Conditional DNA repair mutants enable highly precise genome engineering

Nucleic Acids Res. 2014 Apr;42(8):e62. doi: 10.1093/nar/gku105. Epub 2014 Feb 5.

Abstract

Oligonucleotide-mediated multiplex genome engineering is an important tool for bacterial genome editing. The efficient application of this technique requires the inactivation of the endogenous methyl-directed mismatch repair system that in turn leads to a drastically elevated genomic mutation rate and the consequent accumulation of undesired off-target mutations. Here, we present a novel strategy for mismatch repair evasion using temperature-sensitive DNA repair mutants and temporal inactivation of the mismatch repair protein complex in Escherichia coli. Our method relies on the transient suppression of DNA repair during mismatch carrying oligonucleotide integration. Using temperature-sensitive control of methyl-directed mismatch repair protein activity during multiplex genome engineering, we reduced the number of off-target mutations by 85%, concurrently maintaining highly efficient and unbiased allelic replacement.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adenosine Triphosphatases / genetics
  • Alleles
  • DNA Mismatch Repair*
  • Escherichia coli / genetics
  • Escherichia coli Proteins / genetics
  • Genetic Engineering / methods*
  • Genome, Bacterial*
  • MutL Proteins
  • MutS DNA Mismatch-Binding Protein / genetics
  • Mutation*
  • Oligodeoxyribonucleotides
  • Temperature

Substances

  • Escherichia coli Proteins
  • MutL protein, E coli
  • Oligodeoxyribonucleotides
  • Adenosine Triphosphatases
  • MutL Proteins
  • MutS DNA Mismatch-Binding Protein
  • MutS protein, E coli