Sensitive Measurement of ALKBH3 Activity using a Dual DNA Amplifier by Coupling DNA Circuits with CRISPR/Cas12a

Abstract DNA repair enzyme ALKBH3 has shown great potential as diagnostic as well as therapeutic target for several cancers. However, methods for the sensitive measurement of ALKBH3 repair activity are highly desirable but challenging. Herein, we report a dual DNA amplifier by integrating catalytic hairpin assembly (CHA) with CRISPR/Cas12a for highly sensitive monitoring of ALKBH3 activity. A 3MeC‐modified initiator DNA was designed as the substrate of ALKBH3. Upon ALKBH3‐mediated DNA repair to remove the lesion from initiator DNA, it effectively triggered CHA circuits to generate duplexes that could be recognized by CRISPR/Cas12a to activate the trans‐cleavage activity, resulting significant fluorescence increase. We showed that our method could sensitively detection ALKBH3 activity with a calculated detection limit of 30.5 pM. We also showed that it allowed efficient screening of enzyme inhibitors. Our method enabled specific and sensitive measurement of ALKBH3 activity, thus providing a useful tool for disease diagnostics and therapeutic evaluation.