An expanded genetic toolkit for inducible expression and targeted gene silencing in Rickettsia parkeri.

Pathogenic species within the Rickettsia genus are transmitted to humans through arthropod vectors and cause a spectrum of diseases ranging from mild to life-threatening. Despite rickettsiae posing an emerging global health risk, the genetic requirements of their infectious life cycles remain poorly understood. A major hurdle toward building this understanding has been the lack of efficient tools for genetic manipulation, owing to the technical difficulties associated with their obligate intracellular nature. To this end, we implemented the Tet-On system to enable conditional gene expression in Rickettsia parkeri. Using Tet-On, we show inducible expression of antibiotic resistance and a fluorescent reporter. We further used this inducible promoter to screen the ability of R. parkeri to express four variants of the catalytically dead Cas9 (dCas9). We demonstrate that all four dCas9 variants can be expressed in R. parkeri and used for CRISPR interference (CRISPRi)-mediated targeted gene knockdown. We show targeted knockdown of an antibiotic resistance gene as well as the endogenous virulence factor sca2. Altogether, we have developed systems for inducible gene expression and CRISPRi-mediated gene knockdown for the first time in rickettsiae, laying the groundwork for more scalable, targeted mechanistic investigations into their infectious life cycles.IMPORTANCEThe spotted fever group of Rickettsia contains vector-borne pathogenic bacteria that are neglected and emerging threats to public health. Due to the obligate intracellular nature of rickettsiae, the development of tools for genetic manipulation has been stunted, and the molecular and genetic underpinnings of their infectious lifecycle remain poorly understood. Here, we expand the genetic toolkit by introducing systems for conditional gene expression and CRISPR interference (CRISPRi)-mediated gene knockdown. These systems allow for relatively easy manipulation of rickettsial gene expression. We demonstrate the effectiveness of these tools by disrupting the intracellular life cycle using CRISPRi to deplete the sca2 virulence factor. These tools will be crucial for building a more comprehensive and detailed understanding of rickettsial biology and pathogenesis.