Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing

Sci Rep. 2018 Jan 11;8(1):474. doi: 10.1038/s41598-017-18826-5.

Abstract

In this report, we present an improved protocol for CRISPR/Cas9 genome editing in mice. The procedure consists in the electroporation of intact mouse zygotes with ribonucleoprotein complexes prepared in vitro from recombinant Cas9 nuclease and synthetic dual guide RNA. This simple cloning-free method proves to be extremely efficient for the generation of indels and small deletions by non-homologous end joining, and for the generation of specific point mutations by homology-directed repair. The procedure, which avoids DNA construction, in vitro transcription and oocyte microinjection, greatly simplifies genome editing in mice.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • DNA End-Joining Repair
  • Deubiquitinating Enzymes
  • Electroporation
  • Endopeptidases / chemistry
  • Endopeptidases / genetics
  • Female
  • Gene Editing / methods*
  • Genetic Loci
  • Genotyping Techniques
  • INDEL Mutation
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mutation, Missense
  • RNA, Guide / genetics*
  • Zygote / metabolism*

Substances

  • RNA, Guide
  • Endopeptidases
  • BRCC3 protein, mouse
  • Deubiquitinating Enzymes