Wenjing Liu, Luyao Wang, Fei Ma, Chun‐yang Zhang
Abstract 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@MnO 2 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 Mn 2+ 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.