With almost half of all human pregnancies being unwanted, and the persistent problem of limited contraceptive options for women, the need for creative solutions for contraceptive targeting remains higher than ever. In addition, development of effective male contraception has been thwarted by the very limited number of strategies available to male reproductive biologists. The current proposal seeks to mitigate this problem by establishing a novel intervention point for drug targeting: the spermatogonia-to-meiosis transition, a proposal that was established in response to a Gates Grand Challenges Exploration initiative focused on accelerating discovery of novel contraceptive agents by establishing a robust technical platform for investigating sperm maturation and associated drug targets.  Disrupting the entry into meiosis should block spermatogenesis, while at the same time preserving the integrity of the spermatogonial stem cell (SSC) population for future resumption of spermatogenesis at a time when contraception is no longer needed. Moreover, this phase of spermatogenesis is amenable to drug targeting because it lies outside of the blood-testis barrier, while manipulation of this system is unlikely to cause unwanted side effects on male libido. In our Phase II studies, we will optimize a culture method we devised in Phase I for use in “Perturb-seq”, a state-of-the-art method that will allow us to identify key genes that are important for the transition into meiosis. Such genes can be considered strong potential drug targets. Once we have established the Perturb-seq protocol, we will select a subset of genes identified in this screen, as well as in our single cell transcriptome studies in Phase I. These genes will be used in a CRISPR/Cas13-driven RNA knockdown approach to temporarily disrupt their expression in cultured spermatogonia and to investigate whether such knockdown results in the reversible disruption of meiotic entry. Our studies are the first to investigate meiotic entry as a viable target for contraceptive targeting, and to identify genes that regulate this process as potential candidates for drug targeting. Dissemination to the research community of the tools and strategies identified in our Gates Phase I and Phase II studies, along with our continued studies of this potential “switch” point in spermatogenesis, could alter the landscape for future contraceptive drug development and provide a vital breakthrough in the search for viable male contraceptives.