Summary The study of germ cells has shaped our understanding of many basic fundamental processes across different species. Germ cells share a number of features that have long fascinated biologists. These cells undergo meiosis to form haploid gametes, they are exceptionally good at repairing DNA damage, they utilize a number of small RNA pathways to silence transposable elements, and they reprogram their epigenome back to a state that supports totipotency. Here, we proposed to use the Drosophila ovary as a model to continue to gain insights into genome stability, germ cell differentiation, and the cell-specific regulation of mRNA translation and ribosome biogenesis/turnover. Over the last five years, we have adopted and optimized a number of innovative CRISPR-Cas9- and recombineering-based methods for manipulating the Drosophila genome. Using these approaches, we have mutated and/or tagged over 100 genes that exhibit enriched expression in Drosophila gonads. This work provides a solid foundation for our planned efforts over the next five years. We will focus on a number of different but related areas. We will continue to characterize the highly conserved Germ Cell Nuclear Acidic Peptidase (GCNA) gene and its function in protecting the integrity of germ cells across species. We will also continue to characterize how cytoplasmic Rbfox1 controls early germ cell development. Our previous screening efforts have identified a small number of mutations that exhibit germ cell tumor formation or germ cell loss phenotypes. The molecule function of the disrupted genes will be characterized using the tools and methods we have in hand. Lastly, we are in the process of generating a number of innovative tools that will allow us to better assess ribosome biogenesis and turnover during germ cell development and early embryogenesis. We are very excited by this proposed work and believe the successful completion of these projects will have a positive impact on our understanding of germ cell biology and other molecular processes that impact human health.