CRISPR/Cas9-mediated genome-wide kinome screening to identify protein kinases that regulate AQP2 gene transcription

Water balance in the body is controlled in part through water reabsorption in the kidney collecting duct principal cell through regulation of 1) transcription of aquaporin-2 (AQP2) and 2) trafficking of AQP2 to and from the apical plasma membrane. Impaired regulation of AQP2 transcription has been observed in several water balance disorders, including polyuric and water retention conditions. Here, we utilized a CRISPR screening technique targeting the complete set of protein kinases in a mouse cortical collecting duct cell line (mpkCCD) to identify kinases that regulate AQP2 gene transcription. A CRISPR knockout library targeting 713 proteins, with four gRNAs per gene, was employed. The gRNAs were transduced mpkCCD cells in which GFP is expressed as a reporter for AQP2 transcription at sufficient dilution to limit transduction to < gRNA per cell. Grown in the presence of vasopressin analog dDAVP, the cells were flow-sorted based on GFP intensity. DNA sequencing was performed to identify the kinase gRNAs associated with increased or decreased AQP2 transcription. Individual kinase knock-out cell lines were generated to verify the role of each kinase in AQP2 regulation. In total, 29 proteins showed significant bias in GFP abundance including 16 positive regulators and 13 negative regulators of AQP2 gene transcription. PKA catalytic α (Prkaca) which is a known positive regulator of AQP2 transcription was indeed identified as a positive regulator. Conversely, PKA regulatory subunit (Prkar1a), which is known to inhibit PKA catalytic subunit, was identified as a negative regulator. Additional positive regulators include DYRK1A, AKT1, certain cyclin-dependent kinases (CDKs), and the polarity kinase MARK2. Negative regulators include both subunits of the TGF-β receptor (previously implicated in the vasopressin escape phenomenon) and several components of the stress-activated MAP kinase pathway (Map3k1, Map2k4, and Map2k7). DYRK1A (“midbrain homolog”), a novel positive regulator of AQP2 transcription, was previously identified as general positive regulator of transcription by phosphorylation of the C-terminal domain of RNA polymerase II. DYRK1A was studied further by generating DRYK1A KO cell lines. Indeed, Dyrk1a knock-out cells were unresponsive to dDAVP treatment and showed reduced AQP2 protein abundance. Overall, CRISPR/Cas9 screening identified multiple protein kinases as putative regulators of AQP2 gene transcription. Some of these kinases were already reported to regulate AQP transcription, but most appear to be novel regulators. Among these is DYRK1A, which we showed is necessary for vasopressin-dependent AQP2 gene transcription. This work was funded by the Division of Intramural Research, National Heart, Lung, and Blood Institute (Projects ZIAHL001285 and ZIAHL006129, to M.A.K.). This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.