Short stature corn plants

Abstract

The present invention discloses modified corn plants which have been edited in specific cytokine dehydrogenase genes. These corn plants have a short stature phenotype and have an increased lodging resistance.

Description

[0001] HiNel / ShortStature / 858

[0002] SHORT STATURE CORN PLANTS

[0003] Field of the invention

[0004] The present invention relates to the field of plant molecular biology, more specifically to the field of cultivated crops, more specifically to modified corn plants which plants have a short stature due to specific gene edits in combinations of cytokine dehydrogenase genes.

[0005] Introduction to the invention

[0006] The agricultural Green Revolution is considered one of the most pivotal points in the transformation of global agriculture, having a tremendous impact on worldwide food production, socio-economic conditions, and environmental sustainability. This revolution was realized by the parallel implementation of advanced agronomic practices, modern fertilizers, and a new plant ideotype that dramatically improved lodging tolerance. In small grain crops like wheat and rice, increased inputs of fertilizers and increased planting densities promoted rapid stem elongation that made crops more prone to lodging, which caused significant economic losses associated with reduced yields, quality, and harvesting efficiency. To counter the unintended impacts of lodging, a reduced stature plant ideotype was conceptualized that dramatically improved overall plant sturdiness and enabled higher planting densities. Among the first crops that were developed to harness the power of these new architectural ideotypes were wheat and rice. Since their introduction, both crops have been readily accepted by growers and have contributed to saving millions of hectares of land from being brought into agricultural production. Maize is one of the world's most widely cultivated crops. As future demands for maize will continue to rise, fields will face ever more frequent and extreme weather patterns that directly affect crop productivity. Gene editing is the preferred tool for the creation of novel alleles for plant genetic research and breeding. Subsequent phenotypic analysis is used to associate genes to (molecular) functions and traits such as here short stature. Short stature is controlled by a complex interaction between many different genes and requires the identification of the right combinations of alleles to have pronounced and desired effects. Hence, the ability to stack multiple gene edits in the same plant is crucial for the engineering of complex traits. Multiplexing, i.e. the simultaneous targeting of many genes, is one of the major advantages of the CRISPR / Cas9 gene editing system. Delivery of the Cas9 protein can be combined with a single T-DNA containing multiple guide RNAs (gRNAs), only differing in the spacer sequence and targeting a variety of genes, including multiple members of gene families. Short stature corn plants have been described (see for example in WO2019161149) by gene editing of specific subtypes of GA20 oxidase genes but there is a need for additional gene edit combinations which can lead to an ideal short stature corn plant. An ideal short stature corn plant (in the context of maize hybrids) desirably has the following characteristics: i) a decrease of plant height of about one third (2.74-3.66 HiNel / ShortStature / 858 meters to < 2.13 meters), ii) the ear height should be above 60cm (to maintain compatibility with harvesting equipment), iii) the ear size should be similar to other hybrids and the corn plant should have an increase in standability. In the present invention we disclose combinations of gene disruptions in cytokine dehydrogenases (CKX) which lead to a short stature phenotype. Cytokine dehydrogenase mutants in rice have been described by Zheng X et al (2023) Plant Genome 16:e20283) but the translatability from rice to corn is not possible since the expression pattern of CKX genes is not conserved between rice and corn. In addition, the CKX genes we have identified for editing in corn to obtain a short stature are different than those described in Zheng X et al (2023).

[0007] Summary of the invention

[0008] In a first embodiment the invention provides a modified corn plant, or plant part thereof, comprising a mutant allele at the CKX4b locus and a mutant allele at the CKX12 locus.

[0009] In a particular embodiment only one of said CKX4b or CKX12 loci comprise homozygous mutant alleles.

[0010] In another particular embodiment the invention provides a modified corn plant wherein both of said CKX4b and CKX12 loci comprise homozygous mutant alleles.

[0011] In another particular embodiment the corn plant as described herein comprises in one or in loci of CKX4b and CKX12 a heteroallelic combination or two identical mutant alleles.

[0012] In another particular embodiment the invention provides a modified corn plant, or plant part thereof, according to the plants described herein, wherein the mutant allele exhibits an at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% reduction of expression or enzymatic activity relative to an unmodified, wild type CKX4b or CKX12 allele.

[0013] In yet another embodiment the invention provides a modified corn plant, or plant part thereof, as described herein wherein the mutant allele at the CKX4b and / or CKX12 locus comprises a mutation in a sequence region selected from the group consisting of a promoter, 5'UTR, exon, intron, 3'UTR, terminator, or any combination thereof.

[0014] In yet another embodiment the invention provides a modified corn plant, or plant part thereof, as described herein before, wherein the mutant allele at the CKX4b and / or CKX12 locus comprises one or more mutation types selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice-site mutation, and any combination thereof.

[0015] In yet another embodiment the invention provides a modified corn plant, or plant part thereof, as described herein before, further comprising a mutant allele at the CKX10 locus. HiNel / ShortStature / 858

[0016] In yet another embodiment the invention provides a modified corn plant, or plant part thereof, wherein CKX10 locus comprises homozygous mutant alleles.

[0017] In yet another embodiment the invention provides a modified corn plant, or plant part wherein the mutant allele at the CKX10 locus comprises a mutation in a sequence region selected from the group consisting of a promoter, 5'UTR, exon, intron, 3'UTR, terminator, or any combination thereof and wherein said mutation is selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice-site mutation, and any combination thereof wherein said mutant allele exhibits an at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% reduction of expression or enzymatic activity relative to an unmodified, wild type CKX10 allele.

[0018] In yet another embodiment the invention provides a modified corn plant, or plant part thereof, as described herein before wherein the CKX4b locus comprises a nucleotide sequence encoding for a polypeptide sequence with at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity over the total length of SEQ ID NO: 1 and wherein the CKX12 locus comprises a nucleotide sequence encoding for a polypeptide sequence with at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity over the total length of SEQ ID NO: 2.

[0019] In yet another embodiment the invention provides a modified corn plant, or plant part thereof, wherein the CKX10 locus comprises a nucleotide sequence encoding for a polypeptide sequence with at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity over the total length of SEQ ID NO: 3 and wherein the CKX12 locus comprises a nucleotide sequence encoding for a polypeptide with at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity over the total length of SEQ ID NO: 2 and wherein the CKX4b locus comprises a nucleotide sequence encoding for a polypeptide sequence with at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity over the total length of SEQ ID NO: 1.

[0020] Figure legends

[0021] Figure 1: picture showing the most important parameters which are taken into account when developing a short stature corn plant.

[0022] Figure 2: Schematic representation of the variability generated with the BREEDIT pipeline. A) Transformation constructs able to express 12 gRNA, called SCRIPTS, were used to transform Cas9 expressing plants, called EDITOR. In every transformation event, the editing efficiency can vary generating plants that bear edits in different genes. B) Plants transformed with different SCRIPTS can be crossed generating different combinations of edits in one plant. Stacking multiple SCRIPTS in a single plant is possible, with potential to stack up to 60 gene edits if using 5 SCRIPTS. HiNel / ShortStature / 858 re 3 Short stature ideotype observed in plants with both SCRIPT 2 and SCRIPT 4 (S2S4). A. Compact phenotype of seedlings containing SCRIPT 2 and SCRIPT 4. B. reduced sheath length of plants with SCRIPT 2 and SCRIPT 4. C. Increased leaf blade width of plants with SCRIPT 2 and SCRIPT 4. D. Short stature observed in mature plants containing SCRIPT 2 and SCRIPT 4. (ED: EDITOR line expressing Cas9; S: SCRIPT).

[0023] Representation of the potential combination of edits present in each population in Experiment

[0024] #1 (A) and #2 (B). Columns represent the genotype of the parental plants of each population, they are named based on the possible edits that they can bear in concordance to the Script used for transformation. Colored in green, genes that were not edited, in orange, genes that were edited in heterozygous state and therefore they will segregate producing alleles that are not edited, or edited in heterozygous and homozygous state, and in blue genes that were mutated into a homozygous state and therefore will be consistently edited in the descendant plants. Transgenerational editing due to the presence of Cas9 and SCRIPTS could also increase the number of potential combinations of edits.

[0025] Representation of the variability on the short stature phenotypes observed. EDI (Non-edited control plants), S2 (Plants edited in CKX gene family), S2S4 (Plants edited in members of CKX, TCP and GRF gene families). regression models using XGBoost on plants obtained from experiment #1 (left panel) and experiment #2 (right panel) confirm 3 CKX proteins as targets for gene editing to reduce the rest height:

[0026] CKX4b, CKX10 and CKX12. left panel represents the wild type corn plant, right panel depicts the short stature corn plants which have gene edits in ckx4b and ckxl2 genes.

