Novel 'CLEVER' method accelerates engineering and genetic study of SARS-CoV-2 and its variants biorxivpreprint UniBasel_en genetics geneticengineering covid SARSCoV2 variants reversegentics
By Tarun Sai LomteMay 16 2023Reviewed by Lily Ramsey, LLM A recent study posted to the bioRxiv*preprint server described rapid mutagenesis and rescue of severe acute respiratory syndrome coronavirus 2 variants without cloning, using a novel reverse genetics strategy.
The study and findings In the present study, researchers developed and described an ISA-based strategy termed ‘cloning-free and exchangeable system for virus engineering and rescue’ , using SARS-CoV-2. Additionally, the team tested the impact of transfection using four fragments on reconstitution fidelity by comparing replication competence between wild-type isolates and recombinant viruses. The size and shape of the wild-type virus and rCOV2 generated from four fragments were similar.
The researchers introduced a genetic marker site to differentiate recombinant viruses from accidental contamination with clinical isolates. This marker was present in all reconstituted viruses. The amplification step could be exploited for mutagenesis without de novo synthesis or cloning. The team designed an oligonucleotide pair that introduced N501Y or G614D. The fragments harboring these point mutations were co-transfected with other fragments required for genome assembly.
One hundred base pairs of viral 3’ and 5’ untranslated regions flanked the linker fragment on either side. It was designed for successful recombination into a circular DNA product. Eight SARS-CoV-2 genomic fragments were amplified in a single step from the viral RNA. The amplicons were co-transfected with the linker fragment. Viable viruses were rescued on seven DPT.
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