A one-pot method for universal Dengue virus detection by combining RT-RPA amplification and CRISPR/Cas12a assayZhang, Xiang, Hou
et alBMC Microbiol (2025) 25 (1), 163
Abstract: Dengue Virus (DENV) is a life-threatening pathogen leading to dengue fever, which brings about huge public health challenges globally. However, traditional detection methods currently fail to meet the increasing demands of clinic practice in terms of speed, simplicity, and accuracy. To address these limitations, we developed a novel, rapid, and highly sensitive diagnostic method for universal DENV detection by integrating recombinase polymerase amplification (RPA) assay and the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) and associated (Cas) protein 12a (CRISPR/Cas12a) system into one-pot. This approach achieves exceptional sensitivity and specificity for DENV detection, with the entire process completed within 40 min, without the need for sophisticated equipment. The limit of detection (LOD) was determined to be 91.7 copies/test. Using this one-pot RT-RPA CRISPR/Cas12a detection system, all four serotypes of DENV (1 to 4) were successfully identified. In terms of specificity, the assay accurately detected DENV-infected positive samples without cross-reactivity with four other interfering viruses-infected samples (VSV, SeV, HSV-1 and IAV). Furthermore, we established a universal DENV RT-RPA-CRISPR/Cas12a-lateral flow dipstick (LFD) platform, which successfully identified all four serotypes of DENV with a sensitivity of approximately 250 copies/test. Collectively, our method not only provides a robust alternative for universal DENV detection but also offers valuable insights for the identification of other viruses.© 2025. The Author(s).
Portable DNA extraction integrated with LAMP-CRISPR/Cas12a technology for on-site detection of Salmonella TyphimuriumZhang, Zhang, Liu
et alNPJ Sci Food (2025) 9 (1), 39
Abstract: The infection and outbreak of Salmonella typhimurium (S. typhimurium) highlight the need for developing a reliable on-site detection strategy fitting to various settings. However, due to the requirement of specialized instruments and trained personnel, traditional detection methods have to be implemented in laboratories and are not ideal for on-site applications. To achieve a sample-to-answer and field-deployable detection for S. typhimurium, we developed an integrated nucleic acid detection platform combining single-vial of loop-mediated isothermal amplification (LAMP)-clustered regularly interspaced short palindromic repeat (CRISPR)/Cas12a system, portable device and smartphone app. This platform enables the extraction, concentration, and purification of DNA, amplification of the target, and output of visual fluorescent signals within 1 h. With this detection platform, 102 CFU/mL of S. typhimurium in food and environmental matrix was able to be accurately detected. This method demonstrated excellent selectivity. Also, an auxiliary smartphone application was developed to achieve simplified result interpretation. Our method exhibited potential to better control and respond to outbreaks of foodborne diseases, especially in low-resource settings.© 2025. The Author(s).
Structure-switching G-quadruplex: An efficient CRISPR/Cas12a signal reporter for label-free colorimetric biosensingChen, Lv, Wang
et alInt J Biol Macromol (2025)
Abstract: G-quadruplex is widely used as a signal reporter for colorimetric biosensor construction. However, the effectiveness of CRISPR/Cas12a in trans-cleaving G-quadruplexes is significantly influenced by their resistance to nuclease, resulting in a weak colorimetric signal response. Herein, a structure-switching G-quadruplex regulated by transducer DNA is used as a signal reporter to construct CRISPR/Cas12a-based biosensors. The transducer DNA lacks a stable secondary structure, enabling efficient cleavage by CRISPR/Cas12a, which subsequently affects the catalytic activity of the G-quadruplex/hemin DNAzyme. We used microRNAs (miRNAs) and ATP as model targets to develop a label-free colorimetric detection platform. By optimizing the DNA sequences and reaction conditions, the biosensors exhibit excellent detection selectivity and sensitivity. The reliability of the proposed method was validated by its consistency with RT-qPCR for miRNAs detection and a commercial chemiluminescence kit for ATP assay, demonstrating its potential in clinical diagnosis and bioanalytical studies. The assay is concise and cost-effective because it does not require DNA labeling, magnetic separation, or enzymatic DNA amplification. Our design strategy avoids the use of G-quadruplex as a cleavage substrate for CRISPR/Cas12a while ensuring an efficient response of the G-quadruplex/hemin DNAzyme to CRISPR/Cas12a system, addressing the issue of G-quadruplex resistance to CRISPR/Cas12a nuclease activity.Copyright © 2025. Published by Elsevier B.V.
General and robust sample preparation strategies for cryo-EM studies of CRISPR-Cas9 and Cas12 enzymesOmura, Nureki
Methods Enzymol (2025) 712, 23-39
Abstract: Cas9 and Cas12 are RNA-guided DNA endonucleases derived from prokaryotic CRISPR-Cas adaptive immune systems that have been repurposed as versatile genome-engineering tools. Computational mining of genomes and metagenomes has expanded the diversity of Cas9 and Cas12 enzymes that can be used to develop versatile, orthogonal molecular toolboxes. Structural information is pivotal to uncovering the precise molecular mechanisms of newly discovered Cas enzymes and providing a foundation for their application in genome editing. In this chapter, we describe detailed protocols for the preparation of Cas9 and Cas12 enzymes for cryo-electron microscopy. These methods will enable fast and robust structural determination of newly discovered Cas9 and Cas12 enzymes, which will enhance the understanding of diverse CRISPR-Cas effectors and provide a molecular framework for expanding CRISPR-based genome-editing technologies.Copyright © 2025. Published by Elsevier Inc.
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