階層的ゲノム・エピゲノム編集法を用いた疾患発症モデリング技術の開発

2016 Fiscal Year Research-status Report

• Project/Area Number: 16K18478
• Research Institution: Hiroshima University
• Principal Investigator: 佐久間 哲史 広島大学, 理学研究科, 特任講師 (90711143)
• Project Period (FY): 2016-04-01 – 2018-03-31
• Keywords: がん / ゲノム編集 / エピゲノム編集 / マイクロホモロジー媒介性末端結合 / MMEJ

Outline of Annual Research Achievements

TALENやCRISPR-Cas9を用いたゲノム編集技術、ならびにゲノム編集から派生した転写制御／エピゲノム改変技術の開発は、近年世界中で競って進められ、疾患モデル細胞の作製にも広く利用されつつある。一方で、がんの原因となる変異やエピゲノム異常は、階層的に蓄積されていくことが知られている。そこで本研究では、申請者らが確立したPlatinum TALEプラットフォームやマルチガイドCRISPR-Cas9システム、簡便・正確・高効率な遺伝子ノックインを可能にするPITChシステム等を駆使し、ゲノム・エピゲノム編集ツールを単一細胞内で段階的に作用させることで、ヒト疾患の階層的発症メカニズムのモデル化を実現することを目的とする。
本年度は、階層的ゲノム編集技術の基盤整備のため、培養細胞におけるPITCh法の効率化と、PITCh法による遺伝子ノックインの自動設計ツールの確立を進めた。PITCh法は、相同組換えに依存しない遺伝子ノックイン法であり、マイクロホモロジーと呼ばれる数十bp程度の短い相同配列を利用して、マイクロホモロジー媒介性末端結合（MMEJ）に基づいて外来遺伝子を挿入することが可能である。本研究では、MMEJに関わる因子のスクリーニングを実施し、特定の因子の過剰発現によって、PITCh法による遺伝子ノックインの効率が上昇することを明らかにした。更に、PITCh法に利用するsgRNAおよびマイクロホモロジー配列の設計を自動化するウェブツールを開発することで、ハイスループットなPITCh法の設計を可能にした。

Current Status of Research Progress

2: Research has progressed on the whole more than it was originally planned.

Reason

研究概要に記載した培養細胞におけるPITCh法の効率化の研究成果を、共同責任著者および共同筆頭著者の一人としてBMC Genomics誌に論文発表した。また、同じく研究概要に記載したPITCh法による遺伝子ノックインの自動設計ツールの開発についても、共同責任著者の一人としてBioengineered誌に論文がin pressの状態となっている。これらの成果は、次年度の研究開発の基盤となるものであり、現時点において研究開発が順調に進展していると考えられる。

Strategy for Future Research Activity

次年度は、本年度に確立した技術基盤を更に拡大すると共に、階層的ゲノム・エピゲノム編集への展開を進める予定である。具体的には、PITCh法による遺伝子ノックインの更なる効率化と、PITCh法によってノックインできるドナーDNAのキャパシティーの拡大を予定している。また、Platinum TALEプラットフォームやマルチガイドCRISPR-Cas9システムをエピゲノム編集技術に応用する試みを既に開始しており、本成果についての論文化を予定している。これらの改良型技術基盤を利用し、複数のゲノム編集およびエピゲノム編集を同時または段階的に作用させ、階層的ゲノム・エピゲノム編集を実現する計画である。

