The emergence and evolution of new gene functions is central to biology, yet experimental tools that allow us to prospectively probe and understand this process are lacking. While systems for continuous directed evolution have successfully compressed gene evolution onto laboratory timeframes at scale, past work largely focused on the evolution of single genes towards single functions. In contrast, when nature searches for new gene functions, it does so by exposing a vast repertoire of genes to a multitude of complex selective pressures in vivo, multiplying the possibilities for innovation. To mimic natural gene evolution in laboratory time, we introduce OrthoRep Assisted Continuous Library Evolution (ORACLE). In ORACLE, high-diversity function-agnostic gene libraries (e.g., open reading frames from Saccharomyces cerevisiae and Escherichia coli, cDNA from Arabidopsis thaliana and human cells) are encoded onto OrthoRep’s p1 plasmid in yeast where library members durably mutate up to one-million times faster than the host genome. As a result, ORACLE provides an opportunity to quickly observe the evolutionary emergence and optimization of new gene functions that satisfy complex in vivo selection pressures from an abundance of sources. Using ORACLE, we discovered a wealth of novel gene variants conferring new cellular functions, each experimentally evolved in fewer than ~100 generations corresponding to less than one month of serial passaging. In several cases, genes that initially exhibited little to no observable phenotype rapidly evolved to confer transcription factor activity, chaperone activity, inhibition of specific targets, etc. These findings demonstrate the relative ease with which existing genetic material can enter the view of selection and evolve new function in vivo. Moreover, our work establishes ORACLE as an effective system for exploring the fundamental mechanisms of gene innovation and discovering bespoke biomolecules for diverse applications.
