Figure 6.

DOX-inducible gene editing in skeletal muscle of DMSXL mice

(A) Scheme of AAV constructs containing Cas9 and sgRNAs. ITR, inverted terminal repeat; CK8, creatine kinase 8 promoter; TetO, tetracycline operator; TetR, tetracycline repressor; UbC, ubiquitin C promoter. (B) Editing of genomic DNA extracted from TA muscles of DMSXL hemizygous mice, injected with both the AAV vectors (AAV-CK8-Crispr), and treated with DOX as indicated. Primers DMPK F2 (F2) and DMPK R2 (R2) were used for PCR amplification. In the diagram the expected outcomes in the absence (−DOX) or presence (+DOX) of DOX are indicated. The black triangle indicates the expected CTG-deleted products and the gray triangle indicates non-specific PCR products. (C) qRT-PCR showing fold induction of sg34 and sg589 RNAs in untreated or DOX-treated mice, normalized to mCherry expression; ∗∗p < 0.01, n = 5 (−DOX); n = 12 (+DOX). (D) Western blot analysis of Cas9 and TetR proteins in TA of DMSXL mice, treated as indicated. PBS-injected TA muscle is shown as negative control; p38 is shown as loading control. (E) Dot plots represent qRT-PCR expression analysis of Cas9, sg34, and sg589 RNAs in TA muscles of DMSXL mice injected with the AAV vectors and treated with DOX. RNA expression in samples either with or without CTG editing was normalized to GAPDH expression: ∗p < 0.05, n = 6 (No Editing); n = 11 (Editing).