Acetyl-CoA carboxylase inhibitor resistant sorghum

Description

1 / 60 “ACETYL-CoA CARBOXYLASE INHIBITOR-RESISTANT SORGHUM” REFERENCE TO THE LIST OF SEQUENCES SUBMITTED ELECTRONICALLY

[0001] The official copy of the sequence listing is submitted electronically through the Patent Center as a sequence listing in XML format with a file named 9389_SequenceListing, created on February 21, 2023, and which has a size of 46282 bytes and is filed simultaneously with the descriptive report. The sequence listing included in this document in XML format forms part of the descriptive report and is incorporated herein by reference in its entirety. BACKGROUND

[0002] The development of herbicide resistance in plants offers significant production and economic advantages; as such, the use of herbicides to control weeds or plants in crops has become almost universal practice. Of particular interest to farmers is the use of herbicides with high potency, broad-spectrum efficacy against weeds, and rapid soil degradation. One such class of broad-spectrum herbicides are those compounds that inhibit the activity of the enzyme acetyl-CoA carboxylase (ACCase) in a plant. These herbicides are included in the aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) chemical families.

[0003] Sorghum is susceptible to many ACCase-inhibiting herbicides that target monocotyledonous species, making it nearly impossible to use these herbicides to control weeds in sorghum fields. The development of sorghum varieties resistant to the inhibitory effects of Petition 870250096193, dated 10 / 21 / 2025, page 9 / 70 2 / 60 ACCase-inhibiting herbicides would allow for higher crop yields when these herbicides are used to control weeds. Therefore, due to the importance of sorghum as a cultivated crop worldwide, sorghum varieties resistant to the inhibitory effects of ACCase-inhibiting herbicides are needed, along with methods for producing and using these hybrids. SUMMARY

[0004] A genome-edited sorghum variety is provided comprising a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2 and comprises a non-tryptophan (e.g., arginine, histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, cysteine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, or tyrosine) at a position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the modified ACCase polypeptide is at least 90% identical to SEQ ID NO: 2 and comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the variety of Edited sorghum has greater tolerance to at least one ACCase-inhibiting herbicide compared to a control sorghum variety that does not include the introduced genetic modification.

[0005] Also provided is a genome-edited sorghum hybrid comprising a genetic modification introduced into an endogenous ACCase gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2 and comprises a non-tryptophan (e.g., arginine, Petition 870250096193, dated 10 / 21 / 2025, page 10 / 70 3 / 60 histidine, lysine, aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, cysteine, alanine, valine, isoleucine, leucine, methionine, phenylalanine, or tyrosine) at a position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the modified ACCase polypeptide is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the edited sorghum hybrid has greater tolerance to at least one ACCase-inhibiting herbicide compared to a control sorghum hybrid that does not comprise the introduced genetic modification.

[0006] A method is provided for producing an ACCase inhibitor-tolerant sorghum hybrid, wherein the method is characterized in that it comprises crossing a first sorghum variety comprising a genetic modification introduced into an endogenous ACCase gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a non-tryptophan at a position corresponding to position 2032 of SEQ ID NO: 2, with a second sorghum variety. In certain embodiments, the modified ACCase polypeptide of the first sorghum variety is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2.In certain embodiments, the second sorghum variety comprises a genetic modification introduced into an ACCase gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a non-tryptophan at a position corresponding to position 2032 of SEQ ID NO: 2 in an allele. In certain embodiments, the modified ACCase polypeptide of the second sorghum variety is at least... Petition 870250096193, dated 10 / 21 / 2025, page 11 / 70 4 / 60% identical to SEQ ID NO: 2, and comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2.

[0007] A method is also provided for producing an ACCase inhibitor-tolerant sorghum variety, comprising introducing into a regenerable sorghum plant cell a genetic modification in an endogenous ACCase gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2, and generating a sorghum plant from the plant cell, wherein the generated plant comprises the introduced genetic modification and has improved tolerance to ACCase inhibitor herbicides compared with a control plant.

[0008] Furthermore, a method is provided for producing an ACCase inhibitor-tolerant variety of sorghum, comprising providing a plant cell comprising an ACCase gene sequence with a guide RNA, a polynucleotide modification template comprising at least one nucleotide modification of the ACCase gene sequence to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprising a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2, and a Cas endonuclease, the guide RNA and the Cas endonuclease being able to form a complex that enables the Cas endonuclease to introduce a double-strand break in a target site sequence in the ACCase gene of said plant cell, obtaining a plant from the plant cell,To evaluate the plant for the presence of at least one nucleotide modification and select a parent sorghum plant that includes the modified ACCase gene and has greater tolerance to ACCase-inhibiting herbicides. Petition 870250096193, dated 10 / 21 / 2025, page 12 / 70 5 / 60 compared to a control plant that does not include the modification. In certain embodiments, the method further involves crossing the selected parent sorghum plant with a second sorghum plant to produce an F1 parent plant.

[0009] A method is provided for introgressing a modified ACCase allele into an elite sorghum variety comprising crossing a first sorghum variety with a second sorghum variety to produce a parent population, wherein the first sorghum variety comprises a genetic modification introduced into an endogenous ACCase gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a non-tryptophan at a position corresponding to position 2032 of SEQ ID NO: 2, genotyping the parent population for the presence of the introduced genetic modification and selecting progeny that compare to the introduced genetic modification to obtain soybean plants comprising the modified ACCase allele and having improved tolerance to ACCase-inhibiting herbicides.In certain embodiments, the modified ACCase polypeptide of the first sorghum variety is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the second sorghum variety comprises a genetic modification introduced into an ACCase gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a non-tryptophan at a position corresponding to position 2032 of SEQ ID NO: 2 in an allele. In certain embodiments, the modified ACCase polypeptide of the second sorghum variety is at least 90% identical to SEQ ID NO: 2. Petition 870250096193, dated 10 / 21 / 2025, page 13 / 70 6 / 60 and comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2.

[0010] A method is also provided for controlling weeds in a cultivated area, comprising planting a cultivated area with seeds and / or plants comprising a genetic modification introduced into an ACCase gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a non-tryptophan at a position corresponding to position 2032 of SEQ ID NO: 2, and applying to the seeds and / or plants a sufficient amount of an ACCase-inhibiting herbicide to control the weeds without significantly affecting the seeds and / or plants. In certain embodiments, the modified ACCase polypeptide is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2. BRIEF DESCRIPTION OF THE DRAWING AND SEQUENCE LIST

[0011] The disclosure can be more fully understood from the following detailed description and the attached drawing and sequence listing, which form part of this application.

[0012] The sequence descriptions (Table 1) and the sequence listing attached to this invention comply with the rules governing the disclosure of nucleotide and amino acid sequences in patent applications, as set forth in Title 37 of the CFR §§ 1,831 to 1,835.

[0013] Figure 1 provides the location of the target site in the ACCase gene sequence (SEQ ID NO: 1) for ACC-TS1, ACC-TS2, and ACC-TS3. The nucleotide sequence shown corresponds to positions 14411 to 14458 of SEQ ID NO: 1. The location of Petition 870250096193, dated 10 / 21 / 2025, p. 14 / 70 7 / 60 The nucleotide sequence to be modified is highlighted in bold. Table 1: Description of sequence listing SEQ ID NOS Description 1 Wild-type ACCase - genomic sequence 2 Wild-type ACCase - amino acid sequence 3 Genome-edited ACCase - genomic sequence 4 Genome-edited ACCase - amino acid sequence 5 ACC-TS1 6 ACC-TS2 7 ACC-TS3 8 ACC-CR1 9 ACC-CR2 10 ACC-CR3 11 ACC-CR3.1 12 ACC-TS3.1 13 Guide RNA 14 Polynucleotide Modification Template 15 ACC-TS3F - Primer 16 ACC-TS3R - Primer 17 ACC-editF - Primer 18 ACC-editR - Primer 19 Zm-U6 Pol III Promoter DETAILED DESCRIPTION

[0014] Acetyl-CoA carboxylase (ACCase) is a biotinylated enzyme that catalyzes the carboxylation of acetyl-CoA to produce malonyl-CoA. This carboxylation is a reversible two-step reaction consisting of carboxylation Petition 870250096193, dated 10 / 21 / 2025, page 15 / 70 8 / 60 ATP-dependent transfer of the biotin group to the carboxylic domain by biotin carboxylase activity, followed by the transfer of the carboxylic group from biotin to acetyl-CoA by carboxyltransferase activity (Nikolau et al., 2003, Arch. Biochem. Biophys. 414:211-22). Acetyl-CoA carboxylase is not only an essential enzyme in plants for fatty acid biosynthesis, a process that occurs in chloroplasts and mitochondria, but ACCase also plays a role in the formation of long-chain fatty acids and flavonoids, and in malonylation that occurs in the cytoplasm. There are two isoforms of ACCase, with chloroplast ACCase accounting for more than 80% of the total ACCase activity (Herbert et al., 1996, Biochem. J. 318:997-1006). Aryloxyphenoxypropionate (FOP) and cyclohexanedione (DIM) are two classes of chemicals known to selectively inhibit chloroplastic ACCase in grasses (Rendina et al., 1990, J. Agric. Food Chem. 38:1282-1287).

[0015] Cultivated sorghum [Sorghum bicolor (L.) Moench] is susceptible to many acetyl-CoA carboxylase (ACCase) inhibitor herbicides that target monocotyledonous or grassy weed species, which greatly limits the use of these herbicides to control grassy weeds in sorghum fields. Thus, the development of sorghum varieties resistant to the inhibitory effects of ACCase inhibitor herbicides would allow for higher crop yields when these herbicides are used to control weeds. Consequently, the present disclosure provides genome-edited sorghum varieties with greater resistance to ACCase inhibitor herbicides compared to a variety Petition 870250096193, dated 10 / 21 / 2025, page 16 / 70 9 / 60 of control sorghum that does not involve genome editing, and methods for producing and using genome-edited varieties.

[0016] Genome-edited sorghum varieties and seeds that produce genome-edited sorghum varieties comprising a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least, or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2 and comprises a non-tryptophan (e.g., arginine, histidine, lysine, acid). aspartic acid, glutamic acid, serine, threonine, asparagine, glutamine, glycine, proline, cysteine, alanine, valine, isoleucine, leucine, methionine, phenylalanine or tyrosine) in a position corresponding to position 2032 of SEQ ID NO: 2.In certain embodiments, genome-edited sorghum varieties and the seeds that produce genome-edited sorghum varieties comprise a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least or at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, The endogenous ACCase gene comprises a polynucleotide sequence that is at least, or at least approximately, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 1. In certain embodiments, Petition 870250096193, dated 10 / 21 / 2025, p. 17 / 70 10 / 60 The modified ACCase polypeptide comprises the amino acid sequence of SEQ ID NO: 4. In certain embodiments, the genome-edited sorghum variety has greater tolerance to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) ACCase-inhibiting herbicide compared to a control sorghum variety that does not comprise the introduced genetic modification, such as, for example, a sorghum variety comprising an ACCase gene encoding an ACCase polypeptide comprising SEQ ID NO: 2. In certain embodiments, the genome-edited sorghum variety has greater tolerance to at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more) ACCase-inhibiting herbicide compared to a control sorghum variety comprising an ACCase gene encoding an ACCase polypeptide comprising a cysteine ​​at a position corresponding to the position 2032 of SEQ ID NO: 2 introduced by crossing with an exotic variety of sorghum.

[0017] As used in this document, “gene” includes a nucleic acid fragment that expresses a functional molecule such as, but not limited to, a specific protein coding sequence and regulatory elements, such as those preceding (5' non-coding sequences) and following (3' non-coding sequences) the coding sequence.

