title: Seamless gene correction of β-thalassemia mutations in patient-specific iPSCs using CRISPR/Cas9 and piggyBac.
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.
authors.affiliation: Department of Medicine, University of California, San Francisco, California 94143, USA;
authors.forename: Fei
authors.initials: F
authors.lastname: Xie
authors.affiliation: Department of Medicine, University of California, San Francisco, California 94143, USA;
authors.forename: Lin
authors.initials: L
authors.lastname: Ye
authors.affiliation: Department of Medicine, University of California, San Francisco, California 94143, USA;
authors.forename: Judy C
authors.initials: JC
authors.lastname: Chang
authors.affiliation: Blood Systems Research Institute, San Francisco, California 94118, USA;
authors.forename: Ashley I
authors.initials: AI
authors.lastname: Beyer
authors.affiliation: Department of Medicine, University of California, San Francisco, California 94143, USA; Department of Laboratory Medicine, University of California, San Francisco, California 94143, USA;
authors.forename: Jiaming
authors.initials: J
authors.lastname: Wang
authors.affiliation: Blood Systems Research Institute, San Francisco, California 94118, USA; Department of Laboratory Medicine, University of California, San Francisco, California 94143, USA;
authors.forename: Marcus O
authors.initials: MO
authors.lastname: Muench
authors.affiliation: Department of Medicine, University of California, San Francisco, California 94143, USA; Department of Laboratory Medicine, University of California, San Francisco, California 94143, USA; Institute for Human Genetics, University of California, San Francisco, California 94143, USA yw.kan@ucsf.edu.
authors.forename: Yuet Wai
authors.initials: YW
authors.lastname: Kan
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems; D004251:DNA Transposable Elements / Q000235:genetics; D004900:Erythroblasts / Q000166:cytology / Q000378:metabolism; D004920:Erythropoiesis; D057809:HEK293 Cells; D006801:Humans; D057026:Induced Pluripotent Stem Cells / Q000166:cytology / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D052416:Targeted Gene Repair / Q000379:methods*; D055544:beta-Globins / Q000235:genetics*; D017086:beta-Thalassemia / Q000235:genetics
chemical_list: D004251:DNA Transposable Elements; D055544:beta-Globins
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