Production of the hurler syndrome cellular model using CRISPR-Cas system

سال انتشار: 1397
نوع سند: مقاله کنفرانسی
زبان: انگلیسی
مشاهده: 398

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شناسه ملی سند علمی:

CIGS15_415

تاریخ نمایه سازی: 13 بهمن 1398

چکیده مقاله:

Background: The Hurler syndrome has clinical manifestations including coarse faces, corneal opacity, mental retardation, and enlargement of the liver and spleen. The bone marrow transplantation (HCST) and enzymatic treatment (ERT) are two common treatments for this disorder. Efforts to carry out gene therapy for the genetic modification of the disease continue on the basis of viral carriers while the results are not very promising. The mentioned treatments have limitations in the field of feasibility and implementation, and the treatment of this disease requires alternative methods to overcome these constraints. With the CRISPR-Cas technique discovery as a genome editing technique, hopes for treatment of the monocytes in metabolic disorders have increased. In this study, we optimized the CRISPR-Cas technique to provide the defective gene modification to a healthy and active version in the patients.Method: The sgRNA sequences were designed with an online software tool (http://crispr.mit.edu/). The sgRNAs were cut off by the bbsI enzyme and sub-cloned into pX335 CRISPR-Cas vector. The pX335/sgRNA vector was transfected into the NIH3T3 cell line to knock out the IDUA gene; then, the modified cells were selected using the High Resolution Melting (HRM) technique and enzymatic activity level was evaluated by the ELISA.Results: Recombinant pX335/sgRNA vector was confirmed by bbsI and smaI digestion. Accuracy of the transfection into the NIH3T3 cell line was confirmed by the sequencing, and enzymatic activity level was evaluated by ELISA.

نویسندگان

m Heydari

Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

a hatami bardar

Department of Molecular Genetics, Faculty of Sciences, Marvdasht Branch, Islamic Azad University, Marvdasht, Iran

a eslahi

Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

s Bozorg Ghomi

Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

y jafari

Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran

f alizadeh

Department of Medical Genetics, Faculty of Medicine, Mashhad University of Medical Sciences, Mashhad, Iran