The CRISPR/Cas9 genome editing platform is a promising technology to correct the genetic basis of hereditary diseases. gene editing in DMD individual myoblasts dystrophin Sennidin A expression is usually restored Human dystrophin is also detected after transplantation of genetically corrected individual 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. Introduction Genome editing technologies use synthetic nucleases to induce cellular DNA repair mechanisms and expose site-specific predefined genetic modifications in complex genomes1. These designed enzymes are commonly based on zinc finger nucleases (ZFNs)1 transcription activator-like effector nucleases (TALENs)2 meganucleases3 and most recently the RNA-guided CRISPR/Cas9 system4-10. The nucleases produce site-specific double-strand Sennidin A breaks at predefined genomic sites that stimulate either non-homologous end joining (NHEJ) for targeted gene disruption or homologous recombination for highly efficient gene targeting. The simplicity and versatility of the CRISPR/Cas9 genome editing system has led to quick adoption and growth of this technology that has proven to be Sennidin A amazingly strong for manipulating gene sequences in human cells. This has enabled new possibilities such as efficient multiplex gene editing for simultaneously inactivating multiple genes5 6 11 In this study we apply the CRISPR system to repair genes mutated in hereditary disease including capitalizing on the unique multiplex capacity of this technology to produce large genomic deletions that restore gene expression. CRISPR/Cas9 systems have been adapted from Rabbit polyclonal to PLAC1. multiple bacterial species including CRISPR system have defined the PAM sequence for this Cas9 (SpCas9) as 5′-NGG-3′ and characterized the specificity of this system in human cells13-19. A unique capability of the CRISPR/Cas9 system is the straightforward ability to simultaneously target multiple unique genomic Sennidin A loci by co-expressing a single Cas9 protein with two or more sgRNAs5 6 11 20 One of the most encouraging applications of genome editing is the correction of genetic mutations associated with hereditary disease1-4. Duchenne muscular dystrophy (DMD) is the most common hereditary disease and no effective treatments exist for this disorder. DMD is usually a severe X-linked disease that presents with progressive muscle mass losing that typically prospects to loss of ambulation in the second decade and death within the third decade of life due to respiratory complications or heart failure. The molecular basis of DMD is usually a mutation in the dystrophin gene21 that leads to the complete lack of function of this essential skeletal muscle mass protein. These mutations are most commonly frameshifts generated by large intragenic deletions of one or more exons. DMD is the prototypical example of a group of monogenetic hereditary diseases that can be corrected by removing internal but unessential regions of the mutated gene to restore the proper reading frame22 23 For example 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. This has led to significant desire Sennidin A for developing an oligonucleotide-mediated exon skipping strategy that will restore the dystrophin reading frame during mRNA processing and convert DMD to a Becker-like phenotype22. Whereas early clinical trials in this area have focused on skipping exon 5124 25 which is applicable to 13% of DMD patients other preclinical efforts have exhibited multi-exon skipping of the complete exon 45-55 coding region with a combination treatment of up to 10 oligonucleotides26 27 that could potentially address greater than 60% of known DMD patient mutations23. However you will find significant technical and practical hurdles to designing and developing this type of complex combination therapy in addition to the general difficulties of developing any oligonucleotide-based therapy that must be continuously administered for the lifetime of the patient. In contrast to these transient mRNA-targeted oligonucleotide-mediated exon skipping strategies genome editing has the ability to make.