Reporter-expressing recombinant virus represents an excellent option and a powerful tool to investigate, among others, viral infection, pathogenicity, and transmission, as well as to identify therapeutic compounds that inhibit viral infection and prophylactic vaccines. To combat the ongoing coronavirus disease 2019 (COVID-19) pandemic, we have established a robust bacterial artificial chromosome (BAC)-based reverse genetics (RG) system to rapidly generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) to study the contribution of viral proteins in viral pathogenesis. In addition, we have engineered reporter-expressing recombinant viruses in which we placed the reporter genes upstream of the viral nucleocapsid (N) gene to promote high levels of reporter gene expression, which facilitates the study of SARS-CoV-2 in vitro and in vivo. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the entire SARS-CoV-2 genome (~30,000 nt) is challenging. Herein, we provide the technical details to engineer rSARS-CoV-2 using the BAC-based RG approach. We describe (i) assembly of the full-length (FL) SARS-CoV-2 genome sequences into the empty pBeloBAC, (ii) verification of pBeloBAC-FL, (iii) cloning of a Venus reporter gene into pBeloBAC-FL, and (iv) recovery of the Venus-expressing rSARS-CoV-2. By following this protocol, researchers with knowledge of basic molecular biology and gene engineering techniques will be able to generate wild-type (WT) and reporter-expressing rSARS-CoV-2. IMPORTANCE We have established a bacterial artificial chromosome (BAC)-based RG system to generate recombinant severe acute respiratory syndrome coronavirus 2 (rSARS-CoV-2) and to engineer reporter-expressing recombinant viruses to assess viral infection in vitro and in vivo. To date, we have shared our BAC-based RG system with more than 100 laboratories around the world, which has helped to expedite investigations with SARS-CoV-2. However, genetic manipulation of the BAC containing the full-length SARS-CoV-2 genome of ~30,000 nucleotides is challenging. Here, we provide all the detailed experimental steps required for the successful generation of wild-type (WT) recombinant SARS-CoV-2 (rSARS-CoV-2). Likewise, we provide a comprehensive protocol on how to generate and rescue rSARS-CoV-2 expressing high levels of a Venus fluorescent reporter gene from the locus of the viral nucleocapsid (N) protein. By following these protocols, researchers with basic knowledge in molecular biology will be able to generate WT and Venus-expressing rSARS-CoV-2 within 40 days.
Keywords: BAC; COVID-19; SARS-CoV-2; bacterial artificial chromosome; coronavirus; pBeloBAC; recombinant virus; reporter genes; reverse genetics.