Genome editing without DNA double-strand breaks mediated by CRISPR-Cas9 nickases

• Project/Area Number: 17K07263
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: System genome science
• Research Institution: Aichi Medical University
• Principal Investigator: Konishi Hiroyuki 愛知医科大学, 医学部, 教授 (20344335)
• Co-Investigator(Kenkyū-buntansha): 兵頭 寿典 愛知医科大学, 医学部, 講師 (40710645) ; シバスンダラン カルナン 愛知医科大学, 医学部, 講師 (30557096)
• Project Period (FY): 2017-04-01 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2019: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2018: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2017: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000)
• Keywords: CRISPR / Cas9 / nickase / ゲノム編集 / ノックイン / tandem paired nicking / ニック / CRISPR/Cas9 / CRISPR-Cas9 / ゲノム生物工学

Outline of Final Research Achievements

Targeted knock-in assisted by genome editing is a promising technology for genome engineering which will be applicable to a broad range of research fields in life science. However, the precision, specificity, and safety of this technology need to be improved to expedite its practical applications. In the present study, we characterized a methodology for targeted knock-in, namely tandem paired nicking (TPN), which leverages Cas9 nickases in a manner distinct from their regular use. By empirically addressing the properties of TPN, we found that TPN achieves the efficiency of targeted knock-in largely equivalent to those achieved by the conventional Cas9 nuclease-based approach, while drastically decreasing the incidence of random undesired insertions and deletions occurring at the targeted genomic loci. Moreover, TPN-based targeted knock-in neither activated the p53 signaling pathway as known as “the guardian of the genome,” nor was restricted by the function of wild-type p53.

Academic Significance and Societal Importance of the Research Achievements

本研究の結果、TPN法の利用によってノックインに伴う標的ゲノム部位の予期せぬランダム変異を抑えうることがわかった。また、TPN法では標的外部位（オフターゲット部位）のランダム変異が少ないことも示唆されており、ゲノム編集細胞のがん化などの危険性を抑制しうるものと期待される。さらに、DNA損傷反応を制御するp53シグナル経路がTPN法によって攪乱されないことは、同法の安全性を裏付けるものと考えられる。従来法に比肩するノックイン効率が保持されていることも鑑み、TPN法は今後の技術的な洗練を経たのち、将来的には臨床医学、畜産学、植物科学などに応用可能な有用なノックイン法になりうるものと期待される。

