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

Abstract

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.

Single-cycle SARS-CoV-2 vaccine elicits high protection and sterilizing immunity in hamsters

“…1A, fig. S1A, from design to vaccine virus in ~4 weeks) (Kipfer et al, 2023;Melade et al, 2022). ΔE G 68 and ΔE G were efficiently rescued in Ecomplementing HEK293T cells (HEK293T-indE) and propagated in a Vero E6-based cell line stably expressing the E protein (VeroE2T).…”

“…S2). SCVs were monitored by antigen quick-tests (Kipfer et al, 2023) and quantified in focus formation assays (FFA). The precise deletion of the three intended genes in vaccine virus candidate ΔE G 68 and of the E-gene in ΔE G and their stable functional elimination were verified after repeated passage in VeroE2T cells by NGS and Sanger sequencing .…”