Simple knockout by electroporation of engineered endonucleases into intact rat embryos

Sci Rep. 2014 Oct 1;4:6382. doi: 10.1038/srep06382.

Abstract

Engineered endonucleases, such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system, provide a powerful approach for genome editing in animals. However, the microinjection of endonucleases into embryos requires a high skill level, is time consuming, and may cause damage to embryos. Here, we demonstrate that the electroporation of endonuclease mRNAs into intact embryos can induce editing at targeted loci and efficiently produce knockout rats. It is noteworthy that the electroporation of ZFNs resulted in an embryonic survival rate (91%) and a genome-editing rate (73%) that were more than 2-fold higher than the corresponding rates from conventional microinjection. Electroporation technology provides a simple and effective method to produce knockout animals.

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Cells, Cultured
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Electroporation / methods*
  • Embryo Transfer
  • Embryo, Mammalian / cytology
  • Embryo, Mammalian / metabolism*
  • Endodeoxyribonucleases / genetics
  • Endodeoxyribonucleases / metabolism*
  • Female
  • Gene Targeting / methods*
  • Genetic Engineering / methods*
  • Male
  • Rats
  • Rats, Inbred F344
  • Zinc Fingers

Substances

  • Endodeoxyribonucleases