Genomic editing in by CRISPR/Cas9.

complex bacteria have emerged as opportunistic pathogens in patients with cystic fibrosis and immunocompromised individuals, causing life-threatening infections. Because of the relevance of these microorganisms, genetic manipulation is crucial for explaining the genetic mechanisms leading to pathogenesis. Despite the availability of allelic exchange tools to obtain unmarked gene deletions in , these require a step of merodiploid formation and another of merodiploid resolution through two independent homologous recombination events, making the procedure long-lasting. The CRISPR/Cas9-based system could ease this constraint, as only one step is needed for allelic exchange. Here, we report the modification of a two-plasmid system (pCasPA and pACRISPR) for genome editing in . Several modifications were implemented, including selection marker replacement, the optimization of promoter induction for the expression of Cas9 and λ-Red system encoding genes, and the establishment of plasmid curing procedures based on the gene or growth at a sub-optimal temperature of 18°C-20°C with serial passages. We have shown the efficiency of this CRISPR/Cas9 method in the precise and unmarked deletion of different genes (, , , and ) from two strains of , as well as its usefulness in the targeted insertion of the gene encoding the green fluorescence protein into a precise genome location. As pCasPA was successfully introduced in other complex species, this study opens up the possibility of using CRISPR/Cas9-based systems as efficient tools for genome editing in these species, allowing faster and more cost-effective genetic manipulation.IMPORTANCE encompasses different species of bacteria, some of them pathogenic to animals and plants, but others are beneficial by promoting plant growth through symbiosis or as biocontrol agents. Among these species, , a member of the complex, is one of the predominant species infecting the lungs of cystic fibrosis patients, often causing respiratory chronic infections that are very difficult to eradicate. Since the species is understudied, we have developed a genetic tool based on the CRISPR/Cas9 system to delete genes efficiently from the genomes of these strains. We could also insert foreign genes that can be precisely placed in a chosen genomic region. This method, faster than other conventional strategies based on allelic exchange, will have a major contribution to understanding the virulence mechanisms in , but it can likely be extended to other species.