CRISPR/Cas13a: Compensatory Target Activation Mechanism

ABSTRACT CRISPR/Cas13a is a powerful RNA‐targeting platform for molecular diagnostics, but conventional single‐effector systems typically require contiguous RNA targets longer than ∼20–28 nt, limiting sensitivity and target flexibility. CRISPR/Cas13a‐CTAM is presented as a compensatory target activation mechanism that facilitates synergistic Cas13a activation through two independently programmable short RNA effectors. By functionally decoupling allosteric activation and binding stabilization, CRISPR/Cas13a‐CTAM supports robust activation by ultra‐short RNA targets as short as 13 nt, substantially expanding the detectable target range. Compared with traditional single‐effector Cas13a assays, CRISPR/Cas13a‐CTAM achieves a detection limit of 1 fM for a 13‐nt RNA target, representing an approximately tenfold sensitivity improvement. Notably, a single‐nucleotide mismatch within the 13‐nt target induces up to a 35‐fold reduction in apparent cleavage rate, corresponding to a sevenfold enhancement in mismatch discrimination. The dual‐effector architecture further enables simultaneous dual‐target detection, demonstrated by dual miRNA profiling related to COVID‐19 and combined detection of exosome membrane proteins. Moreover, the weakly activating effector was utilized as an anchoring module to achieve the first functional immobilization of Cas13a on a sensing surface, enabling in situ electrochemical miRNA detection. By overcoming the reliance on long RNA targets, CRISPR/Cas13a‐CTAM provides a sensitive, programmable platform for RNA diagnostics and integrated biosensor development.