Patent ID: 11920151
Assignee: TOOLGEN INCORPORATED
Field: Biotechnology (Chemistry)
Classification: CPC C  G | IPC C  G

Claim 5:
6. A method of identifying a base editing site of 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;
(iii) analyzing nucleic acid sequence of the cleaved DNA fragment; and
(iv) identifying the double strand cleavage site in the nucleic acid sequence read obtained by the analysis,
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.