A Dual-Plasmid CRISPR/Cas9-Based Genome Editing System for Efficient Gene Knockout and Large DNA Integration in Cronobacter malonaticus

Species of Cronobacter are emerging foodborne pathogens that pose a significant threat to neonates. Functional genomic studies in Cronobacter have been hindered by the lack of efficient genetic manipulation tools. Here, we established a CRISPR/Cas9-based genome editing platform for Cronobacter. We developed a dual-plasmid platform, pAmpCRISPR/pCasCm, by integrating CRISPR/Cas9 with the λ-Red recombination machinery. This platform enables scarless genome editing in Cronobacter malonaticus and proved effective for gene knockout and large-fragment integration under the tested conditions. Furthermore, we validated this platform in another clinically relevant species, Cronobacter sakazakii. The development of this genome editing toolkit provides a useful approach for fundamental research into Cronobacter pathogenesis, such as bacterial physiology studies, drug target exploration, and metabolic engineering.