Fig. 4. A competition-based selection to identify RBD mutations within S of SARS-CoV-2 that preferentially bind WT or engineered ACE2 receptors.

(A) Expi293F cells were transfected with WT myc-S and incubated with competing sACE2₂(WT)-IgG1 (25 nM) and sACE2₂.v2.4-8h (20 nM). Bound protein was detected by flow cytometry after immunostaining for the respective epitope tags. (B) As in (A), except cells were transfected with the RBD SSM library. A population of cells expressing S variants with increased specificity toward sACE2₂.v2.4 is apparent (cells shifted to the upper left of the main population). (C) Gates used for FACS of cells expressing the RBD SSM library. After excluding cells without bound protein, the top 20% of cells for bound sACE2₂.v2.4-8h (magenta gate) and for bound sACE2₂(WT)-IgG1 (green gate) were collected. (D and E) Agreement between log₂ enrichment ratios from two independent FACS selections for cells expressing S variants with increased specificity for (D) sACE2₂(WT) or (E) sACE2₂.v2.4. R² values are calculated for nonsynonymous mutations (black). Nonsense mutations are red. (F and G) Conservation scores are calculated from the mean of the log₂ enrichment ratios for all nonsynonymous substitutions at a given residue position. Correlation plots show agreement between conservation scores for two independent selections for cells within the (D) sACE2₂(WT)- or (E) sACE2₂.v2.4-specific gates.