (TALs), argonautes, and / or CRISPR-Cas effector DNA binding domains.

[0217] In some embodiments, a method for editing an endogenous CLE gene in a corn plant or a plant part is provided, the method comprising contacting a target site in a CLE gene in the 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 the target site in the CLE gene, wherein the CLE gene (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, thereby editing the endogenous CLE gene in the corn plant or a part thereof and producing a corn plant or a part thereof comprising at least one cell having a mutation in the endogenous CLE gene.

[0218] In some embodiments, a method for editing an endogenous CLE gene in a corn plant or a plant part is provided, the method comprising contacting a target site in a CLE gene in the corn plant or a plant part with an adenosine base editing system, the adenosine base editing system comprising an adenosine deaminase and a nucleic acid binding domain that binds to the target site in the CLE gene, wherein the CLE gene (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, thereby editing an endogenous CLE gene in a corn plant or a part thereof and producing a plant or a part thereof comprising at least one cell having a mutation in the endogenous CLE gene.

[0219] In some embodiments, a method for detecting a mutant CLE gene (a mutation in an endogenous CLE gene) is provided, the method comprising detecting in the genome of a plant a nucleic acid encoding an amino acid sequence of SEQ ID NOs: 72-77, wherein the amino acid sequence of SEQ ID NOs: 72-77 comprises a mutation of one or more amino acid residues at positions 3, 5, 6, 8, or 9 relative to the amino acid position numbering of any one of SEQ ID NOs: 75, SEQ ID NOs: 76, or SEQ ID NOs: 77. In some specific embodiments, the mutation is the result of a nucleotide substitution of C to T (C>T), G to A (G>A), A to G (A>G), or T to C (T>C). In some embodiments, the mutant CLE peptide comprises at least one mutation comprising a mutation at position 3 from V to I or M, at position 5 from E to K, at position 6 from G to T, at position 8 from D to A or G, and / or at position 9 from P to L relative to the amino acid position numbering of any one of SEQ ID NOs: 75, SEQ ID NOs: 76, or SEQ ID NOs: 77.

[0220] In some embodiments, the present invention provides a method for detecting a mutation in an endogenous CLE gene, comprising detecting a mutated CLE gene in the genome of a plant. As an example, the detected mutated CLE gene may comprise a sequence having at least 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 105-109, or the detected mutated CLE gene may encode a processed polypeptide of any one of the amino acid sequences of SEQ ID NOs: 84-100.

[0221] In some embodiments, the present invention provides a method for producing a plant comprising a mutation in an endogenous CLE gene and at least one polynucleotide of interest, the method comprising crossing a plant of the present invention (a first plant) comprising at least one mutation in an endogenous CLE gene with a second plant comprising the at least one polynucleotide of interest to produce offspring plants; and selecting offspring plants comprising at least one mutation in the CLE gene and the at least one polynucleotide of interest, thereby producing a plant comprising a mutation in an endogenous CLE gene and at least one polynucleotide of interest.

[0222] The present invention also provides a method for producing a plant comprising a mutation in an endogenous CLE gene and at least one polynucleotide of interest, the method comprising introducing at least one polynucleotide of interest into a plant of the present invention comprising at least one mutation in a CLE gene, thereby producing a plant comprising at least one mutation in a CLE gene and at least one polynucleotide of interest. In some embodiments, the plant is a corn plant.

[0223] In some embodiments, the present invention provides a method for producing a plant comprising a mutation in an endogenous CLE gene and at least one polynucleotide of interest, the method comprising introducing at least one polynucleotide of interest into a plant of the present invention comprising at least one mutation in an endogenous CLE gene, thereby producing a plant comprising at least one mutation in a CLE gene and at least one polynucleotide of interest. In some embodiments, the plant is a corn plant.

[0224] The polynucleotide of interest may be any polynucleotide that can confer a desired phenotype or otherwise modify 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, yield increase, nutrient use efficiency increase, or abiotic stress resistance.

[0225] CLEs useful in the present invention include any CLE wherein a mutation as described herein can confer increased ear row number in a plant or part thereof comprising the mutation. In some embodiments, a CLE gene useful in the present invention encodes a FCP1 protein, a CLE7 protein, or a CLE14 protein.

[0226] In some embodiments, the endogenous precursor CLE polypeptide comprises an amino acid sequence that is at least 95% identical (e.g., about 95, 96, 97, 98, 99, 99.5, 100% sequence identity) to any one of the amino acid sequences of SEQ ID NOs: 72-74. In some embodiments, the mature endogenous CLE peptide comprises a sequence that is at least 95% identical (e.g., about 95, 96, 97, 98, 99, 99.5, 100% sequence identity) to any one of the amino acid sequences of SEQ ID NOs: 75-77. In some embodiments, the CLE 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 any one of the nucleotide sequences of SEQ ID NOs: 78-80, or the CLE gene may comprise a sequence (region) therein having at least 90% sequence identity (e.g., about 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 100% sequence identity) to any one of the nucleotide sequences of SEQ ID NOs: 81-83 and 101-105.

[0227] In some embodiments, the at least one non-natural mutation in the endogenous CLE gene in the corn plant can be a base substitution, base deletion and / or base insertion or transversion. In some embodiments, the at least one non-natural mutation in the endogenous CLE gene in the corn plant can be a substitution, deletion, insertion and / or transversion that results in a dominant negative mutation, a semi-dominant mutation, a hypomorphic mutation, or a weak loss-of-function mutation, and the plant has a phenotype of increased ear row number, compared to a control corn plant that does not contain the edit / mutation. For example, the mutation may 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 or 10 amino acids of a CLE polypeptide), or the mutation may be a base substitution, base deletion and / or base insertion of at least 1 nucleotide to about 15 nucleotides (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 nucleotides, or any range or value thereof) in a gene encoding a CLE polypeptide, or may be a base substitution, base deletion and / or base insertion of at least 10 consecutive nucleotides to about 2000 consecutive nucleotides (e.g., about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 2010, 2110, 2120, 2130, 2140, 2150, 2160, 2170, 2180, 2190, 2211 0, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100, 1110, 1120, 1130, 1140, 1150, 1160, 1170, 1180, 1190, 12 In some embodiments, the at least one non-natural mutation may be a base substitution to A, T, G or C. In some embodiments, the at least one non-natural mutation may be a base substitution from C to T (C>T), G to A (G>A), A to G (A>G) or T to C (T>C). The mutation may be a point mutation.In some embodiments, the mutation produces one or more substituted amino acid residues at position 3, 5, 6, 8, or 9 (e.g., V3I, V3M, E5K, G6T, D8A, D8G, and / or P9L), relative to the amino acid position numbering of SEQ ID NO:75, SEQ ID NO:76, or SEQ ID NO:77.

[0228] In some embodiments, mutations in an endogenous CLE gene can be generated after cleavage by an editing system comprising a nuclease and a nucleic acid binding domain that binds to a target site within a target nucleic acid (e.g., a CLE gene), the target nucleic acid comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-83, or encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-77 (e.g., a CLE gene). In some embodiments, the nuclease cleaves the endogenous CLE gene and introduces a mutation into the endogenous CLE gene.

[0229] Also provided herein are guide nucleic acids (e.g., gRNA, gDNA, crRNA, crDNA) that bind to a target site in a CLE gene, wherein the endogenous CLE gene: (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77. In some embodiments, the guide nucleic acid comprises a spacer having a nucleotide sequence of any one of SEQ ID NOs: 101-104 and / or SEQ ID NOs: 110-117.

[0230] In some embodiments, a corn plant or plant part thereof is provided, comprising at least one non-natural mutation in at least one endogenous CLAVATA3 / EMBRYO SURROUNDING REGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene located within a chromosomal interval on chromosome 2 defined by base pair (bp) position 235,093,752 to base pair position 235,094,276 and / or within a chromosomal interval on chromosome 2 defined by base pair (bp) position 40,126,538 to base pair position 40,127,030 and wherein each chromosomal interval corresponds to the reference corn genome of B73 version 4 (B73RefGen_v4), AGPv4) of the MaizegDB internet resource (maizegdb.org / assembly). In some embodiments, a corn plant or plant part thereof is provided that comprises at least one non-natural mutation located in at least one endogenous CLE gene, wherein the endogenous CLE gene is located on chromosome 2 at a chromosomal interval defined by base pair (bp) position 235,093,752 to base pair position 235,094,276 and including them, and wherein the chromosomal interval corresponds to the reference corn genome of B73 version 3 (B73RefGen_v3) of the MaizeGDB internet resource (maizegdb.org / assembly).

[0231] In some embodiments, a guide nucleic acid is provided that binds to a target nucleic acid in a CLAVATA3 / EMBRYO SURROUNDINGREGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene in a corn plant, wherein the target nucleic acid is located on chromosome 2 within a chromosomal interval defined by base pair (bp) position 235,093,752 to base pair position 235,094,276 and including them (Zm00001d007576 (cle14-clavata3 / esr-related14)), or within a chromosomal interval defined by base pair (bp) position 40,126,538 to base pair position 40,127,030 and including them (Zm00001d003320 (fcp1-fon2-like cle14)). protein1)), and wherein each chromosomal interval corresponds to the B73 reference maize genome version 4 (B73_RefGen_v4), AGPv4) of the MaizeGDB internet resource (maizegdb.org / assembly). In some embodiments, a guide nucleic acid that binds to a target nucleic acid in a CLE gene in a corn plant is provided, wherein the target nucleic acid is located in a chromosomal interval defined by and including base pair (bp) position 7,570,324 to base pair position 7,571,104 on chromosome 4 where an endogenous CLE gene is located (GRMZM2G372364 (cle7-clavata3 / esr-related7)), and wherein the chromosomal interval corresponds to the B73 reference maize genome version 3 (B73_RefGen_v3) of the MaizeGDB internet resource (maizegdb.org / assembly).

[0232] In some embodiments, a system comprising a guide nucleic acid is provided, the guide nucleic acid comprising a spacer (e.g., one or more spacers) having a nucleotide sequence of any one of SEQ ID NO: 101-104 and / or SEQ ID NO: 110-117, and a CRISPR-Cas effector protein associated with the guide nucleic acid. In some embodiments, the system may also include a tracr nucleic acid and a CRISPR-Cas effector protein associated with the guide nucleic acid, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked. As used herein, "CRISPR-Cas effector protein associated with a guide nucleic acid" refers to a complex formed between a CRISPR-Cas effector protein and a guide nucleic acid to guide the CRISPR-Cas effector protein to a target site in a gene.

[0233] The present invention also provides a 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 CLE gene, wherein the CLE gene (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77. In some embodiments, the spacer sequence of the guide nucleic acid may comprise a nucleotide sequence of any one of SEQ ID NOs: 101-104 and / or SEQ ID NOs: 110-117. 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.

[0234] The present invention also provides a complex comprising a CRISPR-Cas effector protein, wherein the CRISPR-Cas effector protein comprises a cleavage domain and a guide nucleic acid, wherein the guide nucleic acid binds to a target site in an endogenous CLE gene, wherein the endogenous CLE gene, wherein the endogenous CLE gene: (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, wherein the cleavage domain cleaves the target strand in the CLE gene.

[0235] In some embodiments, 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 in an endogenous CLE gene encoding a precursor CLE peptide, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (i) a sequence comprising a nucleotide sequence having at least 90% sequence identity to any one of SEQ ID NOs: 78-80; (ii) a region comprising a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs: 81-83; (iii) encoding a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 72-74 amino acid sequences; and / or (iv) encoding a domain having a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 75-77.

[0236] Also provided herein are nucleic acids encoding a mutated CLE gene which, when present in a corn plant or plant part, results in a corn plant comprising a phenotype of increased ear row number compared to a corn plant or plant part that does not comprise the CLE mutation.

[0237] An exemplary mutant CLE protein may comprise a sequence having at least 95% sequence identity to an amino acid sequence of any one of SEQ ID NOs: 84- 100. An exemplary mutant CLE gene may comprise a sequence having at least 90% sequence identity to a nucleotide sequence of any one of SEQ ID NOs: 105-109.

[0238] The nucleic acid constructs of the present invention (e.g., constructs comprising a sequence-specific nucleic acid binding domain (e.g., a sequence-specific DNA 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 a target nucleic acid (e.g., an endogenous CLE gene) and / or its expression.

