Gene

Cell biology

Molecular biology

Biology

Transgene

Cas9

Zygote

Genome editing

Gene knockout

CRISPR

Genetically modified mouse

Gene knockin

Immunology

Genetics

Embryonic stem cell

C57BL/6

Embryogenesis

Mutant

Mutagenesis

Gene targeting

Allele

BMC Biotechnology

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.

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