Development of a CRISPR/Cas9-induced gene editing system for Pseudoalteromonas fuliginea and its applications in...

Pseudoalteromonas has been used as a model system to study cold adaptation and is of widespread interest in biotechnology and ecology. To explore its physiological responses to extreme cold, uncover functional genes, and clarify their ecological roles, efficient genetic tools are essential. However, existing genetic manipulation methods in Pseudoalteromonas rely on traditional homology-based recombination, which is laborious and time-consuming in this bacterial system. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we established a CRISPR/Cas9 system in Pseudoalteromonas and carried out an extensive investigation of the Type II CRISPR/Cas9 platform for gene editing in Pseudoalteromonas fuliginea, a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system in P. fuliginea, multiple genes were selected as targets, and the gene editing effects were confirmed through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions in P. fuliginea, encompassing the deletion of genes such as fliJ, indA, and genes encoding Pf sRNAs, as well as the in vivo insertion of 3×FLAG and the gfp gene. The average CRISPR/Cas9 gene editing efficiency in P. fuliginea exceeded 70%. In summary, we developed an efficient CRISPR/Cas9-based editing system in P. fuliginea, which can be utilized to accelerate the development of Pseudoalteromonas as a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications.IMPORTANCEPseudoalteromonas fuliginea is a marine bacterium with great potential for ecological and biotechnological research, yet its genetic manipulation has long been a technical challenge. In this study, we developed a gene editing system based on CRISPR technology that enables efficient and precise genome modification in this organism. Using this system, we successfully deleted, inserted, and tagged multiple genes, including regulatory and non-coding elements, with high success rates. Notably, several of these genes are linked to key traits such as motility and stress response, which contribute to microbial adaptation in polar environments. This tool allows researchers to directly test gene function and study microbial adaptation in cold marine environments. The ability to perform reliable genetic edits in P. fuliginea opens new possibilities for its use as a model organism and will support future advances in microbial ecology, environmental microbiology, and marine biotechnology.