# US 11,111,500 B2 · Mutated protoporphyrinogen IX oxidase (PPX) genes

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- Number: US 11,111,500 B2
- Document ID: US11111500B2
- Status: Granted
- Kind: B2
- Application number: 16/042,985
- Filed: 2018-07-23
- Published: 2018-11-15
- Granted: 2021-09-07
- Earliest claimed: 2010-08-03
- Assignees: Cibus US LLC; Cibus Europe B.V.
- Applicants: Cibus US LLC; Cibus Europe B.V.
- Inventors: Gregory F. W. Gocal; Peter R. Beetham; Aura Estela Gonzalez Schopke; Sarah Dumm; James Pearce; Christian Schopke; Keith A. Walker
- Web page: https://patentgrep.com/patent/US11111500B2

## CPC

- C12N 15/8274: {for herbicide resistance}
- C12N 9/001: {acting on the CH-CH group of donors (1.3)}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

Provided are compositions and methods relating to gene and/or protein mutations in transgenic or non-transgenic plants. In certain embodiments, the disclosure relates to mutations in the protoporphyrinogen IX (PPX) gene. In some embodiments the disclosure relates to plants that are herbicide resistant.

## Claims

### Claim 1

A non-transgenic plant cell, resistant to one or more PPX-inhibiting herbicides, comprising a mutated protoporphyrinogen IX oxidase (PPX) gene, wherein said gene encodes a protein comprising a mutation at an amino acid position corresponding to position 426 of SEQ ID NO: 1, wherein the mutation is a Tyr to Phe substitution.

### Claim 2

A non-transgenic plant, resistant to one or more PPX-inhibiting herbicides, comprising a mutated protoporphyrinogen IX oxidase (PPX) gene, wherein said gene encodes a protein comprising a mutation at an amino acid position corresponding to position 426 of SEQ ID NO: 1, wherein the mutation is a Tyr to Phe substitution.

### Claim 3 (depends on 2)

The non-transgenic plant of claim 2, wherein said plant is selected from the group consisting of potato, sunflower, sugar beet, maize, cotton, soybean, wheat, rye, oats, rice, canola, tobacco, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, carrot, lettuce, onion, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, flax, oilseed rape, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, petunia, tulip, iris, and lily.

### Claim 4

A method for producing a non-transgenic plant cell with a mutated PPX gene, comprising introducing into a plant cell a gene repair oligonucleobase (GRON) encoding a mismatch with a protoporphyrinogen IX oxidase (PPX) gene of the plant cell under conditions that introduce a targeted mutation in the PPX gene resulting from the mismatch, to thereby produce a plant cell that expresses a PPX protein from the PPX gene comprising a mutation at an amino acid position corresponding to position of SEQ ID NO: 1, wherein the mutation is a Tyr to Phe substitution.

### Claim 5 (depends on 4)

The method of claim 4, wherein said plant cell is of a plant selected from the group consisting of potato, sunflower, sugar beet, maize, cotton, soybean, wheat, rye, oats, rice, canola, tobacco, barley, sorghum, tomato, mango, peach, apple, pear, strawberry, banana, melon, carrot, lettuce, onion, sugar cane, pea, field beans, poplar, grape, citrus, alfalfa, rye, oats, flax, oilseed rape, cucumber, morning glory, balsam, pepper, eggplant, marigold, lotus, cabbage, daisy, carnation, petunia, tulip, iris, and lily.

### Claim 6 (depends on 4)

The method of claim 4, wherein said plant is resistant to one or more PPX-inhibiting herbicides.

### Claim 7 (depends on 4)

The method of claim 4, further comprising:
identifying a plant cell comprising the Tyr to Phe substitution and having substantially normal growth and catalytic activity as compared to a corresponding wild-type plant cell in the presence of an herbicide; and
regenerating a non-transgenic herbicide-resistant plant having a mutated PPX gene from said plant cell.

### Claim 8 (depends on 6)

The method of claim 6, wherein the herbicide is a PPX-inhibiting herbicide.