Topologically constrained DNA-mediated one-pot CRISPR assay for rapid detection of viral RNA with single nucleotide resolution

作者：Yanan Li, Ru Xu, Fenglei Quan, Yonghua Wu, Yige Wu, Yongyuan Zhang, Yan Liang, Zhenzhong Zhang, Hua Gao, Ruijie Deng, Kaixiang Zhang, Jinghong Li · 发表于：EBioMedicine · 年份：2025 · DOI：10.1016/j.ebiom.2025.105564 · 被引用次数：10 · 研究领域：CRISPR and Genetic Engineering、SARS-CoV-2 detection and testing、Advanced biosensing and bioanalysis techniques

Background The widespread and evolution of RNA viruses, such as the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), highlights the importance of fast identification of virus subtypes, particularly in non-laboratory settings. Rapid and inexpensive at-home testing of viral nucleic acids with single-base resolution remains a challenge. Methods Topologically constrained DNA ring is engineered as substrates for the trans-cleavage of Cas13a to yield an accelerated post isothermal amplification. The capacity of CRISPR/Cas13a for discriminating single nucleotide variant (SNV) in viral genome is leveraged by designing synthetic mismatches and hairpin structure in CRISPR RNA (crRNA), enabling robust discrimination of different SARS-CoV-2 variants. Via optimisation of CasTDR 3pot to be one-pot assay, CasTDR 1pot can detect Omicron and its subvariants, with only a few copies in clinical samples in less than 30 min without pre-amplification. Findings The detection system boasts high sensitivity (0.1 aM), single-base specificity, and the advantage of a rapid "sample-to-answer" process, which takes only 30 min. In the detection of SARS-CoV-2 clinical samples and their variant strains, CasTDR 1pot has achieved 100% accuracy. Furthermore, the design of a portable signal-reading device facilitates user-friendly result interpretation. For the detection needs of different RNA viruses, the system can be adapted simply by designing the corresponding crRNA. Interpretation Our study pr...