Abstract
Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a new member of the Coronaviridae family responsible for the coronavirus disease 19 (COVID-19) pandemic. To date, SARS-CoV-2 has been accountable for over 624 million infection cases and more than 6.5 million human deaths. The development and implementation of SARS-CoV-2 reverse genetics approaches have allowed researchers to genetically engineer infectious recombinant (r)SARS-CoV-2 to answer important questions in the biology of SARS-CoV-2 infection. Reverse genetics techniques have also facilitated the generation of rSARS-CoV-2 expressing reporter genes to expedite the identification of compounds with antiviral activity in vivo and in vitro. Likewise, reverse genetics has been used to generate attenuated forms of the virus for their potential implementation as live-attenuated vaccines (LAV) for the prevention of SARS-CoV-2 infection. Here we describe the experimental procedures for the generation of rSARS-CoV-2 using a well-established and robust bacterial artificial chromosome (BAC)-based reverse genetics system. The protocol allows to produce wild-type and mutant rSARS-CoV-2 that can be used to understand the contribution of viral proteins and/or amino acid residues in viral replication and transcription, pathogenesis and transmission, and interaction with cellular host factors.