Efficient generation of sheep carrying biallelic mutations in dual gene via the CRISPR/Cas9 system

(A) Schematic of sgRNAs specific to exon 3 of the sheep MSTN locus. The crRNA sequences are highlighted in blue typeface and the PAM in red. (B) Schematic of sgRNAs specific to exon 3 of the sheep FGF5 locus. The crRNA sequences are highlighted in blue typeface and the PAM in red. (C) T7EI assay for sgRNAs of MSTN and FGF5 in sheep fetal fibroblasts. The cleavage bands are marked with an red asterisk (*) and the indel frequencies were calculated using the expected fragments. (D) Summary of the generation of sheep carrying biallelic mutations in dual gene via zygote injection of Cas9 mRNA/sgRNAs. (E) Analysis of genome sequence and amino acid sequence of MSTN-modified sheep. The location of sgRNA and PAM are highlighted in blue and red, respectively. The deletions are indicated by a dashed line (-). (F) Analysis of genome sequence and amino acid sequence of FGF5-modified sheep. The location of sgRNA and PAM are highlighted in blue and red, respectively. The deletions are indicated by a dashed line (-).

The MSTNDel273C mutation with FGF5 knockout sheep highlights a dominant “double-muscle” phenotype and muscle fiber hyperplasia

(A) The 6-month-old WT and MF-/- sheep. The genome-edited sheep displayed an obvious “double-muscle” phenotype compared with the WT. (B) The CT scanning image of the brisket and hip of WT and MF-/- sheep. (C) HE sections of gluteus medius and longissimus dorsi of WT and MF-/- sheep. Scale bar 100 μm. (D) Quantification of muscle fibre cell number of per unit area in WT and MF-/- sheep. (E-F) The percentage of cross-sectional area of different size muscle fibers in all muscle fibers. (G) The proportion of different muscles in carcass in WT and MF+/− sheep. (H) HE sections of gluteus medius in WT and MF+/− sheep. Scale bar 100 μm. (I) Quantification of muscle fibre cell area of gluteus medius in WT and MF+/− sheep. (J) Quantification of muscle fibre cell number of per unit area in WT and MF+/− sheep. (K) The percentage of cross-sectional area of different size muscle fibers.

The slaughter traits of muscles in WT and MF+/− sheep

The MSTNDel273C mutation with FGF5 knockout promote proliferation and inhibit differentiation of skeletal muscle satellite cells

(A) The number of cells was detected by CCK-8 at 12h, 24h, 36h, 48h, and 60h in GM. (B-C) EdU assay showed that the number of EdU positive cells and EdU labeling index were significantly increased in MF+/− cells. Scale bar 130 μm. (D-E) PI staining to detect cell cycle and showed a significant reduce in the proportion of G1 phase and a significant increase in the proportion of S phase in MF+/− cells. (F) The mRNA expression levels of cell cycle marker genes and cell proliferation marker genes. (G) The mRNA expression levels of myogenic differentiation marker genes MyoG, MyoD1, and MyHC. (H-I) The protein expression levels of myogenic differentiation marker genes MyoG, MyoD1, and MyHC. (J) The MyoG and MyHC immunofluorescence staining of myotubes in DM2. Scale bar 130 μm. (K) The myotube fusion index, which was represented by the number of cell nuclei in myotubes/total cell nuclei. (L) The number of myotubes, which was the number of all myotubes in the field of view. (M) The number of nuclei per myotube. (N) The myotube diameter. To reflect the myotube diameter as accurately as possible, the vertical line at the thinnest position of the myotube is taken as the minimum measured (Min), the mid-perpendicular line of the long myotube axis is taken as the middle measured (Middle), and the vertical line at the widest position of the myotube is taken as the maximum measured (Max).

Identification of potential regulators by RNA-seq

(A) Principal component analysis (PCA) of six gluteus medius samples in WT and MF+/− sheep. (B) Volcano plot of differentially expressed genes (DEGs) between WT and MF+/− sheep. The up- and down-regulated DEGs are shown in red and blue, respectively. (C) The heat map of DEGs between WT and MF+/− sheep. (D) Pearson correlation analysis between samples. (E) Go enrichment analysis of DEGs. Among them, the top 20 entries with significant enrichment are listed in biological process (BP). CC, cellular component; MF, molecular function. (F) KEGG enrichment analysis of DEGs. (G) The expression verification of DEGs by RT-qPCR.

FOSL1 may regulate myogenesis by binding to the MyoD1 promoter region

(A) The expression level of FOSL1 mRNA during myogenic differentiation. (B) The mRNA expression levels of FOSL1 both at GM and DM2 in WT and MF+/− cells. (C) The protein-protein interaction (PPI) analysis of FOSL1, c-Fos and MyoD1. (D) The mRNA expression level of c-Fos and MyoD1 at GM in WT and MF+/− myoblasts. (E) Schematic diagram of MyoD1 gene body, promoter region and binding sites. (F-G) FOSL1 recognition motif in the MyoD1 promoter region. (H) FOSL1 ChIP-qPCR of motif 1 recognition region. (I) FOSL1 ChIP-qPCR of motif 2 recognition region.

