Methods and compositions for modifying cytokinin receptor histidine kinase genes in plants

Patent metadata

Field	Value
Publication number	US-12553061-B2
Application number	US-202217819975-A
Country	US
Kind code	B2
Filing date	Aug 16, 2022
Priority date	Aug 17, 2021
Publication date	Feb 17, 2026
Grant date	Feb 17, 2026

Abstract

This invention relates to compositions and methods for modifying Cytokinin Receptor Histidine Kinase (HK) genes in plants, optionally to improve yield traits, the HK genes encoding a histidine kinase (HK) polypeptide. The invention further relates to plants having increased improved yield traits produced using the methods and compositions of the invention.

First claim

That which is claimed is:

1 . A corn plant or plant part thereof comprising at least one mutation in an endogenous Cytokinin Receptor Histidine Kinase (HK) gene encoding a histidine kinase (HK) polypeptide, wherein the encoded HK polypeptide comprises an extracellular cytokinin binding domain,
wherein the encoded HK polypeptide has at least 85% sequence identity to SEQ ID NO:189, and wherein the at least one mutation is a deletion of about 1 base pair to about 27 consecutive base pairs or wherein the at least one mutation results in a substitution of any one or more amino acid residues located in a region having at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs: 183-186 or 210-211.

2 . The corn plant or plant part thereof of claim 1 , wherein the at least one mutation is the deletion of about 1 base pair to about 27 consecutive base pairs.

3 . The corn plant or plant part thereof of claim 1 , wherein the endogenous HK gene comprises a nucleotide sequence having at least 85% sequence identity to SEQ ID NO:187 or 188.

4 . The corn plant or plant part thereof of claim 1 , wherein the substitution is located at position 160 and/or 161 with reference to amino acid position numbering of SEQ ID NO: 189.

5 . The corn plant or plant part thereof of claim 4 , wherein the substitution is threonine (T) to isoleucine (I) (T>I), glutamic acid (E) to lysine (K) (E>K), leucine (L) to phenylalanine (F) (L>F), proline (P) to leucine (L) (P>L), and/or serine(S) to leucine (L) (S>L).

6 . The corn plant or plant part thereof of claim 1 , wherein the corn plant comprising the at least one mutation has a phenotype of one or more improved yield traits as compared to a corn plant that is devoid of the at least one mutation.

7 . The corn plant or plant part thereof of claim 1 , wherein the at least one mutation results in a mutated HK gene comprising a nucleotide sequence having at least 90% sequence identity to SEQ ID NO:247, SEQ ID NO:248, or SEQ ID NO:258.

8 . A corn plant cell comprising at least one mutation within an endogenous Cytokinin Receptor Histidine Kinase (HK) gene, wherein the at least one mutation is a substitution or deletion that is introduced using an editing system that comprises a nucleic acid binding domain that binds to a target site in the endogenous HK gene, wherein the target site is within a region of the endogenous HK gene (a) having at least 95% sequence identity to any one of the nucleotide sequences of SEQ ID NOs:190-206 and/or (b) encoding an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs:183-186 or 207-211,
wherein the at least one mutation is a deletion of about 1 base pair to about 27 consecutive base pairs or wherein the at least one mutation results in a substitution of any one or more amino acid residues located in a region having at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs:183-186 or 210-211, and wherein the at least one mutation results in a mutated HK gene comprising a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs:247-259.

9 . The corn plant cell of claim 8 , wherein the editing system further comprises a nuclease, wherein the target site is within a sequence having least 80% sequence identity to a sequence encoding of any one of SEQ ID NO:187 or a sequence having at least 80% sequence identity to a sequence encoding any one of SEQ ID NOs:190-206, and wherein the at least one mutation is made following cleavage by the nuclease.

10 . The corn plant cell of claim 8 , wherein the at least one mutation results in the substitution of any one or more amino acid residues located in a region having at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs:183-186 or 210-211.

11 . The corn plant cell of claim 10 , wherein the substitution is an amino acid substitution of S160 and/or L161 with reference to amino acid position numbering of SEQ ID NO:189.

12 . A method for producing a corn plant or plant part thereof comprising at least one cell having a mutated endogenous Cytokinin Receptor Histidine Kinase (HK) gene, the method comprising:
contacting a target site in an endogenous HK gene in the corn plant or plant part with a nuclease comprising a cleavage domain and a nucleic acid binding domain, wherein the nucleic acid binding domain binds to a target site in the endogenous HK gene, wherein the endogenous HK gene: (a) comprises a sequence having at least 95% sequence identity to a nucleotide sequence of any one of SEQ ID NOs:187 or 188; (b) comprises a region having at least 95% identity to any one of SEQ ID NOs: 190-206; (c) encodes an amino acid sequence having at least 95% sequence identity to any one of SEQ ID NOs:189; and/or (d) encodes an amino acid sequence comprising a region having at least 95% identity to any one of SEQ ID NOs:207-211, thereby producing the corn plant or plant part thereof comprising at least one cell having a mutation in the endogenous HK gene, wherein the mutation is a deletion of about 1 base pair to about 27 consecutive base pairs or wherein the mutation results in a substitution of any one or more amino acid residues located in a region having at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs:183-186 or 210-211, and wherein the mutated endogenous HK gene comprises a nucleotide sequence having at least 95% sequence identity to any one of SEQ ID NOs:247-259.

13 . The method of claim 12 , wherein the mutation is the substitution of any one or more amino acid residues located in a region having at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs:183-186 or 210-211.

14 . The method of claim 12 , wherein the mutation is the deletion of about 1 base pair to about 27 consecutive base pairs.

15 . The method of claim 12 , wherein the mutation results in a modified amino acid residue located in a region having at least 80% sequence identity to any one of the amino acid sequences of SEQ ID NOs:183-186 or 207-211.

16 . The corn plant or plant part thereof of claim 1 , wherein the at least one mutation results in a mutation in the encoded HK polypeptide that is in or adjacent to the extracellular cytokinin binding domain of the HK polypeptide.

17 . A corn plant or plant part thereof comprising at least one mutation in an endogenous Cytokinin Receptor Histidine Kinase (HK) gene encoding a histidine kinase (HK) polypeptide, wherein the encoded HK polypeptide comprises an extracellular cytokinin binding domain,
wherein the endogenous HK gene comprises a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NOs:159, 160, 187, or 188 and encodes a HK polypeptide having at least 85% sequence identity to SEQ ID NO:161 or SEQ ID NO:189, and wherein the at least one mutation is a deletion of about 1 base pair to about 27 consecutive base pairs or wherein the at least one mutation results in a substitution of any one or more amino acid residues located in a region having at least 85% sequence identity to any one of the amino acid sequences of SEQ ID NOs:180, 181, 183-186 or 210-211.

Assignees

• Pairwise Plants Services Inc

Inventors

• Mathew Lolita George
• Zhang Xiaoyu
• O'Connor Devin

Classifications

• C12N15/8213

  Targeted insertion of genes into the plant genome by homologous recombination · CPC title

• C12N15/11

  DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity} · CPC title

• C12N9/22

  Ribonucleases {[RNase]; Deoxyribonucleases [DNase]} · CPC title

• C12N2310/20

  involving clustered regularly interspaced short palindromic repeats [CRISPR] · CPC title

• Y02A40/146

  Genetically Modified [GMO] plants, e.g. transgenic plants · CPC title