Structure-switchable dsDNA promoter regulates the activity of CRISPR-Cas12a for APE1 detection

Apurinic/apyrimidinic endonuclease 1 (APE1) has been considered as a promising biomarker since it is associated with numerous human diseases, involving neurodegenerative diseases and cancer. However, current APE1 detection methods mainly rely on immunology-based methods, which are burdened by time-consuming and procedural complexity. To overcome these shortcomings, we have developed an innovative all-in-one technique that simplifies APE1 detection by integrating enzyme-responsive elements structure-switchable dsDNA promoter with CRISPR/AsCas12a methodology, namely EDC. In this work, the structure-switchable dsDNA promoter has been well-designed to trigger the site-directed incision of APE1 and then release the split activator to illumine the CRISPR/AsCas12a catalyst system by coupling it with another truncated activator. Under optimal circumstances, the proposed strategy enables sensitive detection of the target APE1 with a detection limit of 4.8 × 10^-5 U/mL and a wide linear range from 5.0 × 10^-5 to 1.0 × 10^-1 U/mL. Moreover, this strategy could be gratifyingly applied to screen APE1 inhibitors and monitor APE1 in lysates from cell extractions or clinical serum samples. Overall, this study presents a novel approach that utilizes dsDNA promoter as programmable switching components, effectively enhancing CRISPR/Cas12a-based diagnostic platforms and demonstrating the significant potential for clinical translation.