Controllable crRNA Self-Transcription Aided Dual-Amplified CRISPR-Cas12a Strategy for Highly Sensitive Biosensing of FEN1 Activity

A controllable crRNA self-transcription aided dual-amplified CRISPR-Cas12a strategy (termed CST-Cas12a) was developed for highly sensitive and specific biosensing of flap endonuclease 1 (FEN1), a structure-selective nuclease in eukaryotic cells. In this strategy, a branched DNA probe with a 5′ overhanging flap was designed to serve as a hydrolysis substrate of FEN1. The flap cut by FEN1 was annealed with a template probe and functioned as a primer for an extension reaction to produce a double-stranded DNA (dsDNA) containing a T7 promoter and crRNA transcription template. Assisting the T7 RNA polymerase, abundant crRNA was generated and assembled with Cas12a to form a Cas12a/crRNA complex, which can be activated by a dsDNA trigger and unlock the indiscriminate fluorophore–quencher reporter cleavage. The highly efficient dual signal amplification and near-zero background enabled CST-Cas12a with extraordinarily high sensitivity. Under optimized conditions, this method allowed highly sensitive biosensing of FEN1 activity in the range of 1 × 10^–5 U μL^–1 to 5 × 10^–2 U μL^–1 with a detection limit of 5.2 × 10^–6 U μL^–1 and achieved excellent specificity for FEN1 in the presence of other interfering enzymes. The inhibitory capabilities of chemicals on FEN1 were also investigated. Further, the newly established CST-Cas12a strategy was successfully applied to FEN1 biosensing in complex biological samples, which might be a reliable biosensing platform for highly sensitive and specific detection of FEN1 activity in clinical applications.