# US 2019/0169653 A1 · Method for preparing gene knock-in cells

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- Number: US 2019/0169653 A1
- Document ID: US20190169653A1
- Status: Published application
- Kind: A1
- Application number: 16/324,213
- Filed: 2017-08-01
- Published: 2019-06-06
- Earliest claimed: 2016-08-10
- Assignees: National University Corporation Tokyo Medical and Dental University
- Applicants: National University Corporation Tokyo Medical and Dental University
- Inventors: Tomomi AIDA; Kohichi TANAKA
- Web page: https://patentgrep.com/patent/US20190169653A1

## CPC

- C12N 15/907: {in mammalian cells}
- A01K 67/027: New or modified breeds of vertebrates
- A01K 67/0278: Knock-in vertebrates, e.g. humanised vertebrates
- C12N 5/10: Cells modified by introduction of foreign genetic material
- C12N 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 15/09: Recombinant DNA-technology
- C12N 15/102: {Mutagenizing nucleic acids}
- A01K 2217/072: maintaining or altering function, i.e. knock in
- A01K 2217/206: Animal model comprising tissue-specific expression system, e.g. tissue specific expression of transgene, of Cre recombinase
- A01K 2227/105: Murine
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- C12N 2800/80: Vectors containing sites for inducing double-stranded breaks, e.g. meganuclease restriction sites
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS

## Abstract

The present inventors tried to prepare a knock-in animal using a genome editing system containing a nuclease in the form of protein and a DNA-targeting RNA and using a single-stranded DNA as a donor, and found that it is possible to obtain a cell in which the donor DNA has been knocked in with extremely high efficiency.

## Claims

### Claim at position 1

1-9. (canceled)

### Claim 10

A method for preparing a cell in which a desired DNA has been inserted into a target DNA region, comprising the step of introducing
(a) a Cas9 protein,
(b) a combination consisting of two molecules: a crRNA fragment and a tracrRNA fragment, and
(c) a single-stranded donor DNA which contains the desired DNA and has a nucleotide sequence of 600 bases or more
into a cell.

### Claim 11 (depends on 10)

The method according to claim 10, wherein
the single-stranded donor DNA (c) is prepared by the following method (i) or (ii):
(i) a method including a step of amplifying DNA by PCR using a pair of primers in different amounts and a step of specifically degrading double-stranded DNA contained in an amplified product; and
(ii) a method including a step of amplifying DNA by PCR using a phosphorylated primer as one of the pair of primers and a step of specifically degrading a phosphorylated strand in an amplified product.

### Claim 12 (depends on 10)

The method according to claim 10, wherein the cell is an oocyte.

### Claim 13 (depends on 12)

The method according to claim 12, wherein the oocyte is a fertilized egg.

### Claim 14 (depends on 10)

A method for preparing a non-human individual containing a cell in which a desired DNA has been inserted into a target DNA region, the method comprising the steps of:
(a) carrying out the method according to claim 10; and
(b) preparing a non-human individual from a cell obtained in the step (a).

### Claim 15 (depends on 14)

The method according to claim 14, wherein the non-human mammal is a rodent.

### Claim 16 (depends on 10)

A kit for use in the method according to claim 10, comprising at least one molecule selected from the group consisting of the following (a) to (c):
(a) a Cas9 protein,
(b) a combination consisting of two molecules: a crRNA fragment and a tracrRNA fragment, and
(c) a single-stranded donor DNA which contains the desired DNA and has a nucleotide sequence of 600 bases or more.