[0027] Visual representation of the protein sequence alignments of CKX4b (A), CKX10 (B) and CKX12

[0028] (C). The first line of each graph represents the consensus sequence indicating the AA position and length of the longest protein sequence. The second line indicates the average pairwise identity % value (Green, 100% average identity; brown, <100-30% average identity; red, <30% average identity). The different genotypes are listed based on higher similarity to the consensus identity. AA display different colors based on their polarity, even though mismatches to the consensus identity are present, often the AA mismatched is replaced for another AA with similar polarity. HiNel / ShortStature / 858

[0029] Detailed description of the invention

[0030] Definitions

[0031] As used herein, "locus" is a chromosomal locus or region where a polymorphic nucleic acid, trait determinant, gene, or marker is located. A "locus" can be shared by two homologous chromosomes to refer to their corresponding locus or region. As used herein, "allele" refers to an alternative nucleic acid sequence of a gene or at a particular locus ( e.g. a nucleic acid sequence of a gene or locus that is different than other alleles for the same gene or locus). Such an allele can be considered (i) wild-type or (ii) mutant if one or more mutations or edits are present in the nucleic acid sequence of the mutant allele relative to the wild-type allele. A mutant allele for a gene may have a reduced or eliminated activity or expression level for the gene relative to the wild-type allele. For diploid organisms such as com, a first allele can occur on one chromosome, and a second allele can occur at the same locus on a second homologous chromosome. If one allele at a locus on one chromosome of a plant is a mutant allele and the other corresponding allele on the homologous chromosome of the plant is wild-type, then the plant is described as being heterozygous for the mutant allele. However, if both alleles at a locus are mutant alleles, then the plant is described as being homozygous for the mutant alleles. A plant homozygous for mutant alleles at a locus may comprise the same mutant allele or different mutant alleles if heteroallelic or biallelic.

[0032] As used herein, a "wild-type gene" or "wild-type allele" refers to a gene or allele having a sequence or genotype that is most common in a particular plant species, or another sequence or genotype with natural variations, polymorphisms, or other silent mutations relative to the most common sequence or genotype that do not significantly impact the expression and activity of the gene or allele. Indeed, a "wild-type" gene or allele contains no variation, polymorphism, or any other type of mutation that substantially affects the normal function, activity, expression, or phenotypic consequence of the gene or allele.

[0033] The terms "percent identity" or "percent identical" as used herein in reference to two or more nucleotide or protein sequences is calculated by (i) comparing two optimally aligned sequences (nucleotide or protein) over a window of comparison, (ii) determining the number of positions at which the identical nucleic acid base (for nucleotide sequences) or amino acid residue (for proteins) occurs in both sequences to yield the number of matched positions, (iii) dividing the number of matched positions by the total number of positions in the window of comparison, and then (iv) multiplying this quotient by 100% to yield the percent identity. HiNel / ShortStature / 858

[0034] As used herein, "modified" in the context of a plant, plant seed, plant part, plant cell, and / or plant genome, refers to a plant, plant seed, plant part, plant cell, and / or plant genome comprising an engineered change in the expression level and / or coding sequence of one or more cytokinin dehydrogenase gene(s) (CKX gene(s)) relative to a wild-type or control plant, plant seed, plant part, plant cell, and / or plant genome, such as via a genome editing event or mutation affecting (e.g., reducing or eliminating) the expression level or activity of one or more endogenous cytokinin dehydrogenase genes. Indeed, the term "modified" may further refer to a plant, plant seed, plant part, plant cell, and / or plant genome having one or more mutations affecting expression of one or more endogenous CKX genes, introduced through chemical mutagenesis, transposon insertion or excision, or any other known mutagenesis technique, or introduced through genome editing. For clarity, therefore, a modified plant, plant seed, plant part, plant cell, and / or plant genome includes a mutated and / or edited plant, plant seed, plant part, plant cell, and / or plant genome having a modified expression level, expression pattern, and / or coding sequence of one or more CKX gene(s) relative to a wild-type or control plant, plant seed, plant part, plant cell, and / or plant genome. Modified plants may be homozygous or heterozygous for any given mutation or edit, and / or may be bi-allelic at a CKX locus. A modified plant is bi-allelic for a CKX gene if each copy of the CKX gene is modified by a different allele (i.e., different mutation(s) and / or edit(s)), wherein each allele lowers the expression level and / or activity of the CKX gene. Modified plants or seeds may contain various molecular changes that affect expression of CKX gene(s), such as CKX4b, CKX10 or CKX12 gene(s), including genetic and / or epigenetic modifications. Modified plants, plant parts, seeds, etc., may have been subjected to mutagenesis, genome editing or site-directed integration (e.g., without being limiting, via methods using site-specific nucleases), genetic transformation (e.g., without being limiting, via methods of Agrobacterium transformation or microprojectile bombardment), or a combination thereof. Such "modified" plants, plant seeds, plant parts, and plant cells include plants, plant seeds, plant parts, and plant cells that are offspring or derived from "modified" plants, plant seeds, plant parts, and plant cells that retain the molecular change (e.g., change in expression level and / or activity) to the one or more CKX genes. A modified seed provided herein may give rise to a modified plant provided herein. A modified plant, plant seed, plant part, plant cell, or plant genome provided herein may comprise a recombinant DNA construct or vector or genome edit as provided herein. A "modified plant product" may be any product made from a modified plant, plant part, plant cell, or plant chromosome provided herein, or any portion or component thereof.

[0035] As used herein, the term "homozygous" refers to a genotype comprising two identical alleles at a given locus in a diploid genome, or a genotype comprising two non-identical mutant alleles at a given locus in a diploid genome. The latter genotype comprising two non-identical mutant alleles is also referred to as being heteroallelic or as a heteroallelic combination. As used herein, "heterozygous" describes a HiNel / ShortStature / 858 genotype comprising a mutant allele and a wild-type allele at a given locus in a diploid genome. As used herein, the term "control plant" (or likewise a "control" plant seed, plant part, plant cell and / or plant genome) refers to a plant (or plant seed, plant part, plant cell and / or plant genome) that is used for comparison to a modified plant ( or modified plant seed, plant part, plant cell and / or plant genome) and has the same or similar genetic background (e.g., same parental lines, hybrid cross, inbred line, testers, etc.) as the modified plant (or plant seed, plant part, plant cell and / or plant genome), except for a genome editing event(s) affecting one or more CKX genes. For example, a control plant may be an inbred line that is the same as the inbred line used to make the modified plant, or a control plant may be the product of the same hybrid cross of inbred parental lines as the modified plant, except for the absence in the control plant of any genome editing event(s) affecting one or more CKX genes. Similarly, an unmodified control plant refers to a plant that shares a substantially similar or essentially identical genetic background as a modified plant, but without the one or more engineered changes to the genome (e.g., transgene, mutation or edit) of the modified plant. For purposes of comparison to a modified plant, plant seed, plant part, plant cell and / or plant genome, a "wild-type plant" ( or likewise a "wild-type" plant seed, plant part, plant cell and / or plant genome) refers to a non-transgenic and non-genome edited control plant, plant seed, plant part, plant cell and / or plant genome. As used herein, a "control" plant, plant seed, plant part, plant cell and / or plant genome may also be a plant, plant seed, plant part, plant cell and / or plant genome having a similar (but not the same or identical) genetic background to a modified plant, plant seed, plant part, plant cell and / or plant genome, if deemed sufficiently similar for comparison of the characteristics or traits to be analyzed. As used herein, a "target site" for genome editing refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by a site-specific nuclease introducing a double stranded break ( or single-stranded nick) into the nucleic acid backbone of the polynucleotide sequence and / or its complementary DNA strand. A target site may comprise at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, at least 23, at least 24, at least 25, at least 26, at least 27, at least 29, or at least 30 consecutive nucleotides. A "target site" for a RNA- guided nuclease may comprise the sequence of either complementary strand of a double-stranded nucleic acid (DNA) molecule or chromosome at the target site. A site-specific nuclease may bind to a target site, such as via a non-coding guide RNA (e.g., without being limiting, a CRISPR RNA (crRNA) or a single-guide RNA (sgRNA) as described further below). A non-coding guide RNA provided herein may be complementary to a target site ( e.g., complementary to either strand of a double-stranded nucleic acid molecule or chromosome at the target site). It will be appreciated that perfect identity or complementarity may not be required for a non-coding guide RNA to bind or 25 hybridize to a target site. For example, at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, or at least 8 HiNel / ShortStature / 858 mismatches ( or more) between a target site and a non-coding RNA may be tolerated. A "target site" also refers to the location of a polynucleotide sequence within a plant genome that is bound and cleaved by another site-specific nuclease that may not be guided by a non-coding RNA molecule, such as a meganuclease, zinc finger nuclease (ZFN), or a transcription activator-like effector nuclease (TALEN), to introduce a double stranded break ( or single-stranded nick) into the polynucleotide sequence and / or its complementary DNA strand. As used herein, a "target region" or a "targeted region" refers to a polynucleotide sequence or region that is flanked by two or more target sites. Without being limiting, in some embodiments a target region may be subjected to a mutation, deletion, insertion or inversion. As used herein, "flanked" when used to describe a target region of a polynucleotide sequence or molecule, refers to two or more target sites of the polynucleotide sequence or molecule surrounding the target region, with one target site on each side of the target region. Apart from genome editing, the term "target site" may also be used in the context of gene suppression to refer to a portion of a mRNA molecule ( e.g., a "recognition site") that is complementary to at least a portion of a non-coding RNA molecule ( e.g., a miRNA, siRNA, etc.) encoded by a suppression construct.

