Supplementary Figure 3: Paired Cas9^FL-gRNAs excise intervening genomic sequences in DNA-electroporated skeletal muscles.

(a) Schematic of gRNAs targeting the Mstn and Acvr2b loci in vivo. DNA vectors encoding Cas9^FL, gRNA pairs (targeting adjacent sites), and GFP were co-electroporated into the tibialis anterior (TA) muscle of adult mice. Deep-sequencing of isolated single GFP+ myofibers indicated that Cas9^FL-gRNAs modified 0.21-6.6% of Mstn and 0.18-7.6% of Acvr2b alleles in these multi-nucleated cells, with frequent precise genomic excision delimited by the two predicted cut-sites (2-4 bp 5’ from the PAM^3,7,64,65). Each bar depicts data from a single myofiber, colored according to fractions of mutation types. pSp, plasmid-SpCas9; MCSp, minicircle-SpCas9; pSpPG, plasmid-SpCas9-P2A-turboGFP; Horizontal dashed lines, sequencing error rate. n denotes mice injected. Representative deep-sequencing alignments are shown with dotted vertical lines demarcating Cas9 cut-sites. The fractions of sequencing reads harbouring precise excisions from each myofiber are shown in the histograms with grey bars. (b) Schematic of the Ai9 allele and in situ detection of gene-edited cells with the excision-dependent Ai9 reporter mice. (c) Transverse muscle sections from mice electroporated with Cas9^FL-gRNAs targeting the 3×Stop cassette (n = 4 mice), Cas9^FL-only no-gRNA control (n = 4 mice), or Cre (n = 3 mice). TdTomato+ cells were induced by Cas9^FL-gRNAs or Cre, and not by Cas9^FL only (no-gRNA). All conditions included 15 μg of co-electroporated pCAG-GFP to demarcate transduced myofibers. Red = tdTomato; Green = GFP; Blue = DAPI. Each image comprises 4 × 4 tiles. Scale bar, 500 μm. (d) TdTomato intensity correlates with GFP intensity in GFP+ myofibers of muscles electroporated with DNA encoding Cas9^FL-gRNAs and GFP (n = 4 mice), or Cre and GFP (n = 3 mice). Dots depict individual transduced myofibers, color-coded to each mouse; all transduced myofibers within the transverse sections were quantified.

3 Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816-821, doi:10.1126/science.1225829 (2012).

7 Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186-191, doi:10.1038/nature14299 (2015).

64 Maddalo, D. et al. In vivo engineering of oncogenic chromosomal rearrangements with the CRISPR/Cas9 system. Nature 516, 423-427, doi:10.1038/nature13902 (2014).

65 Xu, L. et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol Ther, doi:10.1038/mt.2015.192 (2015).