CRISPR/Cas9 screening for protein kinases that regulate aquaporin-2 gene transcription in collecting duct

Authors

• E Park
• L Chen
• MA Knepper

Abstract

Water transport in the renal collecting duct is controlled partly through the regulation of Aqp2 gene transcription, which codes for the aquaporin-2 (AQP2) water channel. Defective control of Aqp2 transcription is seen in both polyuric and water-retention disorders. Here, we have used kinome CRISPR screening to identify protein kinases that regulate Aqp2 transcription.
A mouse kinome CRISPR knockout library was utilized, targeting 713 proteins each with 4 gRNAs (Addgene). These gRNAs were simultaneously transduced into mpkCCD cells expressing Cas9 and GFP in the Aqp2 locus. The gRNA-transduced cells were sorted into four groups according to GFP levels. DNA sequencing was performed to identify the targeted kinases.
Twenty-nine protein kinases showed significant bias in GFP abundance including 16 positive and 13 negative regulators of Aqp2 transcription. PRKACA, a known AQP2 positive regulator, was confirmed as a positive regulator, while PRKAR1A, which inhibits the PKA catalytic subunit, was confirmed as a negative regulator. These provide positive controls for the methodology. Novel positive regulators include AKT1, PIM3 and MARK2. Negative regulators include all three TGFBR isoforms (known mediators of vasopressin escape) and several components of the stress-activated MAP kinase pathway (MAP3K1, MAP2K4, and MAP2K7).
Overall, kinome screening identified 29 kinases as putative regulators of Aqp2 transcription. Pending one-by-one systematic validation of the roles for each kinase as determinants of AQP2 protein abundance, the data will provide a basis for understanding mechanisms involved in defective transcription of Aqp2 in water balance disorders and a valuable information resource guiding future studies.

Scientific Focus Area: Systems Biology

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