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

TLDR

β‑thalassemia is a common genetic disease caused by mutations in the HBB gene. The study aims to generate patient‑derived iPSCs and correct HBB mutations to restore normal hemoglobin function and supply cells for transplantation. CRISPR/Cas9 combined with piggyBac transposon was used to efficiently correct HBB mutations in patient‑derived iPSCs without leaving a residual footprint. Corrected iPSCs showed no off‑target effects, retained pluripotency and normal karyotype, and when differentiated into erythroblasts restored HBB expression, demonstrating an effective, footprint‑free approach toward stem‑cell gene therapy for monogenic diseases.

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.