Rapid in situ mutation detection in extracellular vesicle DNA.

Abstract

Aim: 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-min DNA mutation detection technique using Cas12a-loaded liposomes in a microplate reader, a fundamental laboratory tool. Methods: 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. Results: This method enables the detection of EGFR L858R mutation in EV DNA within 30 min. Laborious extraction, purification, and other preparation steps for EV DNA are eliminated. The need for advanced data processing is also dispensed with. In a cohort study involving 10 healthy donors and 30 patients with advanced non-small cell lung cancer (NSCLC), the assay achieved a sensitivity of 86.7%, a specificity of 90%, and an accuracy of 87.5%. Conclusion: The limit of detection of our Cas12 assay was ~ 8 × 10⁵ EVs, corresponding to a mutation allele frequency (MAF) of ~ 10%. The MAF in late-stage cancers varies widely but often falls within 5%-50%. Therefore, without amplification of targets, this Cas12 assay can detect mutations in patients with advanced lung cancer. Future advancements in multiplex and high-throughput mutation detection using this assay will streamline self-diagnosis and treatment monitoring at home.