Highly efficient ESC genome editing with CRISPR/Cas۹ for production of laboratory models
- سال انتشار: 1402
- محل انتشار: فصلنامه ژنتیک و ژنومیک انسانی، دوره: 7، شماره: 1
- کد COI اختصاصی: JR_JHGG-7-1_002
- زبان مقاله: انگلیسی
- تعداد مشاهده: 184
نویسندگان
Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Shahroud University of Medical Sciences, Shahroud, Iran
Faculty of Paramedical Sciences, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Shahroud University of Medical Sciences, Shahroud, Iran
Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran.
Department of Medical Laboratory Sciences, Faculty of Paramedical Sciences, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran.
Department of Hematology, Faculty of Paramedical Sciences, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran
چکیده
Background: Beta-thalassemia is a group of hereditary blood disorders caused by mutations in the β-globin gene cluster resulting in variable phenotypes ranging from severe anemia to clinically asymptomatic individuals. This study aimed to produce an in vitro model of β-thalassemia using CRISPR/Cas۹ as an easily programmable, fast, more powerful, and efficient technique. Materials and Methods: Guide RNA (gRNA)-Cas۹ co-expression vectors were used for embryonic stem (ES) cell nucleofection. PCR, T۷EI, and Hbb-b۱ gene sequencing tests were done on extracted DNA to evaluate gene mutation. Following erythroid differentiation of ES cells, analysis of hemoglobin genes and erythroid transcription factors were assessed using a quantitative reverse transcription-polymerase chain reaction. Results: Sequencing data associated with clone ۳۱ confirmed the deletion of ۸۵۱ nucleotides between exon ۲ and ۳ in an Hbb-b۱ allele in this clone and Indel mutation in exon ۲ (-۴۰bp/+۳۸bp) from another allele of Hbb-b۱. Significant expression of erythroid transcription factors was observed in wild type, Hbb-b۱+/- and Hbb-b۱-/- groups. The hbb-b۱ gene expression in the Hbb-b۱+/- group significantly decreased, although the Hbb-b۱-/- group had zero expression. Conclusion: Utilizing an efficient erythroid differentiation method on the CRISPR/Cas۹-mediated Hbb-b۱ knock-out in ES cells provides accessibility to the laboratory thalassemia model. This method could be used to produce a mouse model of β-thalassemia intermedia (Hbbth۱/th۱ mice), which are required for the identification of the molecular basis of β-thalassemia and enable testing of the therapeutic approaches such as the recovery of functional β or γ hemoglobin chain.کلیدواژه ها
Beta‐thalassemia, CRISPR‐Cas۹ system, Hbb‐b۱, Mouse embryonic stem cellاطلاعات بیشتر در مورد COI
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