Guide ssDNA Modulates the Recognition Selectivity of CRISPR-Cas12a for Direct miRNA Detection

Abstract Owing to its robust trans-cleavage activity, CRISPR-Cas12a is a preferred tool for biosensing, yet its native DNA-only recognition limits RNA detection. Herein, we report a simple strategy requiring neither protein mutagenesis nor exogenous factors that enables wild-type Cas12a to directly detect RNA targets. We repurposed the Cas12a recognition unit from a full-length ssDNA into a cooperative assembly of two short ssDNAs. In this assembly, a 10−11 nucleotide primer segment relieves the inherent DNA restriction, enabling robust dual-DNA/RNA recognition. Notably, this primer ssDNA modulates substrate preference solely by remodeling the ribonucleoprotein complex conformation without directly triggering trans-cleavage, which remains strictly dependent on target−crRNA complementarity. Molecular docking simulations further reveal that the crRNA−target heteroduplex induces a conformational rearrangement in the Cas12a Nuc domain, forming a catalytic channel that supports trans-cleavage. Further, we constructed a primer ssDNA-mediated Cas12a platform that detects miR-122, miR-92a, and miR-192 with high specificity and sensitivity, rendering it suitable for early clinical diagnosis. Collectively, this work establishes a minimalist, low-cost strategy to overcome Cas12a’s intrinsic RNA detection limitation, elucidates the underlying conformational mechanism, and provides a practical technological tool for rapid RNA diagnostics and integrated molecular diagnostic platforms.