1. MutaT7 GDE : A Single Chimera for the Targeted, Balanced, Efficient, and Processive Installation of All Possible Transition Mutations In Vivo .
- Author
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Mengiste AA, McDonald JL, Nguyen Tran MT, Plank AV, Wilson RH, Butty VL, and Shoulders MD
- Subjects
- Mutation, Mutagenesis, Directed Molecular Evolution methods, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Escherichia coli genetics, Viral Proteins genetics, Viral Proteins metabolism, DNA-Directed RNA Polymerases genetics, DNA-Directed RNA Polymerases metabolism
- Abstract
Deaminase-T7 RNA polymerase fusion (MutaT7) proteins are a growing class of synthetic biology tools used to diversify target genes during in vivo laboratory evolution. To date, MutaT7 chimeras comprise either a deoxyadenosine or deoxycytidine deaminase fused to a 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, and parity of deamination for four different MutaT7
GDE constructs. We identify a single chimera that installs all possible transition mutations more efficiently than preexisting, more cumbersome MutaT7 tools. The optimized MutaT7GDE chimera reported herein is a next-generation hypermutator capable of mediating efficient and uniform target-gene diversification during in vivo directed evolution.- Published
- 2024
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