Evolutionary developmental study by using genome-editing technologies for in vivo reconstruction of gene networks

• Project/Area Number: 25650080
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Developmental biology
• Research Institution: The University of Tokushima
• Principal Investigator: Mito Taro 徳島大学, ソシオテクノサイエンス研究部, 助教 (80322254)
• Project Period (FY): 2013-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥4,030,000 (Direct Cost: ¥3,100,000、Indirect Cost: ¥930,000); Fiscal Year 2015: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2014: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2013: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000)
• Keywords: ゲノム編集 / 昆虫 / 発生 / 転写調節

Outline of Final Research Achievements

It remains unclear how the gene networks regulating development in multicellular organisms evolved. The experimental variation of such evolutionary processes has been possible only in limited cases. In the present study, we aimed to establish a method for in vivo reconstruction of gene networks using genome-editing technologies. We used a model insect, Gryllus bimaculatus, and achieved targeted gene knockout and knockin with CRISPR/Cas9 system. In addition, we produced genetic strains expressing a marker gene in a similar pattern of a targeted gene via enhancer trap. Thus, we established a technical basis for experimental modification of gene networks by modifying cis-regulatory sequences.

Research Products

• [Journal Article] Knockout crickets for the study of learning and memory: Dopamine receptor Dop1 
mediates aversive but not appetitive reinforcement in crickets (2015)
  • Author(s): Awata H, Watanabe T, Hamanaka Y, Mito T, Noji S, Mizunami M.
  • Journal Title: Scientific Reports Volume: 5 Issue: 1 Pages: 15885-15885
  • DOI: 10.1038/srep15885
  • NAID: 120006875677
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Gene knockout by targeted mutagenesis in a hemimetabolous insect, the two-spotted cricket Gryllus bimaculatus, using TALENs (2014)
  • Author(s): T. Watanabe, S. Noji and T. Mito
  • Journal Title: Methods Volume: 69 Issue: 1 Pages: 17-21
  • DOI: 10.1016/j.ymeth.2014.05.006
  • Peer Reviewed / Open Access
• [Journal Article] Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems (2014)
  • Author(s): A. Yasue, S. N. Mitsui, T. Watanabe, T. Sakuma, S. Oyadomari, T. Yamamoto, S. Noji, T. Mito and E. Tanaka
  • Journal Title: Sci Rep Volume: 4 Issue: 1 Pages: 5705-5705
  • DOI: 10.1038/srep05705
  • Peer Reviewed / Open Access
• [Journal Article] コオロギにおけるZFN, TALEN, CRISPR/Cas9を用いた遺伝子改変 (2014)
  • Author(s): 渡辺崇人，三戸太郎，大内淑代，野地澄晴
  • Journal Title: 実験医学別冊 Volume: -
• [Journal Article] ZFN/TALENを用いたコオロギの遺伝子ノックアウト (2013)
  • Author(s): 渡辺崇人，三戸太郎，野地澄晴
  • Journal Title: 細胞工学 Volume: 32 Pages: 543-549
• [Presentation] CRISPR/Cas9システムを用いた遺伝子ノックアウトによるコオロギ胚発生制御メカニズムの解析 (2015)
  • Author(s): 友成さゆり、川本晃平、松岡佑児、渡辺崇人、石丸善康、野地澄晴、三戸太郎
  • Organizer: 第38回日本分子生物学会
  • Place of Presentation: 兵庫県神戸市、神戸国際展示場
  • Year and Date: 2015-12-01
• [Presentation] CRISPR/Casシステムを用いた昆虫ゲノム改変技術の開発 (2015)
  • Author(s): 三戸太郎
  • Organizer: 第86回日本動物学会大会
  • Place of Presentation: 新潟県新潟市、新潟コンベンションセンター
  • Year and Date: 2015-09-18
  • Invited
• [Presentation] Functional analysis of a Hox gene, abdominal-A, using CRISPR/Cas9 system in the cricket Gryllus bimaculatus (2015)
  • Author(s): Yuji Matsuoka, Takahito Watanabe, Sayuri Tomonari, Sumihare Noji, Taro Mito
  • Organizer: International Tribolium Meeting
  • Place of Presentation: 米国、カリフォルニア州バークレー
  • Year and Date: 2015-08-04
  • Int'l Joint Research
• [Presentation] Genome sequencing and annotation of the cricket Gryllus bimaculatus, a hemimetabolous insect model (2015)
  • Author(s): Taro Mito, Takehiko Itoh, Hiroya Morimoto, Ray Kajitani, Atsushi Toyoda, Sayuri Tomonari, Masao Fuketa, Takahito Watanabe, Yuji Matsuoka, Sumihare Noji
