Establishment of next generation chromosome engineering

• Project/Area Number: 26670165
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Pathological medical chemistry
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: Takumi Toru 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, シニアチームリーダー (00222092)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,770,000 (Direct Cost: ¥2,900,000、Indirect Cost: ¥870,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: コピー数多型 / 染色体工学 / ゲノム編集技術 / 自閉症 / ES細胞 / ゲノム編集 / CRISPR / TALEN

Outline of Final Research Achievements

In the CRISPR-Cas9 system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. The sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. We evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal region of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we demonstrated that the genomic contexts of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency.

Research Products

• [Journal Article] Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system (2016)
  • Author(s): Liu X, Homma A, Sayadi J, Yang S, Ohashi J, Takumi T
  • Journal Title: Sci Rep Volume: 6 Issue: 1 Pages: 19675-19675
  • DOI: 10.1038/srep19675
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Consistency between the neuroanatomical and behavioural phenotype in the 15q11-13 duplication mouse model (2015)
  • Author(s): Ellegood, J. Nakai, N., Nakatani, J., Henkelman, R. M., Takumi, T. and Lerch, J.
  • Journal Title: Autism Res. Volume: in press Issue: 5 Pages: 545-555
  • DOI: 10.1002/aur.1469
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Altered Microglia in the Amygdala Are Involved in Anxiety-related Behaviors of a Copy Number Variation Mouse Model of Autism (2015)
  • Author(s): Shigemori, T., Sakai, A., Takumi, T., Itoh, Y. and Suzuki H.
  • Journal Title: Journal of Nippon Medical School Volume: 82 Issue: 2 Pages: 92-99
  • DOI: 10.1272/jnms.82.92
  • NAID: 130005068463
  • ISSN: 1345-4676, 1347-3409
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] MOdel mice for 15q11-13 duplication syndrome exhibit late-onset obesity and altered lipid metabolism (2015)
  • Author(s): Kishimoto R, Tamada K, Liu X, Okubo H, Ise S, Ohta H, Ruf S, Nakatani J, Kohno N, Spitz F, Takumi T
  • Journal Title: Hum Mol Genet Volume: 24 Issue: 16 Pages: 4559-4572
  • DOI: 10.1093/hmg/ddv187
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Cerebllar associative sensory learning defects in five mouse autism models (2015)
  • Author(s): Kloth AD, Badura A, Li A, Cherskov A, Connolly SG, Giovannucci A, Bangash MA, Grasselli G, Penagarikano O, Piochon C, Tsai PT, Geschwind DH, Hansel C, Sahin M, Takumi T, Worley PF, Wang SS
  • Journal Title: Elife Volume: 9
  • DOI: 10.7554/elife.06085
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Autism spectrum disorder model mice: Focus on copy number variation and epigenetics (2015)
  • Author(s): Nakai N, Otsuka S, Myung J, Takumi T
  • Journal Title: Sci China Life Sci Volume: 58 Issue: 10 Pages: 976-984
  • DOI: 10.1007/s11427-015-4891-7
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Transcriptome profiling of white adipose tissue in a mouse model for 15q duplication syndrome (2015)
  • Author(s): Liu X, Tamada K, Kishimoto R, Okubo H, Ise S, Ohta H, Ruf S, Nakatani J, Kohno N, Spitz F, Takumi T
  • Journal Title: Genom Data Volume: 5 Pages: 394-396
  • DOI: 10.1016/j.gdata.2015.06.035
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] Modeling neurodevelopmental disorders (2015)
  • Author(s): Toru Takumi
  • Organizer: EMBO Conference on Neural Development-Function and Dysfunction
  • Place of Presentation: Taipei, Taiwan
  • Year and Date: 2015-12-04
  • Int'l Joint Research / Invited
• [Presentation] Modeling Autism (2015)
  • Author(s): Toru Takumi
  • Organizer: Asia Pacific Regional IMFAR
  • Place of Presentation: Shanghai, China
  • Year and Date: 2015-11-06
  • Int'l Joint Research / Invited
• [Presentation] The development of copy number variation (CNV) database for autism spectrum disorder (ASD) (2015)
  • Author(s): Xiaoxi Liu, Jun Nomura, Yoko Yamaguchi, Toru Takumi
  • Organizer: American Society of Human Genetics Annual Meeting (ASHG) 2015
  • Place of Presentation: Baltimore, MD, USA
  • Year and Date: 2015-10-06
  • Int'l Joint Research
• [Presentation] Modeling Autism (2015)
  • Author(s): Toru Takumi
  • Organizer: 第５８回日本神経化学会大会
  • Place of Presentation: さいたま
  • Year and Date: 2015-09-11
  • Invited
• [Presentation] Modeling Autism (2015)
  • Author(s): Toru Takumi
  • Organizer: ISN Satellite Meeting 20015, The Frontiers in Neurodevelopmental Disorders (FiND)
  • Place of Presentation: Sydney, Australia
  • Year and Date: 2015-08-21
  • Int'l Joint Research / Invited
• [Presentation] Biological analysis of autism model mice that have 15q11-13 chromosome duplication (2015)
  • Author(s): Jin Nakatani, Natsu Koyama, Seiji Hitoshi, Toru Takumi, Akihiko Shiino, Shigehiro Morikawa, Toshiro Inubushi
  • Organizer: 第３８回日本神経科学大会
  • Place of Presentation: 神戸
  • Year and Date: 2015-07-28
• [Presentation] Cortical excitatory/inhibitory imbalance in a mouse model for human 15q11-13 duplication (2015)
  • Author(s): Nobuhiro Nakai, Yasuhito Watanabe, Yoshinobu Kawamura, Taisuke Miyazaki, Masahiko Watanabe, Shigeo Okabe, Masanobu Kano, Kouichi Hashimoto, Toru Takumi
  • Organizer: 第３８回日本神経科学大会
  • Place of Presentation: 神戸
  • Year and Date: 2015-07-28
• [Presentation] Increased ratio of cortical excitatory/inhibitory and modulation by serotonin in a mouse model for human 15q11-13 duplication seen in autism (2015)
  • Author(s): Nobuhiro Nakai, Yasuhito Watanabe, Yoshinobu Kawamura, Taisuke Miyazaki, Masahiko Watanabe, Masanobu Kano, Kouichi Hashimoto, Toru Takumi
  • Organizer: 2015 Bridging Biomedical Worlds Conference, From Neural Circuitry to Neurotechnology
  • Place of Presentation: Tokyo
  • Year and Date: 2015-05-11
  • Int'l Joint Research
• [Presentation] ゲノム編集技術による簡便迅速かつ高効率な次世代染色体工学の開発 (2014)
  • Author(s): 野村淳、佐久間哲史、神田暁史、岸本恵子、前田知花、外丸祐介、山本卓、内匠透
  • Organizer: 第３７回日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（横浜）
  • Year and Date: 2014-11-25 – 2014-11-27
  • Invited
• [Presentation] 次世代染色体工学 (2014)
  • Author(s): 内匠透
  • Organizer: 第８７回日本生化学会大会
  • Place of Presentation: 京都国際会館（京都）
  • Year and Date: 2014-10-15 – 2014-10-18
  • Invited
• [Book] 今すぐ始めるゲノム編集 (2014)
  • Author(s): 野村 淳、内匠 透
  • Total Pages: 205
  • Publisher: 羊土社