High-efficiency genome editing in naturally isolated Aeromonas hydrophila and Edwardsiella piscicida using the CRISPR-Cas9 system

Authors: FENG, JUN - Auburn University; MA, YUECHAO - Auburn University; Zhang, Dunhua; WANG, YI - Auburn University

Submitted to: Biotechnology and Bioengineering
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/25/2024
Publication Date: 11/4/2024
Citation: Feng, J., Ma, Y., Zhang, D., Wang, Y. 2024. High-efficiency genome editing in naturally isolated Aeromonas hydrophila and Edwardsiella piscicida using the CRISPR-Cas9 system. Biotechnology and Bioengineering. 122:606-614. https://doi.org/10.1002/bit.28889.
DOI: https://doi.org/10.1002/bit.28889

Interpretive Summary: CRISPR-Cas9 system was modified and developed from a naturally occurring genome editing mechanism that many bacteria use as an immune defense. The system has been adapted to manipulate (delete/insert/modify) targeted genes in various organisms to understand functions of specific genes. In this study, we seeked to develop a high-efficiency, marker-less genome editing method based on the CRISPR-Cas9 system for several aquaculture important bacterial pathogens, including Aeromonas hydrophila and Edwardsiella piscicida. We successfully developed a highly efficient genetic manipulation tool, enabling us to achieve gene deletions and insertions. Our work represents the first application of the CRISPR-Cas9 system in genome editing of these naturally isolated aquaculture pathogens, providing valuable tools for future research in this field.

Technical Abstract: Aeromonas hydrophila and Edwardsiella piscicida are significant bacterial pathogens in aquaculture, causing severe diseases and tremendous economic losses worldwide. 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 can 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.