Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology
Abstract Background Thalassemia is a genetic blood disorder characterized by decreased hemoglobin production. Severe anemia can damage organs and severe threat to life safety. Allogeneic transplantation of bone marrow-derived hematopoietic stem cell (HSCs) at present represents a promising therapeut...
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| Format: | Article |
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BMC
2022-03-01
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| Series: | Stem Cell Research & Therapy |
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| Online Access: | https://doi.org/10.1186/s13287-022-02768-5 |
| _version_ | 1818245558384459776 |
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| author | Lingli Li Hongyan Yi Zheng Liu Ping Long Tao Pan Yuanhua Huang Yongsheng Li Qi Li Yanlin Ma |
| author_facet | Lingli Li Hongyan Yi Zheng Liu Ping Long Tao Pan Yuanhua Huang Yongsheng Li Qi Li Yanlin Ma |
| author_sort | Lingli Li |
| collection | DOAJ |
| description | Abstract Background Thalassemia is a genetic blood disorder characterized by decreased hemoglobin production. Severe anemia can damage organs and severe threat to life safety. Allogeneic transplantation of bone marrow-derived hematopoietic stem cell (HSCs) at present represents a promising therapeutic approach for thalassemia. However, immune rejection and lack of HLA-matched donors limited its clinical application. In recent years, human-induced pluripotent stem cells (hiPSCs) technology offers prospects for autologous cell-based therapy since it could avoid the immunological problems mentioned above. Methods In the present study, we established a new hiPSCs line derived from amniotic cells of a fetus with a homozygous β41-42 (TCTT) deletion mutation in the HBB gene and a heterozygous Westmead mutation (C > G) in the HBA2 gene. We designed a CRISPR-Cas9 to target these casual mutations and corrected them. Gene-corrected off-target analysis was performed by whole-exome capture sequencing. The corrected hiPSCs were analyzed by teratoma formation and erythroblasts differentiation assays. Results These mutations were corrected with linearized donor DNA through CRISPR/Cas9-mediated homology-directed repair. Corrections of hiPSCs were validated by sequences. The corrected hiPSCs retain normal pluripotency. Moreover, they could be differentiated into hematopoietic progenitors, which proves that they maintain the multilineage differentiation potential. Conclusions We designed sgRNAs and demonstrated that these sgRNAs facilitating the CRISPR-Cas9 genomic editing system could be applied to correct concurrent α- and β-thalassemia in patient-derived hiPSCs. In the future, these corrected hiPSCs can be applied for autologous transplantation in patients with concurrent α- and β-thalassemia. |
| first_indexed | 2024-12-12T14:34:49Z |
| format | Article |
| id | doaj.art-0a1d161a3fdc400d9fa4fdc1b0ec8ebf |
| institution | Directory Open Access Journal |
| issn | 1757-6512 |
| language | English |
| last_indexed | 2024-12-12T14:34:49Z |
| publishDate | 2022-03-01 |
| publisher | BMC |
| record_format | Article |
| series | Stem Cell Research & Therapy |
| spelling | doaj.art-0a1d161a3fdc400d9fa4fdc1b0ec8ebf2022-12-22T00:21:23ZengBMCStem Cell Research & Therapy1757-65122022-03-0113111210.1186/s13287-022-02768-5Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technologyLingli Li0Hongyan Yi1Zheng Liu2Ping Long3Tao Pan4Yuanhua Huang5Yongsheng Li6Qi Li7Yanlin Ma8Hainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Reproductive Medical Center, International Technology Cooperation Base “China-Myanmar Joint Research Center for Prevention and Treatment of Regional Major Disease” By the Ministry of Science and Technology of China, The First Affiliated Hospital of Hainan Medical University, Hainan Medical UniversityHainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Reproductive Medical Center, International Technology Cooperation Base “China-Myanmar Joint Research Center for Prevention and Treatment of Regional Major Disease” By the Ministry of Science and Technology of China, The First Affiliated Hospital of Hainan Medical University, Hainan Medical UniversityCollege of Medical Laboratory Science, Guilin Medical UniversityHainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Reproductive Medical Center, International Technology Cooperation Base “China-Myanmar Joint Research Center