CRISPR-Cas9 homology-independent targeted integration of exons 1-19 restores full-length dystrophin in mice

Duchenne muscular dystrophy is an X-linked disorder typically caused by out-of-frame mutations in the DMD gene. Most of these are deletions of one or more exons, which can theoretically be corrected through CRISPR-Cas9-mediated knockin. Homology-independent targeted integration is a mechanism for achieving such a knockin without reliance on homology-directed repair pathways, which are inactive in muscle. We de-signed a system based on insertion into intron 19 of a DNA fragment containing a pre-spliced mega-exon encoding DMD exons 1-19, along with the MHCK7 promoter, and delivered it via a pair of AAV9 vectors in mice carrying a Dmd exon 2 duplication. Maximal efficiency was achieved using a Cas9:do-nor adeno-associated virus (AAV) ratio of 1:5, with Cas9 under the control of the SPc5-12 promoter. This approach achieved editing of 1.4% of genomes in the heart, leading to 30% correc-tion at the transcript level and restoration of 11% of normal dystrophin levels. Treatment efficacy was lower in skeletal mus-cles. Sequencing additionally revealed integration of fragmen-tary and recombined AAV genomes at the target site. These data provide proof of concept for a gene editing system that could restore full-length dystrophin in individuals carrying mutations upstream of intron 19, accounting for approxi-mately 25% of Duchenne muscular dystrophy patients.