CRISPR/Cas9 Screening for Protein Kinases That Regulate Aquaporin-2 Gene Transcription in Collecting Ducts

Background: Water transport in the collecting duct is controlled in part through regulation of transcription of the gene that codes for aquaporin-2 (AQP2). Animal models of water balance disorders have revealed defective control of AQP2 transcription in both polyuric and water retention disorders. Here, we have used CRISPR screening of the entire kinome in a mouse cortical collecting duct cell line (mpkCCD) to identify protein kinases that regulate AQP2 gene transcription. Methods: A CRISPR knockout pooled library was utilized, targeting 713 protein kinases with 4 guides (gRNAs) for each gene. These gRNAs were simultaneously transduced into GFP-reporting mpkCCD cells, allowing GFP as a reporter for AQP2 transcription. In the presence of vasopressin analog dDAVP, the gRNA-transduced cells were sorted into four groups (GFP-negative, low, mid, and high) based on GFP intensity. RNA sequencing was then performed to identify the targeted kinases. Results: Among 713 targets, 29 protein kinases showed significant bias in GFP abundance including 16 positive regulators of AQP2 gene transcription and 13 negative regulators. One positive regulator was PKA catalytic α (Prkaca) that had been previously identified as a positive regulator. Conversely, PKA regulatory subunit (Prkar1a), is known to inhibit PKA catalytic subunit, was identified as a negative regulator. These findings provide positive controls for the methodology. Novel positive regulators include AKT1, PIM3 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). Conclusion: Whole kinome CRISPR screening identified 29 protein kinases as putative regulators of AQP2 gene transcription. Pending one-by-one systematic validation of roles for each kinase as determinants of AQP2 protein abundance, the data will provide a basis for understanding mechanisms involved in defective transcription of the AQP2 gene in water balance disorders and a valuable information resource guiding future studies of regulation in collecting duct principal cells.