An expanded CRISPR–Cas9-assisted recombineering toolkit for engineering genetically intractable Pseudomonas aeruginosa isolates

Much of our current understanding of microbiology is based on the application of genetic engineering procedures. Since their inception (more than 30 years ago), methods based largely on allelic exchange and two-step selection processes have become a cornerstone of contemporary bacterial genetics. While these tools are established for adapted laboratory strains, they have limited applicability in clinical or environmental isolates displaying a large and unknown genetic repertoire that are recalcitrant to genetic modifications. Hence, new tools allowing genetic engineering of intractable bacteria must be developed to gain a comprehensive understanding of them in the context of their biological niche. Herein, we present a method for precise, efficient and rapid engineering of the opportunistic pathogen Pseudomonas aeruginosa . This procedure relies on recombination of short single-stranded DNA facilitated by targeted double-strand DNA breaks mediated by a synthetic Cas9 coupled with the efficient Ssr recombinase. Possible applications include introducing single-nucleotide polymorphisms, short or long deletions, and short DNA insertions using synthetic single-stranded DNA templates, drastically reducing the need of PCR and cloning steps. Our toolkit is encoded on two plasmids, harboring an array of different antibiotic resistance cassettes; hence, this approach can be successfully applied to isolates displaying natural antibiotic resistances. Overall, this toolkit substantially reduces the time required to introduce a range of genetic manipulations to a minimum of five experimental days, and enables a variety of research and biotechnological applications in both laboratory strains and difficult-to-manipulate P. aeruginosa isolates.