PDF(Pubmed)
Abstract：
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.
摘要：
CRISPR-Cas9短向导RNA(sgRNA)文库筛选是理解生物现象分子机制的有力方法。然而,其体内应用目前有限。这里,我们将先前建立的体外恢复筛选方法发展为体内方法，以利用精子获能作为指标来鉴定参与精子发生完整性的因素。通过将sgRNA文库引入睾丸细胞，我们成功地确定了视网膜变性3（Rd3）基因是精子发生的重要因素。单细胞RNA测序（scRNA-seq）分析强调了Rd3在圆形精子细胞中的高表达，蛋白质组学分析表明Rd3与线粒体相互作用。为了寻找基于scRNA-seq和蛋白质组学分析的细胞类型特异性信号通路，我们开发了一个计算工具，Hub浏览器。通过这个,我们发现Rd3在纤毛发生诱导时通过调节线粒体分布来调节氧化应激。总的来说，我们的筛选系统提供了一种有价值的体内方法来破译生物过程中的分子机制。
