Efficient Dual-Negative Selection for Bacterial Genome Editing

We describe a versatile method for chromosomal gene editing based on classical consecutive single-crossovers. The method exploits rapid plasmid construction using Gibson assembly, a convenient E. coli donor strain, and efficient dual-negative selection for improved suicide vector resolution. We used this method to generate in frame deletions, insertions and point mutations in Salmonella enterica with limited hands-on time. Similar strategies allowed efficient gene editing also in Pseudomonas aeruginosa and multi-drug-resistant (MDR) Escherichia coli clinical isolates.