CRISPR/Cas9-based SLC12A3 gene knock in: a model for cellular feature analysis in Gitelman syndrome

Background: This study aims to investigate the effects of complete SLC12A3 gene knock-in using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) in human-induced pluripotent stem cells (hiPSCs), focusing on cellular characteristics and functional implications in the context of Gitelman syndrome. Methods: Employing CRISPR/Cas9 gene-editing technology, the SLC12A3 gene was integrated into WTC-11 hiPSCs, resulting in the creation of the WTC-11SLC12A3-KI cell line. Successful integration was confirmed through green fluorescence protein (GFP) reporter gene expression, and SLC12A3 expression was verified via polymerase chain reaction, flow cytometry, immunoblotting, and immunofluorescence in kidney organoids. Functional analysis with the intracellular sodium indicator CoroNa Green dye (Invitrogen) assessed the impact of SLC12A3 gene knock-in and thiazides. Results: The WTC-11SLC12A3-KI hiPSCs exhibited successful integration of the SLC12A3 gene, with enhanced SLC12A3 expression evidenced by GFP and SLC12A3 proteins compared to WTC-11 hiPSCs. Upon differentiation into kidney organoids, the WTC-11SLC12A3-KI organoids displayed increased GFP, SLC12A3 expression, and the associated WNK-SPAK/OSR1 signaling cascade relative to WTC- 11 organoids. The intracellular sodium flux was stimulated by angiotensin II treatment in kidney organoid cells and attenuated by thiazides in both WTC-11 and WTC-11SLC12A3-KI groups. Conclusion: This study indicates that the knock-in of the SLC12A3 gene could serve as a therapeutic strategy for Gitelman syndrome.