title: A possible aid in targeted insertion of large DNA elements by CRISPR/Cas in mouse zygotes.
abstract: The CRISPR/Cas system has rapidly emerged recently as a new tool for genome engineering, and is expected to allow for controlled manipulation of specific genomic elements in a variety of species. A number of recent studies have reported the use of CRISPR/Cas for gene disruption (knockout) or targeted insertion of foreign DNA elements (knock-in). Despite the ease of simple gene knockout and small insertions or nucleotide substitutions in mouse zygotes by the CRISPR/Cas system, targeted insertion of large DNA elements remains an apparent challenge. Here the generation of knock-in mice with successful targeted insertion of large donor DNA elements ranged from 3.0 to 7.1 kb at the ROSA26 locus using the CRISPR/Cas system was achieved. Multiple independent knock-in founder mice were obtained by injection of hCas9 mRNA/sgRNA/donor vector mixtures into the cytoplasm of C57BL/6N zygotes when the injected zygotes were treated with an inhibitor of actin polymerization, cytochalasin. Successful germ line transmission of three of these knock-in alleles was also confirmed. The results suggested that treatment of zygotes with actin polymerization inhibitors following microinjection could be a viable method to facilitate targeted insertion of large DNA elements by the CRISPR/Cas system, enabling targeted knock-in readily attainable in zygotes.
authors.affiliation: Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Bunkyo-Ku, Tokyo, 113-0033, Japan.
authors.forename: Harumi
authors.initials: H
authors.lastname: Nakao
authors.affiliation: Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Bunkyo-Ku, Tokyo, 113-0033, Japan.
authors.forename: Takeshi
authors.initials: T
authors.lastname: Harada
authors.affiliation: Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Bunkyo-Ku, Tokyo, 113-0033, Japan.|Animal Resource Development Unit, Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe, Hyogo, 650-0047, Japan.
authors.forename: Kazuki
authors.initials: K
authors.lastname: Nakao
authors.affiliation: Animal Resource Development Unit, Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe, Hyogo, 650-0047, Japan.|Genetic Engineering Team, Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe, Hyogo, 650-0047, Japan.
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Kiyonari
authors.affiliation: Animal Resource Development Unit, Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe, Hyogo, 650-0047, Japan.
authors.forename: Kenichi
authors.initials: K
authors.lastname: Inoue
authors.affiliation: Animal Resource Development Unit, Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe, Hyogo, 650-0047, Japan.|Genetic Engineering Team, Division of Bio-Function Dynamics Imaging, RIKEN Center for Life Science Technologies, Kobe, Hyogo, 650-0047, Japan.
authors.forename: Yasuhide
authors.initials: Y
authors.lastname: Furuta
authors.affiliation: Laboratory of Animal Resources, Center for Disease Biology and Integrative Medicine, Faculty of Medicine, The University of Tokyo, Bunkyo-Ku, Tokyo, 113-0033, Japan.
authors.forename: Atsu
authors.initials: A
authors.lastname: Aiba
mesh_terms: D000199:Actins / Q000737:chemistry; D000595:Amino Acid Sequence; D000818:Animals; D064113:CRISPR-Cas Systems; D003571:Cytochalasin B / Q000494:pharmacology*; D015638:Cytochalasin D / Q000494:pharmacology; D004247:DNA; D055879:Gene Knock-In Techniques / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains / Q000235:genetics*; D008845:Microinjections; D008969:Molecular Sequence Data; D022661:RNA, Untranslated / Q000235:genetics; D015053:Zygote
chemical_list: D000199:Actins; C110853:Gt(ROSA)26Sor non-coding RNA, mouse; D022661:RNA, Untranslated; D015638:Cytochalasin D; D003571:Cytochalasin B; D004247:DNA
keywords: cytochalasin; genome editing; zygote injection