# US 11,299,754 B2 · Gene targeting method

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- Number: US 11,299,754 B2
- Document ID: US11299754B2
- Status: Granted
- Kind: B2
- Application number: 15/570,517
- Filed: 2016-04-29
- Published: 2018-12-13
- Granted: 2022-04-12
- Earliest claimed: 2015-04-30
- Assignees: Hangzhou Genekine Biotech Co., Ltd
- Applicants: Hangzhou Genekine Biotech Co., Ltd
- Inventors: Youwei Jiang; Yunfei Li
- Web page: https://patentgrep.com/patent/US11299754B2

## CPC

- C12N 15/905: {in yeast}
- C12N 1/16: Yeasts; Culture media therefor
- C12N 9/1241: {Nucleotidyltransferases (2.7.7)}
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/11: DNA or RNA fragments; Modified forms thereof (DNA or RNA not used in recombinant technology, C07H21/00); {Non-coding nucleic acids having a biological activity}
- C12N 15/902: {using homologous recombination}
- C12P 21/00: Preparation of peptides or proteins (single cell protein C12N1/00)
- C12N 2800/30: Vector systems comprising sequences for excision in presence of a recombinase, e.g. loxP or FRT
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12P: FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE {(brewing of beer C12C; producing vinegar C12J; producing specific peptides or proteins C07K; producing enzymes C12N9/00; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification C12N15/00; measuring or testing processes involving enzymes or microorganisms C12Q; measuring or testing processes involving nucleic acid amplification reactions C12Q1/6844; fermentation processes to form a food composition, A21 or A23; compounds in general, see the relevant compound class, e.g. C01, C07)}

## Abstract

Provided are a novel two-step gene targeting method and a nucleotide construct for gene targeting. The method can improve the gene targeting efficiency and accurately identify a target gene knock-out mutant. The method of the present invention comprises: firstly, efficiently replacing a target gene in a genome with a targeting box by homologous recombination, the targeting box consisting of a target gene activity variant, a marker gene and site-specific recombination sites; and secondly, resecting the targeting box by recombinase, leaving a site-specific recombination site on the target gene to generate a target gene knock-out mutant, and removing a recombinase expression vector from the knock-out mutant by using a counter selection marker in the recombinase expression vector.

## Claims

### Claim 1

A plasmid construct for altering a genome by gene targeting, comprising the following structure:
5′-A1-B1-C1-C2-X-B2-A2-3′;
5′-A1-B1-X-C1-C2-B2-A2-3′;
5′-A1-B1-C1-X-B2-C2-A2-3′; or
5′-A1-B1-X-C1-B2-C2-A2-3′
wherein
B1 and B2 are site-specific recombination sites;
C1 and C2 are marker genes and cannot be absent at the same time;
X is an active variant of a target gene in the genome;
the active variant possesses a functional activity of the target gene;
A1 is a sequence that is homologous to a region upstream of the target gene in the genome; and
A2 is a sequence that is homologous to a region downstream of the target gene in the genome.

### Claim 2 (depends on 1)

The plasmid construct of claim 1, wherein the site-specific recombination sites are loxP site or FRT site.

### Claim 3 (depends on 2)

The plasmid construct of claim 2, wherein the site-specific recombination sites are loxP site.

### Claim 4 (depends on 1)

The plasmid construct of claim 1, wherein the active variant comprises a promoter and/or terminator.

### Claim 5 (depends on 1)

A composition for gene targeting, wherein the composition comprises the plasmid construct of claim 1 and an expression vector for expressing a recombinase.

### Claim 6 (depends on 5)

The composition of claim 5, wherein the recombinase is Cre recombinase or Flp recombinase.

### Claim 7 (depends on 6)

The composition of claim 6, wherein the recombinase is Cre recombinase.

### Claim 8 (depends on 1)

The plasmid construct of claim 1, wherein C1 and C2 are both present.

### Claim 9 (depends on 1)

A host cell comprising the plasmid construct of claim 1.

### Claim 10 (depends on 9)

The host cell of claim 9, wherein the host cell is a yeast.

### Claim 11 (depends on 1)

A method for gene targeting, comprising:
a) constructing the plasmid construct of claim 1;
b) introducing the plasmid construct obtained in step a) into a cell, thereby replacing the target gene in the genome of the cell with the plasmid construct via homogeneous recombination;
c) excising the plasmid construct by a recombinase, thereby leaving behind a site-specific recombination site on the target gene and yielding a mutant of the target gene.

### Claim 12 (depends on 11)

The method of claim 11, wherein the recombinase is expressed by an expression vector, and the method further comprises removing the expression vector via a counter-selection marker gene of the expression vector.

### Claim 13 (depends on 1)

A method for engineering a strain, comprising:
a) constructing the plasmid construct of claim 1;
b) introducing the plasmid construct obtained in step a) into a strain to be engineered, thereby replacing the target gene in the genome of the strain to be engineered with the plasmid construct via homogeneous recombination;
c) introducing an expression vector for expressing a recombinase in the strain to be engineered, and excising the plasmid construct by the recombinase, thereby leaving behind a site-specific recombination site on the target gene and yielding a disruption mutant of the target gene.

### Claim 14 (depends on 13)

The method of claim 13, wherein the method further comprises removing the expression vector via a counter-selection marker gene of the expression vector.

### Claim 15 (depends on 13)

A method for producing a recombinant protein, comprising a step of growing a strain engineered according to the method of claim 13 to produce the recombinant protein.

### Claim 16 (depends on 13)

A method for producing a metabolite, comprising a step of growing a strain engineered according to the method of claim 13 to obtain the metabolite.

### Claim 17 (depends on 13)

A method for performing a biocatalytic reaction, comprising a step of using a strain engineered according to the method of claim 13 in a biocatalytic reaction.