Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen <i>Piscirickettsia salmonis</i>

Salmonid rickettsial septicemia (SRS) is an infectious disease caused by the marine bacterium <i>Piscirickettsia salmonis</i>. This Gamma-proteobacteria is a fastidious and facultative intracellular pathogen that has a nearly worldwide distribution, particularly impacting Chilean salmonid aquaculture. Its fastidious nature has made it hard to grow in labs, hindering research into its virulence and treatment, especially because of the lack of molecular techniques to study gene function. We show here the successful implementation of the Mobile-CRISPRi system for gene silencing. Significantly, we have adapted this technique for use with the marine pathogen <i>P. salmonis</i>, inserting exogenous genes into the bacterium's chromosome to ensure their constitutive and inducible expression and silencing both exogenous and endogenous gene expression. The Mobile-CRISPRi system was also used to study the iron regulator Fur, confirming Fur's relevance to the iron metabolism in the pathogen.