Research Products

• [Journal Article] Detailed analysis of targeted gene mutations caused by the Platinum-Fungal TALENs in Aspergillus oryzae RIB40 strain and a ligD disruptant (2017)
  • Author(s): Mizutani O, Arazoe T, Toshida K, Hayashi R, Ohsato S, Sakuma T, Yamamoto T, Kuwata S, Yamada O
  • Journal Title: Journal of Bioscience and Bioengineering Volume: 123 Pages: 287-293
  • DOI: 10.1016/j.jbiosc.2016.09.014
  • Peer Reviewed
• [Journal Article] Cas9, Cpf1 and C2c1/2/3-What's next? (2017)
  • Author(s): Nakade S, Yamamoto T, Sakuma T
  • Journal Title: Bioengineered Volume: - Pages: -
  • DOI: 10.1080/21655979.2017.1282018
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Germ cell regeneration-mediated, enhanced mutagenesis in the ascidian Ciona intestinalis reveals flexible germ cell formation from different somatic cells (2017)
  • Author(s): Yoshida K, Hozumi A, Treen N, Sakuma T, Yamamoto T, Shirae-Kurabayashi M, Sasakura Y
  • Journal Title: Developmental Biology Volume: 423 Pages: 111-125
  • DOI: 10.1016/j.ydbio.2017.01.022
  • Peer Reviewed
• [Journal Article] Hox-mediated endodermal identity patterns the pharyngeal muscle formation in the chordate pharynx (2017)
  • Author(s): Yoshida K, Nakahata A, Treen N, Sakuma T, Yamamoto T, Sasakura Y
  • Journal Title: Development Volume: - Pages: -
  • DOI: 10.1242/dev.144436
  • Peer Reviewed
• [Journal Article] Highly efficient biallelic genome editing of human ES/iPS cells using a CRISPR/Cas9 or TALEN system (2017)
  • Author(s): Takayama K, Igai K, Hagihara Y, Hashimoto R, Hanawa M, Sakuma T, Tachibana M, Sakurai F, Yamamoto T, Mizuguchi H
  • Journal Title: Nucleic Acids Research Volume: - Pages: -
  • DOI: 10.1093/nar/gkx130
  • Peer Reviewed / Open Access
• [Journal Article] Establishment of expanded and streamlined pipeline of PITCh knock-in - a web-based design tool for MMEJ-mediated gene knock-in, PITCh designer, and the variations of PITCh, PITCh-TG and PITCh-KIKO (2017)
  • Author(s): Nakamae K, Nishimura Y, Takenaga M, Nakade S, Sakamoto N, Ide H, Sakuma T, Yamamoto T
  • Journal Title: Bioengineered Volume: - Pages: -
  • DOI: 10.1080/21655979.2017.1313645
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Culture time of vitrified/warmed zygotes before microinjection affects the production efficiency of CRISPR-Cas9-mediated knock-in mice (2017)
  • Author(s): Nakagawa Y, Sakuma T, Nishimichi N, Yokosaki Y, Takeo T, Nakagata N, Yamamoto T
  • Journal Title: Biology Open Volume: - Pages: -
  • DOI: 10.1242/bio.025122
  • Peer Reviewed / Open Access
• [Journal Article] Systematic Cellular Disease Models Reveal Synergistic Interaction of Trisomy 21 and GATA1 Mutations in Hematopoietic Abnormalities (2016)
  • Author(s): Banno K, Omori S, Hirata K, Nawa N, Nakagawa N, Nishimura K, Ohtaka M, Nakanishi M, Sakuma T, Yamamoto T, Toki T, Ito E, Yamamoto T, Kokubu C, Takeda J, Taniguchi H, Arahori H, Wada K, Kitabatake Y, Ozono K
  • Journal Title: Cell Reports Volume: 15 Pages: 1228-1241
  • DOI: 10.1016/j.celrep.2016.04.031
  • Peer Reviewed / Open Access
• [Journal Article] Single-Cell-State Culture of Human Pluripotent Stem Cells Increases Transfection Efficiency (2016)
  • Author(s): Nii T, Kohara H, Marumoto T, Sakuma T, Yamamoto T, Tani K
  • Journal Title: Bioresearch Open Access Volume: 5 Pages: 127-136
