Regulating the trans-Cleavage Activity of CRISPR/Cas12a by Using an Elongation-Caged Single-Stranded DNA Activator and the Biosensing Applications

The CRISPR/Cas12a system exhibits extraordinary capabilityin thefield of biosensing and molecular diagnosis due to its trans-cleavage ability. However, it is still desirable for precise controland programmable regulation of Cas12a trans-cleavageactivity to promote the in-depth studies and application expansionof Cas12a-based sensing platforms. In this work, we have developeda new and robust CRISPR/Cas12a regulation mechanism by endowing theactivator with the function of caging crRNA ingeniously. Specifically,we constructed an integrated elongation-caged activator (EL-activator)by extending the ssDNA activator on the 3 & PRIME;-end. We found thatappending only about 8 nt that is complementary to the crRNA repeatregion is enough to cage the crRNA spacer/repeat region, thus effectivelyinhibiting Cas12a trans-cleavage activity. The innerinhibition mechanism was further uncovered after a thorough investigation,demonstrating that the EL-activator works by impeding the conformationof crRNA required for Cas12a recognition and destroying its affinitywith Cas12a. By further switching on the elongated moiety on the EL-activatorusing target biomarkers, the blocked trans-cleavageactivity of Cas12a can be rapidly recovered. Finally, a versatilesensing platform was established based on the EL-activator regulationmechanism, expanding the conventional Cas12a system that only directlyrecognizes DNA to the direct detection of enzymes and RNA biomarkers.This work has enriched the CRISPR/Cas12a regulation toolbox and expandedits sensing applications.