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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.    Electronic supplementary material  The online version of this article (doi:10.1186/s12896-016-0234-4) contains supplementary material, which is available to authorized users.

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