Efficient genome editing for Pseudomonas aeruginosa using CRISPR-Cas12a

•A two-plasmid CRISPR-Cas12a genome editing system for Pseudomonas aeruginosa.•The two plasmids can be easily cured for serial genome editing.•Efficient gene deletion, insertion and replacement in P. aeruginosa.•Deletion of genes involved in the biosynthesis of the virulence factor pyocyanin. The CRISPR-Cas12a system has been demonstrated as an attractive tool for bacterial genome engineering. In particular, FnCas12a recognizes protospacer-adjacent motif (PAM) sites with medium or low GC content, which complements the Cas9-based systems. Here we explored Francisella novicida Cas12a (FnCas12a) for genome editing in Pseudomonas aeruginosa. By using a two-plasmid system expressing the constitutive FnCas12a nuclease, the inducible λRed recombinase, a CRISPR RNA (crRNA), we achieved gene deletion, insertion and replacement with high efficiency (in most cases > 75%), including the deletion of large DNA fragments up to 15 kb and the serial deletion of duplicate gene clusters. This work should provide a useful and complementary addition to the genome engineering toolbox for the study of P. aeruginosa biology and physiology.