Advances in Orthogonal DNA Replication and Application to Small-Molecule Biosensor Evolution

Directed evolution applies Darwinian evolution to engineering new and optimized proteins, pathways, or cells. Traditional directed evolution techniques rely on labor-intensive ex vivo mutagenesis, transformation, and screening steps. Unlike natural evolution, however, this scheme is neither continuous nor capable of substantial parallelization. Our group has developed OrthoRep, an orthogonal DNA polymerase-plasmid pair in yeast that stably mutates ~100,000-fold faster than the host genome in vivo. User-defined genes in OrthoRep can be continuously and rapidly evolved under selection, enabling a new paradigm of high-throughput evolution of biomolecular and cellular function. Here, I describe technological advances to OrthoRep and an application towards evolving a small-molecule biosensor for new ligand specificity. First, I cover efforts to rationally engineer the OrthoRep DNA polymerase for higher mutation rates and determine the spontaneous recombination frequency of the OrthoRep plasmid. Next, I demonstrate facile transfer of OrthoRep plasmids between yeast strains via cytoduction. Third, I show the strain generality of OrthoRep and a CRISPR/Cas9 method for expedient genetic manipulations. Finally, I demonstrate successful evolution towards reprogramming the specificity of a transcription-based biosensor towards a non-native ligand