CRISPR/Cas9-mediated SLC12A3 gene knock-in restores molecular and functional defects in Gitelman syndrome hiPSC-derived kidney organoids

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This study aims to determine whether knock-in of full-length SLC12A3 with ubiquitous promoter into safe harbor locus has functional rescue for Gitelman syndrome (GS) in terms of Na⁺–Cl⁻ cotransporter (NCC) expression and functional recovery, using patient-derived human induced pluripotent stem cells (hiPSCs) and a kidney organoid system. GS1 and GS2 hiPSCs were generated from the peripheral blood of two patients with GS with different mutations at the SLC12A3 . CRISPR/Cas9-mediated SLC12A3 knock-in (GS1 SLC12A3−KI and GS2 SLC12A3−KI hiPSCs) was performed at the AAVS1 safe harbor locus in GS1 and GS2 lines. WTC-11 (wild-type), GS1, GS2, GS1 SLC12A3−KI , GS2 SLC12A3−KI hiPSCs were differentiated into kidney organoids. The expression of NCC and related kinases was analyzed using qRT-PCR, immunoblotting, and immunofluorescence. Sodium transport and thiazide responses were assessed with CoroNa™ Green dye and flow cytometry. GS1 and GS2 hiPSCs were successfully generated, and after SLC12A3 knock-in, its expression was restored in both patient-derived hiPSCs. The generated GS1 SLC12A3−KI and GS2 SLC12A3−KI hiPSCs exhibited normalized SLC12A3 mRNA and NCC protein levels compared to their parental cells (GS1 and GS2, respectively). Kidney organoids derived from GS1 SLC12A3−KI and GS2 SLC12A3−KI hiPSCs displayed restored NCC localization in the tubular epithelium, recovered activation of WNK4–SPAK–OSR1 signaling in response to angiotensin II, and reinstated thiazide-sensitive sodium transport, whereas the GS1 and GS2 kidney organoids did not. These results demonstrate that targeted SLC12A3 knock-in rescues molecular and functional defects of NCC in GS patient-derived kidney organoids, establishing a proof-of-concept for functional correction of SLC12A3 deficiency in a human organoid model.