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Further development of a CRISPR/Cas9 platform for 60% of Duchenne muscular dystrophy patients
Author(s) -
Fernandez Karla
Publication year - 2017
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.31.1_supplement.909.1
Subject(s) - crispr , duchenne muscular dystrophy , cas9 , dystrophin , biology , genome editing , guide rna , muscular dystrophy , genetics , gene , exon , computational biology , bioinformatics
Duchenne muscular dystrophy (DMD) is a fatal genetic disorder that affects approximately 1 in 3,500–5,000 males worldwide, and is caused by mutations in the DMD gene causing loss of dystrophin protein expression. This results in progressive muscle degeneration, leading to lethal conditions of cardiac and respiratory function. Since approximately 60% of the out of frame mutations in DMD occur between exons 45–55, the Spencer lab developed a platform utilizing CRISPR/Cas9 gene editing to restore the DMD reading frame by creating a large deletion of exons 45–55. To expand on this platform we are analyzing DNA homology between humans and larger animal models to design and test new guide RNAs (gRNAs) that would be specific across multiple species. Homology is being analyzed using BLAST to check for previously designed gRNAs that are conserved across dog, pig, and monkey genomes and to design new gRNAs. Secondly, we are analyzing muscle specific promoters. We hypothesize that putting Cas9 behind a muscle specific promoter would restrict Cas9 expression to solely muscle tissue and reduce potential off target effects. By analyzing gene expression and immunostaining of candidate promoters in muscle sections, we expect to determine a candidate promoter to drive Cas9 expression only in muscle tissue and eliminate non‐specificity. In conclusion, these studies allow us to identify gRNAs for large animal models as well as a muscle specific promoter for Cas9, to expand the development and safety of the CRISPR/Cas9 platform which has the potential to serve as a therapy for 60% of DMD patients.