2024/03/01 by Yuki Noguchi, Yasuhito Onodera, Tatsuo Miyamoto +3 · 1 voice · 3 citations
Biochemistry, Genetics and Molecular Biology · #CRISPR and Genetic Engineering #Single-cell and spatial transcriptomics #Retinal Development and Disorders
paper · doi:10.1016/j.xgen.2024.100510
openalex publication_date 2024/03/01 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/28
CRISPR-Cas9 short guide RNA (sgRNA) library screening is a powerful approach to understand the molecular mechanisms of biological phenomena. However, its in vivo application is currently limited. Here, we developed our previously established in vitro revival screening method into an in vivo one to identify factors involved in spermatogenesis integrity by utilizing sperm capacitation as an indicator. By introducing an sgRNA library into testicular cells, we successfully pinpointed the retinal degeneration 3 (Rd3) gene as a significant factor in spermatogenesis. Single-cell RNA sequencing (scRNA-seq) analysis highlighted the high expression of Rd3 in round spermatids, and proteomics analysis indicated that Rd3 interacts with mitochondria. To search for cell-type-specific signaling pathways based on scRNA-seq and proteomics analyses, we developed a computational tool, Hub-Explorer. Through this, we discovered that Rd3 modulates oxidative stress by regulating mitochondrial distribution upon ciliogenesis induction. Collectively, our screening system provides a valuable in vivo approach to decipher molecular mechanisms in biological processes.