(798b) Eukaryotic Reprogramming By Directed Genome Evolution

Genome-wide cellular reprogramming will not only uncover fundamental biological mechanisms of cell differentiation and development but also result in a broad range of applications such as gene therapy, regenerative medicine, microbial production of chemicals and fuels, and transgenic crops with herbicide, salt, and drought tolerance.  However, the current ability to reprogram a cell on the genome scale is limited to bacterial cells and in most cases is confined to modifications of a limited number of gene targets.  Here we report an efficient, genome-scale and generally applicable method for eukaryotic reprogramming, by applying directed evolution strategies to genome engineering.  Iterative cycles of genome-wide trackable mutagenesis coupled with high-throughput selection or screening enable the accumulation of beneficial modifications in an evolving eukaryotic genome.  We show the application of directed genome evolution to Saccharomyces cerevisiae for improved acetic acid (HAc) tolerance, a key trait for microbial production of chemicals and fuels.  Three genetic modification targets were identified which acted synergistically to confer the highest acetic acid tolerance ever achieved in an engineered yeast strain.  Our strategy may greatly accelerate the design and engineering of organisms with desired traits and provide new insights on genome structure, function, and evolution.