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

Abstract

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.

CRISPR/Cas12a-enhanced single-molecule counting for sensitive detection of flap endonuclease 1 activity at the single-cell level

“…16 To further improve the assay sensitivity, a series of nucleic acid amplification strategies have been introduced for amplified sensing of FEN1, but they often require the cooperation of multiple enzymes ( e.g. , ligase, DNA polymerase, nicking enzyme, and RNA polymerase) 17–23 and suffer from high background derived from target-independent amplification. The development of simple, selective, and sensitive methods for FEN1 assay is still highly required.…”

“…However, many fluorescence FEN1 assays require complicated procedures for the synthesis of functional nanomaterials such as graphene oxide, 13 gold nanostars, 14 metal-organic frameworks, 15 and silver nanoclusters. 16 To further improve the assay sensitivity, a series of nucleic acid amplification strategies have been introduced for amplified sensing of FEN1, but they often require the cooperation of multiple enzymes (e.g., ligase, DNA polymerase, nicking enzyme, and RNA polymerase) [17][18][19][20][21][22][23] and suffer from high background derived from target-independent amplification. The development of simple, selective, and sensitive methods for FEN1 assay is still highly required.…”

“…This biosensor exploits single CRISPR/Cas12a enzyme to amplify the target signal directly and efficiently, avoiding the complicated reaction schemes and non-specific amplification involved in the nucleic acid amplification-based FEN1 assays. [17][18][19][20][21][22][23] Moreover, the assembly of multiple HP1/HP2 duplexes on a single MB can enhance the local concentration of probes, 34 greatly improving the FEN1 catalytic efficiency. In addition, the introduction of a magnetic separation strategy can significantly avoid the interferences from the uncleaved probes and complex biological samples.…”