Validation of microinjection methods for generating knockout mice by CRISPR/Cas-mediated genome engineering

Sci Rep. 2014 Mar 28;4:4513. doi: 10.1038/srep04513.

Abstract

The CRISPR/Cas system, in which the Cas9 endonuclease and a guide RNA complementary to the target are sufficient for RNA-guided cleavage of the target DNA, is a powerful new approach recently developed for targeted gene disruption in various animal models. However, there is little verification of microinjection methods for generating knockout mice using this approach. Here, we report the verification of microinjection methods of the CRISPR/Cas system. We compared three methods for injection: (1) injection of DNA into the pronucleus, (2) injection of RNA into the pronucleus, and (3) injection of RNA into the cytoplasm. We found that injection of RNA into the cytoplasm was the most efficient method in terms of the numbers of viable blastocyst stage embryos and full-term pups generated. This method also showed the best overall knockout efficiency.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Base Sequence
  • Blastocyst / metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism
  • Female
  • Gene Targeting / methods*
  • Genome*
  • Male
  • Mice
  • Mice, Knockout
  • Microinjections*
  • Molecular Sequence Data
  • Mutation
  • Reproducibility of Results
  • Sequence Alignment