Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes

  • BMC Biotechnol. 2016 Jan 16:16:4. doi: 10.1186/s12896-016-0234-4.
Van Trung Chu  1 ,  Timm Weber  2 ,  Robin Graf  3 ,  Thomas Sommermann  4 ,  Kerstin Petsch  5 ,  Ulrike Sack  6 ,  Pavel Volchkov  7 ,  Klaus Rajewsky  8 ,  Ralf Kühn  9  10
Affiliations
  • 1. Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. [email protected].
  • 2. Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. [email protected].
  • 3. Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. [email protected].
  • 4. Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. [email protected].
  • 5. Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. [email protected].
  • 6. Present Address: Bayer Pharma AG Building S107, 13353, Berlin, Germany. [email protected].
  • 7. Harvard University, Cambridge, MA, 02138, USA. [email protected].
  • 8. Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. [email protected].
  • 9. Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. [email protected].
  • 10. Berlin Institute of Health, Kapelle-Ufer 2, 10117, Berlin, Germany. [email protected].
PMID: 26772810 DOI: 10.1186/s12896-016-0234-4
Abstract

Background: The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homology-directed mutagenesis and use of inbred embryos are less established. In particular, Rosa26 knock-in alleles for the insertion of transgenes in a genomic 'safe harbor' site, have not been produced. Here we applied CRISPR/Cas9 for the knock-in of 8-11 kb inserts into Rosa26 of C57BL/6 zygotes.

Results: We found that 10-20 % of live pups derived from microinjected zygotes were founder mutants, without apparent off-target effects, and up to 50 % knock-in embryos were recovered upon coinjection of Cas9 mRNA and protein. Using this approach, we established a new mouse line for the Cre/loxP-dependent expression of Cas9.

Conclusions: Altogether, our protocols and resources support the fast and direct generation of new Rosa26 knock-in alleles and of Cas9-mediated in vivo Gene Editing in the widely used C57BL/6 inbred strain.