Direct RNA Triggering of Cas12a through the Native crRNA Architecture Enables Clinical Nucleic Acids Diagnostics

CRISPR/Cas12a has emerged as a powerful platform for nucleic acid diagnostics, yet its activity is widely considered to be restricted to DNA targets, limiting its applicability for direct RNA detection. Here we report a manganese-ion (Mn 2+ )-empowered Cas12a (MEC) platform that overcomes this constraint by allowing the robust RNA-mediated activation of Cas12a. Structural analyses reveal that Mn 2+ strengthens RNA engagement and reorganizes the catalytic center by coordinating RNA phosphates, resulting in an enhancement of trans-cleavage efficiency by 60-fold relative to the Mg 2+ conditions, without compromising sequence specificity. This Mn 2+ -dependent activation mechanism is conserved across multiple Cas12a orthologues (LbCas12a, AsCas12a, FnCas12a), permitting amplification-free detection of RNA with femtomolar sensitivity across diverse targets, particularly the ultrashort abortive transcripts (7 nt). Analysis of clinical serum samples further demonstrates that MEC quantitatively measures circulating miR-21 with performance concordant with reference clinical assays and effectively distinguishes lung cancer patients from healthy individuals. These results reveal an unrecognized role for Mn 2+ in Cas12a biochemistry and establish a simple, versatile, and highly sensitive framework for RNA diagnostics.