[0239] Any corn plant comprising an endogenous CLE gene capable of conferring an increased number of ear rows when modified as described herein can be modified (e.g., mutated, e.g., base edited, cleaved, cut, etc.) as described herein (e.g., using a polypeptide, polynucleotide, RNP, nucleic acid construct, expression cassette and / or vector of the invention) to increase the number of ear rows in the corn plant. The number of ear rows in a plant (e.g., a corn plant) exhibiting an increased number of ear rows can be increased by about 5% to about 100% (e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 0, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76 %, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 or 100% or more, 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 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 20% to about 50%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 50%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 50% to about 100%, about 75% to about 100% or more, or any range or value therein).

[0240] The editing system that can be used for the present invention can be any site-specific (sequence-specific) genome editing system that is now known or developed later, and the system can introduce mutations in a target-specific manner. For example, 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, which, when expressed as a system in a cell, can modify (mutate) a target nucleic acid in a sequence-specific manner. 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.

[0241] 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 can comprise one or more polynucleotides, including but not limited to CRISPR array (CRISPR guide) nucleic acids, extended guide nucleic acids, and / or reverse transcriptase templates.

[0242] In some embodiments, a method of modifying or editing a CLE gene may include contacting a target nucleic acid (e.g., a nucleic acid encoding CLE) with a base editing fusion protein (e.g., a sequence-specific DNA binding protein (e.g., a CRISPR-Cas effector protein or domain)) fused to a deaminase domain (e.g., adenine deaminase and / or cytosine deaminase) and a guide nucleic acid, wherein the guide nucleic acid is capable of directing / targeting the base editing fusion protein to a target nucleic acid, thereby encoding 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 a base editing fusion protein and an expression cassette comprising a guide nucleic acid. In some embodiments, the sequence-specific nucleic acid binding fusion protein and the guide nucleic acid may be provided in the form of a ribonucleoprotein (RNP). In some embodiments, a cell may be contacted with more than one base editing fusion protein and / or one or more guide nucleic acids, which may target one or more target nucleic acids in a cell.

[0243] In some embodiments, a method for modifying or editing a CLE gene may include contacting a target nucleic acid (e.g., a nucleic acid encoding CLE) with a sequence-specific nucleic acid binding fusion protein fused to a peptide tag (e.g., a sequence-specific DNA binding protein (e.g., a CRISPR-Cas effector protein or domain), a deaminase fusion protein (which comprises 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, thereby editing the CLE gene. 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 contained in one or more expression cassettes. In some embodiments, the target nucleic acid can be contacted with a sequence-specific binding fusion protein, a deaminase fusion protein, and an expression cassette comprising a guide nucleic acid. In some embodiments, the sequence-specific nucleic acid binding fusion protein, the deaminase fusion protein, and the guide nucleic acid can be provided in the form of a ribonucleoprotein (RNP).

[0244] In some embodiments, methods such as prime editing can be used to generate mutations in endogenous CLE genes. In prime editing, RNA-dependent DNA polymerase (reverse transcriptase, RT) and reverse transcriptase template (RT template) are used in combination with sequence-specific DNA binding domains, which confer the ability to recognize and bind to targets in a sequence-specific manner, and can also cause incisions 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 nucleic acid comprising an extension portion, the extension portion comprising a primer binding site (primer binding site, PSB) and an editor to be integrated into the genome (template). Similar to base editing, prime editing can utilize various methods for recruiting proteins for editing to target sites, including non-covalent and covalent interactions between proteins and nucleic acids used in selected processes of genome editing.

[0245] As used herein, a "CRISPR-Cas effector protein" is a protein or polypeptide or a domain thereof that cuts or cleaves a nucleic acid, binds a nucleic acid (e.g., a target nucleic acid and / or a guide nucleic acid), and / or identifies, recognizes, or binds a guide nucleic acid as defined herein. In some embodiments, the CRISPR-Cas effector protein can be an enzyme (e.g., a nuclease, an endonuclease, a nickase, etc.) or a portion thereof, and / or can function as an enzyme. In some embodiments, the 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 has been reduced or eliminated, and / or comprises single-stranded DNA cleavage activity (ssDNAse activity) or wherein the ssDNAse activity has been reduced or eliminated, and / or comprises self-processing RNAse activity or wherein the self-processing RNAse activity has been reduced or eliminated. The CRISPR-Cas effector protein can bind to a target nucleic acid.

[0246] In some embodiments, the sequence-specific nucleic acid 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.

[0247] In some embodiments, the CRISPR-Cas effector protein may include, but is not limited to, Cas9, C2c1, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4 (dinG) and / or Csf5 nuclease, optionally wherein the CRISPR-Cas effector protein can be Cas9, Cas12a (Cpf1), Cas12b, Cas12c (C2c3), Cas12d (CasY), Cas12e (CasX), Cas12g, Cas12h, Cas12i, C2c4, C2c5, C2c8, C2c9, C2c10, Cas14a, Cas14b and / or Cas14c effector protein.

[0248] In some embodiments, the CRISPR-Cas effector protein used 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 having a mutation in its nuclease active site and thus no longer comprising nuclease activity is often referred to as "dead," such as dCas. In some embodiments, a CRISPR-Cas effector protein domain or polypeptide having a mutation in its nuclease active site may have impaired activity or reduced activity compared to the same CRISPR-Cas effector protein without a mutation (e.g., a nickase, e.g., a Cas9 nickase, a Cas12a nickase).

[0249] CRISPR Cas9 effector proteins or CRISPR Cas9 effector domains useful in the present invention may be any known or later identified Cas9 nuclease. In some embodiments, the CRISPR Cas9 polypeptide may be a Cas9 polypeptide from, for example, Streptococcus spp. (e.g., S. pyogenes, S. thermophilus), Lactobacillus spp., Bifidobacterium spp., Kandleria spp., Leuconostoc spp., Oenococcus spp., Pediococcus spp., Weissella spp., and / or Olsenella spp. Exemplary Cas9 sequences include, but are not limited to, the amino acid sequences of SEQ ID NO: 59 and SEQ ID NO: 60 or the nucleotide sequences of SEQ ID NO: 61-71.

[0250] In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Streptococcus pyogenes, which 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 thermophilus, which 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 that 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 Staphylococcus aureus that 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 Staphylococcus aureus that recognizes the PAM sequence motifs NGRRT (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Staphylococcus aureus that recognizes the PAM sequence motifs NGRRV (R=A or G). In some embodiments, the CRISPR-Cas effector protein can be a Cas9 polypeptide derived from Neisseria meningitidis, which recognizes a PAM sequence motif NGATT or NGCTT (R=A or G, V=A, G or C) (see, e.g., Hou et al., 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, which can be located within the target nucleic acid.

[0251] In some embodiments, the CRISPR-Cas effector protein may be derived from Cas12a, which is a V-type clustered regularly interspaced short palindromic repeats (CRISPR)-Cas nuclease. See, e.g., SEQ ID NOs: 1-20. Cas12a differs from the more well-known type II CRISPR Cas9 nuclease in several aspects. For example, Cas9 recognizes a G-rich protospacer adjacent motif (PAM) (3'-NGG) located 3' to its guide RNA (gRNA, sgRNA, crRNA, crDNA, CRISPR array) binding site (protospacer, target nucleic acid, target DNA), while Cas12a recognizes a T-rich PAM (5'-TTN, 5'-TTTN) located 5' to the target nucleic acid. In fact, Cas9 and Cas12a bind to their guide RNAs in almost opposite directions 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 cut two DNA chains, while Cas9 uses the HNH domain and the RuvC domain to cut.

[0252] The CRISPR Cas12a effector protein / domain that can be used in the present invention can be any known or later identified Cas12a polypeptide (formerly known as Cpf1) (see, e.g., U.S. Pat. No. 9,790,490, which is incorporated herein by reference for the disclosure of the Cpf1 (Cas12a) sequence therein). The term "Cas12a", "Cas12a polypeptide" or "Cas12a domain" refers to an RNA-guided nuclease comprising a Cas12a polypeptide or a fragment thereof, which comprises a guide nucleic acid binding domain of Cas12a and / or an active, inactive or partially active DNA cleavage domain of Cas12a. In some embodiments, the Cas12a that can be used in the present invention may include a mutation in a nuclease active site (e.g., a RuvC site of a Cas12a domain). A Cas12a domain or Cas12a polypeptide that has a mutation in its nuclease active site and therefore no longer comprises nuclease activity is generally referred to as deadCas12a (e.g., dCas12a). In some embodiments, a Cas12a domain or Cas12a polypeptide having a mutation in its nuclease active site may have impaired activity, for example, may have nickase activity.

[0253] Any deaminase domain / polypeptide that can be used for base editing can be used in the present invention. In some embodiments, the deaminase domain can be a cytosine deaminase domain or an adenine deaminase domain. The cytosine deaminase (or cytidine deaminase) that can be used in the present invention can be any known or later identified cytosine deaminase from any organism (see, for example, U.S. Patent No. 0,167,457 and Thuronyi et al. Nat. Biotechnol. 37: 1070-1079 (2019), each of which is incorporated herein by reference for the disclosure of its cytosine deaminase). Cytosine deaminase can catalyze the hydrolysis and deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. Therefore, in some embodiments, the deaminase or deaminase domain that can be used in the present invention can be a cytidine deaminase domain, catalyzing the hydrolysis and deamination of cytosine to uracil. In some embodiments, the cytosine deaminase may 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 may have about 70% to about 100% identity with 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% identity with a naturally occurring cytosine deaminase, and any range or value therein).

[0254] In some embodiments, the cytosine deaminase useful in the present invention may be an apolipoprotein B mRNA editing complex (APOBEC) family deaminase. In some embodiments, the cytosine deaminase may be an APOBEC1 deaminase, an APOBEC2 deaminase, an APOBEC3A deaminase, an APOBEC3B deaminase, an APOBEC3C deaminase, an APOBEC3D deaminase, an APOBEC3F deaminase, an APOBEC3G deaminase, an APOBEC3H deaminase, an APOBEC4 deaminase, a human activation-induced deaminase (hAID), rAPOBEC1, FERNY and / or CDA1, optionally pmCDA1, atCDA1 (e.g., At2g 19570) and an evolved form thereof (e.g., SEQ ID NO: 27, SEQ ID NO: 28 or SEQ ID NO: 29). In some embodiments, the cytosine deaminase may be an APOBEC1 deaminase having an amino acid sequence of SEQ ID NO: 23. In some embodiments, the cytosine deaminase may be an APOBEC3A deaminase having the amino acid sequence of SEQ ID NO: 24. In some embodiments, the cytosine deaminase may be a CDA1 deaminase, optionally a CDA1 having the amino acid sequence of SEQ ID NO: 25. In some embodiments, the cytosine deaminase may be a FERNY deaminase, optionally a FERNY having the amino acid sequence of SEQ ID NO: 26. In some embodiments, a cytosine deaminase useful in the present invention can have about 70% to about 100% 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%, or 100% identity) 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 invention may have about 70% to about 99.5% identity (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% identity) to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26 (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 99.5% identity) to the amino acid sequence of SEQ ID NO: 23, SEQ ID NO: 24, SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28, or SEQ ID NO: NO:29 has 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% identity). In some embodiments, the polynucleotide encoding the cytosine deaminase can be codon-optimized for expression in plants, and the codon-optimized polypeptide can have about 70% to 99.5% identity to the reference polynucleotide.

[0255] In some embodiments, the nucleic acid construct of the present invention may also encode a uracil glycosylase inhibitor (UGI) (e.g., a uracil-DNA glycosylase inhibitor) polypeptide / domain. Thus, in some embodiments, a nucleic acid construct encoding a CRISPR-Cas effector protein and a cytosine deaminase domain (e.g., encoding a fusion protein comprising a CRISPR-Cas effector protein domain fused to a cytosine deaminase domain, and / or a CRISPR-Cas effector protein domain fused to a peptide tag or an affinity polypeptide capable of binding to a peptide tag, and / or a deaminase protein domain fused to a peptide tag or an affinity polypeptide capable of binding to a peptide tag) may also encode a uracil-DNA glycosylase inhibitor (UGI), optionally wherein UGI may be codon optimized for expression in plants. In some embodiments, the present invention provides a fusion protein comprising a CRISPR-Cas effector polypeptide, a deaminase domain, and UGI and / or one or more polynucleotides encoding the same, optionally wherein the one or more polynucleotides may be codon optimized for expression in plants. In some embodiments, the present invention provides fusion proteins, wherein the CRISPR-Cas effector polypeptide, deaminase domain and UGI can be fused with any combination of peptide tags and affinity polypeptides described herein, thereby recruiting the deaminase domain and 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 deaminase domains and / or UGI can be fused to an affinity polypeptide capable of interacting with the recruitment RNA motif, thereby recruiting the deaminase domain and UGI to the target nucleic acid.

