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. This study employed a Mobile-CRISPRi system to genetically manipulate Piscirickettsia salmonis, which was first cultured under specific conditions (18°C in NPB/NPA media) with purity confirmed by PCR-RFLP. The system was delivered via tri-parental conjugation, where E. coli SM10 donor strains—one carrying the CRISPRi plasmid (pJMP2754, pJMP2774, or a sgRNA-targeting plasmid) and another providing the Tn7 transposase (pTNS3)—were mixed with a stationary-phase P. salmonis recipient culture on a filter. After incubation, transconjugants were selected on antibiotic-containing plates, and successful genomic integration of the plasmid into the glmS locus was confirmed by PCR screening, yielding genetically modified P. salmonis strains for subsequent functional assays.