2020 Fiscal Year Final Research Report
CRISPR-mediated 4D nucleome analysis
| Project/Area Number |
17H01407
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Genome biology
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| Research Institution | Kyushu University |
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Principal Investigator |
Ito Takashi 九州大学, 医学研究院, 教授 (90201326)
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| Project Period (FY) |
2017-04-01 – 2021-03-31
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| Keywords | 核内高次構造 / dCas9 / Dam / 生細胞可視化 / BiFC |
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| Outline of Final Research Achievements |
To explore the higher order structure of the nucleus, we intended to develop a method to detect any genomic regions within a certain spatial distance from a genomic locus of interest and a method for live-cell imaging of a single-copy genomic locus For the former, we succeeded in methylation of the vicinity of a target site bound by a dCas9 to which Dam methylase is tethered to the gRNA through the MS2-MCP interaction. However, we have failed to obtain evidence for methylation at genomic regions spatially proximal to the dCas9-bound site. For the latter, we improved the method for dCas9-mediated bimolecular fluorescence complementation, which enables live cell imaging on chromatin with high singal-to-noise ratio.
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| Free Research Field |
ゲノム科学
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| Academic Significance and Societal Importance of the Research Achievements |
新しい核内高次構造解析技術の開発のための貴重な基盤情報が整備された。関連技術として開発された新規6mA解析法、BiFC可能な蛍光タンパク質の拡張、およびBiFCシグナル増強法は、本課題を越えて幅広い分野への波及効果が期待できるものである。また、本課題の過程で、従来、安全と考えられていたdCas9が、複製フォークの進行を停止し、局所的なゲノム不安定性を誘導することを見い出した。この知見は、dCas9の様々な応用において留意すべき点であり、特に社会的影響の大きな医療応用については重要な警鐘となる可能性がある。
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