One-Pot Detection of Biomarker Apurinic/Apyrimidinic Endonuclease 1 Based on the Modified-crRNA Regulated Trans-Cleavage Activity of CRISPR/Cas12a.

Apurinic/apyrimidinic endonuclease 1 (APE1), a critical protein in DNA repair, plays indispensable roles in the maintenance of cellular homeostasis, thereby garnering significant attention as a biomarker and therapeutic target for various disorders. Current APE1 sensing methods always require multiple enzymes or complex signal amplification. The high programmability of the CRISPR/Cas12-based signal amplifier provides a new chance for developing biosensors. In this study, we introduce a novel method for the detection of APE1 by leveraging the discovery that modulating the length of modified DNA within CRISPR RNA (crRNA) enables precise control over the trans-cleavage activity of CRISPR/Cas12a. By designing a specific crRNA, the APE1-mediated activity recovery of Cas12a (ARC) was developed for rapid, specific, and one-pot detection of APE1. ARC presented a detection limit of 1.74 × 10 U/μL with high specificity in detecting APE1 in biological samples. Besides, this simple method was feasible for APE1 inhibition assays, highlighting its potential for inhibitor screening and evaluation. Collectively, our findings present an innovative approach for APE1 activity analysis and expand the CRISPR-based non-nucleic acid target sensing toolbox through a novel crRNA design.