2024/09/01 by Yuki Noguchi, Masahiro Maruoka, Jun Suzuki · 1 voice
Biochemistry, Genetics and Molecular Biology · Agricultural and Biological Sciences · #CRISPR and Genetic Engineering #Pluripotent Stem Cells Research #Chromosomal and Genetic Variations
paper · doi:10.1016/j.xpro.2024.103306
openalex publication_date 2024/09/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/23
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 • Protocol for in vivo genome-wide screening targeting murine testicular cells • Method for introducing lentivirus-based sgRNA library into male germ cells • Instructions for applying the revival screening method to testis • Pipeline for analyzing sgRNA library results Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. 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.