Iterative in vivo cut'n'paste of chromosomal loci in Escherichia coli K-12 using synthetic DNA

Abstract

We have developed an easy-to-use in vivo method to iteratively relocate functional chromosomal loci onto an episome in Escherichia coli by utilizing synthetic DNA fragments. In this in vivo cutnpaste procedure, "cutting" is executed by the RNA-guided DNA endonuclease Cas9 and a set of guides, while "pasting" is facilitated by the phage {lambda} Red recombinase, which are all synthesized on easily curable helper plasmids. To demonstrate the utility of in vivo cutnpaste, we commercially obtained synthetic DNA fragments containing locus-specific homology regions, antibiotic marker cassettes, and standardized Cas9 target sequences, and successfully relocated seven functional chromosomal loci. Scarless relocation mutants of the trg, aer, tsr, malHM, malQT, macB-nadA, and folA chromosomal 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 chromosomal loci. In vivo cutnpaste should be widely useful, especially in genome (re-)engineering efforts of E. coli and other bacteria because the procedure can be performed by non-specialists in unmodified wild-type cells. SignificanceEditing of genomes is crucial to understand the inner workings of bacteria and to alter them for biotechnological applications. In vivo cutnpaste, in analogy to the frequently used keyboard shortcuts Ctrl-X and Ctrl-V which allow to move text, images, or files on computers, makes it easy to relocate large chunks of the bacterial genome without requiring skills to manipulate DNA outside of the bacterial cell. Like Ctrl-X and Ctrl-V, in vivo cutnpaste can be executed several times to iteratively edit multiple genomic locations in the same bacterium. Since in vivo cutnpaste entirely takes place inside the bacterial cell, it is particularly well suited to edit hard-to-tackle genomic features such as essential genes.