Droplet-based single-cell pairing for high-throughput interaction mapping of antigen-receptor combinations

Mapping the interaction potential of different variant combinations of viral antigens and human cell receptors and understanding how viral antigen mutations interact with human genetic polymorphisms are critical for predicting infection susceptibility and informing precision public health strategies. Here, we develop a droplet-based single-cell pairing and library-on-library interaction screening (SPLIS) system for high-throughput profiling of the syncytium-formation landscapes of various spike-angiotensin-converting enzyme 2 (ACE2) variant combinations. This system uses combined droplet sorting and merging to deterministically encapsulate one antigen-presenting sender cell and one receptor-expressing receiver cell into each drop, followed by selection and sequencing of the fused DNA readouts to characterize the syncytium-formation potential of each combination. We applied SPLIS to characterize both fusion-enhancing and -inhibiting variant pairs, comprehensively profiling how ACE2 single-nucleotide polymorphisms modulate susceptibility to emerging severe acute respiratory syndrome coronavirus 2 spike mutations. Our system emerges as a powerful tool to interrogate the interactions between two libraries of variants, offering valuable insights into host susceptibility patterns and viral infectivity trends. © 2025 the Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a creative commons Attribution Non commercial license 4.0 (cc BY-Nc).