[0018] The terms “polypeptide”, “peptide” and “protein” are used interchangeably in this document to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Petition 870250096193, dated 10 / 21 / 2025, page 18 / 70 11 / 60

[0019] As used in this document, “encoding,” “encoded,” or similar terms, in relation to a specific nucleic acid, mean comprising information for translation into the specified protein. A nucleic acid that encodes a protein may comprise untranslated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intermediate untranslated sequences (e.g., as in cDNA). The information by which a protein is encoded is specified by the use of codons. Typically, the amino acid sequence is encoded by the nucleic acid using a “universal” genetic code. However, variants of the universal code, such as that present in some plant, animal, and fungal mitochondria, the bacterium Mycoplasma capricolum (Yamao, et al., (1985) Proc. Natl. Acad. Sci. USA 82:2306-9) or the ciliate Macronucleus, may be used when the nucleic acid is expressed using these organisms.

[0020] As used in this document, “percentage (%) of sequence identity with respect to a reference sequence (database sequence) is determined as the percentage of amino acid or nucleotide residues in a candidate sequence (search sequence) that are identical to the respective amino acid or nucleotide residues in the reference sequence, after sequence alignment and the introduction of gaps, if necessary, to achieve the maximum percentage of sequence identity, and not considering any conservative amino acid substitutions as part of the sequence identity. Alignment for the purpose of determining the percentage of sequence identity can be achieved in several ways that Petition 870250096193, dated 10 / 21 / 2025, p. 19 / 70 12 / 60 are within the skill of the technique, for example, using publicly available computer programs such as BLAST, BLAST-2. People skilled in the technique can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment along the entire length of the sequences being compared. The percentage of identity between the two sequences is a function of the number of identical positions shared by the sequences (e.g., percentage of identity of the query sequence = number of identical positions between the query and object sequences / total number of positions of the query sequence χ 100).

[0021] Unless otherwise indicated, the sequence identity / similarity values ​​provided herein refer to the value obtained using the BLAST 2.0 program suite using standard parameters (Altschul, et al., (1997) Nucleic Acids Res. 25:338 9-4 02).

[0022] Genetic modification of the endogenous ACCase gene can be introduced using any genome editing technique known in the art or described herein. In certain embodiments, the targeted DNA modification is believed to be introduced using a genome editing technique employing an enzyme selected from the group consisting of a polynucleotide-guided endonuclease, CRISPR-Cas endonucleases, base-editing deaminases, a zinc finger nuclease, a transcription activator-type effector nuclease (TALEN), or a genetically engineered site-specific meganuclease.

[0023] In certain modalities, genome modification can be facilitated by inducing a double-strand break. Petition 870250096193, dated 10 / 21 / 2025, page 20 / 70 13 / 60 strand (DSB) or single-strand break, at a defined position in the genome near the desired alteration. DSBs can be induced using any available DSB-inducing agent including, but not limited to, TALENs, meganucleases, zinc finger nucleases, Cas9gRNA systems (based on bacterial CRISPR-Cas systems), cpf1-guided endonuclease systems, Cas12f endonuclease systems, and the like. In certain embodiments, the introduction of a DSB can be combined with the introduction of a polynucleotide modification template.

[0024] As used in this document, the term “variety” includes whole plants, plant organs, plant tissues, seeds, plant cells, seeds, grains and progeny thereof.

[0025] In certain embodiments, genome editing is introduced into an elite sorghum variety, thereby producing an elite sorghum variety with an edited genome. As used herein, an “elite line,” “elite variety,” or similar term, is an agronomically superior line that has resulted from many cycles of breeding and selection for superior agronomic performance. Numerous elite lines are available and known to those skilled in the art of sorghum breeding. As used herein, an “exotic sorghum line,” “exotic sorghum variety,” or similar term, is a strain or germplasm derived from a sorghum that does not belong to an available elite sorghum line or germplasm strain. In the context of a cross between two sorghum plants or germplasm strains, an exotic germplasm is not closely related by descent to the germplasm of Petition 870250096193, dated 10 / 21 / 2025, page 21 / 70 14 / 60 elite with which it is crossed. More commonly, exotic germplasm is not derived from any known elite soybean line, but instead is selected in order to introduce new genetic elements (typically new alleles) into a breeding program.

[0026] In certain embodiments, the elite variety is an elite inbred variety. As used in this document, an “elite inbred” refers to an elite line or variety that has been created for genetic homogeneity. In certain embodiments, the elite variety is an elite hybrid variety. As used in this document, an “elite hybrid” refers to the offspring obtained through the crossing of at least two genetically different parents, such as, for example, two elite inbred parents. Methods for developing elite inbred and hybrid sorghum lines are known in the art, and numerous elite lines are available and known to those skilled in the art of sorghum breeding.

[0027] As used in this document, the term “germplasm” refers to the genetic material of an individual (e.g., a plant), a group of individuals (e.g., a plant lineage, variety, or family), or a clone derived from a lineage, variety, species, or culture. Germplasm may be part of an organism or cell, or it may be separate from the organism or cell. Germplasm provides genetic material with a specific molecular constitution that provides a physical basis for some or all of the heritable qualities of an organism or cell culture. Germplasm in the context of this disclosure includes cells, seeds, or Petition 870250096193, dated 10 / 21 / 2025, page 22 / 70 15 / 60 tissues from which new plants can be grown, or plant parts, such as leaves, stems, pollen, or cells, that can be grown into a whole plant.

[0028] In certain embodiments, the genome-edited variety (e.g., elite inbred variety and / or elite hybrid variety) has a yield that is greater than, equal to, or within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, compared to the corresponding control plant, for example, one that has a similar genetic background but lacks the introduced genetic modification. In certain embodiments, the genome-edited variety (e.g., elite inbred variety and / or elite hybrid variety) has a yield that is increased by at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, compared to a control sorghum variety comprising an ACCase gene encoding an ACCase polypeptide comprising a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2 introduced by crossing with an exotic sorghum variety.

[0029] In certain embodiments, the genome-edited sorghum variety (e.g., elite inbred variety and / or elite hybrid variety) has a yield that is increased by at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 10% or more when grown in the presence of an ACCase-inhibiting herbicide, compared with a control sorghum variety comprising an ACCase gene encoding an ACCase polypeptide comprising a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2 introduced by crossing with an exotic sorghum variety. In certain embodiments, the edited elite hybrid variety Petition 870250096193, dated 10 / 21 / 2025, page 23 / 70 16 / 60 per genome presents a productivity of at least 4,340 kg per hectare (80 bushels per acre), 4,610 kg / ha (85 bu / A), 4,880 kg / ha (90 bu / A), 5,150 kg / ha (95 bu / A), 5,420 kg / ha (100 bu / A), 5,690 kg / ha (105 bu / A), 5,960 kg / ha (110 bu / A), 6,230 kg / ha (115 bu / A), 6,500 kg / ha (120 bu / A), 6,770 kg / ha (125 bu / A), 7,040 kg / ha (130 bu / A), 7,580 kg / ha (140 bu / A), 8,120 kg / ha (150 bu / A), 9,470 kg / ha (175 bu / A) or 10,820 kg / ha (200 bu / A), when grown in the presence of at least one ACCase inhibitor herbicide. In certain embodiments, the at least one ACCase inhibitor is selected from the aryloxyphenoxypropionate (FOP) herbicide family or the cyclohexanedione (DIM) herbicide family. In certain embodiments, the at least one ACCase inhibitor is quizalofop-p-ethyl.In certain applications, the ACCase-inhibiting herbicide, such as quizalofop-p-ethyl, is applied at a rate of at least approximately 44.8 grams of active ingredient per hectare (0.04 lb ai / A), 50.3 g / ha (0.045 lb ai / A), 56.0 g / ha (0.05 lb ai / A), 61.6 g / ha (0.055 lb ai / A), 67.2 g / ha (0.06 lb ai / A), 72.8 g / ha (0.065 lb ai / A), 78.4 g / ha (0.07 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 224.0 g / ha (0.2 lb ai / A), or 280.0 g / ha (0.25 lb ai / A), and less than 336.0 g / ha (0.3 lb ai / A), 280.0 g / ha (0.25 lb ai / A), 224.0 g / ha (0.2 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 78.4 g / ha (0.07 lb ai / A), or 67.2 g / ha (0.06 lb ai / A).In certain modalities, at least one ACCase inhibitor is quizalofop-p-ethyl, applied at a rate in the range. Petition 870250096193, dated 10 / 21 / 2025, p. 24 / 70 17 / 60 from about 56.0 g / ha (0.05 lb ai / A) to 168.0 g / ha (0.15 lb ai / A).

[0030] As used in this document, “yield” refers to the amount of agricultural production harvested per unit of land and may include reference to sacks per acre or kilograms per hectare of a crop at harvest, as adjusted for grain moisture. Grain moisture is measured in the grain at harvest. The adjusted test weight of the grain is determined as the weight in pounds per sack or kilogram, adjusted for the grain moisture level at harvest.

[0031] In certain embodiments of the genome-edited elite sorghum inbred varieties and / or elite hybrid varieties or seeds thereof described in this document, the introduced genetic modification is present in a single allele of the ACCase gene, such that the genome-edited elite sorghum inbred variety and / or elite hybrid variety is heterozygous for the introduced genetic modification. In certain embodiments of the genome-edited elite sorghum inbred varieties and / or elite hybrid varieties or seeds thereof described in this document, the introduced genetic modification is present in both alleles of the ACCase gene, such that the genome-edited elite sorghum inbred variety and / or elite hybrid variety is homozygous for the introduced genetic modification.

[0032] In certain embodiments, genome-edited elite sorghum inbred varieties and / or elite hybrid varieties described in this document have tolerance to at least one member of the aryloxyphenoxypropionate (FOP) herbicide family. In certain embodiments, the Petition 870250096193, dated 10 / 21 / 2025, p. 25 / 70 18 / 60 genome-edited elite inbred sorghum varieties and / or elite hybrid varieties described in this document have tolerance to quizalofop-p-ethyl when applied at a rate of at least about 44.8 g / ha (0.04 lb ai / A), 50.3 g / ha (0.045 lb ai / A), 56.0 g / ha (0.05 lb ai / A), 61.6 g / ha (0.055 lb ai / A), 67.2 g / ha (0.06 lb ai / A), 72.8 g / ha (0.065 lb ai / A), 78.4 g / ha (0.07 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 224.0 g / ha (0.2 lb ai / A) or 280.0 g / ha (0.25 lb ai / A), and less than 336.0 g / ha (0.3 lb ai / A), 280.0 g / ha (0.25 lb ai / A), 224.0 g / ha (0.2 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 78.4 g / ha (0.07 lb ai / A), or 67.2 g / ha (0.06 lb ai / A).In certain embodiments, quizalofop-p-ethyl, applied at a rate in the range of about 56.0 g / ha (0.05 lb ai / A) to 168.0 g / ha (0.15 lb ai / A). In certain embodiments, the genome-edited elite sorghum inbred varieties and / or elite hybrid varieties described in this document have tolerance to at least one member of the cyclohexanedione (DIM) herbicide family. In certain embodiments, the genome-edited elite sorghum inbred varieties and / or elite hybrid varieties described in this document exhibit tolerance to clethodim when applied at a rate of at least 44.8 g / ha (0.04 lb ai / A), 50.3 g / ha (0.045 lb ai / A), 56.0 g / ha (0.05 lb ai / A), 61.6 g / ha (0.055 lb ai / A), 67.2 g / ha (0.06 lb ai / A), 72.8 g / ha (0.065 lb ai / A), 78.4 g / ha (0.07 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 89.6 g / ha (0.08 lb ai / A). Petition 870250096193, dated 10 / 21 / 2025, p. 26 / 70 19 / 60 ai / A), 95.2 g / ha (0.085 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 224.0 g / ha (0.2 lb ai / A) or 280.0 g / ha (0.25 lb ai / A), and less than 336.0 g / ha (0.3 lb ai / A), 280.0 g / ha (0.25 lb ai / A), 224.0 g / ha (0.2 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 78.4 g / ha (0.07 lb ai / A) or 67.2 g / ha (0.06 lb ai / A). In certain embodiments, clethodim is applied at a rate in the range of about 56.0 g / ha (0.08 lb ai / A) to 168.0 g / ha (0.2 lb ai / A).In certain embodiments, the genome-edited elite sorghum inbred varieties and / or elite hybrid varieties described herein have tolerance to a combination herbicide comprising at least one member of the aryloxyphenoxypropionate (FOP) herbicide family and at least one member of the cyclohexanedione (DIM) herbicide family. Members of the FOP herbicide family are known in the art and include, but are not limited to, clodinafop-propargyl, cy-halofop-butyl, diclofop-methyl, fenoxaprop-p-ethyl, fluazifop-b-utilyl, haloxifop-ethoxyethyl, haloxifop-ethotyl, haloxifop-R-methyl, propaquizafop, quizalofop-p-ethyl, and quizalo-prefuryl compounds. Similarly, members of the DIM family of herbicides are known in the art and include, but are not limited to, alloxidime, butroxidime, clefoxidime, clethodim, cyclooxidime, profoxidime, sethoxidime, tepralooxidime, and tralcoxidime compounds.

