	US 11,920,151 B2
	Method for identifying DNA base editing by means of cytosine deaminase
Daesik Kim, Seoul (KR)
Assigned to TOOLGEN INCORPORATED, Seoul (KR); SEOUL NATIONAL UNIVERSITY R&Db FOUNDATION, Seoul (KR); and INSTITUTE FOR BASIC SCIENCE, Daejeon (KR)
Appl. No. 16/332,036
Filed by TOOLGEN INCORPORATED, Seoul (KR); Seoul National University R&DB Foundation, Seoul (KR); and INSTITUTE FOR BASIC SCIENCE, Daejeon (KR)
PCT Filed Sep. 13, 2017, PCT No. PCT/KR2017/010056 § 371(c)(1), (2) Date Oct. 25, 2019, PCT Pub. No. WO2018/052247, PCT Pub. Date Mar. 22, 2018.
Claims priority of provisional application 62/445,310, filed on Jan. 12, 2017.
Claims priority of provisional application 62/393,682, filed on Sep. 13, 2016.
Prior Publication US 2020/0131536 A1, Apr. 30, 2020
Int. Cl. C12N 15/90 (2006.01); C12N 9/22 (2006.01); C12N 9/78 (2006.01); C12N 15/113 (2010.01); C12Q 1/6806 (2018.01); C12Q 1/6869 (2018.01); G16B 30/00 (2019.01)

CPC C12N 15/90 (2013.01) [C12N 9/22 (2013.01); C12N 9/78 (2013.01); C12N 15/113 (2013.01); C12Q 1/6806 (2013.01); C12Q 1/6869 (2013.01); G16B 30/00 (2019.02); C12Y 305/04004 (2013.01)]

10 Claims

1. A method of analyzing nucleic acid sequence of DNA in which a base editing is introduced by cytosine deaminase, comprising:

(i) introducing or contacting (a) a cytosine deaminase and an inactivated target-specific endonuclease, or (b) a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, or (c) a plasmid comprising a cytosine deaminase coding gene and an inactivated target-specific endonuclease coding gene, into a cell or with DNA isolated from a cell, together with a guide RNA;

(ii) treating the DNA with a uracil-specific excision reagent (USER) and generating double strand cleavage in DNA; and

(iii) analyzing nucleic acid sequence of the cleaved DNA fragment,

wherein the DNA isolated from a cell in step (i) is a genomic DNA, and the nucleic acid sequence analysis of step (iii) is performed by whole genome sequencing,

wherein the uracil-specific excision reagent (USER) comprises uracil DNA glycosylase (UDG) and endonuclease VIII, and

wherein the inactivated target-specific endonuclease is a Cas9 protein derived from Streptococcus pyogenes wherein amino acid residue D10 is substituted with alanine.