Figure 2.

Design of RBD mutagenesis libraries and screening by yeast surface display and deep sequencing

(A) Shown is the amino acid usage in the combinatorial libraries (libraries 3C, 1C, and 2C). Degenerate codons are derived from DMS data for ACE2 binding (Starr et al., 2020).

(B) Representative examples of degenerate codons tiled across RBM-2, which are pooled together to comprise library 2T.

(C) Flow cytometry dot plots depict yeast display screening of combinatorial (1C, 2C, 2CE, and 3C) and tiling (1T, 2T, and 3T) RBD libraries and control RBD (Wu-Hu-1); gating schemes correspond to selection of ACE2-binding and non-binding variants.

(D) Amino acid logo plots of the RBD are based on deep sequencing data from ACE2-binding and non-binding selections.

(E) Flow cytometry dot plots depict yeast display screening of pooled RBD libraries (2C and 2CE) after selection for ACE2 binding; gating schemes correspond to selection of variants for binding and escape (non-binding) to monoclonal antibodies (mAbs).

See also Figures S1 and S2 and Tables S1–S3.