Figure 1.

Prolonged expression of Cas9 protein has no obvious impact on SC function

(A) Generation of mice expressing Cas9 in SCs. KI, knock in.

(B and C) Cas9 and GFP expressions were examined in heterozygous (B) and some homozygous mice (C).

(D and E) No obvious difference was detected between Ctrl and Pax7^Cas9 littermates (TA, tibialis anterior; Gas, gastrocnemius; Quad, quadriceps). Representative images (D) and quantification (E) are shown. n = 12 mice.

(F) The gating strategy to isolate Cas9-GFP positive SCs. The phycoerythrin (PE) channel (biexponential scale) was used to separate GFP+ and GFP− cells.

(G) Left: IF staining of PAX7. n = 5 mice (an average of six fields/mouse). Scale bar, 50 μm.

(H) Cas9 mRNAs were detected (normalized to 18S mRNA). n = 9 independent experiments.

(I) Cas9 expression was examined and the band intensity was quantified. n = 3 independent experiments.

(J) Left: immunostaining of PAX7 and LAMININ on the TA muscle from 8-week-old mice. Right: the number of PAX7+ cells per 100 fibers. n = 12 mice (an average of six fields/mouse). Scale bar, 50 μm.

(K) Average fiber size. n = 12 mice.

(L and M) H&E staining on day 5 and 7 post injury (L). Myofibers with CLN per field (M). n = 6 mice (an average of five fields/mouse). Scale bar, 100 μm.

(N) Left: immunostaining of eMyHC and LAMININ at day 5 post injury. Right: the number of eMyHC+ myofibers. n = 6 mice (an average of six fields/mouse). Scale bar, 50 μm.

(O) CSA of the fibers with CLN at day 5 post injury. n = 6 mice.

(P) Left: immunostaining of LAMININ at day 7 post injury. Right: CSA of the fibers with CLN. n = 6 mice (an average of three fields/mouse). All the bar graphs are presented as mean ± SD. ^∗∗p < 0.01. ns, no significance.