Identification of SARS-CoV-2 S RBD escape mutants using yeast screening and deep mutational scanning

• Published in: STAR protocols Vol. 2; no. 4; p. 100869
• Main Authors: Haas, Cyrus M., Francino-Urdaniz, Irene M., Steiner, Paul J., Whitehead, Timothy A.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 17-12-2021; Elsevier
• Subjects: Angiotensin-Converting Enzyme 2 - genetics; Angiotensin-Converting Enzyme 2 - metabolism; Antibody; Binding Sites; COVID-19 - genetics; COVID-19 - metabolism; COVID-19 - virology; DNA Mutational Analysis - methods; Humans; Immunology; Microbiology; Molecular Biology; Mutation; Protein Biochemistry; Protocol; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - metabolism; SARS-CoV-2 - genetics; Sequence analysis; Sequencing; Spike Glycoprotein, Coronavirus - genetics; Spike Glycoprotein, Coronavirus - metabolism; Protein Biochemistry; Immunology; Microbiology; Molecular Biology; Antibody; Sequence analysis; Sequencing
• ISSN: 2666-1667; 2666-1667
• DOI: 10.1016/j.xpro.2021.100869

Here, we describe a protocol to identify escape mutants on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) receptor-binding domain (RBD) using a yeast screen combined with deep mutational scanning. Over 90% of all potential single S RBD escape mutants can be identified for monoclonal antibodies that directly compete with angiotensin-converting enzyme 2 for binding. Six to 10 antibodies can be assessed in parallel. This approach has been shown to determine escape mutants that are consistent with more laborious SARS-CoV-2 pseudoneutralization assays.
For complete details on the use and execution of this protocol, please refer to Francino-Urdaniz et al. (2021).
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•A protocol for identifying S escape mutations for anti-S monoclonal antibodies•All anti-S antibodies that competitively inhibit ACE2 can be tested•Protocol uses yeast display screening of freely available S RBD libraries•Open-source software used to analyze sequencing data and identify escape mutants
Here, we describe a protocol to identify escape mutants on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike (S) receptor-binding domain (RBD) using a yeast screen combined with deep mutational scanning. Over 90% of all potential single S RBD escape mutants can be identified for monoclonal antibodies that directly compete with angiotensin-converting enzyme 2 for binding. Six to 10 antibodies can be assessed in parallel. This approach has been shown to determine escape mutants that are consistent with more laborious SARS-CoV-2 pseudoneutralization assays.