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Generation of a RIP1 Knockout U937 Cell Line Using the CRISPR-Cas9 System
Published on: April 11, 2025
Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing
Ankita Roy1, Joshua H Goodman1, Gulnaz Begum2
1Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;
Abstract:
Sodium-coupled SLC12 cation chloride cotransporters play important roles in cell volume and chloride homeostasis, epithelial fluid secretion, and renal tubular salt reabsorption. These cotransporters are phosphorylated and activated indirectly by With-No-Lysine (WNK) kinases through their downstream effector kinases, Ste20- and SPS1-related proline alanine-rich kinase (SPAK) and oxidative stress-responsive kinase 1 (OSR1). Multiple WNK kinases can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Deletion of specific WNKs from cells that natively express a functional WNK-SPAK/OSR1 network will help resolve these knowledge gaps. Here, we outline a simple method to selectively knock out full-length WNK1 expression from mammalian cells using RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 endonucleases. Two clonal cell lines were generated by using a single-guide RNA (sgRNA) targeting exon 1 of the WNK1 gene, which produced indels that abolished WNK1 protein expression. Both cell lines exhibited reduced endogenous WNK4 protein abundance, indicating that WNK1 is required for WNK4 stability. Consistent with an on-target effect, the reduced WNK4 abundance was associated with increased expression of the KLHL3/cullin-3 E3 ubiquitin ligase complex and was rescued by exogenous WNK1 overexpression. Although the morphology of the knockout cells was indistinguishable from control, they exhibited low baseline SPAK/OSR1 activity and failed to trigger regulatory volume increase after hypertonic stress, confirming an essential role for WNK1 in cell volume regulation. Collectively, our data show how this new, powerful, and accessible gene-editing technology can be used to dissect and analyze WNK signaling networks.
Insights
Researchers used CRISPR-Cas9 gene editing to knock out WNK1, revealing its crucial role in cell volume regulation and WNK4 protein stability. This method aids in dissecting WNK signaling networks.
Area of Science:
- Molecular Biology
- Cell Physiology
- Genetics
Background:
- Sodium-coupled SLC12 cation chloride cotransporters are vital for cell volume and chloride homeostasis, regulated by With-No-Lysine (WNK) kinases via SPAK/OSR1.
- The specific roles of individual WNK kinases in activating downstream effectors and influencing salt transport are not fully understood.
- Investigating WNK kinase contributions requires selective knockout methods in cells with intact WNK-SPAK/OSR1 signaling.
Purpose of the Study:
- To develop a method for selectively knocking out full-length WNK1 expression in mammalian cells.
- To elucidate the role of WNK1 in WNK4 protein stability and SPAK/OSR1 activation.
- To analyze the function of WNK1 in cell volume regulation and response to osmotic stress.
Main Methods:
- Utilized RNA-guided clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) endonucleases to target exon 1 of the WNK1 gene.
- Generated two clonal mammalian cell lines with WNK1 knockout, confirmed by the absence of WNK1 protein expression due to indels.
- Assessed WNK4 protein abundance, KLHL3/cullin-3 E3 ubiquitin ligase complex expression, and SPAK/OSR1 activity in knockout versus control cells.
Main Results:
- WNK1 knockout cell lines showed reduced WNK4 protein abundance, indicating WNK1 is necessary for WNK4 stability.
- Reduced WNK4 was linked to increased KLHL3/cullin-3 E3 ubiquitin ligase expression, and this effect was reversible by WNK1 re-expression.
- Knockout cells displayed diminished baseline SPAK/OSR1 activity and an inability to perform regulatory volume increase following hypertonic stress.
Conclusions:
- WNK1 plays an essential role in maintaining WNK4 protein stability and is critical for cell volume regulation.
- The developed CRISPR/Cas9 method provides a powerful tool for dissecting WNK signaling pathways.
- This study highlights the importance of WNK1 in cellular responses to osmotic challenges.
