2015 Fiscal Year Final Research Report
Multiple-gene editing system using a non-inheritable maternal Cas9 in mice
| Project/Area Number |
26670153
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| Research Category |
Grant-in-Aid for Challenging Exploratory Research
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| Allocation Type | Multi-year Fund |
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| Research Field |
Pathological medical chemistry
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| Research Institution | Shinshu University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
SHINDO Takayuki 信州大学, 学術研究院医学系, 教授 (90345215)
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| Project Period (FY) |
2014-04-01 – 2016-03-31
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| Keywords | 疾患モデル動物 / 遺伝子改変マウス / CRISPR / Cas9 / ゲノム編集 / 発生工学 / アドレノメデュリン / RAMP |
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| Outline of Final Research Achievements |
The aim of this study was to develop new strategies to generate multiple-gene edited mice by using CRISPR/Cas9 system. First, we established a single blastocyst-based assay for rapidly determining whether CRISPR/Cas9-mediated genome editing worked in mouse preimplantation embryos. Then, we established transgenic (Tg) mice with systemic Cas9 overexpression (sCAT) to use non-inheritable maternal Cas9 (maCas9) protein derived from the zygotes. The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome, and modified nine target genes simultaneously after injection with nine different gRNAs alone. Furthermore, we demonstrated the creation of “Cas9 transgene-free” gene-edited mice using non-Tg (+/+) zygotes carrying maCas9. These results suggest that sCAT zygotes have the potential to create multiple gene-modified mice both with and without the Cas9 transgene, enabling repeatable genome engineering.
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| Free Research Field |
発生工学、 発生生物学
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