次世代染色体工学の確立

2015 Fiscal Year Annual Research Report

• Project/Area Number: 26670165
• Research Institution: Institute of Physical and Chemical Research
• Principal Investigator: 内匠 透 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
• Keywords: ゲノム編集技術 / コピー数多型 / 染色体工学 / ES細胞

Outline of Annual Research Achievements

コピー数多型（CNV）は、ヒトの多様性のみならず、癌や精神疾患などの様々な疾患の原因と考えられ、それらの病態解明、治療法の開発のためにはCNVモデルの開発が必須である。本研究では、TALENやCRISPR/Casといった最新のゲノム編集技術と組み替え用セレクションマーカーを組み合わせ、簡便、高速、高効率を兼ね備えた「次世代」染色体工学の確立を目指す。
CRISPR/Casは、細菌の適応免疫システムで、その構成因子は、Casヌクレアーゼ及びCRISPR-RNA (crRNA)、trans-activating crRNA(trancrRNAs) とよばれるnon-coding RNAsである。標的DNA領域に相補的なcrRNAが標的DNAに結合し、それを目印としたCasヌクレアーゼが標的領域を破壊する。CRISPR/Casは、非常に高効率に二重鎖切断を導入でき、また複数の変異を一度に導入できる。上記TALEN同様に、ベクターにCNV両末端をターゲットとして組み込む事でヒト染色体15q25.2-25.3の480Kb欠失をえることに成功した。CRISPRベクターと上記TALEN法の際に用いた相同組み替え用セレクションマーカーを組み合わせたものを用いて、フィーダーを必要としないmES細胞株（EBRTcH3）で効率を確認した。5’アーム、3’アームそれぞれで複数の候補選び、ES細胞に導入して、surveyorアッセイで調べた。二本鎖切断の確認できたもの、かつoff-targetの可能性が少ないものの組み合わせを用いて、ES細胞に導入し、欠失をPCRでスクリーニングし、サザンブロティング法で確認した。昨年度までに確立したこれらの方法論の効率を挙げることに成功した。
以上のように、CRISPR/Cas9を用いた「次世代」染色体工学の開発に成功した。

Research Products

• [Journal Article] Sequence features asscoaited with the clevage 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 Pages: 19675
  • DOI: 10.1038/srep19675
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Neuroanatomical phenotypes are consistent with autism-like beahvioral phenotypes in the 15q11-13 duplication mouse model (2015)
  • Author(s): Ellegood J, Nakai N, Nakatani J, Henkelman M, Takumi T, Lerch J
  • Journal Title: Autism Res Volume: 8 Pages: 545-555
  • DOI: 10.1002/aur.1469
  • Peer Reviewed / Int'l Joint Research / 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, Suzuki H
  • Journal Title: J Nippon Med Sch Volume: 82 Pages: 92-99
  • DOI: 10.1272/jnms.82.92
  • 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 Pages: 4559-4572
  • DOI: 10.1093/hmg/ddv187
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Cerebellar associative sensory learning defects in the 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: 4 Pages: e06085
  • 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 Pages: 976-984
  • DOI: 10.1007/s11427-015-4891-7
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Trasncriptome 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 – 2015-12-08
  • 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 – 2015-11-08
  • 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 – 2015-10-10
  • 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 – 2015-09-13
  • 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 – 2015-07-31
• [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 – 2015-07-31
• [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 – 2015-05-12
  • Int'l Joint Research