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Rational design of genome editing protein toward fast and accurate editing

Research Project

Project/Area Number 20K06571
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Review Section Basic Section 43040:Biophysics-related
Research InstitutionTohoku University

Principal Investigator

Kamagata Kiyoto  東北大学, 多元物質科学研究所, 准教授 (90432492)

Project Period (FY) 2020-04-01 – 2023-03-31
Project Status Completed (Fiscal Year 2022)
Budget Amount *help
¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000)
Fiscal Year 2022: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000)
Fiscal Year 2021: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
Fiscal Year 2020: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Keywordsゲノム編集 / タンパク質工学 / 単分子計測 / スライディング / 合理的設計
Outline of Research at the Start

ゲノム編集とは、特殊なDNA結合タンパク質を用いて、遺伝情報を含むDNAを自在に書き換える技術である。将来的に遺伝子治療での使用が期待されるが、編集に時間がかかる、標的ではないDNA配列を誤って編集するなどの問題がある。本研究では、単分子計測とタンパク質工学を組み合わせ、DNAに沿ったスライディング運動を促進させることで、速くて正確なゲノム編集タンパク質を合理的に設計する。

Outline of Final Research Achievements

Genome editing is a technology that freely rewrites DNA containing genetic information using a special DNA-binding protein. It is expected to be used in gene therapy in the future, but there are problems such as time-consuming editing and mistakenly editing non-target DNA sequences. In this study, we combined single-molecule measurement and protein engineering to modify the fast and accurate genome-editing protein Cas9 by promoting the sliding motion along the DNA. We identified sites that inhibit the sliding movement of Cas9 and sites that promote the sliding of other proteins, and modified Cas9. Single-molecule fluorescence measurements revealed that the modified Cas9 accelerated the sliding movement by about 8-fold.

Academic Significance and Societal Importance of the Research Achievements

遺伝子治療でゲノム編集タンパク質の使用が期待されている。しかし、既存のゲノム編集タンパク質Cas9には、編集時間がかかる、標的ではないDNA配列を誤って編集するなどの問題がある。遺伝子治療への応用を考えたとき、DNAの誤編集は致命的です。本研究で改変したCas9は、DNA上を素早く移動できるため、編集にかかる時間が短くなり、誤編集も減ると考えられるため、遺伝子治療などのゲノム編集での使用が期待される。

Report

(4 results)
  • 2022 Annual Research Report   Final Research Report ( PDF )
  • 2021 Research-status Report
  • 2020 Research-status Report
  • Research Products

    (59 results)

All 2022 2021 2020 Other

All Int'l Joint Research (8 results) Journal Article (15 results) (of which Int'l Joint Research: 7 results,  Peer Reviewed: 15 results,  Open Access: 13 results) Presentation (28 results) (of which Int'l Joint Research: 10 results,  Invited: 16 results) Book (3 results) Remarks (5 results)

  • [Int'l Joint Research] フランス国立科学研究センター(フランス)

    • Related Report
      2022 Annual Research Report
  • [Int'l Joint Research] UCLA(米国)

    • Related Report
      2021 Research-status Report
  • [Int'l Joint Research] ワイスマン研究所(イスラエル)

    • Related Report
      2021 Research-status Report
  • [Int'l Joint Research] Centre de Biophysique Moleculaire(フランス)

    • Related Report
      2021 Research-status Report
  • [Int'l Joint Research] UCLA(米国)

    • Related Report
      2020 Research-status Report
  • [Int'l Joint Research] ワイスマン研究所(イスラエル)

    • Related Report
      2020 Research-status Report
  • [Int'l Joint Research] Masaryk University(チェコ)

    • Related Report
      2020 Research-status Report
  • [Int'l Joint Research] Centre de Biophysique Moleculaire(フランス)

    • Related Report
      2020 Research-status Report
  • [Journal Article] Rational peptide design for regulating liquid-liquid phase separation on the basis of residue-residue contact energy2022

    • Author(s)
      Kamagata Kiyoto、Ariefai Maulana、Takahashi Hiroto、Hando Atsumi、Subekti Dwiky Rendra Graha、Ikeda Keisuke、Hirano Atsushi、Kameda Tomoshi
    • Journal Title

      Scientific Reports

      Volume: 12 Issue: 1 Pages: 13718-13718

    • DOI

      10.1038/s41598-022-17829-1

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access
  • [Journal Article] Giant Carbon Nano-Test Tubes as Versatile Imaging Vessels for High-Resolution and In Situ Observation of Proteins2022

