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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.

SUMMARYMalaria-causing P. vivax parasites can linger in the human liver for weeks to years, and then reactivate to cause recurrent blood-stage infection. While an important target for malaria eradication, little is known about the molecular features of the replicative and non-replicative states of intracellular P. vivax parasites, or the human host-cell responses to them. Here, we leverage a bioengineered human microliver platform to culture Thai clinical isolates of P. vivax in primary human hepatocytes and conduct transcriptional profiling of infected cultures. By coupling enrichment strategies with bulk and single-cell analyses, we captured both parasite and host transcripts in individual hepatocytes throughout the infection course. We defined host- and state-dependent transcriptional signatures and identified previously unappreciated populations of replicative and non-replicative parasites, sharing features with sexual transmissive forms. We found that infection suppresses transcription of key hepatocyte function genes, and that P. vivax elicits an innate immune response that can be manipulated to control infection. Our work provides an extendible framework and resource for understanding host-parasite interactions and reveals new insights into the biology of malaria dormancy and transmission.