A photocontrolled one-pot isothermal amplification and CRISPR-Cas12a assay for rapid detection of SARS-CoV-2 Omicron variants

作者：Qian Sun, Hongqing Lin, Yuan Li, Liping Yuan, Baisheng Li, Yunan Ma, Haiying Wang, Xiaoling Deng, Hongliang Chen, Shixing Tang · 发表于：Microbiology Spectrum · 年份：2024 · DOI：10.1128/spectrum.03645-23 · 被引用次数：18 · 研究领域：CRISPR and Genetic Engineering、Advanced biosensing and bioanalysis techniques、Mosquito-borne diseases and control

CRISPR-Cas technology has widely been applied to detect single-nucleotide mutation and is considered as the next generation of molecular diagnostics. We previously reported the combination of nucleic acid amplification (NAA) and CRISPR-Cas12a system to distinguish major severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants. However, the mixture of NAA and CRISPR-Cas12a reagents in one tube could interfere with the efficiency of NAA and CRISPR-Cas12a cleavage, which in turn affects the detection sensitivity. In the current study, we employed a novel photoactivated CRISPR-Cas12a strategy integrated with recombinase polymerase amplification (RPA) to develop one-pot RPA/CRISPR-Cas12a genotyping assay for detecting SARS-CoV-2 Omicron sub-lineages. The new system overcomes the potential inhibition of RPA due to early CRISPR-Cas12a activation and cleavage of the target template in traditional one-pot assay using photocleavable p-RNA, a complementary single-stranded RNA to specifically bind crRNA and precisely block Cas12a activation. The detection can be finished in one tube at 39℃ within 1 h and exhibits a low limit of detection of 30 copies per reaction. Our results demonstrated that the photocontrolled one-pot RPA/CRISPR-Cas12a assay could effectively identify three signature mutations in the spike gene of SARS-CoV-2 Omicron variant, namely, R346T, F486V, and 49X, and distinguish Omicron BA.1, BA.5.2, and BF.7 sub-lineages. Furthermore, the assay achieved a sensit...