Endogenous HClO-Activatable CRISPR−Cas12a System for Inflammation-Associated MicroRNA Imaging in Living Cells

Precise imaging of inflammation-associated microRNAs could prompt early disease diagnosis and guide therapeutic interventions. However, developing molecular tools capable of specifically sensing these miRNAs in inflamed cells remains a significant challenge. Herein, we leverage endogenous hypochlorous acid (HClO)a characteristic reactive oxygen species that preferentially accumulates in inflamed cellsas a chemical trigger to construct an activatable CRISPR−Cas12a system for imaging of microRNA-155 (miR-155) with high specificity. This system is engineered by attaching a hairpin-shaped steric hindrance effector to the 3′-terminus of the crRNA scaffold through a phosphorothioate (PS) linker. In the absence of HClO, the hairpin structure sterically hinders the assembly of the functional Cas12a ribonucleoprotein complex, maintaining the system in a dormant state. Upon exposure to HClO, the PS linker undergoes specific oxidative cleavage, releasing the steric blockade and restoring Cas12a-mediated trans-cleavage. This system works only when HClO and miR-155 are simultaneously present, enabling high-fidelity imaging in inflamed cells while minimizing background noise in normal cells. This PS-based chemical gating strategy offers a versatile paradigm for endowing CRISPR−Cas12a systems with responsiveness to endogenous oxidative stress, significantly expanding the toolbox for imaging inflammation-associated biomarkers in living systems.