Figure 1.

In vivo mutagenesis of the Mstn gene in skeletal muscle cells with AAV-SaCRISPR/Cas9. (a) A schematic diagram of adapted AAV-SaCRISPR backbone with SaCas9 driven by a skeletal muscle cell-specific dMCK promoter. (b) Surveyor assays performed at the targeting loci with genomic DNA from NIH-3T3 cells transfected with SaCas9 and different guide RNAs (gRNAs) targeting the mouse Mstn gene. (c) Surveyor assays performed at the top genome-wide off-target sites (Supplementary Table S1) with genomic DNA from NIH-3T3 cells transfected with SaCas9 and gRNA-2 targeting the mouse Mstn gene. For duplicated region (OT2*), the first genomic locus listed in the table was analyzed for potential off-target effect. (d) Schematic description of the in vivo targeting experiment. (e) Surveyor assays performed with genomic DNA from mouse gastrocnemius muscle tissues treated with AAV-SaCRISPR-Mstn or AAV-GFP-2a-Luci at the Mstn targeting locus at week 8. (f) Indel rates at the on-target sites from next-generation DNA sequencing of muscle samples from post-treatment animals receiving either AAV-SaCRISPR-Mstn or AAV-GFP-2a-Luci viruses. In b and e, arrows show the cleavage products resulting from the Surveyor assays; the intensity of the cleavage product bands relative to the uncleaved product band corresponds to the mutagenesis rate. Statistical analyses in f were conducted using the two-tailed Student's t-test; ****P < 0.0001. dMCK promoter, double muscle creatine kinase promoter; ITR, inverted terminal repeat; NLS, nuclear localization signal.