LUCas: Light-Uncaged Cas13a using photocleavable interfering guide RNAs.

CRISPR diagnostics enable sensitive detection of infectious diseases, with the RNA endonuclease Cas13a providing specific, amplification-free RNA detection through collateral trans-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. Here, we introduce Light-Uncaged Cas13a (LUCas), a light-controllable system that directly blocks Cas13a trans-cleavage activity using a photocleavable interfering guide RNA, even in the presence of target RNA. Brief UV illumination releases this suppression, restoring full activity. Quantitative kinetic analysis reveals an ~100-fold suppression of trans-cleavage activity prior to photo-uncaging, including suppression of target-independent background activity. Using measured kinetic parameters, we predict and experimentally validate the limit of detection of the LUCas system for direct detection. We further demonstrate a multiplexed detection strategy termed "temporal barcoding," enabling quantitative detection of viral co-infections in a single bulk reaction. Finally, LUCas is shown to be compatible with one-pot isothermal amplification for enhanced sensitivity and direct detection of target RNA spiked into blood plasma. Together, these results establish LUCas as a general framework for mechanistically informed, light-based control of Cas13a activity.