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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.

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Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas12a, which exhibits excellent target DNA-activated trans-cleavage activity under the guidance of a programmable CRISPR RNA (crRNA), has shown great promise in next-generation biosensing technology. However, current CRISPR-Cas12a-based biosensors usually improve sensitivity by the initial nucleic acid amplification, while the distinct programmability and predictability of the crRNA-guided target binding process has not been fully exploited. Herein, we, for the first time, propose a modular and sensitive CRISPR-Cas12a fluorometric aptasensor by integrating an enzyme-free and robust crRNA-mediated catalytic nucleic acid network, namely, Cas12a-CMCAN, in which crRNA acts as an initiator to actuate cascade toehold-mediated strand displacement reactions (TM-SDRs). As a proof of concept, adenosine triphosphate (ATP) was selected as a model target. Owing to the multiturnover of CRISPR-Cas12a trans-cleavage and the inherent recycling amplification network, this method achieved a limit of detection value of 0.16 μM (20-fold lower than direct Cas12a-based ATP detection) with a linear range from 0.30 to 175 μM. In addition, Cas12a-CMCAN can be successfully employed to detect ATP levels in diluted human serum samples. Considering the simplicity, sensitivity, and easy to tune many targets by changing aptamer sequences, the Cas12a-CMCAN sensing method is expected to offer a heuristic idea for the development of CRISPR-Cas12a-based biosensors and unlock its potential for general and convenient molecule diagnostics.

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