LUCas: Light-Uncaged Cas13a using photocleavable interfering guide RNAs

CRISPR diagnostics have emerged as powerful tools for detecting infectious diseases, with the RNA endonuclease Cas13a enabling sensitive and specific, amplification-free RNA detection through collateral <i>trans</i> -cleavage of fluorescent reporters. However, background cleavage from unbound enzyme, contaminating nucleases, and unsynchronized initiation of reactions limits assay sensitivity and interpretability. A strategy to precisely control the onset of Cas13a catalytic activity, essentially a molecular "starting gun", would address these challenges and expand assay design space. Here, we introduce Light-Uncaged Cas13a (LUCas), a light controllable system that directly gates Cas13a using a photocleavable interfering guide RNA (pc-igRNA) that suppresses <i>trans</i> -cleavage activity even in the presence of target RNA. Brief UV illumination releases this suppression, restoring full activity. Quantitative kinetic analysis reveals an approximately 100-fold suppression of <i>trans</i> -cleavage activity prior to photo-activation. Importantly, LUCas also suppresses target-independent background activity, enabling a predictive, background-limited determination of assay sensitivity. Using measured kinetic parameters, we predict and experimentally validate the limit-of-detection of the LUCas system. Finally, we demonstrate a multiplexed detection strategy termed "temporal barcoding," which enables quantitative detection of viral co-infections in a single bulk reaction. Together, these results establish LUCas as a general framework for mechanistically informed, light-based control of Cas13a activity.