Single-nucleotide variant profiling in liquid biopsy with RECO-Cas.

Mutation detection of cell-free DNA (cfDNA) through liquid biopsy is essential for precision oncology, resistance profiling, and informed clinical decision-making. However, its clinical application has remained limited by the lack of simple, rapid, accurate, and cost-effective detection approaches. Here, we report a recombined DNA construct-activated Cas12a (RECO-Cas) assay for profiling cfDNA mutations. The RECO-Cas assay uses a recombined DNA construct generated from Argonaute-nicked mutant DNA and an artificial DNA activator to selectively trigger CRISPR-Cas12a, enabling 0.01% variant allele frequency sensitivity and single-nucleotide resolution. Using the assay, we detect KRAS, EGFR, and PIK3CA point mutations in cfDNA from clinical plasma samples, demonstrating high sensitivity (90.48%) and excellent specificity (100%). We also apply it to successfully classify and identify KRAS missense variants. RECO-Cas provides a simple, rapid, and affordable solution that is compatible with a compact, wirelessly powered point-of-care diagnostic platform incorporating smartphone-based fluorescence detection. This assay enables highly sensitive and specific detection of low-frequency mutations, facilitating early cancer diagnosis and supporting the development of personalized treatment strategies.