Highly specific enrichment of rare nucleic acids using Thermus thermophilus Argonaute

Characterization of disease-associated, cell-free nucleic acids (liquid biopsy) provides a powerful, minimally-invasive means for early disease detection, genotyping, and personalized therapy. Detection of alleles of clinical interest is often challenged by their low concentration and sequence homology with the much more abundant wild type nucleic acids. We describe a new multiplexed enrichment assay, dubbed NAVIGATER, that utilizes the short nucleic acid-guided endonucleases Argonaute (Ago), derived from the bacterium Thermus thermophilus (TtAgo), to specifically cleave perfectly complementary DNA and RNA while sparing alleles of interest. NAVIGATER greatly increases the fractions of rare alleles with single nucleotide precision enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrate 60-fold enrichment of KRAS G12D in blood samples from pancreatic cancer patients and over ten-fold improved sensitivity of clamped-PCR (PNA and XNA-PCR), enabling multiplex detection of KRAS and EGFR mutants at 0.01% fractions.