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

Abstract

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.

Enhanced single‐base mutation diversity by the combination of cytidine deaminase with DNA‐repairing enzymes in yeast

“…[ 22 ] Recently, Kim and coworkers established eMutaT7 transition which containing cytidine and adenosine deaminase and could generate all transition mutations. [ 27 ] These mutagenesis tools mainly generate transition mutations, while MutaT7 trans could increase the proportion of transversion mutations (C → G, G → T, etc.) to about 10%–20%, and could play a complementary role with the above‐mentioned tools.…”

“…Existing deaminase-based evolutionary techniques are difficult to achieve transversion mutations, and most of them are biased toward generating specific types of transition mutations. [21,22,26,27] Therefore, our mutagenesis tools with dual T7 promoters can further enlarge the mutation libraries, thus promoting the process of evolution. Because this tool could efficiently expand the mutation spectrum, especially increasing the frequency of transversion mutations, we named it MutaT7 trans .…”

“…This strategy has been widely used in bacteria, mammalian cells, and yeasts, playing an important role in many fields such as resistant strain screening and protein engineering. [21][22][23][24][25][26][27][28] With cytidine/adenosine deaminase or both of them, targeted mutagenesis tools mainly generate C → T or A → G mutations at rates greater than 10 −3 s. p. b. However, other types of mutations, especially transversion mutations, are difficult to access.…”