Correction of Hemoglobin E/Beta-Thalassemia Patient-Derived iPSCs Using CRISPR/Cas9

• Main Author: Methichit Wattanapanitch
• Other Authors: Faculty of Medicine Siriraj Hospital, Mahidol University
• Format: Book Chapter
• Published: 2022
• Subjects: Biochemistry, Genetics and Molecular Biology
• Online Access: https://repository.li.mahidol.ac.th/handle/123456789/76423
• Institution: Mahidol University

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.