ArticlebioRxiv : the preprint server for biology2022
An Optimized Circular Polymerase Extension Reaction-based Method for Functional Analysis of SARS-CoV-2.
Article in bioRxiv : the preprint server for biology, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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Abstract
Reverse genetics systems have been crucial for studying specific viral genes and their relevance in the virus lifecycle, and become important tools for the rational attenuation of viruses and thereby for vaccine design. Recent rapid progress has been made in the establishment of reverse genetics systems for functional analysis of SARS-CoV-2, a coronavirus that causes the ongoing COVID-19 pandemic that has resulted in detrimental public health and economic burden. Among the different reverse genetics approaches, CPER (circular polymerase extension reaction) has become one of the leading methodologies to generate recombinant SARS-CoV-2 infectious clones due to its accuracy, efficiency, and flexibility. Here, we report an optimized CPER methodology which, through the use of a modified linker plasmid and by performing DNA nick ligation and direct transfection of permissive cells, overcomes certain intrinsic limitations of the 'traditional' CPER approaches for SARS-CoV-2, allowing for efficient virus rescue. This optimized CPER system may facilitate research studies to assess the contribution of SARS-CoV-2 genes and individual motifs or residues to virus replication, pathogenesis and immune escape, and may also be adapted to other viruses.
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