PANCS-Binders: a rapid, high-throughput binder discovery platform.

Proteins that selectively bind to a target of interest are foundational in research, diagnostics and therapeutics. Current approaches for discovering binders are laborious and time-consuming, taking months or more, and have a high failure rate. Here we establish phage-assisted noncontinuous selection of protein binders (PANCS-Binders), an in vivo selection platform that links the life cycle of M13 phage to target protein binding through proximity-dependent split RNA polymerase biosensors, allowing for comprehensive screening of whether a variant binds a target with high fidelity. We showcase PANCS-Binders by screening multiple protein libraries each against a panel of 95 separate targets, thereby individually assessing more than 10(11) protein-protein interaction pairs, in 2 days. These selections yielded large, high-quality datasets and hundreds of novel binders, which can be affinity matured or directly used in mammalian cells to inhibit or degrade targets. We believe that PANCS-Binders accelerates and simplifies the binder discovery process, which will help unlock new creative potential in proteome targeting with engineered binder-based biotechnologies.