Single-mutant library synthesis and screening revealed the sequence–function relationship between CIS43 antibody mutants and antimalarial antigen recognition. (a) Distribution of single-mutation VH_NNK and VL_MNN DNA codons in presort SSM libraries, as quantified by NGS. (b) FACS gating strategy for affinity-based sorting of SSM libraries. (c) CIS43 single-mutation heat maps showing library frequencies for mutants expressed in the VL⁺ Fab-expressing VH-SSM and VL-SSM libraries, prior to sorting for antimalarial antigen recognition. (d) Flow cytometric analysis of CIS43 wild-type surface-displayed Fab and pre-sort single-mutation Fab libraries, stained with Pep21 and fl_PfCSP. (e) Flow cytometric analysis of CIS43 wild-type surface-displayed Fab and Round 3 single-mutation Fab libraries sorted against Pep21 and fl_PfCSP for low-affinity (upper) and medium-affinity (lower) population phenotypes.