2023 Fiscal Year Final Research Report
Investigation of molecular insight into genome editing tools by single-molecule imaging
| Project/Area Number |
21H01771
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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) |
2021-04-01 – 2024-03-31
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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 |
In this study, we employed high-speed atomic force microscopy (HS-AFM) to examine various CRISPR/Cas genome editing tools, visualizing their interactions with RNA and their binding and cleavage of target DNA in real space and time. Our aim was to achieve a comprehensive understanding of the DNA cleavage mechanism of Cas proteins in action. We successfully visualized the flexible structure of SaCas9 and FnCas9 in their nucleic acid-free states, the stable and rigid two-lobe structure in their RNA-bound states, the fluctuations of the REC domain, and the displacements of the nuclease domain in the RNA-DNA bound states. Additionally, by comparing these results with previous data of SpCas9, we proposed a common structural framework and a distinct molecular mechanism for the operation of Cas9 proteins. We believe that this research is expected to serve as a fundamental basis for the further advancement of genome editing tools.
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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 |
学術的意義として、ほとんどのCas9タンパク質におけるRNAの役割は、標的DNAへのガイド役だけでなく、Cas9の立体構造の安定化に重要であることを明らかにした。さらに、DNA切断時のエンドヌクレアーゼの動きは、標的DNAの方向へプレスするような動きや、ななめにスライドする動きがあり、個々のCas9タンパク質は異なる切断作動機構をもつことを明らかにした。社会的意義として、ゲノム編集ツールは現代のバイオテクノロジーの中心ともいえる存在であり、その統一的なDNA切断作動機構の理解は、ゲノム編集技術のさらなく高度化を可能にし、人類の生命科学の発展に大きく貢献できると考えられる。
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