[0256] The "uracil glycosylase inhibitor" useful in the present invention may be any protein capable of inhibiting uracil-DNA glycosylase base-excision repair enzymes. In some embodiments, the UGI domain comprises a wild-type UGI or a fragment thereof. In some embodiments, the UGI domain useful in the present invention may have about 70% to about 100% 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% or 100% identity to the amino acid sequence of a naturally occurring UGI domain, 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% identity 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, which fragment has 100% identity with a portion of consecutive nucleotides (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides; e.g., about 10, 15, 20, 25, 30, 35, 40, 45 to about 50, 55, 60, 65, 70, 75, 80 consecutive nucleotides) of the amino acid sequence of SEQ ID NO:41. In some embodiments, the UGI domain can be a variant of a known UGI (e.g., SEQ ID NO: 41) having about 70% to about 99.5% sequence identity (e.g., 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% sequence identity, and any range or value therein). In some embodiments, a polynucleotide encoding a UGI can be codon-optimized for expression in a plant (e.g., a plant), and the codon-optimized polypeptide can have about 70% to about 99.5% identity to the reference polynucleotide.

[0257] 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, which is incorporated herein by reference for its disclosure of 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.

[0258] In some embodiments, adenosine deaminase can be a variant of a naturally occurring adenine deaminase. Thus, in some embodiments, adenosine deaminase can have about 70% to 100% identity with 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% identity with a naturally occurring adenine deaminase, and any range or value therein). In some embodiments, the deaminase is not naturally occurring and can be referred to as an engineered, mutated or evolved adenosine deaminase. Thus, for example, an engineered, mutated or evolved adenine deaminase polypeptide or adenine deaminase domain can have about 70% to 99.9% identity to a naturally occurring adenine deaminase polypeptide / domain (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 110%, 111%, 112%, 11 1%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8% or 99.9% identity, and any range or value therein). In some embodiments, the adenosine deaminase can be from bacteria (e.g., Escherichia coli, Staphylococcus aureus, Haemophilus influenzae, Caulobacter crescentus, etc.). In some embodiments, the polynucleotide encoding the adenine deaminase polypeptide / domain can be codon optimized for expression in plants.

[0259] 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 E. coli. In some embodiments, TadA can be modified, such as truncated, and one or more N-terminal and / or C-terminal amino acids can be deleted relative to the full-length TadA (for example, 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 can be deleted relative to the 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 NOs: 31-40 (e.g., SEQ ID NOs: 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.

[0260] Cytosine deaminase catalyzes the deamination of cytosine and produces thymidine (via a uracil intermediate), causing a C to T transition in the genome or a G to A transition within the complementary strand. Thus, in some embodiments, the cytosine deaminase encoded by the polynucleotides 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.

[0261] In some embodiments, the adenine deaminase encoded by the nucleic acid constructs of the invention produces an A→G transition in the sense (e.g., "+"; template) strand of a target nucleic acid or a T→C transition in the antisense (e.g., "-", complementary) strand of a target nucleic acid.

[0262] The nucleic acid construct of the present invention encoding a base editor comprising a sequence-specific nucleic acid binding protein and a cytosine deaminase polypeptide, and the nucleic acid construct / expression cassette / vector encoding the base editor can be combined with a guide nucleic acid to modify a target nucleic acid, including but not limited to generating a C→T or G→A mutation in the target nucleic acid, wherein the target nucleic acid includes but is not limited to a plasmid sequence; generating a C→T or G→A mutation in the coding sequence to change the amino acid identity; generating a C→T or G→A mutation in the coding sequence to generate a stop codon; generating a C→T or G→A mutation in the coding sequence to destroy the start codon; generating a point mutation in the genomic DNA to destroy the function; and / or generating a point mutation in the genomic DNA to destroy the splice point.

[0263] The nucleic acid constructs of the present invention encoding a base editor comprising a sequence-specific nucleic acid binding protein and an adenine deaminase polypeptide, and the expression cassettes and / or vectors encoding the base editors can be combined with guide nucleic acids for modifying target nucleic acids, including but not limited to generating A→G or T→C mutations in target nucleic acids, wherein the target nucleic acids include but are 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.

[0264] The nucleic acid constructs of the present invention comprising CRISPR-Cas effector proteins or fusion proteins thereof can be used in combination with guide RNAs (gRNA, CRISPR arrays, CRISPR RNAs, crRNAs) to modify target nucleic acids, wherein the guide RNAs are designed to function with the encoded CRISPR-Cas effector proteins or domains. The guide nucleic acids useful in the present invention comprise at least one spacer sequence and at least one repeat sequence. The guide nucleic acid is capable of forming a complex with the CRISPR-Cas nuclease domain encoded and expressed by the nucleic acid constructs of the present invention, and the spacer sequence is capable of hybridizing with the target nucleic acid, thereby guiding the complex (e.g., CRISPR-Cas effector fusion protein (e.g., CRISPR-Cas effector domain fused to a deaminase domain, and / or CRISPR-Cas effector domain fused to a peptide tag or affinity polypeptide to recruit a deaminase domain and optionally a UGI) to the target nucleic acid, wherein the target nucleic acid can be modified (e.g., cut or edited) or regulated (e.g., regulated transcription) by the deaminase domain.

[0265] For example, a nucleic acid construct encoding a Cas9 domain (e.g., a fusion protein) linked to a cytosine deaminase domain can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the cytosine deaminase domain of the fusion protein deaminates cytosine bases in the target nucleic acid, thereby editing the target nucleic acid. In another example, a nucleic acid construct encoding a Cas9 domain (e.g., a fusion protein) linked to an adenine deaminase domain can be used in combination with a Cas9 guide nucleic acid to modify a target nucleic acid, wherein the adenine deaminase domain of the fusion protein deaminates adenosine bases in the target nucleic acid, thereby editing the target nucleic acid.

[0266] Similarly, encoding a Cas12a domain (or other selected CRISPR-Cas nucleases, such as C2c1, C2c3, Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm 2, 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) (e.g., fusion proteins) can be used in combination with a Cas12a guide nucleic acid (or a guide nucleic acid for other selected CRISPR-Cas nucleases) to modify a target nucleic acid, wherein the cytosine deaminase domain or the adenine deaminase domain of the fusion protein deaminates a cytosine base in the target nucleic acid, thereby editing the target nucleic acid.

[0267] As used herein, "guide nucleic acid", "guide RNA", "gRNA", "CRISPR RNA / DNA", "crRNA" or "crDNA" means a CRISPR RNA / DNA comprising at least one spacer sequence (e.g., a protospacer) that is complementary to (and hybridizes with) a target DNA 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 C2c1 CRISPR-Cas system of type V or a portion thereof). Repeat sequences or fragments thereof of the Cas system; for example, C2c3, Cas12a (also known as Cpf1), Cas12b, Cas12c, Cas12d, Cas12e, Cas13a, Cas13b, Cas13c, Cas13d, Cas1, Cas1B, Cas2, Cas3, Cas3', Cas3", Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Cs The gRNA of the present invention can be based on a type I, type II, type III, type IV, type V or type VI CRISPR-Cas system.

[0268] 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'.

[0269] 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 sequence-repeat sequence, e.g., repeat-spacer sequence-repeat sequence-spacer sequence-repeat sequence-spacer sequence-repeat sequence-spacer sequence-repeat sequence-spacer sequence, etc.). The guide nucleic acids of the invention are synthetic, artificial, and not found in nature. The gRNA can be quite long and can be used as an aptamer (as in the MS2 recruitment strategy) or other RNA structure hanging from the spacer.

[0270] 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 functions 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 of a CRISPR-Cas locus (e.g., type I, type II, type III, type IV, type V, or type VI), or it can be a synthetic repetitive sequence designed to function in a type I, type II, type III, type IV, type V, or type 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 release): W52-7). In some embodiments, the repetitive sequence or a portion thereof is connected at its 3' end to the 5' end of the spacer sequence to form a repetitive sequence-spacer sequence (e.g., guide nucleic acid, guide RNA / DNA, crRNA, crDNA).

[0271] In some embodiments, the repeat sequence comprises, consists essentially of, or consists of at least 10 nucleotides, depending on whether the particular repeat sequence and the guide nucleic acid comprising it 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.

[0272] The repeat sequence linked to the 5' end of the spacer sequence can include a portion of the repeat sequence (e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or more consecutive nucleotides of 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 5 to about 10 contiguous nucleotides (e.g., about 5, 6, 7, 8, 9, 10 nucleotides) of the wild-type CRISPR Cas repeat nucleotide sequence in length 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., the 5' end) of the 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.

[0273] As used herein, a "spacer sequence" is a nucleotide sequence that is complementary to a target nucleic acid (e.g., a target DNA) (e.g., a protospacer) (e.g., a contiguous nucleotide sequence of the following sequence), wherein the sequence (a) comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) encodes a domain having a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77. In some embodiments, the spacer sequence (e.g., one or more spacers) may include, but is not limited to, the nucleotide sequence of any one of SEQ ID NOs: 101-104 and / or SEQ ID NOs: 110-117. The spacer sequence can be fully complementary or substantially complementary to the target nucleic acid (e.g., at least about 70% (e.g., about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more) (e.g., 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8, 99.9 or 100%) complementary). Thus, in some embodiments, the spacer sequence can have one, two, three, four or five mismatches compared to the target nucleic acid, and the mismatches can be continuous or discontinuous. In some embodiments, the spacer sequence can have 70% complementarity with the target nucleic acid. In other embodiments, the spacer nucleotide sequence can have 80% complementarity with the target nucleic acid. In other embodiments, the spacer nucleotide sequence can have 85%, 90%, 95%, 96%, 97%, 98%, 99% or 99.5% complementarity with the target nucleic acid (protospacer), etc. 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 substantial complementarity in the region where the length of the target nucleic acid (e.g., protospacer) is at least about 15 nucleotides to about 30 nucleotides. In some embodiments, the spacer length is about 20 nucleotides. In some embodiments, the spacer is about 21, 22, or 23 nucleotides in length.

[0274] In some embodiments, the 5' region of the spacer sequence of the guide nucleic acid can be identical to the target DNA, while the 3' region of the spacer can be substantially complementary to the target DNA (e.g., type V CRISPR-Cas), or the 3' region of the spacer sequence of the guide nucleic acid can be identical to the target DNA, while the 5' region of the spacer can be substantially complementary to the target DNA (e.g., type II CRISPR-Cas), and thus, the overall complementarity of the spacer sequence to the target DNA can be less than 100%. Thus, for example, in the guide nucleic acid 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., seed region) of the 20-nucleotide spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary to the target DNA (e.g., at least about 70% complementary). In some embodiments, the first 1 to 8 nucleotides (e.g., the first 1, 2, 3, 4, 5, 6, 7, 8 nucleotides and any range therein) at the 5' end of the spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 3' region of the spacer sequence are substantially complementary to the target DNA (e.g., at least about 50% (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) complementary).

[0275] As another example, in a guide nucleic acid 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., the seed region) of, for example, a 20-nucleotide spacer sequence can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary 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 can be 100% complementary to the target DNA, while the remaining nucleotides in the 5' region of the spacer sequence are substantially complementary to the target DNA (e.g., at least about 50% (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) complementary).

[0276] In some embodiments, the seed region of a spacer can be about 8 to about 10 nucleotides in length, can be about 5 to about 6 nucleotides in length, or can be about 6 nucleotides in length.

[0277] As used herein, "target nucleic acid," "target DNA," "target nucleotide sequence," "target region," or "target region in a genome" refers to a region in a plant genome that is fully complementary (100% complementary) or substantially complementary (e.g., at least 70% (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) complementary) to a spacer sequence in a guide nucleic acid of the invention. The target region useful for a CRISPR-Cas system can be immediately 3' (e.g., a type V CRISPR-Cas system) or 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.

[0278] A "protospacer sequence" refers to a target double-stranded DNA, specifically a portion of a target DNA (e.g., or a target region in a genome) that is fully complementary or substantially complementary (and hybridizes) to a spacer sequence of a CRISPR repeat-spacer sequence (e.g., a guide nucleic acid, a CRISPR array, a crRNA).

[0279] In the case of type V CRISPR-Cas (e.g., Cas12a) systems and type II CRISPR-Cas (Cas9) systems, the original spacer sequence is flanked by (e.g., immediately adjacent to) the original spacer 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 (e.g., see below).

