A <i>MSTN</i>^Del73C mutation with <i>FGF5</i> knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia.

Abstract

Mutations in the well-known Myostatin (<i>MSTN</i>) 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 <i>MSTN</i> 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 <i>MSTN</i>^Del73C mutation with <i>FGF5</i> knockout sheep, in which the <i>MSTN</i> and <i>FGF5</i> dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon of <i>MSTN</i>, resulting in cysteine-depleted at amino acid position 73, and the <i>FGF5</i> double allele mutation led to inactivation of <i>FGF5</i> gene. The <i>MSTN</i>^Del73C mutation with <i>FGF5</i> 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 <i>MSTN</i>^Del73C mutation with <i>FGF5</i> knockout mediated the activation of <i>FOSL1</i> via the MEK-ERK-FOSL1 axis. The activated <i>FOSL1</i> 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 Ca²⁺-dependent CaMKII activation pathway, leading to myoblasts fusion to form smaller myotubes.