PT  - JOURNAL ARTICLE
AU  - Daeje Seo
AU  - Ga-eul Eom
AU  - Hye Won Kim
AU  - Seokhee Kim
TI  - A dual gene-specific mutator system installs all transition mutations at similar rates <em>in vivo</em>
AID  - 10.1101/2022.06.09.495438
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.06.09.495438
4099  - http://biorxiv.org/content/early/2022/06/12/2022.06.09.495438.short
4100  - http://biorxiv.org/content/early/2022/06/12/2022.06.09.495438.full
AB  - 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 × 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.Competing Interest StatementThe authors have declared no competing interest.