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Bacterial Artificial Chromosome Reverse Genetics Approaches for SARS-CoV-2

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Reverse Genetics of RNA Viruses

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2733))

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.

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Acknowledgments

Research on SARS-CoV-2 in L.M-S laboratory was partially supported by grants W81XWH2110103 and W81XWH2110095 from the Department of Defense (DoD) Peer Reviewed Medical Research Program (PRMRP); 1R43AI165089-01, 1R01AI161363-01, and 1R01AI161175-01A1 from the National Institutes of Health (NIH); the Center for Research on Influenza Pathogenesis and Transmission (CRIPT), one of the National Institute of Allergy and Infectious Diseases (NIAID)-funded Centers of Excellence for Influenza Research and Response (CEIRR; contract # 75N93021C00014); the Antiviral Countermeasures Development Center (AC/DC) (1U19AI171403-01), the Center for Antiviral Medicines & Pandemic Preparedness (CAMPP) (1U19AI171443-01), and the QCRG Pandemic Response Program (1U19AI171110-01), three of the National Institutes of Health (NIH)-funded Antiviral Drug Discovery Centers for Pathogens of Pandemic Concern; the San Antonio Partnership for Precision Therapeutics; and the San Antonio Medical Foundation. Research in L.M-S was also partially supported by NIH R01AI145332, R01AI142985, and R01AI141607 grants and by DoD W81XWH1910496 PRMRP grant.

Materials Availability

The pBeloBAC11-SARS-CoV-2 BAC clone described in this book chapter for the generation of rSARS-CoV-2 is available at this website: https://www.txbiomed.org/services-2/reverse-genetics-plasmids. Other BAC clones as well as their respective rSARS-CoV-2 are also available at the same website.

Conflict of Interest

C.Y, F.A, and L. M.-S are co-inventors on a patent application directed to BAC-based reverse genetics approaches to generate rSARS-CoV-2.

Author information

Authors and Affiliations

  1. Texas Biomedical Research Institute, San Antonio, TX, USA

    Kevin Chiem, Chengjin Ye & Luis Martínez-Sobrido

  2. Centro de Investigación en Sanidad Animal (CISA-INIA/CSIC), Madrid, Spain

    Aitor Nogales

  3. Department of Molecular and Cell Biology, Centro Nacional de Biotecnología (CNB), CSIC, Madrid, Spain

    Fernando Almazán

Authors
  1. Kevin Chiem
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  2. Aitor Nogales
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  3. Fernando Almazán
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  4. Chengjin Ye
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  5. Luis Martínez-Sobrido
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Corresponding authors

Correspondence to Chengjin Ye or Luis Martínez-Sobrido .

Editor information

Editors and Affiliations

  1. College of Veterinary Medicine, University of Georgia, Athens, GA, USA

    Daniel R. Pérez

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Chiem, K., Nogales, A., Almazán, F., Ye, C., Martínez-Sobrido, L. (2024). Bacterial Artificial Chromosome Reverse Genetics Approaches for SARS-CoV-2. In: Pérez, D.R. (eds) Reverse Genetics of RNA Viruses. Methods in Molecular Biology, vol 2733. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3533-9_9

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  • DOI: https://doi.org/10.1007/978-1-0716-3533-9_9

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-3532-2

  • Online ISBN: 978-1-0716-3533-9

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