Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac
- Fei Xie1,
- Lin Ye1,
- Judy C. Chang1,
- Ashley I. Beyer2,
- Jiaming Wang1,3,
- Marcus O. Muench2,3 and
- Yuet Wai Kan1,3,4
- 1Department of Medicine, University of California, San Francisco, California 94143, USA;
- 2Blood Systems Research Institute, San Francisco, California 94118, USA;
- 3Department of Laboratory Medicine, University of California, San Francisco, California 94143, USA;
- 4Institute for Human Genetics, University of California, San Francisco, California 94143, USA
- Corresponding author: yw.kan{at}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.
Footnotes
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[Supplemental material is available for this article.]
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Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.173427.114.
- Received January 30, 2014.
- Accepted June 25, 2014.
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