  • DOI: 10.1089/biores.2016.0009
  • Peer Reviewed / Open Access
• [Journal Article] Functional consequence of fibulin-4 missense mutations associated with vascular and skeletal abnormalities and cutis laxa (2016)
  • Author(s): Sasaki T, Hanisch FG, Deutzmann R, Sakai LY, Sakuma T, Miyamoto T, Yamamoto T, Hannappel E, Chu ML, Lanig H, von der Mark K
  • Journal Title: Matrix Biology Volume: 56 Pages: 132-149
  • DOI: 10.1016/j.matbio.2016.06.003
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Generation of a Nonhuman Primate Model of Severe Combined Immunodeficiency Using Highly Efficient Genome Editing (2016)
  • Author(s): Sato K, Oiwa R, Kumita W, Henry R, Sakuma T, Ito R, Nozu R, Inoue T, Katano I, Sato K, Okahara N, Okahara J, Shimizu Y, Yamamoto M, Hanazawa K, Kawakami T, Kametani Y, Suzuki R, Takahashi T, Weinstein EJ, Yamamoto T, Sakakibara Y, Habu S, Hata J, Okano H, Sasaki E
  • Journal Title: Cell Stem Cell Volume: 19 Pages: 127-138
  • DOI: 10.1016/j.stem.2016.06.003
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Ultra-superovulation for the CRISPR-Cas9-mediated production of gene-knockout, single-amino-acid-substituted, and floxed mice (2016)
  • Author(s): Nakagawa Y, Sakuma T, Nishimichi N, Yokosaki Y, Yanaka N, Takeo T, Nakagata N, Yamamoto T
  • Journal Title: Biology Open Volume: 5 Pages: 1142-1148
  • DOI: 10.1242/bio.019349
  • Peer Reviewed / Open Access
• [Journal Article] Highly multiplexed CRISPR-Cas9-nuclease and Cas9-nickase vectors for inactivation of hepatitis B virus (2016)
  • Author(s): Sakuma T, Masaki K, Abe-Chayama H, Mochida K, Yamamoto T, Chayama K
  • Journal Title: Genes to Cells Volume: 21 Pages: 1253-1262
  • DOI: 10.1111/gtc.12437
  • Peer Reviewed
• [Journal Article] Non-RVD mutations that enhance the dynamics of the TAL repeat array along the superhelical axis improve TALEN genome editing efficacy (2016)
  • Author(s): Tochio N, Umehara K, Uewaki JI, Flechsig H, Kondo M, Dewa T, Sakuma T, Yamamoto T, Saitoh T, Togashi Y, Tate SI
  • Journal Title: Scientific Reports Volume: 6 Pages: 37887
  • DOI: 10.1038/srep37887
  • Peer Reviewed / Open Access
• [Journal Article] C-Type Lectin Receptor DCAR Recognizes Mycobacterial Phosphatidyl-Inositol Mannosides to Promote a Th1 Response during Infection (2016)
  • Author(s): Toyonaga K, Torigoe S, Motomura Y, Kamichi T, Hayashi JM, Morita YS, Noguchi N, Chuma Y, Kiyohara H, Matsuo K, Tanaka H, Nakagawa Y, Sakuma T, Ohmuraya M, Yamamoto T, Umemura M, Matsuzaki G, Yoshikai Y, Yano I, Miyamoto T, Yamasaki S
  • Journal Title: Immunity Volume: 45 Pages: 1245-1257
  • DOI: 10.1016/j.immuni.2016.10.012
  • Peer Reviewed
• [Journal Article] Gene cassette knock-in in mammalian cells and zygotes by enhanced MMEJ (2016)
  • Author(s): Aida T, Nakade S, Sakuma T, Izu Y, Oishi A, Mochida K, Ishikubo H, Usami T, Aizawa H, Yamamoto T, Tanaka K
  • Journal Title: BMC Genomics Volume: 17 Pages: 979
  • DOI: 10.1186/s12864-016-3331-9
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] Transcriptional regulation of a horizontally transferred gene from bacterium to chordate (2016)
  • Author(s): Sasakura Y, Ogura Y, Treen N, Yokomori R, Park SJ, Nakai K, Saiga H, Sakuma T, Yamamoto T, Fujiwara S, Yoshida K
  • Journal Title: Proceedings of the Royal Society B: Biological Sciences Volume: 283 Pages: -