[0033] As used in this document, “herbicide tolerance,” “herbicide resistance,” and the like refer to plants that exhibit few or no lesions. Petition 870250096193, dated 10 / 21 / 2025, page 27 / 70 20 / 60 blanched, necrotic, lytic, chlorotic, or other lesions and do not become stunted, wilted, or deformed when subjected to the herbicide at concentrations and rates that are normally used by the agricultural community to exterminate weeds in the field. As used in this document, “greater tolerance,” “greater resistance,” or similar refers to any detectable decrease in blanched, necrotic, lytic, chlorotic, or other lesions or any detectable decrease in the number of stunted, wilted, or deformed plants compared to an appropriate control when subjected to the herbicide. When comparing two plants, the plant that shows the least number of blanched, necrotic, lytic, chlorotic, or other lesions, or the fewest number of stunted, wilted, or deformed plants, will be considered to have greater tolerance compared to the other plant.Greater tolerance can be demonstrated when plants exhibiting higher tolerance to a herbicide are subjected to an ACCase inhibitor, and a dose-response curve is shifted to the right when compared to that provided by an appropriate control plant.

[0034] In certain embodiments, the genome-edited sorghum varieties (e.g., elite inbred varieties and / or elite hybrid varieties) and seeds thereof described herein may further comprise one or more additional genetic modifications of the endogenous ACCase gene that are associated with tolerance or increased tolerance to ACCase-inhibiting herbicides. In certain embodiments, the genome-edited sorghum varieties (e.g., elite inbred varieties and / or elite hybrid varieties) and seeds thereof described herein Petition 870250096193, dated 10 / 21 / 2025, page 28 / 70 21 / 60 document, may further comprise one or more additional genetic modifications of the endogenous ACCase gene to increase the expression and / or activity of the encoded polypeptide. In certain embodiments, the one or more additional genetic modifications of the endogenous ACCase gene comprise a modification of a regulatory element. In certain embodiments, the regulatory element is the promoter of the endogenous ACCase gene. In certain embodiments, the promoter of the endogenous ACCase gene is replaced by a promoter sequence that results in increased expression of the ACCase gene. In certain embodiments, the one or more additional genetic modifications of the endogenous ACCase gene are introduced at the same time as the editing resulting in the W2032C mutation in the encoded polypeptide.

[0035] A “regulatory sequence” generally refers to a transcriptional regulatory element involved in regulating the transcription of a nucleic acid molecule, such as a gene or a target gene. The regulatory element is a nucleic acid and may include a promoter, an enhancer, an intron, a 5' untranslated region (5'-UTR also known as a leader sequence) or a 3'-UTR, or a combination thereof. A “promoter” refers to a region of DNA upstream of the transcription start and involved in the recognition and binding of RNA polymerase and other proteins to initiate transcription. An “enhancer” is any nucleic acid molecule that increases the transcription of a nucleic acid molecule when functionally bound to a promoter regardless of its relative position. An “intron” is an intervening sequence in a gene that is transcribed into RNA but is then removed in the process of generating mature mRNA.The term is also used for the removed RNA sequences. A. Petition 870250096193, dated 10 / 21 / 2025, page 29 / 70 The 5' untranslated region (5' UTR) (also known as a translation leader sequence or leader RNA) is the region of an mRNA that is directly upstream of the initiation codon. This region is involved in regulating the translation of a transcript by different mechanisms in viruses, prokaryotes, and eukaryotes. "3' non-coding sequences" refer to DNA sequences located downstream of a coding sequence and include polyadenylation recognition sequences and other sequences that encode regulatory signals capable of affecting mRNA processing or gene expression. The polyadenylation signal is typically characterized by affecting the addition of poly(adenylic acid) tails to the 3' end of the precursor mRNA.

[0036] In certain embodiments, the promoter of the endogenous ACCase gene is replaced by a heterologous promoter with a constitutive promoter or a tissue-preferred promoter.

[0037] Also provided in this document are genome-edited inbred and hybrid varieties of sorghum and seeds thereof, which have increased tolerance to ACCase-inhibiting herbicides and which comprise a genetic modification that introduces a modified acetyl-CoA carboxylase (ACCase) gene encoding a modified ACCase polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and comprises a non-tryptophan (e.g., a cysteine) in a position corresponding to position 2032 of SEQ ID NO: 2, the modified ACCase gene being introduced into a genomic locus different from the locus of the endogenous ACCase gene. In certain embodiments, the modified ACCase gene is operationally linked to an element Petition 870250096193, dated 10 / 21 / 2025, p. 30 / 70 23 / 60 heterologous regulator such as, for example, a heterologous promoter. In certain modalities, inbred and hybrid varieties are elite inbred and hybrid varieties.

[0038] In certain embodiments, the genome-edited sorghum varieties (e.g., elite inbred varieties and / or elite hybrid varieties) and seeds thereof described herein may further comprise one or more additional traits of interest. A trait, as used herein, refers to the phenotype derived from a particular sequence or groups of particular sequences. In certain embodiments, the genome-edited sorghum varieties and seeds thereof comprise at least one additional polynucleotide conferring tolerance to at least one additional herbicide, such as, for example, one or more sequences conferring tolerance to: an ALS inhibitor; an HPPD inhibitor; 2,4-D; other phenoxy auxin herbicides; glyphosate; dicamba; glufosinate herbicides; herbicides targeting the protox enzyme (also called “protox inhibitors”).In certain embodiments, the genome-edited sorghum varieties (e.g., elite inbred varieties and / or elite hybrid varieties) and seeds thereof described herein further comprise at least one additional polynucleotide that confers herbicide tolerance. In certain embodiments, at least one additional polynucleotide confers tolerance to glyphosate, 2,4-D, glufosinate, or any combination thereof. In certain embodiments, at least one additional polynucleotide confers tolerance to glyphosate, 2,4-D, and glufosinate. In certain embodiments, the genome-edited sorghum varieties (e.g., elite inbred varieties and / or elite hybrid varieties). Petition 870250096193, dated 10 / 21 / 2025, page 31 / 70 The 24 / 60 elite hybrids) and seeds thereof described herein further comprise a polynucleotide conferring glyphosate tolerance, a polynucleotide conferring 2,4-D tolerance, and a polynucleotide conferring glufosinate tolerance. At least one additional polynucleotide may be introduced into the sorghum variety using any method known in the art, such as backcrossing, transformation, genome editing, or locus conversion.

[0039] The genome-edited sorghum varieties (e.g., elite inbred varieties and / or elite hybrid varieties) and seeds thereof described herein may also be combined with at least one other trait to produce plants comprising a further variety of desired trait combinations. For example, the genome-edited sorghum variety may be stacked with polynucleotides encoding polypeptides that have pesticidal and / or insecticidal activity, or a plant, plant cell, plant part, seed and / or grain comprising the genome modification provided herein may be combined with a plant disease resistance gene.

[0040] In certain embodiments, the genome-edited sorghum variety has at least one additional polynucleotide that confers increased seed protein or oil content. For example, a modified polynucleotide encoding a diacylglycerol acyltransferase (DGAT) polypeptide, such as those described in document no. WO19 / 232182, or a high trace of oleic acid, such as those described in US Patent no. 8,609,935.

[0041] These combinations can be created through any method including, but not limited to, improvement of Petition 870250096193, dated 10 / 21 / 2025, page 32 / 70 25 / 60 plants through any conventional methodology or genetic transformation. If the sequences are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. Traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences are introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained in the same transformation cassette (cis). The expression of the sequences can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of the polynucleotide of interest.This can be combined with any combination of other suppression cassettes or overexpression cassettes to generate the desired combination of traits in the plant. It is further recognized that polynucleotide sequences can be stacked at a desired genomic location using a site-specific recombination system. See, for example, documents no. WO99 / 25821, WO99 / 25854, WO99 / 25840, WO99 / 25855, and WO99 / 25853, which are incorporated herein by reference.

[0042] A method is also provided for producing an ACCase inhibitor-tolerant sorghum hybrid, comprising crossing a first inbred sorghum variety comprising any inbred sorghum variety described in this document with a second variety Petition 870250096193, dated 10 / 21 / 2025, page 33 / 70 26 / 60 inbreeding of sorghum. In certain embodiments, the method also includes collecting seeds from a progeny population produced from the cross and cultivating the collected seeds. In certain embodiments, the first inbred sorghum variety comprises a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and comprises a non-tryptophan (e.g., a cysteine) at a position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the non-tryptophan is a cysteine ​​such that the modified ACCase polypeptide comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2.In certain embodiments, the introduced genetic modification is present in a single allele of the ACCase gene in the first sorghum variety. In certain embodiments, the introduced genetic modification is present in both alleles of the ACCase gene in the first sorghum variety. In certain embodiments, the second inbred variety of sorghum comprises a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and comprises a non-tryptophan (e.g., a cysteine) at a position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the non-tryptophan is a cysteine ​​so that the ACCase polypeptide is modified in the second variety of. Petition 870250096193, dated 10 / 21 / 2025, page 34 / 70 27 / 60 sorghum comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the introduced genetic modification is present in a single allele of the ACCase gene in the second sorghum variety. In certain embodiments, the introduced genetic modification is present in both alleles of the ACCase gene in the second sorghum variety. In certain embodiments, the second inbred sorghum variety does not comprise the introduced genetic modification.

