AbstractTargetedin vivohypermutation accelerates directed evolution of proteins through concurrent DNA diversification and selection. Although systems employing a fusion protein of a nucleobase deaminase and T7 RNA polymerase present gene-specific targeting, their mutational spectra have been limited to exclusive or dominant C:G→T:A mutations. Here we describe eMutaT7transition, a new gene-specific hypermutation system, that installs all transition mutations (C:G→T:A and A:T→G:C) at comparable frequencies. 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 substitutions at a sufficiently high frequency (∼6.7 substitutions in 1.3 kb gene during 80-hin vivomutagenesis). Through eMutaT7transition-mediated TEM-1 evolution for antibiotic resistance, we generated many mutations found in clinical isolates. Overall, with a high mutation frequency and wider mutational spectrum, eMutaT7transitionis a potential first-line method for gene-specificin vivohypermutation.