# US 10,501,748 B2 · Methods and compositions for integration of an exogenous sequence within the genome of plants

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- Number: US 10,501,748 B2
- Document ID: US10501748B2
- Status: Granted
- Kind: B2
- Application number: 14/243,553
- Filed: 2014-04-02
- Published: 2014-10-09
- Granted: 2019-12-10
- Earliest claimed: 2013-04-05
- Assignees: Dow AgroSciences LLC; Sangamo Therapeutics, Inc.
- Applicants: Dow AgroSciences LLC; Sangamo BioSciences, Inc.
- Inventors: W. Michael Ainley; Dmitry Y. Guschin; Matthew Hayden; Daniel Isenegger; John Mason; Jeffrey C. Miller; Joseph F. Petolino; Yidong Ran; Tim Sawbridge; German Spangenberg; Steven R. Webb
- Web page: https://patentgrep.com/patent/US10501748B2

## CPC

- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8274: {for herbicide resistance}
- C12N 15/8278: {Sulfonylurea}
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- 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)
- Y02A: TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE

## Abstract

Disclosed herein are methods and compositions for parallel or sequential transgene stacking in plants to produce plants with selected phenotypes.

## Claims

### Claim 1

A plant cell comprising a targeted genomic modification to one or more alleles of an endogenous acetohydroxyacid synthase (AHAS) gene in the plant cell, wherein:
(i) the endogenous AHAS gene comprises any one of SEQ ID NO:35-56 or 263-278;
(ii) the plant cell comprises a site-specific nuclease that binds to a target site as shown in any of SEQ ID NO:35-56 or 263-278 and cleaves the endogenous AHAS gene; and
(iii) the genomic modification comprises an insertion and/or deletion in the endogenous AHAS sequence of the plant cell within any of SEQ ID NO:37-56 or 263-278;
between SEQ ID NO:35 and 36; between SEQ ID NO:37 and 39; between SEQ ID NO:38 and 39 or 40; between SEQ ID NO:41 and 42; between SEQ ID NO:43 and 45; between SEQ ID NO:44 and 45; between SEQ ID NO:46 and 47; between SEQ ID NO:48 and 29; between SEQ ID NO:50 and 51; between SEQ ID NO:52 or 53 and 54; between SEQ ID NO:55 and 56; between SEQ ID NO:263 and 264; between SEQ ID NO:265 and 266; between SEQ ID NO:267 and 268; between SEQ ID NO:269 and 270; between SEQ ID NO:271 and 272; between SEQ ID NO:273 and 274; between SEQ ID NO:275 and 276; or between SEQ ID NO:277 and 278 of the endogenous AHAS gene following cleavage of the endogenous AHAS gene by the site-specific nuclease.

### Claim 2 (depends on 1)

The plant cell of claim 1, wherein the genomic modification comprises integration of one or more exogenous sequences.

### Claim 3 (depends on 1)

The plant cell of claim 1, wherein the genomic modification comprises introduction of one or more indels that disrupt expression of the endogenous gene.

### Claim 4 (depends on 2)

The plant cell of claim 2, wherein the exogenous sequence does not comprise a sequence comprising a promoter operably linked to a sequence encoding a transgenic selectable marker.

### Claim 5 (depends on 2)

The plant cell of claim 2, wherein the exogenous sequence encodes a protein selected from the group consisting of a protein that increases crop yield, a protein encoding disease resistance, a protein that increases growth, a protein encoding insect resistance, a protein encoding herbicide tolerance, and combinations thereof.

### Claim 6 (depends on 5)

The plant cell of claim 5, wherein the increased crop yield comprises an increase in fruit yield, grain yield, biomass, fruit flesh content, size, dry weight, solids content, weight, color intensity, color uniformity, altered chemical characteristics, or combinations thereof.

### Claim 7 (depends on 1)

The plant cell of claim 1, wherein two or more exogenous sequences are integrated into the endogenous gene.

### Claim 8 (depends on 1)

The plant cell of claim 1, wherein the plant cell is a polyploid plant cell.

### Claim 9 (depends on 1)

The plant cell of claim 1, wherein the site specific nuclease comprises a zinc finger DNA-binding domain, and a FokI cleavage domain.

### Claim 10 (depends on 9)

The plant cell of claim 9, wherein the FokI cleavage domain is a wild-type or engineered cleavage domain.

### Claim 11 (depends on 1)

The plant cell of claim 1, wherein the plant is selected from the group consisting of wheat, soy, maize, potato, alfalfa, rice, barley, sunflower, tomato, Arabidopsis, cotton, Brassica species, and timothy grass.

### Claim 12 (depends on 2)

A plant, plant part, seed, or fruit comprising one or more plant cells of claim 2 or progeny of the plant, plant part, seed or fruit that comprises a plant cell comprising the integrated exogenous sequence.

### Claim 13 (depends on 1)

The plant cell of claim 1, further comprising one or more transgenes integrated into the genome of the plant cell at one or more loci different from the endogenous gene.

### Claim 14 (depends on 1)

A method for making a plant cell according to claim 1, the method comprising:
expressing the site specific nuclease in the plant cell; and
modifying one or more alleles of an endogenous AHAS gene across multiple genomes of a polyploid plant cell.

### Claim 15 (depends on 14)

The method of claim 14, wherein the modification disrupts expression of the endogenous gene.

### Claim 16 (depends on 14)

The method of claim 14, wherein the modification comprises integration of one or more exogenous sequences into one or more alleles of the endogenous gene.