Jan 17, 2016

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

BMC Biotechnology
Van Trung ChuRalf Kühn

Abstract

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. 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. 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.

  • References30
  • Citations55

Citations

Mentioned in this Paper

Embryo
In Vivo
Microinjections
Genome
RNA, Untranslated
CRISPR-Cas Systems
C57BL/6 Mouse
Gene Editing
Gt(ROSA)26Sor non-coding RNA, mouse
Inbred Strain

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