Construction of a CRISPR-Cas9 System for Pig Genome Targeting

Anim Biotechnol. 2015;26(4):279-88. doi: 10.1080/10495398.2015.1027774.

Abstract

A Cas9/sgRNA RNA-guided endonuclease expression system including a codon-optimized Streptococcus pyogenes A20 Cas9 recombinant protein expression vector and a spacer-guide chimeric RNA expression vector using the porcine U6 promoter was constructed for application in pigs. Only the Flag2-NLS1-Cas9-NLS2 recombinant protein in complex with sgRNA was translocated into the nucleus; the Flag2-NLS1-Cas9-NLS2 protein alone was excluded from the nucleus. Up to 13% of porcine PK1 cells targeted in vitro were observed, regardless of transfection efficiency.

Keywords: CRISPR; Cas9; sgRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Bacterial Proteins / genetics
  • Base Sequence
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems / genetics*
  • Cell Line
  • Endonucleases / genetics
  • Genetic Engineering / methods*
  • Genome / genetics*
  • HeLa Cells
  • Humans
  • Molecular Sequence Data
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Sus scrofa / genetics*
  • Swine
  • Transfection

Substances

  • Bacterial Proteins
  • RNA, Guide, CRISPR-Cas Systems
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases