Temporal inactivation of DNA repair enables highly precise genome

engineering in Escherichia coli

Ákos Nyerges, Bálint CsörgÅ?, György Pósfai, Csaba Pál

Synthetic and Systems Biology Unit, Institute of Biochemistry, Biological Research Centre of the Hungarian

Academy of Sciences, Szeged, Hungary http://group.szbk.u-szeged.hu/sysbiol/

The emergence of advanced in vivo genome engineering technologies is transforming biotechnology, systems, and evolutionary biology. By exploiting the customized engineering of living organisms, these techniques have redefined the repertoire of experimental protocols. One of the methods, Multiplex Automated Genome Engineering (MAGE), uniquely allows simultaneous targeting of many genomic loci, and thereby enables the generation of huge sequence diversity within a practical timescale and at a reasonable cost.
However, MAGE has one major â??Achillesâ?? heelâ?, which limits its practical application. As the methyl-directed mismatch repair system acts against the incorporation of desired genomic modifications, MAGE requires mutants with endogenous mismatch repair system deficiency. As a by-product, inactivation of the hostâ??s mismatch repair system results in a dramatically elevated general mutation rate and therefore leads to the accumulation of background mutations across the whole genome. Accumulation of such undesired genetic alterations can dramatically alter the studied effects of the targeted modifications.
Our work presents 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, which enables the transient suppression of DNA repair during ssDNA- recombineering in Escherichia coli. By restricting the cells mutator state only to the relatively brief oligonucleotide integration period, the number of off-target mutations was reduced by
85%, concurrently maintaining highly efficient and unbiased allelic replacement. This novel
strategy therefore enables a more precise in vivo genome-editing approach with reduced off-target effects. This will help future genome engineering endeavors and allow for the investigation of uncharted cellular interactions and several central evolutionary issues.

Citation:

Conditional DNA repair mutants enable highly precise genome engineering

Ákos Nyerges, Bálint CsörgÅ?, István Nagy, Dóra Latinovics, Béla Szamecz, György Pósfai,

Csaba Pál, Nucl. Acids Res. 2014,