Establishment of a reverse genetics system for SARS-CoV-2 using circular polymerase extension reaction.

Cell Rep

Department of Molecular Virology, Research Institute for Microbial Diseases, Osaka University, Suita, Osaka 565-0871, Japan; Center for Infectious Diseases Education and Research, Osaka University, Suita, Osaka 565-0871, Japan. Electronic address:

Published: April 2021

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has been identified as the causative agent of coronavirus disease 2019 (COVID-19). Although multiple mutations have been observed in SARS-CoV-2, functional analysis of each mutation of SARS-CoV-2 has been limited by the lack of convenient mutagenesis methods. In this study, we establish a PCR-based, bacterium-free method to generate SARS-CoV-2 infectious clones. Recombinant SARS-CoV-2 could be rescued at high titer with high accuracy after assembling 10 SARS-CoV-2 cDNA fragments by circular polymerase extension reaction (CPER) and transfection of the resulting circular genome into susceptible cells. The construction of infectious clones for reporter viruses and mutant viruses could be completed in two simple steps: introduction of reporter genes or mutations into the desirable DNA fragments (∼5,000 base pairs) by PCR and assembly of the DNA fragments by CPER. This reverse genetics system may potentially advance further understanding of SARS-CoV-2.

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Source
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8015404PMC
http://dx.doi.org/10.1016/j.celrep.2021.109014DOI Listing

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