[0280]

[0281] In the case of type II CRISPR-Cas (e.g., Cas9) systems, the PAM is immediately adjacent to the 3' of the target region. The PAM of type I CRISPR-Cas systems is located at the 5' of the target strand. Type III CRISPR-Cas systems have no known PAM. Makarova et al. describe the nomenclature of all classes, types, and subtypes of CRISPR systems (Nature Reviews Microbiology 13: 722-736 (2015)). Guide structures and PAMs are described by R. Barrangou (Genome Biol. 16: 247 (2015)).

[0282] Typical Cas12a PAM is rich in T. In some embodiments, the typical Cas12a PAM sequence can be 5'-TTN, 5'-TTTN or 5'-TTTV. In some embodiments, the typical Cas9 (e.g., Streptococcus pyogenes) PAM can be 5'-NGG-3'. In some embodiments, atypical PAMs can be used, but may be less efficient.

[0283] Those skilled in the art can determine other PAM sequences by established experimental and computational methods. Thus, for example, experimental methods include targeting sequences flanked by all possible nucleotide sequences and identifying sequence members that do not undergo targeting, such as by transformation of target plasmid DNA (Esvelt et al. 2013. Nat. Methods 10: 1116-1121; Jiang et al. 2013. Nat. Biotechnol. 31: 233-239). In some aspects, computational methods may include performing BLAST searches on natural spacers to identify the original target DNA sequence in a 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).

[0284] 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 on the same expression cassette or vector as the expression cassette or vector comprising the one or more guide nucleic acids or on another expression cassette or vector. When a nucleic acid construct encoding a base editor or a component for base editing is contained on an expression cassette or vector separate from an 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 a component for base editing and the guide nucleic acid in any order relative to each other, e.g., before, simultaneously with, or after providing (e.g., contacting with) the expression cassette comprising the guide nucleic acid.

[0285] As known in the art, the fusion protein of the present invention may comprise a sequence-specific nucleic acid binding domain (e.g., a sequence-specific DNA binding domain), a CRISPR-Cas polypeptide, and / or a deaminase domain fused to a peptide tag 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 (which is 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 the target nucleic acid.

[0286] 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 tags), c-Myc affinity tags, HA affinity tags, His affinity tags, S affinity tags, methionine-His affinity tags, RGD-His affinity tags, FLAG octapeptides, strep tags or strep tags II, V5 tags, and / or VSV-G epitopes. Any epitope that can be linked to a polypeptide and for which there is a corresponding affinity polypeptide that can be linked to another polypeptide can be used as a peptide tag in the present invention. In some embodiments, the peptide tag may comprise 1 or 2 or more copies of a peptide tag (e.g., repeating unit, multimeric epitope (e.g., tandem repeat)) (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 Opin Struc Biol 22(4):413-420 (2013)), U.S. Patent No. 9,982,053, which is incorporated herein by reference in its entirety for its teachings related to affibodies, anti-anticalins, monobodies, and / or DARPins. Examples of peptide tag sequences and affinity polypeptides thereof include, but are not limited to, amino acid sequences SEQ ID NOs: 45-47.

[0287] 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 nucleic acid binds to the target nucleic acid, and the RNA recruitment motif binds to the affinity polypeptide, thereby recruiting the polypeptide to the guide nucleic acid 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). Examples of RNA recruitment motifs and affinity polypeptides thereof include, but are not limited to, sequences of SEQ ID NOs: 48-58.

[0288] 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 sequence-spacer-extended portion (RT template-primer binding site)-RNA recruitment motif). In some embodiments, the RNA recruitment motif can be embedded in the extended portion.

[0289] 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, such as 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-3 mRNA binding factor (PUF).

[0290] In some embodiments, the components used to recruit polypeptides and nucleic acids can be those that function through chemical interactions, including but not limited to: dimerization of FRB-FKBP induced by rapamycin; biotin-streptavidin; SNAP tag; Halo tag; CLIP tag; DmrA-DmrC heterodimer induced by a compound; bifunctional ligands (e.g., two protein binding chemicals fused together, such as dihydrofolate reductase (DHFR)).

[0291] In some embodiments, a nucleic acid construct, expression cassette or vector of the invention optimized for expression in plants can be about 70% to 100% (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%) identical to a nucleic acid construct, expression cassette or vector comprising the same polynucleotide but which has not been codon-optimized for expression in plants.

[0292] Also provided herein are cells comprising one or more polynucleotides, guide nucleic acids, nucleic acid constructs, expression cassettes, or vectors of the invention.

[0293] The nucleic acid constructs of the present invention (e.g., constructs comprising a sequence-specific DNA 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 a target nucleic acid and / or its expression.

[0294] The polypeptides, polynucleotides, ribonucleoproteins (RNPs), nucleic acid constructs, expression cassettes and / or vectors of the present invention can be used to modify (e.g., mutate, e.g., base edit, cut, nick, etc.) any plant or plant part (or grouping of plants, e.g., into genus or higher classification) target nucleic acid, the plants including angiosperms, gymnosperms, monocots, dicots, C3 plants, C4 plants, CAM plants, bryophytes, ferns and / or pseudoferns, microalgae and / or macroalgae. The plants and / or plant parts that can be modified as described herein can be plants and / or plant parts of any plant species / variety / cultivar. In some embodiments, the plants that can be modified as described herein are monocots. In some embodiments, the plants that can be modified as described herein are dicots.

[0295] As used herein, the term "plant part" includes, but is not limited to, reproductive tissues (e.g., petals, sepals, stamens, pistils, receptacles, anthers, pollen, flowers, fruits, flower buds, ovules, seeds, embryos, nuts, grains, ears, cobs, and husks); vegetative tissues (e.g., petioles, stems, roots, root hairs, root tips, pith, coleoptiles, stalks, buds, 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, "shoots" refer to the above-ground parts including leaves and stems. As used herein, the term "tissue culture" includes cultures of tissues, cells, protoplasts and callus.

[0296] 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 a separated single cell, or can be a cultured cell, or can be a part of a higher organizational unit, such as a plant tissue (including callus) or a plant organ. In some embodiments, the plant cell can be an algae cell." 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 nucleic acid molecules of the present invention and / or nucleotide sequences 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 can not be regenerated into a plant.

[0297] "Plant cell culture" means cultures of plant units such as protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes, and embryos at various stages of development.

[0298] 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.

[0299] As used herein, "plant tissue" refers to a group of plant cells organized into structural and functional units. Included is any plant tissue in a plant 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 structural and / or functional units. The term, when used in conjunction with any specific type of plant tissue listed above or otherwise encompassed by this definition, or when used without reference to any specific type of plant tissue listed above or otherwise encompassed by this definition, is not intended to exclude any other type of plant tissue.

[0300] 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 plant can be hybridized with a non-transgenic plant and selected in the offspring for a plant comprising the desired gene edit but not comprising the transgenic for producing the edit, the transgene can be eliminated from the plant developed from the transgenic tissue or cell.

[0301] Any plant comprising an endogenous CLE gene can be modified as described herein to increase the number of ear rows, optionally without substantially reducing the length of the ear (e.g., the ear length is not reduced by more than 5-30%), and optionally to improve yield traits in the plant. Non-limiting examples of plants that can be modified as described herein can include, but are not limited to, turfgrasses (e.g., bluegrass, bentgrass, ryegrass, fescue), feather reed grass (e.g., grass), bunch grass, miscanthus, reed, switchgrass, vegetable crops including artichokes, kohlrabi, arugula, leeks, asparagus, lettuce (e.g., head, leaf, romaine), dark green malanga, melons (e.g., cantaloupe, watermelon, crenshaw, honeydew, cantaloupe), rape crops (e.g., Brussels sprouts, cabbage, cauliflower, broccoli, kale, kale, Chinese cabbage, pakchoy), cardoon, carrot, napa, okra, onion, celery, parsley, chickpea, parsnip, endive, pepper, potato, cucurbits (e.g., zucchini, cucumber, zucchini, pumpkin, honeydew, watermelon, cantaloupe), radish, bulb onion, rutabaga, eggplant, salsify, endive, shallot, chicory, garlic, spinach, onions, pumpkin, leafy greens, beets (sugar and fodder), sweet potatoes, chard, horseradish, tomatoes, turnips, and spices; fruit crops such as apples, apricots, cherries, nectarines, peaches, pears, plums, prunes, cherries, quince, figs, nuts (e.g., chestnuts, pecans, pistachios, hazelnuts, pistachios, peanuts, walnuts, macadamia nuts, almonds, etc.), citrus (e.g., clementines, kumquats, oranges, grapefruits, tangerines, mandarins, lemons, limes, etc.), blueberries, black raspberries, boysenberries, cranberries, currants, gooseberries, loganberries, raspberries, strawberries, blackberries, grapes (wine and table), avocados, bananas, kiwis, persimmons, pomegranates, pineapples, tropical fruits, pomegranates, melons, mangoes, papayas, and lychees, field crops such as clover, alfalfa, timothy, evening primrose, meadowfoam, foam), corn / maize (field, sweet corn, popcorn), hops, jojoba, buckwheat, safflower, quinoa, wheat, rice, barley, rye, millet, sorghum, oats, triticale, sorghum, tobacco, kapok, legumes (beans (e.g., green and dry), lentils, peas, soybeans), oil plants (rape, canola, mustard, olives, sunflower, coconut, castor oil plant, cocoa, peanuts, oil palm), duckweed, Arabidopsis, fiber plants (cotton, flax, jute), Lauraceae (cinnamon, camphor), or plants such as coffee, sugar cane, tea, and natural rubber plants;and / or such as flowering plants, cacti, succulents and / or such as bedding plants and / or ornamental plants (e.g., roses, tulips, violets), and trees such as forest trees (broadleaf trees and evergreens, such as conifers; e.g., elm, ash, oak, maple, fir, spruce, cedar, pine, birch, cypress, eucalyptus, willow) and shrubs and other seedlings. In some embodiments, the nucleic acid constructs of the invention and / or the expression cassettes and / or vectors encoding the same can be used to modify corn, soybean, wheat, canola, rice, tomato, pepper or sunflower. ;

[0302] In some embodiments, plants that can be modified as described herein may include, but are not limited to, corn, soybean, rapeseed, wheat, rice, cotton, sugarcane, sugar beet, barley, oats, alfalfa, sunflower, safflower, oil palm, sesame, coconut, tobacco, potato, sweet potato, cassava, coffee, apple, plum, apricot, peach, cherry, pear, fig, banana, citrus, cocoa, avocado, olive, almond, walnut, strawberry, watermelon, pepper, grape, tomato, cucumber, or certain species of Brassica (e.g., European rapeseed (B. napus), cabbage (B. oleracea), turnip (B. rapa), mustard rapeseed (B. juncea) and / or black mustard (B. nigra)). In some embodiments, plants that can be modified as described herein are dicots. In some embodiments, plants that can be modified as described herein are monocots. In some embodiments, plants that can be modified as described herein are corn (i.e., maize). In some embodiments, the plant that can be modified as described herein is wheat (eg, various species of the genus Triticum).

[0303] Thus, plants or plant cultivars that are preferentially treated according to the invention include all plants that have acquired, by genetic modification, genetic material that imparts particularly advantageous useful properties ("traits") to these plants. Examples of such properties are better plant growth, vigor, stress tolerance, ability to stand, 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.

[0304] A further example of such properties is an enhanced 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 that confer tolerance to such animal and microbial pests, in particular insects, special mention will be made of the genetic material encoding Bt proteins from Bacillus thuringiensis, which are widely described in the literature and are well known to those skilled in the art. Mention will also be made of proteins extracted from bacteria such as Photorhabdus (WO97 / 17432 and WO98 / 08932). In particular, 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 also 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 protein 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, VIP3Aa protein or toxic fragments thereof (e.g., Estruch et al. (1996), Proc Natl Acad Sci US A.28; 93 (11): 5389-94), Cry proteins as described in WO2001 / 47952, insecticidal proteins from Xenorhabdus (as described in WO98 / 50427), Serratia (especially from S. entomophila) or Photorhabdus species, 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 sequences (especially the sequences of their toxic fragments) in certain amino acids (1-10, preferably 1-5) or fused with a transit peptide (such as a plastid transit peptide) or another protein or peptide is also included in this article.

