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

  1. Yuet Wai Kan1,3,4
  1. 1Department of Medicine, University of California, San Francisco, California 94143, USA;
  2. 2Blood Systems Research Institute, San Francisco, California 94118, USA;
  3. 3Department of Laboratory Medicine, University of California, San Francisco, California 94143, USA;
  4. 4Institute for Human Genetics, University of California, San Francisco, California 94143, USA
  1. 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

  • Received January 30, 2014.
  • Accepted June 25, 2014.

This article is distributed exclusively by Cold Spring Harbor Laboratory Press for the first six months after the full-issue publication date (see http://genome.cshlp.org/site/misc/terms.xhtml). After six months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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