Project Summary/Abstract More than 1.8 million cases of Chlamydia trachomatis infections are reported to the CDC each year, making it the most commonly reported infectious disease in the country. This pathogen is an obligate intracellular bacterium that can only reproduce in human cells. As a result of this dependence on a host cell, Chlamydia has undergone reductive evolution and has one of the smallest of bacterial genomes. Many of the remaining genes, including Chlamydia-specific genes, are essential genes whose functions have not been elucidated because of the limitations of current genetic methods. To study chlamydial gene function, we are developing a novel protein depletion method that exploits the ability of small RNAs (sRNAs) to downregulate expression of specific target proteins. In proof-of-principle experiments, we have shown that we can inducibly knockdown protein levels for two chlamydial proteins, IncA and IncG. In Aim 1, we will knockdown additional C. trachomatis genes, including non-essential and essential genes. In Aim 2, we will compare this sRNA knockdown method to another protein depletion method, CRISPRi. We will compare the ability of each method to control the level and timing of knockdown and will investigate whether they cause polar effects on genes within the same operon. Successful completion of these studies will lead to an innovative genetic approach for selectively depleting a Chlamydia protein. This new tool for studying chlamydial gene function will have a sustained impact on the field.