Comprehensive studies of working mechanism of Cas proteins using HS-AFM

• Project/Area Number: 18H01836
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Basic Section 28040:Nanobioscience-related
• Research Institution: Kanazawa University
• Principal Investigator: Shibata Mikihiro 金沢大学, ナノ生命科学研究所, 教授 (80631027)
• Project Period (FY): 2018-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥17,810,000 (Direct Cost: ¥13,700,000、Indirect Cost: ¥4,110,000); Fiscal Year 2020: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000); Fiscal Year 2019: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000); Fiscal Year 2018: ¥11,830,000 (Direct Cost: ¥9,100,000、Indirect Cost: ¥2,730,000)
• 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

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.

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。

Research Products

• [Journal Article] Histone variant H2A.B-H2B dimers are spontaneously exchanged with canonical H2A-H2B in the nucleosome (2021)
  • Author(s): Rina Hirano, Yasuhiro Arimura, Tomoya Kujirai, Mikihiro Shibata, Aya Okuda, Ken Morishima, Rintaro Inoue, Masaaki Sugiyama, Hitoshi Kurumizaka
  • Journal Title: Communications Biology Volume: 4 Issue: 1 Pages: 1-11
  • DOI: 10.1038/s42003-021-01707-z
  • NAID: 120006960298
  • Peer Reviewed / Open Access
• [Journal Article] Applicability of Styrene-Maleic Acid Copolymer for Two Microbial Rhodopsins, RxR and HsSRI. (2020)
  • Author(s): Tetsuya Ueta, Keiichi Kojima, Tomoya Hino, Mikihiro Shibata, Shingo Nagano, Yuki Sudo*
  • Journal Title: Biophysical journal Volume: 119 Issue: 9 Pages: 1760-1770
  • DOI: 10.1016/j.bpj.2020.09.026
  • Peer Reviewed
• [Journal Article] Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor. (2019)
  • Author(s): Sakai K, Passioura T, Sato H, Ito K, Furuhashi H, Umitsu M, Takagi J, Kato Y, Mukai H, Warashina S, Zouda M, Watanabe Y, Yano S, Shibata M, Suga H, Matsumoto K
  • Journal Title: Nature Chemical Biology Volume: 15 Issue: 6 Pages: 598-606
  • DOI: 10.1038/s41589-019-0285-7
  • Peer Reviewed / Open Access
• [Journal Article] Crystal structure of heliorhodopsin (2019)
  • Author(s): W. Shihoya, K. Inoue, M. Singh, M. Konno, S. Hososhima, K. Yamashita, K. Ikeda, A. Higuchi, T. Izume, Okazaki S, Hashimoto M, Mizutori R, Tomida S, Yamauchi Y, Abe-Yoshizumi R, K. Katayama, SP. Tsunoda, M. Shibata, Y. Furutani, A. Pushkarev, O. Béjà, T. Uchihashi, H. Kandori, O. Nureki
  • Journal Title: Nature Volume: 574 Issue: 7776 Pages: 132-136
  • DOI: 10.1038/s41586-019-1604-6
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Macrocyclic peptide-based inhibition and imaging of hepatocyte growth factor. (2019)
  • Author(s): Katsuya Sakai, Toby Passioura, Hiroki Sato, Kenichiro Ito, Hiroki Furuhashi, Masataka Umitsu, Junichi Takagi, Yukinari Kato, Hidefumi Mukai, Shota Warashina, Maki Zouda, Yasuyoshi Watanabe, Seiji Yano, Mikihiro Shibata, Hiroaki Suga, and Kunio Matsumoto
  • Journal Title: Nat. Chem. Biol. Volume: 印刷中
  • Peer Reviewed
• [Journal Article] Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy (2018)
  • Author(s): Shibata Mikihiro、Inoue Keiichi、Ikeda Kento、Konno Masae、Singh Manish、Kataoka Chihiro、Abe-Yoshizumi Rei、Kandori Hideki、Uchihashi Takayuki
  • Journal Title: Scientific Reports Volume: 8 Issue: 1 Pages: 8262-8262
  • DOI: 10.1038/s41598-018-26606-y
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] High thermal stability of oligomeric assemblies of Thermophilic rhodopsin in a lipid environment. (2018)
  • Author(s): Tomomi Shionoya, Misao Mizuno, Takashi Tsukamoto, Kento Ikeda, Hayato Seki, Keiichi Kojima, Mikihiro Shibata, Izuru Kawamura, Yuki Sudo, and Yasuhisa Mizutani
  • Journal Title: J. Phys. Chem. B Volume: 122 Issue: 27 Pages: 6945-6953
  • DOI: 10.1021/acs.jpcb.8b04894
  • Peer Reviewed
• [Presentation] 高速AFMを用いたCaMKII多量体のCa2+信号積算メカニズムの解明 (2020)
  • Author(s): 柴田幹大, 村越秀治
  • Organizer: 日本生体エネルギー研究会 第46回討論会
• [Presentation] 高速原子間力顕微鏡による柔軟なタンパク質動態のナノスケール撮影 (2020)
  • Author(s): 柴田幹大
  • Organizer: 第43回日本分子生物学会年会
