Fig. 1: Overview of SARS-CoV-2 reverse genetic system.

The SARS-CoV-2 infectious clone model contains seven cDNA fragments to cover the complete viral genome, to disrupt toxic elements, and to aid in genetic manipulation. The SARS-CoV-2 plasmids are amplified in E. coli and sequentially ligated following digestion with type II restriction enzymes to remove the plasmid backbone. The full-length viral DNA is then in vitro transcribed using T7 polymerase to generate full-length genomic SARS-CoV-2 RNA and electroporated into cells with N-protein transcript expressed in trans. Following electroporation, cells are seeded into cell culture flasks and virus recovered 2–5 d post-electroporation.