Conditional DNA repair mutants enable highly precise genome engineering.
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.
Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary and Symbiosis and Functional Genomics Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged H-6726, Hungary.
Ákos
Á
Nyerges
Bálint
B
Csorgő
István
I
Nagy
Dóra
D
Latinovics
Béla
B
Szamecz
György
G
Pósfai
Csaba
C
Pál
D000251:Adenosine Triphosphatases / Q000235:genetics; D000483:Alleles; D053843:DNA Mismatch Repair; D004926:Escherichia coli / Q000235:genetics; D029968:Escherichia coli Proteins / Q000235:genetics; D005818:Genetic Engineering / Q000379:methods*; D016680:Genome, Bacterial; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein / Q000235:genetics; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D013696:Temperature
D029968:Escherichia coli Proteins; C074770:MutL protein, E coli; D009838:Oligodeoxyribonucleotides; D000251:Adenosine Triphosphatases; D000070956:MutL Proteins; D051719:MutS DNA Mismatch-Binding Protein; C497175:MutS protein, E coli