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CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman’s Disease Phenotype in a Patient-Derived Kidney Organoid System
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 CMCi0...
Autores principales: | , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2023
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9917614/ https://www.ncbi.nlm.nih.gov/pubmed/36769335 http://dx.doi.org/10.3390/ijms24033019 |
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author | 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 |
author_facet | 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 |
author_sort | Lim, Sun Woo |
collection | PubMed |
description | 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-001(corr). Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001(corr) group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm(2) vs. 3.7 ± 0.2/cm(2), p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001(corr) group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001(corr) group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001(corr) 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. |
format | Online Article Text |
id | pubmed-9917614 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2023 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-99176142023-02-11 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 Int J Mol Sci Article 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-001(corr). Both hiPSCs were differentiated into kidney organoids, and we analyzed the GIT phenotype. The number of matured kidney organoids from the CMC-GIT-001(corr) group was significantly higher, 3.3-fold, than that of the CMC-GIT-001 group (12.2 ± 0.7/cm(2) vs. 3.7 ± 0.2/cm(2), p < 0.05). In qRT-PCR, performed using harvested kidney organoids, relative sodium chloride cotransporter (NCCT) mRNA levels (normalized to each iPSC) were increased in the CMC-GIT-001(corr) group compared with the CMC-GIT-001 group (4.1 ± 0.8 vs. 2.5 ± 0.2, p < 0.05). Consistently, immunoblot analysis revealed increased levels of NCCT protein, in addition to other tubular proteins markers, such as LTL and ECAD, in the CMC-GIT-001(corr) group compared to the CMC-GIT-001 group. Furthermore, we found that increased immunoreactivity of NCCT in the CMC-GIT-001(corr) 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. MDPI 2023-02-03 /pmc/articles/PMC9917614/ /pubmed/36769335 http://dx.doi.org/10.3390/ijms24033019 Text en © 2023 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article 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 CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman’s Disease Phenotype in a Patient-Derived Kidney Organoid System |
title | CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman’s Disease Phenotype in a Patient-Derived Kidney Organoid System |
title_full | CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman’s Disease Phenotype in a Patient-Derived Kidney Organoid System |
title_fullStr | CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman’s Disease Phenotype in a Patient-Derived Kidney Organoid System |
title_full_unstemmed | CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman’s Disease Phenotype in a Patient-Derived Kidney Organoid System |
title_short | CRISPR-Cas9-Mediated Correction of SLC12A3 Gene Mutation Rescues the Gitelman’s Disease Phenotype in a Patient-Derived Kidney Organoid System |
title_sort | crispr-cas9-mediated correction of slc12a3 gene mutation rescues the gitelman’s disease phenotype in a patient-derived kidney organoid system |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9917614/ https://www.ncbi.nlm.nih.gov/pubmed/36769335 http://dx.doi.org/10.3390/ijms24033019 |
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