[0305] Another particularly emphasized example of such properties is the conferring of tolerance to one or more herbicides (e.g., imidazolinones, sulfonylureas, glyphosate or glufosinate). Among the DNA sequences encoding proteins that confer tolerance to certain herbicides on transformed plant cells and plants (i.e., target polynucleotides), particular mention will be made of the bar or PAT genes described in WO 2009 / 152359 or the Streptomyces coelicolor gene (which confer tolerance to glufosinate herbicides), genes encoding suitable EPSPS (5-enolpyruvylshikimate-3-phosphate synthase) (which confer tolerance to herbicides targeting EPSPS, in particular herbicides such as glyphosate and its salts), genes encoding glyphosate-n-acetyltransferases or genes encoding glyphosate oxidoreductases. Other 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 a 2,4-D-monooxygenase that confers tolerance to 2,4-D (2,4-dichlorophenoxyacetic acid), and a gene encoding a dicamba monooxygenase that confers tolerance to dicamba (3,6-dichloro-2-methoxybenzoic acid).

[0306] Further examples of such properties are enhanced resistance to phytopathogenic fungi, bacteria and / or viruses due to, for example, systemic acquired resistance (SAR), systemins, phytoalexins, elicitors and also resistance genes and correspondingly expressed proteins and toxins.

[0307] Particularly useful transgenic events in transgenic plants or plant cultivars that can be preferentially treated according to the present invention include event 531 / PV-GHBK04 (cotton, insect control, described in WO2002 / 040677), event 1143-14A (cotton, insect control, not deposited, described in WO2006 / 128569); event 1143-51B (cotton, insect control, not deposited, described in WO2006 / 128570); event 1445 (cotton, herbicide tolerance, not deposited, described in US-A 2002-120964 or WO2002 / 034946); Event 17053 (rice, herbicide tolerance, deposited as PTA-9843, described in WO2010 / 117737); Event 17314 (rice, herbicide tolerance, deposited as PTA-9844, described in WO2010 / 117735); Event 281-24-236 (cotton, insect control-herbicide tolerance, deposited as PTA-6233, described in WO2005 / 103266 or US-A 2005-216969); Event 3006-210-23 (cotton, insect control-herbicide tolerance, deposited as PTA-6233, described in US-A 2007-143876 or WO2005 / 103266); event 3272 (corn, quality traits, deposited as PTA-9972, described in WO2006 / 098952 or US-A 2006-230473); event 33391 (wheat, herbicide tolerance, deposited as PTA-2347, described in WO2002 / 027004), event 40416 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-11508, described in WO 11 / 075593); event 43A47 (corn, insect control-herbicide tolerance, deposited as ATCC PTA-11509, described in WO2011 / 075595); event 5307 (corn, insect control, deposited as ATCC PTA-9561 deposited, 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 (corn, 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 (Brassica napus, restoration of male sterility, deposited as NCIMB 41193, described in WO2005 / 074671),Event CE43-67B (cotton, insect control, deposited as DSMACC2724, 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 described in 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 deposited, described in WO2011 / 022469); event DAS-44406-6 / pDAB8264.44.06.1 (soybean, herbicide tolerance, deposited as PTA-11336, described in WO2012 / 075426), event DAS-14536-7 / pDAB8291.45.36.2 (soybean, herbicide tolerance, deposited as PTA-11335, described in WO2012 / 075429), event DAS-59122-7 (corn, insect control - herbicide tolerance, deposited as ATCC PTA 11384, described in US-A 2006-070139), corn event DAS-59132 (corn, insect control - herbicide tolerance, not deposited, described in WO2009 / 100188); event DAS68416 (soybean, herbicide tolerance, deposited as ATCC PTA-10442, described in WO2011 / 066384 or WO2011 / 066360); event DP-098140-6 (corn, herbicide tolerance, deposited as ATCC PTA-8296, described in US-A 2009-137395 or WO08 / 112019); event DP-305423-1 (soybean, quality traits, not deposited, described in US-A 2008-312082 or described in WO2008 / 054747); event DP-32138-1 (maize, hybrid system,009-0210970 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 (brinjal, insect control, not deposited, described in WO07 / 091277); event Fil 17 (corn, herbicide tolerance, deposited as ATCC 209031, described in US-A 2006-059581 or WO98 / 044140); event FG72 (soybean, herbicide tolerance, deposited as PTA-11041, described in WO2011 / 063413), event GA21 (corn, herbicide tolerance, deposited as ATCC 209033, described in US-A 2005-086719 or WO98 / 044140); event GG25 (corn, herbicide tolerance, deposited as ATCC 209032, described in US-A 2005-188434 or WO98 / 044140); event GHB119 (cotton, insect control-herbicide tolerance, deposited as ATCC PTA-8398 deposited, described in WO2008 / 151780); event GHB614 (cotton, herbicide tolerance, deposited as ATCC PTA-6878, described in US-A 2010-050282 or WO2007 / 017186); event GJ11 (maize, 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 tolerance, not deposited, described in US-A 2008-064032); event LL27 (soybean, herbicide tolerance, deposited as NCIMB 41658, described in WO2006 / 108674 or US-A 2008-320616); event LL55 (soybean, herbicide tolerance, deposited as NCIMB 41660, described in WO2006 / 108675 or US-A 2008-196127); event LLcotton25 (cotton, herbicide tolerance, deposited as ATCC PTA-3343, described in2003 / 013224 or USA 2003-097687); event LLRICE06 (rice, herbicide tolerance, deposited as ATCC 203353, described in U.S. Pat. No. 6,468,747 or WO2000 / 026345); event LLRice62 (rice, herbicide tolerance, deposited as ATCC 20335, 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, not deposited, described in ATCC 2004 / 011601 or US-A 2006-095986); event MON87427 (maize, pollination control, deposited as ATCC PTA-7899, described in WO 2011 / 062904); event MON87460 (maize, stress tolerance, deposited as ATCC PTA-8910, described in WO 2009 / 111263 or US-A 2011-0138504); event MON87701 (soybean, insect control, deposited as ATCC PTA-8194, described in US-A 2009-130071 or WO 2009 / 064652); event MON87705 (soybean, quality trait - herbicide tolerance, deposited as ATCC PTA-9241, described in US-A 2010-0080887 or described in WO2010 / 037016); event MON87708 (soybean, herbicide tolerance, deposited as ATCC PTA-9670, described in WO2011 / 034704); event MON87712 (soybean, yield, deposited as PTA-10296, described in2012 / 051199), event MON87754 (soybean, quality trait, deposited as ATCC PTA-9385, described in WO2010 / 024976); event MON87769 (soybean, quality trait, 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 (Brassica napus, 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 MS11 (Brassica napus, pollination control-herbicide tolerance, deposited as ATCC PTA-850 or PTA-2485, described in WO2001 / 031042); event MS8 (Brassica napus, 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 (Brassica napus, pollination control-herbicide tolerance, deposited as ATCC PTA-730, described in WO2001 / 041558 or US-A 2003-188347); event RT73 (Brassica napus, herbicide tolerance, not deposited, described in WO2002 / 036831 or US-A 2008-070260); event SYHT0H2 / SYN-000H2-5 (soybean, herbicide tolerance, deposited as PTA-11226,2001-029014 or WO2001 / 051654); event T304-40 (cotton, insect control - herbicide tolerance, deposited as ATCC PTA-8171, described in US-A 2010-077501 or described in 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, described in ATCC PTA-3925 deposited, 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), optionally with event EE-GM1 / LL2 7 or event EE-GM2 / LL55 (WO2011 / 063413A2) stacked with event EE-GM3 / FG72 (soybean, herbicide tolerance, ATCC accession number PTA-11041), event DAS-68416-4 (soybean, herbicide tolerance, ATCC accession number PTA-10442, WO2011 / 066360A1), event DAS-68416-4 (soybean, herbicide tolerance, A TCC Accession No. PTA-10442, WO2011 / 066384A1), Event DP-040416-8 (corn, insect control, ATCC Accession No. PTA-11508, WO2011 / 075593A1), Event DP-043A47-3 (corn, insect control, ATCC Accession No. PTA-11509, WO2011 / 075595A1), Event DP-004114-3 (corn, insect control, ATCC Accession No. PTA-11506, WO2011 / 084621A1), event DP-032316-8 (corn, insect control, ATCC Accession No. PTA-11507, WO2011 / 084632A1), event MON-88302-9 (Brassica napus, 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 (large beans, stacked herbicide tolerance, ATCC accession number PTA-11335, WO2012 / 075429A1), event SYN-000H2-5 (soybean, herbicide tolerance, ATCC accession number PTA-11226, WO2012 / 082548A2), event DP-061061-7 (Brassica napus, herbicide tolerance, no deposit number available, WO2012071039A1), event DP-073496-4 (Brassica napus, herbicide tolerance, no deposit number available, US201 2131692), event 8264.44.06.1 (soybean, stacked herbicide tolerance, accession number PTA-11336, WO2012075426A2), event 8291.45.36.2 (soybean, stacked herbicide tolerance, accession number PTA-11335, WO2012075429A2), event SYHT0H2 (soybean, ATCC accession number PTA-11226, WO2012 / 082548A2), event MON88701 (cotton, ATCC C accession number PTA-11754, WO2012 / 134808A1), event KK179-2 (alfalfa, ATCC accession number PTA-11833, WO2013 / 003558A1), event pDAB8264.42.32.1 (soybean, stacked herbicide tolerance, ATCC accession number PTA-11993, WO2013 / 010094A1), event MZDT09Y (corn, ATCC accession number PTA-13025, WO2013 / 012775A1). ,

[0308] The genes / events (e.g., target polynucleotides) that confer the desired traits 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 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 enhanced resistance of the plants to insects, arachnids, nematodes, slugs and snails, as well as the enhanced resistance of the plants to one or more herbicides.

[0309] Commercially available examples of such plants, plant parts or plant seeds that may be preferentially treated according to the present invention include RIB ROUNDUP VTDOUBLE VT TRIPLE BOLLGARD ROUNDUP READY 2 ROUNDUP 2XTEN DTM 、INTACTA RR2 VISTIVE and / or XTENDFLEX TM A commodity sold or distributed by a trade name, such as plant seeds.

[0310] 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 skilled person fall within the scope of the present invention.

[0311] Example

[0312] Example 1 Design of editing constructs for CLE editing

[0313] The genomic sequences of CLE7, CLE14, and FCP1 were identified in a proprietary maize line. Based on these reference sequences, spacer sequences (SEQ ID NOs: 101-104 and 110-117) were designed for editing constructs. The spacers were placed together with Cas-effector cleavage enzymes or Cas-effector base editing complexes. Editing constructs containing CRISPR-Cas effectors and spacer sequences were designed to produce four different editing outcomes. 1) The Cas-effector base editing complex was placed to modify the amino acid sequence of the mature CLE peptide, generating D>G changes in CLE7, V>I or M and E>K changes in CLE14, and V>I or M and P>L changes in FCP1. 2) The Cas-effector cleavage enzyme complex was deployed to generate out-of-frame base pair deletions, resulting in premature termination loss-of-function mutations in the CLE7, CLE14, and FCP1 coding regions by disrupting the mature CLE peptide sequence. 4) Deployment of the Cas effector cleavage complex to generate in-frame deletions in the sequences encoding the mature peptides of CLE7, CLE14, and FCP1. 5) Deployment of the Cas effector cleavage complex to generate small DNA transversions within the CLE7, CLE14, and FCP1 coding regions, resulting in dominant knockdown of gene transcription from both edited and unedited gene copies when in the het state. These small DNA rearrangements result in a dominant reduction in CLE function, which may be a result of multiple CLE genes being targeted for RNA interference-based transcriptional reduction.

[0314] Example 2 Transformation and selection of edited E0 plants

[0315] The vector encoding the spacer produced in Example 1 and the selected CRISPR-Cas effector is introduced into the dried excised corn embryo using Agrobacterium. The transformed tissue is selected in vitro with antibiotics to regenerate positive transformants. Healthy non-chimeric plants (E0) are selected and inserted into growth trays. Tissues (E0 generation) are collected from regenerated plants for DNA extraction, followed by molecular screening to identify the edits. The selected E0 plants are self-pollinated to produce the E1 generation. The selected E1 is self-pollinated to produce the E2 generation, backcrossed to produce the E2 progeny, or hybridized with a proprietary heterozygous test line to test in a heterozygous state.

[0316] Example 3 Phenotypic Assessment of Trait Activity

[0317] Seeds of E1, E2, backcrossed and hybridized materials were planted on flat plates and then transferred to pots after emergence. All materials were cultivated and grown to reproductive maturity under standard greenhouse conditions. According to standard practice, emerging ears were covered with small paper bags before silk appeared, and ears were covered during the flowering period to capture pollen on a plant-by-plant basis.

[0318] After the ears were harvested and dried, the ear rows were counted manually for all ears. The data represent the average of three row counts per ear taken from the middle portion of the ear with the most defined row line. To prevent double counting of rows, markers (e.g., paper clips) were inserted between the rows where counting began and designated where the row count should stop.

