CRISPR/Cas12a-based hypochlorous acid and myeloperoxidase biosensors designed on RESET effect

Both hypochlorous acid (HOCl) and HOCl-related myeloperoxidase (MPO) play important roles in disease diagnosis and treatment. Herein, CRISPR/Cas12a-based biosensors were developed for the facile, sensitive and specific detection of HOCl and MPO. Based on the RESET effect recently reported by our group, a hairpin DNA with optimized sequence and a phosphorothioate-modified site was elaborately designed. Via specific recognition and cleavage of phosphorothioate-modified site by HOCl, the hairpin DNA without Cas12a activation capability was converted to highly efficient Cas12a activator, activating the trans -cleavage activity of Cas12a to achieve the fluorescence turn-on detection of HOCl. The introduction of RESET effect endowed the sensing system with extremely low background and thus high detection sensitivity. By further utilizing MPO-catalyzed H2O2 -Cl - reaction to produce HOCl, the proposed sensing system could be easily extended to the MPO quantitation. Due to the dual signal amplification of cascaded catalytic reactions (MPO-catalyzed HOCl production and CRISPR/Cas12a-catalyzed fluorescence enhancement), the MPO biosensor gave a high sensitivity with a detection limit as low as 0.67 ng/mL, and was demonstrated to work well for screening MPO inhibitors and evaluating their inhibition capabilities, thus showing great promise in clinical diagnosis and drug screening applications.