Elucidation of molecular mechanisms of RNA silencing

• Project/Area Number: 26291010
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Partial Multi-year Fund
• Section: 一般
• Research Field: Structural biochemistry
• Research Institution: The University of Tokyo
• Principal Investigator: Nishimasu Hiroshi 東京大学, 大学院理学系研究科(理学部), 助教 (00467044)
• Co-Investigator(Renkei-kenkyūsha): NUREKI Osamu 東京大学, 大学院理学系研究科, 教授 (10272460) ; SIOMI Mikiko 東京大学, 大学院理学系研究科, 教授 (20322745) ; SIOMI Haruhiko 慶應義塾大学, 医学部, 教授 (60202107) ; SHIBATA Mikihiro 金沢大学, 新学術創成研究機構, 准教授 (80631027) ; KODERA Noriyuki 金沢大学, 理工研究域, 准教授 (30584635)
• Research Collaborator: Zhang Feng Broad Institute, Associate Professor
• Project Period (FY): 2014-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥16,640,000 (Direct Cost: ¥12,800,000、Indirect Cost: ¥3,840,000); Fiscal Year 2016: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2015: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2014: ¥7,020,000 (Direct Cost: ¥5,400,000、Indirect Cost: ¥1,620,000)
• Keywords: 非コードRNA / CRISPR / Cas9 / Argonaute / piRNA / ゲノム編集 / RNA / 結晶構造 / Maelstrom / Crystal structure / Ribonuclease / CRISPR-Cas9

Outline of Final Research Achievements

We purified the PIWI protein from silkworm (Siwi) and determined its crystal structure. The structure revealed the molecular mechanism by which Siwi binds piRNA to protect germ cells. In addition, we determined the crystal structures of Cas9 and Cpf1 from various bacteria, thereby improving our mechanistic understanding of diverse CRISPR-Cas enzymes and paving the way for further development of new genome-editing technologies.

