Evaluation of homologous recombination enhancers in genome editing

• Project/Area Number: 19K06472
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Review Section: Basic Section 42040:Laboratory animal science-related
• Research Institution: Kurume University (2020-2021); Keio University (2019)
• Principal Investigator: Shiozawa Seiji 久留米大学, 医学部, 准教授 (10447039)
• 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 / 相同組み換え / 発生工学

Outline of Research at the Start

我々は小型霊長類であるコモンマーモセットの胚性幹（ES）細胞を用いた研究から、この細胞ではヒトiPS細胞などに比べ、相同組み換えの効率が極めて高いことを見出した。さらに、トランスクリプトームデータの解析から、ヒトES/iPS細胞と比較し、複数の相同組み換え関連遺伝子の発現が高いことを見出した。これらの遺伝子をヒトiPS細胞に強制発現させたところ、相同組み換え効率の有意な上昇を認めた。本申請研究では、この効果をさらに実証するため、マウスES細胞及びマウス受精卵におけるこれらの遺伝子の有効性を検証することを目的とする。

Outline of Final Research Achievements

Genome editing technology is an excellent technology that allows genetic modification easily. However, compared to simple knockout, homologous recombination-mediated knock-in is still less efficient and remains a major challenge.
In this research subject, we have constructed a system that enables us to easily evaluate the knock-in efficiency in mouse ES cells and mouse embryo genome editing. Using this system, we found genes and small molecules that improve the knock-in efficiency.

Academic Significance and Societal Importance of the Research Achievements

ゲノム編集技術は簡便に遺伝子を改変できる優れた技術であり、生命科学研究のみならず医療や畜産分野などへの応用も期待されている。しかしながら、遺伝子改変の精度や効率の点では未だ改良の余地も多い。本研究課題では、ノックインによる遺伝子改変効率の改善を目的とし、まずその効率を簡便に評価する実験系を構築した。さらにこの実験系を用い、いくつかの遺伝子及び低分子化合物がノックイン効率を改善することを見出した。これにより、ゲノム編集技術の高度化に寄与することが期待される。また、今回確立した実験系を用いることで、さらに高い効果を持つ因子の発見に役立つことが期待される。

