High Sensitivity Detection of Mutant cf-ctDNA with DNA-Guided Argonaut Enzymes

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Liquid biopsy is emerging as a powerful, cost effective tool for genotyping cancer cells, individualizing therapy to match the growing arsenal of drugs with cancer genetics, and monitoring genetic shifts in cancer cells in nearly real time. Liquid biopsy has the potential of significantly improving therapy outcomes while reducing cost. The detection of cancer-related mutant DNA in body fluids, such as blood and urine, often requires a “needle in a haystack” approach. Excess wild-type (WT) DNA exhausts essential reagents during polymerase amplification, introduces false positives, and masks mutation alleles’ signals. Common strategies to enrich very low abundance aberrant nucleic acids include selective digestion of WT DNA with guided cleaving enzymes such as CRISPR Cas9 and/or peptide nucleic acid (PNA) polymerase amplification clamping technique that suppresses WT DNA amplification. Although these strategies have been successful in some cases, they suffer from a number of shortcomings and often require multiple, time-consuming unit operations that increase contamination risk. To augment and overcome the limitations of available enrichment tools, we propose a new enrichment method that utilizes the DNA-guided, argonaute protein cleaving enzyme isolated from Thermus thermophilus (TtAgo). TtAgo is highly specific, efficient, and versatile. Unlike CRISPR cas9, TtAgo does not require presence of any specific sequences such as the PAM motif (that is essential for CRISPR Cas9) to enable cleaving. TtAgo is amenable to multiplexing and operates at > 65oC, which minimizes non specific hybridization of DNA guides and makes TtAgo ideal for integration with various isothermal amplification schemes. At the conclusion of this project, we will have developed a TtAgo-based multiplexed assay for rare alleles’ enrichment that would greatly enhance the sensitivity of downstream mutant allele detection schemes such as NGS and ddPCR with and without multiplexed pre-amplification. The method developed here is also useful to enhance detection of other rare mutant alleles such as associated with fetal abnormalities.