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

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 resource

SUBMITTER: Chu VT 

PROVIDER: S-EPMC4715285 | biostudies-literature | 2016 Jan

REPOSITORIES: biostudies-literature