US 12,305,218 B2
Optimization of in vitro isolation of nucleic acids using site-specific nucleases
Jimmy Ouellet, Lardy (FR); and Jerome Maluenda, Saint Michel S/orge (FR)
Assigned to DEPIXUS, Paris (FR)
Appl. No. 17/282,683
Filed by DEPIXUS, Paris (FR)
PCT Filed Nov. 18, 2019, PCT No. PCT/EP2019/081625
§ 371(c)(1), (2) Date Apr. 2, 2021,
PCT Pub. No. WO2020/099675, PCT Pub. Date May 22, 2020.
Claims priority of application No. 18306507 (EP), filed on Nov. 16, 2018.
Prior Publication US 2021/0388414 A1, Dec. 16, 2021
This patent is subject to a terminal disclaimer.
Int. Cl. C12Q 1/6806 (2018.01); C12N 9/22 (2006.01)
CPC C12Q 1/6806 (2013.01) [C12N 9/22 (2013.01)] 20 Claims
 
1. A method of preparing a nucleic acid molecule comprising a target region for isolation from a sample comprising the steps of:
a) contacting a population of nucleic acid molecules with:
a Class 2 Type V Cas protein-gRNA complex, wherein the gRNA comprises a guide segment that is complementary to a first sequence within a nucleic acid molecule, the first sequence being located adjacent to the target region, thereby forming a Class 2 Type V Cas protein-gRNA-nucleic acid complex, and
a first protector molecule,
to form a Class 2 Type V Cas protein-gRNA-nucleic acid complex,
b) contacting the population of nucleic acid molecules containing the Class 2 Type V Cas protein-gRNA-nucleic acid complex formed in a) with at least one enzyme having exonuclease activity, thus degrading unprotected nucleic acid molecules,
c) recovering the nucleic acid molecule treated with exonuclease in step b) comprising the target region from the Class 2 Type V Cas protein-gRNA-nucleic acid complex formed in step a), and
d) contacting the recovered nucleic acid molecule of step c) with a processive polymerase, thus performing end repair on the nucleic acid molecule.