[0043] In certain embodiments, the hybrid produced is heterozygous for the modified ACCase gene. In certain embodiments, the hybrid produced is homozygous for the modified ACCase gene. In certain embodiments, the hybrid produced has tolerance to at least one member of the aryloxyphenoxypropionate (FOP) herbicide family.In certain varieties, the resulting hybrid exhibits tolerance to quizalofop-p-ethyl when applied at a rate of at least approximately 44.8 g / ha (0.04 lb ai / A), 50.3 g / ha (0.045 lb ai / A), 56.0 g / ha (0.05 lb ai / A), 61.6 g / ha (0.055 lb ai / A), 67.2 g / ha (0.06 lb ai / A), 72.8 g / ha (0.065 lb ai / A), 78.4 g / ha (0.07 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 224.0 g / ha (0.2 lb ai / A) or 280.0 g / ha (0.25 lb ai / A), and less than 336.0 g / ha (0.3 lb ai / A), 280.0 g / ha (0.25 lb ai / A), 224.0 g / ha (0.2 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 78.4 g / ha (0.07 lb ai / A) or 67.2 g / ha (0.06 lb ai / A). In certain embodiments, quizalofop-p-ethyl, applied at a rate in the range of about 56.0 g / ha (0.05 lb. Petition 870250096193, dated 10 / 21 / 2025, p. 35 / 70 28 / 60 ai / A) to 168.0 g / ha (0.15 lb ai / A). In certain embodiments, the hybrid produced has tolerance to at least one member of the cyclohexanedione (DIM) herbicide family.In certain varieties, the resulting hybrid exhibits tolerance to clethodim when applied at a rate of at least 44.8 g / ha (0.04 lb ai / A), 50.3 g / ha (0.045 lb ai / A), 56.0 g / ha (0.05 lb ai / A), 61.6 g / ha (0.055 lb ai / A), 67.2 g / ha (0.06 lb ai / A), 72.8 g / ha (0.065 lb ai / A), 78.4 g / ha (0.07 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 224.0 g / ha (0.2 lb ai / A) or 280.0 g / ha (0.25 lb ai / A), and less than 336.0 g / ha (0.3 lb ai / A), 280.0 g / ha (0.25 lb ai / A), 224.0 g / ha (0.2 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 78.4 g / ha (0.07 lb ai / A), or 67.2 g / ha (0.06 lb ai / A).In certain embodiments, clethodim is applied at a rate in the range of about 56.0 g / ha (0.08 lb ai / A) to 168.0 g / ha (0.2 lb ai / A). In certain embodiments, the hybrid produced has tolerance to a combination herbicide comprising at least one member of the aryloxyphenoxypropionate (FOP) herbicide family and at least one member of the cyclohexanedione (DIM) herbicide family. In certain embodiments, the hybrid produced has a yield that is greater than, equal to, or within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, compared to the corresponding control plant, for example, one that has a similar genetic background but lacks the introduced genetic modification. In certain embodiments, the hybrid produced has a yield that... Petition 870250096193, dated 10 / 21 / 2025, p. 36 / 70 29 / 60 is increased by at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, compared with a control sorghum variety comprising an ACCase gene encoding an ACCase polypeptide comprising a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2 introduced by crossing with an exotic sorghum variety.

[0044] Sorghum varieties are primarily self-pollinated; therefore, self-pollination of parental varieties must be controlled to make the development of hybrid varieties viable. An effective pollination control and pollen transfer system from one parent to another offers better plant reproduction and an effective method for producing hybrid seeds and plants. For example, the milo or A1 cytoplasmic male sterility (CMS) system, developed through a cross between milo and kafir cultivars, is one of the most frequently used CMS systems in hybrid sorghum production (Stephens JC & Holland PF, Cytoplasmic Male Sterility for Hybrid Sorghum Seed Production, Agron. J. 46:20-23 (1954)). Other CMS systems for sorghum include, but are not limited to, A2, isolated from IS 12662c (Schertz KF, Registration of A2Tx 2753 and BTx 2753 Sorghum Germplasm, Crop Sci.17: 983 (1977)), A3, isolated from IS 1112c or converted Nilwa (Quinby JR, Interactions of Genes and Cytoplasms in Male-Sterility in Sorghums, Proc. 35th Corn Sorghum Res. Conf. Am. Seed Trade Assoc. Chicago, Ill., pp. 5-8 (1980)), A4, isolated from IS 7920c (Worstell et al, Relationship among MaleSterility Inducing Cytoplasms of Sorghum, Crop Sci. 24:186-189 (1984)).

[0045] In the development of new improved varieties of sorghum, breeders can use a CMS plant as Petition 870250096193, dated 10 / 21 / 2025, page 37 / 70 30 / 60 female parental. By using these plants, breeders attempt to improve seed production efficiency and the quality of F1 hybrids, as well as reduce breeding costs. When hybridization is conducted without the use of CMS plants, it is more difficult to obtain and isolate the desired traits in the progeny (F1 generation) because the parents are capable of both cross-pollination and self-pollination. If one of the parents is a CMS plant incapable of producing pollen, only cross-pollination will occur. By eliminating pollen from a parental variety in a cross, the plant breeder is guaranteed to obtain seeds of uniform quality, provided the parents are of uniform quality and the breeder conducts a single cross.

[0046] In certain embodiments, the production of hybrid varieties described in this document includes crossing a CMS female parent with a pollen-producing male parent. In certain embodiments, the CMS female parent comprises any of the genetic modifications of the ACCase gene described in this document. In certain embodiments, the pollen-producing male parent comprises any of the genetic modifications of the ACCase gene described in this document. In certain embodiments, both the CMS female parent and the pollen-producing male parent comprise any of the genetic modifications of the ACCase gene described in this document. To reproduce effectively, however, the male parent of the F1 hybrid has a fertility-restoring gene (Rf gene). The presence of an Rf gene means that the F1 generation will not be completely or partially sterile, so self-pollination or cross-pollination can occur.Self-pollination of the F1 generation to produce several subsequent generations ensures... Petition 870250096193, dated 10 / 21 / 2025, p. 38 / 70 31 / 60 that a desired trait is heritable and stable and that a new variety has been isolated.

[0047] Methods for producing an ACCase inhibitor-tolerant sorghum variety are further provided in this document, comprising introducing into a regenerable sorghum plant cell a genetic modification in an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and comprising a non-tryptophan (e.g., a cysteine) at a position corresponding to the position 2032 of SEQ ID NO: 2, and generate a sorghum plant from the plant cell, wherein the generated plant comprises the introduced genetic modification and has better tolerance to ACCase-inhibiting herbicides compared to a control plant.In certain embodiments, the non-tryptophan is a cysteine, so the modified ACCase polypeptide comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the genetic modification is introduced using a genome modification technique selected from the group consisting of a polynucleotide-guided endonuclease, CRISPR-Cas endonucleases, base-editing deaminases, a transcription activator-like effector nuclease (TALEN), and site-specific genetically engineered endonucleases.

[0048] In certain modalities, genome editing can be facilitated by inducing a double-strand break (DSB) or single-strand break at a defined position in the genome near the desired alteration. DSBs can be induced Petition 870250096193, dated 10 / 21 / 2025, pp. 39 / 70 32 / 60 with the use of any available DSB induction agent including, but not limited to, TALENs, meganucleases, zinc finger nucleases, CRISPR-Cas systems (e.g., Cas9-gRNA systems, cpf1-guided endonuclease systems, Cas12f endonuclease systems and the like). In some embodiments, the introduction of a DSB may be combined with the introduction of a polynucleotide modification template.

[0049] The process of editing a genomic sequence that combines DSB and modification templates generally comprises: providing a host cell with a DSB-inducing agent, or a nucleic acid encoding a DSB-inducing agent, that recognizes a target sequence in the chromosomal sequence and is capable of inducing a DSB in the genomic sequence, and at least one polynucleotide modification template comprising at least one nucleotide change compared to the nucleotide sequence to be edited. The polynucleotide modification template may further comprise nucleotide sequences flanking at least one nucleotide change, wherein the flanking sequences are substantially homologous to the chromosomal region flanking the DSB.

[0050] Endonuclease can be delivered to a cell by any method known in the art, for example, but not limited to, transient introduction methods, transfection, microinjection and / or topical application, or indirectly through recombinant constructs. Endonuclease can be delivered as a protein or as a guided polynucleotide complex directly to a cell or indirectly through recombinant constructs. Endonuclease can be introduced into a cell in a manner Petition 870250096193, dated 10 / 21 / 2025, pp. 40 / 70 33 / 60 transient or can be incorporated into the host cell genome using any method known in the art. In the case of a CRISPR-Cas system, the uptake of the endonuclease and / or guided polynucleotide into the cell can be facilitated with a Cell-Penetrating Peptide (CPP), as described in document no. WO2016073433.

[0051] TAL effector nucleases (TALEN) are a class of sequence-specific nucleases that can be used to make double-strand breaks in specific target sequences in the genome of a plant or other organism (Miller et al. (2011) Nature Biotechnology 29:143 — 148).

[0052] Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Endonucleases include restriction endonucleases, which cleave DNA at specific sites without damaging the bases, and meganucleases, also known as homing endonucleases (HEases), which, like restriction endonucleases, bind and cut at a specific recognition site; however, the recognition sites for meganucleases are typically longer, about 18 bp or more (patent application PCT / US12 / 30061). Meganucleases have been classified into four families based on conserved sequence motifs. These motifs participate in the coordination of metal ions and hydrolysis of phosphodiester bonds. HEases are notable for their long recognition sites and for tolerating some sequence polymorphisms in their DNA substrates. The naming convention for meganucleases is similar to the convention for other restriction endonucleases.Meganucleases are also characterized by the prefix F-, I-, or PI- for enzymes encoded by ORFs, introns, and inteins. Petition 870250096193, dated 10 / 21 / 2025, page 41 / 70 34 / 60 independent, respectively. One step in the recombination process involves polynucleotide cleavage at or near the recognition site. The cleavage activity can be used to produce a double-strand break. For reviews on site-specific recombinases and their recognition sites, see Sauer (1994) Curr Op Biotechnol 5:521-527; and Sadowski (1993) FASEB 7:760-767. In some instances, the recombinase is from the Integrase or Resolvase families.

[0053] Zinc finger nucleases (ZFNs) are modified double-strand break induction agents comprising a zinc finger DNA-binding domain and a double-strand break induction agent domain. Site recognition specificity is conferred by the zinc finger domain, which typically comprises two, three, or four zinc fingers, for example, having a C2H2 structure; however, other zinc finger structures are known and have been modified. Zinc finger domains are amenable to designing polypeptides that specifically bind to a selected polynucleotide recognition sequence. ZFNs include a modified DNA-binding zinc finger domain linked to a non-specific endonuclease domain, for example, the nuclease domain of a Type II endonuclease, such as FokI.Additional functionalities can be fused with the zinc finger binding domain, including transcription activator domains, transcription repressor domains, and methylases. In some examples, dimerization of the nuclease domain is required for cleavage activity. Each zinc finger recognizes three consecutive base pairs in the target DNA. For example, a 3-finger domain recognized a sequence of 9 contiguous nucleotides, with a requirement of... Petition 870250096193, dated 10 / 21 / 2025, page 42 / 70 In the 35 / 60 dimerization of the nuclease, two sets of zinc finger triplets are used to link an 18-nucleotide recognition sequence.

[0054] Genome editing using DSB-inducing agents, such as Cas9 and gRNA complexes, has been described, for example in Patent Applications No. US 2015-0082478 A1, WO2015 / 026886 A1, WO2016007347 and WO201625131, all of which are incorporated by reference herein.

[0055] In certain modalities, genetic modification is introduced without introducing a double-strand break using base editing technology, see, for example, Gaudelli et al., (2017) Programmable base editing of A*T to G*C in genomic DNA without DNA cleavage. Nature 551(7681):464-471; Komor et al., (2016) Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage, Nature 533(7603):420-424.

[0056] In certain embodiments, base editing comprises (i) a catalytically altered CRISPR-Cas9 mutant that is mutated such that one of its nuclease domains cannot perform DSBs; (ii) a single-strand-specific cytidine / adenine deaminase that converts C to U or A to G within a suitable nucleotide gap in the single-stranded DNA bubble created by Cas9; (iii) a uracil glycosylase inhibitor (UGI) that prevents uracil excision and downstream processes that diminish base editing efficacy and product purity; or (iv) nickase activity to cleave the unedited DNA strand, followed by cellular DNA repair processes to replace the G-containing DNA strand.

[0057] A method for producing a is also provided. Petition 870250096193, dated 10 / 21 / 2025, page 43 / 70 36 / 60 ACCase inhibitor-tolerant sorghum variety, comprising providing a sorghum plant cell comprising an endogenous ACCase gene sequence, a guide polynucleotide, a polynucleotide modification template comprising at least one nucleotide modification of the ACCase gene sequence to encode a modified ACCase polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and comprising a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2, and a Cas endonuclease, the guide polynucleotide and the Cas endonuclease capable of forming a complex that allows the Cas endonuclease to introduce a double-strand break in a target site sequence in the ACCase gene of said plant cell, to obtain a plant from the plant cell,To evaluate the plant for the presence of at least one nucleotide modification and to select a parent sorghum plant containing the modified ACCase gene and having greater tolerance to ACCase-inhibiting herbicides compared to a control plant that does not contain the modification.

