Figure 4.

Genome-Editing Strategies for Individuals with Duplication of DMD Exons 18–30

(A) Electropherogram of the junction of the duplication of DMD exons 18–30; highlighted in blue is the insertion of AAAT at the junction.

(B) Schematic of the position of DMD sgRNA 1 and the duplication-removal strategy.

(C) Schematic of the three-primer duplication-removal strategy.

(D) Targeted deletion of a 139 kb duplication in DMD. PCR was performed on DNA from three replicate experiments in which affected myoblasts were transduced with LentiGFP or lentiCRISPR Cas9 nuclease with DMD sgRNA 1. The top band was amplified with universal primers (P1 + P3) to both an allele with the duplication and a control. The bottom band is specific to alleles harboring the duplication (P1 + P2). A decrease in the bottom band, indicating removal of the duplicated region, was only observed when Cas9 and sgRNA 1 were present.

(E) Western blot with antibodies against dystrophin, α-dystroglycan, and tubulin as a loading control. The amount of dystrophin was normalized to that of tubulin by densitometric analysis. ^∗p < 0.05, ^∗∗p < 0.01 (Student’s t test from three independent experiments).