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Scalable continuous evolution of genes at mutation rates above genomic error thresholds

By Arjun Ravikumar, Garri A. Arzumanyan, Muaeen K.A. Obadi, Alex A. Javanpour, Chang C. Liu

Posted 03 May 2018
bioRxiv DOI: 10.1101/313338 (published DOI: 10.1016/j.cell.2018.10.021)

Directed evolution is a powerful approach for engineering biomolecules and understanding adaptation. However, experimental strategies for directed evolution are notoriously low-throughput, limiting access to demanding functions, multiple functions in parallel, and the study of molecular evolution in replicate. Here, we report OrthoRep, a yeast orthogonal DNA polymerase-plasmid pair that stably mutates ~100,000-fold faster than the host genome in vivo, exceeding error thresholds of genomic replication that lead to single-generation extinction. User-defined genes in OrthoRep continuously and rapidly evolve through serial passaging, a highly scalable process. Using OrthoRep, we evolved drug resistant malarial DHFRs 90 times and uncovered a more complex fitness landscape than previously realized. We find rare fitness peaks that resist the maximum soluble concentration of the antimalarial pyrimethamine (these resistant variants support growth at pyrimethamine concentrations >40,000-fold higher than the wild-type enzyme can tolerate) and also find that epistatic interactions direct adaptive trajectories to convergent outcomes. OrthoRep enables a new paradigm of routine, high-throughput evolution of biomolecular and cellular function.

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