Rapid in situ mutation detection in extracellular vesicle-DNA

Abstract

A PCR- and sequencing-free mutation detection assay facilitates cancer diagnosis and reduces over-reliance on specialized equipment. This benefit was highlighted during the pandemic when high demand for viral nucleic acid testing often sidelined mutation analysis. This shift led to substantial challenges for patients on targeted therapy in tracking mutations. Here, we report a 30-minute DNA mutation detection technique using Cas12a-loaded liposomes in a microplate reader, a fundamental laboratory tool. CRISPR-Cas12a complex and fluorescence-quenching (FQ) probes are introduced into tumor-derived extracellular vesicles (EV) through membrane fusion. When CRISPR-RNA hybridizes with the DNA target, activated Cas12a can trans-cleave FQ probes, resulting in fluorescence signals for the quantification of DNA mutation. Future advancements in multiplex and high-throughput mutation detection using this assay will streamline self-diagnosis and treatment monitoring at home.