We recently developed an orthogonal replication system (OrthoRep) in yeast that allows for the rapid continuous mutagenesis of a special plasmid without mutating the genome. Although OrthoRep has been successfully applied to evolve several proteins and enzymes, the generality of OrthoRep has not yet been systematically studied. Here, we show that OrthoRep is fully compatible with all Saccharomyces cerevisiae strains tested, demonstrate that the orthogonal plasmid can encode genetic material of at least 22 kb, and report a CRISPR/Cas9-based method for expedient genetic manipulations of OrthoRep. It was previously reported that the replication system upon which OrthoRep is based is only stable in respiration-deficient S. cerevisiae strains that have lost their mitochondrial genome (ρ0 strains). However, here we trace this biological incompatibility to the activity of the dispensable toxin/antitoxin system encoded on the wild-type orthogonal plasmid. Since the toxin/antitoxin system is replaced by genes of interest in any OrthoRep application, OrthoRep is a generally compatible platform for continuous in vivo evolution in S. cerevisiae.
Keywords: mutagenesis; CRISPR/Cas9; OrthoRep; continuous evolution; orthogonal replication; protein-primed replication.