A mutation with knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia.

Mutations in the well-known Myostatin () produce a 'double-muscle' phenotype, which makes it commercially invaluable for improving livestock meat production and providing high-quality protein for humans. However, mutations at different loci of the often produce a variety of different phenotypes. In the current study, we increased the delivery ratio of Cas9 mRNA to sgRNA from the traditional 1:2 to 1:10, which improves the efficiency of the homozygous mutation of biallelic gene. Here, a mutation with knockout sheep, in which the and dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon of , resulting in cysteine-depleted at amino acid position 73, and the double allele mutation led to inactivation of gene. The mutation with knockout sheep highlights a dominant 'double-muscle' phenotype, which can be stably inherited. Both F0 and F1 generation mutants highlight the excellent trait of high-yield meat with a smaller cross-sectional area and higher number of muscle fibers per unit area. Mechanistically, the mutation with knockout mediated the activation of via the MEK-ERK-FOSL1 axis. The activated promotes skeletal muscle satellite cell proliferation and inhibits myogenic differentiation by inhibiting the expression of MyoD1, and resulting in smaller myotubes. In addition, activated ERK1/2 may inhibit the secondary fusion of myotubes by Ca-dependent CaMKII activation pathway, leading to myoblasts fusion to form smaller myotubes.