Supplementary Figure 9: Enrichment of immune cells in Cas9-expressing muscles.

(a) Full Western blot image corresponding to Figure 2a. (b) Cas9 induces lymphocyte infiltration in both the draining lymph nodes and Cas9-expressing muscles (n = 4 mice per condition) (*, P < 0.05; ***, P < 0.001; n-way ANOVA). Checkmarks denote injected vectors and conditions. (c) CD45+ immunostaining is enriched around transgene-expressing myofibers. Mice were electroporated with minicircle-Cas9^FL and pCAG-GFP. Part of a histological section is shown to depict quantification method. Mean CD45 fluorescence intensity on the edge of myofibers was calculated for GFP+ myofibers, 1° and 2° neighboring myofibers, and distal (> 2°) myofibers, followed by normalization to the mean intensity around distal (> 2°) myofibers within the same section (n = 4 mice, 2 sections from each). (*, P < 0.001, Wilcoxon rank-sum against distal myofibers, Bonferroni corrected). Black lines, means. (d) Schematic of FACS gating for immune cell surface markers. (e) Fractions of immune cell types within all live cells in injected muscles as assessed by FACS (n = 4 mice per condition). Myeloid and T-cell fractions increased in Cas9-treated muscles (*, P < 0.05, **, P < 0.01; ***, P < 0.001; n.s., not significant; one-way ANOVA, followed by Dunnett’s test against vehicle-injected muscles). (f) AAV9-Cas9-VPR-gRNAs elicit immune cell infiltration/expansion irrespective of gRNAs employed (n = 3 mice per condition). gRNA set 1 targets Mstn, Fst, Pd-l1, and Cd47, while gRNA set 2 targets Mstn and Fst, all at 4E12 vg of 1:1 AAV9-Cas9^N-gRNAs:AAV9-Cas9^C-VPR and 1E11 vg of AAV9-turboRFP (*, P < 0.05; n.s., not significant; one-way ANOVA, followed by Dunnett’s test against AAV9-turboRFP-injected muscles). Error bars denote s.e.m.