ArticleJournal of microbiology (Seoul, Korea)2025
Efficient and modular reverse genetics system for rapid generation of recombinant severe acute respiratory syndrome coronavirus 2.
Article in Journal of microbiology (Seoul, Korea), 2025. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
The global spread of COVID-19 has underscored the urgent need for advanced tools to study emerging coronaviruses. Reverse genetics systems have become indispensable for dissecting viral gene functions, developing live-attenuated vaccine candidates, and identifying antiviral targets. In this study, we describe a robust and efficient reverse genetics platform for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). The system is based on the assembly of a full-length infectious cDNA clone from seven overlapping fragments, each flanked by homologous sequences to facilitate seamless assembly using the Gibson assembly method. Individual cloning of each fragment into plasmids enables modular manipulation of the viral genome, allowing rapid site-directed mutagenesis by fragment exchange. Infectious recombinant virus was successfully recovered from the assembled cDNA, exhibiting uniform plaque morphology and genetic homogeneity compared to clinical isolates. Additionally, fluorescent reporter viruses were generated to enable real-time visualization of infection, and the effects of different mammalian promoters on viral rescue were evaluated. This reverse genetics platform enables efficient generation and manipulation of recombinant SARS-CoV-2, providing a valuable resource for virological research and the development of preventive and therapeutic antiviral measures.
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