MnO ₂ Nanosponge‐Accelerated Cas12a <i>Trans</i> ‐Cleavage: Breaking the Kinetic Barrier for In Vivo RNA Imaging

CRISPR/Cas12a system has emerged as a promising tool for in vitro biosensing, but its in vivo applications are hindered by its inefficient intracellular delivery and suboptimal trans-cleavage kinetics. To address these challenges, a Cas12a@MnO₂ nanosponge (hMNS) nanoprobe is constructed, in which hMNS as both a degradable carrier and an accelerator of CRISPR/Cas12a system for efficient imaging of RNA in living cells. The Cas12a@hMNS nanoprobe is obtained via a one-step co-assembly process. It not only facilitates synchronous cellular uptake and glutathione (GSH)-responsive release of CRISPR/Cas12a components, but also supplies adequate Mn²⁺ cofactors to improve the trans-cleavage activity of Cas12a. This dual-function probe can break the kinetic barrier of conventional CRISPR/Cas12a systems due to its unique characteristics of effective cellular internalization, rapid intracellular release, and accelerated signal gain, enabling sensitive detection of mRNA down to 63.6 pM without pre-amplification. Moreover, the Cas12a@hMNS nanoprobe can profile endogenous mRNA at the single-cell level, discriminate breast cancer tissues from healthy counterparts, and real-time visualize mRNA dynamics in living cells with exceptional spatiotemporal precision. Importantly, the elongation-blocked (EB) activator-modulated CRISPR/Cas12a system can be extended to detect various intracellular biomarkers, holding promising applications in clinical diagnosis, treatment, and surveillance.