Abstract

Background The clinical translation of microRNAs as cancer biomarkers is challenging due to the lack of detection assays that are simple, rapid, and sensitive enough for point-of-care use. Methods Here, we present a one-pot, isothermal CRISPR-Cas12a platform for miRNA detection that integrates rolling circle amplification (RCA) with the CRISPR-Cas12a system for the direct detection of miRNAs. Our assay uniquely leverages the cis-cleavage activity of Cas12a to fragment RCA amplicons, enabling an exponential amplification cycle, while simultaneously employing its trans-cleavage activity for real-time fluorescent signal readout. This true one-step reaction does not require separate pre-amplifications. Results When applied to plasma-derived extracellular vesicles, the assay detected the lung cancer-associated miRNA miR-451a at a sensitivity of 8 fM with single-nucleotide specificity. The assay is rapid, and its compatibility with a smartphone-based fluorimeter demonstrates its potential for point-of-care testing. In a clinical validation study using over 100 plasma samples, the assay distinguished lung cancer patients from healthy controls with an area under the curve (AUC) of 0.90. Conclusion The 451a-CRISPR platform represents a significant advancement towards a simple and effective liquid biopsy tool for early cancer detection. Data access statement This publication does not involve research data.