[0036] A modified plant, plant part, cell, or explant provided herein may be of an elite variety or an elite line. An elite variety or an elite line refers to a variety that has resulted from breeding and selection for superior agronomic performance. A edited plant, cell, or explant provided herein may be a hybrid plant, cell, or explant. As used herein, a "hybrid" is created by crossing two plants from different varieties, lines, inbreds, or species, such that the progeny comprises genetic material from each parent. Skilled artisans recognize that higher order hybrids can be generated as well. For example, a first hybrid can be made by crossing Variety A with Variety B to create a A x B hybrid, and a second hybrid can be made by crossing Variety C with Variety D to create an C x D hybrid. The first and second hybrids can be further crossed to create the higher order hybrid (A x B) x (C x D) comprising genetic information from all four parent varieties.

[0037] Short-stature corn plants are gaining attention in the agricultural sector due to their significant economic relevance. These hybrids, which are typically one-third shorter than traditional corn, offer several advantages that contribute to their economic importance. Shorter corn varieties allow for higher planting densities, meaning more plants can be grown per acre. This can potentially lead to increased yields without the risk of lodging (when plants fall over due to weak stalks), which is a common issue with taller corn varieties. By mitigating this risk, short-stature corn can maintain or even enhance overall productivity on the same land area. The reduced height of these corn plants enables easier and more precise application of agricultural inputs, such as fertilizers and pesticides, throughout the growing season. Farmers can use ground-based equipment later in the season without damaging the crops, which improves input efficiency and can lower overall production costs. Short-stature corn is less susceptible HiNel / ShortStature / 858 to damage from extreme weather events like high winds and storms, which are becoming more common due to climate change. This resilience reduces the likelihood of crop loss and contributes to a more stable and predictable yield, which is economically beneficial for farmers. These hybrids require fewer resources, such as water and nutrients, to grow effectively. This makes them particularly valuable in regions with limited water availability or poor soil conditions. The ability to produce more with less is a key factor in improving the sustainability of corn production, which has economic implications as demand for sustainable farming practices grows. By allowing for more precise and targeted management practices, short-stature corn can help reduce input costs (e.g., fertilizers, pesticides) and minimize the need for additional labor or specialized equipment, leading to overall cost savings for farmers. Overall, the economic relevance of short-stature corn lies in its ability to increase yields, improve management practices, enhance resilience to environmental stressors, and contribute to sustainable agriculture, all of which have significant financial benefits for farmers and the agricultural industry at large.

[0038] This means in order to screen for genes that contribute to a short statue phenotype that one does not only can focus on genes that reduce the total plant height when knocked out (see Figure 1 for the nomenclature on height measurements) but one needs to focus on genes and combinations that reduce the rest height more than the height to ear (small reductions in height to ear can be tolerated when the reduction in rest height is much higher). Ideally, the diameter of the stem should also remain constant (or increase) as this organ is important for lodging resistance (small stem widths will increase the sensitivity to lodging and greensnap). Likewise, root architecture should not change in significant amounts (as this could increase the probability of root lodging. This decrease in plant height should also not translate to significant decreases in plant yield (the increase in yield due to a higher plant density should be larger than the decrease in the yield per plant).

[0039] Targeted mutations (or edits) in the genome of a plant can be made by introducing a double strand break (DSB) or nick. According to this approach, mutations, such as deletions, insertions, inversions and / or substitutions maybe introduced at a target site via imperfect repair of the DSB or nick to produce a knock-out or knock-down of a CKX gene. Such mutations may be generated by imperfect repair of the targeted locus even without the use of a donor template molecule. A "knock-out" of a CKX gene may be achieved by inducing a DSB or nick at or near the endogenous locus of the CKX gene that results in nonexpression of the CKX protein or expression of a non-functional protein, whereas a "knock-down" of a CKX gene may be achieved in a similar manner by inducing a DSB or nick at or near the endogenous locus of the CKX gene that is repaired imperfectly at a site that does not affect the coding sequence of the CKX gene in a manner that would eliminate the function of the encoded CKX protein. For example, the site of the DSB or nick within the endogenous locus may be in the upstream or 5' region of the CKX gene (e.g., a promoter and / or enhancer sequence) to affect or reduce its level of expression. Similarly, such HiNel / ShortStature / 858 targeted knock-out or knock-down mutations of a CKX gene may be generated with a donor template molecule to direct a particular or desired mutation at or near the target site via repair of the DSB or nick. The donor template molecule may comprise a homologous sequence with or without an insertion sequence and comprising one or more mutations, such as one or more deletions, insertions, inversions and / or substitutions, relative to the targeted genomic sequence at or near the site of the DSB or nick. For example, targeted knock-out mutations of a CKX gene may be achieved by deleting or inverting at least a portion of the gene or by introducing a frame shift or premature stop codon into the coding sequence of the gene. A deletion of a portion of a CKX gene may also be introduced by generating DSBs or nicks at two target sites and causing a deletion of the intervening target region flanked by the target sites.

[0040] A site-specific nuclease provided herein may be selected from the group consisting of a zinc-finger nuclease (ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, a transposase, or any combination thereof. A recombinase may be a serine recombinase attached to a DNA recognition motif, a tyrosine recombinase attached to a DNA recognition motif or other recombinase enzyme known in the art. A recombinase or transposase may be a DNA transposase or recombinase attached to a DNA binding domain. A tyrosine recombinase attached to a DNA recognition motif may be selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase. According to some embodiments, a Cre recombinase or a Gin recombinase provided herein is tethered to a zinc-finger DNA binding domain. In another embodiment, a serine recombinase attached to a DNA recognition motif provided herein is selected from the group consisting of a PhiC31 integrase, an R4 integrase, and a TP-901 integrase. In another embodiment, a DNA transposase attached to a DNA binding domain provided herein is selected from the group consisting of a TALE-piggyBac and TALE-Mutator.

[0041] According to embodiments of the present disclosure, an RNA-guided endonuclease may be selected from the group consisting of Casl, CasIB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl 7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, Csf3, Csf4, Cpfl, CasX, CasY, and homologs or modified versions thereof, Argonaute (non-limiting examples of Argonaute proteins include Thermus thermophilus Argonaute (TtAgo ), Pyrococcus furiosus Argonaute (PfAgo ), Natronobacterium gregoryi Argonaute (NgAgo) and homo logs or modified versions thereof. According to some embodiments, an RNA-guided endonuclease may be a Cas9 or Cpfl enzyme. HiNel / ShortStature / 858

[0042] In an aspect, a site-specific nuclease provided herein is selected from the group consisting of a zinc-finger nuclease, a meganuclease, an RNA-guided nuclease, a TALE-nuclease, a recombinase, a transposase, or any combination thereof. In another aspect, a site-specific nuclease provided herein is selected from the group consisting of a Cas9 or a Cpfl . In another aspect, a site-specific nuclease provided herein is selected from the group consisting of a Casl, a CasIB, a Cas2, a Cas3, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas9, a Casio, a Csyl, a Csy2, a Csy3, a Csel, a Cse2, a Cscl, a Csc2, a Csa5, a Csn2, a Csm2, a Csm3, a Csm4, a Csm5, a Csm6, a Cmrl, a Cmr3, a Cmr4, a Cmr5, a Cmr6, a Csbl, a Csb2, a Csb3, a Csxl 7, a Csxl4, a CsxlO, a

