Figure 5.

Expression of dystrophin in treated muscle samples

(A) Western blotting of muscle sample lysates reveals increased near-full-length dystrophin expression over background/revertant levels in samples receiving CRISPR treatment (Dys (CT), 427 kDa band). (B) Expression of μDys5 (∼147 kDa) was visualized using antibodies raised against H1/R1 of dystrophin [Dys (Ex10/11)]. The slightly weaker product seen between the 147 kDa μDys5 band and the 427 kDa full-length dystrophin band (possibly reflecting dystrophin-revertant fibers) likely represents μDys5 duplexes that were insufficiently reduced during sample preparation. (C) Expression of SaCas9 was observed only in samples receiving CRISPR/Cas9, visualized using antibodies against a triple HA epitope fused at the 3′ end of SaCas9. (D) Detection of GAPDH expression to visualize total protein load for the various samples. Note that CRISPR-treated sample 1 from the 8-year-old dog exhibits reduced expression of near-full-length dystrophin and almost undetectable expression of SaCas9, likely reflecting a poorly transduced sample from the IM injection protocol applied. The positive control (WT) lanes were loaded with normal (wild-type) canine muscle extracts. The negative control sample (−) was acquired from the vastus lateralis muscle of the 3-year-old dog. Select additional molecular size markers are provided in gray italics (130 and 250 kDa).