    • Author(s)
      Tracy T Chuong, Toshihiko Ogura, Norihito Hiyoshi, Kazuma Takahashi, Sangho Lee, Keita Hiraga, Hiroki Iwase, Akira Yamaguchi, Kiyoto Kamagata, Eriko Mano, Satoshi Hamakawa, Hirotomo Nishihara, Takashi Kyotani, Galen D Stucky, Tetsuji Itoh
    • Journal Title

      ACS Applied Materials & Interfaces

      Volume: 14 Issue: 23 Pages: 26507-26516

    • DOI

      10.1021/acsami.2c06318

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Structure-dependent recruitment and diffusion of guest proteins in liquid droplets of FUS2022

    • Author(s)
      Kamagata Kiyoto、Iwaki Nanako、Kanbayashi Saori、Banerjee Trishit、Chiba Rika、Gaudon Virginie、Castaing Bertrand、Sakomoto Seiji
    • Journal Title

      Scientific Reports

      Volume: 12 Issue: 1 Pages: 7101-7101

    • DOI

      10.1038/s41598-022-11177-w

    • Related Report
      2022 Annual Research Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Development of peptide binder design method for disease-related phase separation proteins2022

    • Author(s)
      鎌形清人
    • Journal Title

      Folia Pharmacologica Japonica

      Volume: 157 Issue: 6 Pages: 392-395

    • DOI

      10.1254/fpj.22016

    • ISSN
      0015-5691, 1347-8397
    • Related Report
      2022 Annual Research Report
    • Peer Reviewed
  • [Journal Article] Engineering of the genome editing protein Cas9 to slide along DNA2021

    • Author(s)
      Banerjee Trishit、Takahashi Hiroto、Subekti Dwiky Rendra Graha、Kamagata Kiyoto
    • Journal Title

      Scientific Reports

      Volume: 11 Issue: 1 Pages: 14165-14165

    • DOI

      10.1038/s41598-021-93685-9

    • Related Report
      2021 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Testing mechanisms of DNA sliding by architectural DNA-binding proteins: dynamics of single wild-type and mutant protein molecules <i>in vitro</i> and <i>in vivo</i>2021

    • Author(s)
      Kamagata Kiyoto, Itoh Yuji, Tan Cheng, Mano Eriko, Wu Yining, Mandali Sridhar, Takada Shoji, Johnson Reid C
    • Journal Title

      Nucleic Acids Research

      Volume: 49 Issue: 15 Pages: 8642-8664

    • DOI

      10.1093/nar/gkab658

    • Related Report
      2021 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Molecular principles of recruitment and dynamics of guest proteins in liquid droplets2021

    • Author(s)
      Kamagata Kiyoto、Iwaki Nanako、Hazra Milan Kumar、Kanbayashi Saori、Banerjee Trishit、Chiba Rika、Sakomoto Seiji、Gaudon Virginie、Castaing Bertrand、Takahashi Hiroto、Kimura Michiko、Oikawa Hiroyuki、Takahashi Satoshi、Levy Yaakov
    • Journal Title

      Scientific Reports

      Volume: 11 Issue: 1 Pages: 19323-19323

    • DOI

      10.1038/s41598-021-98955-0

    • Related Report
      2021 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Single-Molecule Microscopy Meets Molecular Dynamics Simulations for Characterizing the Molecular Action of Proteins on DNA and in Liquid Condensates2021

    • Author(s)
      Kamagata Kiyoto
    • Journal Title

      Frontiers in Molecular Biosciences

      Volume: 8 Pages: 795367-795367

    • DOI

      10.3389/fmolb.2021.795367

    • Related Report
      2021 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Characterization of design grammar of peptides for regulating liquid droplets and aggregates of FUS2021

    • Author(s)
      Kamagata Kiyoto、Chiba Rika、Kawahata Ichiro、Iwaki Nanako、Kanbayashi Saori、Maeda Kana、Takahashi Hiroto、Hirano Atsushi、Fukunaga Koji、Ikeda Keisuke、Kameda Tomoshi
    • Journal Title

      Scientific Reports

      Volume: 11 Issue: 1 Pages: 6643-6643

    • DOI

      10.1038/s41598-021-86098-1

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] The disordered DNA-binding domain of p53 is indispensable for forming an encounter complex to and jumping along DNA2021

    • Author(s)
      Graha Subekti Dwiky Rendra、Kamagata Kiyoto
    • Journal Title

      Biochemical and Biophysical Research Communications

      Volume: 534 Pages: 21-26

    • DOI

      10.1016/j.bbrc.2020.12.006

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] The HMGB chromatin protein Nhp6A can bypass obstacles when traveling on DNA2020

