US 12,286,727 B2
Assessing nuclease cleavage
Joshua C. Tycko, Redwood City, CA (US); Barrett Ethan Steinberg, Cambridge, MA (US); Nick Huston, New Haven, CT (US); and Hariharan Jayaram, San Mateo, CA (US)
Assigned to Editas Medicine, Inc., Cambridge, MA (US)
Appl. No. 16/470,870
Filed by Editas Medicine, Inc., Cambridge, MA (US)
PCT Filed Dec. 19, 2017, PCT No. PCT/US2017/067444
§ 371(c)(1), (2) Date Jun. 18, 2019,
PCT Pub. No. WO2018/119010, PCT Pub. Date Jun. 28, 2018.
Claims priority of provisional application 62/436,239, filed on Dec. 19, 2016.
Prior Publication US 2020/0190699 A1, Jun. 18, 2020
Int. Cl. C12Q 1/68 (2018.01); C12N 9/22 (2006.01); C12N 15/63 (2006.01); C12Q 1/44 (2006.01); C12Q 1/6869 (2018.01); C40B 40/06 (2006.01)
CPC C40B 40/06 (2013.01) [C12N 9/22 (2013.01); C12N 15/63 (2013.01); C12Q 1/44 (2013.01); C12Q 1/6869 (2013.01)] 18 Claims
OG exemplary drawing
 
1. A method of assessing cleavage of a target cleavage site of a plurality of variant nucleic acid templates by a CRISPR associated DNA endonuclease, wherein each of the plurality of variant nucleic acid templates comprises degenerate sequences in its candidate target site for the CRISPR associated DNA endonuclease, the method comprising the steps of:
(a) obtaining a first cleavage composition by incubating a plurality of variant nucleic acid templates with a CRISPR associated DNA endonuclease and a plurality of guide RNAs under conditions favorable for cleavage of at least one variant nucleic acid template of the plurality of variant nucleic acid templates by the CRISPR associated DNA endonuclease in complexes formed by the CRISPR associated DNA endonuclease and at least one guide RNA from the plurality of guide RNAs,
(i) wherein each variant nucleic acid template of the plurality of nucleic acid templates comprises:
(A) a candidate target site for the CRISPR associated DNA endonuclease, wherein the candidate target site for the CRISPR associated DNA endonuclease is degenerate target sites comprising the degenerate nucleotide sequences,
(B) a protospacer adjacent motif (PAM) site situated adjacent to the candidate target site, and
(C) a restriction enzyme target site; and
(ii) wherein the first cleavage composition comprises:
a first plurality of cleaved variant nucleic acid templates, wherein each of the cleaved variant nucleic acid templates comprises a cleaved end generated by the CRISPR associated DNA endonuclease and the degenerate target sites uncleaved by the CRISPR associated DNA endonuclease but lacks the restriction enzyme target site, and
a first plurality of uncleaved variant nucleic acid templates, wherein each of the uncleaved variant nucleic acid templates comprises the restriction enzyme target site and the degenerate target sites that are not capable of cleaving by the CRISPR associated DNA endonuclease but lacks the cleaved end generated by the CRISPR associated DNA endonuclease;
(b) incubating the first cleavage composition with the restriction enzyme under conditions to allow cleavage of the first plurality of uncleaved variant nucleic acid templates by the restriction enzyme, thereby obtaining a second cleavage composition comprising:
(i) the first plurality of cleaved variant nucleic acid templates, and
(ii) a second plurality of cleaved variant nucleic acid templates comprising a cleaved end generated by the restriction enzyme and the degenerate target sites that are not capable of cleaving by the CRISPR associated DNA endonuclease but lacking the restriction enzyme target site and the cleaved end generated by the CRISPR associated DNA endonuclease; and
(c) ligating the second cleavage composition with one or more oligonucleotide capture probes and obtaining:
(i) a first plurality of ligation products comprising the one or more oligonucleotide capture probes and the first plurality of cleaved variant nucleic acid templates, and
(ii) a second plurality of ligation products comprising the one or more oligonucleotide capture probes and the second plurality of cleaved variant nucleic acid templates; and
(d) detecting presence of the first plurality of ligation products and the second plurality of ligation products, thereby assessing the cleavage of the target cleavage site of the plurality of variant nucleic acid templates by the CRISPR associated DNA endonuclease.