直接针对睾丸细胞的体内 CRISPR 筛选。

IF 11.1 Q1 CELL BIOLOGY
Cell genomics Pub Date : 2024-03-13 Epub Date: 2024-03-05 DOI:10.1016/j.xgen.2024.100510
Yuki Noguchi, Yasuhito Onodera, Tatsuo Miyamoto, Masahiro Maruoka, Hidetaka Kosako, Jun Suzuki
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引用次数: 0

摘要

CRISPR-Cas9 短向导 RNA(sgRNA)文库筛选是了解生物现象分子机制的有力方法。然而,它在体内的应用目前还很有限。在这里,我们将之前建立的体外复苏筛选方法发展为体内筛选方法,利用精子获能作为指标,鉴定参与精子发生完整性的因素。通过将 sgRNA 文库导入睾丸细胞,我们成功地将视网膜变性 3(Rd3)基因定位为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。为了在 scRNA-seq 和蛋白质组学分析的基础上寻找细胞类型特异性信号通路,我们开发了一种计算工具 Hub-Explorer。通过这一工具,我们发现 Rd3 在诱导纤毛虫发生时通过调节线粒体的分布来调节氧化应激。总之,我们的筛选系统为破译生物过程中的分子机制提供了一种宝贵的体内方法。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
In vivo CRISPR screening directly targeting testicular cells.

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.

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CiteScore
7.10
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