    • Author(s)
      Kamagata Kiyoto、Ouchi Kana、Tan Cheng、Mano Eriko、Mandali Sridhar、Wu Yining、Takada Shoji、Takahashi Satoshi、Johnson Reid C
    • Journal Title

      Nucleic Acids Research

      Volume: 48 Issue: 19 Pages: 10820-10831

    • DOI

      10.1093/nar/gkaa799

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] Dependence of DNA length on binding affinity between TrpR and trpO of DNA2020

    • Author(s)
      Nobuo Shimamoto, Mikito Toda, Shigetoshi Nara, Tamiki Komatsuzaki, Kiyoto Kamagata, Takashi Kinebuchi, Jun-ichi Tomizawa
    • Journal Title

      Scientific Reports

      Volume: 10 Issue: 1 Pages: 15624-15624

    • DOI

      10.1038/s41598-020-71598-3

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Transient binding and jumping dynamics of p53 along DNA revealed by sub-millisecond resolved single-molecule fluorescence tracking2020

    • Author(s)
      Subekti D. R. G., Murata A., Itoh Y., Takahashi S., Kamagata K.
    • Journal Title

      Scientific Reports

      Volume: 10 Issue: 1 Pages: 13697-13697

    • DOI

      10.1038/s41598-020-70763-y

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Journal Article] A synthetic ion channel with anisotropic ligand response2020

    • Author(s)
      Muraoka Takahiro, Noguchi Daiki, Kasai Rinshi, Sato Kohei, Sasaki Ryo, Tabata Kazuhito, Ekimoto Toru, Ikeguchi Mitsunori, Kamagata Kiyoto, Hoshino Norihisa, Noji Hiroyuki, Akutagawa Tomoyuki, Ichimura Kazuaki, Kinbara Kazushi
    • Journal Title

      Nature Communications

      Volume: 11 Issue: 1 Pages: 2924-2924

    • DOI

      10.1038/s41467-020-16770-z

    • NAID

      120006878529

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] DNA結合タンパク質研究の最前線2020

    • Author(s)
      鎌形 清人
    • Journal Title

      Precision Medicine

      Volume: 3 Pages: 547-553

    • Related Report
      2020 Research-status Report
    • Peer Reviewed
  • [Presentation] Single-molecule characterization of DNA-binding protein action on DNA and engineering of genome editing protein Cas9 for improving its function2022

    • Author(s)
      Kamagata K.
    • Organizer
      European Nucleic Acids 2022
    • Related Report
      2022 Annual Research Report
    • Int'l Joint Research / Invited
  • [Presentation] 天然変性タンパク質を標的としたペプチドバインダーの合理的設計2022

    • Author(s)
      鎌形清人
    • Organizer
      第7回タタバイオ分子クラブ
    • Related Report
      2022 Annual Research Report
    • Invited
  • [Presentation] がん疾患関連タンパク質p53の液-液相分離と人工ペプチドによる制御2022

    • Author(s)
      鎌形清人
    • Organizer
      2022年度 日本分光学会NMR分光部会 集中講義
    • Related Report
      2022 Annual Research Report
    • Invited
  • [Presentation] 液-液相分離会合体の分子取り込みと 並進拡散運動に関する分子文法解析2022

    • Author(s)
      鎌形清人
    • Organizer
      第60回 日本生物物理学会年会
    • Related Report
      2022 Annual Research Report
    • Invited
  • [Presentation] 疾患関連相分離タンパク質を標的としたペプチドバインダーの設計法の開発2022

    • Author(s)
      鎌形清人
    • Organizer
      第73回日本薬理学会北部会
    • Related Report
      2022 Annual Research Report
    • Invited
  • [Presentation] 液-液相分離会合体の分子取り込みと並進拡散運動に関する分子文法解析2022

    • Author(s)
      鎌形清人
    • Organizer
      第6回LLPS研究会
    • Related Report
      2022 Annual Research Report
    • Invited
  • [Presentation] 液-液相分離会合体の分子取り込みと並進拡散運動に関する分子文法解析2022

    • Author(s)
      鎌形清人、岩城奈々子、Yaakov Levy
    • Organizer
      第22回蛋白質科学会年会
    • Related Report
      2022 Annual Research Report
  • [Presentation] Rational design of phase-separating peptides based on natural phase-separating protein disordered sequence2022

