Abstract
HbE/β-thalassemia is one of the most common thalassemic syndromes in Southeast Asia and Thailand. Patients have mutations in β hemoglobin (HBB) gene resulting in decreased and/or abnormal production of β hemoglobin. Here, we describe a protocol for CRISPR/Cas9-mediated gene correction of the mutated hemoglobin E from one allele of the HBB gene by homology-directed repair (HDR) in HbE/β-thalassemia patient-derived induced pluripotent stem cells (iPSCs) using a CRISPR/Cas9 plasmid-based transfection method and a single-stranded DNA oligonucleotide (ssODN) repair template harboring the correct nucleotides. Our strategy allows the seamless HbE gene correction with the editing efficiency (HDR) up to 3%, as confirmed by Sanger sequencing. This protocol provides a simple one-step genetic correction of HbE mutation in the patient-derived iPSCs. Further differentiation of the corrected iPSCs into hematopoietic stem/progenitor cells will provide an alternative renewable source of cells for the application in autologous transplantation in the future.
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Acknowledgments
M.W. is supported by Chalermphrakiat Grant, Faculty of Medicine Siriraj Hospital, Mahidol University, Thailand.
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Wattanapanitch, M. (2021). Correction of Hemoglobin E/Beta-Thalassemia Patient-Derived iPSCs Using CRISPR/Cas9. In: Narayanan, K. (eds) Bio-Carrier Vectors. Methods in Molecular Biology, vol 2211. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-0943-9_14
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DOI: https://doi.org/10.1007/978-1-0716-0943-9_14
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-0943-9
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