AbstractsBiology & Animal Science

CRISPR/Cas9 for the Correction of Mutated Dystrophin Gene

by Priyanka 1988- Thapa




Institution: University of Iceland
Department:
Year: 2016
Keywords: Lyfjafræði
Posted: 02/05/2017
Record ID: 2066348
Full text PDF: http://hdl.handle.net/1946/24117


Abstract

Introduction: The recent discovery of a CRISPR/Cas adaptive immunity in bacteria and archaea has led to the development of a eukaryotic-optimized RNA-guided Cas9 system, which has various potential clinical applications. Amongst candidate diseases is X-linked recessive Duchenne Muscular Dystrophy, a degenerative muscular disease that leads to muscle wasting and limited life span. Aim: The overall main aim of this project was to induce a frame-shift in the DMD gene using CRISPR/Cas9 in hope to restore a lost reading frame resulting from deletion of exons 8-12. Specific aims of the Master´s project were to locate deletion breakpoints, design the CRISPR/Cas system and to get a viable cell lineage from a subject for experimental testing. Methods: Deletion breakpoint in the DMD gene were mapped in the subject using Comparative Genomic Hybridization microarray and Polymerase Chain Reaction with Sanger sequencing. Whole Genome sequencing of the subject genome were obtained by deCODE. The CRISPR/Cas9 system was designed to target the genome using various softwares. Expression vectors for the system were transformed into E.coli DH5α cells. Immortalized myoconverted fibroblasts were derived from the subject in collaboration with the AFM-Myobank and expanded. Transfection was done with lipofection (Viafect). Results: Analysis of the location of the breakpoint showed that the location was inside a fused 7/12 intron, and the estimated DNA deletion size was around 196 kb. A transformation efficiency of 1.5 x 107 cfu/μg was obtained in the E.coli DH5α cells. The observed transfection efficiency of myoconverted fibroblast was 5% and lowest toxic dose of blasticidin S was 3 μg/ml. Discussion/Conclusion: The results will be useful in future experiments to correct the DMD gene by frame shift with short indels at the repaired cut site. This project will add valuable specific and general knowledge to genome editing can in treating genetic disease. Inngangur: Uppgötvunin á CRISPR/Cas sérhæfða ónæmiskerfinu í bakteríum og fornbakteríum hefur leitt til þróunar á RNA-miðluðu endónúkleasa kerfi fyrir heilkjörnunga sem mögulega er hægt að nota í meðferð sjúklinga. Einn álitssjúkdóma er Duchenne vöðvarýrnum, sem er eingena og erfist kynbundið víkjandi. Markmið: Heildarmarkmið verkefnisins var að nota CRISPR/Cas9 til að laga lesrammahliðrun vegna brottfalls á útröðum 8-12. Undirmarkmið meistaraverkefnis var að staðsetja rofstað brottfallsins, hanna CRISPR/Cas9 kerfi og að fá frumulínu úr viðfanginu til tilrauna. Aðferðir: Rofstaður brottfallsins innan DMD gensins var ákvarðaður í þátttakanda með því að nota örflögugreiningu og keðjuverkandi fjölliðun á DNA ásamt Sanger raðgreiningu. Gögn frá heildarerfðamengisraðgreiningu úr viðfanginu voru fengin frá deCODE. CRISPR/Cas9 kerfið var hannað til að miða á erfðamengið með ýmsum forritum. Tjáningaferjur fyrir kerfið voru fluttar inn í DH5α frumur. Ódauðlegar vöðvalíkir fíbroblastar voru fengir frá viðfanginu í samvinnu við AFM-vöðvabankann og ræktaðir upp. Gena flutningur í frumur var gerð með…