[0319] All spikes were photorecorded with a Canon digital camera and EOS application. The images were subsequently imported into ImageJ and all spikes were measured using the line track function. Spike length was determined in centimeters by setting a scale in the image analysis program to output the distance in centimeters after tracing the spike with a line along the length of the spike from its tip to the base of the spike. Unedited germplasm and lines transformed with the Gus plasmid were used as wild-type controls for phenotyping.

[0320] The present invention relates to the following embodiments:

[0321] 1. A plant or plant part thereof comprising at least one non-natural mutation in an endogenous CLAVATA3 / EMBRYO SURROUNDING REGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene encoding a wild-type precursor CLE polypeptide.

[0322] 2. The plant or part thereof according to embodiment 1, wherein the mutation is within a portion of an endogenous CLE gene encoding a mature CLE peptide, and the mutation results in the mature CLE peptide having an amino acid substitution compared to a wild-type mature CLE peptide.

[0323] 3. The plant or part thereof according to embodiment 1 or 2, wherein the CLE gene encodes a FON2-LIKE CLEPROTEIN1 (FCP1) FCP1 protein, a CLE7 protein or a CLE14 protein.

[0324] 4. The plant or part thereof according to any one of the preceding embodiments, wherein the at least one non-natural mutation produces a dominant negative allele, a semi-dominant allele, a weak loss-of-function allele, a null allele or a hypomorphic mutation.

[0325] 5. The plant or part thereof according to any one of the preceding embodiments, wherein the at least one non-natural mutation is a base substitution, a base deletion, a base insertion and / or an inversion.

[0326] 6. The plant or part thereof according to any one of the preceding embodiments, wherein the at least one non-natural mutation comprises a base substitution to A, T, G or C.

[0327] 7. The plant or part thereof according to any one of the preceding embodiments, wherein the at least one non-natural mutation is a substitution of at least one base pair (eg, 1, 2, 3, 4 or 5 base pairs).

[0328] 8. The plant or part thereof according to any one of the preceding embodiments, wherein the at least one non-natural mutation produces one or more substituted amino acid residues at positions 3, 5, 6, 8 or 9, as numbered relative to the amino acid position of SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 77.

[0329] 9. The plant or part thereof according to any one of the preceding embodiments, wherein the at least one non-natural mutation results in a substitution of valine (V) for isoleucine (I) or methionine (M) at position 3, a substitution of glutamate (E) for lysine (K) at position 5, a substitution of glycine (G) for threonine (T) at position 6, a substitution of aspartic acid (D) for alanine (A) at position 8, a substitution of aspartic acid (D) for glycine (G) at position 8, and / or a substitution of proline (P) for leucine (L) at position 9, the positions being numbered relative to the amino acid positions of SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 77.

[0330] 10. The plant or part thereof of any preceding embodiment, wherein the endogenous CLE gene comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 78, SEQ ID NO: 79 or SEQ ID NO: 80, comprises a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NO: 81, SEQ ID NO: 82 or SEQ ID NO: 82; encodes a sequence having at least 95% sequence identity to SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74, or encodes a domain having a sequence having at least 95% sequence identity to SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 77.

[0331] 11. The plant or part thereof of any one of embodiments 2-9, wherein the wild-type mature peptide comprises an amino acid sequence having a length of 12 amino acids, and R at position 1, V at position 3, P at position 4, G at position 6, P at position 9, H at position 11, and H at position 12, optionally wherein the wild-type processed peptide comprises any one of the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77.

[0332] 12. The plant or part thereof according to any one of the preceding embodiments, wherein the CLE gene comprising at least one non-natural mutation comprises the sequence of any one of SEQ ID NOs: 105-109.

[0333] 13. The plant or part thereof according to any one of the preceding embodiments, wherein the mature CLE peptide comprising at least one non-natural mutation comprises any one of the amino acid sequences of SEQ ID NOs: 84-100.

[0334] 14. The plant or part thereof according to any one of the preceding embodiments, wherein the plant is corn.

[0335] 15. The corn plant or part thereof according to any one of the preceding embodiments, wherein the corn plant comprising the at least one non-natural mutation in the endogenous CLE gene has an increased number of ear rows, optionally without substantially reducing ear length.

[0336] 16. A plant cell comprising an editing system, the editing system comprising:

[0337] (a) CRISPR-Cas effector proteins; and

[0338] (b) a guide nucleic acid having a spacer sequence having complementarity to a region of an endogenous target gene encoding a precursor CLE polypeptide.

[0339] 17. The plant cell of embodiment 16, wherein the endogenous target gene comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 78, SEQ ID NO: 79 or SEQ ID NO: 80.

[0340] 18. The plant cell of embodiment 16 or 17, wherein the endogenous target gene encodes a precursor CLE polypeptide comprising a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 72, SEQ ID NO: 73 or SEQ ID NO: 74.

[0341] 19. The plant cell of any one of embodiments 16-18, wherein the guide nucleic acid comprises a spacer sequence comprising any one of the nucleotide sequences of SEQ ID NOs: 101-104 and / or SEQ ID NOs: 110-117.

[0342] 20. The plant cell of any one of embodiments 16-19, wherein the plant cell is a corn cell.

[0343] 21. A plant regenerated from the plant part of any one of embodiments 1-15 or the plant cell of any one of embodiments 16-20.

[0344] 22. The plant of embodiment 21, wherein the plant is a corn plant and comprises a phenotype of increased ear row number, optionally without substantially reducing ear length.

[0345] 23. A corn plant cell comprising at least one non-natural mutation within a CLE gene encoding a precursor CLE polypeptide, 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 CLE gene.

[0346] 24. The corn plant cell of embodiment 23, wherein the CLE gene encodes a FCP1 protein, a CLE7 protein, or a CLE14 protein.

[0347] 25. A corn plant cell according to embodiment 23 or embodiment 24, wherein the at least one non-natural mutation is a dominant negative allele, a semi-dominant allele, a weak loss-of-function allele, or a hypomorphic allele.

[0348] 26. The corn plant cell of any one of embodiments 23-25, wherein the target site is within a region of a CLE gene, the region comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83.

[0349] 27. The corn plant cell of any one of embodiments 23-26, wherein the target site encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77.

[0350] 28. A corn plant cell according to any one of embodiments 23-27, wherein the mutation is generated after cutting by an editing system, wherein the editing system comprises a nuclease and a nucleic acid binding domain, and the nucleic acid binding domain binds to a target site within a sequence having at least 90% sequence identity with any one of the nucleotide sequences in SEQ ID NOs: 81-83.

[0351] 29. The corn plant cell of embodiment 28, 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.

[0352] 30. The corn plant cell of any one of embodiments 23-29, 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.

[0353] 31. The corn plant cell of any one of embodiments 23-30, wherein the at least one non-natural mutation within the CLE gene is a point mutation.

[0354] 32. A corn plant cell according to any one of embodiments 23-31, wherein the at least one non-natural mutation results in one or more substituted amino acid residues located at positions 3, 5, 6, 8 or 9, wherein the positions are numbered with reference to the amino acid position of SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 77.

[0355] 33. A corn plant regenerated from the corn plant cell of any one of embodiments 23-32, wherein the plant comprises the at least one non-natural mutation and the mutation produces a dominant negative allele.

[0356] 34. A corn plant according to embodiment 33, wherein the corn plant exhibits a phenotype of increased ear row number, optionally without substantially reducing ear length, when compared to a wild-type plant not comprising the allele (e.g., an isogenic wild-type plant).

[0357] 35. A method of producing / growing a transgene-free edited corn plant comprising:

[0358] Crossing a non-transgenic corn plant with the corn plant of any one of embodiments 1-15, 21, 22, 33, or 34, thereby introducing the at least one non-natural mutation into the non-transgenic corn plant; and

[0359] Progeny corn plants are selected that contain the at least one non-natural mutation and are free of the transgene, thereby producing transgene-free edited corn plants.

[0360] 36. A method for providing a plurality of corn plants having an increased number of ear rows, the method comprising planting two or more plants of any one of embodiments 1-15, 21, 22 or 33-35 in a growing area, thereby providing a plurality of corn plants having an increased number of ear rows compared to a plurality of control corn plants not comprising the mutation.

[0361] 37. A method for generating a variation in a mature corn CLE peptide, the method comprising:

[0362] introducing an editing system into a maize plant cell, wherein the editing system targets a region of a maize CLE gene encoding a mature maize CLE peptide protein, wherein the region comprises a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NO: 75, SEQ ID NO: 76, or SEQ ID NO: 77, or the region is encoded by a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; and

[0363] A region of the maize CLE gene is exposed to the editing system, thereby introducing a mutation in a mature maize CLE peptide in a maize plant cell; and generating a variation in the mature maize CLE peptide.

[0364] 38. A method according to embodiment 37, wherein the mutation is introduced after cutting by the editing system, wherein the editing system comprises a nuclease and a nucleic acid binding domain, and the nucleic acid binding domain binds to a target site within a sequence having at least 90% sequence identity with any one of the nucleotide sequences in SEQ IDNO: 81-83.

[0365] 39. A method for editing a specific site in the genome of a corn plant cell, the method comprising: cleaving a target site within an endogenous CLE gene in the corn plant cell in a site-specific manner, the endogenous CLE gene:

[0366] (a) comprising (i) a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80, and / or (ii) a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; and / or

[0367] (b) encoding (i) a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (ii) a domain having a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, thereby producing an edit in a portion of an endogenous gene of a corn plant cell encoding a mature CLE peptide and producing a plant cell comprising the edit in the portion of the endogenous gene.

[0368] 40. The method of any one of embodiments 38 to 39, further comprising regenerating a corn plant from a corn plant cell comprising the edit or mutation in the endogenous CLE gene to produce a corn plant comprising the edit or mutation in its endogenous CLE gene.

[0369] 41. The method of any one of embodiments 37-40, wherein the CLE gene encodes FCP1 protein, CLE7 protein or CLE14 protein.

[0370] 42. A method according to any one of embodiments 39-41, wherein the editing results in a non-natural mutation.

[0371] 43. A method according to embodiment 42, wherein the non-natural mutation results in one or more substituted amino acid residues located at positions 3, 5, 6, 8 or 9, and the positions are numbered with reference to the amino acid position of SEQ ID NO: 75, SEQ ID NO: 76 or SEQID NO: 77.

[0372] 44. A method for producing a corn plant comprising:

[0373] (a) contacting a population of maize plant cells comprising an endogenous CLE gene encoding a precursor CLE polypeptide with a nuclease (e.g., an editing system) linked to a nucleic acid binding domain that binds to a sequence that (i) has at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80, (ii) has at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83, (iii) encodes a sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74, or (ii) encodes a sequence that has at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77;

[0374] (b) selecting plant cells from the population in which the endogenous CLE gene has been mutated, thereby producing plant cells comprising a mutation in the endogenous CLE gene; and

[0375] (c) growing the selected plant cells into plants.

[0376] 45. A method for increasing the number of ear rows in a corn plant comprising:

[0377] (a) contacting a corn plant cell comprising an endogenous CLE gene with a nuclease targeted to the endogenous CLE 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 CLE gene, wherein the endogenous CLE gene:

[0378] (i) comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80;

[0379] (ii) comprising a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83;

[0380] (iii) encoding a sequence having at least 95% sequence identity to any one of SEQ ID NOs: 72-74; and / or

[0381] (iv) encoding a domain having a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, to produce a corn plant cell comprising a mutation in an endogenous CLE gene; and

[0382] (b) growing the corn plant cell into a corn plant comprising the mutation in the endogenous CLE gene, thereby producing a corn plant having a mutated endogenous CLE gene and an increased number of ear rows.

[0383] 46. ​​A method of producing a corn plant or part thereof comprising at least one cell having a mutated endogenous CLE gene, the method comprising:

[0384] contacting a target site in an endogenous CLE gene in the 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 CLE gene,

[0385] Wherein the endogenous CLE gene:

[0386] (a) comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80;

[0387] (b) comprising a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83;

[0388] (c) encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or

[0389] (d) a domain encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77,

[0390] Thereby producing said corn plant or part thereof comprising at least one cell having a mutation in said endogenous CLE gene.

[0391] 47. A method for producing a corn plant or part thereof comprising a mutated endogenous CLE gene and exhibiting increased ear row number, the method comprising:

[0392] contacting a target site in an endogenous CLE gene in the 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 CLE gene,

[0393] Wherein the endogenous CLE gene:

[0394] (a) comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80;

[0395] (b) comprising a region having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83;

[0396] (c) encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or

[0397] (d) encoding a domain having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77,

[0398] Thereby producing a corn plant or part thereof comprising an endogenous CLE gene having a mutation and exhibiting an increased number of ear rows.

