Kinetics Accelerated CRISPR-Cas12a Enabling Live-Cell Monitoring of Mn 2+ 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 Mn to accelerate cleavage kinetics of Cas12a and deployed for live-cell Mn sensing by leveraging the accelerated trans-cleavage for signal reporting. In this work, we found that Mn 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 Mn, which achieved robust Mn 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 Mn 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 Mn in living nerve cells, offering a valuable tool to reveal the cellular response of nerve cells to Mn disorder and homeostasis.