Abstract
The CRISPR/Cas system has become an indispensable tool for programmable and accurate biosensing, with its performance critically dependent on precise activity control. While most regulatory strategies have focused on engineering Cas proteins or modifying CRISPR RNAs, relatively little attention has been given to the design of substrate probes. Here, we systematically characterize the trans-cleavage activity of split CRISPR/Cas12a on structured substrates and leverage this insight to engineer a tunable "Hooded" probe with switchable properties. This probe architecture confers protection against trans-cleavage, and its activity can be progressively modulated by varying the probe length. Utilizing this design, we constructed a multiplexed logic-gated detection platform for direct and simultaneous analysis of miRNA and PSA, which demonstrated high sensitivity and specificity. Furthermore, we validated the robust performance of this system for logic-operated imaging in diverse cellular models, confirming its reliability in complex biological settings. Overall, our Hooded probe strategy not only broadens the applicability of CRISPR/Cas12a in molecular diagnostics, but also provides a novel design principle for the multiplexed biosensing.