Dr. Paula Cohen has developed a co-culture system involving isolated spermatogonial cells and testicular somatic cells to induce entry of sperm precursors to resume meiosis as part of their maturation process. Additionally, the project has performed initial studies on decellularized testicular scaffolds and extracellular matrix analogs in order to provide a physical environment to support viability and maturation of sperm, with the goal of establishing a relevant micro physiological system replicating key stages of sperm development. Genetic tools for unbiased target identification (e.g. PerturbSeq) and targeted reverse genetics (e.g. CRISPR-based approaches) are being optimized for use in parallel with this co-culture system to provide a genetic pipeline for target identification and validation. This grant will focus on final optimization and integration of these various tools, including a deep characterization of their coculture system using sequencing and transcriptomics approaches and development of reporters to allow facile monitoring of sperm meiosis. The project will leverage scRNAseq data, PerturbSeq data, and genes identified from the literature (including human genetic studies) to build a pipeline of targets for validation via CRISPR/Cas13d and in vivo characterization. This will yield novel targets for male contraception that can enter drug discovery activities as part of the foundation’s broader program.