Efficient and iterative retron-mediated in vivo recombineering in E. coli

Recombineering is an important tool in gene editing, enabling fast, precise, and highly specific in vivo modification of microbial genomes. Oligonucleotide-mediated recombineering via the in vivo production of ssDNA can overcome the limitations of traditional recombineering methods which rely on the exogenous delivery of editing template. By modifying a previously reported plasmid-based system for fully in vivo ssDNA recombineering, we demonstrate iterative editing of independent loci by utilizing a temperature-sensitive origin of replication for easy curing of the editing plasmid from recombinant cells. Combined with targeted counter-selection against unedited cells with Cas9-nuclease, editing efficiencies of 90-100% are achieved. Requiring only a single cloning step for re-targeting, our system provides an easy-to-use method for rapid, efficient construction of desired mutants.