posted on 2022-10-14, 17:37authored byYang Song, Ke Gao, Xiaoying Cai, Wei Cheng, Shijia Ding, Decai Zhang, Shixiong Deng
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.