Rapid and sensitive RNA detection is of great value in diverse areas, ranging from biomedical research to clinical diagnostics. Existing methods for RNA detection often rely on reverse transcription (RT) and DNA amplification or involve a time-consuming procedure and poor sensitivity. Herein, we proposed a CRISPR/Cas12a-enabled amplification-free assay for rapid, specific, and sensitive RNA diagnostics. This assay, which we termed T7/G4-CRISPR, involved the use of a T7-powered nucleic acid circuit to convert a single RNA target into numerous DNA activators via toehold-mediated strand displacement reaction and T7 exonuclease-mediated target recycling amplification, followed by activating Cas12a trans-cleavage of the linker strands inhibiting split G-Quadruplex (G4) assembly, thereby inducing fluorescence attenuation proportion to the input RNA target. We first performed step-by-step validation of the entire assay process and optimized the reaction parameters. Using the optimal conditions, T7/G4-CRISPR was capable of detecting as low as 3.6 pM target RNA, obtaining ∼100-fold improvement in sensitivity compared with the most direct Cas12a assays. Meanwhile, its excellent specificity could discriminate single nucleotide variants adjacent to the toehold region and allow species-specific pathogen identification. Furthermore, we applied it for analyzing bacterial 16S rRNA in 40 clinical urine samples, exhibiting a sensitivity of 90% and a specificity of 100% when validated by RT-quantitative PCR. Therefore, we envision that T7/G4-CRISPR will serve as a promising RNA sensing approach to expand the toolbox of CRISPR-based diagnostics.
A controllable crRNA self-transcription aided dual-amplified CRISPR-Cas12a strategy (termed CST-Cas12a) was developed for high-senitive and specific biosensing of flap endonuclease 1 (FEN1), a structure-selective nuclease in eukaryotic cells. In this strategy, a branced DNA probe with a 5′ overhanging flap was designed to serve as a hydrolysis substrate for FEN1. The flap cut by FEN1 annealed with a template strand and functioned as a primer for extension reaction to produce a double-stranded DNA (dsDNA) containing T7 promoter and crRNA transcription template. Assisting the T7 RNA polymerase, abundant crRNA was generated and assembled with Cas12a to form Cas12a/crRNA complex, which can be activated by dsDNA trigger and unlock the indiscriminate fluorescent-labeled reporter cleavage. The highly efficient dual signal amplification and near-zero background enabled CST-Cas12a with extraordinary high sensitivity (limit of detection, LOD, 5.2 × 10-6 U μL-1) and 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 high-sensitive and specific detection of FEN1 activity in clinical applications.
A controllable crRNA self-transcription aided dual-amplified CRISPR-Cas12a strategy (termed CST-Cas12a) was developed for high-senitive and specific biosensing of flap endonuclease 1 (FEN1), a structure-selective nuclease in eukaryotic cells. In this strategy, a branced DNA probe with a 5′ overhanging flap was designed to serve as a hydrolysis substrate of FEN1. The flap cut by FEN1 annealed with a template probe and functioned as a primer for extension reaction to produce a double-stranded DNA (dsDNA) containing T7 promoter and crRNA transcription template. Assisting the T7 RNA polymerase, abundant crRNA was generated and assembled with Cas12a to form Cas12a/crRNA complex, which can be activated by dsDNA trigger and unlock the indiscriminate fluorescent-labeled reporter cleavage. The highly efficient dual signal amplification and near-zero background enabled CST-Cas12a with extraordinary high sensitivity. Under optimized conditions, this method allowed the 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 high-sensitive and specific detection of FEN1 activity in clinical applications.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a-based detection strategies with a fluorophore quencher-labeled ssDNA reporter or gold nanoparticle ssDNA reporter have been widely used in point-of-care (POC) molecular diagnostics. However, the potential of these CRISPR/Cas12a strategies for POC molecular diagnostics is often compromised due to the complex labeling, high cost, and low signal-to-noise ratio. Herein, we show a pre-folded G-quadruplex (G4) structure with tunable tolerance to CRISPR/Cas12a trans-cleavage and explore its mechanism. Two G4 structures (i.e., Tel22-10 and G16C) sensitive or tolerant to CRISPR/Cas12a trans-cleavage are designed and used as signal elements to fabricate a label-free visible fluorescent strategy or "signal-on" colorimetric strategy, respectively. These two strategies facilitate an ultrasensitive visual nucleic acid determination of Group B Streptococci with a naked-eye limit of detection of 1 aM. The feasibility of the developed G4-assisted CRISPR/Cas12a strategies for real-world applications is demonstrated in clinical vaginal/anal specimens and further verified by a commercial qPCR assay. This work suggests that the proposed G4 structures with tunable tolerance can act as promising signal reporters in the CRISPR/Cas12a system to enable ultrasensitive visible nucleic acid detection.
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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