Highly-sensitive and logic platform based on spatially-constrained T7 transcription enhanced Cas13a for DNA repair enzyme detection and intracellular imaging.

The activity of DNA repair enzymes, particularly Flap endonuclease 1 (FEN1) and apurinic/apyrimidinic endonuclease 1 (APE1), plays a critical role in disease prevention, diagnosis, and prognosis. Accurate detection of these enzymes is therefore essential. Recent advancements in CRISPR-Cas technology, particularly its programmable and trans-cleavage activity, have paved the way for the development of innovative detection methods. However, there is a need for a simple, low-background, highly sensitive detection platform with logical capabilities for FEN1 and APE1. In this study, we present a novel detection platform that integrates spatially constrained T7 transcription with the CRISPR-Cas13a system. This biosensor minimizes background interference and achieves high sensitivity, with limits of detection as low as 5 × 10 U/μL for FEN1 and 2 × 10 U/μL for APE1, making it one of the most sensitive methods available for detecting these enzymes. The platform supports both OR and logic detection, offering enhanced versatility. It demonstrates robustness by detecting FEN1 activity at concentrations as low as 1 cell/μL and screening enzyme inhibitors. Additionally, the system was successfully used for intracellular imaging of FEN1 activity in cells and reliably measured APE1 activity in ovarian tissue samples, confirming its clinical applicability. This biosensor represents a promising tool for detecting FEN1 and APE1, further expanding the potential of CRISPR-Cas13a in diagnostic applications.