[0058] As used in this document, the term “guide polynucleotide” refers to a polynucleotide sequence that can form a complex with a Cas endonuclease, including the Cas endonuclease described in this document, and allow the Cas endonuclease to recognize, optionally bind to, and optionally cleave a target DNA site. The guide polynucleotide sequence can be an RNA sequence, a DNA sequence, or a combination thereof (an RNA combination sequence). Petition 870250096193, dated 10 / 21 / 2025, p. 44 / 70 37 / 60 In certain embodiments, the guide polynucleotide is a guide RNA (gRNA) comprising a CRISPR RNA (crRNA) and a transactivation CRISPR RNA (tracrRNA) to guide the Cas endonuclease to its DNA target. The crRNA comprises a spacer region complementary to a strand of the double-stranded DNA target and a region that forms base pairs with the tracrRNA, forming an RNA duplex. In certain embodiments, the gRNA is a “single guide RNA” (sgRNA) comprising a synthetic fusion of crRNA and tracrRNA.

[0059] In certain embodiments of the methods described in this document, the guide RNA comprises a crRNA-tracrRNA fusion transcript. In certain embodiments, the tracrRNA comprises a nucleotide sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 13. In certain embodiments, the crRNA comprises a nucleotide sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the SEQ ID Nos: 8, 9, or 10.

[0060] The term “polynucleotide modification template” includes a polynucleotide comprising at least one nucleotide modification when compared to the nucleotide sequence to be edited. A nucleotide modification may be at least one nucleotide substitution, addition, or deletion. Optionally, the polynucleotide modification template may additionally comprise homologous nucleotide sequences flanking at least one nucleotide modification, wherein the flanking homologous nucleotide sequences provide sufficient homology with the Petition 870250096193, dated 10 / 21 / 2025, page 45 / 70 38 / 60 desired nucleotide sequence to be edited. In certain embodiments of the methods described herein, the polynucleotide modification template comprises a nucleotide sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 14.

[0061] The terms target site, target sequence, “sequence of target sites”, target DNA, target locus, genomic target site, genomic target sequence, genomic target locus and protospacer are used interchangeably in this document and refer to a polynucleotide sequence, such as, but not limited to, a nucleotide sequence on a chromosome, episome, locus or any other DNA molecule in the genome (including chromosomal, chloroplast, mitochondrial, plasmid DNA) of a cell, where a guide polynucleotide / Cas endonuclease complex can recognize, bind and optionally cut or cleave. The target site may be an endogenous site in the genome of a cell or, alternatively, the target site may be heterologous to the cell and thus not naturally occurring in the cell's genome, or the target site may be found at a genomically heterologous location compared to where it occurs in nature.As used in this document, the terms endogenous target sequence and native target sequence are used interchangeably herein to refer to a target sequence that is endogenous or native to the genome of a cell and is in the endogenous or native position of that target sequence in the cell's genome. An artificial target site or artificial target sequence is used interchangeably herein and refers to... Petition 870250096193, dated 10 / 21 / 2025, pp. 46 / 70 39 / 60 a target sequence that has been introduced into the genome of a cell. Such an artificial target sequence may be identical, in sequence terms, to an endogenous or native target sequence in the genome of a cell, but may be located at a different position (i.e., a non-endogenous or non-native position) in the genome of a cell. In certain embodiments, the target site sequence in the endogenous ACCase gene comprises a nucleotide sequence that is at least 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to any of the SEQ ID NOs: 5, 6, 7, or 12.

[0062] The Cas endonuclease for use in the methods described herein is not particularly limited and may be any Cas endonuclease known in the art. In certain embodiments, the Cas endonuclease is a Class 2 Type II. In Class 2 Type II systems, the Cas endonuclease acts in complex with a guide RNA (gRNA) that directs the Cas endonuclease to cleave the target DNA to enable target recognition, binding, and cleavage by the Cas endonuclease. The gRNA comprises a Cas endonuclease recognition domain (CER) that interacts with the Cas endonuclease, and a Variable Targeting (VT) domain that hybridizes with a nucleotide sequence in a target DNA. In certain embodiments, the Cas endonuclease is selected from the group consisting of Cas9, Cpf1, and Cas12f (also known in the art as Cas-alpha 10).

[0063] In certain embodiments, the parent plant comprises the modification in a single allele of the ACCase gene. In certain embodiments, the parent plant comprises the modification in both alleles of the ACCase gene. In certain Petition 870250096193, dated 10 / 21 / 2025, pp. 47 / 70 In the 40 / 60 modalities, the parent plant has tolerance to at least one member of the aryloxyphenoxypropionate (FOP) herbicide family.In certain modalities, the parent plant has tolerance to quizalofop-p-ethyl when applied at a rate of at least approximately 44.8 g / ha (0.04 lb ai / A), 50.3 g / ha (0.045 lb ai / A), 56.0 g / ha (0.05 lb ai / A), 61.6 g / ha (0.055 lb ai / A), 67.2 g / ha (0.06 lb ai / A), 72.8 g / ha (0.065 lb ai / A), 78.4 g / ha (0.07 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 100.8 g / ha (0.09 lb ai / A). ai / A), 106.4 g / ha (0.095 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 224.0 g / ha (0.2 lb ai / A) or 280.0 g / ha (0.25 lb ai / A), and less than 336.0 g / ha (0.3 lb ai / A), 280.0 g / ha (0.25 lb ai / A), 224.0 g / ha (0.2 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 78.4 g / ha (0.07 lb ai / A) or 67.2 g / ha (0.06 lb ai / A).In certain embodiments, quizalofop-p-ethyl is applied at a rate in the range of about 56.0 g / ha (0.05 lb ai / A) to 168.0 g / ha (0.15 lb ai / A). In certain embodiments, the parent plant has tolerance to at least one member of the cyclohexanedione (DIM) herbicide family. In certain modalities, the parent plant has tolerance to clethodim when applied at a rate of at least 44.8 g / ha (0.04 lb ai / A), 50.3 g / ha (0.045 lb ai / A), 56.0 g / ha (0.05 lb ai / A), 61.6 g / ha (0.055 lb ai / A), 67.2 g / ha (0.06 lb ai / A), 72.8 g / ha (0.065 lb ai / A), 78.4 g / ha (0.07 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 224.0 g / ha (0.2 lb ai / A) or 280.0 g / ha (0.25. Petition 870250096193, dated 10 / 21 / 2025, pp. 48 / 70 41 / 60 lb ai / A) 106.4 g / ha (0.095 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 78.4 g / ha (0.07 lb ai / A) or 67.2 g / ha (0.06 lb ai / A). In certain embodiments, clethodim is applied at a rate in the range of about 56.0 g / ha (0.08 lb ai / A) to 168.0 g / ha (0.2 lb ai / A). In certain embodiments, the parent plant has tolerance to a combination herbicide comprising at least one member of the aryloxyphenoxypropionate (FOP) herbicide family and at least one member of the cyclohexanedione (DIM) herbicide family.In certain modalities, the parent plant has a yield that is greater than, equal to, or within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, compared to the corresponding control plant, for example, one that has a similar genetic background but lacks the introduced genetic modification. In certain embodiments, the parent plant has a yield that is increased by at least about 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, compared with a control sorghum variety comprising an ACCase gene encoding an ACCase polypeptide comprising a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2 introduced by crossing with an exotic sorghum variety.

[0064] In certain embodiments, the method further comprises crossing the selected parent sorghum plant with a second sorghum plant to produce an F1 hybrid parent plant. In certain embodiments, the second sorghum plant comprises an ACCase polynucleotide encoding a modified ACCase polypeptide that is at least 50%, 55%, 60%, Petition 870250096193, dated 10 / 21 / 2025, pp. 49 / 70 42 / 60 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the F1 hybrid parent plant is homozygous for the modified ACCase polynucleotide. In certain embodiments, the F1 hybrid parent plant is heterozygous for the modified ACCase polynucleotide. In certain embodiments, the F1 hybrid parent plant has tolerance to at least one member of the aryloxyphenoxypropionate (FOP) herbicide family.In certain embodiments, the F1 hybrid parent plant is tolerant to quizalofop-p-ethyl when applied at a rate of at least approximately 44.8 g / ha (0.04 lb ai / A), 50.3 g / ha (0.045 lb ai / A), 56.0 g / ha (0.05 lb ai / A), 61.6 g / ha (0.055 lb ai / A), 67.2 g / ha (0.06 lb ai / A), 72.8 g / ha (0.065 lb ai / A), 78.4 g / ha (0.07 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 224.0 g / ha (0.2 lb ai / A) or 280.0 g / ha (0.25 lb ai / A), and less than 336.0 g / ha (0.3 lb ai / A), 280.0 g / ha (0.25 lb ai / A), 224.0 g / ha (0.2 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 78.4 g / ha (0.07 lb ai / A), or 67.2 g / ha (0.06 lb ai / A).In certain embodiments, quizalofop-petila is applied at a rate in the range of about 56.0 g / ha (0.05 lb ai / A) to 168.0 g / ha (0.15 lb ai / A). In certain embodiments, the F1 hybrid parent plant has tolerance to at least one member of the cyclohexanedione herbicide family. Petition 870250096193, dated 10 / 21 / 2025, pp. 50 / 70 43 / 60 (DIM). In certain embodiments, the F1 hybrid parent plant is tolerant to clethodim when applied at a rate of at least 44.8 g / ha (0.04 lb ai / A), 50.3 g / ha (0.045 lb ai / A), 56.0 g / ha (0.05 lb ai / A), 61.6 g / ha (0.055 lb ai / A), 67.2 g / ha (0.06 lb ai / A), 72.8 g / ha (0.065 lb ai / A), 78.4 g / ha (0.07 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 224.0 g / ha (0.2 lb ai / A) or 280.0 g / ha (0.25 lb ai / A), and less than 336.0 g / ha (0.3 lb ai / A), 280.0 g / ha (0.25 lb ai / A), 224.0 g / ha (0.2 lb ai / A), 168.0 g / ha (0.15 lb ai / A), 112.0 g / ha (0.1 lb ai / A), 106.4 g / ha (0.095 lb ai / A), 100.8 g / ha (0.09 lb ai / A), 95.2 g / ha (0.085 lb ai / A), 89.6 g / ha (0.08 lb ai / A), 84.0 g / ha (0.075 lb ai / A), 78.4 g / ha (0.07 lb ai / A), or 67.2 g / ha (0.06 lb ai / A).In certain embodiments, clethodim is applied at a rate in the range of about 56.0 g / ha (0.08 lb ai / A) to 168.0 g / ha (0.2 lb ai / A). In certain embodiments, the F1 hybrid parent plant has tolerance to a combination herbicide comprising at least one member of the aryloxyphenoxypropionate (FOP) herbicide family and at least one member of the cyclohexanedione (DIM) herbicide family. In certain embodiments, the F1 hybrid parent plant has a yield that is greater than, equal to, or within 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, compared to the corresponding control plant, for example, one that has a similar genetic background but lacks the introduced genetic modification. In certain variations, the F1 hybrid parent plant has a yield that is increased by at least approximately 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%, compared to a control sorghum variety. Petition 870250096193, dated 10 / 21 / 2025, pp. 51 / 70 44 / 60 comprising an ACCase gene encoding an ACCase polypeptide comprising a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2 introduced by crossing with an exotic variety of sorghum.

