2020 Fiscal Year Final Research Report
Comprehensive studies of working mechanism of Cas proteins using HS-AFM
| Project/Area Number |
18H01836
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Review Section |
Basic Section 28040:Nanobioscience-related
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| Research Institution | Kanazawa University |
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Principal Investigator |
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| Project Period (FY) |
2018-04-01 – 2021-03-31
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| Keywords | １分子イメージング・ナノ計測 / ゲノム工学 / タンパク質・酵素化学 / １分子計測・操作 / バイオイメージング |
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| Outline of Final Research Achievements |
The purpose of this study was to understand the mechanism of DNA cleavage reaction of Cas proteins, which are used as genome editing tools. Using high-speed atomic force microscopy (HS-AFM), we tried to visualize the dynamics of Cas proteins during the binding to and cleaving a target site of DNA in real-time with nano-meter resolution. During the three years research period, HS-AFM observations were performed on SaCas9, AsCas12a, and LbCas12a. HS-AFM videos of three Cas proteins showed that structures of Cas proteins without nucleic acids were flexible, while structures of RNA-binding Cas proteins were stable and rigid. Furthermore, when RNA-Cas complex bound to the target site of DNA, a complex remained tightly bound to the target site of DNA and did not slide on the DNA. Since this molecular dynamics was observed in all Cas proteins, we concluded that it could be a common molecular binding mechanism to the target site of DNA of Cas proteins.
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| Free Research Field |
ナノバイオサイエンス
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| Academic Significance and Societal Importance of the Research Achievements |
本研究成果における学術的意義として、Casタンパク質におけるRNAの役割が、標的DNA配列へのガイド役だけでなく、立体構造の安定化に寄与することを明らかにした点や、RNA-Casタンパク質がDNA配列へひとたび結合すると、DNA上をスライドすることなく強固に結合したままであることを明らかにした点が挙げられる。また、現代のバイオテクノロジーの中心であるゲノム編集を担うCasタンパク質群のDNA切断分子機構の統一的な理解は、人類にとって重要であり、ゲノム編集技術のさらなく高度化を促す研究成果と位置付けられ、社会的意義は大きいと考えられる。
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