Research Products

• [Int'l Joint Research] Broad Institute/NIH(米国)
• [Int'l Joint Research] Broad Institute of MIT and Harvard(米国)
• [Journal Article] Structure of Cpf1 in Complex with Guide RNA and Target DNA (2016)
  • Author(s): Yamano T, Nishimasu H, Zetsche B, Hirano H, Slaymaker IM, Li Y, Fedorova I, Nakane T, Makarova KS, Koonin EV, Ishitani R, Zhang F, Nureki O
  • Journal Title: Cell Volume: 165 Issue: 4 Pages: 946-962
  • DOI: 10.1016/j.cell.2016.04.003
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Crystal structure of silkworm PIWI-clade Argonaute Siwi bound to piRNA (2016)
  • Author(s): Matsumoto N, Nishimasu H, Sakakibara K, Nishida KM, Hirano T, Ishitani R, Siomi H, Siomi MC, Nureki O
  • Journal Title: Cell Volume: 167 Issue: 2 Pages: 484-497
  • DOI: 10.1016/j.cell.2016.09.002
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Crystal Structure of the Minimal Cas9 from Campylobacter jejuni Reveals the Molecular Diversity in the CRISPR-Cas9 Systems (2016)
  • Author(s): Yamada M, Watanabe Y, Gootenberg JS, Hirano H, Ran FA, Nakane T, Ishitani R, Zhang F, Nishimasu H, Nureki O
  • Journal Title: Mol Cell Volume: 65 Issue: 6 Pages: 1109-1121
  • DOI: 10.1016/j.molcel.2017.02.007
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Structure and Engineering of Francisella novicida Cas9 (2016)
  • Author(s): Hisato Hirano, Jonathan S. Gootenberg, Takuro Horii, Omar O. Abudayyeh, Mika Kimura, Takanori Nakane, Ryuichiro Ishitani, Izuho Hatada, Feng Zhang, Hiroshi Nishimasu, Osamu Nureki
  • Journal Title: Cell Volume: 164 Issue: 5 Pages: 950-961
  • DOI: 10.1016/j.cell.2016.01.039
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Structural Basis for the Altered PAM Specificities of Engineered CRISPR-Cas9 (2016)
  • Author(s): Seiichi Hirano, Hiroshi Nishimasu, Ryuichiro Ishitani, Osamu Nureki
  • Journal Title: Molecular Cell Volume: 61 Issue: 6 Pages: 886-894
  • DOI: 10.1016/j.molcel.2016.02.018
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Crystal Structure of Staphylococcus aureus Cas9 (2015)
  • Author(s): Hiroshi Nishimasu, Le Cong, Winston X. Yan, F. Ann Ran, Bernd Zetsche, Yinqing Li, Arisa Kurabayashi, Ryuichiro Ishitani, Feng Zhang, Osamu Nureki
  • Journal Title: Cell Volume: 162 Issue: 5 Pages: 1113-1126
  • DOI: 10.1016/j.cell.2015.08.007
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex (2015)
  • Author(s): Konermann S*, Brigham MD*, Trevino AE, Joung J, Abudayyeh OO, Barcena C, Hsu PD, Habib N, Gootenberg JS, Nishimasu H, Nureki O, Zhang F
  • Journal Title: Nature Volume: 517 Issue: 7536 Pages: 583-588
  • DOI: 10.1038/nature14136
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Crystal Structure and Activity of the Endoribonuclease Domain of the piRNA Pathway Factor Maelstrom (2015)
  • Author(s): Naoki Matsumoto, Kaoru Sato, Hiroshi Nishimasu, Yurika Namba, Kana Miyakubi, Naoshi Dohmae, Ryuichiro Ishitani, Haruhiko Siomi, Mikiko C. Siomi, Osamu Nureki
  • Journal Title: Cell Reports Volume: 11 Issue: 3 Pages: 1-10
  • DOI: 10.1016/j.celrep.2015.03.030
  • Peer Reviewed / Open Access
• [Presentation] Crystal structure of silkworm PIWI, Siwi, in complex with piRNA (2016)
  • Author(s): Hiroshi Nishimasu, Naoki Matsumoto, Kazuhiro Sakakibara, Kazumichi Nishida, Ryuichiro Ishitani, Haruhiko Siomi, Mikiko C. Siomi, Osamu Nureki
  • Organizer: RNA2016
  • Place of Presentation: 京都国際会館（京都府京都市）
  • Year and Date: 2016-06-28
  • Int'l Joint Research / Invited
• [Presentation] 小型CRISPR-Cas9の結晶構造 (2015)
  • Author(s): 西増弘志、石谷隆一郎、濡木 理
  • Organizer: BMB2015
  • Place of Presentation: 神戸国際会議場（兵庫県神戸市）
  • Year and Date: 2015-12-03
• [Presentation] CRISPR-Cas9の結晶構造 (2015)
  • Author(s): 西増弘志
  • Organizer: 第53回日本生物物理学会年会
  • Place of Presentation: 金沢大学（石川県金沢市）
  • Year and Date: 2015-09-15
  • Invited
• [Presentation] Cas9-ガイド鎖RNA-標的DNAの結晶構造 (2014)
  • Author(s): 西増弘志
  • Organizer: 平成26年度日本結晶学会年会
  • Place of Presentation: 東京大学（東京）
  • Year and Date: 2014-11-03
  • Invited
• [Presentation] Crystal structure of Cas9 in complex with guide RNA and target DNA (2014)
  • Author(s): Nishimasu H, Ran FA, Ishitani R, Zhang F, Nureki O
  • Organizer: 平成26年度日本結晶学会年会
  • Place of Presentation: 東京大学（東京）
  • Year and Date: 2014-11-01
• [Presentation] Cas9-ガイド鎖RNA-標的DNA三者複合体の結晶構造 (2014)
  • Author(s): 西増弘志
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 京都国際会館（京都）
  • Year and Date: 2014-10-16
• [Presentation] Crystal structure of Cas9 in complex with guide RNA and target DNA (2014)
  • Author(s): Nishimasu H, Ran FA, Ishitani R, Zhang F, Nureki O
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 京都国際会館（京都）
  • Year and Date: 2014-10-16
• [Presentation] 立体構造から迫る酵素の作動機構 (2014)
  • Author(s): 西増弘志
  • Organizer: 第87回日本生化学会大会
  • Place of Presentation: 京都国際会館（京都）
  • Year and Date: 2014-10-15
  • Invited
• [Presentation] Cas9を応用した新規技術 (2014)
  • Author(s): 西増弘志
  • Organizer: 第14回日本蛋白質科学会年会
  • Place of Presentation: ワークピア横浜（横浜）
  • Year and Date: 2014-06-25
  • Invited
• [Presentation] Crystal structure of Cas9 in complex with guide RNA and target DNA (2014)
  • Author(s): Nishimasu H, Ran FA, Ishitani R, Zhang F, Nureki O
  • Organizer: 第14回日本蛋白質科学会年会
  • Place of Presentation: ワークピア横浜（横浜）
  • Year and Date: 2014-06-25
• [Presentation] Crystal structure of Cas9 in complex with guide RNA and target DNA (2014)
  • Author(s): Hiroshi Nishimasu
  • Organizer: CRISPR2014
  • Place of Presentation: Berlin (Germany)
  • Year and Date: 2014-05-16
• [Presentation] Crystal structure of Cas9 in complex with guide RNA and target DNA (2014)
  • Author(s): 西増弘志
  • Organizer: The 14th Tokyo RNA Club
  • Place of Presentation: 東京大学（東京）
  • Year and Date: 2014-05-09
  • Invited
• [Remarks] ゲノム編集のための「小さなはさみ」のかたち
  • URL: http://www.s.u-tokyo.ac.jp/ja/press/2015/36.html
• [Remarks] ゲノム編集のための新たな「はさみ」-立体構造にもとづくCRISPR-Cas9の改変-
  • URL: https://www.s.u-tokyo.ac.jp/ja/press/2016/4599/