Project Summary: Development of Tools for Conditional Knockouts in Chlamydia Chlamydia is an obligate intracellular bacterial pathogen that causes a range of serious diseases in humans. In developed countries, Chlamydia trachomatis is the primary cause of bacterial sexually transmitted infections (STI). In developing countries, C. trachomatis is not only a significant cause of STI, but it is also responsible for the primary cause of infectious preventable blindness, trachoma. The major concern of chlamydial infections is that they are often asymptomatic and undiagnosed, which can lead to chronic sequelae. These include pelvic inflammatory disease, tubal factor infertility, and reactive arthritis for C. trachomatis. Consequently, chlamydial diseases remain a significant burden on health care systems around the world. In adapting to obligate intracellular growth, Chlamydia has significantly reduced its genome size and eliminated genes from various pathways as it relies on the host cell for its metabolic needs. This, combined with its obligate intracellular dependence, makes Chlamydia a difficult organism with which to work and study. Recent development of genetic tools to study chlamydiae mechanistically have significantly enhanced our understanding of this pathogen. However, because most chlamydial genes are likely to be essential, the current toolset is insufficient to study these critical genes. We have demonstrated the feasibility of CRISPR interference to inducibly knock down chlamydial gene expression, in other words, to create conditional knockouts. The proposal is designed to leverage our early successes to better characterize and refine the utility of the system as well as to develop complementary strategies for blocking chlamydial protein function. Results will lead to the identification of novel diagnostic and therapeutic targets that have the potential to identify and treat asymptomatic chlamydial infections.