# US 2014/0196169 A1 · Methods and means to modify a plant genome

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 2014/0196169 A1
- Document ID: US20140196169A1
- Status: Published application
- Kind: A1
- Application number: 14/201,577
- Filed: 2014-03-07
- Published: 2014-07-10
- Earliest claimed: 2011-08-22
- Assignees: Bayer CropScience AG
- Inventors: Kathleen D'HALLUIN; Jan DITTGEN; Stefan JANSENS; Frank MEULEWAETER; Koen WETERINGS; Hal MOSER; Thomas WILDE; Ruediger HAIN; Udo BICKERS; Linda TROLINDER; Gary HENNIGER
- Web page: https://patentgrep.com/patent/US20140196169A1

## CPC

- C12N 15/8201: {Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation}
- C12N 9/0069: {acting on single donors with incorporation of molecular oxygen, i.e. oxygenases (1.13)}
- C12N 15/01: Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8245: {involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis}
- C12N 15/8247: {involving modified lipid metabolism, e.g. seed oil composition}
- C12N 15/8271: {for stress resistance, e.g. heavy metal resistance}
- C12N 15/8274: {for herbicide resistance}
- C12N 15/8279: {for biotic stress resistance, pathogen resistance, disease resistance}
- C12N 15/8282: {for fungal resistance}
- C12N 15/8286: {for insect resistance}
- C12Y 113/11027: 4-Hydroxyphenylpyruvate dioxygenase (1.13.11.27)
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Y: ENZYMES

## Abstract

Methods and means are provided to modify in a targeted manner the genome of a plant in close proximity to an existing elite event using a double stranded DNA break inducing enzyme. Also provided are plants, in particular cotton plants showing tolerance to a field dose of at least 1× of at least one HPPD inhibitor, and methods for making such plants.

## Claims

### Claim 1

A method for modifying the genome of a plant cell at a predefined site comprising the steps of
a. inducing a double stranded DNA break in the vicinity of or at said predefined site, said double stranded break being induced by the introduction into said cell of a double stranded DNA break inducing (DSBI) enzyme which recognizes a recognition sequence in the vicinity of or at said predefined site;
b. selecting a plant cell wherein said double stranded DNA break has been repaired resulting in a modification in the genome at said preselected site, wherein said modification is selected from
i. a replacement of at least one nucleotide;
ii. a deletion of at least one nucleotide;
iii. an insertion of at least one nucleotide; or
iv. any combination of i.-iii.;
characterized in that said predefined site is located in close proximity to an existing elite event.

### Claim 2 (depends on 1)

The method of claim 1, wherein said event is GHB119.

### Claim 3 (depends on 1, 2)

The method of claim 1 or 2, wherein said recognition sequence is comprised within SEQ ID NO: 3 or SEQ ID NO: 4.

### Claim 4 (depends on 1, 2, 3)

The method of any one of claims 1-3, wherein said recognition sequence comprises the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

### Claim 5

A method for modifying the genome of a plant cell at a predefined site comprising the steps of
a. inducing a double stranded DNA break in the vicinity of or at said predefined site, said double stranded break being induced by the introduction into said cell of a double stranded DNA break inducing (DSBI) enzyme which recognizes a recognition sequence in the vicinity of or at said predefined site;
b. selecting a plant cell wherein said double stranded DNA break has been repaired resulting in a modification in the genome at said preselected site, wherein said modification is selected from
i. a replacement of at least one nucleotide;
ii. a deletion of at least one nucleotide;
iii. an insertion of at least one nucleotide; or
iv. any combination of i.-iii.;
characterized in that said recognition site comprises the nucleotide sequence of SEQ ID No. 1 or SEQ ID No. 2.

### Claim 6 (depends on 1, 2, 3, 4, 5)

The method of any one of claims 1-5, wherein said DSBI enzyme is introduced into said cell by the delivery into said cell of a nucleic acid molecule comprising one or more chimeric genes encoding said endonuclease enzyme.

### Claim 7 (depends on 1, 2, 3, 4, 5, 6)

The method of any one of claims 1-6, wherein said DSBI enzyme is a single chain meganuclease or a pair of meganucleases which recognizes or recognize in concert said predefined site and induces or induce said double stranded break.