  • Organizer: Ninth Annual Arthropod Genomics Symposium
  • Place of Presentation: 米国、カンザス州マンハッタン
  • Year and Date: 2015-06-17
  • Int'l Joint Research
• [Presentation] Genome editing in the two-spotted cricket, Gryllus bimaculatus, using CRISPR/Cas9 system (2015)
  • Author(s): Takahito Watanabe, Yuji Matsuoka, Sayuri Tomonari, Chinami Kurita, Sumihare Noji, Taro Mito
  • Organizer: Insect Genetic Technologies Workshop
  • Place of Presentation: 米国、カンザス州マンハッタン
  • Year and Date: 2015-06-17
  • Int'l Joint Research
• [Presentation] フタホシコオロギにおけるCRISPR/Cas9システムを用いたHox遺伝子abdominal-Aの機能解析 (2015)
  • Author(s): 松岡佑児，渡辺崇人，栗田千波，友成さゆり，野地澄晴，三戸太郎
  • Organizer: 第48回日本発生生物学会
  • Place of Presentation: 茨城県つくば市、つくば国際会議場
  • Year and Date: 2015-06-02
• [Presentation] Whole-genome sequencing and targeted genome editing in the cricket G. bimaculatus (2014)
  • Author(s): Taro Mito
  • Organizer: Hokkaido Neuroethology Workshops 2014
  • Place of Presentation: 北海道大学（北海道札幌市）
  • Year and Date: 2014-06-27
  • Invited
• [Presentation] In vivo study of Msx1 gene in mice using CRISPR/Cas system (2014)
  • Author(s): Silvia Mitsui, Akihiro Yasue, Issei Imoto, Seiichi Oyadomari, Sumihare Noji, Taro Mito, Eiji Tanaka
  • Organizer: 第47回日本発生生物学会
  • Place of Presentation: WINC AICHI（愛知県名古屋市）
  • Year and Date: 2014-05-27 – 2014-05-30
• [Presentation] Functions of Polycomb group gene in regulation of Hox gene expression in a primitive mode of insect embryogenesis in the cricket Gryllus bimaculatus (2014)
  • Author(s): Yuji Matsuoka, Tetsuya Bando, Takahito Watanabe, Sumihare Noji, Taro Mito
  • Organizer: 第47回日本発生生物学会
  • Place of Presentation: WINC AICHI（愛知県名古屋市）
  • Year and Date: 2014-05-27 – 2014-05-30
• [Presentation] Targeted gene disruption in the cricket, Gryllus bimaculatus, using CRISPR/Cas9 system (2014)
  • Author(s): Takahito Watanabe, Yuji Matsuoka, Taro Mito, Sumihare Noji
  • Organizer: 第47回日本発生生物学会
  • Place of Presentation: WINC AICHI（愛知県名古屋市）
  • Year and Date: 2014-05-27 – 2014-05-30
• [Presentation] Genome editing in a cricket using a CRISPR/Cas system (2014)
  • Author(s): Taro Mito, Takahito Watanabe, Sumihare Noji
  • Organizer: 1st Asian Invertebrate Immunity Symposium
  • Place of Presentation: Pusan National University (Busan, Korea)
  • Invited
• [Presentation] CRISPR/Casシステムを用いたフタホシコオロギにおけるゲノム編集 (2014)
  • Author(s): 三戸太郎・渡辺崇人・野地澄晴
  • Organizer: 第５８回 日本応用動物昆虫学会大会（昆虫ホルモン談話会）
  • Place of Presentation: 高知大学（高知県高知市）
  • Invited
• [Presentation] Targeted genome modifications using ZFNs and TALENs in the cricket Gryllus bimaculatus (2013)
  • Author(s): Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, Taro Nakamura, Taro Mito, Takashi Yamamoto, Sumihare Noji
  • Organizer: 第46回日本発生生物学会年会
  • Place of Presentation: くにびきメッセ（島根県松江市）
• [Presentation] Targeted genome modifications in the cricket, Gryllus bimaculatus (2013)
  • Author(s): Takahito Watanabe, Hiroshi Ochiai, Tetsushi Sakuma, Satoshi Ishihara, Taro Nakamura, Takashi Yamamoto, Sumihare Noji, and Taro Mito
  • Organizer: Conference of Transposition & Genome Engineering 2013
  • Place of Presentation: Danubius Thermal Hotel Margitsziget (Budapest, Hungary)
• [Presentation] ゲノム編集技術によるノックアウトコオロギの作製 (2013)
  • Author(s): 渡辺崇人，松岡佑児，石原聡，山本卓，野地澄晴，三戸太郎
  • Organizer: 第１５回 日本進化学会大会
  • Place of Presentation: 筑波大学（茨城県つくば市）
  • Invited
• [Presentation] ゲノム編集技術によるノックアウトコオロギの作製 (2013)
  • Author(s): 三戸太郎，渡辺崇人，松岡佑児，山本卓，野地澄晴
  • Organizer: 第36回日本分子生物学会年会
  • Place of Presentation: 神戸ポートアイランド（兵庫県神戸市））
  • Invited
• [Book] 進化するゲノム編集技術 (2015)
  • Author(s): 渡辺崇人, 松岡佑児, 野地澄晴, 三戸太郎
  • Total Pages: 341
  • Publisher: エヌ・ティー・エス
• [Book] TALENs: Methods and Protocols (2015)
  • Author(s): Watanabe T, Noji S, Mito T
  • Total Pages: 272
  • Publisher: Springer
• [Book] 実験医学別冊 今すぐ始めるゲノム編集（山本卓編）第１０章 コオロギにおけるZFN,TALEN,CRISPR/Cas9を用いた遺伝子改変 (2014)
  • Author(s): 渡辺崇人，三戸太郎，大内淑代，野地澄晴
  • Total Pages: 205
  • Publisher: 羊土社