Click-chemistry-mediated modulation of CRISPR-Cas12a activity through activator modification

Chemical modification strategies offer a promising route for spatiotemporal regulation of CRISPR-Cas12a activity in molecular diagnostics. However, existing methods involve CRISPR RNA with photolabile groups that suffer from complexity and RNA instability. To address these limitations, we report a simple and robust strategy using dibenzocyclooctyne (DBCO)-mediated click chemistry to modulate CRISPR-Cas12a activity. The copper-free strain-promoted azide-alkyne cycloaddition reaction enables CRISPR-Cas12a modulation with low toxicity, biocompatibility, and high selectivity. Utilizing azide-modified non-target DNA strand sequences at different locations to react with DBCO, we show that DBCO-modified activators can regulate CRISPR-Cas12a cleavage in three distinct states: maintain, enhance, and suppress. Mechanistic studies through cleavage kinetics and molecular docking reveal that the regulatory outcome depends on the modification position, protospacer adjacent motif composition, and DBCO concentration. We further employ asymmetric polymerase chain reaction to generate azide-modified DNA for click-chemistry-mediated modulation of CRISPR-Cas12a. This strategy could be a promising tool for regulating CRISPR-Cas12a activity in molecular diagnostics.