DOI: 10.1101/2023.05.11.540343

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

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

E. T.Kipfer D. Hauser M. J. Lett ...+6 T. Klimkait

摘要

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.

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