Depixus

Jerome Maluenda

Jimmy Ouellet

The present invention relates to a method of preparing a nucleic acid molecule comprising a target region for isolation from a sample, said method comprising the steps of contacting a population of nucleic acid molecules with a first 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, said first sequence being located adjacent to said target region, thereby forming a Class 2 Type V Cas protein-gRNA-nucleic acid complex, and a protector molecule, contacting the population of nucleic acid molecules with at least one enzyme having exonuclease activity, recovering said nucleic acid molecule comprising the target region from the Class 2 Type V Cas protein-gRNA-nucleic acid complex formed in step a), and contacting the nucleic acid molecule of step c) with a processive polymerase, preferably a mesophilic or thermophilic polymerase, even more preferably a T4 DNA polymerase.

Optimization of in vitro isolation of nucleic acids using site-specific nucleases

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.

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

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&#x2032; exonuclease activity, thereby forming a 5&#x2032; 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.

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Bretigny, France

Jerome Maluenda