A Universal Light-Activated CRISPR-RNA Based on Split Direct Repeat for One-Pot Cas12a Nucleic Acid Detection

Spatiotemporal regulation of CRISPR-Cas systems holds significant promise for precision gene editing and molecular diagnostics. While photochemical strategies for CRISPR activity control have advanced, a universal regulatory approach remains elusive. Here, we report a modular light-activated CRISPR-RNA design through splitting conventional crRNA within the direct repeat (DR) into two functional domains: a conserved 5' split direct repeat (5' SDR) and a variable 3' split direct repeat (SDR) + spacer (3' SDR-Spacer) module. Double-stranded extensions were introduced at the cleavage site to preserve functional integrity. Through screening of light-sensitive caging group modification sites in the universal 5' SDR, a novel light-activated CRISPR-RNA system was developed. This system only requires spacer redesign of the 3' SDR-Spacer for new targets, while the caged 5' SDR is universal. Thereupon, we established a universal light-activated CRISPR-RNA assisted one-pot RAA-Cas12a detection system (UniLight-CRISPR). When applied to <i>Mycoplasma pneumoniae</i> detection using qPCR-validated clinical samples, UniLight-CRISPR demonstrated 95.45% sensitivity and 100% specificity, matching the performance of conventional two-step Cas12a assays. This universal photo regulation strategy not only addresses current limitations in CRISPR diagnostics but also provides a blueprint for adapting other Cas enzymes. We anticipate broad applications of our universal light-activated CRISPR-RNA system, extending from molecular diagnostics to gene-editing research.