	US 11,987,837 B2
	Nucleic acid sequence enrichment by defined nucleic acid-directed endonuclease digestion
Emily D. Crawford, San Francisco, CA (US); and Jenai Quan, San Francisco, CA (US)
Assigned to CZ Biohub SF, LLC, San Francisco, CA (US)
Appl. No. 17/265,499
Filed by CZ Biohub SF, LLC, San Francisco, CA (US)
PCT Filed Aug. 6, 2019, PCT No. PCT/US2019/045352 § 371(c)(1), (2) Date Feb. 2, 2021, PCT Pub. No. WO2020/033438, PCT Pub. Date Feb. 13, 2020.
Claims priority of provisional application 62/715,180, filed on Aug. 6, 2018.
Prior Publication US 2021/0198719 A1, Jul. 1, 2021
Int. Cl. C12Q 1/6806 (2018.01); C12Q 1/6855 (2018.01)

CPC C12Q 1/6806 (2013.01) [C12Q 1/6855 (2013.01)]

14 Claims

1. A method of sample analysis, comprising:

(a) providing a sample comprising a plurality of end-blocked polynucleotides;

(b) digesting the sample with one or more defined nucleic acid-directed endonucleases that target one or more sequences of interest to produce a digested sample of polynucleotide fragments, wherein one or more of the fragments in the digested sample comprise:

one or more sequences of interest and

at least one ligatable end that has been generated by endonuclease cleavage;

(c) enriching for fragments that contain the one or more sequences of interest, wherein the one or more sequences of interest are enriched greater than 55 times their relative abundance in the sample, thereby generating an enriched sample; and

(d) analyzing the enriched sample,

wherein

(i) in step (a), a quantity of the end-blocked polynucleotides in the sample is 10 picograms to 100 nanograms, and

(ii) in step (b), an amount of at least one of the one or more defined nucleic acid-directed endonucleases is 0.0032 pmole to 2 pmoles per nanogram of the end-blocked polynucleotides.