MutaT7GDE: A single chimera for the targeted installation of all possible transition mutations in vivo

Deaminase-T7 RNA polymerase fusion (MutaT7) enzymes are a growing class of synthetic biology tools used to diversify target genes during in vivo laboratory evolution. Currently, MutaT7 chimeras comprise either an adenine or cytosine deaminase fused to T7 RNA polymerase. Their expression drives targeted deoxyadenosine-to-deoxyguanosine or deoxycytidine-to-deoxythymidine mutagenesis, respectively. Here, we repurpose recently engineered, substrate-promiscuous general deaminases (GDEs) to establish a substantially simplified system based on a single chimeric enzyme capable of targeting both deoxyadenosine and deoxycytidine. We assess on- and off-target mutagenesis, strand and context preference, as well as parity of deamination for four different MutaT7GDE constructs and identify a single chimera that installs all possible transition mutations more efficiently than preexisting tools. The optimized MutaT7GDE tool reported herein is a next-generation hypermutator capable of mediating efficient and uniform target-gene diversification during in vivo directed evolution.