Methods and compositions for identifying and enriching for cells comprising site specific genomic modifications

Patent metadata

Field	Value
Publication number	US-9963710-B2
Application number	US-201514974247-A
Country	US
Kind code	B2
Filing date	Dec 18, 2015
Priority date	Dec 23, 2014
Publication date	May 8, 2018
Grant date	May 8, 2018

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.

First claim

What is claimed is:

1. A method of integrating a transgene into a genomic nuclease cleavage site in a maize genome, comprising introducing into a maize cell:
a) a first nucleic acid molecule comprising a genomic target site, wherein the nucleic acid molecule comprises at least 100 contiguous nucleotides with at least 90% identity to at least 100 contiguous nucleotides of SEQ ID NO:2, 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,
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 cleavage site in the maize genome.

2. The method of claim 1 , wherein the genomic nuclease cleavage site is located within a chromosome interval on chromosome 1 defined by and including base pair (bp) position 38,860,000 to base pair (bp) position 39,015,000 as defined by Maize B73 RefGen_V2.

3. The method of claim 1 , wherein the first nucleic acid molecule and the second nucleic acid molecule are introduced into the maize cell by biolistic nucleic acid delivery.

4. The method of claim 1 , wherein the first nucleic acid molecule and the second nucleic acid molecule are introduced into the maize cell via an Agrobacterium.

5. The method of claim 1 , wherein the first nucleic acid molecule and the second nucleic acid molecule are present on a single nucleic acid construct.

6. The method of claim 1 , wherein the first nucleic acid molecule and the second nucleic acid molecule are present on separate nucleic acid constructs.

7. The method of claim 1 , wherein the first nucleic acid molecule and/or the second nucleic acid molecule are transiently expressed in the maize cell.

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

9. The method of claim 1 , wherein the genomic nuclease cleavage site is located within a maize chromosome that comprises at least 100 contiguous nucleotides with at least 90% identity to SEQ ID NO: 2.

10. The method of claim 1 , wherein the genomic nuclease cleavage site comprises SEQ ID NO: 3-28, SEQ ID NO: 31-34, SEQ ID NO: 40-67, or a complement thereof.

11. The method of claim 1 , wherein the first nucleic acid molecule comprises SEQ ID NO: 38, SEQ ID NO: 39, or a complement thereof.

Assignees

• Syngenta Participations Ag

Inventors

• Chen Zhongying
• Kim Myoung
• Chilton Mary-Dell
• Zhong Heng
• Gu Weining
• Jiang Yaping
• Que Qiudeng

Classifications

• C12N15/8213 Primary

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

• C12N15/8207

  by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers · CPC title

• C12N15/1058

  Directional evolution of libraries, e.g. evolution of libraries is achieved by mutagenesis and screening or selection of mixed population of organisms · CPC title

• C12N15/8205

  Agrobacterium mediated transformation · CPC title