### Claim 8 (depends on 7)

The method of claim 7, wherein said meganuclease or pair of meganucleases is/are derived from I-CreI and wherein the following amino acids are present in meganuclease unit 1:
a. S at position 32;
b. Y at position 33;
c. Q at position 38;
d. Q at position 80;
e. S at position 40;
f. Tat position 42;
g. R at position 77;
h. Y at position 68;
i. Q at position 70;
j. H at position 75;
k. T at position 44;
l. I at position 24;
m. Q at position 26;
n. K at position 28;
o. N at position 30.
and wherein the following amino acids are present in meganuclease unit 2:
p. S at position 70;
q. Q at position 44;
r. K at position 24;
s. A at position 26;
t. K at position 28;
u. N at position 30;
v. S at position 32;
w. Y at position 33;
x. Q at position 38;
y. Q at position 80;
z. S at position 40;
aa. T at position 42;
bb. Q at position 77;
cc. Y at position 68.

### Claim 9 (depends on 7, 8)

The method of claim 7 or 8, wherein said meganuclease or pair of meganucleases comprises the amino acid sequence of SEQ ID NO. 6 from amino acid position 11-165 and from position 204-360.

### Claim 10 (depends on 7, 8, 9)

The method of any one of claims 7-9, wherein said meganuclease or pair of meganucleases is encoded by one or more nucleotide sequences which comprises or comprise together the nucleotide sequences of SEQ ID NO. 5 from nucleotide position 3120-3584 and from position 3698-4169.

### Claim 11 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10)

The method of any one of claims 1-10, wherein prior to step b. a repair DNA molecule is delivered into said cell, said repair DNA molecule being used as a template for repair of said double stranded DNA break.

### Claim 12 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11)

The method according to any one of claims 1-11, wherein said repair DNA comprises at least one flanking region comprising a nucleotide sequence having sufficient homology to the upstream or downstream DNA region of said predefined site to allow recombination with said upstream or downstream DNA region.

### Claim 13 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12)

The method according to any one of claims 1-12, wherein said repair DNA comprises two flanking regions located on opposite ends of said repair DNA, one of said flanking regions comprising a nucleotide sequence having sufficient homology to the upstream DNA region of said predefined site, the other flanking region comprising a nucleotide sequence having sufficient homology to the downstream sequence of said predefined site to allow recombination between said flanking nucleotide sequences and said upstream and downstream DNA regions.

### Claim 14 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13)

The method according to any one of claims 1-13, wherein said repair DNA comprises a selectable marker gene.

### Claim 15 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14)

The method according to any one of claims 1-14, wherein said repair DNA comprises a plant expressible gene of interest.

### Claim 16 (depends on 15)

The method according to claim 15, wherein said plant expressible gene of interest is selected from the group of a herbicide tolerance gene, an insect resistance gene, a disease resistance gene, an abiotic stress resistance gene, an enzyme involved in oil biosynthesis, carbohydrate biosynthesis, an enzyme involved in fiber strength or fiber length, an enzyme involved in biosynthesis of secondary metabolites.

### Claim 17 (depends on 16)

The method according to claim 16, wherein said plant expressible gene of interest encodes a protein having HPPD activity.

### Claim 18 (depends on 16, 17)

The method according to claim 16 or 17, wherein said plant expressible gene of interest encodes a protein having HPPD activity, wherein said protein has a tryptophan at a position corresponding to position 336 of SEQ ID NO: 19, wherein said protein provides to said plant tolerance to a field dose of at least 1× of at least one HPPD inhibitor.

### Claim 19 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

The method according to any one of claims 1-18, wherein said plant cell is further regenerated into a plant.

### Claim 20 (depends on 19)

The method according to claim 19, wherein said plant is further crossed with another plant.

### Claim 21

A cotton plant cell, plant part, plant, or seed comprising a chimeric gene comprising
(a) a nucleic acid sequence encoding a protein having HPPD activity, wherein said protein has a tryptophan at a position corresponding to position 336 of SEQ ID NO: 19, wherein said protein provides to said plant tolerance to a field dose of at least 1× of at least one HPPD inhibitor, operably linked to
(b) a plant expressible promoter and optionally
(c) a translational termination and polyadenylation region.

### Claim 22 (depends on 21)

The cotton plant cell, plant part, plant, or seed of claim 21, wherein said protein has at least 95% sequence identity to the amino add sequence of SEQ ID NO: 21.

### Claim 23 (depends on 21, 22)

The cotton plant cell, plant part, plant, or seed of claim 21 or 22, wherein said nucleic acid sequence encoding a protein having HPPD activity is optimized for expression in cotton.

### Claim 24 (depends on 21, 22, 23)

The cotton plant cell, plant part, plant, or seed of any one of claims 21 to 23, wherein said protein comprises the amino acid sequence of SEQ ID NO: 21 or is encoded by a nucleic acid comprising the nucleotide sequence of SEQ ID NO: 20 from nt 949 to nt 2025.

