An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

PLoS One. 2018 May 3;13(5):e0196891. doi: 10.1371/journal.pone.0196891. eCollection 2018.

Abstract

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6 mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Electroporation / economics
  • Electroporation / instrumentation
  • Electroporation / methods*
  • Endonucleases / genetics
  • Endonucleases / metabolism
  • Female
  • Gene Editing / methods*
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Zygote / growth & development
  • Zygote / metabolism*

Substances

  • RNA, Guide
  • Endonucleases

Grant support

This work was supported by the Köln Fortune Program (F01) of the University of Cologne (www.uni-koeln.de) to B.Z., and the German Research Foundation (SCHE1562/6; www.dfg.de) and the German Federal Ministry of Research and Education (BMBF grant 01GM1515; NEOCYST consortium; www.bmbf.de) to B.S. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.