Disruption of FGF5 in Cashmere Goats Using CRISPR/Cas9 Results in More Secondary Hair Follicles and Longer Fibers

PLoS One. 2016 Oct 18;11(10):e0164640. doi: 10.1371/journal.pone.0164640. eCollection 2016.

Abstract

Precision genetic engineering accelerates the genetic improvement of livestock for agriculture and biomedicine. We have recently reported our success in producing gene-modified goats using the CRISPR/Cas9 system through microinjection of Cas9 mRNA and sgRNAs targeting the MSTN and FGF5 genes in goat embryos. By investigating the influence of gene modification on the phenotypes of Cas9-mediated goats, we herein demonstrate that the utility of this approach involving the disruption of FGF5 results in increased number of second hair follicles and enhanced fiber length in Cas9-mediated goats, suggesting more cashmere will be produced. The effects of genome modifications were characterized using H&E and immunohistochemistry staining, quantitative PCR, and western blotting techniques. These results indicated that the gene modifications induced by the disruption of FGF5 had occurred at the morphological and genetic levels. We further show that the knockout alleles were likely capable of germline transmission, which is essential for goat population expansion. These results provide sufficient evidences of the merit of using the CRISPR/Cas9 approach for the generation of gene-modified goats displaying the corresponding mutant phenotypes.

MeSH terms

  • Alleles
  • Animals
  • Base Sequence
  • CRISPR-Cas Systems / genetics*
  • Embryo, Mammalian / metabolism
  • Fibroblast Growth Factor 5 / chemistry
  • Fibroblast Growth Factor 5 / deficiency
  • Fibroblast Growth Factor 5 / genetics*
  • Germ-Line Mutation
  • Goats
  • Hair Follicle / chemistry*
  • Hair Follicle / pathology
  • Hair Follicle / physiology
  • Male
  • Microscopy, Electron, Transmission
  • Microscopy, Fluorescence
  • Phenotype
  • RNA, Messenger / metabolism
  • Skin / pathology
  • Testis / metabolism

Substances

  • RNA, Messenger
  • Fibroblast Growth Factor 5

Grants and funding

This work is supported by grants from the Major Projects for New Varieties of Genetically Modified Organisms of China (2016ZX08008-002), and by National Natural Science Foundation of China (31372279, 31402038, 31171377, 31572369), and China Agriculture Research System (CARS-40-13), as well as a Provincial Key Projects of Shaanxi (2014KTDZ02-01).