Nicking enzyme assisted amplification combined with CRISPR-Cas12a system for one-pot sensitive detection of APE1†

Abstract

Apurinic/apyrimidinic endonuclease 1 (APE1) is a critical enzyme in the base excision repair (BER) pathway, essential for preserving cellular equilibrium. Variations in APE1 activity within blood or tissues can provide significant insights for clinical cancer screening and disease diagnosis. Consequently, the detection of APE1 activity is critical for clinical diagnostics. However, there is currently a deficiency in rapid, straightforward, and sensitive methods for APE1 detection. To address this issue, we developed a method that integrates Nicking Enzyme Assisted Amplification (NEAA) with CRISPR-Cas12a signal amplification, enabling one-pot detection of APE1 activity. This method utilizes NEAA to produce a substantial quantity of target DNA that is complementary to the crRNA, thereby triggering the trans-cleavage activity of Cas12a. The activated Cas12a then amplifies and emits signals by cleaving the reporter probe. Our strategy allows for the swift and precise detection of APE1, in only 3 h, with a detection threshold of 1 × 10⁻⁶ U mL⁻¹ and a linear detection range of 5 × 10⁻⁶ to 0.1 U mL⁻¹. It has been effectively utilized for the detection of APE1 in biological samples.