Rapid continuous evolution of gene libraries towards arbitrary functions

Abstract Understanding the evolution of new gene functions is a central challenge in biology, yet the experimental tools to prospectively probe this process remain limited. We introduce OrthoRep Assisted Continuous Library Evolution (ORACLE), a scalable in vivo platform that accelerates the evolution of diverse, multi-length gene libraries. By encoding function-agnostic gene libraries from Saccharomyces cerevisiae , Escherichia coli , Arabidopsis thaliana , and human sources onto the hypermutating p1 plasmid of OrthoRep, ORACLE enables search for new gene function at error rates up to one-million times higher than that of the genome. Under a variety of selection pressures, evolution experiments yielded novel gene variants with new function within fewer than 100 generations, corresponding to less than one month of serial passaging. Notably, genes that initially exhibited little to no observable phenotype rapidly evolved to confer transcription factor activity, chaperone activity, and inhibition of specific proteins. These findings demonstrate that continuous directed evolution via ORACLE not only enables the rapid discovery and optimization of new biomolecular functions but also provides a powerful experimental system to probe fundamental mechanisms of gene innovation. ORACLE thus holds promise for advancing both our understanding of evolutionary dynamics and the engineering of bespoke biomolecules for diverse applications.