Research Products

• [Journal Article] ヒト及び非ヒト霊長類多能性幹細胞におけるナイーブ型多能性状態の獲得 (2021)
  • Author(s): 1)塩澤 誠司
  • Journal Title: 九州実験動物雑誌 Volume: 37 Pages: 3-7
• [Journal Article] Generation and validation of a common marmoset embryonic stem cell line ActiCre-B1 that ubiquitously expresses a tamoxifen-inducible Cre-driver (2021)
  • Author(s): Yoshimatsu Sho、Ohtsu Kanae、Sato Tsukika、Yamamoto Masafumi、Sasaki Erika、Shiozawa Seiji、Okano Hideyuki
  • Journal Title: Stem Cell Research Volume: 51 Pages: 102164-102164
  • DOI: 10.1016/j.scr.2021.102164
  • Peer Reviewed / Open Access
• [Journal Article] Generation of a male common marmoset embryonic stem cell line DSY127-BV8VT1 carrying double reporters specific for the germ cell linage using the CRISPR-Cas9 and PiggyBac transposase systems (2020)
  • Author(s): Yoshimatsu Sho、Sato Tsukika、Yamamoto Masafumi、Sasaki Erika、Nakajima Mayutaka、Nakamura Mari、Shiozawa Seiji、Noce Toshiaki、Okano Hideyuki
  • Journal Title: Stem Cell Research Volume: 44 Pages: 101740-101740
  • DOI: 10.1016/j.scr.2020.101740
  • Peer Reviewed
• [Journal Article] Primed to Naive-Like Conversion of the Common Marmoset Embryonic Stem Cells. (2020)
  • Author(s): Shiozawa S*, Nakajima M, Okahara J, Kuortaki Y, Kisa F, Yoshimatsu S, Nakamura M, Koya I, Yoshimura M, Sasagawa Y, Nikaido I, Sasaki E, Okano H.
  • Journal Title: Stem Cells Dev. Volume: 29 Issue: 12 Pages: 761-773
  • DOI: 10.1089/scd.2019.0259
  • Peer Reviewed
• [Journal Article] Direct Neuronal Reprogramming of Common Marmoset Fibroblasts by ASCL1, microRNA-9/9*, and microRNA-124 Overexpression (2020)
  • Author(s): Nemoto Akisa、Kobayashi Reona、Yoshimatsu Sho、Sato Yuta、Kondo Takahiro、Yoo Andrew S.、Shiozawa Seiji、Okano Hideyuki
  • Journal Title: Cells Volume: 10 Issue: 1 Pages: 6-6
  • DOI: 10.3390/cells10010006
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Evaluating the efficacy of small molecules for neural differentiation of common marmoset ESCs and iPSCs (2019)
  • Author(s): Yoshimatsu Sho、Nakamura Mari、Nakajima Mayutaka、Nemoto Akisa、Sato Tsukika、Sasaki Erika、Shiozawa Seiji、Okano Hideyuki
  • Journal Title: Neuroscience Research Volume: 19 Pages: 30262-7
  • DOI: 10.1016/j.neures.2019.09.005
  • Peer Reviewed
• [Journal Article] Robust and efficient knock-in in embryonic stem cells and early-stage embryos of the common marmoset using the CRISPR-Cas9 system (2019)
  • Author(s): Yoshimatsu Sho、Okahara Junko、Sone Takefumi、Takeda Yuta、Nakamura Mari、Sasaki Erika、Kishi Noriyuki、Shiozawa Seiji、Okano Hideyuki
  • Journal Title: Scientific Reports Volume: 9 Issue: 1 Pages: 1528-1528
  • DOI: 10.1038/s41598-018-37990-w
  • Peer Reviewed / Open Access
• [Journal Article] Establishment of induced pluripotent stem cells from common marmoset fibroblasts by RNA-based reprogramming (2019)
  • Author(s): Nakajima Mayutaka、Yoshimatsu Sho、Sato Tsukika、Nakamura Mari、Okahara Junko、Sasaki Erika、Shiozawa Seiji、Okano Hideyuki
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 515 Issue: 4 Pages: 593-599
  • DOI: 10.1016/j.bbrc.2019.05.175
  • Peer Reviewed / Open Access
• [Journal Article] Tau isoform expression and phosphorylation in marmoset brains (2019)
  • Author(s): Sharma Govinda、Huo Anni、Kimura Taeko、Shiozawa Seiji、Kobayashi Reona、Sahara Naruhiko、Ishibashi Minaka、Ishigaki Shinsuke、Saito Taro、Ando Kanae、Murayama Shigeo、Hasegawa Masato、Sobue Gen、Okano Hideyuki、Hisanaga Shin-ichi
  • Journal Title: Journal of Biological Chemistry Volume: 294 Issue: 30 Pages: 11433-11444
  • DOI: 10.1074/jbc.ra119.008415
  • Peer Reviewed / Open Access
• [Journal Article] A versatile toolbox for knock-in gene targeting based on the Multisite Gateway technology (2019)
  • Author(s): Yoshimatsu Sho、Sone Takefumi、Nakajima Mayutaka、Sato Tsukika、Okochi Ryotaro、Ishikawa Mitsuru、Nakamura Mari、Sasaki Erika、Shiozawa Seiji、Okano Hideyuki
  • Journal Title: PLOS ONE Volume: 14 Issue: 8 Pages: e0221164-e0221164
  • DOI: 10.1371/journal.pone.0221164
  • Peer Reviewed / Open Access
• [Journal Article] Pathological Progression Induced by the Frontotemporal Dementia-Associated R406W Tau Mutation in Patient-Derived iPSCs (2019)
  • Author(s): Nakamura Mari、Shiozawa Seiji、Tsuboi Daisuke、Amano Mutsuki、Watanabe Hirotaka、Maeda Sumihiro、Kimura Taeko、Yoshimatsu Sho、Kisa Fumihiko、Karch Celeste M.、Miyasaka Tomohiro、Takashima Akihiko、Sahara Naruhiko、Hisanaga Shin-ichi、Ikeuchi Takeshi、Kaibuchi Kozo、Okano Hideyuki
  • Journal Title: Stem Cell Reports Volume: 13 Issue: 4 Pages: 684-699
  • DOI: 10.1016/j.stemcr.2019.08.011
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Developing a 3D hiPSC-derived culture model system for inducing endogenous tau aggregation (2021)
  • Author(s): Sumihiro Maeda, Hayato Hiramine, Mitsuru Ishikawa, Ikuko Koya, Ryosuke Nagashima, Seiji Shiozawa, Mari Nakamura Manabu Itoh, Hideyuki Okano
  • Organizer: International Society for Stem Cell Research 19th annual meeting
  • Int'l Joint Research
• [Presentation] hiPSC3D培養モデルを用いた内在性タウタンパク質凝集誘導系の開発 (2021)
  • Author(s): 平峯勇人、前田純宏、石川充、古家育子、永島崚甫、塩澤誠司、中村真理、伊藤学、岡野栄之
  • Organizer: 第40回日本認知症学会学術集会
• [Presentation] hiPSC3D培養モデルを用いた内在性タウタンパク質凝集誘導系の開発 (2021)
  • Author(s): Hayato Hiramine, Sumihiro Maeda, Mitsuru Ishikawa, Ikuko Koya, Ryosuke Nagashima, Seiji Shiozawa, Mari Nakamura, Manabu Itoh, Hideyuki Okano
  • Organizer: 第64回日本神経化学会大会
• [Presentation] IN VITRO DISEASE MODELING OF THE FTDP-17 TAU R406W MUTATION USING PATIENT-DERIVED IPSCS (2019)
  • Author(s): Mari Nakamura, Seiji Shiozawa, Daisuke Tsuboi, Mutsuki Amano, Hirotaka Watanabe, Sumihiro Maeda, Taeko Kimura, Sho Yoshimatsu, Fumihiko Kisa, Celeste Karch, Tomohiro Miyasaka, Akihiko Takashima, Naruhiko Sahara, Shinichi Hisanaga, Takeshi Ikeuchi, Kozo Kaibuchi, Hideyuki Okano
  • Organizer: International Society for Stem Cell Research 17th annual meeting
  • Int'l Joint Research
• [Remarks] 久留米大学医学部動物実験センター
  • URL: https://animal.med-kurume.jp/