[0043] 5 Csxl6, a CsaX, a Csx3, a Csxl, a Csxl5, a Csfl, a Csf2, a Csf3, a Csf4, a Cpfl, CasX, CasY, a homolog thereof, or a modified version thereof. In another aspect, an RNA-guided nuclease provided herein is selected from the group consisting of a Cas9 or a Cpfl. In another aspect, an RNA guided nuclease provided herein is selected from the group consisting of a Casl, a CasIB, a Cas2, a Cas3, a Cas4, a Cas5, a Cas6, a Cas7, a Cas8, a Cas9, a Casio, a Csyl, a Csy2, a Csy3, a Csel, a Cse2, a Cscl, a Csc2, a Csa5, a Csn2, a Csm2, a Csm3, a Csm4, a Csm5, a Csm6, a Cmrl, a Cmr3, a Cmr4, a Cmr5, a Cmr6, a Csbl, a Csb2, a Csb3, a Csxl 7, a Csxl4, a CsxlO, a Csxl6, a CsaX, a Csx3, a Csxl, a Csxl5, a Csfl, a Csf2, a Csf3, a Csf4, a Cpfl, CasX, CasY, a homolog thereof, or a modified version thereof. In another aspect, a method and / or a composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific nucleases. In yet another aspect, a method and / or a composition provided herein comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten polynucleotides encoding at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten site-specific nucleases. For RNA-guided endonucleases, a guide RNA (gRNA) molecule is further provided to direct the endonuclease to a target site in the genome of the plant via base-pairing or hybridization to cause a DSB or nick at or near the target site. The gRNA may be transformed or introduced into a plant cell or tissue (perhaps along with a nuclease, or nuclease- encoding DNA molecule, construct or vector) as a gRNA molecule, or as a recombinant DNA molecule, construct or vector comprising a transcribable DNA sequence encoding the guide RNA operably linked to a plant-expressible promoter. As understood in the art, a "guide RNA" may comprise, for example, a CRISPR RNA (crRNA), a single-chain guide RNA (sgRNA), or any other RNA molecule that may guide or direct an endonuclease to a specific target site in the genome. A "single-chain guide RNA" ( or "sgRNA") is a RNA molecule comprising a crRNA covalently linked a tracrRNA by a linker sequence, which may be expressed as a single RNA transcript or molecule. The guide RNA comprises a guide or targeting sequence that is identical or complementary to a target site within the plant genome, such as at or near a CKX gene. A protospacer-adjacent motif (PAM) may be present in the genome immediately adjacent and HiNel / ShortStature / 858 upstream to the 5' end of the genomic target site sequence complementary to the targeting sequence of the guide RNA - i.e., immediately downstream (3') to the sense (+) strand of the genomic target site (relative to the targeting sequence of the guide RNA) as known in the art. The genomic PAM sequence on the sense(+) strand adjacent to the target site (relative to the targeting sequence of the guide RNA) may comprise 5'-NGG-3'. However, the corresponding sequence of the guide RNA (i.e., immediately downstream (3') to the targeting sequence of the guide RNA) may generally not be complementary to the genomic PAM sequence.

[0044] For knockout (and possibly knockdown) mutations through genome editing, an RNA-guided endonuclease may be targeted to a coding and / or intron sequence of a CKX gene to potentially eliminate expression and / or activity of a functional CKX protein from the gene. However, a knockout of a CKX gene expression may also be achieved in some cases by targeting the upstream and / or 5 'UTR sequence(s) of the gene, or other sequences at or near the genomic locus of the gene. Thus, a knockout of a CKX gene expression may be achieved by targeting a genomic sequence at or near the site or locus of a targeted CKX4b or CKX12 or CKX10 gene, an upstream or downstream sequence, such as a promoter and / or enhancer sequence, or an intron, 5'UTR, and / or 3'UTR sequence, of a CKX4b or CKX12 or CKX10 gene, as described above for knockdown of a CKX4b or CKX12 or CKX10 gene.

[0045] In addition to the guide sequence, a guide RNA may further comprise one or more other structural or scaffold sequence(s), which may bind or interact with an RNA-guided endonuclease. Such scaffold or structural sequences may further interact with other RNA molecules (e.g. tracrRNA). Methods and techniques for designing targeting constructs and guide RNAs for genome editing and site-directed integration at a target site within the genome of a plant using an RNA-guided endonuclease are known in the art.

[0046] According to some embodiments, recombinant DNA constructs and vectors are provided comprising a polynucleotide sequence encoding a site-specific nuclease, such as a zinc-finger nuclease (ZFN), a meganuclease, an RNA-guided endonuclease, a TALE-endonuclease (TALEN), a recombinase, or a transposase, wherein the coding sequence is operably linked to a plant expressible promoter. For RNA- guided endonucleases, recombinant DNA constructs and vectors are further provided comprising a polynucleotide sequence encoding a guide RNA, wherein the guide RNA comprises a guide sequence of sufficient length having a percent identity or complementarity to a target site within the genome of a plant, such as at or near a targeted CKX gene. According to some embodiments, a polynucleotide sequence of a recombinant DNA construct and vector that encodes a site-specific nuclease or a guide RNA may be operably linked to a plant expressible promoter, such as an inducible promoter, a constitutive promoter, a tissue-specific promoter, etc. HiNel / ShortStature / 858

[0047] In another aspect, a modified corn plant does not have any significant off-types in at least one female organ or ear. In an aspect, a modified corn plant exhibits essentially no reproductive abnormality. In a further aspect, an off-type or reproductive abnormality is selected from the group consisting of male (tassel or anther) sterility, reduced kernel or seed number, and the presence of one or more masculinized or male (or male-like) reproductive structures in the female organ or ear ( e.g., anther ear). In another aspect, a modified com plant comprises one or more traits, relative to an unmodified control plant, selected from the group consisting of shorter plant height, increased stalk / stem diameter, improved lodging resistance, reduced green snap, deeper roots, increased leaf area, earlier canopy closure, higher stomata! conductance, lower ear height, increased foliar water content, improved drought tolerance, improved nitrogen use efficiency, reduced anthocyanin content and area in leaves under normal or nitrogen-limiting or water-limiting stress conditions, increased ear weight, increased harvest index, increased yield, preserved yield, commercially acceptable seed yield, increased seed number, increased seed weight, and increased prolificacy.

[0048] In an aspect, a modified corn plant is an inbred. In another aspect, a modified com plant is a hybrid. In an aspect, a modified corn plant is a plant modified by a targeted genome editing technique.

[0049] According to some embodiments, a recombinant DNA construct or vector may comprise a first polynucleotide sequence encoding a site-specific nuclease and a second polynucleotide sequence encoding a guide RNA that may be introduced into a plant cell together via plant transformation techniques. Alternatively, two recombinant DNA constructs or vectors may be provided including a first recombinant DNA construct or vector and a second DNA construct or vector that may be introduced into a plant cell together or sequentially via plant transformation techniques, wherein the first recombinant DNA construct or vector comprises a polynucleotide sequence encoding a site-specific nuclease and the second recombinant DNA construct or vector comprises a polynucleotide sequence encoding a guide RNA. According to some embodiments, a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a site-specific nuclease may be introduced via plant transformation techniques into a plant cell that already comprises (or is transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a guide RNA. Alternatively, a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a guide RNA may be introduced via plant transformation techniques into a plant cell that already comprises (or is transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a site-specific nuclease. According to yet further embodiments, a first plant comprising (or transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a site-specific HiNel / ShortStature / 858 nuclease may be crossed with a second plant comprising (or transformed with) a recombinant DNA construct or vector comprising a polynucleotide sequence encoding a guide RNA. Such recombinant DNA constructs or vectors may be transiently transformed into a plant cell or stably transformed or integrated into the genome of a plant cell. In an aspect, vectors comprising polynucleotides encoding a site-specific nuclease, and optionally one or more, two or more, three or more, or four or more gRNAs are provided to a plant cell by transformation methods known in the art (e.g., without being limiting, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation). In an aspect, vectors comprising polynucleotides encoding a Cas9 nuclease, and optionally one or more, two or more, three or more, or four or more gRNAs are provided to a plant cell by transformation methods known in the art (e.g., without being limiting, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation). In another aspect, vectors comprising polynucleotides encoding a Cpfl and, optionally one or more, two or more, three or more, or four or more crRNAs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).

[0050] Several site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, are not RNA-guided and instead rely on their protein structure to determine their target site for causing the DSB or nick, or they are fused, tethered or attached to a DNA-binding protein domain or motif. The protein structure of the site-specific nuclease (or the fused / attached / tethered DNA binding domain) may target the site-specific nuclease to the target site. According to many of these embodiments, non-RNA-guided site-specific nucleases, such as recombinases, zinc finger nucleases (ZFNs), meganucleases, and TALENs, may be designed, engineered and constructed according to known methods to target and bind to a target site at or near the genomic locus of an endogenous CKX gene of a com plant, such as the CKX4b gene or the CKX10 gene in corm, to create a DSB or nick at such genomic locus to knockout or knockdown expression of the CKX genes via repair of the DSB or nick.

