A MSTNDel73C mutation with FGF5 knockout sheep by CRISPR/Cas9 promotes skeletal muscle myofiber hyperplasia

Elife. 2024 Oct 4:12:RP86827. doi: 10.7554/eLife.86827.

Abstract

Mutations in the well-known Myostatin (MSTN) 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 MSTN 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 MSTNDel73C mutation with FGF5 knockout sheep, in which the MSTN and FGF5 dual-gene biallelic homozygous mutations were produced via the deletion of 3-base pairs of AGC in the third exon of MSTN, resulting in cysteine-depleted at amino acid position 73, and the FGF5 double allele mutation led to inactivation of FGF5 gene. The MSTNDel73C mutation with FGF5 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 MSTNDel73C mutation with FGF5 knockout mediated the activation of FOSL1 via the MEK-ERK-FOSL1 axis. The activated FOSL1 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.

Keywords: FGF5; FOSL1; MSTN; developmental biology; dual-gene biallelic mutation; myogenesis.

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Fibroblast Growth Factor 5* / genetics
  • Fibroblast Growth Factor 5* / metabolism
  • Gene Knockout Techniques
  • Hyperplasia / genetics
  • Muscle Fibers, Skeletal / metabolism
  • Muscle Fibers, Skeletal / pathology
  • Muscle, Skeletal / metabolism
  • Muscle, Skeletal / pathology
  • Mutation
  • Myostatin* / genetics
  • Myostatin* / metabolism
  • Sheep

Substances

  • Myostatin
  • Fibroblast Growth Factor 5