Methods and compositions for generating dominant short stature alleles using genome editing
AssigneeMonsanto Technology LLC
InventorsSivalinganna Manjunath, Linda A. Rymarquis, Thomas L. Slewinski
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Abstract
The present disclosure provides compositions and methods for altering gibberellin (GA) content in corn or other cereal plants. Methods and compositions are also provided for altering the expression of genes related to gibberellin biosynthesis through editing of specific subtypes of GA20 oxidase genes to introduce an antisense sequence or segment into the gene. Modified plant cells and plants having a dominant allele reducing the expression or activity of one or more GA oxidase genes are further provided comprising reduced gibberellin levels and improved characteristics, such as reduced plant height and increased lodging resistance, but without off-types.
Claims44
A modified corn plant, or plant part thereof, comprising a mutant allele of the endogenous GA20 oxidase_3 locus, wherein the mutant allele comprises a DNA segment inserted into the endogenous GA20 oxidase_3 locus, wherein the DNA segment encodes an antisense RNA sequence that is at least 70% complementary to at least 20 consecutive nucleotides of one or more of SEQ ID NOs:
- 1-3 and 5-7, and wherein the mutant allele of the endogenous GA20 oxidase_3 locus produces a RNA transcript comprising the antisense RNA sequence.
A method for producing a mutant allele of the endogenous GA20 oxidase_3 locus, the method comprising:
- a. generating a double-stranded break in the endogenous GA20 oxidase_3 locus in a corn cell using a targeted editing technique;
- and
- b. inserting at the double-stranded break a DNA segment, wherein the DNA segment encodes an antisense RNA sequence that is at least 70% complementary to at least 20, consecutive nucleotides of one or more of SEQ ID NOs:
- 1-3 and 5-7, and wherein the mutant allele of the endogenous GA20 oxidase_3 locus produces a RNA transcript comprising the antisense RNA sequence.
A method for producing a mutant allele of the endogenous GA20 oxidase_3 locus, the method comprising:
- a. generating a first double-stranded break and a second double strand break in the endogenous GA20 oxidase_3 locus in a corn cell using a targeted editing technique;
- and
- b. inserting a DNA segment between the first double-stranded break and the second double-stranded break, wherein the DNA segment encodes an antisense RNA sequence that is consecutive nucleotides of one or more of SEQ ID NOs:
- 1-3 and 5-7, and wherein the mutant allele of the endogenous GA20 oxidase_3 locus produces a RNA transcript comprising the antisense RNA sequence.
A modified corn plant, or plant part thereof, comprising a mutant allele of the endogenous GA20 oxidase_5 locus, wherein the mutant allele comprises a DNA segment inserted into the endogenous GA20 oxidase_5 locus, wherein the DNA segment encodes an antisense RNA sequence that is at least 70% complementary to at least 20 consecutive nucleotides of one or more of SEQ ID NOs:
- 1-3 and 5-7, and wherein the mutant allele of the endogenous GA20 oxidase_5 locus produces a RNA transcript comprising the antisense RNA sequence.
A method for producing a mutant allele of the endogenous GA20 oxidase_5 locus, the method comprising:
- a. generating a double-stranded break (DSB) in the endogenous GA20 oxidase_5 locus in a corn cell using a targeted editing technique;
- and
- b. inserting at the DSB a DNA segment using a targeted editing technique, wherein the DNA segment encodes an antisense RNA sequence that is at least 70% complementary to at least 20 consecutive nucleotides of one or more of SEQ ID NOs:
- 1-3 and 5-7, and wherein the mutant allele of the endogenous GA20 oxidase_5 locus produces a RNA transcript comprising the antisense RNA sequence.
A method for producing a mutant allele of the endogenous GA20 oxidase_5 locus, the method comprising:
- a. generating a first double-stranded break and a second double strand break in the endogenous GA20 oxidase_5 locus in a corn cell using a targeted editing technique;
- and
- b. inserting a DNA segment between the first double-stranded break and the second double-stranded break, wherein the DNA segment encodes an antisense RNA sequence that is consecutive nucleotides of one or more of SEQ ID NOs:
- 1-3 and 5-7, and wherein the mutant allele of the endogenous GA20 oxidase_5 locus produces a RNA transcript comprising the antisense RNA sequence.
A method for generating a corn plant comprising:
- a. fertilizing at least one female corn plant with pollen from a male corn plant, wherein the at least one female corn plant and/or the male corn plant comprise(s) a mutant allele of an endogenous GA20 oxidase locus, wherein the mutant allele comprises a DNA segment inserted into the endogenous GA20 oxidase locus, wherein the DNA segment encodes an antisense RNA sequence that is at least 70% complementary to at least 20 consecutive nucleotides of one or more of SEQ ID NOs:
- 1-3 or 5-7, and wherein the mutant allele of the endogenous GA20 oxidase locus produces a RNA transcript comprising the antisense RNA sequence;
- and
- b. obtaining at least one seed produced by said fertilizing of step (a).
A modified corn plant part, corn cell, or corn tissue comprising a mutant allele of the endogenous GA20 oxidase_3 locus, wherein the mutant allele comprises a DNA segment inserted into the endogenous GA20 oxidase_3 locus, wherein the DNA segment encodes an antisense RNA sequence that is at least 70% complementary to at least 20 consecutive nucleotides of one or more of SEQ ID NOs:
- 1-3 and 5-7, and wherein the mutant allele of the endogenous GA20 oxidase_3 locus produces a RNA transcript comprising the antisense RNA sequence.
A modified corn plant part, corn cell, or corn tissue comprising a mutant allele of the endogenous GA20 oxidase_5 locus, wherein the mutant allele comprises a DNA segment inserted into the endogenous GA20 oxidase_5 locus, wherein the DNA segment encodes an antisense RNA sequence that is at least 70% complementary to at least 20 consecutive nucleotides of one or more of SEQ ID NOs:
- 1-3 and 5-7, and wherein the mutant allele of the endogenous GA20 oxidase_5 locus produces a RNA transcript comprising the antisense RNA sequence.
Dependencies are inferred from the claim text.