Protocol for in vivo CRISPR screening targeting murine testicular cells

Abstract

Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• CRISPR
• Genomics
• Molecular Biology