title: 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 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.
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. VanTrung.Chu@mdc-berlin.de.
authors.forename: Van Trung
authors.initials: VT
authors.lastname: Chu
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Timm.Weber@mdc-berlin.de.
authors.forename: Timm
authors.initials: T
authors.lastname: Weber
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Robin.graf@mdc-berlin.de.
authors.forename: Robin
authors.initials: R
authors.lastname: Graf
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Thomas.sommermann@mdc-berlin.de.
authors.forename: Thomas
authors.initials: T
authors.lastname: Sommermann
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Kerstin.Petsch@mdc-berlin.de.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Petsch
authors.affiliation: Present Address: Bayer Pharma AG Building S107, 13353, Berlin, Germany. ulrike.sack@bayer.com.
authors.forename: Ulrike
authors.initials: U
authors.lastname: Sack
authors.affiliation: Harvard University, Cambridge, MA, 02138, USA. vpwwww@gmail.com.
authors.forename: Pavel
authors.initials: P
authors.lastname: Volchkov
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. Klaus.Rajewsky@mdc-berlin.de.
authors.forename: Klaus
authors.initials: K
authors.lastname: Rajewsky
authors.affiliation: Max-Delbrück-Center for Molecular Medicine, 13125, Berlin, Germany. ralf.kuehn@mdc-berlin.de.|Berlin Institute of Health, Kapelle-Ufer 2, 10117, Berlin, Germany. ralf.kuehn@mdc-berlin.de.
authors.forename: Ralf
authors.initials: R
authors.lastname: Kühn
mesh_terms: D000818:Animals; D064113:CRISPR-Cas Systems / Q000235:genetics*; D003001:Cloning, Molecular; D004622:Embryo, Mammalian; D055879:Gene Knock-In Techniques / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008845:Microinjections; D022661:RNA, Untranslated / Q000235:genetics*
chemical_list: C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D022661:RNA, Untranslated