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Elucidation of DNA binding modes of TALEs and creation of artificial DNA binding proteins

Research Project

Project/Area Number 24510295
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field Living organism molecular science
Research InstitutionKyoto University

Principal Investigator

IMANISHI Miki  京都大学, 化学研究所, 助教 (80362391)

Project Period (FY) 2012-04-01 – 2015-03-31
Project Status Completed (Fiscal Year 2014)
Budget Amount *help
¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000)
Fiscal Year 2014: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
Fiscal Year 2012: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
KeywordsDNA binding protein / Directed evolution / DNA結合タンパク質 / TALE / 転写因子 / DNA結合蛋白質
Outline of Final Research Achievements

Transcription activator-like effectors (TALEs) are useful templates for genome editing at specific sites. However, most TALE binding sites are preceded by a highly conserved 5’-terminal T nucleotide (5’-T), resulting in restriction of target sites. To remove this constraint, tryptophan 232 in the repeat-1 loop region of the dHax3 N-terminal domain was substituted for other 19 amino acids. Furthermore, whole hairpin loop region of repeat-1 was randomized. Although point mutation was insufficient to remove the constraint, directed evolution from the randomized library yielded repeat-1 mutants with unbiased targeting sites for 5’-terminal bases. It was indicated that the repeat-1 loop region of dHax3 is important for 5’-terminal base accommodation, and that molecular evolution of repeat-1 of TALEs is an efficient strategy to remove the constraint and thus allow targeting of any DNA sequences.

Report

(4 results)
  • 2014 Annual Research Report   Final Research Report ( PDF )
  • 2013 Research-status Report
  • 2012 Research-status Report
  • Research Products

    (11 results)

All 2015 2014 2013 Other

All Journal Article (1 results) (of which Peer Reviewed: 1 results) Presentation (10 results) (of which Invited: 2 results)

  • [Journal Article] Creating a TALE Protein with Unbiased 5'-T Binding2013

    • Author(s)
      Tsuji, S.; Futaki, S.; Imanishi, M.
    • Journal Title

      Biochem. Biophys. Res. Commun.

      Volume: 441 Issue: 1 Pages: 262-265

    • DOI

      10.1016/j.bbrc.2013.10.060

    • Related Report
      2013 Research-status Report
    • Peer Reviewed
  • [Presentation] Design of Artificial DNA Binding Proteins towards Manipulation of Biological Rhythms2015

    • Author(s)
      Imanishi, M.
    • Organizer
      RNA & CLOCK 2015
    • Place of Presentation
      淡路市
    • Year and Date
      2015-03-26
    • Related Report
      2014 Annual Research Report
    • Invited
  • [Presentation] 分子進化法を用いたTALEタンパク質の機能拡張2014

    • Author(s)
      辻将吾,今西未来,二木史朗
    • Organizer
      「細胞を創る」研究会7.0
    • Place of Presentation
      東京都
    • Year and Date
      2014-11-14
    • Related Report
      2014 Annual Research Report
  • [Presentation] Creating a TALE Binding non-5’-T Sequence2014

    • Author(s)
      Tsuji, S., Imanishi, M., Futaki, S.
    • Organizer
      FASEB SRC "Genome engineering: Cutting-edge research and application"
    • Place of Presentation
      Nassau (Bahamas)
    • Year and Date
      2014-06-25
    • Related Report
      2014 Annual Research Report
  • [Presentation] 分子進化法を用いた5'末塩基非選択性TALEタンパク質の創製

    • Author(s)
      辻将吾、今西未来、二木史朗
    • Organizer
      日本薬学会第134年会
    • Place of Presentation
      熊本大学(熊本市)
    • Related Report
      2013 Research-status Report
  • [Presentation] 分子進化法を用いたTALE蛋白質の標的DNA条件の解除

    • Author(s)
      今西未来、辻将吾、二木史朗
    • Organizer
      細胞を創る研究会6.0
    • Place of Presentation
      鶴岡メタボロームキャンパス(鶴岡市)
    • Related Report
      2013 Research-status Report
  • [Presentation] Molecular evolution of a TALE protein to change DNA binding manner

    • Author(s)
      今西未来、辻将吾、二木史朗
    • Organizer
      CBI学会2013年大会 -生命医薬情報学連合大会-
    • Place of Presentation
      タワーホール船堀(東京都)
    • Related Report
      2013 Research-status Report
    • Invited
  • [Presentation] Analysis of 5’-thymine recognition by TAL effectors

    • Author(s)
      Tsuji, S.; Imanishi, M.; Futaki, S.
    • Organizer
      国際ケミカルバイオロジー学会(ICBS)第2回年会(ICBS2013)
    • Place of Presentation
      京都大学(京都市)
    • Related Report
      2013 Research-status Report
  • [Presentation] TAL effectorによる5'末端チミン認識におけるrepeat -1の役割

    • Author(s)
      辻将吾、今西未来、二木史朗
    • Organizer
      第7回バイオ関連化学シンポジウム
    • Place of Presentation
      名古屋大学(名古屋市)
    • Related Report
      2013 Research-status Report
  • [Presentation] 5'-Tthymine Recognition by TAL Effectors

    • Author(s)
      Tsuji S., Imanishi M., Futaki S.
    • Organizer
      The 39th Inernational Symposium on Nucleic Acids Chemistry
    • Place of Presentation
      名古屋大学(名古屋市)
    • Related Report
      2012 Research-status Report
  • [Presentation] TAL effector による5’-チミン認識機構の解明

    • Author(s)
      辻 将吾、今西未来、二木史朗
    • Organizer
      日本薬学会第133年会
    • Place of Presentation
      パシフィコ横浜(横浜市)
    • Related Report
      2012 Research-status Report

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Published: 2013-05-31   Modified: 2019-07-29  

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