[0399] 48. A method according to any one of embodiments 44-47, wherein the nuclease cleaves the endogenous CLE gene, thereby introducing the mutation into the endogenous CLE gene.

[0400] 49. The method of any one of embodiments 44-48, wherein the CLE gene encodes an FCP1 protein, a CLE7 protein, or a CLE14 protein.

[0401] 50. A method according to any one of embodiments 44-49, wherein the mutation is a dominant negative mutation, a semi-dominant mutation, a weak loss-of-function mutation, a hypomorphic mutation, or a null mutation.

[0402] 51. A method according to any one of embodiments 44-50, wherein the mutation is a base substitution, a base deletion and / or a base insertion.

[0403] 52. A method according to any one of embodiments 44-51, wherein the mutation comprises a point mutation.

[0404] 53. A method according to any one of embodiments 43-52, wherein the mutation produces one or more substituted amino acid residues located at position 3, 5, 6, 8 or 9, wherein the positions are numbered relative to the amino acid position of SEQ ID NO: 75, SEQ ID NO: 76 or SEQ ID NO: 77.

[0405] 54. A method according to any one of embodiments 44-53, 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.

[0406] 55. A method according to any one of embodiments 44-54, 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.

[0407] 56. A guide nucleic acid that binds to a portion of a target site in a CLE gene encoding a precursor CLE peptide, wherein the target site comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83 or encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77.

[0408] 57. The guide nucleic acid according to embodiment 56, wherein the guide nucleic acid comprises a spacer comprising a nucleotide sequence of any one of SEQ ID NOs: 101-104 and / or SEQ ID NOs: 110-117.

[0409] 58. A system comprising the guide nucleic acid of embodiment 56 or embodiment 57 and a CRISPR-Cas effector protein associated with the guide nucleic acid.

[0410] 59. The system of embodiment 58, further comprising a tracr nucleic acid and a CRISPR-Cas effector protein associated with the guide nucleic acid, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

[0411] 60. 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 CLE gene encoding a precursor CLE peptide.

[0412] 61. A gene editing system according to embodiment 60, wherein the CLE gene encodes FCP1 protein, CLE7 protein or CLE14 protein.

[0413] 2. The gene editing system of embodiment 60 or 61, wherein the CLE gene:

[0414] (a) comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80;

[0415] (b) comprising a region having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83;

[0416] (c) encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or

[0417] (d) a domain encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77.

[0418] 63. A gene editing system according to any one of embodiments 60-62, wherein the guide nucleic acid comprises a spacer sequence having a nucleotide sequence shown in any one of SEQ ID NOs: 101-104 and / or SEQ ID NOs: 110-117.

[0419] 64. The gene editing system of any one of embodiments 60-63, further comprising 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.

[0420] 65. A complex comprising a CRISPR-Cas effector protein comprising a cleavage domain and a guide nucleic acid, wherein the guide nucleic acid binds to a target site in an endogenous CLE gene encoding a precursor CLE polypeptide, wherein the endogenous CLE gene:

[0421] (a) comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80;

[0422] (b) comprising a region having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83;

[0423] (c) encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or

[0424] (d) a domain encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77,

[0425] The cleavage domain shown therein cleaves the target strand in the CLE gene.

[0426] 66. 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 CLE gene encoding a precursor CLE peptide, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to:

[0427] (i) comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80;

[0428] (ii) comprising a region having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 75-77;

[0429] (iii) encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or

[0430] (iv) a domain encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77.

[0431] 67. A nucleic acid encoding a dominant negative allele of a maize CLE gene, wherein the maize CLE gene encodes a precursor CLE polypeptide.

[0432] 68. The nucleic acid of embodiment 67, wherein the nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 105-109.

[0433] 69. The nucleic acid of embodiment 67, wherein the nucleic acid comprises a sequence encoding any one of the amino acid sequences of SEQ ID NOs: 84-100.

[0434] 70. A corn plant or part thereof comprising the nucleic acid of any one of embodiments 67-69.

[0435] 71. A method for generating a mutation in an endogenous CLE gene in a plant, comprising:

[0436] (a) targeting a gene editing system to a portion of a CLE gene, said portion encoding a domain having a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-71, and

[0437] (b) selecting a plant comprising an amino acid residue substitution at one of positions 3, 5, 6, 8 or 9, wherein the positions are numbered with reference to the amino acid position of SEQ ID NO:75, SEQ ID NO:76 or SEQ ID NO:77.

[0438] 72. A corn plant or plant part thereof comprising at least one non-natural mutation located in at least one endogenous CLE gene, wherein the endogenous CLE gene is located on chromosome 2 at a chromosomal interval defined by base pair (bp) position 235,093,752 to base pair position 235,094,276 and / or on chromosome 2 at a chromosomal interval defined by base pair (bp) position 40,126,538 to base pair position 40,127,030 and including them.

[0439] 73. A corn plant or plant part thereof comprising at least one non-natural mutation in at least one endogenous CLE gene, wherein the endogenous CLE gene is located on chromosome 4 in a chromosomal interval defined by base pair (bp) position 7,570,324 to base pair position 7,571,104 and including them.

[0440] 74. The corn plant or part thereof of embodiment 72, wherein the chromosome interval corresponds to the reference corn genome of B73 version 4 (B73 RefGen_v4).

[0441] 75. The corn plant or part thereof of embodiment 73, wherein the chromosomal interval corresponds to the reference corn genome of B73 version 3 (B73 RefGen_v3).

[0442] 76. A guide nucleic acid that binds to a target nucleic acid in a CLE gene in a corn plant, wherein the target nucleic acid is located on chromosome 2 at a chromosomal interval defined by base pair (bp) position 235,093,752 to base pair position 235,094,276 and / or on chromosome 2 at a chromosomal interval defined by base pair (bp) position 40,126,538 to base pair position 40,127,030 and including them.

[0443] 76. A guide nucleic acid that binds to a target nucleic acid in a CLE gene in a corn plant, wherein the target nucleic acid is located within a chromosomal interval defined by base pair (bp) position 235,093,752 to base pair position 235,094,276 on chromosome 2 and including them.

[0444] 77. A guide nucleic acid according to embodiment 75, wherein the chromosomal interval corresponds to the reference corn genome of B73 version 4 (B73RefGen_v4).

[0445] 78. A guide nucleic acid according to embodiment 76, wherein the chromosomal interval corresponds to the reference maize genome of B73 version 3 (B73RefGen_v3).

[0446] The foregoing descriptions illustrate the present invention and are not to be construed as limiting the present invention. The present invention is defined by the following claims, with equivalents of each claim to be included therein.

Claims

1. A method for producing a plant, comprising: Regenerating a plant from a plant part or plant cell, wherein the plant part or plant cell comprises at least one non-natural mutation in an endogenous CLAVATA3 / EMBRYO SURROUNDINGREGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene encoding a wild-type precursor CLE polypeptide, wherein the CLE gene is an FCP1 gene encoding a FON2-LIKE CLE PROTEIN1 (FCP1) protein.

2. A method for producing plant parts, comprising: A plant part is regenerated from a plant cell, wherein the plant cell comprises at least one non-natural mutation in an endogenous CLE gene encoding a wild-type precursor CLE polypeptide, wherein the CLE gene is an FCP1 gene encoding an FCP1 protein.

3. The method of claim 1 or 2, wherein the mutation is located within the portion of the endogenous FCP1 gene encoding a mature FCP1 peptide, and the mutation results in the mature FCP1 peptide having an amino acid substitution compared to a wild-type mature FCP1 peptide.

4. The method of claim 1 or 2, wherein the at least one non-natural mutation produces a dominant negative allele, a semi-dominant allele, a weak loss-of-function allele, a null allele, or a hypomorphic mutation.

5. The method according to claim 1 or 2, wherein the at least one non-natural mutation is a base substitution, a base deletion, a base insertion and / or an inversion.

6. The method of claim 1 or 2, wherein the at least one non-natural mutation comprises a base substitution to A, T, G or C.

7. The method of claim 1 or 2, wherein the at least one non-natural mutation is a substitution of at least one base pair (eg, 1, 2, 3, 4 or 5 base pairs).

8. The method of claim 1 or 2, wherein the at least one non-natural mutation produces one or more substituted amino acid residues at positions 3, 5, 6, 8 or 9, as numbered relative to the amino acid position of SEQ ID NO:

75.

9. The method of claim 1 or 2, wherein the at least one non-natural mutation results in a substitution of valine (V) for isoleucine (I) or methionine (M) at position 3, a substitution of glutamate (E) for lysine (K) at position 5, a substitution of glycine (G) for threonine (T) at position 6, a substitution of aspartic acid (D) for alanine (A) at position 8, a substitution of aspartic acid (D) for glycine (G) at position 8, and / or a substitution of proline (P) for leucine (L) at position 9, the positions being numbered relative to the amino acid positions of SEQ ID NO:

75.

10. The method of claim 1 or 2, wherein the endogenous FCP1 gene comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 78, comprises a region having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 81; encodes a sequence having at least 95% sequence identity to SEQ ID NO: 72, or encodes a domain having a sequence having at least 95% sequence identity to SEQ ID NO:

75.

11. The method of any one of claims 3-8, wherein the wild-type mature peptide comprises an amino acid sequence having a length of 12 amino acids, and R at position 1, V at position 3, P at position 4, G at position 6, P at position 9, H at position 11, and H at position 12, optionally wherein the wild-type processed peptide comprises the amino acid sequence of SEQ ID NO:

75. 12 . The method according to claim 1 or 2 , wherein the FCP1 gene comprising at least one non-natural mutation comprises the sequence of any one of SEQ ID NOs: 105-107.

13. The method of claim 1 or 2, wherein the mature FCP1 peptide comprising at least one non-natural mutation comprises any one of the amino acid sequences of SEQ ID NOs: 84-90.

14. The method of claim 1 or 2, wherein the plant is corn.

15. The method of claim 1 or 2, wherein the corn plant comprising the at least one non-natural mutation in the endogenous FCP1 gene has an increased number of ear rows.

16. The method of claim 15, wherein the corn plant comprising the at least one non-natural mutation in the endogenous FCP1 gene does not have substantially reduced ear length.

17. A method of producing a plant or plant part comprising regenerating the plant or plant part from a plant cell comprising an editing system comprising: (a) CRISPR-Cas effector proteins; and (b) a guide nucleic acid having a spacer sequence having complementarity to a region of an endogenous target gene encoding a precursor FCP1 polypeptide.

18. The method of claim 17, wherein the endogenous target gene comprises a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:

78.

19. The method of claim 17 or 18, wherein the endogenous target gene encodes a precursor FCP1 polypeptide comprising a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

72.

20. The method of any one of claims 17-19, wherein the guide nucleic acid comprises a spacer sequence comprising any one of the nucleotide sequences of SEQ ID NO: 101 and / or SEQ ID NOs: 112-114.

21. The method of any one of claims 17-20, wherein the plant cell is a corn cell.

22. A method of producing a corn plant or plant part, comprising regenerating a corn plant or plant part from a corn plant cell comprising at least one non-natural mutation within an FCP1 gene encoding a precursor FCP1 polypeptide, 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 FCP1 gene.

23. The method of claim 22, wherein the at least one non-natural mutation is a dominant negative allele, a semi-dominant allele, a weak loss-of-function allele, or a hypomorphic allele.

24. The method of claim 22, wherein the target site is within a region of the FCP1 gene, the region comprising a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:

81.

25. The method of claim 22, wherein the target site encodes a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

75.

26. The method of claim 22, wherein the mutation is generated after cleavage by an editing system comprising a nuclease and a nucleic acid binding domain that binds to a target site within a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:

81.

27. The method of claim 26, 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.

28. The method of claim 22, 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.

29. The method of claim 22, wherein the at least one non-natural mutation within the FCP1 gene is a point mutation.

30. The method of claim 22, wherein the at least one non-natural mutation results in one or more substituted amino acid residues at positions 3, 5, 6, 8 or 9, as numbered with reference to the amino acid positions of SEQ ID NO:

75.

31. The method of claim 22, wherein the corn plant or plant part comprises the at least one non-natural mutation, and the mutation produces a dominant negative allele.

32. The method of claim 31, wherein the corn plant exhibits a phenotype of increased ear row number, optionally without substantially reducing ear length, when compared to a wild-type plant not comprising the allele (e.g., an isogenic wild-type plant).

