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المستخلص
B-thalassemia is a genetic disorder caused by mutations in the human haemoglobin beta (HBB) gene. It is a common inherited disease extending from the Mediterranean area through the Middle East to Southeast Asia. Patients homozygous for B-thalassemia mutations have severe anaemia and usually require frequent transfusions and iron chelation. So far, hematopoietic stem cell transplantation is the only cure available when histocompatible donors are available.The ideal approach to cure a genetic disease such as B-thalassemia is to correct the mutations that cause the disease.The mutation-corrected CD34 positive cells could be differentiated into hematopoietic stem and progenitor cells (HSPC) for autologous transplantation. Such an approach would avoid the problems of immune responses to allogeneic transplantation and the possibility of insertional mutations associated with viral gene delivery.Efficiency of mutation correction can be increased by introducing DNA double-stranded breaks (DSBs) at or close to the site of the mutation. Site-specific DSBs can be achieved by introducing engineered nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), as well as the recently developed RNA-directed Cas9 nucleases The RNA-directed Cas9 nucleases from the type II bacterial CRISPR/Cas system have been shown to be robust and versatile tools for generating DSBs in eukaryotic cells.In this study we assessed the role of CRISPR/Cas9 technology, to efficiently correct the HBB mutations in patient-derived CD34 positive cells without leaving any residual footprint. In this study we successfully isolated CD34 cells from peripheral blood of the B-thalassemia patients by magnetic activated cell sorting method, the cells were transfected with cas9 endonuclease together with g RNA by calcium phosphate transfection method to create DSB