High‐Efficiency Genome Editing in Naturally Isolated Aeromonas hydrophila and Edwardsiella Piscicida Using the CRISPR‐Cas9 System

ABSTRACT Aeromonas hydrophila and Edwardsiella piscicida are significant bacterial pathogens in aquaculture, causing severe diseases and tremendous economic losses worldwide. Additionally, both of them can act as opportunistic pathogens in humans, leading to severe infections. Efficient genome editing tools for these pathogens are essential for understanding their pathogenic mechanisms and physiological behaviors, enabling the development of targeted strategies to control and mitigate their effects. In this study, we adapted the CRISPR‐Cas9 system for high‐efficiency, marker‐less genome editing in multiple naturally isolated strains of these two aquaculture pathogens. We developed a streamlined procedure that successfully generated deletion mutants of the aerA gene (encoding for aerolysin, a pore‐forming toxin that plays a critical role in the pathogenicity) and the gfp insertion mutants in three naturally isolated A. hydrophila strains. Additionally, we deleted five putative hemolysin‐encoding genes in both A. hydrophila ML10‐51K and its ∆aerA derivative. The same system was also applied to the naturally isolated E. piscicida S11‐285 strain, successfully deleting the ssaV gene (a component of the Type III Secretion System—a critical virulence mechanism in many pathogenic bacteria). The methodologies developed herein could be broadly applied to other pathogenic strains from natural environments, providing valuable tools for studying bacterial pathogenesis and aiding in the development of effective control strategies.