# US 12,215,315 B2 · Method for modifying target nucleic acid in genome of cell

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- Number: US 12,215,315 B2
- Document ID: US12215315B2
- Status: Granted
- Kind: B2
- Application number: 17/271,971
- Filed: 2019-08-30
- Published: 2021-11-04
- Granted: 2025-02-04
- Earliest claimed: 2018-08-31
- Assignees: Industry-Academic Cooperation Foundation, Yonsei University
- Applicants: Industry-Academic Cooperation Foundation, Yonsei University
- Inventors: Hyong Bum Kim; Hui Kwon Kim
- Web page: https://patentgrep.com/patent/US12215315B2

## CPC

- C12N 15/102: {Mutagenizing nucleic acids}
- 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 9/22: Ribonucleases {[RNase]; Deoxyribonucleases [DNase]}
- C12N 9/226: {Class 2 CAS enzyme complex, e.g. single CAS protein}
- C12N 15/111: {General methods applicable to biologically active non-coding nucleic acids}
- C12N 15/907: {in mammalian cells}
- 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)

## Abstract

Provided are: a method for modifying a target nucleic acid in the genome of a cell using a novel PAM sequence; and a cell in which a target nucleic acid in the genome is modified thereby. Accordingly, genome editing can be performed by targeting a position that could not be previously targeted as a target for genome editing, and thus the range of applications of genome editing can be expanded.

## Claims

### Claim 1

A method for modifying a target nucleic acid in the genome of a cell, the method comprising:
incubating a cell comprising a target nucleic acid; a polynucleotide encoding a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease or a variant thereof, and a guide RNA,
wherein the Cas nuclease or a variant thereof is Streptococcus pyogenes Cas9-NG (SpCas9-NG),
wherein the target nucleic acid comprises a protospacer adjacent motif (PAM) and a target sequence complementary to the guide RNA,
the PAM consists of a sequence selected from the group consisting of 5′-NTG-3′, 5′-GTH-3′, 5′-VCGD-3′, and 5-TCGDG-3′,
the target nucleic acid can be recognized by a complex of the Cas nuclease or a variant thereof with the guide RNA, and
the complex of the Cas nuclease or a variant thereof with the guide RNA modifies the target nucleic acid sequence-specifically.

### Claim 2 (depends on 1)

The method of claim 1, wherein the guide RNA is a polynucleotide complementary to a nucleotide sequence having consecutive 2 to 24 nucleotides in the 5′- or 3′-direction of the PAM in the target nucleic acid.

### Claim 3 (depends on 1)

The method of claim 1, wherein the length of the guide RNA is 17 to 24 nucleotides.

### Claim 4 (depends on 1)

The method of claim 1, wherein the modification is cleavage, insertion, ligation, deamination, or combinations thereof.