The MSTNDel273C mutation with FGF5 knockout contributes to muscle phenotype via MEK-ERK-FOSL1 axis

(A) Western blot of FOSL1, c-Fos, and key kinases of MAPK signaling pathways in GM. (B-E) Quantification of protein expression of FOSL1, c-Fos, and key kinases of MAPK signaling pathways in GM. (F) Western blot of FOSL1, c-Fos, and key kinases of MAPK signaling pathways in DM2. (G-J) Quantification of protein expression of FOSL1, c-Fos, and key kinases of MAPK signaling pathways in DM2.

FOSL1 overexpression promotes the proliferation of skeletal muscle satellite cells

(A) The mRNA expression level of FOSL1 at GM and DM2 after lentivirus infection. (B) The number of cells detected by CCK-8 at 0h, 12h, 24h, 36h, 48h, and 60h after infection with lentivirus. (C) The mRNA expression levels of cell cycle marker genes and cell proliferation marker genes. (D-E) EdU assay showed that the number of EdU positive cells and EdU labeling index were significantly increased after infection with lentivirus. Scale bar 130 μm. (F) The mRNA expression levels of c-Fos and MyoD1 at GM after FOSL1 overexpression. (G-H) The protein expression levels of FOSL1, p-FOSL1 and MyoD1 at GM after FOSL1 overexpression.

Highly expressed or activated FOSL1 inhibits myogenic differentiation

(A) The mRNA expression levels of myogenic differentiation marker genes MyoD1, MyoG and MyHC at DM2 after overexpression of FOSL1. (B-C) The protein expression levels of FOSL1, p-FOSL1 and myogenic differentiation marker genes MyoD1, MyoG and MyHC at DM2 after overexpression of FOSL1. (D) The MyoG and MyHC immunofluorescence staining of myotubes at DM2 after overexpression of FOSL1. Scale bar 130 μm. (E-H) The myotube fusion index, number of myotubes, number of nuclei per myotube and the myotube diameter at DM2 after overexpression of FOSL1. (I) The MyoG and MyHC immunofluorescence staining of myotubes at DM2 after addition of 20 μM TBHQ. Scale bar 130 μm. (J-M) The myotube fusion index, number of myotubes, number of nuclei per myotube and the myotube diameter at DM2 after the addition of 20 μM TBHQ.

Schematic illustration of the regulation of muscle phenotypes by MSTNDel273C mutation with FGF5 knockout

The MSTNDel273C mutation with FGF5 knockout mediated the activation of FOSL1 via MEK-ERK-FOSL1 axis. The activated FOSL1 further promotes skeletal muscle satellite cell proliferation and inhibits myogenic differentiation, and resulting in smaller myotubes, which demonstrates the potential mechanism for smaller muscle fibers of MSTNDel273C mutation with FGF5 knockout sheep.

FGF5 mutation does not affect muscle fiber size

(A) HE sections of gluteus medius in WT and FGF5+/− sheep. Scale bar 100 μm. (B) Quantification of muscle fibre cell area of gluteus medius in WT and FGF5+/− sheep. (C) Quantification of muscle fibre cell number of per unit area in WT and FGF5+/− sheep. (D) The percentage of cross-sectional area of different size muscle fibers

Genotype identification of F1 generation MF+/− sheep

(A) Identification of MSTN mutation type. The ear, liver, skin and muscle tissues of F1 generation MF+/− sheep were selected to identify the genotype of MSTN. (B) MSTN mutation sequencing peak. (C) Identification of FGF5 mutation type. The ear tissue of F1 generation MF+/− sheep was selected to identify the genotype of FGF5. (D-E) FGF5 mutation sequencing peak.

The MSTNDel273C mutation with FGF5 knockout has no potential effect on MSTN expression

(A) MSTN immunohistochemistry of gluteus medius and longissimus dorsi in WT and MF-/- sheep. (B) MSTN mRNA expression level of gluteus medius in WT and MF+/− sheep. (C-D) MSTN protein expression level of gluteus medius in WT and MF+/− sheep. (E) MSTN immunofluorescence staining in myoblasts and myotubes of WT and MF+/− sheep.

The myogenic differentiation ability of MF+/− cells was continuously inhibited

(A) The MyoG and MyHC immunofluorescence staining of myotubes in DM4. Scale bar 130 μm. (B-E) The myotube fusion index, number of myotubes, number of nuclei per myotube and the myotube diameter in DM4. (F) The MyoG and MyHC immunofluorescence staining of myotubes in DM6. Scale bar 130 μm. (G-J) The myotube fusion index, number of myotubes, number of nuclei per myotube and the myotube diameter in DM6. (K) The protein expression levels of myogenic differentiation marker genes and potential regulator FOSL1 and its family member c-Fos during myogenic differentiation. (L-N) Quantification of protein levels of myogenic differentiation marker genes.

The expression of DEGs at different levels

(A) The expression level of DEGs in the longissimus dorsi. (B) The expression level of PDPN mRNA during myogenic differentiation. (C) The expression level of ANKRD2 mRNA during myogenic differentiation.