Conditional DNA repair mutants enable highly precise genome engineering

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.

Figure 1.

(A) Mutation rate measurement of employed strains at various temperatures. A rifampicin resistance assay was used to calculate mutation rates as described in ‘Materials and Methods’ section. Error bars represent 95% confidence intervals of pooled samples of two independent measurements of 12 parallel samples each. (B) Allelic replacement efficiencies of oligos generating various types of single base pair modifications in the chromosome, designated by chromosomal base:oligo base in MGλ, MGλ-ΔmutS and MGλ-tMMR. The efficiency of allelic replacement was estimated by the number of mutant cells per total colony-forming units. The values are the means of two independent measurements each, error bars represent standard errors. An A:G (G:A) mismatch was created two separate times using two different oligos. (C)Allelic replacement efficiencies of oligos generating modifications of increasing size in the genome of MGλ, MGλ-ΔmutS and MGλ-tMMR. The values are the means of two independent measurements each, error bars represent standard errors. For details, see ‘Materials and Methods’ section.

Figure 2.

General scheme of the modified MAGE protocol. ssDNA oligos are incorporated into the bacterial genomes in a cyclical manner. The orange arrow represents the main novelty of the modified procedure, a recovery period at 36°C to which the mutator state is restricted to. See main text for details. Adapted from Wang et al. (2).

Figure 3.

Effects of temperature increase on growth parameters of MGλ-tMMR. Relative growth rate of MGλ-tMMR, compared with wild-type E. coli MG1655 (wild-type = 1), at gradually elevating temperatures. Values shown are the mean of 30 replicates each, error bars represent 95% confidence intervals.

Figure 4.

Optimization of recovery time at 36°C. Allelic replacement efficiency of a representative oligo that introduces a nonsense mutation by two consecutive mismatches in lacZ. Values shown are the mean of four independent experiments, error bars represent 95% confidence intervals.