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In vivo CRISPR screening directly targeting testicular cells
Yuki Noguchi1, Yasuhito Onodera2, Tatsuo Miyamoto3
1Graduate School of Biostudies, Kyoto University, Konoe-cho, Yoshida, Sakyoku, Kyoto 606-8501, Japan; Institute for Integrated Cell-Material Sciences (WPI-iCeMS), Kyoto University, Yoshida-Honmachi, Sakyoku, Kyoto 606-8501, Japan.
Cell Genomics
|March 6, 2024
Summary
We developed an in vivo CRISPR-Cas9 screening method to identify genes crucial for sperm development. This method identified the Rd3 gene as vital for spermatogenesis integrity and oxidative stress regulation.
Area of Science:
- Molecular Biology
- Genetics
- Reproductive Biology
Background:
- CRISPR-Cas9 (Clustered Regularly Interspaced Short Palindromic Repeats-Cas9) short guide RNA (sgRNA) library screening is effective for in vitro studies but limited in vivo.
- Understanding molecular mechanisms in complex biological processes like spermatogenesis requires robust in vivo screening tools.
Purpose of the Study:
- To adapt an in vitro revival screening method for in vivo application.
- To identify novel genetic factors regulating spermatogenesis integrity using sperm capacitation as a readout.
- To elucidate the molecular function of identified genes in male germ cell development.
Main Methods:
- Development of an in vivo CRISPR-Cas9 sgRNA library screening system.
- Introduction of the sgRNA library into testicular cells to screen for genes affecting spermatogenesis.
- Utilization of sperm capacitation as an indicator for successful spermatogenesis.
- Single-cell RNA sequencing (scRNA-seq) and proteomics analysis to identify and characterize gene function.
- Development of the computational tool Hub-Explorer for pathway analysis.
Main Results:
- Successfully adapted the in vitro revival screening method for in vivo application.
- Identified the retinal degeneration 3 (Rd3) gene as a critical factor in maintaining spermatogenesis.
- scRNA-seq revealed high Rd3 expression in round spermatids.
- Proteomics indicated Rd3 interacts with mitochondria.
- Hub-Explorer analysis showed Rd3 modulates oxidative stress via mitochondrial regulation during ciliogenesis.
Conclusions:
- The developed in vivo CRISPR-Cas9 screening system is a valuable tool for deciphering molecular mechanisms in biological processes.
- The retinal degeneration 3 (Rd3) gene plays a significant role in spermatogenesis integrity by regulating mitochondrial function and oxidative stress.
- This study provides new insights into the genetic regulation of male fertility and opens avenues for future research in reproductive biology.