    • Author(s)
      Atsumi Hando、Maulana Ariefai、Nanako Iwaki、Saori Kanbayashi、Keisuke Ikeda、Kiyoto Kamagata
    • Organizer
      第60回 日本生物物理学会年会
    • Related Report
      2022 Annual Research Report
  • [Presentation] Single-molecule characterization of target search of DNA-binding proteins inside liquid DNA droplets2022

    • Author(s)
      Ryo Kusano、Trishit Banerjee、Saori Kambayashi、Kiyoto Kamagata
    • Organizer
      第60回 日本生物物理学会年会
    • Related Report
      2022 Annual Research Report
  • [Presentation] 相分離タンパク質を標的としたペプチドバインダーの設計法の開発と神経変性疾患関連タンパク質FUSへの応用2022

    • Author(s)
      鎌形清人
    • Organizer
      第95回日本薬理学会年会
    • Related Report
      2021 Research-status Report
    • Invited
  • [Presentation] Single-molecule characterization of DNA-binding protein action on DNA and engineering of genome editing protein Cas9 for improving its function2022

    • Author(s)
      Kiyoto Kamagata
    • Organizer
      European Nucleic Acids 2022
    • Related Report
      2021 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] DNA上や相分離複合体内でのタンパク質ダイナミクスの単分子計測と分子動力学解析2021

    • Author(s)
      鎌形清人
    • Organizer
      第59回 日本生物物理学会年会
    • Related Report
      2021 Research-status Report
    • Invited
  • [Presentation] Rational and experiment-guided peptide design for disease-related intrinsically disordered proteins2021

    • Author(s)
      Kiyoto Kamagata
    • Organizer
      Virtual Conference on Nanomedicine & Drug Delivery
    • Related Report
      2021 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] 液―液相分離の単分子計測と制御分子設計2021

    • Author(s)
      鎌形清人
    • Organizer
      第21回蛋白質科学会年会
    • Related Report
      2021 Research-status Report
    • Invited
  • [Presentation] 天然変性タンパク質p53液滴内への分子取込と1分子ダイナミクス観測2021

    • Author(s)
      岩城奈那子 、上林さおり、バネルジー トリシット 、千葉梨佳 、木村美智子 、小井川浩之 、高橋聡 、鎌形清人
    • Organizer
      第59回 日本生物物理学会年会
    • Related Report
      2021 Research-status Report
  • [Presentation] タンパク質の液-液相分離会合体への分子取り込みとダイナミクス解析2021

    • Author(s)
      岩城奈那子、上林さおり、Trishit Banerjee、小井川浩之、高橋聡、Bertrand Castaing、Koby Levy、鎌形清人
    • Organizer
      第21回 東北大学多元物質科学研究所研究発表会
    • Related Report
      2021 Research-status Report
  • [Presentation] Characterization of molecular uptake and single-molecule dynamics in liquid droplet2021

    • Author(s)
      岩城奈那子、鎌形清人
    • Organizer
      発動分子科学研究会
    • Related Report
      2021 Research-status Report
  • [Presentation] DNA結合タンパク質の機能を観る-単分子計測の挑戦-2021

    • Author(s)
      鎌形 清人
    • Organizer
      第29回生命科学交流ミーティング
    • Related Report
      2020 Research-status Report
    • Invited
  • [Presentation] Engineering of Genome Editing Protein Cas9 that Slides Along DNA Faster and Might Enable Efficient Target Search2021

    • Author(s)
      Trishit Banerjee, Dwiky Rendra Graha Subekti, Hiroto Takahashi, Kiyoto Kamagata
    • Organizer
      the 65th Biophysical Society Annual Meeting (USA)
    • Related Report
      2020 Research-status Report
    • Int'l Joint Research
  • [Presentation] Rational design of peptide targeting intrinsically disordered protein p532020

    • Author(s)
      Kiyoto Kamagata
    • Organizer
      第3回多元研ー台北科技大ジョイントシンポジウム
    • Related Report
      2020 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] Watching DNA-binding proteins on DNA and regulating the function by designed peptide2020

    • Author(s)
      鎌形 清人
    • Organizer
      令和2年度化学系学協会東北大会
    • Related Report
      2020 Research-status Report
    • Invited
  • [Presentation] Watching, controlling, and designing of function and phase separation of DNA-binding protein2020

    • Author(s)
      Kiyoto Kamagata
    • Organizer
      第58回 日本生物物理学会年会
    • Related Report
      2020 Research-status Report
    • Int'l Joint Research / Invited
  • [Presentation] タンパク質/DNA複合系の単分子ダイナミクスと液液相分離と人工ペプチドによる制御2020

