Abstract: FR-PO565
Single-Cell CRISPR Screening Is a Powerful Tool for Gene Regulatory Network Studies in Kidney Collecting Ducts
Session Information
- Fluid, Electrolyte, and Acid-Base Disorders: Basic
October 25, 2024 | Location: Exhibit Hall, Convention Center
Abstract Time: 10:00 AM - 12:00 PM
Category: Fluid, Electrolytes, and Acid-Base Disorders
- 1101 Fluid, Electrolyte, and Acid-Base Disorders: Basic
Authors
- Cao, Shuang, Medizinische Hochschule Hannover, Hannover, Niedersachsen, Germany
- López-Cayuqueo, Karen I., Medizinische Hochschule Hannover, Hannover, Niedersachsen, Germany
- Schmidt-Ott, Kai M., Medizinische Hochschule Hannover, Hannover, Niedersachsen, Germany
Background
The renal collecting duct plays a crucial role in maintaining the body’s fluid and electrolyte balance, which is essential for overall physiological homeostasis. However, the molecular mechanisms governing the functions of the medullary collecting duct remain insufficiently understood. Recent advancements in single-cell genomics and CRISPR-based genetic tools have opened new avenues for dissecting the complex regulatory networks. By identifying and characterizing the molecular regulators of the renal collecting ducts, we can gain deeper insights into their physiological mechanisms and potentially uncover novel understandings of collecting duct disorders.
Methods
1. SCENIC analysis: We used established snRNA-seq data from mouse kidneys and applied SCENIC methods to construct cell type-specific regulatory networks, and identified regulons based on gene co-expression and binding motifs.
2. Identification of transcription factors: From the constructed networks, we identified 24 specific transcription factors associated with collecting duct principal cells that exhibited significant gene regulatory network activities.
3. CRISPR-based single cell screen: To investigate the biological functions of these 24 TFs, we performed a CRISPR-based single cell screen experiment in an in vitro mIMCD3 cell model, perturbing TF expression and assessing the resulting transcriptomic phenotypes to understand their impact on gene expression profiles.
Results
The single-cell CRISPR screen effectively reproduced the transcriptional regulatory influence of the established transcription factor Grhl2 on downstream target genes, including Cldn4. The perturbation of the transcriptome profile of this transcription factor, which served as a central quality control parameter for the entire screen experiment, demonstrated the reliability of the study conducted. Among 24 candidates, we identified the transcription factor Ilf2 as a novel potential functional regulator of medullary collecting duct cell homeostasis.
Conclusion
Our study highlights the potential of CRISPR single cell screening as a powerful tool for uncovering the functions of novel genes in kidney cells. This approach improves our understanding of the molecular mechanisms involved in collecting duct cell physiology and provides a promising framework for future kidney disease research in nephrology.