[0051] In an aspect, a targeted genome editing technique described herein may comprise the use of a recombinase. In some embodiments, a tyrosine recombinase attached, etc., to a DNA recognition domain or motif may be selected from the group consisting of a Cre recombinase, a Flp recombinase, and a Tnpl recombinase. In an aspect, a Cre recombinase or a Gin recombinase provided herein may be tethered to a zinc-finger DNA binding domain. The Flp-FRT site-directed recombination system may come from the 2p plasmid from the baker's yeast Saccharomyces cerevisiae. In this system, Flp recombinase (flippase) may recombine sequences between flippase recognition target (FRT) sites. FRT sites comprise 34 nucleotides. Flp may bind to the "arms" of the FRT sites (one arm is in reverse orientation) and cleaves the FRT site at either end of an intervening nucleic acid sequence. After HiNel / ShortStature / 858 cleavage, Flp may recombine nucleic acid sequences between two FRT sites. Cre-lox is a site-directed recombination system derived from the bacteriophage P 1 that is similar to the Flp-FRT recombination system. Cre-lox can be used to invert a nucleic acid sequence, delete a nucleic acid sequence, or translocate a nucleic acid sequence. In this system, Cre recombinase may recombine a pair of lox nucleic acid sequences. Lox sites comprise 34 nucleotides, with the first and last 13 nucleotides (arms) being palindromic. During recombination, Cre recombinase protein binds to two lox sites on different nucleic acids and cleaves at the lox sites. The cleaved nucleic acids are spliced together (reciprocally translocated) and recombination is complete.

[0052] ZFNs are synthetic proteins consisting of an engineered zinc finger DNA-binding domain fused to a cleavage domain ( or a cleavage half-domain), which may be derived from a restriction endonuclease (e.g., Fok / ). The DNA binding domain may be canonical (C2H2) or non-canonical ( e.g., C3H or C4). The DNA-binding domain can comprise one or more zinc fingers (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or more zinc fingers) depending on the target site. Multiple zinc fingers in a DNA-binding domain may be separated by linker sequence(s). ZFNs can be designed to cleave almost any stretch of double-stranded DNA by modification of the zinc finger DNA-binding domain. ZFNs form dimers from monomers composed of a non-specific DNA cleavage domain (e.g., derived from the Fok / nuclease) fused to a DNA-binding domain comprising a zinc finger array engineered to bind a target site DNA sequence. The DNA-binding domain of a ZFN may typically be composed of 3-4 (or more) zinc-fingers. The amino acids at positions -1, +2, +3, and +6 relative to the start of the zinc finger a-helix, which contribute to site-specific binding to the target site, can be changed and customized to fit specific target sequences. The other amino acids may form a consensus backbone to generate ZFNs with different sequence specificities. Methods and rules for designing ZFNs for targeting and binding to specific target sequences are known in the art.

[0053] Meganucleases, which are commonly identified in microbes, such as the LAGLIDADG family of homing endonucleases, are unique enzymes with high activity and long recognition sequences (> 14 bp) resulting in site-specific digestion of target DNA. Engineered versions of naturally occurring meganucleases typically have extended DNA recognition sequences (for example, 14 to 40 bp ). According to some embodiments, a meganuclease may comprise a scaffold or base enzyme selected from the group consisting of l-Crel, l-Ceul, l-Msol, l-Seel, l-Anil, and I-Dnwl. The engineering of meganucleases can be more challenging than ZFNs and TALENs because the DNA recognition and cleavage functions of meganucleases are intertwined in a single domain. Specialized methods of mutagenesis and high- throughput screening have been used to create novel meganuclease variants that recognize unique sequences and possess improved nuclease activity. Thus, a meganuclease may be selected or engineered to bind to a genomic target sequence in a plant, such as at or near the genomic locus of a CKX gene. In an aspect, a method and / or composition provided herein comprises one or more, two or more, three or HiNel / ShortStature / 858 more, four or more, or five or more meganucleases. In another aspect, a meganuclease provided herein is capable of generating a targeted DSB. In an aspect, vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more meganucleases are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobocter / um-mediated transformation).

[0054] TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a nuclease domain (e.g., Fok). When each member of a TALEN pair binds to the DNA sites flanking a target site, the Fok monomers dimerize and cause a double-stranded DNA break at the target site. Besides the wild-type Fok cleavage domain, variants of the Fok cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The Fok domain functions as a dimer, requiring two constructs with unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the Fok cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity.

[0055] TALENs are artificial restriction enzymes generated by fusing the transcription activator-like effector (TALE) DNA binding domain to a nuclease domain. In some aspects, the nuclease is selected from a group consisting of Pvull, },JutH, Tevl, Fok!, Alwl Aflyl, Sbjl, Sdal, St.sf, CleDORF, Clo051, and Pept071. When each member of a TALEN pair binds to the DNA sites flanking a target site, the F okl monomers dimerize and cause a double-stranded DNA break at the target site. The term TALEN, as used herein, is broad and includes a monomeric TALEN that can cleave double stranded DNA without assistance from another TALEN. The term TALEN is also refers to one or both members of a pair of TALENs that work together to cleave DNA at the same site.

[0056] Transcription activator-like effectors (TALEs) can be engineered to bind practically any DNA sequence, such as at or near the genomic locus of a CKX gene in a plant. TALE has a central DNA-binding domain composed of 13-28 repeat monomers of 33-34 amino acids. The amino acids of each monomer are highly conserved, except for hypervariable amino acid residues at positions 12 and 13. The two variable amino acids are called repeat-variable diresidues (RVDs). The amino acid pairs Nl, NG, HD, and NN of RVDs preferentially recognize adenine, thymine, cytosine, and guanine / adenine, respectively, and modulation of RVDs can recognize consecutive DNA bases. This simple relationship between amino acid sequence and DNA recognition has allowed for the engineering of specific DNA binding domains by selecting a combination of repeat segments containing the appropriate RVDs. Besides the wild-type Fokl cleavage domain, variants of the Fokl cleavage domain with mutations have been designed to improve cleavage specificity and cleavage activity. The Fokl domain functions as a dimer, requiring two constructs with HiNel / ShortStature / 858 unique DNA binding domains for sites in the target genome with proper orientation and spacing. Both the number of amino acid residues between the TALEN DNA binding domain and the Fokl cleavage domain and the number of bases between the two individual TALEN binding sites are parameters for achieving high levels of activity. Pvull MutH, and Tevl cleavage domains are useful alternatives to Fokl and Fokl variants for use with T ALEs. Pvull functions as a highly specific cleavage domain when coupled to a TALE (see Yank etal. 2013. PLoS One. 8: e82539). AlutH is capable of introducing strand-specific nicks in DNA (see Gabsalilow et al. 2013. Nucleic Acids Research. 41: e83). Tevl introduces doublestranded breaks in DNA at targeted sites (see Beurdeley et al., 2013. Nature Communications. 4: 1762).

[0057] The relationship between amino acid sequence and DNA recognition of the TALE binding domain allows for designable proteins. Software programs such as DNA Works can be used to design TALE constructs. Other methods of designing TALE constructs are known to those of skill in the art. In an aspect, a method and / or composition provided herein comprises one or more, two or more, three or more, four or more, or five or more TALENs. In another aspect, a TALEN provided herein is capable of generating a targeted DSB. In an aspect, vectors comprising polynucleotides encoding one or more, two or more, three or more, four or more, or five or more TALENs are provided to a cell by transformation methods known in the art (e.g., without being limiting, viral transfection, particle bombardment, PEG-mediated protoplast transfection or Agrobacterium-mediated transformation).

[0058] As used herein, a "targeted genome editing technique" refers to any method, protocol, or technique that allows the precise and / or targeted editing of a specific location in a genome of a plant (i.e., the editing is largely or completely non-random) using a site-specific nuclease, such as a meganuclease, a zinc-finger nuclease (ZFN), an RNA-guided endonuclease (e.g., the CRISPR / Cas9 system), a TALE-endonuclease (TALEN), a recombinase, or a transposase. As used herein, "editing" or "genome editing" refers to generating a targeted mutation, deletion, inversion or substitution of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 1000, at least 2500, at least 5000, at least 10,000, or at least 25,000 nucleotides of an endogenous plant genome nucleic acid sequence. As used herein, "editing" or "genome editing" also encompasses the targeted insertion or site-directed integration of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 35, at least 40, at least 45, at least 50, at least 75, at least 100, at least 250, at least 500, at least 750, at least 1000, at least 1500, at least 2000, at least 2500, at least 3000, at least 4000, at least 5000, at HiNel / ShortStature / 858

[0059] 20 least 10,000, or at least 25,000 nucleotides into the endogenous genome of a plant. An "edit" or "genomic edit" in the singular refers to one such targeted mutation, deletion, inversion, substitution or insertion, whereas "edits" or "genomic edits" refers to two or more targeted mutation (s), deletion (s), inversion (s), substitution (s) and / or insertion (s), with each "edit" being introduced via a targeted genome editing technique. In an aspect, targeted gene editing approaches are used to modify the sequence of the promoter and / or regulatory region(s) of one or more of the CKX4b, 12 or 10 genes to knock-down or knock-out expression of these gene(s), such as through targeted deletions, insertions, mutations, or other sequence changes. Indeed, the promoter and / or regulatory region(s) or sequence(s), or the 5'-UTR, 3'UTR, and / or intron sequence(s), of one or more of the CKX4b, 12 or 10 genes may be largely deleted or mutated.