    • Author(s)
      鎌形 清人
    • Organizer
      有機・生命・計測科学研究交流セミナー
    • Related Report
      2020 Research-status Report
    • Invited
  • [Presentation] タンパク質・DNA間の結合に新機構を発見:分子の揺らぎを利用するラチェットの可能性2020

    • Author(s)
      嶋本 伸雄, 戸田 幹人, 奈良 重俊, 小松崎 民樹, 鎌形 清人, 杵淵 隆, 富澤 純一
    • Organizer
      第20回東北大学多元物質科学研究所発表会
    • Related Report
      2020 Research-status Report
  • [Presentation] Rational design of peptide targeting intrinsically disordered protein p53 and discovery of liquid-liquid phase separation of p532020

    • Author(s)
      Kiyoto Kamagata and Tomoshi Kameda
    • Organizer
      The 4th Symposium for The Core Research Cluster for Materials Science and the 3rd Symposium on International Joint Graduate Program in Materials Science
    • Related Report
      2020 Research-status Report
    • Int'l Joint Research
  • [Presentation] Transient binding and non-rotational coupled motion of p53 revealed by sub-millisecond resolved single-molecule fluorescence tracking2020

    • Author(s)
      Dwiky Rendra Graha Subekti , Satoshi Takahashi, Kiyoto Kamagata
    • Organizer
      第58回日本生物物理学会年会
    • Related Report
      2020 Research-status Report
    • Int'l Joint Research
  • [Presentation] Search for peptides to control liquid-liquid phase separation of RNA binding protein FUS2020

    • Author(s)
      千葉 梨佳, 岩城 奈那子, 上林 さおり, 池田  恵介, 亀田 倫史, 鎌形 清人
    • Organizer
      第58回日本生物物理学会年会
    • Related Report
      2020 Research-status Report
    • Int'l Joint Research
  • [Presentation] Engineering of genome editing protein Cas9 that slides along DNA faster and might enable efficient target search2020

    • Author(s)
      Trishit Banerjee, Dwiky Rendra Graha Subekti, Hiroto Takahashi, Satoshi Takahashi, Kiyoto Kamagata
    • Organizer
      第58回日本生物物理学会年会
    • Related Report
      2020 Research-status Report
    • Int'l Joint Research
  • [Book] "A Study of p53 Action on DNA at the Single Molecule Level" in P53 - A Guardian of the Genome and Beyond [Working Title]2021

    • Author(s)
      Kiyoto Kamagata
    • Total Pages
      13
    • Publisher
      IntechOpen
    • Related Report
      2021 Research-status Report
  • [Book] "DNA結合タンパク質研究の最前線-がん抑制タンパク質p53の動態-" in BIO Clinica2021

    • Author(s)
      鎌形清人
    • Total Pages
      7
    • Publisher
      北隆館
    • Related Report
      2021 Research-status Report
  • [Book] 相分離生物学の全貌(現代化学増刊46)2020

    • Author(s)
      白木 賢太郎、鎌形 清人 他
    • Total Pages
      400
    • Publisher
      東京化学同人
    • ISBN
      9784807913466
    • Related Report
      2020 Research-status Report
  • [Remarks] 改変されたゲノム編集Cas9、DNA 上を動く! ― 遺伝子治療への応用に期待 ―

    • URL

      https://www.tohoku.ac.jp/japanese/2021/07/press20210719-02-dna.html

    • Related Report
      2021 Research-status Report
  • [Remarks] DNA結合タンパク質はDNA上でのタンパク質渋滞をすり抜けて移動できる

    • URL

      http://www2.tagen.tohoku.ac.jp/lab/news_press/20201117/

    • Related Report
      2020 Research-status Report
  • [Remarks] タンパク質・DNA間の結合に新機構を発見 分子の揺らぎを利用するラチェットの可能性

    • URL

      http://www2.tagen.tohoku.ac.jp/lab/news_press/20201009/

    • Related Report
      2020 Research-status Report
  • [Remarks] DNA結合タンパク質のジャンプまで観察できるサブミリ秒分解単分子蛍光計測法の提案

    • URL

      http://www2.tagen.tohoku.ac.jp/lab/news_press/20200825/

    • Related Report
      2020 Research-status Report
  • [Remarks] タンパク質の液-液相分離を制御するペプチド設計法を開発 ー 神経変性疾患などの創薬に期待 ー

    • URL

      http://www2.tagen.tohoku.ac.jp/lab/news_press/20210324/

    • Related Report
      2020 Research-status Report

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Published: 2020-04-28   Modified: 2024-01-30  

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