Development of universal method for genome modification using zinc finger nuclease

• Project/Area Number: 24657156
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Developmental biology
• Research Institution: Hiroshima University
• Principal Investigator: YAMAMOTO Takashi 広島大学, 理学(系)研究科(研究院), 教授 (90244102)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2014: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2013: ¥1,170,000 (Direct Cost: ¥900,000、Indirect Cost: ¥270,000); Fiscal Year 2012: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
• Keywords: バイオテクノロジー / ゲノム編集 / 発生・分化 / 人工ヌクレアーゼ

Outline of Final Research Achievements

In this study, I tried to establish the method for gene modifications such as targeted gene knockout and gene knock-in in various animal species using artificial endonucleases (ZFN and TALEN). Using ZFN protein targeting sea urchin HesC gene generated by E. coli, I observed mutations in early stage of sea urchin embryo, indicating that microinjection of ZFN protein can efficiently introduce mutations in target gene compared to that of ZNF mRNA. Moreover, I established construction system for a highly active TALEN (Platinum TALEN) and demonstrated efficient targeted gene disruption using Platinum TALENs in various animal species such as roundworm (C. elegans), fruit fly (D. melanogaster), sea squirt (C. intestinalis), sea urchin (H. pulcherrimus), frog (Xenopus laevis) and newt (P. waltl)

