Project Summary/Abstract Coxiella burnetii is an obligate intracellular bacterial pathogen of mammals, and the causative agent of the zoonotic disease Q fever. Upon entry into mammalian cells, C. burnetii passively traffics through the endolysosomal pathway before directing the establishment of a spacious, lysosome-derived organelle called the Coxiella-containing vacuole (CCV) that allows bacterial replication. The process of CCV biogenesis and maturation is driven by the collective activity of the roughly 130 effector proteins that are translocated by the bacterial Dot/Icm type IVB secretion system into the host cell cytosol during infection. Despite several years of intense research, the cellular targets, biochemical activities, and functional relationships among each other remain unknown for most of the Dot/Icm effector proteins encoded by C. burnetii. Genetic manipulations in C. burnetii, such as the generation of targeted gene deletion mutants, have proven to be a challenging, lengthy, and highly inefficient undertaking. In this project we propose to develop an inducible and reversible CRISPR interference (CRISPRi) platform to efficiently silence C. burnetii effector gene expression during intracellular replication. In Aim 1, we will construct a comprehensive CRISPRi strain library targeting all genes that are predicted to encode C. burnetii Dot/Icm effector proteins, individually. This targeted approach will allow us to assess the contributions of individual effector proteins to vacuole biogenesis and intracellular replication. In Aim 2, we will leverage the powerful advantage of CRISPRi to simultaneously reduce the expression of multiple genes in the same strain. The goal is to begin teasing apart genetic interactions between effectors. We will express pairwise combinations of sgRNAs targeting a set of 17 effector genes that have previously been identified as important for CCV biogenesis and/or intracellular replication. Together, these aims will greatly enhance the toolkit available for studying virulence genes involved in C. burnetii intracellular replication and pathogenesis, and will start to shed light on epistatic relationships among effector proteins important for the formation of the vacuolar niche.