新規CRISPR-Cas系酵素の構造解析および作動機構の解明

• Project/Area Number: 22KJ0608
• Project/Area Number (Other): 21J20951 (2021-2022)
• Research Category: Grant-in-Aid for JSPS Fellows
• Allocation Type: Multi-year Fund (2023); Single-year Grants (2021-2022)
• Section: 国内
• Review Section: Basic Section 38020:Applied microbiology-related
• Research Institution: The University of Tokyo
• Principal Investigator: 中川 綾哉 東京大学, 理学系研究科, 特別研究員(DC1)
• Project Period (FY): 2023-03-08 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥2,200,000 (Direct Cost: ¥2,200,000); Fiscal Year 2023: ¥700,000 (Direct Cost: ¥700,000); Fiscal Year 2022: ¥700,000 (Direct Cost: ¥700,000); Fiscal Year 2021: ¥800,000 (Direct Cost: ¥800,000)
• Keywords: CRISPR-Cas / CRISPR-Cas system / RNA編集 / cryo-EM

Outline of Research at the Start

最小のRNA依存性RNAヌクレアーゼとして注目を集めているCas13bt3の立体構造を解明することで、Cas13bt3がどのようにしてRNAを特異的に認識し、切断機構を解明する。また、得られた立体構造を基にした合理的な改変によって、RNA編集効率が向上した高活性変異体の作製を目指す。RNA編集技術は、ゲノムを直接書き換えることなく発現タンパク質の形質を変えることができるという点でゲノム編集技術よりも安全であり、本研究は遺伝性疾患に対する革新的な治療法の確立に繋がる。

Outline of Annual Research Achievements

CRISPR-Cas systems in prokaryotes provide adaptive immunity against foreign nucleic acids, and are divided into two classes (classes 1 and 2) and six types (type I-VI). In the class 1 systems, multiple Cas proteins associate with a guide RNA to de-grade invading nucleic acids. By contrast, in the class 2 systems, single multidomain Cas proteins, Cas9 (type II), Cas12 (type V), and Cas13 (type VI), associate with their guide RNA to form effector complexes responsible for target nucleic acid cleavage. Since class 2 Cas proteins operate as single components, they have been harnessed for various innovative techniques, such as genome editing. I performed biochemical and structural analyses on three effectors associated with the class 2 CRISPR-Cas system: Cas9 (type II), Cas12 (type V), and Cas13 (type VI), elu-cidating the mechanistic details of the RNA-guided DNA/RNA cleavage. These find-ings advance our understanding of the conservation and divergence among the class 2 CRISPR-Cas systems, and shed light on their evolutionary scenario originating from transposon-encoded proteins. Furthermore, I rationally engineered com-pact Cas ef-fectors with enhanced activities. These results expand the CRISPR-Cas toolbox for therapeutic genome engineering.

Research Products

• [Journal Article] Cryo-EM structure of the transposon-associated TnpB enzyme (2023)
  • Author(s): Nakagawa Ryoya、Hirano Hisato、Omura Satoshi N.、Nety Suchita、Kannan Soumya、Altae-Tran Han、Yao Xiao、Sakaguchi Yuriko、Ohira Takayuki、Wu Wen Y.、Nakayama Hiroshi、Shuto Yutaro、Tanaka Tatsuki、Sano Fumiya K.、Kusakizako Tsukasa、Kise Yoshiaki et al.
  • Journal Title: Nature Volume: 616 Issue: 7956 Pages: 390-397
  • DOI: 10.1038/s41586-023-05933-9
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] An AsCas12f-based compact genome-editing tool derived by deep mutational scanning and structural analysis (2023)
  • Author(s): Hino Tomohiro、Omura Satoshi N.、Nakagawa Ryoya、Togashi Tomoki、Takeda Satoru N.、Hiramoto Takafumi、Tasaka Satoshi、Hirano Hisato、Tokuyama Takeshi、Uosaki Hideki、Ishiguro Soh、Kagieva Madina、Yamano Hiroyuki、Ozaki Yuki、Motooka Daisuke、Mori Hideto、Kirita Yuhei、Kise Yoshiaki et al.
  • Journal Title: Cell Volume: 186 Issue: 22 Pages: 4920-4935
  • DOI: 10.1016/j.cell.2023.08.031
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Structure and engineering of the minimal type VI CRISPR-Cas13bt3 (2022)
  • Author(s): Nakagawa Ryoya、Kannan Soumya、Altae-Tran Han、Takeda Satoru N.、Tomita Atsuhiro、Hirano Hisato、Kusakizako Tsukasa、Nishizawa Tomohiro、Yamashita Keitaro、Zhang Feng、Nishimasu Hiroshi、Nureki Osamu
  • Journal Title: Molecular Cell Volume: 82 Issue: 17 Pages: 3178-3192
  • DOI: 10.1016/j.molcel.2022.08.001
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Engineered Campylobacter jejuni Cas9 variant with enhanced activity and broader targeting range (2022)
  • Author(s): Nakagawa Ryoya、Ishiguro Soh、Okazaki Sae、Mori Hideto、Tanaka Mamoru、Aburatani Hiroyuki、Yachie Nozomu、Nishimasu Hiroshi、Nureki Osamu
  • Journal Title: Communications Biology Volume: 5 Issue: 1 Pages: 1-8
  • DOI: 10.1038/s42003-022-03149-7
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Amplification-free RNA detection with CRISPR?Cas13 (2021)
  • Author(s): Shinoda Hajime、Taguchi Yuya、Nakagawa Ryoya、Makino Asami、Okazaki Sae、Nakano Masahiro、Muramoto Yukiko、Takahashi Chiharu、Takahashi Ikuko、Ando Jun、Noda Takeshi、Nureki Osamu、Nishimasu Hiroshi、Watanabe Rikiya
  • Journal Title: Communications Biology Volume: 4 Issue: 1 Pages: 476-476
  • DOI: 10.1038/s42003-021-02001-8
  • Peer Reviewed / Open Access