# US 11,999,946 B2 · Methods for controlling meristem size for crop improvement

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- Number: US 11,999,946 B2
- Document ID: US11999946B2
- Status: Granted
- Kind: B2
- Application number: 17/212,665
- Filed: 2021-03-25
- Published: 2021-09-30
- Granted: 2024-06-04
- Earliest claimed: 2020-03-26
- Assignees: Pairwise Plants Services, Inc.
- Applicants: Pairwise Plants Services, Inc.
- Inventors: Dale Karlson; Devin O'Connor; Nathaniel Graham
- Web page: https://patentgrep.com/patent/US11999946B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- C07K 14/415: from plants
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8261: {with agronomic (input) traits, e.g. crop yield}
- A01H 6/4684: {Zea mays [maize]}
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- Y02A 40/146: Genetically Modified [GMO] plants, e.g. transgenic plants
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- A01H: NEW PLANTS OR {NON-TRANSGENIC} PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

This invention relates to compositions and methods for modifying FACIATED EAR2 (FEA2) genes in plants, optionally to modify meristem size. The invention further relates to plants having increased kernel row number produced using the methods and compositions of the invention.

## Claims

### Claim 1

A corn plant comprising at least one mutation in an endogenous FACIATED EAR2 (FEA2) gene, wherein the at least one mutation is a deletion, the endogenous FEA2 gene comprises a nucleotide sequence having at least 90% sequence identity to the nucleotide sequence SEQ ID NO:72 or SEQ ID NO:73 and/or encodes an amino acid sequence having at least 90% sequence identity to the amino acid sequence SEQ ID NO:74 and the at least one mutation is within a region of the endogenous FEA2 gene, the region comprising a sequence having at least 90% sequence identity to the nucleotide sequence SEQ ID NO:77 or SEQ ID NO:78 and/or encoding a sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO:75 or SEQ ID NO:76, and an ear of the corn plant comprising the deletion comprises an increased kernel row number and a length that is decreased by less than 30% as compared to an ear of a control corn plant not comprising the deletion and grown under the same environmental conditions, wherein the at least one mutation results in a mutated gene comprising the nucleotide sequence SEQ ID NOs: 88, 91, 92, 96, 97, 99, 105-107, 112-114, 120-122, 127, 128, or 153 and/or encoding the amino acid sequence SEQ ID NOs:134, 140-142, 147, 148, 158, 163, 165, 166, 169, 170, 172,178-180, 185, or 186.

### Claim 2

A method for producing a corn plant or part thereof comprising at least one cell having a mutated endogenous FEA2 gene, the method comprising contacting a target site in an endogenous FEA2gene 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 FEA2 gene,
wherein the endogenous FEA2 gene
(a) encodes a sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO:74;
(b) comprises a sequence having at least 90% sequence identity to the nucleotide sequence SEQ ID NO:72 or SEQ ID NO:73;
(c) comprises a sequence having at least 90% sequence identity to the nucleotide sequence SEQ ID NO:77 or SEQ ID NO:78; and/or
(d) encodes a sequence having at least 90% sequence identity to the amino acid sequence SEQ ID NO:75 or SEQ ID NO:76, thereby producing the corn plant or part thereof comprising at least one cell having a mutation in the endogenous FEA2 gene,
wherein the target site is within a region of the endogenous FEA2 gene, the region comprising a sequence having at least 90% sequence identity to the nucleotide sequence SEQ ID NO:77 or SEQ ID NO:78 and/or encoding a sequence having at least 95% sequence identity to the amino acid sequence SEQ ID NO:75 or SEQ ID NO:76, and following cleavage by the nuclease, an edit is made that results in a deletion within the region of the endogenous FEA2 gene of the at least one cell of the corn plant or part thereof, and the corn plant comprising the at least one cell having the deletion produces an ear that comprises an increased kernel row number and a length that is decreased by less than 30% as compared to an ear of a control corn plant not comprising the deletion and grown under the same environmental conditions, and wherein the mutation results in a mutated FEA2 gene comprising the nucleotide sequence SEQ ID NOs:88, 91, 92, 96, 97, 99, 105-107, 112-114, 120-122, 127, 128, or 153 and/or encoding the amino acid sequence SEQ ID NOs:134, 140-142, 147,148,158,163,165, 166, 169, 170, 172, 178-180, 185, or 186.

### Claim 3

A nucleic acid encoding a mutated corn FEA2 protein, wherein the nucleic acid comprises the sequence SEQ ID NOs: 88, 91, 92, 96, 97, 99, 105-107, 112-114, 120-122, 127, 128, or 153 and/or encodes the amino acid sequence SEQ ID NOs:134, 140-142, 147, 148, 158, 163, 165, 166, 169, 170, 172, 178-180, 185 or 186.

### Claim 4

A modified corn FEA2 protein comprising the amino acid sequence SEQ ID NOs: 134, 140-142, 147, 148, 158, 163, 165, 166, 169, 170, 172, 178-180, 185 or 186.