Fig. 3: A size-exclusion selection-based strategy for high-throughput screening of syncytium formation.
a, Workflow for DMS in sender cells using a size-exclusion selection-based system. Large syncytia are collected using the cell strainer, while small GFP+ syncytia are collected by FACS. The two populations containing the fusion-competent spike variants are subjected to NovaSeq-based sequencing. b, Heat maps depicting how all single mutations affect the syncytium-forming potential of spike’s FPPR and furin cleavage site regions. Squares are coloured by mutational effect (that is, FC) according to colour bars on the right, with red and purple indicating syncytium-enhancing and inhibiting substitutions, respectively. FC represents each variant’s relative abundance in the syncytia collected using the cell strainer or FACS vs the cell pool before mixing and is normalized to WT. Spike variants with increased syncytium-formation potential were enriched (with fold change >1), while those with decreased syncytium-formation potential were depleted (with fold change <1). Grey cross, mutations with no measurement; black dot, SARS-CoV-2 amino acid; *, stop codon. c, Correlation of the profiling results (that is, FC) obtained using droplet microfluidics-based and size-exclusion selection-based strategies. Validated syncytium-enhancing and inhibiting mutations/residues are highlighted and labelled. d, Mutational effects on syncytium formation revealed by the pooled screen using the size-exclusion selection-based system and individual validation assay for the spike variants.
