title: Generation of WNK1 knockout cell lines by CRISPR/Cas-mediated genome editing.
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.
authors.affiliation: Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;
authors.forename: Ankita
authors.initials: A
authors.lastname: Roy
authors.affiliation: Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;
authors.forename: Joshua H
authors.initials: JH
authors.lastname: Goodman
authors.affiliation: Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;
authors.forename: Gulnaz
authors.initials: G
authors.lastname: Begum
authors.affiliation: Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;
authors.forename: Bridget F
authors.initials: BF
authors.lastname: Donnelly
authors.affiliation: Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania;
authors.forename: Gabrielle
authors.initials: G
authors.lastname: Pittman
authors.affiliation: Department of Medicine, University of Maryland Medical School, Baltimore, Maryland; and.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Weinman
authors.affiliation: Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania.
authors.forename: Dandan
authors.initials: D
authors.lastname: Sun
authors.affiliation: Department of Medicine, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Department of Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania; Veterans Affairs Pittsburgh Healthcare System, Pittsburgh, Pennsylvania ars129@pitt.edu.
authors.forename: Arohan R
authors.initials: AR
authors.lastname: Subramanya
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D064113:CRISPR-Cas Systems; D002352:Carrier Proteins / Q000235:genetics / Q000378:metabolism; D048430:Cell Shape; D048429:Cell Size; D044842:Cullin Proteins / Q000235:genetics / Q000378:metabolism; D015536:Down-Regulation; D005091:Exons; D055785:Gene Knockdown Techniques / Q000379:methods*; D015894:Genome, Human; D005838:Genotype; D057809:HEK293 Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000172:deficiency* / Q000235:genetics; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; D009997:Osmotic Pressure; D010641:Phenotype; D017346:Protein Serine-Threonine Kinases / Q000172:deficiency* / Q000235:genetics / Q000378:metabolism; D055550:Protein Stability; D015398:Signal Transduction; D013997:Time Factors; D014162:Transfection; D000076204:WNK Lysine-Deficient Protein Kinase 1
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C503645:CUL3 protein, human; D002352:Carrier Proteins; D044842:Cullin Proteins; D047908:Intracellular Signaling Peptides and Proteins; C489837:KLHL3 protein, human; D008840:Microfilament Proteins; D015778:Minor Histocompatibility Antigens; C118135:OXSR1 protein, human; D017346:Protein Serine-Threonine Kinases; C486409:STK39 protein, human; D000076204:WNK Lysine-Deficient Protein Kinase 1; C498743:WNK1 protein, human; C433806:WNK4 protein, human
keywords: CRISPR/Cas system; SLC12 cotransporters; WNK1; genome editing