Method for identifying DNA base editing by means of cytosine deaminase
Provided are: a composition for DNA double-strand breaks (DSBs), comprising (1) a cytosine deaminase and an inactivated target-specific endonuclease, (2) a guide RNA, and (3) a uracil-specific excision reagent (USER); a method for producing DNA double-strand breaks by means of a cytosine deaminase using the composition; a method for analyzing a DNA nucleic acid sequence to which base editing has been introduced by means of a cytosine deaminase; and a method for identifying (or measuring or detecting) base editing, base editing efficiency at an on-target site, an off-target site, and/or target specificity by means of a cytosine deaminase.
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.
2. The method of claim 1 , wherein the cytosine deaminase and inactivated target-specific endonuclease are in a form of a fusion protein, or the cytosine deaminase coding gene and inactivated target-specific endonuclease coding gene encode a fusion protein comprising the cytosine deaminase and inactivated target-specific endonuclease.
3. The method of claim 1 , wherein amino acid residue H840 of the inactive target-specific endonuclease is substituted with alanine, and wherein generating double strand cleavage in DNA comprises treating the DNA with an endonuclease specifically cleaving a single strand region of DNA.
4. The method of claim 1 , wherein the guide RNA is a crRNA:tracrRNA duplex in which crRNA and tracrRNA is coupled to each other, or a single-strand guide RNA (sgRNA).
5. The method of claim 1 , which is performed in vitro.
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.
7. The method of claim 6 , wherein the cytosine deaminase and inactivated target-specific endonuclease are in a form of a fusion protein, or the cytosine deaminase coding gene and inactivated target-specific endonuclease coding gene encode a fusion protein comprising the cytosine deaminase and inactivated target-specific endonuclease.
8. The method of claim 6 , wherein amino acid residue H840 of the inactive target-specific endonuclease is substituted with alanine, and wherein generating double strand cleavage in DNA comprises treating the DNA with an endonuclease specifically cleaving a single strand region of DNA.
9. The method of claim 6 , wherein the guide RNA is a crRNA:tracrRNA duplex in which crRNA and tracrRNA is coupled to each other, or a single-strand guide RNA (sgRNA).
10. The method of claim 6 , which is performed in vitro.