Method of nucleic acid enrichment using site-specific nucleases followed by capture

US11473124B2 · kind B2 · utility

Assignee

• Depixus · FR

Inventors

• Jimmy Ouellet · Lardy, FR
• Jerome Maluenda · Saint-Michel-sur-Orge, FR

Key dates

• Filing date: Dec 12, 2019
• Grant date: Oct 18, 2022
• Expiry date: Dec 12, 2039

Classification

• Technology area (CPC C): Chemistry; Metallurgy
• CPC primary: C12Q1/683
• WIPO field: Biotechnology
• WIPO sector: Chemistry

Abstract

The present invention relates to a method of isolating a nucleic acid target region from a population of nucleic acid molecules, said method comprising the steps of a) contacting said 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 site adjacent to said target region, thereby forming a Class 2 Type V Cas protein-gRNA-nucleic acid complex, b) contacting the population of nucleic acid molecules comprising said Class 2 Type V Cas protein-gRNA-nucleic acid complex with at least one enzyme having single-strand 3′ to 5′ exonuclease activity, thereby forming a 5′ single-stranded overhang in said first site, c) removing the Class 2 Type V Cas protein-gRNA complex from the population of step b), d) contacting the population of step c) with an oligonucleotide probe, said probe comprising a sequence that is at least partially complementary to said overhang, thereby forming a duplex between said probe and said overhang, and e) isolating said duplex from the population of nucleic acid molecules of step d), thereby isolating said nucleic acid target region.