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BMC Biotechnol. 2016 Jan 16;16:4. doi: 10.1186/s12896-016-0234-4.

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

Author information

1
Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. VanTrung.Chu@mdc-berlin.de.
2
Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Timm.Weber@mdc-berlin.de.
3
Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Robin.graf@mdc-berlin.de.
4
Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Thomas.sommermann@mdc-berlin.de.
5
Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Kerstin.Petsch@mdc-berlin.de.
6
Present Address: Bayer Pharma AG Building S107, 13353, Berlin, Germany. ulrike.sack@bayer.com.
7
Harvard University, Cambridge, MA, 02138, USA. vpwwww@gmail.com.
8
Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Klaus.Rajewsky@mdc-berlin.de.
9
Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. ralf.kuehn@mdc-berlin.de.
10
Berlin Institute of Health, Kapelle-Ufer 2, 10117, Berlin, Germany. ralf.kuehn@mdc-berlin.de.

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.

PMID:
26772810
PMCID:
PMC4715285
DOI:
10.1186/s12896-016-0234-4
[Indexed for MEDLINE]
Free PMC Article

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