Breakthrough in LbCas12a Chemical Inhibitor Screening: The Firefly Probe and Its Application in Dual-Mode Biosensors

Biosensors employing anti-CRISPR (Acr) proteins have been designed, and prohibitive manufacturing expenses and unfavorable storage conditions still restrict Acr protein applications. Since chemical inhibitors have lower production costs and are relatively insensitive to storage conditions, they are more accessible and easier to store and transport than protein inhibitors. Furthermore, they could be efficiently manufactured industrially for future applications. To screen the chemical inhibitors of CRISPR/Cas12a, we constructed a label-free fluorescent probe, dubbed the firefly probe. Then, three highly effective chemical inhibitors (H₂O₂, catechol, and hydroquinone) against LbCas12a were discovered based on this probe. These small molecule inhibitors could preferentially bind with LbCas12a to interrupt the assembly of the LbCas12a/crRNA binary complex. Thus, the cis- and trans-cleavage activities of LbCas12a were simultaneously inhibited. The inhibitory mechanisms were also explored through electrophoresis and molecular docking experiments. Subsequently, a universal sensing platform based on the inhibitor H₂O₂ was built, which enabled fluorescence analysis of catalase and glucose. The limits of detection for catalase and glucose in the fluorescence mode were as low as 0.080 U/mL and 0.0059 mM, respectively. Furthermore, the platform supported visual analysis by integrating smartphones with the RGB measurement software. In summary, small molecule inhibitors of LbCas12a were found, and their inhibition mechanism was identified in this research. These inhibitors can serve as efficient tools for blocking the CRISPR/Cas12a system and facilitate the application of LbCas12a inhibitors in biosensors.