Efficient dual-negative selection for bacterial genome editing

Gene editing is key for elucidating gene function. Traditional

Our method is time-effective and allows facile manipulation of multiple bacterial species including MDR clinical isolates. We anticipate that this method might be broadly applicable to additional bacterial species, including those for which recombineering has been difficult to implement.

Here, we established a time-effective variant of consecutive single-crossovers. This 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. Adapted versions enabled efficient gene editing also in Pseudomonas aeruginosa and multi-drug resistant (MDR) Escherichia coli clinical isolates. Conclusions: Our method is time-effective and allows facile manipulation of multiple bacterial species including MDR clinical isolates. We anticipate that this method might be broadly applicable to additional bacterial species, including those for which recombineering has been difficult to implement.