Ultrasensitive Detection of FEN1 Activity for Cancer Diagnosis Using a CRISPR/Cas13a-Based Triple Cascade Amplification System.

Abstract

Flap endonuclease 1 (FEN1) is closely associated with tumor progression and proliferation, making it a promising biomarker for cancer diagnosis. However, developing a sensitive, reliable, and user-friendly method for quantitative FEN1 detection remains technically challenging. In this study, an ultrasensitive FEN1 biosensor is established using a target-induced cleavage-ligation-transcription-activation cascade strategy (LTACas13a) to enhance the cleavage ability of CRISPR/Cas13a. The LTACas13a method has shown excellent performance in screening FEN1 inhibitors and detecting endogenous FEN1 activity in living cells, as well as in clinical biological samples such as human serum and tissue samples. Additionally, using a universal dumbbell probe derived from FEN1, a multiplex LTACas13a strategy is developed for detecting various DNA glycosylases, including formamidopyrimidine DNA glycosylase, uracil DNA glycosylase, and human alkyl adenine DNA glycosylase. This straightforward approach provides a reliable and effective diagnostic tool for early-stage cancer detection and offers significant opportunities for FEN1 biosensing and related drug discovery.