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High-Fidelity and cost-effective engineering of SARS-CoV-2

Efficient reverse genetics systems are essential for understanding SARS-CoV-2 pathogenesis, host-virus interactions, and potential therapeutic interventions. Here, we developed a cost-effective PCR-based reverse genetics platform that splits the SARS-CoV-2 genome into only six bacterial plasmids, en...

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Main Authors: Olguin-Nava, Marco (Author) , Hennig, Thomas (Author) , Börtlein, Charlene (Author) , Bohn, Patrick (Author) , Ambi, Uddhav B. (Author) , Gabel, Alexander (Author) , Günter, Lina M. (Author) , Gribling-Burrer, Anne-Sophie (Author) , Schmidt, Nora (Author) , Caliskan, Neva (Author) , Dölken, Lars (Author) , Munschauer, Mathias (Author) , Smyth, Redmond P. (Author)
Format: Article (Journal)
Language:English
Published: 11 December 2025
In: Viruses
Year: 2025, Volume: 17, Issue: 12, Pages: 1-31
ISSN:1999-4915
DOI:10.3390/v17121604
Online Access:Verlag, kostenfrei, Volltext: https://doi.org/10.3390/v17121604
Verlag, kostenfrei, Volltext: https://www.mdpi.com/1999-4915/17/12/1604
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Author Notes:Marco Olguin-Nava, Thomas Hennig, Charlene Börtlein, Patrick Bohn, Uddhav B. Ambi, Alexander Gabel, Lina M. Günter, Anne-Sophie Gribling-Burrer, Nora Schmidt, Neva Caliskan, Lars Dölken, Mathias Munschauer and Redmond P. Smyth
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Summary:Efficient reverse genetics systems are essential for understanding SARS-CoV-2 pathogenesis, host-virus interactions, and potential therapeutic interventions. Here, we developed a cost-effective PCR-based reverse genetics platform that splits the SARS-CoV-2 genome into only six bacterial plasmids, enabling cloning, manipulation, and the rescue of recombinant SARS-CoV-2 (rSARS-CoV-2) with high fidelity and high viral titers after a single passage. Using this system, we generated and characterized spike protein mutants Y453F and N501Y, as well as a U76G mutation in the 5′-UTR. Y453F showed reduced replication kinetics, lower cell binding, and diminished fitness, while N501Y exhibited comparable replication and fitness, highlighting the distinct effects of these spike protein mutations. The U76G mutation is located within a novel NSP9 binding site in the 5′-UTR and leads to impaired RNA synthesis and reduced viral replication efficiency, suggesting an important role in transcription and replication. Our findings highlight the robustness and adaptability of this reverse genetics system, providing a versatile, cost-effective tool for studying SARS-CoV-2 mutations and their effects on replication and fitness, with potential applications in vaccine and therapeutic development.
Item Description:Gesehen am 02.06.2026
Physical Description:Online Resource
ISSN:1999-4915
DOI:10.3390/v17121604

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