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

TLDR

CRISPR/Cas9 is widely used for gene disruption in mouse zygotes, yet efficient knock‑in of large transgenes at the Rosa26 safe‑harbor locus in C57BL/6 embryos remains unestablished. The authors performed CRISPR/Cas9‑mediated insertion of 8–11 kb transgenes into the Rosa26 locus of C57BL/6 zygotes. The approach yielded 10–20 % founder mutants among live pups, up to 50 % knock‑in embryos with Cas9 mRNA/protein, and enabled creation of a Cre/loxP‑dependent Cas9 line, demonstrating rapid generation of Rosa26 knock‑ins in C57BL/6 mice.

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.