Creating a Modular Activatable CRISPR–Cas12a System by Engineering crRNA Scaffold with a Steric Hindrance Effector

CRISPR-Cas systems inherently lack spatiotemporal control over cleavage events, compromising their accuracy and efficiency in biotechnological applications. Although advances have been made in regulating these systems, developing modular activatable platforms adaptable to diverse stimuli remains challenging. To address this limitation, we developed an innovative modular activatable CRISPR-Cas12a system by engineering a crRNA scaffold with a steric hindrance effector (SHE) motif at its 3'-terminus, attached via a stimulus-cleavable linker. Through systematic evaluation of SHEs with varied sizes and structures─including linear, duplex, hairpin, and triplex─we identified their steric hindrance-dependent inhibitory effects on CRISPR-Cas12a functionality. Notably, the effective SHEs capable of completely inhibiting CRISPR-Cas12a functionality were successfully released upon ultraviolet light exposure, restoring system functionality. Furthermore, the crRNA scaffold-engineering strategy proved applicable to other type V-A Cas12a orthologs. Importantly, the system was adapted to respond to enzymatic stimuli (such as human apurinic/apyrimidinic endonuclease 1 [APE1]) and chemical stimuli (such as glutathione [GSH]). Finally, the modular activatable CRISPR-Cas12a system enabled light-activatable, one-pot nucleic acid diagnostics and APE1-activatable, tumor cell-selective microRNA imaging. Our study highlights the innovative use of steric hindrance to manipulate CRISPR-Cas12a functionality and provides valuable insights for the rational design of modular activatable CRISPR-Cas systems.