[0060] Alternatively, all or a portion of the coding (exon), 5-UTR, 3'UTR, and / or intron sequence(s) of one or more of the CKX4b, 12 and 10 genes may be edited, deleted, mutated, or otherwise modified to knockdown or knock-out expression or activity of these gene(s). Such targeted modifications to the CKX4b, 12 and / 10 genes loci may be achieved using any suitable genome editing technology known in the art, such as via repair of a double strand break (DSB) or nick introduced by a site-specific nuclease, such as, for example, a zinc-finger nuclease, an engineered or native meganuclease, a TALE-endonuclease, or an RNA-guided endonuclease (e.g., Cas9 or Cpfl). Such repair of the DSB or nick may introduce spontaneous or stochastic deletions, additions, mutations, etc., at the targeted site where the DSB or nick was introduced, or repair of the site may involve the use of a donor template molecule to direct or cause a preferred or specific deletion, addition, mutation, etc., at the targeted site.

[0061] In certain embodiments, the population of crop plant cells, parts, or plants which are screened for the presence of a loss-of-function allele in the endogenous crop gene have been subjected to one or more mutagenesis treatments. Loss-of-function alleles of the endogenous crop gene can be generated by mutagenesis methods known in the art, such as chemical mutagenesis or radiation mutagenesis. Suitable chemical mutagens include ethyl methanesulfonate (EMS), sodium azide, methylnitrosourea (MNU), and diepoxybutane (DEB). Suitable radiation includes x-rays, fast neutron radiation, and gamma radiation. Crop plant cells, parts, or plants comprising a loss-of-function allele of the endogenous crop gene can be generated using mutagenesis and identified by TILLING (Targeting Induced Local Lesions IN Genomes) or identified using EcoTILLING. TILLING is a general reverse genetics technique that uses mutagenesis methods to create libraries of mutagenized individuals that are later subjected to high throughput screens for the discovery of mutations. In addition to allowing efficient detection of induced mutations, high-throughput TILLING technology is ideal for the detection of natural mutations. EcoTILLING is a method that uses TILLING techniques to look for natural mutations in individuals (Barkley and Wang. Current genomics vol. 9,4 (2008): 212-26. doi:10.2174 / 138920208784533656). Identified mutations can HiNel / ShortStature / 858 then be introduced into desirable genetic backgrounds by crossing the mutant with a plant of the desired genetic background and performing a suitable number of backcrosses to cross out the originally undesired parent background. A more detailed description of methods and compositions for TILLING are disclosed in US Patent Application Publication 2004 / 0053236 Al, which is incorporated herein by reference in its entirety and can be adapted for use in the methods provided herein for identifying crop plant cells, parts, or plants comprising a loss-of-function allele of the endogenous crop gene.

[0062] This disclosure is also directed to methods for producing a crop plant having a loss-of-function allele of the endogenous crop gene by crossing a first parent crop plant with a second parent crop plant wherein the first or second parent crop plant comprises the loss-of-function allele. Further, both the first and second parent crop plants can comprise the loss-of-function allele. Any such methods using a crop plant comprising the loss-of-function allele are part of this disclosure: selfing, backcrosses, hybrid production, crosses to populations, and the like. All plants produced using a crop plant comprising the loss-of- function allele as a parent are within the scope of this disclosure, including plants derived from a crop plant having the loss-of-function allele. Also provided are the Fi progeny crop plants produced from the crossing of a crop plant comprising the loss-of-function allele with any other crop plant, Fi seed, and various parts of the Fi crop plant. The following describes breeding methods that can be used with crop plants of the disclosure in the development of further crop plants. One such embodiment is a method for developing a progeny crop plant in a crop plant breeding program comprising: obtaining the crop plant, or its parts, comprising a loss-of-function allele of the endogenous crop gene and utilizing the plant or plant parts as a source of breeding material; and selecting a progeny plant having the loss-of-function allele. Breeding steps that can be used in the crop plant breeding program include pedigree breeding, backcrossing, mutation breeding, and recurrent selection. In conjunction with these steps, techniques such as restriction fragment polymorphism enhanced selection, marker-assisted selection (for example SNP or SSR markers), and the making of double haploids can be utilized.

[0063] Field crops are bred through techniques that take advantage of the plant's method of pollination. A crop plant of the disclosure can be self-pollinated, sib-pollinated, or cross pollinated to create a pedigree crop plant. A plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant. A plant is sib-pollinated when individuals within the same family or variety are used for pollination. A plant is cross-pollinated if the pollen comes from a flower on a different plant from a different family or variety. The terms "cross-pollination" and "out-cross" as used herein do not include self-pollination or sib-pollination. For example maize can be bred by both self-pollination and crosspollination techniques. Maize has separate male and female flowers on the same plant, located on the tassel and the ear, respectively. Natural pollination occurs in maize when wind blows pollen from the tassels to the silks that protrude from the tops of the ears. HiNel / ShortStature / 858

[0064] Any other suitable breeding, selection, or growing methods may be used. Choice of the particular breeding or selection method will vary depending on environmental factors, population size, and the like.

[0065] In certain optional embodiments, the crop plant cells disclosed herein are non-regenerable crop plant cells. In certain optional embodiments provided herein, the crop plant cells, plant propagules (e.g., a seed, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, explant, or callus), and plants provided herein are not produced by an exclusively biological process. In certain optional embodiments provided herein, the methods for producing crop plant cells, plant propagules (e.g., a seed, seedling, ovule, embryo, pollen, root, stem, leaf, shoot, explant, or callus), and plants provided herein are not exclusively biological processes.

[0066] The term "yield" as used herein generally refers to a measurable product from a plant, particularly a crop. Yield, yield increase, commercially acceptable seed yield (in comparison to a non-transformed starting or wild-type plant) can be measured in a number of ways, and it is understood that a skilled person will be able to apply the correct meaning in view of the particular embodiments, the particular crop concerned and the specific purpose or application concerned. The terms "improved yield" or "increased yield" can be used interchangeable. As used herein, the term "improved yield" or the term "increased yield" means any improvement in the yield of any measured plant product, such as grain, fruit, leaf, root, cob or fiber. In accordance with the invention, changes in different phenotypic traits may improve yield. For example, and without limitation, parameters such as floral organ development, root initiation, root biomass, seed number, seed weight, harvest index, leaf formation, phototropism, apical dominance, and fruit development, are suitable measurements of improved yield. Increased yield includes higher fruit yields, higher seed yields, higher fresh matter production, and / or higher dry matter production. Any increase in yield is an improved yield in accordance with the invention. For example, the improvement in yield can comprise a 0.1%, 0.5%, 1%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or greater increase in any measured parameter.

[0067] For purposes of the present disclosure, a "plant" includes an explant, plant part, seedling, plantlet or whole plant at any stage of regeneration or development. As used herein, a "plant part" may refer to any organ or intact tissue of a plant, such as a meristem, shoot organ / structure (e.g., leaf, stem or node), root, flower or floral organ / structure (e.g., bract, sepal, petal, stamen, carpel, anther and ovule), seed (e.g., embryo, endosperm, and seed coat), fruit (e.g., the mature ovary), propagule, or other plant tissues ( e.g., vascular tissue, dermal tissue, ground tissue, and the like), or any portion thereof. Plant parts of the present disclosure may be viable, nonviable, regenerable, and / or non-regenerable. A "propagule" may include any plant part that can grow into an entire plant. HiNel / ShortStature / 858

[0068] Examples

[0069] Example 1. Multiplex genome editing strategy

[0070] Multiplex genome editing coupled to a crossing scheme was used to create variability within selected genes considered as growth regulators. Selection of plants after phenotypical seedling screening for plants with broad leaf blades and short leaf sheaths was performed. The selected seedlings were grown to maturity and short stature phenotype was observed. Multiplexed amplicon sequencing was used to determine the edits that occurred to all the experimental plants and with the support of developed machine learning algorithms we identified the causative genes for the short stature phenotype.

[0071] Specifically, by using a previously described method, BREEDIT (Lorenzo CD et al. (2023) The Plant Cell 35, 218-238), which is a pipeline that combines multiplex genome editing, phenotyping and multiplex amplicon sequencing, we generated variability with 5 transformation constructs (called Scripts) for 59 genes that were selected as growth regulators (see Figure 2A). We designed a crossing scheme to further exploit and combine the gene edits generated (Figure 2B).

[0072] During the phenotypic screen of juvenile BREEDIT populations for leaf phenotypes, we observed that 10- 20% of a plant population containing both SCRIPT 2 and SCRIPT 4 (S2S4, Figure 2) displays a compact plant architecture (Figure 2 A), coupled with a reduction of the leaf sheath length (Figure 2 B) and an increased leaf blade width (Figure 1 C). These compact seedlings developed into short stature mature plants (Figure 1 D), an ideotype with high agronomic potential, and thus an attractive target for crop improvement efforts. SCRIPT 2 contains gRN As targeting members of the CKX family and SCRIPT 4 mainly targets members of the TCP and GRF family (for details of the genes targeted see Figure 4). Based on our preliminary analysis (on seedling level) we have identified a potential combination of 11 genes responsible for the short stature trait. Nonetheless, as this was inferred from seedling phenotypes, a proper evaluation at mature stage was required.

