Efficient cell-free evolution of RNA polymerases by droplet microfluidics

Directed evolution enables the rapid creation of biomolecules with new functions, yet linking genotype and phenotype often requires laborious cell-based workflows. Here we integrate a boosted in vitro transcription-translation system capable of protein expression from single-copy DNA with ultrahigh-throughput fluorescence-activated droplet sorting, developing a simple one-day workflow for efficient protein evolution. Using this system, we evolved SP6 RNA polymerase into a highly robust variant that functions in multiple environments, including cell-free reactions and mammalian cells. We further engineered the evolved enzyme into a proximity-dependent split RNA polymerase that converts molecular interactions into transcriptional activity with minimal background. The resulting biosensors detect diverse molecular targets in vitro, including proteins, peptides, RNAs, and molecular glues. This cell-free platform provides versatile routes for evolving diverse functional biomolecules, accelerating the development of advanced biotechnologies such as diagnostics and therapeutics.