RRC ID 54081
Author Ifuku M, Iwabuchi KA, Tanaka M, Lung MSY, Hotta A.
Title Restoration of Dystrophin Protein Expression by Exon Skipping Utilizing CRISPR-Cas9 in Myoblasts Derived from DMD Patient iPS Cells.
Journal Methods Mol. Biol.
Abstract Duchenne muscular dystrophy (DMD) is a congenital X-linked disease caused by mutations in the gene encoding the dystrophin protein, which is required for myofiber integrity. Exon skipping therapy is an emerging strategy for restoring the open reading frame of the dystrophin gene to produce functional protein in DMD patients by skipping single or multiple exons. Although antisense oligonucleotides are able to target pre-mRNA for exon skipping, their half-lives are short and any therapeutic benefit is transient. In contrast, genome editing by DNA nucleases, such as the CRISPR-Cas9 system, could offer permanent correction by targeting genomic DNA. Our laboratory previously reported that disrupting the splicing acceptor site in exon 45 by plasmid delivery of the CRISPR-Cas9 system in iPS cells, derived from a DMD patient lacking exon 44, successfully restored dystrophin protein expression in differentiated myoblasts. Herein, we describe an optimized methodology to prepare myoblasts differentiated from iPS cells by mRNA transfection of the CRISPR-Cas9 system to skip exon 45 in myoblasts, and evaluate the restored dystrophin by RT-PCR and Western blotting.
Volume 1828
Pages 191-217
Published 2018
DOI 10.1007/978-1-4939-8651-4_12
PMID 30171543
MeSH Alternative Splicing CRISPR-Cas Systems* Cell Culture Techniques Cell Cycle Checkpoints / drug effects Cell Cycle Checkpoints / genetics Cell Differentiation / genetics Cells, Cultured Computational Biology / methods Dystrophin / genetics* Dystrophin / metabolism Exons* Gene Editing* Gene Expression Regulation Humans Induced Pluripotent Stem Cells / cytology* Induced Pluripotent Stem Cells / metabolism* Mitomycin / pharmacology Muscle Development Muscular Dystrophy, Duchenne / genetics* Muscular Dystrophy, Duchenne / metabolism Muscular Dystrophy, Duchenne / therapy Mutation MyoD Protein / genetics Myoblasts / cytology Myoblasts / metabolism* RNA, Guide RNA, Messenger / genetics Transduction, Genetic
Human and Animal Cells CiRA00111(HPS0383)