for Prevention and Treatment of Regional Major Disease” By the Ministry of Science and Technology of China, The First Affiliated Hospital of Hainan Medical University, Hainan Medical UniversityKey Laboratory of Tropical Translational Medicine of Ministry of Education, Hainan Medical UniversityHainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Reproductive Medical Center, International Technology Cooperation Base “China-Myanmar Joint Research Center for Prevention and Treatment of Regional Major Disease” By the Ministry of Science and Technology of China, The First Affiliated Hospital of Hainan Medical University, Hainan Medical UniversityKey Laboratory of Tropical Translational Medicine of Ministry of Education, Hainan Medical UniversityHainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Reproductive Medical Center, International Technology Cooperation Base “China-Myanmar Joint Research Center for Prevention and Treatment of Regional Major Disease” By the Ministry of Science and Technology of China, The First Affiliated Hospital of Hainan Medical University, Hainan Medical UniversityHainan Provincial Key Laboratory for Human Reproductive Medicine and Genetic Research, Reproductive Medical Center, International Technology Cooperation Base “China-Myanmar Joint Research Center for Prevention and Treatment of Regional Major Disease” By the Ministry of Science and Technology of China, The First Affiliated Hospital of Hainan Medical University, Hainan Medical UniversityAbstract Background Thalassemia is a genetic blood disorder characterized by decreased hemoglobin production. Severe anemia can damage organs and severe threat to life safety. Allogeneic transplantation of bone marrow-derived hematopoietic stem cell (HSCs) at present represents a promising therapeutic approach for thalassemia. However, immune rejection and lack of HLA-matched donors limited its clinical application. In recent years, human-induced pluripotent stem cells (hiPSCs) technology offers prospects for autologous cell-based therapy since it could avoid the immunological problems mentioned above. Methods In the present study, we established a new hiPSCs line derived from amniotic cells of a fetus with a homozygous β41-42 (TCTT) deletion mutation in the HBB gene and a heterozygous Westmead mutation (C > G) in the HBA2 gene. We designed a CRISPR-Cas9 to target these casual mutations and corrected them. Gene-corrected off-target analysis was performed by whole-exome capture sequencing. The corrected hiPSCs were analyzed by teratoma formation and erythroblasts differentiation assays. Results These mutations were corrected with linearized donor DNA through CRISPR/Cas9-mediated homology-directed repair. Corrections of hiPSCs were validated by sequences. The corrected hiPSCs retain normal pluripotency. Moreover, they could be differentiated into hematopoietic progenitors, which proves that they maintain the multilineage differentiation potential. Conclusions We designed sgRNAs and demonstrated that these sgRNAs facilitating the CRISPR-Cas9 genomic editing system could be applied to correct concurrent α- and β-thalassemia in patient-derived hiPSCs. In the future, these corrected hiPSCs can be applied for autologous transplantation in patients with concurrent α- and β-thalassemia.https://doi.org/10.1186/s13287-022-02768-5ThalassemiaCRISPR-Cas9 technologyHuman-induced pluripotent stem cellsHBB geneHBA2 gene |
| spellingShingle | Lingli Li Hongyan Yi Zheng Liu Ping Long Tao Pan Yuanhua Huang Yongsheng Li Qi Li Yanlin Ma Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology Stem Cell Research & Therapy Thalassemia CRISPR-Cas9 technology Human-induced pluripotent stem cells HBB gene HBA2 gene |
| title | Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology |
| title_full | Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology |
| title_fullStr | Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology |
| title_full_unstemmed | Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology |
| title_short | Genetic correction of concurrent α- and β-thalassemia patient-derived pluripotent stem cells by the CRISPR-Cas9 technology |
| title_sort | genetic correction of concurrent α and β thalassemia patient derived pluripotent stem cells by the crispr cas9 technology |
| topic | Thalassemia CRISPR-Cas9 technology Human-induced pluripotent stem cells HBB gene HBA2 gene |
| url | https://doi.org/10.1186/s13287-022-02768-5 |
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