Mobile-CRISPRi as a tool for genetic manipulation in the intracellular pathogen Piscirickettsia salmonis

Piscirickettsia salmonis is the causative agent of Salmonid Rickettsial Septicemia (SRS), the main bacterial disease affecting the salmon industry in Chile. In this work, we implemented a Mobile-CRISPRi system to generate gene silencing using a catalytically inactive dCas9 protein and an IPTG-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system in P. salmonis by silencing an exogenous gene encoding green fluorescent protein (sfGFP), and the endogenous homolog of the fur gene, whose gene product regulates intracellular iron homeostasis in bacteria. The inducible expression of dcas9 and the sfGFP-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in seven P. salmonis strains from both genogroups. Furthermore, the same system was used to construct fur knockdown strains. A 50-fold decrease in fur expression level was determined in these strains, when the expression of the fur gRNA was induced with IPTG. By RNA-seq we detected a significant increase in the expression of genes encoding the Fe+2 and Fe+3 acquisition systems and iron mobilization in the fur1 knockdown, after IPTG induction. All the genes with over two-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The successful implementation of the Mobile-CRISPRi system in P. salmonis paves the way for systematic analysis of gene function in this pathogen We anticipate that these analyses will be very valuable in identifying genes involved in the mechanisms of pathogenesis of P. salmonis.