# US 2018/0020646 A1 · Gene knockout method

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- Number: US 2018/0020646 A1
- Document ID: US20180020646A1
- Status: Published application
- Kind: A1
- Application number: 15/538,554
- Filed: 2015-12-25
- Published: 2018-01-25
- Earliest claimed: 2014-12-26
- Assignees: Riken
- Applicants: Riken
- Inventors: Hiroki UEDA; Genshiro SUNAGAWA; Kenta SUMIYAMA; Maki UKAI; Dimitri PERRIN
- Web page: https://patentgrep.com/patent/US20180020646A1

## CPC

- A01K 67/0276: Knock-out vertebrates
- C12N 15/113: Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; {Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing (when used in plants C12N15/8218)}
- A01K 67/027: New or modified breeds of vertebrates
- 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 15/87: Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N 15/873: Techniques for producing new embryos, e.g. nuclear transfer, manipulation of totipotent cells or production of chimeric embryos
- A01K 2207/05: Animals modified by non-integrating nucleic acids, e.g. antisense, RNAi, morpholino, episomal vector, for non-therapeutic purpose
- A01K 2217/075: inducing loss of function, i.e. knock out
- C12N 2310/20: involving clustered regularly interspaced short palindromic repeats [CRISPR]
- A01K: ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## Abstract

A method for producing a cell in which a target gene is knocked out, the method including the step of: introducing a CRISPR-Cas system into a cell having one or more kinds of target genes, the CRISPR-Cas system being able to produce (i) three or more kinds of guide RNAs for each of the one or more kinds of target genes and (ii) a Cas protein. The present invention can provide a method that enables highly efficient (90% or more) production of whole-body biallelic knockout animals in a single generation.

## Claims

### Claim 1

A method for producing a cell in which a target gene is knocked out,
the method comprising the step of:
introducing a CRISPR-Cas system into a cell having one or more kinds of target genes, the CRISPR-Cas system being able to produce (i) three or more kinds of guide RNAs for each of the one or more kinds of target genes and (ii) a Cas protein.

### Claim 2 (depends on 1)

The method as set forth in claim 1, wherein
the one or more kinds of target genes are knocked out by (i) causing three kinds of guide RNAs to target each of the one or more kinds of target genes and then (ii) causing a Cas protein to cut each of the one or more kinds of target genes.

### Claim 3 (depends on 1)

The method as set forth in claim 1, wherein
the CRISPR-Cas system is a system including (i) three or more kinds of guide RNAs for each of the one or more kinds of target genes and (ii) RNA that encodes a Cas protein.

### Claim 4 (depends on 1)

The method as set forth in claim 1, wherein
the one or more kinds of target genes are two or more kinds of target genes, and the two or more kinds of target genes are knocked out.

### Claim 5 (depends on 1)

The method as set forth in claim 1, wherein
the Cas protein is a Cas9 protein.

### Claim 6 (depends on 1)

The method as set forth in claim 1, wherein
the cell is an animal cell.

### Claim 7 (depends on 1)

The method as set forth in claim 1, wherein
the cell is a fertilized egg.

### Claim 8 (depends on 1)

The method as set forth in claim 1, wherein
the CRISPR-Cas system is introduced into a cytoplasm of the cell.

### Claim 9 (depends on 1)

A method for producing a knockout non-human organism, comprising the steps of:
obtaining an embryo in which a target gene has been knocked out by a method recited in claim 1; and
transplanting the embryo into a pseudopregnant non-human animal, so as to obtain an offspring.

### Claim 10 (depends on 9)

The method as set forth in claim 9, wherein
the knockout non-human organism is a knockout non-human animal.

### Claim 11 (depends on 9)

The method as set forth in claim 9, wherein
the target gene is a biallelic gene, and
a ratio of whole-body biallelic knockout individuals to all of offspring obtained is 90% or more.

### Claim 12 (depends on 11)

The method as set forth in claim 11, wherein
the target gene is two or more kinds of target genes, and
a ratio of whole-body biallelic knockout individuals for each of the two or more kinds of target genes to all of offspring obtained is 90% or more.

### Claim 13 (depends on 9)

A knockout non-human organism produced by a method recited in claim 9.