### Claim 25 (depends on 21, 22, 23, 24)

The cotton plant cell, plant part, plant, or seed of any one of claims 21 to 24, which has a tolerance to a field dose of at least 1.5× of said at least one HPPD inhibitor.

### Claim 26 (depends on 21, 22, 23, 24, 25)

The cotton plant cell, plant part, plant, or seed of any one of claims 21 to 25, which has a tolerance to a field dose of at least 2× of said at least one HPPD inhibitor.

### Claim 27 (depends on 21, 22, 23, 24, 25, 26)

The cotton plant cell, plant part, plant, or seed of any one of claims 21 to 26, which has a tolerance to a field dose of at least 4× of said at least one HPPD inhibitor.

### Claim 28 (depends on 21, 22, 23, 24, 25, 26, 27)

The cotton plant cell, plant part, plant, or seed of any one of claims 21 to 27, wherein said at least one HPPD inhibitor is selected from mesotrione, isoxaflutole, topramezone, pyrasulfutole and tembotrione.

### Claim 29 (depends on 28)

The cotton plant cell, plant part, plant, or seed of claim 28, which is tolerant to at least two HPPD inhibitors, preferably at least three HPPD inhibitors, more preferably at least four HPPD inhibitors such as at least 5 or at least 6 HPPD inhibitors.

### Claim 30 (depends on 21, 22, 23, 24, 25, 26, 27, 28, 29)

The cotton plant cell, plant part, plant, or seed of any one of claims 21 to 29, wherein said chimeric gene comprises the nucleic acid sequence of SEQ ID NO: 20 from position 88 to position 2714.

### Claim 31 (depends on 21, 22, 23, 24, 25, 26, 27, 28, 29, 30)

The cotton plant cell, plant part, plant or seed of any one of claims 21 to 30, which comprises at least one further chimeric gene comprising a nucleic acid sequence encoding an enzyme providing to the plant tolerance to a herbicide which is not an HPPD inhibitor or providing tolerance to at least one insect or fungal species.

### Claim 32

A method for obtaining a cotton plant or plant cell tolerant to field dose of at least 1× of at least one HPPD inhibitor, comprising
introducing a chimeric gene comprising:
(a) a nucleic acid sequence encoding a protein having HPPD activity, wherein said protein has a tryptophan at a position corresponding to position 336 of SEQ ID NO: 19, wherein said protein provides to said plant tolerance to a field dose of at least 1× of at least one HPPD inhibitor, operably linked to
(b) a plant expressible promoter and optionally
(c) a translational termination and polyadenylation region.

### Claim 33

A method for controlling weeds in the vicinity of a cotton plant or on a plant field comprising
applying at least one HPPD inhibitor to the vicinity of a cotton plant or to a cotton plant field in a field dose of at least 1×.

### Claim 34 (depends on 32, 33)

The method of claim 32 or 33, wherein said at least one HPPD inhibitor is selected from mesotrione, isoxaflutole, topramezone, pyrasulfutole and tembotrione.

### Claim 35 (depends on 32, 33, 34)

The method of any one of claims 32 to 34, wherein said at least one HPPD inhibitor is applied in a field dose of at least 1.5×, at least 2× or at least 4×.

### Claim 36 (depends on 32, 33, 34, 35)

The method of any one of claims 32 to 35, wherein said at least one HPPD inhibitor is isoxaflutole and mesotrione, wherein said isofluxatole is applied pre-emergence and said mesotrione is applied post-emergence.

### Claim 37 (depends on 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18)

A plant cell comprising a modification at a predefined site of the genome, obtained by the method according to any one of claims 1-18.

### Claim 38 (depends on 19, 37)

A plant, plant part, seed or propagating material thereof, comprising a modification at a predefined site of the genome, obtained by the method according to claim 19 or consisting essentially of the plant cells of claim 37.

### Claim 39 (depends on 38)

A method of growing a plant according to claim 38, comprising the step of applying a chemical to said plant or substrate wherein said plant is grown.

### Claim 40 (depends on 37, 38)

A method for producing a plant comprising a modification at a predefined site of the genome, comprising the step of crossing a plant consisting essentially of the plant cells of claim 37 or a plant according to claim 38 with another plant or with itself and optionally harvesting seeds.

### Claim 41

Use of a DSBI enzyme to modify the genome of a plant cell in the proximity of an elite event.

### Claim 42

Use of a nucleic acid sequence encoding a protein having HPPD activity, wherein said protein has a tryptophan at a position corresponding to position 336 of SEQ ID NO: 19, to produce a cotton plant, or plant cell, plant part or seed thereof, said cotton plant being tolerant to a field dose of at least 1× of at least one HPPD inhibitor.