Published in
Oxford University Press, Nucleic Acids Research, 8(42), p. e62-e62, 2014
DOI: 10.1093/nar/gku105
Links
- ORCID
- ORCID
- Oxford University Press
- [www.ncbi.nlm.nih.gov] | PDF
- [real.mtak.hu]
- [real.mtak.hu] | PDF
- [real.mtak.hu] | PDF
- [europepmc.org] | PDF
- [www.researchgate.net] | PDF
- [www.ncbi.nlm.nih.gov] | PDF
Tools
Conditional DNA repair mutants enable highly precise genome engineering
,
B. Csorg,
Bálint Csörgő ,
István Nagy,
Dóra Latinovics,
Béla Szamecz,
György Pósfai,
Csaba Pál
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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.