[0073] Example 2: statistically cytokine dehydrogenase (CKX) edited plants are overrepresented in the short stature subpopulation

[0074] To further characterize which gene edits are responsible for the short stature phenotype we designed 2 experiments to phenotype 2 different populations in mature stage.

[0075] Experiment #1 focused on the analysis of segregating populations that contained gene edits in 22 genes alongside EDI plants (EDI plant is the Cas9 editor line of corn which serves here as a control plant, or a non-gene edited plant). A total of 550 plants were both phenotyped and genotyped. 50 plants from two populations bore edits in S2 (CKX gene family), 50 plants from a single population bore edits in S4 (TCP and GRF gene families and BPC6), 200 plants from 3 populations bore a combination of edits from S2 HiNel / ShortStature / 858 and S4 transformation constructs in 15 out of the 22 gene edits present (Broad gene space), and 200 more from 3 populations, which were the descendants of self-pollinated short stature plants, contained edits in only 11 genes (Condensed gene space) (see Figure 4A).

[0076] In Experiment #2, a wider range of edit combinations was analyzed with a total of 45 gene edits. A total of 1,260 plants were both genotyped and phenotyped. This included 50 plants with edits in each of SCRIPTS 1-5, 50 non-edited (control) plants and 192 plants for each of the following combinations: S1S4, S2S4, S2S5, S3S4, and S3S5 (see Figure 4B).

[0077] Since each population differed in the edited genes, every population could provide different edit combinations (see Figure 4). Furthermore, due to the presence of Cas9 and gRNAs transgenerational gene editing was still possible, increasing the potential edit combinations present in both experiments.

[0078] This setup allowed us to study the effect of different combinations of edits on plant architecture.

[0079] Plants bearing edits in CKX gene family members from S2 and S2S4 populations displayed reduced plant size compared to the non-edited plants (EDI) (see Figure 5). Independent analysis of both experiments with machine learning algorithms indicated that edits CKX4b, CKX10 and CKX12 are the main causative genes reducing plant height (see materials and methods for details of the statistical analysis).

[0080] The plants were grown in a greenhouse with controlled environmental conditions and extra plants were grown surrounding the experimental plants to homogenize the border effect.

[0081] The phenotyping consisted of manual measurements of plant height (considering the height to the primary ear and the distance from the primary ear to the collar of the last leave), measurements of the length and width of the leave of the primary ear, measurements of the stem width of the internode below the primary ear and scoring of the flowering time (in days to anthesis and days to silking). The primary ear was harvested to score the seed yield potential of each plant.

[0082] The genotyping of the plants was performed with Multiplexed Amplicon Sequencing method. A mix of primer pairs was used to simultaneously amplify the 60 genes that were part of the study with a barcoding system that allows us to identify each plant after pooling the samples following Next Generation Sequencing.

[0083] Table 1. SCRIPT (Transformation construct), gene name and gene identifiers (ID) of the genes analyzed. HiNel / ShortStature / 858 HiNel / ShortStature / 858

[0084] Example 3. Phenotype of the double edited corn plant in CKX4b and CKX12 genes

[0085] During the phenotypical screenings performed to the BREEDIT populations, edits in CKX4b and CKX12 were detected as negative regulators of biomass productivity. Backcrossing of selected plants bearing edits in CKX4b and CKX12 with Wild-type B104 allowed us to obtain segregant plants and isolate the HiNel / ShortStature / 858 different edits. Heterozygous plants bearing edits in CKX4b, CKX12 and both together were selfpollinated. The F2 descendants were screened by Sanger sequencing and homozygous plants were selected and self-pollinated. Mature stage phenotyping confirmed that double mutants CKX4b;CKX12 display reduced plant height (see Figure 7) and display an ideal short stature plant with a commercially acceptable seed yield.

[0086] Example 4. Amino acid similarity matrices for the candidate CKX genes in corn germplasm

[0087] To study whether the specific CKX genes causing the short stature phenotype are conserved in different corn cultivars we retrieved the protein sequences of CKX4b, CKX10 and CKX12 from genomically diverse maize genomes that flower under long-day conditions, these cultivars comprise tropical, temperate, sweet corn and popcorn varieties, representing a wide range of the maize genetic variability. Genomes were extracted from MaizeGDB. Reference to the genomes is found in: Cannon et al., 2011 "POPcorn: An Online Resource Providing Access to Distributed and Diverse Maize Project Data". The 25 founder lines were chosen to maximize genetic diversity while still producing inbred lines that flowered under long-day conditions in North Carolina. They include 13 tropical lines, 9 temperate lines, 2 sweet corn lines, and 1 popcorn line.

[0088] We found that these three CKX proteins are highly conserved among the analyzed genomes of the corn germplasm indicating that the three proteins have the same function in the different inbred lines analyzed.

[0089] Pairwise Identity % average (excluding AA insertions or deletions) for the 3 different proteins:

[0090] CKX4b: 99%

[0091] CKX10: 99%

[0092] CKX12: 98.9%

[0093] Identical sites % average (including AA insertions or deletions) for the 3 different proteins:

[0094] CKX4b: 95%

[0095] CKX10: 96.6%

[0096] CKX12: 95.3%

[0097] Maximal dissimilarity (excluding AA insertions or deletions) for the 3 different proteins:

[0098] CKX4b: 96.5%

[0099] CKX10: 97.9%

[0100] CKX12: 97.3% HiNel / ShortStature / 858

[0101] Percentage identity matrices (reflecting the percentages of AA's that match the alignment) are depicted for CKX4b, CKX10 and CKX12 proteins in Figure 8.

[0102] Materials and methods

[0103] Plant material and growth conditions

[0104] Seeds of the maize inbred line B104 were originally obtained from the USDA National Plant Germplasm System (Accession no. PI 594047) and were used for the generation of EDI plants by transformation. Super-transformation of EDI plants, expressing Cas9, with SCRIPT constructs expressing up to 12 gRNAs generated the reported variability as described in Lorenzo et al., 2023.

[0105] Maize seeds were imbibed in water for 24h at room temperature prior to sowing in peat moss substrate in 0.3L pots. For seedling phenotyping plants were watered 3 times a week to reach 1.4 g of water per dry gram of soil potting mix. After 2 weeks the plants were brought to 5 liter pots. Plants were grown in a regime of 16 h light (25°C), and 8 h dark (22°C)) until grown to maturity under well-watered conditions.

[0106] Genomic DNA isolation and multiplex amplicon

[0107] A piece of 1-2 cm of leaf material was placed in 8-strip, 2-mL capacity tubes (National Scientific Supply Co) together with two 3-mm stainless steel ball bearings, snap frozen in liquid nitrogen, and ground using a Mixer Mill MM400 (Retsch®). 0.5 mL DNA extraction buffer (2.5 mL 1 M Tris-HCI pH 8, 3 mL 5 M NaCI, 5 g saccharose, to 50 mL with Milli-Q water) was added, and samples were shaken and incubated at 65°C for 20 min. Tubes were centrifuged (2 min at 1800 x g) and 50 pL of supernatant was mixed with 70 pL magnetic beads (HighPrep™ PCR Clean-up System, Magbio) and put on a magnet. The supernatant was taken off, beads were washed twice with 80% ethanol and dried for further processing. Highly multiplex amplicon sequencing (HiPlex, Floodlight Genomics LLC, Knoxville, TN, USA) was performed as described in Lorenzo et al. (2023).

[0108] Modelling of gene edit impact on plant size

[0109] We trained XGBoost regression models to predict plant phenotypes from genotype data for two datasets: Experiment #1 (Expl) and Experiment #2 (Exp2). The process was implemented in Python using scikit-learn and XGBoost libraries. Training datasets were created from an integrated dataset using the NGS data, automated-phenotyping data captured during growth, and manual phenotyping measured from mature plants. Genotypes were represented as mutation rates per gene for each plant and were the covariates during modeling. The phenotypes are used as dependent variables. Phenotypes included traits such as plant height, leaf area, stem area, and yield. Separate models were trained for each HiNel / ShortStature / 858 phenotype. To detect potential overfitting, we added 5 decoy variables from random normal distributions to the training data.

[0110] The phenotype features were standardized based on EDI control plants. This standardization process involved calculating the median and median absolute deviation (MAD) for each feature in the control group, then applying the formula: (value - control_median) / control_MAD. This approach ensured that phenotype values for control plants typically ranged from -2 to +2, with values outside this range considered unexpected in control plants.

