An Orthogonal Genetic System for Rapid Evolution

We recently developed an orthogonal DNA replication system in the yeast Saccharomyces cerevisiae.¹ This system consists of an orthogonal DNA plasmid–DNA polymerase pair wherein the orthogonal DNA polymerase stably and specifically replicates the orthogonal plasmid in the cytoplasm of yeast. Engineered error-prone DNA polymerases show complete mutational targeting in vivo: per-base mutation rates on the plasmid can be increased substantially with no increase in genomic rates. Orthogonal replication therefore serves as a platform for in vivo continuous evolution of user-selected genes and as a system whose replicative properties can be manipulated independently of the host's.

In this presentation, I will present the design and implementation of orthogonal DNA replication, discuss its use as a system for rapid targeted gene evolution in vivo, present new data on the addition of copy number control and recombination to our orthogonal replication system, and explore the broader implications of orthogonal replication as an unencumbered read/write system in biology that can be freely engineered for recording biological events in vivo.

¹A Ravikumar, A Arrieta, CC Liu. An Orthogonal DNA Replication System in
Yeast. Nature Chemical Biology, 10, 175-177 (2014).