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

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Item Type:Article
Title:Efficient generation of Rosa26 knock-in mice using CRISPR/Cas9 in C57BL/6 zygotes
Creators Name:Chu, V.T. and Weber, T. and Graf, R. and Sommermann, T. and Petsch, K. and Sack, U. and Volchkov, P. and Rajewsky, K. and Kühn, R.
Abstract: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.
Keywords:CRISPR, Cas9, Knock-In Mice, Rosa26, Zygotes, Animals, Mice
Source:BMC Biotechnology
Publisher:BioMed Central
Page Range:4
Date:16 January 2016
Official Publication:https://doi.org/10.1186/s12896-016-0234-4
PubMed:View item in PubMed

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