# US 12,215,363 B2 · Balanced indels

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- Number: US 12,215,363 B2
- Document ID: US12215363B2
- Status: Granted
- Kind: B2
- Application number: 16/811,393
- Filed: 2020-03-06
- Published: 2020-08-27
- Granted: 2025-02-04
- Earliest claimed: 2017-09-08
- Assignees: Keygene N.V.
- Applicants: Keygene N.V.
- Inventors: Paul Bundock
- Web page: https://patentgrep.com/patent/US12215363B2

## CPC

- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/102: {Mutagenizing nucleic acids}
- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C12N 15/8274: {for herbicide resistance}
- C12N 15/90: Stable introduction of foreign DNA into chromosome
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/22: Vectors comprising a coding region that has been codon optimised for expression in a respective host
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

The invention pertains to the targeted alteration of a duplex DNA in a cell, whereby two site-specific nucleases generate an indel, such that the open reading frame is not altered after the second indel. The invention further pertains to the use of such nucleases for the targeted alteration of an open reading frame in duplex DNA and a kit of parts for use in a method of the invention. Using the method of the invention, novel plants were obtained having an improved herbicide resistance. The invention therefore also concerns plants having improved herbicide resistance due to the expression of an altered ALS protein.

## Claims

### Claim 1

A method for producing a protein having a gain-of-function by targeted alteration of a coding sequence (CDS) in between target sites wherein nontargeted CDS is unaltered in duplex DNA in a cell, comprising
exposing the duplex DNA to at least two site-specific nucleases, wherein a first site-specific nuclease targets the DNA at a first position within the CDS and cleaves the DNA generating a first indel at a first location within the CDS, and wherein a second site-specific nuclease targets the DNA at a second position within the CDS and cleaves the DNA generating a second indel at a second location within the same CDS, wherein the first and second indel are frame shift mutations, resulting in an altered part of the CDS, wherein the altered part of the CDS has a length between 2-15 codons, wherein the CDS before the first indel and after the second indel remain in the same reading frame, wherein the altered part of the CDS starts with the first indel up to and including the second indel, wherein the altered part of the CDS does not comprise a stop codon; and
selecting a cell comprising the targeted alteration,
wherein the CDS comprising the targeted alteration encodes the protein having a gain of function, and
wherein the protein having a gain-of-function is produced with a higher efficiency than with an identical method using a single site-specific nuclease.

### Claim 2 (depends on 1)

The method according to claim 1, wherein the CDS is altered by introducing or deleting at least one nucleotide at the first location and by introducing or deleting at least one nucleotide at the second location, wherein the total of introduced nucleotides preferably is 0, 3, 6, 9 or 12 and/or wherein the total of deleted nucleotides preferably is 0, 3, 6, 9 or 12.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the altered part of the CDS has a length between 2-10 or 2-5 codons.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the altered part of the CDS has a length of 2, 3, 4, 5, 6, 7, 8, 9 or 10 codons.

### Claim 5 (depends on 1)

The method according to claim 1, wherein at least one of the nucleases is a CRISPR nuclease and wherein the method further comprises exposing the duplex DNA to:
(i) a first guide RNA that comprises a first guide sequence for targeting the first nuclease to the first location in the duplex DNA; and/or
(ii) a second guide RNA that comprises a second guide sequence for targeting the second nuclease to the second location in the duplex DNA.

### Claim 6 (depends on 5)

The method according to claim 5, wherein the at least one CRISPR nuclease is Cas9 or Cpf1.

### Claim 7 (depends on 1)

The method according to claim 1, wherein at least one of the nucleases is selected from the group consisting of a zinc finger nuclease, a meganuclease and a TALEN.

### Claim 8 (depends on 1)

The method according to claim 1, wherein the duplex DNA is exposed to two, three or four site-specific nucleases and wherein the two, three or four site-specific nucleases cleave the duplex DNA of the same CDS.

### Claim 9 (depends on 1)

The method according to claim 1, wherein at least one of the site-specific nucleases and/or one or more guide RNAs is introduced into the cell.

### Claim 10 (depends on 1)

The method according to claim 1, wherein the cell is transfected with a nucleic acid construct encoding at least one of the site-specific nucleases and/or one or more guide RNAs.

### Claim 11 (depends on 10)

The method according to claim 10, wherein the nucleic acid construct encodes at least two guide RNAs.

### Claim 12 (depends on 1)

The method according to claim 1, further comprising regenerating a plant or descendent thereof comprising the altered part of the CDS.