Research Products

• [Journal Article] Unliganded thyroid hormone receptor alpha regulates developmental timing via gene repression in Xenopus tropicalis (2015)
  • Author(s): J. Choi, K. T. Suzuki, T. Sakuma, L. Shewade, T. Yamamoto and D. R. Buchholz
  • Journal Title: Endocrinology Volume: 156 Issue: 2 Pages: 735-44
  • DOI: 10.1210/en.2014-1554
  • Peer Reviewed / Open Access
• [Journal Article] Germ cell mutations of the ascidian Ciona intestinalis with TALE nucleases. (2014)
  • Author(s): Yoshida K, Treen N, Hozumi A, Sakuma T, Yamamoto T, Sasakura Y.
  • Journal Title: Genesis Volume: in press Issue: 5 Pages: 431-439
  • DOI: 10.1002/dvg.22770
  • Peer Reviewed
• [Journal Article] TALEN-induced gene knock out in Drosophila. (2014)
  • Author(s): Kondo T
  • Journal Title: Development Growth and Differentiation Volume: 56:1 Issue: 1 Pages: 86-91
  • DOI: 10.1111/dgd.12097
  • Peer Reviewed
• [Journal Article] Transcription activator-like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration. (2014)
  • Author(s): Hayashi T, Sakamoto K, Sakuma T, Yokotani N, Inoue T, Kawaguchi E, Agata K, Yamamoto T, Takeuchi T
  • Journal Title: Development, Growth and Differentiation Volume: 56 Issue: 1 Pages: 115-121
  • DOI: 10.1111/dgd.12103
  • Peer Reviewed / Open Access
• [Journal Article] Targeted mutagenesis in sea urchin embryos using TALENs. (2014)
  • Author(s): Hosoi S, Sakuma T, Sakamoto N and Yamamoto T
  • Journal Title: Dev Growth Differ Volume: 56 Issue: 1 Pages: 92-97
  • DOI: 10.1111/dgd.12099
  • Peer Reviewed
• [Journal Article] Targeted mutagenesis of multiple and paralogous genes in Xenopus laevis using two pairs of transcription activator-like effector nucleases (2014)
  • Author(s): Sakane, Y., Sakuma, T., Kashiwagi, K., Kashiwagi, A., Yamamoto, T. and Suzuki, K. T.
  • Journal Title: Dev Growth Differ Volume: 56(1) Issue: 1 Pages: 108-114
  • DOI: 10.1111/dgd.12105
  • Peer Reviewed / Open Access
• [Journal Article] Versatile strategy for isolating TALEN-mediated knockout mutants in Caenorhabditis elegans. (2014)
  • Author(s): Sugi T, Sakuma T, Ohtani T and Yamamoto T
  • Journal Title: Dev Growth Differ Volume: 56 Issue: 1 Pages: 78-85
  • DOI: 10.1111/dgd.12108
  • Peer Reviewed
• [Journal Article] Tissue-specific and ubiquitous gene knockouts in Ciona by electroporating TALENs provide new approaches to investigate gene functions (2014)
  • Author(s): Treen, N., Yoshida, K., Sakuma, T., Sasaki, H., Kawai, N., Yamamoto, T., Sasakura, Y.
  • Journal Title: Development Volume: 141 Issue: 2 Pages: 481-487
  • DOI: 10.1242/dev.099572
  • Peer Reviewed
• [Journal Article] TALEN-mediated single-base-pair editing identification of an intergenic mutation upstream of BUB1B as causative of PCS (MVA) syndrome. (2014)
  • Author(s): Ochiai H, Miyamoto T, Kanai A, Hosoba K, Sakuma T, Kudo Y, Asami K, Ogawa A, Watanabe A, Kajii T, Yamamoto T, Matsuura S.
  • Journal Title: Proc Natl Acad Sci U S A. Volume: 111(4) Issue: 4 Pages: 1461-6
  • DOI: 10.1073/pnas.1317008111
  • Peer Reviewed
• [Journal Article] Efficient TALEN construction and evaluation methods for human cell and animal applications. (2013)
  • Author(s): Sakuma T, Hosoi S, Woltjen K, Suzuki KI, Kashiwagi K, Wada H, Ochiai H, Miyamoto T, Kawai N, Sasakura Y, Matsuura S, Okada Y, Kawahara A, Hayashi S, Yamamoto T.
  • Journal Title: Genes to Cells Volume: (In press) Issue: 4 Pages: 315-326
  • DOI: 10.1111/gtc.12037
  • Peer Reviewed
• [Journal Article] Production of Sry knockout mouse using TALEN via oocyte injection. (2013)
  • Author(s): Kato T, Miyata K, Sonobe M, Yamashita S, Tamano M, Miura K, Kanai Y, Miyamoto S, Sakuma T, Yamamoto T, Inui M, Kikusui T, Asahara H, Takada S.
  • Journal Title: Scientific reports Volume: 3 Issue: 1 Pages: 3136-3136
  • DOI: 10.1038/srep03136
  • Peer Reviewed
• [Journal Article] Repeating pattern of non-RVD variations in DNA-binding modules enhances TALEN activity. (2013)
  • Author(s): Sakuma T, Ochiai H, Kaneko T, Mashimo T, Tokumasu D, Sakane Y, Suzuki K, Miyamoto T, Sakamoto N, Matsuura S, Yamamoto T
