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CRISPR/Cas mediated genome editing reveals new insights into the WNK‐SPAK/OSR1 network
Author(s) -
Roy Ankita,
Goodman Joshua,
Pittman Gabrielle,
Begum Gulnaz,
Donnelly Bridget,
Sun Dandan,
Subramanya Arohan
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.969.20
Subject(s) - microbiology and biotechnology , hek 293 cells , crispr , chemistry , post translational regulation , kinase , effector , phosphorylation , biology , biochemistry , gene
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, SPAK and OSR1. Multiple WNKs can coexist within a single cell type, although their relative contributions to SPAK/OSR1 activation and salt transport remain incompletely understood. Here, we outline a simple method to selectively knock out WNK expression from mammalian cells using single guide RNA (sgRNA) targeted CRISPR/Cas9 endonucleases. Genetically and biochemically validated WNK1 knockout HEK293T cells exhibited low baseline WNK4 and SPAK/OSR1 activity. This was associated with increased expression of KLHL3/CUL3, suggesting that WNK1 regulates WNK4 stability through their cognate E3 ubiquitin ligase. WNK1 KO cells also failed to trigger regulatory volume increase (RVI) during hypertonic stress, proving that WNK1 is necessary for cell volume control. Currently, we are adapting this technology to generate WNK4 and WNK3 KOs. To optimize sgRNA efficiency, we used a surrogate reporter‐based assay that triggers eGFP fluorescence upon target site editing, permitting enrichment of edited cells by FACS. This technique dramatically enhanced detection of genomic modification by T7E1 mismatch screening. Collectively, our data illustrate how this new and powerful technology can be used to dissect and analyze complex signaling networks. Support: NIDDK, VA