Generation and validation of a CRISPR platform for rapid and inducible genome editing in human pluripotent stem cells and kidney organoids
Our current knowledge about the function of human genes is mostly based on data from genetic studies using animal models. However, divergence among species may hamper the understanding of genetic mechanisms underlying human specific traits. Nowadays, an alternative to animal models stands on the gen...
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| Tipo de recurso: | tesis doctoral |
| Estado: | Versión publicada |
| Fecha de publicación: | 2022 |
| País: | España |
| Institución: | CBUC, CESCA |
| Repositorio: | TDR. Tesis Doctorales en Red |
| OAI Identifier: | oai:www.tdx.cat:10803/673798 |
| Acceso en línea: | http://hdl.handle.net/10803/673798 |
| Access Level: | acceso abierto |
| Palabra clave: | Células madre pluripotentes Edició genètica CRISPR-Cas9 Modelatge de malalties Organoide Diferenciació cel.lular Pluripotent stem cells Genome editing Disease Modelling Organoid Cell diferentiation 575 |
| Sumario: | Our current knowledge about the function of human genes is mostly based on data from genetic studies using animal models. However, divergence among species may hamper the understanding of genetic mechanisms underlying human specific traits. Nowadays, an alternative to animal models stands on the generation of organ-like cultures derived from human pluripotent stem cells (hPSCs), the so called organoids. In the last years, the organoids have proved to recapitulate, in a high extent, the development, multicellular architecture, and physiology of human organs. To perform genetic studies in these valuable models, methods for rapid and controllable genetic manipulation of hPSCs are needed. To fulfill this need, the generation of an inducible CRISPR platform (iCRISPR) previously enabled efficient and inducible genome editing in hPSCs. Nevertheless, iCRISPR had important limitations hampering further transgenesis for expanding its possible applications. Based on iCRISPR, this thesis describes the generation of a new iCRISPR2 (iC2) platform with improved versatility for transgenesis. We generated selection-free hPSCs with monoallelic insertions of an inducible Cas9 expression module at the AAVS1 locus. Engineered iC2 hPSCs were then exploited for the high-throughput generation of stable and inducible KO hPSCs lines, precise KI hPSCs lines, and a reporter hPSCs line. In addition, the iC2 platform was repurposed for the generation of hPSCs for controlled gene regulation by CRISPR/dCas9 systems. Finally, some stable and inducible KO hPSCs were differentiated into kidney organoids to study the role of LHX1 and VHL in kidney development and disease. Our results showed that LHX1 function is required for renal vesicle formation prior nephrogenesis, as well as that VHL depletion impairs mitochondrial respiration in tubular cells derived from kidney organoids. On balance, iCRISPR2 technology could be applied in any hPSCs-based differentiation model to systematically dissect gene function in human development, physiology and disease |
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