[0065] A method is also provided for introgressing a modified ACCase allele into an elite sorghum variety, comprising crossing a first sorghum variety with a second sorghum variety to produce a progeny population, wherein the first sorghum variety comprises any of the introduced genetic modifications described herein, genotyping the progeny population for the presence of the introduced genetic modification, and selecting the progeny comprising the introduced genetic modification to obtain sorghum varieties comprising the modified ACCase allele and having improved tolerance to ACCase-inhibiting herbicides. In certain embodiments, the selected progeny comprises a single ACCase allele with the introduced genetic modification. In certain embodiments, the selected progeny comprises the introduced genetic modification in both alleles.In certain modalities, the presence of the introduced genetic modification is detected using primers selected from the group consisting of SEQ IDs 15, 16, 17, and 18.

[0066] A method is also provided for controlling and / or selectively controlling weeds in a cultivated area, comprising planting a cultivated area with seeds and / or plants comprising any introduced genetic modifications described herein and applying to the seeds and / or plants a sufficient quantity of an ACCase inhibitor herbicide to control the weeds. Petition 870250096193, dated 10 / 21 / 2025, pp. 52 / 70 45 / 60 without significantly affecting the seeds and / or plants. In certain embodiments, the seed produces a hybrid elite sorghum variety comprising a genetic modification introduced into the endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to SEQ ID NO: 2, and comprises a non-tryptophan (e.g., a cysteine) at a position corresponding to position 2032 of SEQ ID NO: 2. In certain embodiments, the non-tryptophan is a cysteine ​​such that the modified ACCase polypeptide comprises a cysteine ​​at the position corresponding to position 2032 of SEQ ID NO: 2.

[0067] “Weed” as used in this document refers to a plant that is undesirable in a specific area. In certain embodiments, a weed is a non-cultivated plant or a non-cultivated species, while in some embodiments, a weed is a cultivated species that is intended to be eliminated from a specific area.

[0068] As used in this document, the term “control” and its derivations, for example, as in “control weeds,” refer to one or more of the following: inhibiting the growth, germination, reproduction, and / or proliferation; and / or exterminating, removing, destroying, or otherwise diminishing the occurrence and / or activity of a weed. As used in this document, “selectively controlled” means that the majority of weeds in a cultivated area are significantly damaged or exterminated, while, if crop plants are also present in the field, the majority of the plants Petition 870250096193, dated 10 / 21 / 2025, pp. 53 / 70 46 / 60 of the crop is not significantly damaged. Thus, a method is considered to selectively control weeds when at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more of the weeds are significantly damaged or eradicated, while, if crop plants are also present in the field, less than 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% or 1% of the crop plants are significantly damaged or eradicated.

[0069] As used in this document, a “growing area” comprises any region in which it is desired to cultivate a plant. These growing areas include, but are not limited to, a field in which a plant is grown, a greenhouse, or a growth chamber.

[0070] In certain embodiments, the ACCase-inhibiting herbicide is applied in a pre-emergence application. In certain embodiments, the ACCase-inhibiting herbicide is applied in a post-emergence application. In certain embodiments, the ACCase-inhibiting herbicide is applied in both a pre-emergence and a post-emergence application. In certain embodiments, a non-ACCase-inhibiting herbicide (e.g., glyphosate, 2,4-D, glufosinate) is applied in a pre-emergence application and the ACCase-inhibiting herbicide is applied in a post-emergence application.

[0071] When the sorghum variety further comprises at least one additional polynucleotide that confers tolerance to a herbicide (e.g., glyphosate, 2,4-D, glufosinate), the herbicide may be applied in a pre-emergence application, a post-emergence application, or both pre-emergence and post-emergence, whereas the ACCase-inhibiting herbicide may Petition 870250096193, dated 10 / 21 / 2025, pp. 54 / 70 47 / 60 can be applied simultaneously or sequentially in a pre-emergency application, a post-emergency application, or both pre-emergency and post-emergency.

[0072] Pre-emergent or similar refers to a herbicide that is applied to an area of ​​interest (e.g., a field or cultivated area) before a plant visibly emerges from the soil. Post-emergent or similar refers to a herbicide that is applied to an area after a plant visibly emerges from the soil. In some cases, the terms pre-emergent and post-emergent are used with reference to a weed in an area of ​​interest, and in some cases, these terms are used with reference to a cultivated plant in an area of ​​interest. When used with reference to a weed, these terms may apply only to a specific type of weed or weed species that is present or believed to be present in the area of ​​interest.

[0073] Methods are further provided for detecting the genome-edited sorghum varieties described herein, comprising obtaining a nucleic acid sample from said plant tissues and (i) contacting said nucleic acid sample with a polynucleotide comprising a sequence of at least 8 nucleotides that are identical to a contiguous sequence of SEQ ID NO: 1, or complements thereof; subjecting said sample and said polynucleotide to stringent hybridization conditions; and testing said sample for hybridization of said polynucleotide with said DNA; or (ii) contacting said nucleic acid sample with a first and a second PCR primer, wherein said first and second PCR primers each bind, Petition 870250096193, dated 10 / 21 / 2025, pp. 55 / 70 48 / 60 specifically SEQ ID NO: 1, subject the sample to polymerase chain reaction and analyze an amplicon generated between the first and second primers. In certain embodiments, the first and second primers comprise SEQ ID NO: 15, 16, 17, 18, or any combination thereof.

[0074] As used in this document, “stringing conditions” encompass conditions in which hybridization will only occur if there is less than 20% mismatch between the hybridizing molecule and a sequence within the target nucleic acid molecule. “Stringing conditions” include additional specific levels of stringency. Thus, as used in this document, “moderate stringency” conditions are those under which molecules with more than 20% sequence mismatch do not hybridize; “high stringency” conditions are those under which sequences with more than 10% mismatch do not hybridize; and “very high stringency” conditions are those under which sequences with more than 5% mismatch do not hybridize. Representative non-limiting hybridization conditions are listed below.

[0075] High stringent condition (detects sequences that share at least 90% sequence identity): Hybridization in 5x SSC buffer (where the SSC buffer contains a detergent, such as SDS, and additional reagents such as salmon sperm DNA, EDTA, etc.) at 65°C for 16 hours; washing twice in 2x SSC buffer (where the SSC buffer contains a detergent, such as SDS, and additional reagents such as salmon sperm DNA, EDTA, etc.) at room temperature for 15 minutes each; and washing twice in 0.5x SSC buffer (where the SSC buffer contains a detergent, such as SDS, and reagents Petition 870250096193, dated 10 / 21 / 2025, pp. 56 / 70 49 / 60 additional samples such as salmon sperm DNA, EDTA, etc.) at 65 °C for 20 minutes each.

[0076] Moderate stringent condition (detects sequences that share at least 80% sequence identity): Hybridization in 5x-6x SSC buffer (where the SSC buffer contains a detergent, such as SDS, and additional reagents such as salmon sperm DNA, EDTA, etc.) at 65-70 °C for 16-20 hours; wash twice in 2x SSC buffer (where the SSC buffer contains a detergent, such as SDS, and additional reagents such as salmon sperm DNA, EDTA, etc.) at room temperature for 5-20 minutes each; and wash twice in 1x SSC buffer (where the SSC buffer contains a detergent, such as SDS, and additional reagents such as salmon sperm DNA, EDTA, etc.) at 55-70 °C for 30 minutes each.

[0077] Non-stringing control condition (sequences that share at least 50% sequence identity will hybridize): Hybridization in 6x SSC buffer (where the SSC buffer contains a detergent, such as SDS, and additional reagents such as salmon sperm DNA, EDTA, etc.) at room temperature at 55 °C for 16-20 hours; wash at least twice in 2x-3x SSC buffer (where the SSC buffer contains a detergent, such as SDS, and additional reagents such as salmon sperm DNA, EDTA, etc.) at room temperature at 55 °C for 20-30 minutes each.

[0078] Plants produced by the methods described in this document are also supplied. Seeds produced or generated from any of the plants described in this document are also supplied, wherein the seeds comprise an introduced genetic modification described in this document, as well as seeds for producing Petition 870250096193, dated 10 / 21 / 2025, pp. 57 / 70 50 / 60 any of the plants described herein comprising an introduced genetic modification described herein. Furthermore, any genome-edited sorghum variety disclosed herein may be used to produce a food or food product. Such methods comprise obtaining a plant, explant, seed, plant cell or cell comprising the polynucleotide sequence, and processing the plant, explant, seed, plant cell or cell to produce a food or food product.

[0079] The following are examples of specific embodiments of some aspects of the invention. The examples are offered for illustrative purposes only and are not intended to limit the scope of the invention in any way. EXAMPLE 1

[0080] This example demonstrates the introduction of a targeted genetic modification to produce sorghum resistant to ACCase inhibitor.

[0081] The endogenous ACCase gene was edited to produce an endogenous ACCase allele that has a tryptophan amino acid substitution for cystine at position 2032 using the gRNA / Cas9 site-targeted nuclease system. To modify the endogenous ACCase gene, the following target sites were used: ACCaseTS1, ACCase-TS2, and ACCase-TS3 (SEQ ID NOs: 5, 6, and 7, respectively). The location of each target site in the ACCase genomic sequence (SEQ ID NO: 1) is shown in Figure 1, and the target sequences are listed in Table 2. Table 2: ACCase target site sequence Sorghum genomic target site sequence PAM sequence Petition 870250096193, dated 10 / 21 / 2025, pp. 58 / 70 51 / 60 ACC-TS1 SEQ ID NO: 5 TGG ACC-TS2 SEQ ID NO: 6 TGG ACC-TS3 SEQ ID NO: 7 AGG ACC-TS3.1 SEQ ID NO: 12 AGG

[0082] To generate the Cas9 vector, the maize codon-optimized Cas9 gene from Streptococcus pyogenes M1 GAS (SF370) and the potato ST-LS1 intron were used to eliminate expression in E. coli and A. agrobacterium. To facilitate nuclear localization of the encoded Cas9 protein in maize cells, the monopartite amino-terminal nuclear localization signal from Simian virus 40 (SV40) and the bipartite nuclear localization signal from the VirD2 agrobacterium tumefaciens endonuclease were incorporated into the amino and carboxyl termini of the open Cas9 reading frame, respectively. The Cas9 gene was operationally ligated to a maize ubiquitin promoter (Zm-Ubi pro) and potato intron II (PinII) using standard molecular biological techniques.

[0083] To direct the Cas9 nuclease to designated genomic target sites, a maize U6 polymerase III promoter (SEQ ID NO: 19) and its cognate U6 III polymerase termination sequence were used to direct the initiation and termination of gRNA expression. The guide RNA (SEQ ID NO: 13) was a designed crRNA-tracrRNA fusion transcript comprising a 20 bp variable targeting domain that was chosen from a maize genomic target site. The guide RNA variable targeting domains for ACCase gene editing were identified as ACC-CR1 (SEQ ID NO: 8), ACC-CR2 (SEQ ID NO: 9), and ACC-CR3 (SEQ ID NO: 10). The guide RNA variations ACC-CR3.1 (SEQ ID NO: 11) and ACC-TS3.1 (SEQ ID NO: 12) were also Petition 870250096193, dated 10 / 21 / 2025, pp. 59 / 70 52 / 60 were included to increase cleavage and editing frequency. The DNA encoding each of the variable nucleotide targeting domains was cloned into a gRNA expression cassette via BsbI sites.

[0084] The ACC-TS3 replacement / repair template donor contained the nucleotide substitution to produce an ACCase gene encoding a polypeptide comprising the W2032C amino acid substitution along with a 100 bp homology arm sequence 1 and a 103 bp homology arm sequence 2 flanking ACC-TS3 (SEQ ID NO: 7), ACCTS3.1 was placed outside the homology arms to increase editing efficiency in the stable transformation of Agrobacterium.

