Efficient dual-negative selection for bacterial genome editing

• DOI: 10.1186/s12866-020-01819-2
• Publication Date: 2020-05-24T11:02:52Z
• Authors: Francesca Romana ...; Olivier Cunrath; Dirk Bumann
• Keywords: Gene Editing; 0301 basic medicine; 0303 health sciences; Methodology Article; Genes, Transgenic, Suicide; Salmonella enterica; Microbiology; QR1-502; 3. Good health; 03 medical and health sciences; Salmonella; Mutagenesis; Conjugation, Genetic; Drug Resistance, Multiple, Bacterial; MDR; Mutation; Pseudomonas aeruginosa; Escherichia coli; Homologous recombination; CRISPR-Cas Systems; Gene manipulation; Genome, Bacterial; Plasmids

Abstract Background Gene editing is key for elucidating gene function. Traditional methods, such as consecutive single-crossovers, have been widely used to modify bacterial genomes. However, cumbersome cloning and limited efficiency of negative selection often make this method slower than other methods such as recombineering.

Results 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.