Abstract
The CRISPR/Cas9 genome editing platform is a promising technology to correct the genetic basis of hereditary diseases. The versatility, efficiency, and multiplexing capabilities of the CRISPR/Cas9 system enable a variety of otherwise challenging gene correction strategies. Here we use the CRISPR/Cas9 system to restore the expression of the dystrophin gene in cells carrying dystrophin mutations that cause Duchenne muscular dystrophy (DMD). We design single or multiplexed sgRNAs to restore the dystrophin reading frame by targeting the mutational hotspot at exons 45–55 and introducing shifts within exons or deleting one or more exons. Following gene editing in DMD patient myoblasts, dystrophin expression is restored in vitro. Human dystrophin is also detected in vivo after transplantation of genetically corrected patient cells into immunodeficient mice. Importantly, the unique multiplex gene editing capabilities of the CRISPR/Cas9 system facilitate the generation of a single large deletion that can correct up to 62% of DMD mutations.
Highlights
The CRISPR/Cas[9] genome-editing platform is a promising technology to correct the genetic basis of hereditary diseases
Whereas early clinical trials in this area have focused on skipping exon 51, which is applicable to 13% of Duchenne muscular dystrophy (DMD) patients, other preclinical efforts have demonstrated multi-exon skipping of the complete exon 45–55 coding region with a combination treatment of up to ten oligonucleotides[26,27] that could potentially address greater than 60% of known DMD patient mutations[23]
We did not detect translocations using this assay in HEK293T cells or sorted DMD myoblasts treated with CR36 or CR6/CR36, respectively (Supplementary Fig. 9), that had Genome editing is a powerful tool for correcting genetic disease and the recent development of the CRISPR/Cas[9] system is dramatically accelerating progress in this area
Summary
The CRISPR/Cas[9] genome-editing platform is a promising technology to correct the genetic basis of hereditary diseases. There is a class of common deletions in the exon 45–55 mutation hotspot region of the dystrophin gene that maintain the correct reading frame and lead to the expression of a truncated, but functional, dystrophin protein Patients with this class of mutations are often asymptomatic or display mild symptoms associated with Becker muscular dystrophy, a substantially less severe disease than DMD. There are significant technical and practical hurdles to designing and developing this type of complex combination therapy, in addition to the general challenges of developing any oligonucleotide-based therapy that must be continuously administered for the lifetime of the patient In contrast to these transient mRNA-targeted oligonucleotidemediated exon skipping strategies, genome editing has the ability to make precise and permanent changes to gene sequences that will persist after cell division. This study provides proof-of-principle that the CRISPR/Cas[9] technology is a versatile method for correcting a significant fraction of dystrophin mutations and with continued development may serve as a general platform for treating genetic disease
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