  • DOI: 10.1098/rspb.2016.1712
  • Peer Reviewed
• [Presentation] Recent Development and Application of Genome Editing Tools and Methods (2017)
  • Author(s): Tetsushi Sakuma
  • Organizer: The 20th US-Japan Cellular and Gene Therapy Conference, CRISPR/Cas9 Gene Editing In Vivo
  • Place of Presentation: Silver Spring, MD, USA
  • Year and Date: 2017-03-09 – 2017-03-09
  • Int'l Joint Research / Invited
• [Presentation] Highly practical gene knock-in in mammalian cells and zygotes with MMEJ-dependent strategy (2017)
  • Author(s): Tetsushi Sakuma
  • Organizer: 2nd Annual Genome Editing & Engineering Conference
  • Place of Presentation: San Diego, CA, USA
  • Year and Date: 2017-02-06 – 2017-02-07
  • Int'l Joint Research / Invited
• [Presentation] Advanced method to generate gene cassette knock-in and floxed mice using the PITCh system facilitated with MMEJ enhancer (2017)
  • Author(s): Tetsushi Sakuma, Tomomi Aida, Shota Nakade, Takashi Yamamoto, Kohichi Tanaka
  • Organizer: Keystone Symposia, Precision Genome Engineering
  • Place of Presentation: Breckenridge, CO, USA
  • Year and Date: 2017-01-08 – 2017-01-12
  • Int'l Joint Research
• [Presentation] ゲノム編集の最新技術 (2016)
  • Author(s): 佐久間哲史
  • Organizer: Skeletal Science Retreat 骨代謝スクール
  • Place of Presentation: 熱海
  • Year and Date: 2016-11-26 – 2016-11-27
  • Invited
• [Presentation] Current advances and future prospects of genome editing technology (2016)
  • Author(s): Tetsushi Sakuma
  • Organizer: 10th International Symposium on Nanomedicine (ISNM2016)
  • Place of Presentation: つくば
  • Year and Date: 2016-11-24 – 2016-11-26
  • Int'l Joint Research / Invited
• [Presentation] ゲノム編集の最新方法論 (2016)
  • Author(s): 佐久間哲史
  • Organizer: DNA鑑定学会第9回大会
  • Place of Presentation: 東京
  • Year and Date: 2016-11-10 – 2016-11-11
  • Invited
• [Presentation] ゲノム編集の最新方法論 (2016)
  • Author(s): 佐久間哲史
  • Organizer: 第68回日本生物工学会大会
  • Place of Presentation: 富山
  • Year and Date: 2016-09-28 – 2016-09-30
  • Invited
• [Presentation] ゲノム編集の基礎と最新動向 (2016)
  • Author(s): 佐久間哲史
  • Organizer: 日本ゲノム編集学会第1回大会
  • Place of Presentation: 広島
  • Year and Date: 2016-09-06 – 2016-09-07
• [Presentation] ゲノム編集による遺伝子改変の最前線 (2016)
  • Author(s): 佐久間哲史
  • Organizer: 第58回歯科基礎医学会学術大会
  • Place of Presentation: 札幌
  • Year and Date: 2016-08-24 – 2016-08-26
  • Invited
• [Presentation] Highly practical gene cassette knock-in in mammalian cells and zygotes mediated by MMEJ (2016)
  • Author(s): Tetsushi Sakuma, Tomomi Aida, Shota Nakade, Kohichi Tanaka, Takashi Yamamoto
  • Organizer: Cold Spring Harbor Laboratory Meeting, Genome Engineering: The CRISPR-Cas Revolution
  • Place of Presentation: Cold Spring Harbor, NY, USA
  • Year and Date: 2016-08-17 – 2016-08-20
  • Int'l Joint Research
• [Presentation] MMEJ-directed gene cassette knock-in in mammalian cells and zygotes (2016)
  • Author(s): Tetsushi Sakuma, Shota Nakade, Tomomi Aida, Kohichi Tanaka, Takashi Yamamoto
  • Organizer: FASEB Science Research Conference, Genome Engineering - Cutting-Edge Research and Applications
  • Place of Presentation: Lisbon, Portugal
  • Year and Date: 2016-06-05 – 2016-06-09
  • Int'l Joint Research
• [Book] In Vitro Mutagenesis, Methods in Molecular Biology (2017)
  • Author(s): Sakuma T, Sakamoto T, Yamamoto T
  • Total Pages: 16
  • Publisher: Springer