Efficient generation of gene-modified pigs via injection of zygote with Cas9/sgRNA

Sci Rep. 2015 Feb 5;5:8256. doi: 10.1038/srep08256.

Abstract

Co-injection of zygotes with Cas9 mRNA and sgRNA has been proven to be an efficient gene-editing strategy for genome modification of different species. Genetic engineering in pigs holds a great promise in biomedical research. By co-injection of one-cell stage embryos with Cas9 mRNA and Npc1l1 sgRNA, we achieved precise Npc1l1 targeting in Chinese Bama miniature pigs at the efficiency as high as 100%. Meanwhile, we carefully analyzed the Npc1l1 sgRNA:Cas9-mediated on- and off-target mutations in various somatic tissues and ovaries, and demonstrated that injection of zygotes with Cas9 mRNA and sgRNA is an efficient and reliable approach for generation of gene-modified pigs.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alleles
  • Animals
  • Animals, Genetically Modified*
  • Base Sequence
  • CRISPR-Cas Systems / genetics*
  • Female
  • Gene Targeting / methods
  • Membrane Proteins / chemistry
  • Membrane Proteins / genetics
  • Microinjections* / methods
  • Molecular Sequence Data
  • Mosaicism
  • Organ Specificity / genetics
  • Ovary / metabolism
  • RNA Editing
  • RNA, Guide*
  • RNA, Messenger*
  • Swine*
  • Zygote / metabolism*

Substances

  • Membrane Proteins
  • RNA, Guide
  • RNA, Messenger