Postamplifying Cas12a Activation through Hybridization Chain Reaction-Triggered Fluorescent Nanocluster Formation for Ultrasensitive Nucleic Acid Detection.

CRISPR/Cas12a-based biosensing is advancing rapidly; however, achieving sensitive and cost-effective reporting of Cas12a activation remains a challenge. In response, we have developed a label-free system capable of postamplifying Cas12a activation by integrating hybridization chain reaction (HCR) and DNA-copper nanoclusters (DNA-CuNCs). The trans-cleavage of Cas12a triggers a silenced HCR, leading to the in situ assembly of fluorescent DNA-CuNCs, allowing for the turn-on reporting of Cas12a activation. Without preamplification, this assay can detect DNA with a detection limit of 5 fM. Furthermore, when coupled with preamplification, the system achieves exceptional sensitivity, detecting the monkeypox virus (MPXV) plasmid at 1 copy in human serum. In a MPXV pseudovirus-based validation test, the obtained results are in agreement with those obtained by qPCR, reinforcing the robustness of this method. Our study represents the first effort to manipulate DNA-CuNC formation on HCR for highly sensitive and cost-effective reporting of Cas12a, resulting in an efficient synthetic biology-enabled sensing platform for biosafety applications.

Heparin Specifically Inhibits CRISPR/Cas12 Activation, Enabling Ultrasensitive Heparin Detection and Gene Editing Regulation.

Heparin is a highly sulfated linear glycosaminoglycan that is used as an anticoagulant to prevent and treat thrombotic diseases. Herein, we find that heparin specifically inhibits the activation of the Cas12 protein through the competitive binding of heparin and crRNA to Cas12. Studies illustrate that heparin's high molecular weight and strong negative charge are critical parameters for its inhibitory effect. This unexpected finding was engineered for the detection of heparin, affording a low detection limit of 0.36 ng/mL for fluorometric quantification. We further developed a rapid lateral flow-based system named HepaStrip (heparin strip), allowing heparin monitoring in clinical samples within 20 min. Finally, in vivo investigations revealed that heparin can regulate gene editing with the clusters of the regularly spaced short palindromic repeat (CRISPR)/Cas12 system in Escherichia coli. Heparin blocks the formation of Cas12-crRNA ribonucleoprotein, allowing the application of CRISPR for rapid and field-deployable detection of heparin and unleashing the potential use of heparin in future anti-CRISPR applications.

Active CRISPR-Cas12a on Hydrophilic Metal-Organic Frameworks: A Nanobiocomposite with High Stability and Activity for Nucleic Acid Detection.

CRISPR-Cas12a is an accurate and responsive biosensing technique, but its limited stability has restricted its widespread applications. To address this, we propose a strategy using metal-organic frameworks (MOFs) to protect Cas12a from harsh environments. After screening multiple candidate MOFs, it was found that hydrophilic MAF-7 is highly compatible with Cas12a, and the as-formed Cas12a-on-MAF-7 (COM) not only retains high enzymatic activity but also possesses excellent tolerance to heat, salt, and organic solvents. Further investigation showed that COM can serve as an analytical component for nucleic acid detection, resulting in an ultrasensitive assay for SARS-CoV-2 RNA detection with a detection limit of 1 copy. This is the first successful attempt to create an active Cas12a nanobiocomposite that functions as a biosensor without the need for shell deconstruction or enzyme release.

Advancing sensing technology with CRISPR: From the detection of nucleic acids to a broad range of analytes - A review.

The CRISPR/Cas technology, derived from an adaptive immune system in bacteria, has been awarded the Nobel Prize in Chemistry in 2020 for its success in gene editing. Increasing reports reveal that CRISPR/Cas technology has a wide scope of applications and it could be incorporated into biosensors for detecting critical analytes. CRISPR-powered biosensors have attracted significant research interest due to their advantages including high accuracy, good specificity, rapid response, and superior integrity. Now the CRISPR technology is not only admirable in nucleic acid monitoring, but also promising for other kinds of biomarkers' detection, including metal ions, small molecules, peptides, and proteins. Therefore, it is of great worth to explore the prospect, and summarize the strategies in applying CRISPR technology for the recognition of a broad range of targets. In this review, we summarized the strategies of CRISPR biosensing for non-nucleic-acid analytes, the latest development of nucleic acid detection, and proposed the challenges and outlook of CRISPR-powered biosensors.

DNA-templated silver nanoclusters as an efficient catalyst for reduction of nitrobenzene derivatives: a systematic study.

Nitrobenzene compounds are highly toxic pollutants with good stability, and they have a major negative impact on both human health and the ecological environment. Herein, it was found for the first time that fluorescent DNA-silver nanoclusters (DNA-AgNCs) can catalyze the reduction of toxic and harmful nitro compounds into less toxic amino compounds with excellent tolerance to high temperature and organic solvents. In this study, the reduction of p-nitrophenol (4-NP) as a model was systematically investigated, followed by expending the substrate to disclose the versatility of this reaction. This report not only expanded the conditions for utilizing catalytic reduction conditions of DNA-AgNCs as an efficient catalyst in the control of hazardous chemicals but also widened the substrate range of DNA-AgNCs reduction, providing a new angle for the application of noble metal nanoclusters.

Signal-Amplified Detection of the Tumor Biomarker FEN1 Based on Cleavage-Induced Ligation of a Dumbbell DNA Probe and Rolling Circle Amplification.

Flap endonuclease 1 (FEN1), an endogenous nuclease with the ability to cleave the 5' overhang of branched dsDNA, is of significance in DNA replication and repair. The overexpression of FEN1 is common in cancer because of the ubiquitous upregulation of DNA replication; thus, FEN1 has been recognized as a potential biomarker in oncological investigations. However, few analytical methods targeting FEN1 with high sensitivity and simplicity have been developed. This work developed a signal-amplified detection of FEN1 based on the cleavage-induced ligation of a dumbbell DNA probe and rolling circle amplification (RCA). A flapped dumbbell DNA probe (FDP) was rationally designed with a FEN1 cleavable flap at the 5' end. The cleavage generated a nick site with juxtaposed 5' phosphate and 3' hydroxyl ends, which were linkable by T4 DNA ligase to form a closed dumbbell DNA probe (CDP) with a circular conformation. The CDP functioned as a template for RCA, which produced abundant DNA that could be probed using SYBR Green I. The highly sensitive detection of FEN1 with a limit of detection of 15 fM was achieved, and this method showed high specificity, which enabled the quantification of FEN1 in real samples. The inhibitory effects of chemicals on FEN1 were also evaluated. This study represents the first attempt to develop an FEN1 assay that involves signal amplification, and the novel biosensor method enriches the tools for FEN1-based diagnostics.