| Project/Area Number |
17K07263
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Multi-year Fund |
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| Section | 一般 |
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| Research Field |
System genome science
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| Research Institution | Aichi Medical University |
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Principal Investigator |
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| Co-Investigator(Kenkyū-buntansha) |
兵頭 寿典 愛知医科大学, 医学部, 講師 (40710645)
シバスンダラン カルナン 愛知医科大学, 医学部, 講師 (30557096)
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| Project Period (FY) |
2017-04-01 – 2021-03-31
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| Project Status |
Completed (Fiscal Year 2020)
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| Budget Amount *help |
¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000)
Fiscal Year 2019: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
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| Keywords | CRISPR / Cas9 / nickase / ゲノム編集 / ノックイン / tandem paired nicking / ニック / CRISPR/Cas9 / CRISPR-Cas9 / ゲノム生物工学 |
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| Outline of Final Research Achievements |
Targeted knock-in assisted by genome editing is a promising technology for genome engineering which will be applicable to a broad range of research fields in life science. However, the precision, specificity, and safety of this technology need to be improved to expedite its practical applications. In the present study, we characterized a methodology for targeted knock-in, namely tandem paired nicking (TPN), which leverages Cas9 nickases in a manner distinct from their regular use. By empirically addressing the properties of TPN, we found that TPN achieves the efficiency of targeted knock-in largely equivalent to those achieved by the conventional Cas9 nuclease-based approach, while drastically decreasing the incidence of random undesired insertions and deletions occurring at the targeted genomic loci. Moreover, TPN-based targeted knock-in neither activated the p53 signaling pathway as known as “the guardian of the genome,” nor was restricted by the function of wild-type p53.
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| Academic Significance and Societal Importance of the Research Achievements |
本研究の結果、TPN法の利用によってノックインに伴う標的ゲノム部位の予期せぬランダム変異を抑えうることがわかった。また、TPN法では標的外部位（オフターゲット部位）のランダム変異が少ないことも示唆されており、ゲノム編集細胞のがん化などの危険性を抑制しうるものと期待される。さらに、DNA損傷反応を制御するp53シグナル経路がTPN法によって攪乱されないことは、同法の安全性を裏付けるものと考えられる。従来法に比肩するノックイン効率が保持されていることも鑑み、TPN法は今後の技術的な洗練を経たのち、将来的には臨床医学、畜産学、植物科学などに応用可能な有用なノックイン法になりうるものと期待される。
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