	US 12,467,077 B2
	Compositions and methods for DNA modification detection
John Stamatoyannopoulos, Seattle, WA (US); and Pavel Zrazhevskiy, Seattle, WA (US)
Assigned to Altius Institute for Biomedical Sciences, Seattle, WA (US)
Appl. No. 17/293,869
Filed by Altius Institute for Biomedical Sciences, Seattle, WA (US)
PCT Filed Nov. 18, 2019, PCT No. PCT/US2019/062040 § 371(c)(1), (2) Date May 13, 2021, PCT Pub. No. WO2020/106633, PCT Pub. Date May 28, 2020.
Claims priority of provisional application 62/806,539, filed on Feb. 15, 2019.
Claims priority of provisional application 62/768,991, filed on Nov. 19, 2018.
Prior Publication US 2022/0010366 A1, Jan. 13, 2022
Int. Cl. C12Q 1/6816 (2018.01)

CPC C12Q 1/6816 (2013.01)

23 Claims

1. A method for detecting induction of cleavage in genomic DNA by a nuclease, the method comprising:

contacting a live cell comprising genomic DNA comprising a cleavage site for the nuclease with the nuclease under conditions sufficient for induction of cleavage at the cleavage site;

fixing the cell and contacting the fixed cell with a plurality of oligonucleotides complementary to a single stranded (ss) 3′-overhang created at the cleavage site, wherein the ss 3′-overhang is at least 1 kb in length, wherein the genomic DNA is not denatured; and

detecting hybridization of the plurality of oligonucleotides to the ss 3′-overhang,

wherein hybridization of the oligonucleotides indicates induction of the cleavage by the nuclease.