UC Irvine

Directed evolution is a powerful approach for engineering biomolecules and understanding the basic principles of adaptation. However, experimental strategies for directed evolution are notoriously labor-intensive and low-throughput, limiting access to demanding functions, multiple functions in parallel, and the study of molecular evolution in replicate. Here, I describe OrthoRep, an orthogonal DNA polymerase-plasmid pair in yeast that stably mutates ~100,000-fold faster than the host genome in vivo, exceeding the error threshold of genomic replication that causes single-generation extinction. User-defined genes in OrthoRep continuously and rapidly evolve through serial passaging, a highly straightforward and scalable process. Using OrthoRep, I evolved malarial DHFR to strongly resist the drug pyrimethamine in 90 independent replicates. This large-scale experiment revealed a more complex fitness landscape than previously realized, including new resistant alleles, common adaptive trajectories constrained by epistasis, rare outcomes that avoid a frequent early adaptive mutation, and a suboptimal fitness peak that occasionally traps evolving populations. OrthoRep enables a new paradigm of routine, high-throughput evolution of biomolecular and cellular function.