Development of a CRISPR/Cas9-induced gene editing system forPseudoalteromona fuligineaand its applications in functional genomics

Pseudoalteromonashas 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 inPseudoalteromonasrely on traditional homology-based recombination, which is inefficient and time-consuming. Consequently, improving editing efficiency is crucial for advancing both basic research and applied potential. Here, we introduced the CRISPR/Cas9 system intoPseudoalteromonasfor the first time, and conducted an extensive investigation into the application of the Type II CRISPR/Cas9 system for gene editing inPseudoalteromonas fuliginea, a representative species thriving in the frigid polar oceans. To validate the feasibility of the CRISPR/Cas system inP. fuliginea, multiple genes were selected as targets and confirmed the gene editing effects through phenotypic changes or gene expression. We have successfully achieved both gene knockouts and insertions inP. fuliginea, encompassing the deletion of genes such asfliJ,indA, and genes encoding Pf sRNAs, as well as the invivoinsertion of3×flagand thegfpgene. The average CRISPR/Cas9 gene editing efficiency inP. fuligineaexceeded 70% (range: 73.3%-95.8%), which is significantly higher than the traditional homology-based approach (less than 0.1%). In summary, we developed an efficient CRISPR/Cas9-based editing system inP. fuliginea, which can be utilized to accelerate the development ofPseudoalteromonasas a model system for addressing fundamental questions related to extreme environmental adaptation and to fulfill its potential biotechnological applications.