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bioRxiv · 10.1101/2023.05.11.540343

Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform

Kipfer, E. T.·Hauser, D.·Lett, M. J.·Otte, F.·Urda, L.·Zhang, Y.·Lang, C. M. R.·Chami, M.·Mittelholzer, C.·Klimkait, T.
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Abstract

Reverse genetic systems enable the engineering of RNA virus genomes and are instrumental in studying RNA virus biology. With the recent outbreak of the COVID-19 pandemic, already established methods were challenged by the large genome of SARS-CoV-2. Herein we present an elaborated strategy for the rapid and straightforward rescue of recombinant plus-stranded RNA viruses with high sequence fidelity, using the example of SARS-CoV-2. The strategy called CLEVER (CLoning-free and Exchangeable system for Virus Engineering and Rescue) is based on the intracellular recombination of transfected overlapping DNA fragments allowing the direct mutagenesis within the initial PCR-amplification step. Furthermore, by introducing a linker fragment - harboring all heterologous sequences - viral RNA can directly serve as a template for manipulating and rescuing recombinant mutant virus, without any cloning step. Overall, this strategy will facilitate recombinant SARS-CoV-2 rescue and accelerate its manipulation. Using our protocol, newly emerging variants can quickly be engineered to further elucidate their biology. To demonstrate its potential as a reverse genetics platform for plus-stranded RNA viruses, the protocol has been successfully applied for the cloning-free rescue of recombinant Chikungunya and Dengue virus.

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Kipfer, E. T., Hauser, D., Lett, M. J., Otte, F., Urda, L., Zhang, Y., Lang, C. M. R., Chami, M., Mittelholzer, C., Klimkait, T.. 2023-05-12. Rapid cloning-free mutagenesis of new SARS-CoV-2 variants using a novel reverse genetics platform. https://doi.org/10.1101/2023.05.11.540343

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