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

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

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • CRISPR-Cas Systems / genetics*
  • Cloning, Molecular
  • Embryo, Mammalian
  • Gene Knock-In Techniques / methods*
  • Mice
  • Mice, Inbred C57BL
  • Microinjections
  • RNA, Untranslated / genetics*

Substances

  • Gt(ROSA)26Sor non-coding RNA, mouse
  • RNA, Untranslated