  • Journal Title: Sci Rep. Volume: 3 Issue: 1 Pages: 3379-3379
  • DOI: 10.1038/srep03379
  • Peer Reviewed
• [Journal Article] High efficiency TALENs enable FO functional analysis by targeted gene disruption in Xenopus laevis embryos. (2013)
  • Author(s): Suzuki KI, Isoyama Y, Kashiwagi K, Sakuma T, Ochiai H, Furuno N, Kashiwagi A and Yamamoto T.
  • Journal Title: Biology Open Volume: (In press) Issue: 5 Pages: 448-452
  • DOI: 10.1242/bio.20133855
  • Peer Reviewed
• [Journal Article] Non-transgenic genome modifications in a hemimetabolous insect using zinc-finger and TAL effector nucleases. (2012)
  • Author(s): Watanabe T, Ochiai H, Sakuma T, Horch HW, Hamaguchi N, Nakamura T, Bando T, Ohuchi H, Yamamoto T, Noji S, Mito T.
  • Journal Title: Nature Communications Volume: 3 Issue: 1 Pages: 1017-1017
  • DOI: 10.1038/ncomms2020
  • Peer Reviewed
• [Journal Article] Zinc-finger nuclease-mediated targeted insertion of reporter genes for quantitative imaging of gene expression in sea urchin embryos. (2012)
  • Author(s): Ochiai H, Sakamoto N, Fujita K, Nishikawa M, Suzuki K, Matsuura S, Miyamoto T, Sakuma T, Shibata T, Yamamoto T.
  • Journal Title: Proceedings of the National Academy of Sciences of the United States of America Volume: 109 Issue: 27 Pages: 10915-10920
  • DOI: 10.1073/pnas.1202768109
  • Peer Reviewed
• [Journal Article] Efficient targeted mutagenesis of the chordate Ciona intestinalis genome with zinc-finger nucleases. (2012)
  • Author(s): Kawai N, Ochiai H, Sakuma T, Yamada L, Sawada H, Yamamoto T, Sasakura Y.
  • Journal Title: Development, Growth & Differentiation Volume: 54 Issue: 5 Pages: 535-545
  • DOI: 10.1111/j.1440-169x.2012.01355.x
  • NAID: 10031136923
  • Peer Reviewed
• [Presentation] Genome editing using Platinum TALENs (2014)
  • Author(s): Sakuma T, Suzuki KI and Yamamoto T
  • Organizer: FASEB Science Research Conference
  • Place of Presentation: Nassau, Bahamas
  • Year and Date: 2014-06-22 – 2014-06-27
• [Presentation] ZFN法とTALEN法を用いたゲノム編集時代の到来 (2012)
  • Author(s): 山本卓
  • Organizer: 第35回日本分子生物学会
  • Place of Presentation: 福岡
  • Invited
• [Presentation] 人工ヌクレアーゼ（ZFNやTALEN）を用いたゲノム編集研究の現状と可能性 (2012)
  • Author(s): 山本卓
  • Organizer: 第5回DNA鑑定学会
  • Place of Presentation: 東京
  • Invited
• [Presentation] 人工ヌクレアーゼを基盤とした培養細胞および動物でのゲノム編集 (2012)
  • Author(s): 山本卓
  • Organizer: 日本動物学会 第83回大会
  • Place of Presentation: 大阪
  • Invited
• [Presentation] 人工ヌクレアーゼを基盤とする動物におけるゲノム編集 (2012)
  • Author(s): 山本卓
  • Organizer: 日本学術会議公開シンポジウム
  • Place of Presentation: 東京
  • Invited
• [Presentation] Platinum TALENの開発と様々な動物におけるゲノム編集
  • Author(s): 山本卓
  • Organizer: 理研シンポジウム「ゲノムデザイン技術と疾患モデル研究」
  • Place of Presentation: つくば
  • Invited
• [Presentation] 人工ヌクレアーゼを用いた両生類でのゲノム編集
  • Author(s): 山本卓
  • Organizer: 日本動物学会第84回大会シンポジウム「生物実験材料としてのネッタイツメガエルの長所と有用性」
  • Place of Presentation: 岡山
  • Invited
• [Presentation] 高活性型TALENの開発と哺乳類培養細胞および動物での標的遺伝子改変
  • Author(s): 山本卓
  • Organizer: 第65回日本生物工学会シンポジウム「次世代の植物バイオテクノロジー」
  • Place of Presentation: 広島
  • Invited
• [Presentation] ゲノム編集革命ー人工ヌクレアーゼを利用した遺伝子改変技術の開発
  • Author(s): 山本卓
  • Organizer: 平成25年度広島バイオフォーラム「ここまで進んだゲノム科学とその活用」
  • Place of Presentation: 広島
  • Invited
• [Presentation] ゲノム編集技術の限りない可能性
  • Author(s): 山本卓
  • Organizer: 染色体工学研究センター研究成果発表会
  • Place of Presentation: 東京
  • Invited
• [Remarks] 分子遺伝学研究室
  • URL: http://www.mls.sci.hiroshima-u.ac.jp/smg/index.html
• [Remarks] 分子遺伝学研究室
  • URL: http://www.mls.sci.hiroshima-u.ac.jp/smg/index.html
• [Remarks] ゲノム編集コンソーシアム
  • URL: http://www.mls.sci.hiroshima-u.ac.jp/smg/genome_editing/index.html
• [Remarks] 広島大学大学院理学研究科分子遺伝学研究室ホームページ
  • URL: http://www.mls.sci.hiroshima-u.ac.jp/smg/index.html
• [Remarks] ゲノム編集コンソーシアム
  • URL: http://www.mls.sci.hiroshima-u.ac.jp/smg/genome_editing/index.html
• [Patent(Industrial Property Rights)] 核酸挿入用ベクター (2013)
  • Inventor(s): 鈴木賢一、坂根祐人、佐久間哲史、山本卓
  • Industrial Property Rights Holder: 鈴木賢一、坂根祐人、佐久間哲史、山本卓
  • Industrial Property Rights Type: 特許
  • Filing Date: 2013-11-06