Highly Specific Enrichment of Rare Nucleic Acids using Thermus Thermophilus Argonaute with Applications in Cancer Diagnostics

Detection of disease-associated, cell-free nucleic acids enables early diagnostics, genotyping, and personalized therapy, but is challenged by their low concentration and sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for N ucleic A cid enrichment V ia DNA- G uided A rgonaute from T hermus th er mophilus ( Tt Ago) that allows for specific cleavage of guide-complementary DNA and RNA with single nucleotide precision. NAVIGATER greatly increases the fractions of rare alleles, enhancing the sensitivity of downstream detection such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of KRAS G12D and nearly 100-fold increased sensitivity of clamped-PCR (PNA and XNA-PCR), enabling detection of low-frequency (0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-DASH, identifying more mutation-positive samples when combined with XNA-PCR. Moreover, Tt Ago does not require the target to contain any specific (PAM-like) motifs; is a multi-turnover enzyme; cleaves ssDNA, dsDNA, and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases. The here-described NAVIGATER approach has important advantages over other enrichment methods.