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Genome Res. 2014 Sep;24(9):1526-33. doi: 10.1101/gr.173427.114. Epub 2014 Aug 5.

Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac.

Author information

1
Department of Medicine, University of California, San Francisco, California 94143, USA;
2
Blood Systems Research Institute, San Francisco, California 94118, USA;
3
Department of Medicine, University of California, San Francisco, California 94143, USA; Department of Laboratory Medicine, University of California, San Francisco, California 94143, USA;
4
Blood Systems Research Institute, San Francisco, California 94118, USA; Department of Laboratory Medicine, University of California, San Francisco, California 94143, USA;
5
Department of Medicine, University of California, San Francisco, California 94143, USA; Department of Laboratory Medicine, University of California, San Francisco, California 94143, USA; Institute for Human Genetics, University of California, San Francisco, California 94143, USA yw.kan@ucsf.edu.

Abstract

β-thalassemia, one of the most common genetic diseases worldwide, is caused by mutations in the human hemoglobin beta (HBB) gene. Creation of human induced pluripotent stem cells (iPSCs) from β-thalassemia patients could offer an approach to cure this disease. Correction of the disease-causing mutations in iPSCs could restore normal function and provide a rich source of cells for transplantation. In this study, we used the latest gene-editing tool, CRISPR/Cas9 technology, combined with the piggyBac transposon to efficiently correct the HBB mutations in patient-derived iPSCs without leaving any residual footprint. No off-target effects were detected in the corrected iPSCs, and the cells retain full pluripotency and exhibit normal karyotypes. When differentiated into erythroblasts using a monolayer culture, gene-corrected iPSCs restored expression of HBB compared to the parental iPSCs line. Our study provides an effective approach to correct HBB mutations without leaving any genetic footprint in patient-derived iPSCs, thereby demonstrating a critical step toward the future application of stem cell-based gene therapy to monogenic diseases.

PMID:
25096406
PMCID:
PMC4158758
DOI:
10.1101/gr.173427.114
[Indexed for MEDLINE]
Free PMC Article

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