A dual gene-specific mutator system installs all transition mutations at similar rates in vivo

Targeted in vivo hypermutation accelerates directed evolution of proteins through concurrent DNA diversification and selection. Among recently developed methods, the systems employing a fusion protein of a nucleobase deaminase and T7 RNA polymerase present gene-specific targeting. However, their mutational spectra have been largely limited to exclusive or dominant C:G[->]T:A mutations. Here we describe eMutaT7transition, a new gene-specific mutator system, that installs all the transition mutations (C:G[->]T:A and A:T[->]G:C) at comparable rates. By using two mutator proteins in which two efficient deaminases, PmCDA1 and TadA-8e, are separately fused to T7 RNA polymerase, we obtained similar numbers of C:G[->]T:A and A:T[->]G:C mutations at a sufficiently high rate ([~]3.4 x 10-5 mutations per base per generation or [~]1.3 mutations per 1 kb per day). Through eMutaT7transition-mediated TEM-1 evolution for antibiotic resistance, we generated many mutations also found in clinical isolates. Overall, with a fast mutation rate and wider mutational spectrum, eMutaT7transition is a potential first-line method for gene-specific in vivo hypermutation.