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

Abstract

UNLABELLED: 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 isopropyl β-D-1-thiogalactopyranoside (IPTG)-inducible single-guide RNA (sgRNA). We demonstrate the efficacy of the CRISPRi system inby silencing an exogenous reporter (sfGFP) and an endogenous regulator (Fur) that controls intracellular iron homeostasis in bacteria. The inducible expression ofand the-directed sgRNA caused a 98.7% decrease in fluorescence in the knockdown strain. This silencing system was effective in sevenstrains from both genogroups. Furthermore, the same system was used to constructknockdown strains. A 50-fold decrease inexpression level was determined in these strains when the expression of thegRNA was induced with IPTG. By RNA-seq, we detected a significant increase in the expression of genes encoding the Feand Feacquisition systems and iron mobilization in theknockdown after IPTG induction. All the genes with over 2-fold increased expression in the RNA-seq presented the Fur box consensus sequence in their regulatory region. The implementation of the Mobile-CRISPRi system inhas been demonstrated to be effective, thus providing a tool with potential application for the analysis of gene function in this pathogen. It is anticipated that these analyses will be valuable in identifying genes involved in the mechanisms of pathogenesis of. IMPORTANCE: Salmonid rickettsial septicemia (SRS) is an infectious disease caused by the marine bacterium. 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, 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.