Methods and compositions for generating dominant brachytic alleles using genome editing

A method for producing a mutant allele of an endogenous brachytic2 (Zm.br2) gene, the method comprising:

• (a) generating a double-stranded break (DSB) or nick in the endogenous Zm.br2 gene in a corn cell of an explant using a targeted editing technique;
• and
• (b) selecting a modified corn plant or plant part developed or regenerated from the cell of the explant comprising the mutant allele of the endogenous Zm.br2 gene,
• wherein the mutant allele of the endogenous brachytic2 (br2) gene encodes a truncated Br2 protein that comprises a first transmembrane segment that comprises an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:
• 28 and a second transmembrane segment that comprises an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:
• 29 and does not comprise a Walker A, Q-Loop, ABC Transport, Walker B, D-Loop, or H-Loop motif of a nucleotide binding domain, and wherein the truncated Br2 protein encoded by the mutant allele of the endogenous Zm.br2 gene disrupts the function of a wild-type Br2 protein expressed from an endogenous wild-type Zm.br2 locus, and wherein the mutant allele of the endogenous brachytic2 (br2) gene is dominant or semi-dominant for a shorter plant height and/or improved lodging resistance phenotype or trait relative to an unmodified control plant.

A method for generating a corn plant comprising:

• (a) fertilizing at least one female corn plant with pollen from a male corn plant, wherein said female corn plant or said male corn plant comprises a mutant allele of an endogenous brachytic2 (br2) gene;
• and
• (b) obtaining at least one plant part or seed comprising the mutant allele of the endogenous brachytic2 (br2) gene produced by said fertilizing step (a),
• wherein the mutant allele of the endogenous brachytic2 (br2) gene encodes a truncated Br2 protein that comprises a first transmembrane segment that comprises an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:
• 28 and a second transmembrane segment that comprises an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:
• 29 and does not comprise a Walker A, Q-Loop, ABC Transport, Walker B, D-Loop, or H-Loop motif of a nucleotide binding domain, and wherein the truncated Br2 protein encoded by the mutant allele of the endogenous Zm.br2 gene disrupts the function of a wild-type Br2 protein expressed from an endogenous wild-type Zm.br2 locus, and wherein the mutant allele of the endogenous brachytic2 (br2) gene is dominant or semi-dominant for a shorter plant height and/or improved lodging resistance phenotype or trait relative to an unmodified control plant.

A method for producing a mutant allele of an endogenous brachytic2 (br2) locus or gene, the method comprising:

• (a) generating at least a first double-stranded break (DSB) or nick at or near a first target site and a second DSB or nick at or near a second target site in the endogenous br2 locus or gene in a corn cell using a targeted editing technique;
• and
• (b) identifying at least one corn plant, plant part, plant seed or plant cell developed or regenerated from said corn cell comprising a deletion in the endogenous br2 locus or gene between the first target site and the second target site, wherein the mutant allele of the endogenous brachytic2 (br2) locus or gene encodes a truncated Br2 protein that comprises a first transmembrane segment that comprises an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:
• 28 and a second transmembrane segment that comprises an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:
• 29 and does not comprise a Walker A, Q-Loop, ABC Transport, Walker B, D-Loop, or H-Loop motif of a nucleotide binding domain, and wherein the truncated Br2 protein encoded by the mutant allele of the endogenous Zm.br2 gene disrupts the function of a wild-type Br2 protein expressed from an endogenous wild-type Zm.br2 locus, and wherein the mutant allele of the endogenous brachytic2 (br2) gene is dominant or semi-dominant for a shorter plant height and/or improved lodging resistance phenotype or trait relative to an unmodified control plant.

A method for producing a mutant allele of an endogenous brachytic2 (br2) locus or gene, the method comprising:

• (a) generating a double-stranded break (DSB) or nick at or near a target site in the endogenous br2 locus or gene in a corn cell using a targeted editing technique;
• and
• (b) identifying at least one corn plant, plant part, plant seed or plant cell developed or regenerated from said corn cell comprising a premature stop codon in the coding sequence of the endogenous br2 locus or gene, wherein the premature stop codon in the coding sequence of the endogenous br2 locus or gene results in a truncated Br2 protein that comprises a first transmembrane segment that comprises an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:
• 28 and a second transmembrane segment that comprises an amino acid sequence having at least 95% sequence identity to the sequence of SEQ ID NO:
• 29 and does not comprise a Walker A, Q-Loop, ABC Transport, Walker B, D-Loop, or H-Loop motif of a nucleotide binding domain, and wherein the truncated Br2 protein encoded by the mutant allele of the endogenous Zm.br2 gene disrupts the function of a wild-type Br2 protein expressed from an endogenous wild-type Zm.br2 locus, and wherein the mutant allele of the endogenous brachytic2 (br2) gene is dominant or semi-dominant for a shorter plant height and/or improved lodging resistance phenotype or trait relative to an unmodified control plant.