The CRISPR/Cas9 system is increasingly used for gene inactivation in mouse zygotes, but homologydirected 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. | Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes