Temperature sensitive DNA repair mutants enable highly precise genome manipulationView all posters
Hungarian Academy of Sciences, Hungary
Oligonucleotide mediated allelic replacement is an efficient tool for bacterial genome manipulation (MAGE: Wang et al. Nature 460:894). Mutants with endogenous mismatch repair (MMR) system deficiency have been shown to be a beneficial background for oligonucleotide mediated allelic replacement, due to the increase of mismatch incorporation efficiency and unbiased mutation spectra. However, inactivation of the host’s mismatch repair system results in a dramatically elevated general mutation rate and therefore accumulation of unwanted background mutations across the genome. We present a novel strategy for mismatch repair evasion using temperature sensitive DNA repair mutants and a method for temporal inactivation of the mismatch repair protein complex in Escherichia coli. This method enables the transient suppression of DNA repair during mismatch carrying oligonucleotide integration, but allows normal mismatch correction during cell growth and electrocompetent cell preparation stages. This advanced technique further increases the precision of oligonucleotide mediated allelic replacement and enables more predictable cell programming.