Iterativein vivocut‘n’paste of chromosomal loci inEscherichia coliK-12 using synthetic DNA

We have developed a simple and highly efficientin vivomethod to iteratively relocate functional chromosomal loci onto an episome inEscherichia coliby utilizing synthetic DNA fragments. In thisin vivocut’n’paste procedure, “cutting” is executed by the RNA-guided DNA endonuclease Cas9 and a set of guides, while “pasting” is facilitated by the phage λ Red recombinase, which are all synthesized on easily curable plasmids. To demonstrate the utility of this approach, we commercially obtained synthetic DNA fragments containing locus-specific homology regions, antibiotic marker cassettes, and standardized Cas9 target sequences. By using these locus-specific synthetic DNA fragments, we relocated 7 functional chromosomal loci. Scarless relocation mutants of thetrg,aer,tsr,malHM,malQT,macB-nadA, andfolAchromosomal loci were obtained as antibiotic-resistant isolates by combining Cas9 counterselection with the restoration of an antibiotic marker cassette. The additional antibiotic marker cassettes and standardized Cas9 target sequences present in the synthetic DNA fragments are inherently eliminated upon completion of the procedure, enabling iterative processing of loci. This procedure should be widely useful, especially in genome (re-)engineering ofE. coliand other bacteria because the procedure can be done on wild-type cells.