Human Genetics and Genomics

en Highly efficient ESC genome editing with CRISPR/Cas9 for production of laboratory models تخصصي Gene expression پژوهشي Research <div> <table align="left" hspace="0" vspace="0"> <tbody> <tr> <td align="left" style="padding-top:0in; padding-right:12px; padding-bottom:0in; padding-left:12px" valign="top">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/Cas9 as an easily programmable, fast, more powerful, and efficient technique. Materials and Methods: Guide RNA (gRNA)-Cas9 co-expression vectors were used for embryonic stem (ES) cell nucleofection. PCR, T7EI, and Hbb-b1 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 31 confirmed the deletion of 851 nucleotides between exon 2 and 3 in an Hbb-b1 allele in this clone and Indel mutation in exon 2 (-40bp/+38bp) from another allele of Hbb-b1. Significant expression of erythroid transcription factors was observed in wild type, Hbb-b1+/- and Hbb-b1-/- groups. The hbb-b1 gene expression in the Hbb-b1+/- group significantly decreased, although the Hbb-b1-/- group had zero expression. Conclusion: Utilizing an efficient erythroid differentiation method on the CRISPR/Cas9-mediated Hbb-b1 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 (Hbbth1/th1 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. </td> </tr> </tbody> </table> </div> Beta‐thalassemia, CRISPR‐Cas9 system, Hbb‐b1, Mouse embryonic stem cell 0 0 http://humangeneticsgenomics.ir/browse.php?a_code=A-10-364-1&slc_lang=en&sid=1 Mansoureh Ajami ajami.m@shmu.ac.ir 1003194753284600740 1003194753284600740 No Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Shahroud University of Medical Sciences, Shahroud, Iran Omid Moeini mr.omidmoeini@gmail.com 1003194753284600741 1003194753284600741 No Faculty of Paramedical Sciences, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran Amir Atashi atashia@shmu.ac.ir 1003194753284600742 1003194753284600742 No Department of Medical Laboratory Sciences, School of Allied Medical Sciences, Shahroud University of Medical Sciences, Shahroud, Iran Masoud Soleimani soleim_m@modares.ac.ir 1003194753284600743 1003194753284600743 No Department of Hematology, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran. Hossein Dehghani 1003194753284600744 1003194753284600744 No Department of Medical Laboratory Sciences, Faculty of Paramedical Sciences, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran. Monireh Ajami m.ajami@iautmu.ac.ir 1003194753284600745 1003194753284600745 Yes Department of Hematology, Faculty of Paramedical Sciences, Tehran Medical Sciences, Islamic Azad University, Tehran, Iran