Protocol for in vivo CRISPR screening targeting murine testicular cells

In vivo genome-wide screening elucidates tissue-specific molecular events. Here, we present a protocol for an in vivo genome-wide CRISPR-Cas9 single-guide RNA (sgRNA) library screening technique optimized for mouse testicular cells to investigate spermatogenesis. We describe steps for virus injection, sperm sorting, and primase-based whole-genome amplification. We then detail procedures for library reconstruction using a "revival screening" technique. Our approach reveals intricate spermatogenesis processes and is adaptable for diverse tissue-specific studies. For complete details on the use and execution of this protocol, please refer to Noguchi et al.¹.