Kinetics Accelerated CRISPR-Cas12a Enabling Live-Cell Monitoring of Mn²⁺ Homeostasis

The CRISPR/Cas12a system has been repurposed as a versatile nuclei acid bio-imaging tool, but its utility in sensing non-nucleic acid analytes in living cells has been less exploited. Herein, we demonstrated the ability of Mn²⁺ to accelerate cleavage kinetics of Cas12a and deployed for live-cell Mn²⁺ sensing by leveraging the accelerated trans-cleavage for signal reporting. In this work, we found that Mn²⁺ could significantly boost both the cis-cleavage and trans-cleavage activities of Cas12a. On the basis of this phenomenon, we harnessed CRISPR-Cas12a as a direct sensing system for Mn²⁺, which achieved robust Mn²⁺ detection in the concentration range of 0.5–700 μM within 15 min in complex biological samples. Furthermore, we also demonstrated the versatility of this system to sense Mn²⁺ in the cytoplasm of living cells. With the usage of a conditional guide RNA, this Cas12a-based sensing method was applied to study the cytotoxicity of Mn²⁺ in living nerve cells, offering a valuable tool to reveal the cellular response of nerve cells to Mn²⁺ disorder and homeostasis.