Project 2: Scalable in vitro gametogenesis for identifying genetic and epigenetic infertility factors. This project seeks to advance the understanding of infertility by developing and deploying in vitro gametogenesis platforms to identify underlying genetic and epigenetic causes. The field of reproductive genetics has been severely hindered by challenges in distinguishing causative infertility alleles amongst variants of unknown significance (VUS) identified by genome/exome sequencing. Our extensive use of mouse modeling revealed that purely in silico pathogenicity prediction of infertility VUS have high false positive rates, underscoring a need for scalable in vitro platforms for rapid screening of candidates. Speed and accuracy in identification of causative variants is critical for clinical diagnostics and gene therapies – a goal of this overall P50. Building upon a cytokine-free, transcription factor (TF)-based platform that we developed for simple and scalable in vitro differentiation of mouse embryonic stem cells (ESCs) into primordial germ cell-like cells (PGCLCs), this project proposes three interconnected aims to improve and deploy this system for accelerating infertility genetics and epigenetics. First, missense VUS identified in non-obstructive azoospermia (NOA) patients by Project 1, which correspond to PGC-expressed genes, will be screened in our mouse PGCLC differentiation system. Additionally, we will develop human iPSC lines with TFs that enable similar scalability to enable functional testing of human-specific VUS. Secondly, we will test VUS predicted to impact post-PGC stages of gametogenesis by CRISPR-assisted gene targeting in ESCs that will be differentiated by: 1) conventional methods involving co-culture of testis somatic cells with PGCLCs; 2) by engineering our scalable platform for autonomous and more efficient differentiation of PGCLCs into meiotic stages; and 3) making 100% chimeric mice that can be phenotyped directly. Third, the project will validate the impact of disruptions in epigenetic genes on both mouse and human PGCLC development, with a focus on 25 candidates identified in our CRISPR inhibition (CRISPRi) screen of 701 epigenetic genes. Additionally, we will test whether two widely- used histone deacetylase inhibitors (the anticonvulsant valproic acid and dietary supplement sodium butyrate), both of which mimic the effect of depleting the top hit NCOR2 in the PGCLC differentiation system, disrupt PGC development in mice. Overall, the integrated approach is designed to accelerate the validation of infertility variants and mutations that are flagged as being potentially causative by clinical sequencing. This will aid in training computational models (Project 1) and accelerate the identification actionable targets for gene therapy approaches (Project 3).