# US 11,898,149 B2 · Methods and compositions for identifying and enriching for cells comprising site specific genomic modifications

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- Number: US 11,898,149 B2
- Document ID: US11898149B2
- Status: Granted
- Kind: B2
- Application number: 18/181,704
- Filed: 2023-03-10
- Published: 2023-08-24
- Granted: 2024-02-13
- Earliest claimed: 2014-12-23
- Assignees: Syngenta Participations AG
- Applicants: Syngenta Participations AG
- Inventors: Zhongying Chen; Myoung Kim; Mary-Dell Chilton; Heng Zhong; Weining Gu; Yaping Jiang; Qiudeng Que
- Web page: https://patentgrep.com/patent/US11898149B2

## CPC

- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/1058: {Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms}
- C12N 15/8205: {Agrobacterium mediated transformation}
- C12N 15/8207: {by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The present invention relates to methods and compositions for modifying a target site in the genome of a plant cell. Such modifications include integration of a transgene and mutations. The present invention also relates to methods and compositions for identifying and enriching for cells which comprise a modified target site.

## Claims

### Claim 1

A method of integrating a transgene into a genomic nuclease cleavage site in an event MIR604 transgenic maize genome, comprising introducing into an event MIR604 maize cell:
a) a first nucleic acid molecule comprising at least 100 contiguous nucleotides, wherein said at least 100 contiguous nucleotides have at least 90% identity with a target site in a nucleotide sequence comprising SEQ ID NO: 135, and further comprising a transgene; and
b) a second nucleic acid molecule comprising a nucleotide sequence encoding a nuclease for site-directed cleavage at a genomic nuclease cleavage site adjacent to a nucleotide sequence with at least 90% identity to a nucleotide sequence comprising SEQ ID NO: 135, that corresponds to the at least 100 contiguous nucleotides of (a),
under conditions wherein expression of the second nucleic acid molecule can occur to produce the nuclease and the nuclease can cleave the nucleotide sequence at the genomic nuclease cleavage site, whereby the transgene is integrated at the genomic nuclease target cleavage site in the event MIR604 maize genome.

### Claim 2 (depends on 1)

A method of producing a maize plant, plant part, or progeny thereof comprising a transgene integrated into a genomic nuclease cleavage site in an event MIR604 maize genome, comprising regenerating a maize plant from the maize cell produced by the method of claim 1.

### Claim 3 (depends on 2)

A maize plant, plant part, or progeny thereof comprising a transgene integrated into a genomic nuclease cleavage site in an event MIR604 maize genome, produced by the method of claim 2.