[0111] XGBRegressor models were trained for each phenotype using 10-fold cross-validation with random splits. Hyperparameter optimization was performed using RandomizedSearchCV with 100 iterations, optimizing for negative root mean squared error. The hyperparameter search space included learning rate (0.01 to 0.1), number of estimators (3 to 10), minimum child weight (0.001 to 100), maximum depth (2 to 5), and regularization parameters alpha and lambda (0 to 10). Model interpretation utilized SHAP (SHapley Additive exPlanations) values to understand which genes had the most predictive power for each phenotype. The contribution of decoy variables in the SHAP analysis served as an indicator of potential overfitting.

[0112] The experiment #1 dataset comprised 522 plants and 33 genotypes, and the experiment #2 dataset included 1213 plants and 52 genotypes. We trained models for 6 phenotypes in Experiment #1 and 30+ phenotypes in Experiment #2. Phenotypes related to plant height where total plant height, heigth_to_ear, rest height and height_to_ear_ratio. This data-driven approach allowed us to find genotypic factors influencing various aspect of plant height phenotypes while maintaining safeguards against overfitting through the use of decoy variables and cross-validation.

[0113] Alignment of CKX sequences from a diverse panel of maize inbred lines

[0114] Genomic DNA sequences from all NAM founders were retrieved from MaizeGDB using BLAST (https: / / www.maizegdb.org / popcorn / search / sequence_search / home. php?a=BLAST_UI). Regions aligned to the CDS reference sequence of B104 inbred line were extracted and translated to protein sequences. The translated protein sequences were aligned using CLUSTAL algorithm. The values for Pairwise identity % average, Identical sites % average and Maximal dissimilarity % were obtained using Geneious Prime. HiNel / ShortStature / 858 uence

[0115] SEQ ID NO: 1: CKX4b from Zea mays, Zm00007a00037930

[0116] MTRCLMFM PPLFLVSSLISTVGLPVEPPAELLQLGGDVSGGRLSVDASDIAEASRDFGGLSRAEPMAVFQPRAAGDV

[0117] AGLVRAAFGSARGFRVSARGHGHSISGQAQAPGGVVVDMGHGG—

[0118] AVARALPVHSPALGGHYVDVWGGELWVDVLNWTLSHGGLAPRSWTDYLYLSVGGTLSNAGISGQAFHHGPQISNV

[0119] YELDVVTGKGEVVTCSETENPDLFFGVLGGLGQFGIITRARIALERAPQRVRWIRALYSNFTEFTADQERLISLGSRRFDY

[0120] VEGFVVAAEGLINNWRSSFFSPQNPVKLSSLKHHSGVLYCLEVTKNYDDATAGSVEQDVDALLGELNFIPGTVFTTDLP

[0121] YVDFLDRVHKAELKLRAKGMWEVPHPWLNLFVPASRIADFDRGVFRGVLGGGTAGAGGPILIYPM NKHRWDPRSS

[0122] VVTPDEDVFYLVAFLRSALPGAPESLEALARQNRRVLDFCAEAGIGAKQYLPNHKAPGEWAEHFGAARWERFARLKA

[0123] QFDPRAILAAGQGIFRPPGSPPLVADS

[0124] SEQ ID NO: 2: CKX12 from Zea mays, Zm00007a00035050

[0125] MEGKVLCTYAGIVALLLCSSVNFIQSPSDVFGPVALLEPTASAARDFGGVVSEAAIAVMQPGSPADIARLLGALSSTGP

[0126] GPGPKAAVAARGAGHSLHGQAQARGGIVVETRALPRLVEVVRRGDGDGGGAAYADVGGGALWVEVLEECLRAGL

[0127] APRSWTDYLYLTVGGTLSNGGISGQAFKHGPQISNVLQLEVVTGTGEVVTCSPTQSPELFFAVLGGLGQFGIITRARIPL

[0128] QVAPPKVRWVRAFYDSFETFTKDQELLVSMPELVDYVEGFMVLNEQSLRSSSVAFPAQVNFRPDFGSDDGTNKKVC

[0129] YYYCIEFAVHDFQRQDSAADHVVDLVSGKLSYLRPHAYSVEVAYWDFLNRVRM EEESLRRRGLWDVPHPWLNLFVP

[0130] RHGVARFMDLLMATIAQGDFEGPVLVYPLLTHRWDGNMSAVVPAAPDGVMYVFSVLRSTDPARCGRACMERILEQ

[0131] HRRVADEACRRLGAKQYLARQPSLAHWRDHFGASWDRFVARKARFDPMNVLGPGQGIFPWTDSSSSPM

[0132] SEQ ID NO: 3: CKX10 from Zea mays, Zm00007a00010979

[0133] MMLAYMDRATAAAEPEDAGREPATTAGGCAAAAATDFGGLASAMPAAVVRPASADDVASAIRAAALTPHLTVAA

[0134] RGNGHSVAGQAMAEGGLVLDMRSLAAPSRRAQMQLVVQCPDGGGGRC-

[0135] CFADVPGGALWEEVLHWAVDNHGLAPASWTDYLRLTVGGTLSNGGVSGQSFRYGPQVSNVAELEVVTGDGERRV

[0136] CSPSSHPDLFFAVLGGLGQFGVITRARIPLHRAPQAVRWTRVVYASIADYTADAEWLVTRPPDAAFDYVEGFAFVNSD

[0137] DPVNGWPSVPIPGGARFDPSLLPAGAGPVLYCLEVALYQYAHRPDDVDDDDEEDQAAVTVSRMMAPLKHVRGLEF

[0138] AADVGYVDFLSRVNRVEEEARRNGSWDAPHPWLNLFVSARDIADFDRAVIKGMLADGIDGPM LVYPMLKSKWDP

[0139] NTSVALPEGEVFYLVALLRFCRSGGPAVDELVAQNGAILRACRANGYDYKAYFPSYRGEADWARHFGAARWRRFVD

[0140] RKARYDPLAILAPGQKIFPRVPASVAV

Claims

HiNel / ShortStature / 858Claims1. A modified corn plant, or plant part thereof, comprising a mutant allele at the CKX4b locus and a mutant allele at the CKX12 locus.

2. The modified corn plant, or plant part thereof, of claim 1, wherein only one of said CKX4b and CKX12 loci comprise homozygous mutant alleles.

3. The modified corn plant of claim 1 wherein both of said CKX4b and CKX12 loci comprise homozygous mutant alleles.

4. The modified corn plant, or plant part thereof, of any one of claims 1 to 3, wherein one or both of said CKX4b and CKX12 loci comprise a heteroallelic combination or two identical mutant alleles.

5. The modified corn plant, or plant part thereof, of any one of claims 1 to 3 wherein said mutant allele exhibits an at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% reduction of expression or enzymatic activity relative to an unmodified, wild type CKX4b or CKX12 allele.

6. The modified corn plant, or plant part thereof, of any one of claims 1 to 5, wherein said mutant allele at the CKX4b and / or CKX12 locus comprises a mutation in a sequence region selected from the group consisting of a promoter, 5'UTR, exon, intron, 3'UTR, terminator, or any combination thereof.

7. The modified corn plant, or plant part thereof, of any one of claims 1 to 6, wherein said mutant allele at the CKX4b and / or CKX12 locus comprises one or more mutation types selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice-site mutation, and any combination thereof.

8. The modified corn plant, or plant part thereof, according to any one of claims 1 to 7 further comprising a mutant allele at the CKX10 locus.

9. The modified corn plant, or plant part thereof, of claim 8 wherein CKX10 locus comprises homozygous mutant alleles.

10. The modified corn plant, or plant part thereof of claims 8 or 9 wherein said mutant allele at the CKX10 locus comprises a mutation in a sequence region selected from the group consisting of a promoter, 5'UTR, exon, intron, 3'UTR, terminator, or any combination thereof and wherein said mutation is selected from the group consisting of a nonsense mutation, a missense mutation, a frameshift mutation, a splice-site mutation, and any combination thereof wherein said mutant allele exhibits an at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90% or 100% reduction of expression or enzymatic activity relative to an unmodified, wild type CKX10 allele.

11. The modified corn plant, or plant part thereof, of any one of claims 1 to 7 wherein the CKX4b locus comprises a nucleotide sequence encoding for a polypeptide sequence with at least 95% identity29HiNel / ShortStature / 858 over the total length of SEQ ID NO: 1 and wherein the CKX12 locus comprises a nucleotide sequence encoding for a polypeptide sequence with at least 95% identity over the total length of SEQ ID NO: 2.

12. The modified corn plant, or plant part thereof, of any one of claims 8 to 10 wherein the CKX10 locus comprises a nucleotide sequence encoding for a polypeptide sequence with at least 95% identity over the total length of SEQ ID NO: 3 and wherein the CKX12 locus comprises a nucleotide sequence encoding for a polypeptide sequence with at least 95% identity over the total length of SEQ ID NO: 2 and wherein the CKX4b locus comprises a nucleotide sequence encoding for a polypeptide sequence with at least 95% identity over the total length of SEQ ID NO: 1.

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