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development of genome-editing techniques using engineered nucleases

Research Project

Project/Area Number 26460088
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeMulti-year Fund
Section一般
Research Field Biological pharmacy
Research InstitutionInstitute of Physical and Chemical Research

Principal Investigator

Hisano Yu  国立研究開発法人理化学研究所, 脳科学総合研究センター, 基礎科学特別研究員 (60467636)

Co-Investigator(Renkei-kenkyūsha) KAWAHARA Atsuo  山梨大学, 総合研究部, 教授 (10362518)
KOBAYASHI Takuma  国立研究開発法人理化学研究所, 脳科学総合研究センター, 研究員 (80582288)
Project Period (FY) 2014-04-01 – 2017-03-31
Project Status Completed (Fiscal Year 2016)
Budget Amount *help
¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000)
Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2015: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Fiscal Year 2014: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
Keywords人工ヌクレアーゼ / ゲノム編集 / TALEN / CRISPR/Cas9 system / ゼブラフィッシュ / ノックイン / 逆遺伝学 / ヌクレアーゼ / CRISPR/Cas9
Outline of Final Research Achievements

This study aimed to develop new genome-editing techniques using engineered nucleases such as CRISPR/Cas9 system in zebrafish. CRISPR/Cas9 system causes DNA double strand breaks, which can be repaired by non-homologous end joining or homology directed recombination. We achieved to knock-in exogenous genes into target genomic locus using donor vector harboring short homologous sequences. The donor vector was designed to have gRNA-recognition sites, homology arms, and the exogenous gene to be integrated. By introducing two gRNA-recognition sites in both side of the integrating gene, backbone sequences of donor vector can be eliminated. Furthermore, the precise genome modification was also occurred in germ cells, resulting in the establishment of knocked-in zebrafish line. This method enables efficient and precise genome modification in a homology-dependent manner, which should be able to be applied to other model organisms

Report

(4 results)
  • 2016 Annual Research Report   Final Research Report ( PDF )
  • 2015 Research-status Report
  • 2014 Research-status Report
  • Research Products

    (16 results)

All 2016 2015 2014

All Journal Article (7 results) (of which Peer Reviewed: 7 results,  Open Access: 3 results,  Acknowledgement Compliant: 5 results) Presentation (7 results) (of which Int'l Joint Research: 1 results) Book (2 results)

  • [Journal Article] Site-Specific Integration of Exogenous Genes Using Genome Editing Technologies in Zebrafish2016

    • Author(s)
      Atsuo Kawahara, Yu Hisano, Satoshi Ota, Kiyohito Taimatsu
    • Journal Title

      International Journal of Molecular Sciences

      Volume: 17 Issue: 5 Pages: 727-727

    • DOI

      10.3390/ijms17050727

    • Related Report
      2016 Annual Research Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Journal Article] Comprehensive analysis of sphingosine-1-phosphate receptor mutants during zebrafish embryogenesis.2015

    • Author(s)
      Hisano Y, Inoue A, Taimatsu K, Ota S, Ohga R, Kotani H, Muraki M, Aoki J, Kawahara A
    • Journal Title

      Genes Cells.

      Volume: 20 Issue: 8 Pages: 647-658

    • DOI

      10.1111/gtc.12259

    • Related Report
      2015 Research-status Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Maternal and Zygotic Sphingosine Kinase 2 Are Indispensable for Cardiac Development in Zebrafish.2015

    • Author(s)
      Hisano Y, Inoue A, Okudaira M, Taimatsu K, Matsumoto H, Kotani H, Ohga R, Aoki J, Kawahara A.
    • Journal Title

      J Biol Chem.

      Volume: 290 Issue: 24 Pages: 14841-14851

    • DOI

      10.1074/jbc.m114.634717

    • Related Report
      2015 Research-status Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.2015

    • Author(s)
      Hisano Y, Sakuma T, Nakade S, Ohga R, Ota S, Okamoto H, Yamamoto T, Kawahara A.
    • Journal Title

      Sci Rep.

      Volume: 5 Issue: 1 Pages: 8841-8841

    • DOI

      10.1038/srep08841

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Open Access
  • [Journal Article] Molecular and physiological functions of sphingosine 1-phosphate transporters2014

    • Author(s)
      Nishi, T., Kobayashi, N., Hisano, Y., Kawahara, A. and Yamaguchi, A
    • Journal Title

      Biochimica et Biophysica Acta

      Volume: 1841 Issue: 5 Pages: 759-765

    • DOI

      10.1016/j.bbalip.2013.07.012

    • Related Report
      2014 Research-status Report
    • Peer Reviewed
  • [Journal Article] Multiple genome modifications by the CRISPR/Cas9 system in zebrafish2014

