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Disruption of Sox6 Gene Using Crispr/Cas9 Technology for Gamma-Globin Reactivation: An Approach Towards Gene Therapy of Β-Thalassemia Publisher Pubmed



Shariati L1, 2 ; Rohani F3 ; Heidari Hafshejani N4 ; Kouhpayeh S5 ; Boshtam M2 ; Mirian M6 ; Rahimmanesh I4 ; Hejazi Z4 ; Modarres M4 ; Pieper IL7 ; Khanahmad H4
Authors
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Authors Affiliations
  1. 1. Applied Physiology Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
  2. 2. Isfahan Cardiovascular Research Center, Cardiovascular Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
  3. 3. Department of Clinical Sciences, Faculty of Veterinary Medicine, University of Shahrekord, Shahrekord, Iran
  4. 4. Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran
  5. 5. Isfahan Neurosciences Research Center, Alzahra Research Institute, Isfahan University of Medical Sciences, Isfahan, Iran
  6. 6. Department of Pharmaceutical Biotechnology and Isfahan Pharmaceutical Science Research Center, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, Iran
  7. 7. Institute of Life Science, College of Medicine, Swansea University Medical School, Swansea, United Kingdom

Source: Journal of Cellular Biochemistry Published:2018


Abstract

Elevation of Hemoglobin F ameliorates symptoms of β-thalassemia, a common autosomal recessive disorder. The transcription factor SOX6 plays a key role in the γ to β-globin gene switching. In the current investigation, a mutation was induced using the CRISPR/Cas9 technology in the binding domain region of SOX6 to reactivate γ-globin expression. Three CRISPR/Cas9 cassettes were provided, whose single-guide RNAs targeted different regions in the SOX6 gene-binding domain. After transfection of K562 cells with CRISPR a, b and c, and subsequent erythroid differentiation, the indel percentage of the cells was about 30%, 25%, and 24%, respectively. Relative quantification showed that the γ-globin mRNA level increased to 1.3-, 2.1-, and 1.1-fold in the cells treated with CRISPR/Cas9 a, b, and c, respectively, compared with untreated cells. Our results show that mutation induction in the binding site of the SOX6 gene leads to γ-globin reactivation. These findings support the idea that CRISPR interrupts the SOX6 binding site, and, as a result, SOX6 is incapable of binding the γ-globin promoter. In conclusion, SOX6 disruption could be considered as a therapeutic approach for β-thalassemia treatment. CRISPR/Cas9 was selected for this purpose as it is the most rapidly evolving technology. © 2018 Wiley Periodicals, Inc.