title: A non-inheritable maternal Cas9-based multiple-gene editing system in mice.
abstract: The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection-based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create "Cas9 transgene-free" gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice.
authors.affiliation: Department of Cardiovascular Research, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Sakurai
authors.affiliation: Department of Cardiovascular Research, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
authors.forename: Akiko
authors.initials: A
authors.lastname: Kamiyoshi
authors.affiliation: Department of Cardiovascular Research, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
authors.forename: Hisaka
authors.initials: H
authors.lastname: Kawate
authors.affiliation: Department of Cardiovascular Research, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
authors.forename: Chie
authors.initials: C
authors.lastname: Mori
authors.affiliation: Animal Genome Research Unit, Division of Animal Science, National Institute of Agrobiological Sciences, 2-1-2 Kannondai, Tsukuba, Ibaraki 305-8602, Japan.
authors.forename: Satoshi
authors.initials: S
authors.lastname: Watanabe
authors.affiliation: Department of Cardiovascular Research, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
authors.forename: Megumu
authors.initials: M
authors.lastname: Tanaka
authors.affiliation: Department of Cardiovascular Research, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
authors.forename: Ryuichi
authors.initials: R
authors.lastname: Uetake
authors.affiliation: Section of Gene Expression Regulation, Frontier Science Research Center, Kagoshima University, 8-35-1 Sakuragaoka, Kagoshima, Kagoshima 890-8544, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Sato
authors.affiliation: Department of Cardiovascular Research, Graduate School of Medicine, Shinshu University, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan.
authors.forename: Takayuki
authors.initials: T
authors.lastname: Shindo
mesh_terms: D000818:Animals; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism; D064113:CRISPR-Cas Systems; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D005700:Galactosyltransferases / Q000235:genetics; D000072669:Gene Editing; D015870:Gene Expression; D056426:Genetic Loci; D016678:Genome; D022781:Hepatocytes / Q000378:metabolism; D054643:INDEL Mutation; D008297:Male; D051379:Mice; D008822:Mice, Transgenic; D009154:Mutation; D009865:Oocytes / Q000378:metabolism; D010641:Phenotype; D000094704:RNA, Guide, CRISPR-Cas Systems / Q000235:genetics; D013094:Spermatozoa / Q000378:metabolism; D019076:Transgenes; D015053:Zygote / Q000378:metabolism
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D005700:Galactosyltransferases
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