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

Simon E Tröder; Lena K Ebert; Linus Butt; Sonja Assenmacher; Bernhard Schermer; Branko Zevnik

doi:10.1371/journal.pone.0196891

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.

Authors

Simon E Tröder^{1

2}, Lena K Ebert^{1

3}, Linus Butt^{1

3}, Sonja Assenmacher^{1

2}, Bernhard Schermer^{1

3

4}, Branko Zevnik^{1

2}

Affiliations

¹ Cologne Excellence Cluster for Cellular Stress Responses in Aging-Associated Diseases (CECAD), University of Cologne, Cologne, Germany.
² in vivo Research Facility, Medical Faculty, University of Cologne, Cologne, Germany.
³ Department II of Internal Medicine and Center for Molecular Medicine Cologne (CMMC), University of Cologne, Cologne, Germany.
⁴ Systems Biology of Aging Cologne (Sybacol), University of Cologne, Cologne, Germany.

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, CRISPR-Cas Systems / genetics
RNA, Guide, CRISPR-Cas Systems / metabolism
Zygote / growth & development
Zygote / metabolism*

Substances

RNA, Guide, CRISPR-Cas Systems
Endonucleases

Grants and funding

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.