Figure 3: Targeted frameshifts to restore the dystrophin reading frame using CRISPR/Cas9. | Nature Communications

Figure 3: Targeted frameshifts to restore the dystrophin reading frame using CRISPR/Cas9.

From: Multiplex CRISPR/Cas9-based genome editing for correction of dystrophin mutations that cause Duchenne muscular dystrophy

Figure 3

(a) The 5′ region of exon 51 was targeted using a sgRNA, CR3, that binds immediately upstream of the first out-of-frame stop codon. PAM: protospacer-adjacent motif. (b) The exon 51 locus was PCR amplified from HEK293T cells treated with SpCas9 and CR3 expression cassettes. Sequences of individual clones were determined by Sanger sequencing. The top sequence (bolded, exon in red) is the native, unmodified sequence. The number of clones for each sequence is indicated in parentheses. (c) Summary of total gene editing efficiency and reading frame conversions resulting from gene modification shown in b. (d) Western blot for dystrophin expression in human DMD myoblasts treated with SpCas9 and the CR3 sgRNA expression cassette (Fig. 2c) to create targeted frameshifts to restore the dystrophin reading frame. Dystrophin expression was probed using an antibody against the rod-domain of the dystrophin protein after 6 days of differentiation.

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