Fig 2: The rAAV9-SaCas9 system efficiently targets the Mstn site in C57BL/10 male mice thigh muscle. a A 521 bp region of the sgMstn1 locus including the site targeted for editing was PCR amplified from the genomic DNA and analyzed for genome editing with a T7 endonuclease digestion identification method. The top band on the gel was uncut DNA at 521 bp in length in all groups, but the two smaller bands were edited DNA at 181 and 340 bp in length for sgMstn1 in the treatment groups (red arrow indicated). The nucleic acid marker was a 50 bp DNA ladder. b The percentage distribution of indels at sgMstn1 in thigh muscle in mice with different dose rAAV9-sgMstn1, as measured by sequencing analyses. Data represent means ± SD from three technical replicates. c Mice left thigh muscles were treated with ~ 1 × 1010 vg/1 × 1011 vg of the rAAV9-sgMstn1 and 1and, analyzed by western blotting with anti-MSTN antibody at 6, 8, 10 weeks post infection. Anti-GAPDH was used as a loading control. d Stacked histogram showing the percentage distribution of MSTN expression in mice thigh muscles treated with different dose rAAV9-sgMstn1. The MSTN value of each group was divided by the corresponding GAPDH value, and then the data of each group were divided by the same multiple to normalize by the control group value. Data represent means ± SD from three technical replicates

Fig 3: The cytokines expression were changed by AAV-CN, AAV-sgMstn2, RAAV-sgMstn2 treatment in muscle tissues.RT-PCR analysis was performed on 8 weeks on AAV-CN, AAV-sgMstn2, RAAV-sgMstn2-treated muscles in order to determine kinds of cytokines mRNA levels relative to GAPDH levels. Normalized data were used for generating the a Smad2, b p21, c CDK2, d AKT, e mTOR and f FoxO1 graphs depicted. Data represent means ± SEM (The number of samples was 3 per group, and each sample was repeated three times). *: 0.01 < P < 0.05, **P < 0.01 when AAV-sgMstn2, RAAV-sgMstn2-treated were compared with AAV-CN-treated. g A schematic were marked key cytokines of MSTN-related signaling pathway.

Fig 4: The SaCas9/CRISPR system map depicting EF1α:SaCas9-eGFP-tRNA:sgRNA. a EF1α:SaCas9-eGFP-tRNA:sgRNA is an SaCas9/CRISPR system that consists of an EF1α promoter controlling the expression of the coding regions of SaCas9, followed by a self-cleaving P2A peptide sequence and then an eGFP Flag. The sgRNA expression cassette is under the control of an tRNA promoter. b The approximate location of the sgRNA binding site in the Mstn locus. These three target sites were all located in the conserved N-terminal sequence in the first and second exons of the Mstn gene, which play an important role in MSTN protein synthesis. The sgRNAs, or target sequences are underlined, and the PAM sequence (NNGRRT) is specified

Fig 5: Satellite cells are increased in number and myogenic proteins changed during the Mstn knockout in the left thigh of aged C57BL/10 male mice.a Satellite cells expression. Immunofluorescent staining of muscle sections using a Pax7 antibody (in red). Representative images are shown. The nuclei were stained with DAPI (in blue). The white arrow indicates the satellite cell. b Quantification of the number of Pax7-positive cells/mm2 from a. Data are means ± SEM from five muscles derived from five different aged mice per group. *: 0.01 < P < 0.05, **P < 0.01. Western blot analysis was also performed in order to determine the kinds of cell factors protein levels. c MyoD, MyoG and Pax7 were detected during the Mstn knockout. Normalized data were used for generating the d MyoD, e MyoG and f Pax7 graphs depicted. Data represent means ± SEM (the number of samples was 3 per group, and each sample was repeated three times). *: 0.01 < P < 0.05, **P < 0.01 when AAV-sgMstn2, RAAV-sgMstn2-treated were compared with AAV-CN-treated. g Western blot was used to detect the effect of MSTN on the expression of MuRF1 and MAFbx proteins. Normalized data were used for generating the h MuRF1, i MAFbx graphs depicted. Data represent means ± SEM (the number of samples was 3 per group, and each sample was repeated three times). *: 0.01 < P < 0.05, **P < 0.01 when AAV-sgMstn2, RAAV-sgMstn2-treated were compared with AAV-CN-treated.