Development of easy-to-use genome editing protein synthesis system and one-step genetically modified animal production method

• Project/Area Number: 19K07696
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 50010:Tumor biology-related
• Research Institution: Keio University
• Principal Investigator: Onishi Nobuyuki 慶應義塾大学, 医学部(信濃町), 訪問研究員 (40534540)
• Project Period (FY): 2019-04-01 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2021: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2020: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000); Fiscal Year 2019: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
• Keywords: ゲノム編集 / ゲノム編集タンパク質 / 遺伝子改変動物 / 遺伝子改変マウス作製法 / In vivoエレクトロポレーション / Cas9タンパク質 / Transposaseタンパク質

Outline of Research at the Start

CRISPR/Cas9システムによるゲノム編集の技術革新は他に類を見ない。CRISPR/Cas9ヌクレアーゼの発現ベクターや精製タンパク質が一般に入手できるようになったことで、目的遺伝子ノックアウト細胞の作製が身近になり研究室レベルで頻繁に挑戦されるようになったが、研究室単位で遺伝子改変マウスの作製を行うにはまだまだハードルが高いのが現状である。申請者はこれまでに構築してきたpiggyBac/in vivoエレクトロポレーション法によるin vivo遺伝子発現システムとGONAD法を組み合わせることで迅速かつ簡便な遺伝子改変マウス作製法の樹立を目指す。

Outline of Final Research Achievements

In this study, we developed a method to express HypaCas9 protein, which is a high-precision Cas9, and gRNA at a time using an E. coli protein expression system and purify them as ribonucleoprotein (RNP). By optimizing the selection of E. coli strains, protein expression induction conditions, and E. coli disruption method, HypaCas9 protein could be purified in a single band. Specifically, BL21 (DE3) is used instead of Rosetta2 (DE3), which is commonly used in Cas9 protein synthesis, and expression is induced at 30 °C instead of low temperature. Contaminant proteins could be reduced by disrupting E. coli under mild conditions using freeze-thaw and lysozyme instead of sonication.

Academic Significance and Societal Importance of the Research Achievements

CRISPR/Cas9ヌクレアーゼの精製タンパク質が一般に入手できるようになったことで、目的遺伝子ノックアウト細胞の作製が身近になり研究室レベルで頻繁に挑戦されるようになったが、細胞一つ一つで切断された遺伝子配列の修復様式が異なるため、クローン化する必要がある場合は細胞株樹立までに長い期間を要する。ましてや研究室単位で遺伝子改変マウスの作製を行うにはまだまだハードルが高いのが現状である。本研究を進めていくことで、迅速かつ簡便に遺伝子改変マウスを作製することが可能となり、多様な疾患ならびに治療モデルの構築に挑戦していくことで、有効な治療法が無い疾患に対して新たな治療法開発に貢献することができる。

Research Products

• [Journal Article] Isolation and characterization of neural stem/progenitor cells in the subventricular zone of the naked mole-rat brain (2021)
  • Author(s): Yamamura Y, Kawamura Y, Oiwa Y, Oka K, Onishi N, Saya H, Miura K.
  • Journal Title: Inflamm Regen. Volume: 41 Issue: 1 Pages: 31-31
  • DOI: 10.1186/s41232-021-00182-7
  • Peer Reviewed / Open Access
• [Journal Article] Epigenetic suppression of SLFN11 in germinal center B-cells during B-cell development (2021)
  • Author(s): Moribe F, Nishikori M, Takashima T, Taniyama D, Onishi N, Arima H, Sasanuma H, Akagawa R, Elloumi F, Takeda S, Pommier Y, Morii E, Takaori-Kondo A, Murai J.
  • Journal Title: PLoS One. Volume: 16 Issue: 1 Pages: 0237554-0237554
  • DOI: 10.1371/journal.pone.0237554
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] 2-Nitroimidazoles induce mitochondrial stress and ferroptosis in glioma stem cells residing in a hypoxic niche (2020)
  • Author(s): Koike Naoyoshi、Kota Ryuichi、Naito Yoshiko、Hayakawa Noriyo、Matsuura Tomomi、Hishiki Takako、Onishi Nobuyuki、Fukada Junichi、Suematsu Makoto、Shigematsu Naoyuki、Saya Hideyuki、Sampetrean Oltea
  • Journal Title: Communications Biology Volume: 3 Issue: 1 Pages: 450-462
  • DOI: 10.1038/s42003-020-01165-z
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] The insulin-PI3K-Rac1 axis contributes to terminal adipocyte differentiation through regulation of actin cytoskeleton dynamics (2020)
  • Author(s): Kunitomi H, Oki Y, Onishi N, Kano K, Banno K, Aoki D, Saya H, Nobusue H
  • Journal Title: Genes to Cells Volume: 25 Issue: 3 Pages: 165-174
  • DOI: 10.1111/gtc.12747
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] IMP dehydrogenase-2 Drives Aberrant Nucleolar Activity and Promotes Tumorigenesis in Glioblastoma (2019)
  • Author(s): Kofuji S, Hirayama A, Eberhardt AO, Kawaguchi R, Sakamoto N, Kitahara S, Wolfe K, Lange L, Onishi N, Uematsu M, Saya H, Soga T, Grummt I, Bierhoff H, Sasaki AT.
  • Journal Title: Nat Cell Biol Volume: 21 Issue: 8 Pages: 1003-1014
  • DOI: 10.1038/s41556-019-0363-9
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Rock inhibition suppresses osteosarcoma tumorigenesis by inducing terminal adipocyte differentiation in chemoresistant stemlike cells (2019)
  • Author(s): 1.Takahashi N, Nobusue H, Shimizu T, Sugihara E, Yamaguchi-Iwai S, Onishi N, Kunitomi H, Kuroda T, Saya H
  • Journal Title: Cancer Research Volume: in press Issue: 12 Pages: 3088-3099
  • DOI: 10.1158/0008-5472.can-18-2693
  • Peer Reviewed / Open Access / Int'l Joint Research