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

Reverse genetic systems enable engineering of RNA virus genomes and are instrumental to study 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 template for manipulation and rescue of recombinant mutant virus, without any cloning-step needed. 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 its biology.