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

Autor/innen

  • V.T. Chu
  • T. Weber
  • R. Graf
  • T. Sommermann
  • K. Petsch
  • U. Sack
  • P. Volchkov
  • K. Rajewsky
  • R. Kühn

Journal

  • BMC Biotechnology

Quellenangabe

  • BMC Biotechnol 16 (1): 4

Zusammenfassung

  • 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.


DOI

doi:10.1186/s12896-016-0234-4