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

Author:

Seo Daeje,Eom Ga-eul,Kim Hye Won,Kim SeokheeORCID

Abstract

ABSTRACTTargeted 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.

Publisher

Cold Spring Harbor Laboratory

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