    • Author(s)
      Satoshi Ota, Yu Hisano, Yohshiya Ikawa, Atsuo Kawahara
    • Journal Title

      Genes to Cells

      Volume: 19 Issue: 7 Pages: 555-564

    • DOI

      10.1111/gtc.12154

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Acknowledgement Compliant
  • [Journal Article] Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering2014

    • Author(s)
      Kimura, Y., Hisano, Y., Kawahara, A., and Higashijima, S.
    • Journal Title

      Scientific Reports

      Volume: 4 Issue: 1 Pages: 6545-6545

    • DOI

      10.1038/srep06545

    • Related Report
      2014 Research-status Report
    • Peer Reviewed / Open Access / Acknowledgement Compliant
  • [Presentation] Spatial proteomics analysis for identifying sphingosine 1-phosphate receptor- associated proteins on endothelial cells2016

    • Author(s)
      Hisano Y, Obinata H, Hla T.
    • Organizer
      19th International Vascular Biology Meeting
    • Place of Presentation
      ボストン
    • Year and Date
      2016-10-30
    • Related Report
      2016 Annual Research Report
    • Int'l Joint Research
  • [Presentation] Loss-of-function analysis of S1P signaling molecules in zebrafish.2015

    • Author(s)
      Kotani H, Hisano Y, Inoue A, Okudaira M, Ohga R, Okamoto H, Aoki H, Kawahara A.
    • Organizer
      6th International Conference on Phospholipase A2 and Lipid Mediators (PLM2015)
    • Place of Presentation
      東京 京王プラザホテル
    • Year and Date
      2015-02-10 – 2015-02-12
    • Related Report
      2014 Research-status Report
  • [Presentation] Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering2014

    • Author(s)
      Kimura Y, Hisano Y, Kawahara A, Higashijima S.
    • Organizer
      The 3rd “International Institute for Advanced Studies” Conference of Novel Developments on the Study of Life and Biological Systems Based on Genome Engineering and Imaging Science
    • Place of Presentation
      京都国際高等研究所
    • Year and Date
      2014-10-28 – 2014-10-29
    • Related Report
      2014 Research-status Report
  • [Presentation] Exogenous DNA integration into zebrafish genome without indel mutations at the junctions2014

    • Author(s)
      Hisano Y, Okamoto H, Kawahara A.
    • Organizer
      The 3rd “International Institute for Advanced Studies” Conference of Novel Developments on the Study of Life and Biological Systems Based on Genome Engineering and Imaging Science
    • Place of Presentation
      京都国際高等研究所
    • Year and Date
      2014-10-28 – 2014-10-29
    • Related Report
      2014 Research-status Report
  • [Presentation] CRISPR/Cas9 systemを利用した遺伝子挿入ゼブラフィッシュの作製2014

    • Author(s)
      久野悠、太田聡、岡本仁、川原敦雄
    • Organizer
      第4回ゲノム編集研究会
    • Place of Presentation
      広島国際会議場ダリア
    • Year and Date
      2014-10-06 – 2014-10-07
    • Related Report
      2014 Research-status Report
  • [Presentation] Precise genome-editing using CRISPR/Cas9 system2014

    • Author(s)
      Hisano Y, Ota S, Okamoto H, Kawahara A.
    • Organizer
      20th Japanese Medaka and Zebrafish Meeting
    • Place of Presentation
      東京 慶應義塾大学薬学部(芝共立キャンパス)
    • Year and Date
      2014-09-21
    • Related Report
      2014 Research-status Report
  • [Presentation] Efficient generation of knock-in transgenic zebrafish carrying reporter/driver genes by CRISPR/Cas9-mediated genome engineering2014

    • Author(s)
      Kimura Y, Hisano Y, Kawahara A, Higashijima S.
    • Organizer
      20th Japanese Medaka and Zebrafish Meeting
    • Place of Presentation
      東京 慶應義塾大学薬学部(芝共立キャンパス)
    • Year and Date
      2014-09-20 – 2014-09-21
    • Related Report
      2014 Research-status Report
  • [Book] 実験医学「新しい創薬標的としての脂質メディエーター分泌輸送体」2016

    • Author(s)
      西毅、久野悠、小林直木、山口明人
    • Total Pages
      6
    • Publisher
      羊土社
    • Related Report
      2016 Annual Research Report
  • [Book] 今すぐ始めるゲノム編集-TALEN&CRISPR/Cas9の必須知識と実験プロトコール2014

    • Author(s)
      木下政人、安齋賢、久野悠、川原敦雄
    • Total Pages
      202
    • Publisher
      羊土社
    • Related Report
      2014 Research-status Report

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Published: 2014-04-04   Modified: 2018-03-22  

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