A non-inheritable maternal Cas9-based multiple-gene editing system in mice

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Takayuki Sakurai ; Akiko Kamiyoshi ; Hisaka Kawate ; Chie Mori ; Satoshi Watanabe ; Megumu Tanaka ; Ryuichi Uetake ; Masahiro Sato ; Takayuki Shindo (2016)
  • Publisher: Nature Publishing Group
  • Journal: Scientific Reports, volume 6 (issn: 2045-2322, eissn: 2045-2322)
  • Related identifiers: doi: 10.1038/srep20011, pmc: PMC4730228
  • Subject: Article

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.
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