Genome editing of rodents by electroporation of CRISPR/Cas9 into frozen-warmed pronuclear-stage embryos

Cryobiology. 2020 Feb 1;92:231-234. doi: 10.1016/j.cryobiol.2020.01.016. Epub 2020 Jan 24.

Abstract

Genome edited animals can now be easily produced using the clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9) system. Traditionally, these animals have been produced by the introduction of endonucleases into pronuclear-stage embryos. Recently, a novel electroporation method, the "Technique for Animal Knockout system by Electroporation (TAKE)," has been established as a simple and highly efficient tool to introduce endonucleases into embryos instead of methods such as microinjection. Use of frozen-warmed pronuclear-stage embryos in this method has further contributed to efficient production of genome edited animals. However, early developmental stage embryos, including pronuclear-stage embryos, especially those of rats, sometimes show low resistance to physical damage by vitrification and introduction of endonucleases during microinjection. In this study, we propose an ethanol-free, slow-freezing method to reduce physical damage to pronuclear-stage embryos followed by the TAKE method. All mouse and rat frozen embryos were survived after electroporation, and 18% and 100% of offspring were edited target gene, respectively. The resulting protocol is an efficient method for producing genome edited animals.

Keywords: CRISPR/Cas; Electroporation; Embryo; Genome editing; Mouse; Rat; Slow-freezing.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics
  • Cryopreservation / methods*
  • Electroporation / methods*
  • Embryo, Mammalian / cytology*
  • Embryonic Development
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Freezing
  • Gene Editing / methods*
  • Mice
  • Mice, Knockout
  • Microinjections
  • Rats
  • Vitrification

Substances

  • Endonucleases