# US 11,306,320 B2 · Method for promoting homologous recombination

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- Number: US 11,306,320 B2
- Document ID: US11306320B2
- Status: Granted
- Kind: B2
- Application number: 16/465,697
- Filed: 2017-11-27
- Published: 2020-01-16
- Granted: 2022-04-19
- Earliest claimed: 2016-12-07
- Assignees: Kao Corporation
- Applicants: Kao Corporation
- Inventors: Mayumi Wada; Shintaro Ryo
- Web page: https://patentgrep.com/patent/US11306320B2

## CPC

- C12N 15/8213: {Targeted insertion of genes into the plant genome by homologous recombination}
- C07K 14/405: from algae
- C12N 1/12: Unicellular algae; Culture media therefor (as new plants A01H13/00)
- C12N 1/125: Unicellular algae isolates
- C12N 15/902: {using homologous recombination}
- C12R 2001/89: Algae {; Processes using algae}
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)
- C12R: INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS

## Abstract

A method of producing a transformant, which contains using an alga belonging to the genus Nannochloropsis as a host, wherein function of the following protein (A) or (B) of the alga is suppressed, inhibited or deleted: (A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 50; and (B) a DNA binding protein consisting of an amino acid sequence having 70% or more identity with the amino acid sequence of the protein (A).

## Claims

### Claim 1

A method of producing a transformant, the method comprising:
transforming a Nannochloropsis host with a polynucleotide and inserting the polynucleotide at a target site in the host's genomic DNA by homologous recombination;
wherein, in the host:
a gene encoding the following protein (A) or protein (B) has been deleted or inactivated prior to the transforming, thereby
(i) reducing the host's frequency of non-homologous recombination, and
(ii) increasing the host's frequency of homologous recombination;
wherein, both (i) and (ii) are as compared to that of a Nannochloropsis alga that is the same as the host except that the gene encoding protein (A) or protein (B) is not deleted or inactivated:
wherein protein (A) is:
(A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 50; and
wherein protein (B) is:
(B) a DNA binding protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of protein (A),
and wherein protein (B) recognizes DNA ends generated by a DNA double strand break, and binds to the DNA ends recognized to recruit a DNA-dependent kinase to the DNA ends.

### Claim 2

A method of producing a host, the method comprising deleting or inactivating a gene that encodes the following protein (A) or protein (B) in an alga belonging to the genus Nannochloropsis, thereby producing a host in which the host's frequency of non-homologous recombination is reduced and the host's frequency of homologous recombination is increased, both as compared to that of a Nannochloropsis alga that is the same as the host except that the gene encoding protein (A) or encoding protein (B) is not deleted or inactivated:
wherein protein (A) is:
(A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 50; and
wherein protein (B) is:
(B) a DNA binding protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of protein (A),
and wherein protein (B) recognizes DNA ends generated by a DNA double strand break, and binds to the DNA ends recognized to recruit a DNA-dependent kinase to the DNA ends.

### Claim 3 (depends on 1)

The method according to claim 1, wherein the gene encoding protein (A) or the gene encoding protein (B) is deleted.

### Claim 4 (depends on 1)

The method according to claim 1, wherein the amino acid sequence of protein (B) has 95% or more identity with the amino acid sequence of protein (A).

### Claim 5 (depends on 1)

The method according to claim 1, wherein the gene encoding protein (A) is a gene consisting of the following DNA (a) or the gene encoding protein (B) is a gene consisting of the following DNA (b):
wherein DNA (a) is:
(a) a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 51; and
wherein DNA (b) is:
(b) a DNA consisting of a nucleotide sequence having 85% or more identity with the nucleotide sequence of DNA (a), and encoding the protein (B).

### Claim 6 (depends on 1)

The method according to claim 1, wherein the host is Nannochloropsis oculata.

### Claim 7

An alga belonging to the genus Nannochloropsis, in which
(a) a gene encoding the following protein (A) or protein (B) is deleted or inactivated; and
(b) the alga's frequency of non-homologous recombination is reduced and the alga's frequency of homologous recombination is increased, both as compared to that of a Nannochloropsis alga that is the same except that the gene encoding protein (A) or the gene encoding protein (B) is not deleted or inactivated,
wherein protein (A) is:
(A) a protein consisting of the amino acid sequence set forth in SEQ ID NO: 50; and
wherein protein (B) is:
(B) a DNA binding protein consisting of an amino acid sequence having 90% or more identity with the amino acid sequence of protein (A), and wherein protein (B) is a protein that recognizes DNA ends generated by a DNA double strand break, and binds to the DNA ends recognized to recruit a DNA-dependent kinase to the DNA ends.

### Claim 8 (depends on 7)

The alga according to claim 7, wherein the gene encoding protein (A) or the gene encoding protein (B) is deleted.

### Claim 9 (depends on 7)

The alga according to claim 7, wherein the amino acid sequence of protein (B) has 95% or more identity with the amino acid sequence of protein (A).

### Claim 10 (depends on 7)

The alga according to claim 7, wherein the gene encoding protein (A) is a gene consisting of the following DNA (a) or the gene encoding protein (B) is a gene consisting of the following DNA (b):
wherein DNA (a) is:
(a) a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 51; and
wherein DNA (b) is:
(b) a DNA consisting of a nucleotide sequence having 85% or more identity with the nucleotide sequence of DNA (a), and encoding the protein (B).

### Claim 11 (depends on 7)

The alga according to claim 7, wherein the alga is Nannochloropsis oculata.

### Claim 12 (depends on 2)

The method according to claim 2, wherein the gene encoding protein (A) or protein (B) is deleted.

### Claim 13 (depends on 2)

The method according to claim 2, wherein the amino acid sequence of protein (B) has 95% or more identity with the amino acid sequence of protein (A).

### Claim 14 (depends on 2)

The method according to claim 2, wherein the gene encoding protein (A) is a gene consisting of the following DNA (a) or the gene encoding protein (B) is a gene consisting of the following DNA (b):
wherein DNA (a) is:
(a) a DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 51; and
wherein DNA (b) is:
(b) a DNA consisting of a nucleotide sequence having 85% or more identity with the nucleotide sequence of DNA (a), and encoding the protein (B).

### Claim 15 (depends on 2)

The method according to claim 2, wherein the alga is Nannochloropsis oculata.