Cellular Reprogramming, Genome Editing, and Alternative CRISPR Cas9 Technologies for Precise Gene Therapy of Duchenne Muscular Dystrophy

Figure 1

Dystrophin gene targeting strategies by CRISPR Cas9. (a) Examples of normal and patient dystrophin gene exons. Individual exons are represented by beige, dark blue, light blue, and brown. In healthy patient genomes, the exons are in frame and will lead to the expression of a full protein. In DMD patient genomes, the deletion of the dark blue exon leads to a frame shift, disrupting the ORF and causing a premature stop codon. (b) Four main strategies of genome editing to correct the ORF of the dystrophin gene: (i) for exon skipping, sgRNA is designed to target a splicing acceptor. This disruption would mask the exon as an intron, which would not be included in the final mRNA product; (ii) exon deletion involves the complete deletion of a single or multiple exons from the genome. Exon(s) within the range of two targeting sgRNAs would be excised. Mono-exon deletions could be designed for each dystrophin gene mutation type. For a multiexon deletion strategy, exons 45–55 (or exons 44–54) are deleted and could be applied to up to 60% of DMD patients, although this results in the production of a much smaller dystrophin protein as seen in Becker muscular dystrophy; (iii) another approach to avoid premature stop codons and recover the ORF is by inserting or deleting bases and making frame shifts instead of an exon deletion. NHEJ-mediated insertions or deletions may induce frame shifts and recover the ORF; (iv) dystrophin gene deletion mutations involving one or multiple exons could be rescued by a knock-in strategy of the deleted exon(s) to completely restore full length dystrophin protein expression. In this strategy, a donor template should be delivered in addition to Cas9 and sgRNA.

(a) Example of normal and DMD patient exons

(b) Targeting strategies