Expanded MutaT7 toolkit efficiently and simultaneously accesses all possible transition mutations in bacteria.

Targeted mutagenesis mediated by nucleotide base deaminase-T7 RNA polymerase fusions has recently emerged as a novel and broadly useful strategy to power genetic diversification in the context of in vivo directed evolution campaigns. Here, we expand the utility of this approach by introducing a highly active adenosine deaminase-T7 RNA polymerase fusion protein (eMutaT7A→G), resulting in higher mutation frequencies to enable more rapid directed evolution. We also assess the benefits and potential downsides of using this more active mutator. We go on to show in Escherichia coli that adenosine deaminase-bearing mutators (MutaT7A→G or eMutaT7A→G) can be employed in tandem with a cytidine deaminase-bearing mutator (MutaT7C→T) to introduce all possible transition mutations simultaneously. We illustrate the efficacy of this in vivo mutagenesis approach by exploring mutational routes to antibacterial drug resistance. This work sets the stage for general application of optimized MutaT7 tools able to induce all types of transition mutations during in vivo directed evolution campaigns across diverse organisms.