PROJECT SUMMARY Male infertility is a complicated pathological condition characterized by a heterogeneous spectrum of phenotypic presentations, rendering its underlying causes obscure. In recent years, genetic disorders emerge as one of the leading causes of male infertility, accounting for at least 15% of cases. Therefore, understanding the genetic network that influences various aspects of male fertility such as spermatogenesis (i.e., sperm production) would greatly benefit the diagnosis and treatment of male infertility. However, the estimate that thousands of genes may be involved in spermatogenesis makes it difficult to ascribe specific genetic causes to male infertility. Traditionally the functions of testis-expressing genes can be analyzed by generating knockout mouse lines given the similarities between mouse and human spermatogenesis. However, this approach demands significant time and resources, making it challenging to scale. Emerging technologies such as CRISPR screens coupled with single cell RNA sequencing (scRNA-seq) can examine gene functions at scale, but suffer from two major limitations for dissecting testicular gene functions: (i) the lack of a cell culture model that faithfully recapitulates spermatogenesis makes it difficult to assess whether perturbation of a gene leads to defects in sperm production in vitro; and (ii) while cell intrinsic effects of a gene perturbation may be read out using scRNA-seq, the extracellular effects of a gene perturbation cannot be assessed due to tissue disassociation. This excludes using CRISPR screens to identify genes controlling phenotypes that require spatial resolution to assess such as genes encoding for secreted factors. Therefore, a CRISPR screen approach that retains the spatial context of spermatogenesis is needed to interrogate testicular gene functions at a high throughput. There are currently two main challenges to develop a spatially resolved CRISPR screen approach: (i) to capture mRNA transcripts in situ at scale and at single-cell resolution; and (ii) to read out the identity of each gene perturbation and the mRNA transcripts within a cell simultaneously. To address these two main challenges, we will greatly improve and expand an in situ RNA sequencing protocol we have recently established to spatially profile hundreds of mRNA species directly in testicular samples. We will also perform a proof-of-concept experiment to demonstrate co-capture of CRISPR guide RNA and mRNA in intact testicular tissues using the same in situ sequencing approach. Together, these efforts will enable a highly innovative functional genomics approach to dissect gene functions in the native tissue context at an unprecedented spatial resolution and throughput.