Conditional DNA repair mutants enable highly precise genome engineering

Ákos Nyerges, Bálint Csörgo, István Nagy, Dóra Latinovics, Béla Szamecz, György Pósfai, Csaba Pál

Research output: Contribution to journalArticle

31 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)e62
JournalNucleic acids research
Volume42
Issue number8
DOIs
Publication statusPublished - Apr 2014

ASJC Scopus subject areas

  • Genetics

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