RcisT: A Cas12a cis-cleavage-triggered strand displacement assay for sensitive and single-nucleotide-resolved nucleic acid detection

Abstract

Rapid and accurate detection of bacterial antimicrobial resistance genes and single-base mutations is essential for clinical diagnostics and point-of-care testing (POCT). Most CRISPR/Cas12a-based platforms rely on trans-cleavage activity, which is prone to nonspecific signals and exhibits limited single-nucleotide discrimination, particularly for low-abundance mutations. Here, we report an RcisT system that integrates RDA amplification, Cas12a cis-cleavage to generate double-stranded DNA intermediates containing short 5′ overhangs, followed by a toehold-mediated strand displacement reaction (TSDR) for signal transduction, enabling specific detection and dual-mode readout via visual fluorescence and lateral flow strips. Using the lateral-flow strip readout, the RcisT system achieved a visual detection threshold of 10 copies per reaction for the NDM-1 gene within 50–60 min and enabled detection of the Mycobacterium tuberculosis rpoB S450L mutation at mutant allele frequencies as low as 0.1%. The RcisT probe exhibited a discrimination factor of 11.7, approximately 6.5-fold higher than that of the conventional ssDNA reporter (DF = 1.8). Clinical validation using 30 samples demonstrated 96.0% sensitivity, 100% specificity, and an AUC of 0.992, comparable to qPCR. RcisT enabled low-abundance mutation detection with a visually interpreted lateral-flow endpoint readout and operated through cis-cleavage and TSDR, rather than via Cas12a trans-cleavage activity.