Gene editing in mouse zygotes using the CRISPR/Cas9 system

  • Methods. 2017 May 15:121-122:55-67. doi: 10.1016/j.ymeth.2017.02.008.
Benedikt Wefers  1 ,  Sanum Bashir  2 ,  Jana Rossius  3 ,  Wolfgang Wurst  4 ,  Ralf Kühn  5
Affiliations
  • 1. German Center for Neurodegenerative Diseases (DZNE), Feodor-Lynen Str. 17, 81377 Munich, Germany; Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany. Electronic address: [email protected].
  • 2. Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle Str. 10, 13125 Berlin, Germany; Berlin Institute of Health, Kapelle-Ufer 2, 10117 Berlin, Germany. Electronic address: [email protected].
  • 3. Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle Str. 10, 13125 Berlin, Germany. Electronic address: [email protected].
  • 4. German Center for Neurodegenerative Diseases (DZNE), Feodor-Lynen Str. 17, 81377 Munich, Germany; Helmholtz Zentrum München, German Research Center for Environmental Health, Institute of Developmental Genetics, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Technische Universität München-Weihenstephan, Chair of Developmental Genetics, c/o Helmholtz Zentrum München, Ingolstädter Landstr. 1, 85764 Neuherberg, Germany; Munich Cluster for Systems Neurology (SyNergy), Feodor-Lynen-Str. 17, 81377 Munich, Germany. Electronic address: [email protected].
  • 5. Max-Delbrück-Centrum für Molekulare Medizin, Robert-Rössle Str. 10, 13125 Berlin, Germany; Berlin Institute of Health, Kapelle-Ufer 2, 10117 Berlin, Germany. Electronic address: [email protected].
PMID: 28263886 DOI: 10.1016/j.ymeth.2017.02.008
Abstract

The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, Gene Editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from Gene Editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.

Keywords
CRISPR; Cas9; Gene editing; Mouse; Zygotes.