33. A method of producing / growing a transgene-free edited corn plant comprising: Crossing a non-transgenic corn plant with a corn plant as defined in any one of claims 1, 3-16, 31 or 32, thereby introducing the at least one non-natural mutation into the non-transgenic corn plant; and Progeny corn plants are selected that contain the at least one non-natural mutation and are free of the transgene, thereby producing transgene-free edited corn plants.

34. A method for providing a plurality of corn plants having an increased number of ear rows, the method comprising planting two or more plants as defined in any one of claims 1, 3-16, 31 or 32 in a growing area, thereby providing a plurality of corn plants having an increased number of ear rows compared to a plurality of control corn plants not comprising the mutation.

35. A method for generating a variation in a mature corn FCP1 peptide, the method comprising: introducing an editing system into a maize plant cell, wherein the editing system targets a region of a maize FCP1 gene encoding a mature maize FCP1 peptide protein, wherein the region comprises a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 75, or the region is encoded by a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 81; and The region of the maize FCP1 gene is exposed to the editing system, thereby introducing a mutation in a mature maize FCP1 peptide in a maize plant cell; and generating a variation in the mature maize FCP1 peptide.

36. The method of claim 35, wherein the mutation is introduced after cleavage by the editing system, the editing system comprising a nuclease and a nucleic acid binding domain that binds to a target site within a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO:

81.

37. A method for editing a specific site in the genome of a corn plant cell, the method comprising: cleaves a target site within an endogenous FCP1 gene in the corn plant cell in a site-specific manner, wherein the endogenous FCP1 gene: (a) comprising (i) a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 78, and / or (ii) a region having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 81; and / or (b) a domain encoding (i) a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 72; and / or (ii) a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 75, thereby producing an edit in a portion of an endogenous gene of a corn plant cell encoding a mature FCP1 peptide and producing a plant cell comprising the edit in the portion of the endogenous gene.

38. The method of any one of claims 35-37, further comprising regenerating a corn plant from a corn plant cell comprising the edit or mutation in the endogenous FCP1 gene to produce a corn plant comprising the edit or mutation in its endogenous FCP1 gene.

39. The method of any one of claims 35-37, wherein the editing results in a non-natural mutation.

40. The method of claim 39, wherein the non-natural mutation results in one or more substituted amino acid residues at positions 3, 5, 6, 8 or 9, as numbered with reference to the amino acid positions of SEQ ID NO:

75.

41. A method for producing a corn plant comprising: (a) contacting a population of maize plant cells comprising an endogenous FCP1 gene encoding a precursor FCP1 polypeptide with a nuclease (e.g., an editing system) linked to a nucleic acid binding domain that binds to a sequence that (i) has at least 90% sequence identity to a nucleotide sequence of SEQ ID NO:78, (ii) has at least 90% sequence identity to a nucleotide sequence of SEQ ID NO:81, (iii) encodes a sequence that has at least 95% sequence identity to an amino acid sequence of SEQ ID NO:72, or (ii) encodes a sequence that has at least 95% sequence identity to an amino acid sequence of SEQ ID NO:75; (b) selecting plant cells from the population in which the endogenous FCP1 gene has been mutated, thereby producing plant cells comprising a mutation in the endogenous FCP1 gene; and (c) growing the selected plant cells into plants.

42. A method for increasing the number of ear rows in a corn plant comprising: (a) contacting a corn plant cell comprising an endogenous FCP1 gene with a nuclease targeted to the endogenous FCP1 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 FCP1 gene, wherein the endogenous FCP1 gene: (i) comprising a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 78; (ii) comprising a region having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 81; (iii) encodes a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 72; and / or (iv) a domain encoding a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 75, to produce a corn plant cell comprising a mutation in an endogenous FCP1 gene; and (b) growing the corn plant cell into a corn plant comprising the mutation in the endogenous FCP1 gene, thereby producing a corn plant having a mutated endogenous FCP1 gene and an increased number of ear rows.

43. A method of producing a corn plant or part thereof comprising at least one cell having a mutated endogenous FCP1 gene, the method comprising: contacting a target site in an endogenous FCP1 gene in the 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 FCP1 gene, Wherein the endogenous FCP1 gene: (a) comprising a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 78; (b) comprising a region having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 81; (c) encoding a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 72; and / or (d) encoding a domain having a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 75, Thereby producing said corn plant or part thereof comprising at least one cell having a mutation in said endogenous FCP1 gene.

44. A method for producing a corn plant or part thereof comprising a mutated endogenous FCP1 gene and exhibiting increased ear row number, the method comprising: contacting a target site in an endogenous FCP1 gene in the 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 FCP1 gene, Wherein the endogenous FCP1 gene: (a) comprising a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 78; (b) comprising a region having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 81; (c) encoding a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 72; and / or (d) encoding a domain having a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 75, Thereby a corn plant or part thereof is produced which comprises an endogenous FCP1 gene having a mutation and exhibits an increased number of ear rows.

45. The method of any one of claims 41-44, wherein the nuclease cleaves the endogenous FCP1 gene, thereby introducing the mutation into the endogenous FCP1 gene.

46. ​​The method of any one of claims 41-44, wherein the mutation is a dominant negative mutation, a semi-dominant mutation, a weak loss-of-function mutation, a hypomorphic mutation, or a null mutation.

47. The method according to any one of claims 41-44, wherein the mutation is a base substitution, a base deletion and / or a base insertion.

48. The method of any one of claims 41-44, wherein the mutation comprises a point mutation.

49. The method of any one of claims 41-44, wherein the mutation results in one or more substituted amino acid residues at positions 3, 5, 6, 8 or 9, relative to the amino acid position numbering of SEQ ID NO:

75.

50. The method of any one of claims 41-44, 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.

51. The method of any one of claims 41-44, 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.

52. A guide nucleic acid that binds to a portion of a target site in a CLE gene encoding a precursor CLE peptide, wherein the target site comprises a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83 or encodes a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77.

53. The guide nucleic acid according to claim 52, wherein the guide nucleic acid comprises a spacer comprising the nucleotide sequence of any one of SEQ ID NOs: 101-104 and / or SEQ ID NOs: 110-117.

54. A system comprising the guide nucleic acid of claim 52 or claim 53 and a CRISPR-Cas effector protein associated with the guide nucleic acid.

55. The system of claim 54, further comprising a tracr nucleic acid and a CRISPR-Cas effector protein associated with the guide nucleic acid, optionally wherein the tracr nucleic acid and the guide nucleic acid are covalently linked.

56. 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 CLE gene encoding a precursor CLE peptide, wherein the CLE gene encodes an FCP1 protein, a CLE7 protein, or a CLE14 protein.

57. The gene editing system of claim 56, wherein the CLE gene: (a) comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprising a region having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) a domain encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77.

58. The gene editing system of claim 56, wherein the guide nucleic acid comprises a spacer sequence having a nucleotide sequence as shown in any one of SEQ ID NOs: 101-104 and / or SEQ ID NOs: 110-117.

59. The gene editing system of any one of claims 56-58, 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.

60. A complex comprising a CRISPR-Cas effector protein comprising a cleavage domain and a guide nucleic acid, wherein the guide nucleic acid binds to a target site in an endogenous CLE gene encoding a precursor CLE polypeptide, wherein the endogenous CLE gene: (a) comprising a sequence having at least 90% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 78-80; (b) comprising a region having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs: 81-83; (c) encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 72-74; and / or (d) a domain encoding a sequence having at least 95% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 75-77, The cleavage domain shown therein cleaves the target strand in the CLE gene.

61. 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 FCP1 gene encoding a precursor FCP1 peptide, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (i) comprising a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 78; (ii) comprising a region having at least 95% sequence identity to the nucleotide sequence of SEQ ID NO: 81; (iii) encodes a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 72; and / or (iv) a domain encoding a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

75.

62. A nucleic acid encoding a dominant negative allele of a maize FCP1 gene, wherein the maize FCP1 gene encodes a precursor FCP1 polypeptide.

63. The nucleic acid of claim 62, wherein the nucleic acid comprises the nucleotide sequence of any one of SEQ ID NOs: 105-109.

64. The nucleic acid of claim 62, wherein the nucleic acid comprises a sequence encoding any one of the amino acid sequences of SEQ ID NOs: 84-90.

65. A method for producing a corn plant or plant part, comprising introducing a nucleic acid comprising any one of claims 62-64 into a corn plant cell, plant or plant part, and optionally regenerating the corn plant cell into a corn plant or plant part and / or regenerating the corn plant part into a corn plant.

66. A method for generating a mutation in an endogenous FCP1 gene in a plant, comprising: (a) targeting a gene editing system to a portion of the FCP1 gene, said portion encoding a domain having a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 75, and (b) selecting a plant comprising an amino acid residue substitution at one of positions 3, 5, 6, 8 or 9, wherein said positions are numbered with reference to the amino acid position of SEQ ID NO:

75.

67. A method for producing a corn plant or a plant part thereof, comprising regenerating a corn plant or a plant part thereof from a corn plant part or a corn plant cell comprising at least one non-natural mutation located in at least one endogenous FCP1 gene, wherein the endogenous FCP1 gene is located on chromosome 2 in a chromosomal interval defined by base pair (bp) position 235,093,752 to base pair position 235,094,276 and / or on chromosome 2 in a chromosomal interval defined by base pair (bp) position 40,126,538 to base pair position 40,127,030 and including them.

68. A method for producing a corn plant or a plant part thereof, comprising regenerating a corn plant or a plant part thereof from a corn plant part or a corn plant cell comprising at least one non-natural mutation in at least one endogenous FCP1 gene, wherein the endogenous FCP1 gene is located on chromosome 4 in a chromosomal interval defined by base pair (bp) position 7,570,324 to base pair position 7,571,104 and including them.

69. The method of claim 67, wherein the chromosomal interval corresponds to the reference maize genome of B73 version 4 (B73 RefGen_v4).

70. The method of claim 68, wherein the chromosomal interval corresponds to the reference maize genome of B73 version 3 (B73 RefGen_v3).

71. A guide nucleic acid that binds to a target nucleic acid in a CLE gene in a corn plant, wherein the target nucleic acid is located on chromosome 2 at a chromosomal interval defined by base pair (bp) position 235,093,752 to base pair position 235,094,276 and / or on chromosome 2 at a chromosomal interval defined by base pair (bp) position 40,126,538 to base pair position 40,127,030 and including them.

72. A guide nucleic acid that binds to a target nucleic acid in a CLE gene in a corn plant, wherein the target nucleic acid is located on chromosome 2 within a chromosomal interval defined by base pair (bp) position 235,093,752 to base pair position 235,094,276 and including them.

73. The guide nucleic acid of claim 71, wherein the chromosomal interval corresponds to the reference maize genome of B73 version 4 (B73RefGen_v4).

74. The guide nucleic acid of claim 72, wherein the chromosomal interval corresponds to the reference maize genome of B73 version 3 (B73RefGen_v3).

75. A method of producing a plant comprising: Regenerating a plant from a plant part or plant cell, wherein the plant part or plant cell comprises at least one non-natural mutation in an endogenous CLAVATA3 / EMBRYO SURROUNDINGREGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene encoding a wild-type precursor CLE polypeptide, wherein the CLE gene is a CLE7 gene encoding a CLE7 protein.

76. A method of producing a plant part comprising: Regenerating a plant part from a plant cell, wherein the plant cell comprises at least one non-natural mutation in an endogenous CLAVATA3 / EMBRYO SURROUNDING REGION-RELATED (CLV3 / ESR-RELATED) (CLE) gene encoding a wild-type precursor CLE polypeptide, wherein the CLE gene is a CLE7 gene encoding a CLE7 protein.

77. The method of claim 75 or 76, wherein the mutation is located within the portion of the endogenous CLE7 gene encoding a mature CLE7 peptide, and the mutation results in the mature CLE7 peptide having an amino acid substitution compared to a wild-type mature CLE7 peptide.

78. The method of claim 75 or 76, wherein the endogenous CLE7 gene: (a) comprising a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 80; (b) comprising a region having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 83; (c) encodes a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:74; or (d) a domain encoding a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:

77.

79. 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 CLE7 gene encoding a precursor CLE7 peptide, wherein the guide nucleic acid comprises a spacer sequence that is complementary to and binds to: (i) comprising a sequence having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 80; (ii) comprising a region having at least 90% sequence identity to the nucleotide sequence of SEQ ID NO: 83; (iii) encodes a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO: 74; and / or (iv) a domain encoding a sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO:77.

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