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

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

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.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • DNA Transposable Elements / genetics
  • Erythroblasts / cytology
  • Erythroblasts / metabolism
  • Erythropoiesis
  • HEK293 Cells
  • Humans
  • Induced Pluripotent Stem Cells / cytology
  • Induced Pluripotent Stem Cells / metabolism*
  • Mice
  • Mutation*
  • Targeted Gene Repair / methods*
  • beta-Globins / genetics*
  • beta-Thalassemia / genetics

Substances

  • DNA Transposable Elements
  • beta-Globins