DNA gap repair in Escherichia coli for multiplex site-directed mutagenesis

Site-directed mutagenesis allows the generation of novel DNA sequences that can be used for a variety of important applications such as the functional analysis of genetic variants. To overcome the limitations of existing site-directed mutagenesis approaches, we explored in vivo DNA gap repair. We found that site-specific mutations in plasmid DNA can be generated in Escherichia coli using mutant single-stranded oligonucleotides to target PCR-derived linear double-stranded plasmid DNA. We called this method DeGeRing, and we characterized its advantages, including non-biased multiplex mutagenesis, over existing site-directed mutagenesis methods such as recombineering (recombination-mediated genetic engineering), single DNA break repair (SDBR, introduced by W. Mandecki), and QuikChange (Agilent Technologies, La Jolla, CA). We determined the efficiency of DeGeRing to induce site-directed mutations with and without a phenotype in three K-12 E coli strains using multiple single-stranded oligonucleotides containing homological and heterological parts of various sizes. Virtual lack of background made the isolation of mutants with frequencies up to 10^-6 unnecessary. Our data show that endogenous DNA gap repair in E coli supports efficient multiplex site-directed mutagenesis. DeGeRing might facilitate the generation of mutant DNA sequences for protein engineering and the functional analysis of genetic variants in reverse genetics.