Duchenne muscular dystrophy (DMD), the most prevalent form of muscular dystrophy, is an X-linked inherited lethal disease due to mutations in the DMD gene. Duplications and deletions make up the majority of DMD mutations (65-80%). Multiple studies have successfully used CRISPR-Cas9 to remove DMD duplications by creating double-strand breaks (DSBs) but these create unwanted insertions/deletions (indels) and possible off-target edits. In contrast, Cas9 nickases create a single-strand break, which two adjacent nicks use for editing can lower the chances of off-target editing. In this study, we developed a novel approach using a SaCas9 nickase with a single gRNA (SNSG), to correct the DMD duplications.
We created an all-in-one AAV construct incorporating a miniU6 promoter, dual or single gRNA, together with a CK8e myogenic promoter driven SaCas9 (D10A) nickase. In addition, we created similar lentiviral constructs to initially study editing in patient cells. We conducted lentiviral transduction on two patient myogenic cell lines, harboring exon 7 and exon 20-25 duplications. After transduction and differentiation into myotubes, the cell lines achieved restoration of full-length dystrophin at the genomic, mRNA level, and protein level. We next performed next-generation sequencing (NGS) at the targeted site and the top predicted off-target sites. Surprisingly, SNSG produced no detectable indels at the target site, nor any off-target editing with comparable in vitro editing efficiency to Cas9 nuclease. In contrast, SaCas9 nuclease showed mixtures of indels, while no detectable off-target editing. Similarly, SaCas9 nickase using a pair of adjacent gRNAs also contained unwanted indels. Our SNSG, characterized with no detectable indels, helps avoid uncontrolled and unwanted editing on the genome, e.g. creating cryptic splice sites in introns, or leading frameshift editing in exons.
In conclusion, we developed a novel CRISPR-Cas9 approach, SNSG, to correct DMD duplication-specific mutations without creating any indel or off-target editing, which could potentially be a clinically translatable method to restore full-length dystrophin in patients harboring large DMD duplications.