CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman's Disease Phenotype in a Patient-Derived Kidney Organoid System.

Lim, Sun Woo; Fang, Xianying; Cui, Sheng; Lee, Hanbi; Shin, Yoo Jin; Ko, Eun Jeong; Lee, Kang In; Lee, Jae Young; Chung, Byung Ha; Yang, Chul Woo

The aim of this study is to explore the possibility of modeling Gitelman's disease (GIT) with human-induced pluripotent stem cell (hiPSC)-derived kidney organoids and to test whether gene correction using CRISPR/Cas9 can rescue the disease phenotype of GIT. To model GIT, we used the hiPSC line CMCi002 (CMC-GIT-001), generated using PBMCs from GIT patients with SLC12A3 gene mutation. Using the CRISPR-Cas9 system, we corrected CMC-GIT-001 mutations and hence generated CMC-GIT-001corr. Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001corr group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm2 vs. 3.7 ± 0.2/cm2, p corr group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p corr group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001corr group was colocalized with ECAD (a distal tubule marker) using confocal microscopy. Kidney organoids from GIT patient-derived iPSC recapitulated the Gitelman's disease phenotype, and correction of SLC12A3 mutation utilizing CRISPR-Cas9 technology provided therapeutic insight.

PHENOTYPES; GENETIC mutation; GENOME editing; PLURIPOTENT stem cells; KIDNEYS; CONFOCAL microscopy; SALT

International Journal of Molecular Sciences, 2023, Vol 24, Issue 3, p3019

1661-6596

Academic Journal

10.3390/ijms24033019