  • Invited
• [Presentation] 高速 AFM によるハブ毒液由来脂質分解酵素 PLA2の膜認識機構の解明 (2020)
  • Author(s): 神谷孫斗, 柴田幹大, 上田直子, 角野歩
  • Organizer: 第58回日本生物物理学会年会
• [Presentation] Signal integration mechanism of Ca2+/calmodulin-dependent protein kinase II revealed by High-speed AFM. (2020)
  • Author(s): Mikihiro Shibata and Hideji Murakoshi
  • Organizer: BPS2020 64th Annual Meeting
  • Int'l Joint Research
• [Presentation] Nano-scale imaging of biological samples using High-speed Atomic Force Microscopy. (2019)
  • Author(s): Mikihiro Shibata
  • Organizer: LIIF2019
  • Int'l Joint Research / Invited
• [Presentation] 高速原子間力顕微鏡によるクロマチン動態のナノスケール観察 (2019)
  • Author(s): 柴田幹大
  • Organizer: 第２回クロマチン潜在能領域会議
  • Invited
• [Presentation] Visualizing flexibility in protein structures by high-speed atomic force microscopy. (2019)
  • Author(s): Mikihiro Shibata
  • Organizer: 57th日本生物物理学会年会
  • Invited
• [Presentation] High-speed atomic force microscopy visualization of protein flexibility in action. (2019)
  • Author(s): Mikihiro Shibata
  • Organizer: Joint UBI-NanoLSI workshop TRENDS IN MOLECULAR BIOPHYSICS OF LIVING CELLS
  • Invited
• [Presentation] Physicochemical properties of the microbial rhodopsin RxR in the membrane-mimicking molecule styrene maleic acid (SMA) copolymer. (2019)
  • Author(s): Tetsuya Ueta, Keiichi Kojima, Tomoya Hino, Mikihiro Shibata, Shingo Nagano & Yuki Sudo
  • Organizer: 第11回日本生物物理学会 中国四国支部大会
• [Presentation] Structure and biophysical characterization of the heliorhodopsin. (2019)
  • Author(s): W. Shihoya, K. Inoue, M. Singh, M. Konno, S. Hososhima, K. Yamashita, K. Ikeda, A. Higuchi, S. Okazaki, T. Izume, M. Hashimoto, R. Mizutori, S. Tomida, Y. Yamauchi, R. Abe-Yoshizumi, K. Katayama, S. P.Tsunoda, M. Shibata, Y. Furutani, A. Pushkarev, O. Beja, T. Uchihashi, H. Kandori and O. Nureki
  • Organizer: 57th日本生物物理学会年会
• [Presentation] Application of drift elimination method for high-speed AFM images. (2019)
  • Author(s): Shotaro Tsujioka, Hideji Murakoshi, and Mikihiro Shibata
  • Organizer: 57th日本生物物理学会年会
• [Presentation] Mapping of mechanical property on live cell surface by scanning ion conductance microscope. (2019)
  • Author(s): Satoko Kitazawa, Linhao Sun, Ayako Housaka, Takahiro Watanabe-Nakayama, Hiroki Konno, Mikihiro Shibata, and Shinji Watanabe
  • Organizer: 57th日本生物物理学会年会
• [Presentation] 計測系から見た分子集合体の機能計測-高速AFM計測 (2019)
  • Author(s): 柴田幹大
  • Organizer: 日本化学会第99秋季年会
  • Invited
• [Presentation] High-speed atomic force microscopy shows conformational dynamics of Ca2+/calmodulin-dependent protein kinase II (2019)
  • Author(s): Mikihiro Shibata and Hideji Murakoshi
  • Organizer: BPS19 63th Annual Meeting
  • Int'l Joint Research
• [Presentation] Nanoscale imaging of biomolecules by high-speed atomic force microscopy. (2018)
  • Author(s): Mikihiro Shibata
  • Organizer: International Symposium on Tumor Biology in Kanazawa Joint Symposium
  • Int'l Joint Research / Invited
• [Presentation] Watching single proteins in action using high-speed AFM. (2018)
  • Author(s): Mikihiro Shibata
  • Organizer: 56th生物物理学会年会
  • Invited
• [Presentation] Filming single proteins at work using high-speed atomic force microscopy. (2018)
  • Author(s): Mikihiro Shibata
  • Organizer: Irago Conference 2018
  • Int'l Joint Research / Invited
• [Presentation] High-speed atomic force microscopy for nano-scale imaging of biological samples. (2018)
  • Author(s): Mikihiro Shibata
  • Organizer: The 2nd NanoLSI Symposium in London -Towards Establishment of New Research Field: Nanoprobe Life Science-
  • Int'l Joint Research / Invited
• [Presentation] 高速原子間力顕微鏡による生体分子のナノスケールイメージング (2018)
  • Author(s): 柴田幹大
  • Organizer: 2018日本表面真空学会学術講演会
  • Invited
• [Presentation] 高速原子間力顕微鏡による揺らいだタンパク質構造のナノスケール撮影 (2018)
  • Author(s): 柴田幹大
  • Organizer: 非共有結合系の分子科学：計測技術から探る生体分子科学の新展開
  • Invited
• [Presentation] Nanoscale visualization of biomolecules using high-speed atomic force microscopy. (2018)
  • Author(s): Mikihiro Shibata
  • Organizer: 6th AIST International Imaging Workshop
  • Invited