Methods and compositions for generating dominant brachytic alleles using genome editing
The present disclosure provides compositions and methods for altering auxin accumulation in corn or maize plants. Methods and compositions are also provided for altering the expression of genes related to auxin efflux through editing or mutagenesis of a brachytic2 (br2) gene to introduce a premature stop codon or a deletion into the gene such that a truncated Br2 protein encoded by the mutant allele of the br2 gene, which may be a dominant or semi-dominant allele, has at least part of a transmembrane domain without a nucleotide binding domain or motif. Modified plant, plant parts and cells having such a mutant allele with reduced or altered expression or activity of a br2 gene product can have improved characteristics, such as reduced plant height and increased lodging resistance, but without off-types in the plant.
1 . A modified corn plant, plant part or plant cell comprising a mutant allele of an endogenous brachytic2 (br2) gene encoding a truncated Br2 protein, wherein the truncated Br2 protein encoded by the mutant allele of the endogenous brachytic2 (br2) gene 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.
2 . The modified corn plant, plant part or plant cell of claim 1 , wherein:
a) the transmembrane domain of the truncated Br2 protein comprises three transmembrane segments, four transmembrane segments, five transmembrane segments, or six transmembrane segments of the first transmembrane domain;
b) the truncated Br2 protein comprises 3-6 of the first transmembrane domain of a wild-type Zm.Br2 protein or one or more of transmembrane segments 7-12 of a second transmembrane domain of a wild-type Zm.Br2 protein;
c) the truncated Br2 protein comprises a transmembrane domain that comprises amino acids 137-421 of SEQ ID NO: 3;
d) the truncated Br2 protein further comprises a transmembrane domain comprising:
a third transmembrane region that comprises the sequence of SEQ ID NO: 30
a fourth transmembrane region that comprises the sequence of SEQ ID NO:31,
a fifth transmembrane region that comprises the sequence of SEQ ID NO: 32, or
a sixth transmembrane region that comprises the sequence of SEQ ID NO: 33,
or any combination thereof;
e) the truncated Br2 protein encoded by the mutant allele of the endogenous brachytic2 (br2) gene further comprises a N-terminal region;
f) the mutant allele of the endogenous Zm.br2 gene comprises a premature stop codon;
g) the mutant allele of the endogenous Zm.br2 gene is made using a mutagenesis or targeted editing technique;
h) the modified corn plant has a shorter plant height and/or an improved lodging resistance phenotype relative to an unmodified control plant;
i) the modified corn plant does not have any significant off-types in at least one female organ or ear;
j) the modified corn plant exhibits essentially no reproductive abnormality;
k) the modified corn plant, plant part or plant cell is homozygous for the mutant allele of the endogenous Zm.br2 gene; or
l) the modified corn plant, plant part or plant cell is heterozygous for the mutant allele of the endogenous Zm.br2 gene.
3 . The modified corn plant, plant part or plant cell of claim 2 , wherein:
a) the transmembrane domain of the truncated Br2 protein comprises a polypeptide sequence that comprises the sequence of amino acids 137-197 of SEQ ID NO: 3, amino acids 137-283 of SEQ ID NO: 3, amino acids 137-307 of SEQ ID NO: 3, amino acids 137-392 of SEQ ID NO: 3, amino acids 137-421 of SEQ ID NO: 3, amino acids 180-283 of SEQ ID NO: 3, amino acids 180-307 of SEQ ID NO: 3, amino acids 180-392 of SEQ ID NO: 3, amino acids 180-421 of SEQ ID NO: 3, amino acids 264-307 of SEQ ID NO: 3, amino acids 264-392 of SEQ ID NO: 3, amino acids 264-421 of SEQ ID NO: 3, amino acids 285-392 of SEQ ID NO: 3, amino acids 285-421 of SEQ ID NO: 3, or amino acids 370-421 of SEQ ID NO: 3;
b) the premature stop codon is present in exon 2 or exon 3 of the mutant allele of the endogenous Zm.br2 gene; or
c) the premature stop codon is present within a genomic sequence of the mutant allele of the endogenous Zm.br2 gene corresponding to nucleotides 1477-2717 or 1477-2609 of SEQ ID NO: 1, nucleotides 1591-2717 or 1591-2609 of SEQ ID NO: 1, nucleotides 1591-2717 or 1591-2609 of SEQ ID NO: 1, nucleotides 1992-2717 or 1992-2609 of SEQ ID NO: 1, nucleotides 2064-2717 or 2064-2609 of SEQ ID NO: 1, 2319-2717 or 2319-2609 of SEQ ID NO: 1, or nucleotides 2495-2717 or 2495-2609 of SEQ ID NO: 1.
4 . The modified corn plant, plant part or plant cell of claim 3 , wherein the truncated Br2 protein comprises a polypeptide sequence that comprises the sequence of amino acids 137-197, 137-283, 137-307, 137-392, or 137-421 of SEQ ID NO: 3.
