	US 11,733,248 B2
	High efficiency targeted in situ genome-wide profiling
Steven Henikoff, Seattle, WA (US); Hatice Seda Kaya Okur, Redmond, WA (US); Terri Dawn Bryson, Lynnwood, WA (US); and Peter James Skene, Issaquah, WA (US)
Assigned to Fred Hutchinson Cancer Center, Seattle, WA (US)
Appl. No. 16/650,179
Filed by Fred Hutchinson Cancer Research Center, Seattle, WA (US)
PCT Filed Sep. 25, 2018, PCT No. PCT/US2018/052707 § 371(c)(1), (2) Date Mar. 24, 2020, PCT Pub. No. WO2019/060907, PCT Pub. Date Mar. 23, 2019.
Claims priority of provisional application 62/562,918, filed on Sep. 25, 2017.
Prior Publication US 2022/0214356 A1, Jul. 7, 2022
Int. Cl. C12N 15/62 (2006.01); G01N 33/68 (2006.01); C12Q 1/6804 (2018.01); C12N 15/10 (2006.01); C12Q 1/6841 (2018.01)

CPC G01N 33/6872 (2013.01) [C12N 15/1065 (2013.01); C12Q 1/6804 (2013.01); C12Q 1/6841 (2013.01); C12Q 2522/10 (2013.01)]

30 Claims

1. An in situ method for determining the binding site of a chromatin-associated factor of interest to DNA sequences in a cell or nucleus, comprising:

contacting a permeabilized animal, plant, or bacteria cell or nucleus with a first specific binding agent that specifically binds the chromatin-associated factor interest, wherein the first specific binding agent is coupled to a plurality of transposomes, each of the plurality of transposomes comprising:

at least one transposase; and

a transposon comprising:

a first DNA molecule comprising a first transposase recognition site; and

a second DNA molecule comprising a second transposase recognition site;

activating the transposase, thereby excising and tagging the sequence of DNA bound to the chromatin-associated factor of interest with the DNA tag, wherein the at least one transposase integrates the first and second DNA molecules into chromatin DNA, thereby cleaving and tagging chromatin DNA with the first and second DNA molecules;

isolating the excised DNA; and

determining the sequence of the excised DNA, thereby mapping binding of a chromatin-associated factor of interest to one or more sequences of DNA in the cell or nucleus;

wherein the at least one transposase is or comprises a Tn5 transposase, a hyperactive Tn5 transposase, a Mu transposase, an IS5 transposase, an IS91 transposase, a Tn552 transposase, a Ty1 transposase, a Tn7 transposase, a Tn/O transposase, an IS10 transposase, a Mariner transposase, a Tel transposase, a P Element transposase, a Tn3 transposase, a bacterial insertion sequence transposase, a retrovirus transposase, a yeast retrotransposon transposase, an ISS transposase, a Tn1O transposase, a Tn903 transposase, or a combination thereof.