Conditional DNA repair mutants enable highly precise genome engineering

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

doi:10.1093/nar/gku105

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.

Highlights

Recent breakthroughs in genome engineering have greatly expanded our ability to design organisms in a directed and combinatorial manner
All oligonucleotides for allelic replacement as well as polymerase chain reaction (PCR) primers used in this study are presented in Supplementary File S1
We found that in six of the eight cases, the MG-tMMR strain allowed efficient and mostly unbiased oligo incorporation comparable with that achieved by the isogenic mismatch repair knockout strain MG-ÁmutS (Figure 1B)

Summary

Introduction

Recent breakthroughs in genome engineering have greatly expanded our ability to design organisms in a directed and combinatorial manner. The myriad of novel genome-scale modification technologies offer new opportunities for the construction of biological systems with desired properties [1]. From these techniques, oligonucleotide (oligo)mediated allelic replacement has been optimized toward multiplexing and automation [2]. The power of MAGE has been demonstrated in a wide range of biotechnological applications It allows (i) optimization of metabolic pathways to produce industrially relevant compounds [2,3], (ii) improvement of bacterial growth properties under selected conditions [4] and (iii) genome-wide replacement of a specific codon in Escherichia coli [5,6]. MAGE has the potential to transform basic research by accelerating and expanding the range of protocols for genome editing and analysis [1]

Methods

Results

Conclusion