(502g) Synthetic Genetic Systems for Rapid Mutation and Evolution In Vivo

We are interested in building genetic systems that have extremely high mutation rates in order to speed up the evolution of target proteins and enzymes in vivo as well as to record transient information, such as lineage relationships or exposure to biological stimuli, as genetic information in situ. I will describe a highly error-prone yeast orthogonal DNA replication (OrthoRep) system that mutates user-selected genes at a base pair substitution (bps) rate of 1e-5 without any increase in the genomic mutation rate (1e-10 bps). This ~100,000-fold mutational acceleration allows for the rapid continuous evolution of target biomolecules entirely in vivo using a simple serial passaging process amenable to extensive repetition. I will summarize a large DNA polymerase engineering effort leading to these OrthoRep systems, show that OrthoRep can stably cross the error threshold of haploid yeast, and discuss the use of OrthoRep in a yeast model of Plasmodium falciparum DHFR where we evolved resistance to the antimalarial drug, pyrimethamine, in 90 independent 0.5 mL cultures through serial passaging alone. We find that a highly adaptive first-step mutation constrains path-choice through sign epistasis, leading to convergence, but rare mutations direct trajectories to an alternative smooth fitness peak, illustrating the balance between fate and chance in drug resistance. OrthoRep may enable a paradigm of high-throughput directed evolution, where scores of evolving lines are routinely used to sample adaptive landscapes, evolve multiple functions in parallel, or exploit subpopulation structure in the search for desirable biomolecular function and the study of molecular evolution.