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Updated: Mar 27, 2026
Zygote Microinjection for Creating Gene Cassette Knock-in and Flox Alleles in Mice
Published on: June 24, 2022
Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes
Van Trung Chu1, Timm Weber2, Robin Graf3
1Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. VanTrung.Chu@mdc-berlin.de.
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.