[0085] The auxotrophic strain LBA4404 Thy of Agrobacterium carrying a ternary vector was selected as the delivery system to generate ACCase editing events. The ternary vector system contains the binary T-DNA vector and the pVIR evaluator plasmid. The binary T-DNA vector had multigene cassettes containing Zm-Ubi pro:SpCas9, Zm-U6 pro:guide RNAs, donor repair template with desired editing, additional gene cassettes including Zm-Ubi pro:NPTII (neomycin phosphotransferase II), maize morphogenic genes Zm-Pltp pro:Bbm (Baby boom) and Zm-Axig1 pro:WUS2 (Wuschel2) for transformant selection and to stimulate transformation. The ternary project was initially assembled by mobilizing the accessory plasmid in the auxotrophic strain LBA4404 Thy- of Agrobacterium and selecting it in a medium supplemented with gentamicin (25 mg l-1).Subsequently, the binary constructs were electroporated onto the LBA4404 Thy-Agrobacterium strain containing the accessory plasmid, and recombinant colonies were formed. Petition 870250096193, dated 10 / 21 / 2025, pages 60 / 70 53 / 60 selected samples were taken from media supplemented with gentamicin and spectinomycin. All constructs were then subjected to next-generation sequencing and sequence confirmation before transformation experiments were performed.

[0086] To select a guide for stable transformation, the three guides described above were transiently tested using bombardment of plasmid DNA particles and ribonucleoprotein (RNP). Briefly, the S. pyogenes Cas9 protein with two nuclear localization signals was obtained from Corteva's internal sources, and the sgRNA in the form of RNA molecules was synthesized in Synthego. To generate a guide RNA-ribonucleoprotein Cas9 (RNP) complex, 7 μg of Cas9 protein and 3 μg of gRNA molecules (molar ratio 1:2) were mixed in 1 χ NEB Buffer 3 and 1 μg of RNA inhibitor (Ribo Guard, Epicentre, USA) to a total volume of 20 μg and incubated at room temperature for 15 min. The particle delivery matrix, composed of RNP complexes supplemented with plasmids containing YFP regulated by the ubiquitin promoter (20 ng), ODP2 regulated by PLTP (10 ng), and WUS regulated by the maize auxiliary promoter (10 ng), was delivered into sorghum embryonic cells.In summary, RNPs and DNA were precipitated to 0.6 μm (average diameter) (Bio-Rad, USA) using a water-soluble cationic lipid TransIT-2020 (Mirus, USA) as follows: 50 μm of gold particles (10 mgml-1 water suspension) and 2 μm of TransIT-2020 water solution were added to the premixed RNPs and DNA vectors, gently mixed, and incubated on ice for 10 min. RNP / DNA coated gold particles were then pelleted in a microcentrifuge at 8000 g for 30 seconds, and the supernatant was removed. The pellet was resuspended in 50... Petition 870250096193, dated 10 / 21 / 2025, pp. 61 / 70 54 / 60 μL of sterile water by brief sonication. Immediately after sonication, the coated gold particles were loaded onto a macrocarrier (10 μL each) and allowed to air dry. Immature sorghum embryos, 8–10 days post-pollination, were bombarded using a PDS-1000 / He Gun (Bio-Rad) with a burst pressure of 2,930 kPa (425 pounds per square inch). Embryos were collected 3 days after bombardment.

[0087] Immature embryo explants isolated from sorghum plants were transformed with the auxotrophic strain LBA4404 Thy of Agrobacterium, carrying a ternary vector transformation system, to generate transgenic sorghum plants. The nine-week callus proliferation phase was eliminated due to the morphogenic gene cassettes used in the vector. After Agrobacterium infection and co-culture of immature embryos, the immature embryos were then subcultured in multipurpose medium with selection (250 mg / l-G418) for three weeks to induce somatic embryo formation. After three weeks of G418 selection, the somatic embryos were transferred to maturation medium without selection for four weeks before rooting to produce T0 plants.

[0088] DNA was extracted from embryos after bombardment, which then underwent PCR and deep amplicon sequencing with the primers shown in Table 3. High mutation frequency at the target site was obtained with ACCCR3, and the most dominant type of mutation at the ACCCR3 target site was named ACC-TS3.1, the corresponding ACC-CR3.1 guide RNA to the new target site was included in the agro vector. Table 3: Initiators used in tab selection and editing event screening Petition 870250096193, dated 10 / 21 / 2025, pp. 62 / 70 55 / 60 Initiator Name SEQ ID NO: ACC-TS3F 15 ACC-TS3R 16 ACC-editF 17 ACC-editR 18

[0089] To identify ACCase-edited variants, genomic DNA was extracted from leaf tissue of T0 plants. Next-generation sequencing (NGS) (Illumina) was used to identify the edited T0 variants. The region was amplified by PCR, and the primers used in the primary PCR reaction are shown in Table 3. One T0 plant with the W2032C edit was obtained from the 271 T0 plants analyzed.

[0090] The ACCase-edited T0 plant was transferred to a flat surface in a controlled environment. The T0 plant was self-fertilized to produce T1(S1) seeds; only 24 T1 seeds were produced from the edited T0 plant. The T1(S1) plants underwent comprehensive molecular characterization not only to confirm that the edits observed in the T0 plant were stably inherited, but also to verify that the T1 plants were free of any part of the T-DNA used during the transformation process. The same NGS assay to identify edited variants used in the T0 stage was performed on samples from T1 plants to confirm the presence of the edit. qPCR was performed on all auxiliary genes in the T-DNA, which included Cas9, guide RNA, transformation selection marker (NPTII), and the transformation enhancer genes Bbm and WUS2, to confirm that the genes (T-DNA) segregated from the generated edited alleles.Selected T1 plants with edited and null segregants were analyzed. Petition 870250096193, dated 10 / 21 / 2025, pp. 63 / 70 56 / 60 subsequently with Southern blotting by sequencing (SbS) to give further support to the idea that the plants were free of any foreign DNA. All surviving T1 seeds / seedlings were homozygous for Trp2032Cys editing and free of T-DNA. EXAMPLE 2

[0091] This example demonstrates the tolerance of ACCase inhibitors in plants edited with ACCase.

[0092] ACCase-edited T1 seeds were planted on flat surfaces; all seeds germinated and appeared healthy. Furthermore, all were able to produce T2 seeds with high seed counts. Nineteen homozygous ACCase-edited T1 plants, at the eight-leaf stage, were sprayed with quizalofop-p-ethyl at a rate of 207 ml (7 fl. oz) (15.4–37.7 g (0.034–0.083 lb ai)), which corresponds to the average dose indicated on the Assure II product label. All 19 homozygous ACCase-edited T1 plants showed resistance (tolerance) to the herbicide. A subset of T1 plants after the spraying test was maintained to maturity, and all produced T2 seeds with good seed formation. In contrast, wild-type control plants were all susceptible to quizalofop-p-ethyl spraying and died.

[0093] This example demonstrates that sorghum plants edited with ACCase had tolerance to an ACCase inhibitor. EXAMPLE 3

[0094] This example demonstrates field tolerance of plants edited with ACCase.

[0095] The ACCase-edited plants generated in Example 1 were crossed with 3 sterile male lines in a Petition 870250096193, dated 10 / 21 / 2025, pp. 64 / 70 57 / 60 controlled environment. F1 plants containing a single copy of the W2032C edit in the ACCase gene and the elite edited inbred line containing 2 copies of ACCase W2032C (hereinafter referred to as inbred_W3032C), along with the unedited elite inbred line (control wt) and the 3 male-sterile lines were field-tested for efficacy. Four short rows, each row with ~100 seedlings, were planted for each replicate variety. When the plants reached the 5-6 leaf stage, replicate 1 of each variety was treated with a 1x dose of Assure II (Quizalofop-P-ethyl) and replicate 2 of each variety was treated with a 2x dose of Assure II (Quizalofop-P-ethyl). The plants were then evaluated for tolerance 7 and 14 days after application. The batches were scored by assigning a score from 1 to 9 to each batch, with 1 being dead and 9 being no visible response.As shown in Table 4, the inbred plants_W2032C exhibited tolerance to the herbicide Quizalofop-P-ethyl, while the control plants without the W2032C edition of ACCase showed no tolerance, with all plants dying 10 to 14 days after spraying. The three F1 hybrids with a single copy of the W2032C edition of ACCase showed some tolerance to the herbicide Quizalofop-P-ethyl.

[0096] PH2545MW_W2032C received a score of 7 with a 1x application dose of Quizalofop-P-ethyl (Table 4), as some leaf twisting was observed due to herbicide damage. However, after about a week, the leaf twisting may no longer be visible because the leaves were growing outwards, which may be indicated by the change in score from 5 to 6 on day 7 and day 14 after a 2x application dose of Quizalofop-P-ethyl. Petition 870250096193, dated 10 / 21 / 2025, pp. 65 / 70 58 / 60 Table 4: Field Test Scores for Sorghum Varieties Treated with 1x or 2x Quizalofop-P-ethyl Variety Name | Number of copies of ACCaseW2032 C | Quizalofop-P-ethyl | Dosage | 7-day post-application score | 14-day post-application score | Elite inbred WT | 0 | 3 | 1 | Elite inbred W2032C | 2 | 7 | 7 | Sterile Male | WT | 0 | 2 | 1 | F1 Hybrid | W2032C | 1 | 5 | 4 | Sterile Male | WT | 0 | 2 | 1 | F1 Hybrid | 2 | W2032C | 1 | 5 | 4 | Sterile Male | WT | 0 | 3 | 1 | F1 Hybrid | 3 | W2032C | 1 | 5 | 3 | Elite inbred WT | 0 | 2 | 3 | 1 | Elite inbred W2032C | 2 | 2 | 5 | 6 | Sterile Male | 1 WT 0 2x 2 1 F1 Hybrid l_W2032C 1 2x 4 3 Sterile Male 2 WT 0 2x 2 1 F1 Hybrid 2_W2032C 1 2x 5 3 Sterile Male 3 WT 0 2x 2 1 F1 Hybrid 3_W2032C 1 2x 5 4

[0097] All publications and patent applications in this descriptive report are indicative of the common skill level in the art to which this invention belongs. All publications and patent applications are incorporated herein by reference to the same point as if each individual publication or patent application were specifically and individually cited as a reference. Petition 870250096193, dated 10 / 21 / 2025, pp. 66 / 70 59 / 60

[0098] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person of ordinary skill in the art to which this invention pertains. Except where otherwise stated, the techniques employed or contemplated herein are standard methods well known to persons of ordinary skill in the art. The materials, methods and examples are for illustrative purposes only and are not limiting.

[0099] Many modifications and other embodiments of the inventions set forth herein will come to mind of one skilled in the art to which these inventions relate, having the benefit of the teachings set forth in the preceding descriptions and associated drawings. Therefore, it should be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used only in a generic and descriptive sense and not for purposes of limitation.

[0100] Units, prefixes, and symbols may be denoted in their SI-accepted form. Unless otherwise indicated, nucleic acids are written from left to right in a 5' to 3' orientation; amino acid sequences are written from left to right in the amino to carboxy orientation, respectively. Numerical ranges are inclusive of the numbers that define the range. Amino acids may be referred to herein either by their commonly known three-letter symbols or by the symbols of Petition 870250096193, dated 10 / 21 / 2025, pp. 67 / 70 60 / 60 one-letter codes are recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, similarly, can be referred to by their commonly accepted single-letter codes. Petition 870250096193, dated 10 / 21 / 2025, pp. 68 / 70

Claims

1 / 12 CLAIMS 1. Genome-edited sorghum variety characterized in that it comprises a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2, the edited sorghum variety having greater tolerance to at least one ACCase-inhibiting herbicide compared to a control sorghum variety that does not comprise the introduced genetic modification.

2. Genome-edited sorghum variety according to claim 1, characterized in that the encoded modified ACCase polypeptide comprises SEQ ID NO:

4.

3. Genome-edited sorghum variety according to any one of claims 1 to 2, characterized in that the sorghum variety is an elite sorghum variety.

4. Genome-edited sorghum variety according to claim 3, characterized in that the elite sorghum variety is an elite inbred variety.