5 . The modified corn plant, plant part or plant cell of claim 2 , wherein:
a) the mutant allele of the endogenous Zm.br2 gene comprises a deletion of most or all of the genomic sequence of the endogenous Zm.br2 gene encoding a nucleotide binding domain or a deletion of all genomic sequences of the endogenous Zm.br2 gene encoding nucleotide binding domain motifs of the nucleotide binding domain;
b) the mutant allele of the endogenous Zm.br2 gene comprises a deletion of (i) most or all of the genomic sequence of the endogenous Zm.br2 gene encoding a first nucleotide binding domain or all genomic sequences of the endogenous Zm.br2 gene encoding nucleotide binding domain motifs of the first nucleotide binding domain, and (ii) most or all of the genomic sequence of the endogenous Zm.br2 gene encoding a second nucleotide binding domain or all genomic sequences of the endogenous Zm.br2 gene encoding nucleotide binding domain motifs of the second nucleotide binding domain;
c) the mutant allele of the endogenous Zm.br2 gene further comprises a deletion of all or part of the one or more genomic sequences of the endogenous Zm.br2 gene encoding the second transmembrane domain, and/or the C-terminal region of the Zm.Br2 protein;
d) the mutant allele of the endogenous Zm.br2 gene comprises a deletion of all or part of exon 2, intron 2, exon 3, intron 3, exon 4, intron 4, and/or exon 5 of the endogenous Zm.br2 gene; or
e) the truncated Br2 protein has an amino acid length within a range of 175 amino acids and 495 amino acids.
6 . 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.
7 . The method of claim 6 , wherein:
a) the targeted editing technique comprises the use of at least one site-specific nuclease;
b) the mutant allele of the endogenous Zm.br2 gene comprises a premature stop codon;
c) the mutant allele of the endogenous Zm.br2 gene comprises a deletion of one or more nucleotides within the coding region of the endogenous Zm.br2 gene; or
d) the target site for introducing the double-stranded break (DSB) or nick in the endogenous br2 gene in a corn cell is downstream of a genomic sequence encoding the first transmembrane segment and the second transmembrane segment of a Zm.Br2 protein encoded by the endogenous br2 gene and upstream of a genomic sequence encoding a first nucleotide binding domain or a Walker A motif of the first nucleotide binding domain of the Zm.Br2 protein.
8 . The method of claim 7 , wherein:
a) the at least one site-specific nuclease is selected from the group consisting of a zinc-finger nuclease, a meganuclease, an RNA-guided nuclease, a TALE-nuclease, a recombinase, a transposase, and any combination thereof; or
b) the at least one site-specific nuclease is a RNA-guided nuclease selected from the group consisting of a Cas9 nuclease or a variant thereof and a Cpf1 nuclease or a variant thereof.
9 . The method of claim 6 , further comprising:
(c) regenerating or developing a corn plant or plant part from the corn cell.
10 . The method of claim 6 , wherein the selecting step (b) comprises selecting a modified corn plant having a shorter plant height and/or an improved lodging resistance phenotype or trait relative to an unmodified control plant.
11 . 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.
12 . The method of claim 11 , wherein said method further comprises:
(c) growing or developing at least one progeny corn plant comprising the mutant allele from said at least one seed obtained in step (b).
13 . The method of claim 12 , wherein:
a) said at least one progeny corn plant is heterozygous for said mutant allele;
b) said at least one progeny corn plant has a shorter plant height and/or improved lodging resistance relative to a control plant that does not comprise said mutant allele; or
c) said at least one progeny corn plant has a shorter plant height and/or improved lodging resistance relative to said male corn plant and/or said female corn plant.
14 . The method of claim 11 , wherein:
a) said female corn plant does not comprise said mutant allele;
b) said female corn plant is homozygous for said mutant allele;
c) said female corn plant is heterozygous for said mutant allele;
d) said male corn plant does not comprise said mutant allele;
e) said male corn plant is homozygous for said mutant allele;
f) said male corn plant is heterozygous for said mutant allele;
g) said female corn plant is an inbred corn plant or a hybrid corn plant;
h) said male corn plant is an inbred corn plant or a hybrid corn plant;
i) said female corn plant and/or said male corn plant is an elite corn plant;
j) said female corn plant and said male corn plant are grown in a greenhouse or growth chamber;
k) said female corn plant and said male corn plant are grown outdoors or in the field;
l) said female corn plant is a first inbred corn line or variety and said male corn plant is a second inbred corn line or variety, and wherein said first inbred corn line or variety and said second inbred corn line or variety are different and genetically distinct; or
m) said female corn plant has been detasseled or is a cytoplasmically male sterile corn plant.
15 . 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.
16 . 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.
17 . The method of claim 16 , wherein the method further comprises providing to the at least one corn cell a donor template comprising the premature stop codon.
18 . The method of claim 17 , wherein the donor template further comprises at least one homology arm to direct the integration of a mutation at or near the target site in the endogenous br2 locus.
19 . The method of claim 15 , wherein the method further comprises developing or regenerating at least one corn plant or plant part from the at least one corn cell identified in step (b).
20 . The method of claim 16 , wherein the method further comprises developing or regenerating at least one corn plant or plant part from the at least one corn cell identified in step (b).