5. Genome-edited sorghum variety according to claim 3, characterized in that the elite sorghum variety is an elite hybrid variety.

6. Genome-edited sorghum variety according to any one of claims 1 to 5, characterized in that the genome-edited sorghum variety has a yield that is greater than or equal to the yield of the control sorghum variety when grown under the same environmental conditions.

7. Genome-edited sorghum variety according to claim 5, characterized in that the genome-edited elite hybrid sorghum variety has a yield of at least 5,961 kg per hectare (95 bushels per acre) when grown in the presence of at least one ACCase-inhibiting herbicide.

8. Genome-edited sorghum variety according to any one of claims 1 to 7, characterized in that at least one ACCase inhibitor is quizalofop-p-ethyl.

9. Genome-edited sorghum variety according to any one of claims 1 to 8, characterized in that the introduced genetic modification is present in a single allele of the ACCase gene.

10. Genome-edited sorghum variety according to any one of claims 1 to 8, characterized in that the introduced genetic modification is present in both alleles of the ACCase gene.

11. Genome-edited sorghum variety according to any one of claims 1 to 10, characterized in that the edited sorghum variety has tolerance to the herbicide quizalofop-p-ethyl when applied at a rate of at least 0.02268 kg of active ingredient per hectare (0.05 pounds per acre) (kg / ha; lb ai / A).

12. Seed produced by a genome-edited sorghum variety, as defined in any one of claims 1 to 11, wherein the seed is characterized by the fact that it comprises the introduced genetic modification.

13. Genome-edited sorghum hybrid characterized in that it comprises a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2, the edited sorghum hybrid having greater tolerance to at least one ACCase-inhibiting herbicide compared to a control sorghum hybrid that does not comprise the introduced genetic modification.

14. Genome-edited sorghum hybrid according to claim 13, characterized in that the encoded modified ACCase polypeptide comprises SEQ ID NO:

4.

15. Genome-edited sorghum hybrid according to any one of claims 13 to 14, characterized in that the sorghum hybrid is an elite sorghum hybrid.

16. Genome-edited sorghum hybrid, according to any one of claims 13 to 15, characterized in that the yield is greater than or equal to the yield of the control sorghum hybrid when grown under the same environmental conditions.

17. Genome-edited sorghum hybrid according to any one of claims 13 to 16, characterized in that the yield of the edited sorghum hybrid is at least 5,961 kg per hectare (95 bushels per acre) when grown in the presence of at least one ACCase-inhibiting herbicide. Petition 870250087606, dated 09 / 26 / 2025, pp. 85 / 96 4 / 12 18. Genome-edited sorghum hybrid, according to any one of claims 13 to 17, characterized in that the introduced genetic modification is present in a single allele of the ACCase gene.

19. Genome-edited sorghum hybrid according to any one of claims 13 to 17, characterized in that the introduced genetic modification is present in both alleles of the ACCase gene.

20. Genome-edited sorghum hybrid according to any one of claims 13 to 19, characterized in that the edited sorghum hybrid has tolerance to the herbicide quizalofop-p-ethyl when applied at a rate of at least 0.02268 kg of active ingredient per hectare (0.05 pounds per acre) (kg / ha; lb ai / A).

21. Seed produced by a genome-edited sorghum hybrid, as defined in any one of claims 13 to 20, wherein the seed is characterized in that it comprises the introduced genetic modification.

22. Method for producing an ACCase inhibitor-tolerant sorghum hybrid, wherein the method is characterized in that it comprises crossing a first sorghum variety comprising a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2, with a second sorghum variety.

23. Method, according to claim 22, characterized in that the genetic modification introduced is present in a single allele of the ACCase gene in the first variety of sorghum.

24. Method according to claim 22, characterized in that the genetic modification introduced is present in both alleles of the ACCase gene in the first variety of sorghum.

25. Method, according to any one of claims 22 to 24, characterized in that the second variety of sorghum comprises a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2 in an allele.

26. Method, according to any one of claims 22 to 24, characterized in that the second sorghum variety comprises a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2 in both alleles.

27. A method, according to any one of claims 22 to 24, characterized in that the second variety of sorghum does not comprise the genetic modification introduced.

28. A method according to any one of claims 22 to 27, characterized in that the variety produced is an elite hybrid variety.

29. Method, according to any one of claims 22 to 28, characterized in that the hybrid produced is heterozygous for the modified ACCase gene.

30. A method, according to any one of claims 22 to 28, characterized in that the hybrid produced is homozygous for the modified ACCase gene.

31. Method, according to any one of claims 22 to 30, wherein the method is characterized in that it further comprises collecting the seeds of a progeny population produced from the crossing and cultivating the collected seeds.

32. Method for producing a variety of sorghum tolerant to ACCase inhibitor, wherein the method is characterized in that it comprises: a. introducing into a regenerable sorghum plant cell a genetic modification in an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2; and b. generating a sorghum plant from the plant cell, wherein the generated plant comprises the introduced genetic modification and has improved tolerance to ACCase inhibitor herbicides compared to a control plant.

33. Method according to claim 32, characterized in that the genetic modification is introduced using a genome modification technique selected from the group consisting of a polynucleotide-guided endonuclease, CRISPR-Cas endonucleases, base-editing deaminases, a zinc finger nuclease, a transcription activator-like effector nuclease (TALEN), and genetically engineered site-specific endonucleases.

34. Plant characterized in that it is produced by the method as defined in any one of claims 32 to 33.

35. Method for producing a variety of sorghum tolerant to ACCase inhibitor, wherein the method is characterized in that it comprises: a. providing a plant cell comprising an ACCase gene sequence with a guide RNA, a polynucleotide modification template comprising at least one nucleotide modification of the ACCase gene sequence to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprising a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2, and a Cas endonuclease, the guide RNA and the Cas endonuclease being capable of forming a complex that enables the Cas endonuclease to introduce a double-strand break at a target site sequence in the ACCase gene of said plant cell; b. obtaining a plant from the plant cell of (a); c. evaluating the plant of (b) for the presence of said at least one nucleotide modification; and d.Select a parent sorghum plant that carries the modified ACCase gene and has greater tolerance to ACCase-inhibiting herbicides compared to a control plant that does not carry the modification.

36. Method according to claim 35, characterized in that the parent plant comprises the modification in a single allele of the ACCase gene. Petition 870250087606, dated 09 / 26 / 2025, pp. 89 / 96 8 / 12 37. Method according to claim 35, characterized in that the parent plant comprises the modification in both alleles of the ACCase gene.

38. Method, according to any one of claims 35 to 37, characterized in that the target site sequence in the ACCase gene comprises a nucleotide sequence that is at least 90% identical to any one of the SEQ ID NOS: 5, 6, 7 or 12.

39. Method, according to any one of claims 35 to 38, characterized in that the guide RNA comprises a crRNA-tracrRNA fusion transcript.

40. Method according to claim 39, characterized in that the tracrRNA comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

13.

41. Method, according to any one of claims 39 to 40, characterized in that the crRNA comprises a nucleotide sequence that is at least 90% identical to any one of the SEQ ID NOS: 8, 9 or 10.

42. Method, according to any one of claims 35 to 41, characterized in that the polynucleotide modification template comprises a nucleotide sequence that is at least 90% identical to SEQ ID NO:

14.

43. Method, according to any one of claims 35 to 42, characterized in that it further comprises crossing the selected parent sorghum plant with a second sorghum plant to produce an F1 parent plant. Petition 870250087606, dated 09 / 26 / 2025, pp. 90 / 96 9 / 12 44. Method according to claim 43, characterized in that the second sorghum plant comprises an ACCase polynucleotide encoding a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO:

2.

45. Plant produced by the method as defined in any one of claims 35 to 44, wherein the plant is characterized in that it comprises the modified ACCase gene and has greater tolerance to ACCase-inhibiting herbicides compared with a control plant that does not comprise the modification.

46. ​​Plant according to claim 45, wherein the plant is characterized in that it further comprises a yield that is greater than or equal to the yield of the control sorghum hybrid when grown under the same environmental conditions.

47. Method for introgression of a modified ACCase allele into an elite sorghum variety, wherein the method is characterized in that it comprises: a. crossing a first sorghum variety with a second sorghum variety to produce a parent population, wherein the first sorghum variety comprises a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2; b. genotyping the parent population for the presence of the introduced genetic modification; and Petition 870250087606, dated 09 / 26 / 2025, pp. 91 / 96 10 / 12 c. Select progeny that compares the introduced genetic modification to obtain soybean plants that include the modified ACCase allele and that have better tolerance to ACCase-inhibiting herbicides.

48. Method according to claim 47, characterized in that the selected progeny comprises a single ACCase allele with the introduced genetic modification.

49. Method according to claim 48, characterized in that the selected progeny comprises the genetic modification introduced in both ACCase alleles.

50. A method for controlling weeds in a cultivated area, wherein the method is characterized in that it comprises: a. planting a cultivated area with seeds and / or plants comprising a genetic modification introduced into an endogenous acetyl-CoA carboxylase (ACCase) gene to encode a modified ACCase polypeptide that is at least 90% identical to SEQ ID NO: 2, and comprises a cysteine ​​at a position corresponding to position 2032 of SEQ ID NO: 2; and b. applying to the seeds and / or plants a sufficient amount of an ACCase-inhibiting herbicide to control the weeds without significantly affecting the seeds and / or plants.

51. Method according to claim 50, characterized in that the herbicide is applied in a pre-emergent application, a post-emergent application, or a combination thereof. Petition 870250087606, dated 09 / 26 / 2025, pp. 92 / 96 11 / 12 52. A method for detecting the genome-edited sorghum variety as defined in any one of claims 1 to 11 or the genome-edited sorghum hybrid variety as defined in any one of claims 13 to 20, wherein the method is characterized in that it comprises: obtaining a nucleic acid sample from said plant tissues; (i) contacting said nucleic acid sample with a polynucleotide comprising a sequence of at least 8 nucleotides that are identical to a contiguous sequence of SEQ ID NO: 1, or complements thereof; subjecting said sample and said polynucleotide to stringent hybridization conditions; and testing said sample for hybridization of said polynucleotide with said DNA; or (ii) contacting said nucleic acid sample with a first and a second PCR primer, wherein said first and second PCR primers each bind specifically to SEQ ID NO: 1;subject said sample to a polymerase chain reaction; and test an amplicon generated between said first and second initiators.

53. Method according to claim 52, characterized in that the first and second initiators comprise SEQ ID NO: 15, 16, 17, 18, or any combination thereof.

54. Genome-edited sorghum variety according to any one of claims 1 to 11, wherein the genome-edited sorghum variety is characterized by the fact that it further comprises at least one additional polynucleotide that confers tolerance to a herbicide.

55. Genome-edited sorghum variety according to claim 54, characterized in that at least one additional polynucleotide confers tolerance to glyphosate, 2,4-D, glufosinate or any combination thereof.

56. Genome-edited sorghum variety according to any one of claims 54 to 55, characterized in that at least one additional polynucleotide confers tolerance to glyphosate, 2,4-D and glufosinate.

57. Genome-edited sorghum variety according to any one of claims 54 to 56, characterized in that at least one additional polynucleotide is introduced into the variety by backcrossing, transformation or genome editing.

58. Genome-edited sorghum hybrid according to any one of claims 13 to 20, characterized in that the genome-edited sorghum variety further comprises at least one additional polynucleotide that confers tolerance to a herbicide.

59. Genome-edited sorghum variety according to claim 58, characterized in that at least one additional polynucleotide confers tolerance to glyphosate, 2,4-D, glufosinate or any combination thereof.

60. Genome-edited sorghum variety according to any one of claims 58 to 59, characterized in that at least one additional polynucleotide confers tolerance to glyphosate, 2,4-D and glufosinate. Petition 870250087606, dated 09 / 26 / 2025, pp. 94 / 96

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