Research Products

• [Journal Article] CD52 is a novel target for the treatment of FLT3-ITD-mutated myeloid leukemia (2021)
  • Author(s): 1.Karnan S, Hanamura I, Ota A, Takasugi S, Nakamura A, Takahashi M, Uchino K, Murakami S, Wahiduzzaman M, Vu LQ, Rahman ML, Hasan MN, Hyodo T, Konishi H, Tsuzuki S, Yoshikawa K, Suzuki S, Ueda R, Ejiri M, Hosokawa Y, and Takami A
  • Journal Title: Cell Death Discovery Volume: -
  • Peer Reviewed / Open Access
• [Journal Article] Identification of CD24 as a potential diagnostic and therapeutic target for malignant pleural mesothelioma (2020)
  • Author(s): KarnanS, Ota A, Murakami H, Rahman ML, Hasan MN, Wahiduzzaman MD, Hanamura I, Vu LQ, Inoko A, Hyodo T, Konishi H, Tsuzuki S, Hosokawa Y.
  • Journal Title: Cell Death Discovery Volume: 18 Issue: 1 Pages: 127-139
  • DOI: 10.1038/s41420-020-00364-1
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Novel Interleukin-6 Inducible Gene PDZ-Binding Kinase Promotes Tumor Growth of Multiple Myeloma Cells (2020)
  • Author(s): Ota Akinobu、Hanamura Ichiro、Karnan Sivasundaram、Inaguma Shingo、Takei Norio、Lam Vu Quang、Mizuno Shohei、Kanasugi Jo、Wahiduzzaman Md、Rahman Md Lutfur、Hyodo Toshinori、Konishi Hiroyuki、Tsuzuki Shinobu、Ikeda Hiroshi、Takami Akiyoshi、Hosokawa Yoshitaka
  • Journal Title: Journal of Interferon & Cytokine Research Volume: 40 Issue: 8 Pages: 389-405
  • DOI: 10.1089/jir.2020.0111
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Targeting MEF2D-fusion Oncogenic Transcriptional Circuitries in B-cell Precursor Acute Lymphoblastic Leukemia (2020)
  • Author(s): Shinobu Tsuzuki, Takahiko Yasuda, Shinya Kojima, Masahito Kawazu, Koshi Akahane, Takeshi Inukai, Masue Imaizumi, Takanobu Morishita, Masashi Sanada, Hideki Muramatsu, Yoshiyuki Takahashi, Yasushi Miyazaki, Itaru Matsumura, Hitoshi Kiyoi, Yoshitaka Hosokawa, Hiroyuki Mano, Fumihiko Hayakawa
  • Journal Title: Blood Cancer Discovery Volume: 1 Issue: 1 Pages: 82-95
  • DOI: 10.1158/2643-3230.bcd-19-0080
  • Peer Reviewed / Open Access
• [Journal Article] Discovery of novel molecular characteristics and cellular biological properties in ameloblastoma (2020)
  • Author(s): Kondo Sayuri、Ota Akinobu、Ono Takayuki、Karnan Sivasundaram、Wahiduzzaman Md、Hyodo Toshinori、Lutfur Rahman Md、Ito Kunihiro、Furuhashi Akifumi、Hayashi Tomio、Konishi Hiroyuki、Tsuzuki Shinobu、Hosokawa Yoshitaka、Kazaoka Yoshiaki
  • Journal Title: Cancer Medicine Volume: 9 Issue: 8 Pages: 2904-2917
  • DOI: 10.1002/cam4.2931
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Bi-allelic loss of FAM46C triggers tumor growth with concomitant activation of Akt signaling in multiple myeloma cells (2020)
  • Author(s): Kanasugi J, Hanamura I, Ota A, Karnan S, Vu LQ, Mizuno S, Wahiduzzaman M, Rahman ML, Hyodo T, Konishi H, Tsuzuki S, Hosokawa Y, Takami A
  • Journal Title: Cancer Science Volume: - Issue: 5 Pages: 999-999
  • DOI: 10.1111/cas.14386
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Tandem paired nicking promotes precise genome editing with scarce interference by p53 (2019)
  • Author(s): Hyodo T, Rahman ML, Karnan S, Ito T, Toyoda A, Ota A, Wahiduzzaman M, Tsuzuki S, Okada Y, Hosokawa Y, Konishi H.
  • Journal Title: Cell Rep. Volume: 30 Issue: 4 Pages: 1195-1207
  • DOI: 10.1016/j.celrep.2019.12.064
  • Peer Reviewed / Open Access
• [Journal Article] Establishment and characterization of CRISPR/Cas9-mediated NF2(-/-) human mesothelial cell line: Molecular insight into fibroblast growth factor receptor 2 in malignant pleural mesothelioma (2019)
  • Author(s): Wahiduzzaman Md、Karnan Sivasundaram、Ota Akinobu、Hanamura Ichiro、Murakami Hideki、Inoko Akihito、Rahman Md Lutfur、Hyodo Toshinori、Konishi Hiroyuki、Tsuzuki Shinobu、Hosokawa Yoshitaka
  • Journal Title: Cancer Science Volume: 110 Issue: 1 Pages: 180-193
  • DOI: 10.1111/cas.13871
  • Peer Reviewed / Open Access
• [Journal Article] Novel combined Ato-C treatment synergistically suppresses proliferation of Bcr-Abl-positive leukemic cells in vitro and in vivo (2018)
  • Author(s): Wahiduzzaman Md、Ota Akinobu、Karnan Sivasundaram、Hanamura Ichiro、Mizuno Shohei、Kanasugi Jo、Rahman Md Lutfur、Hyodo Toshinori、Konishi Hiroyuki、Tsuzuki Shinobu、Takami Akiyoshi、Hosokawa Yoshitaka
  • Journal Title: Cancer Letters Volume: 433 Pages: 117-130
  • DOI: 10.1016/j.canlet.2018.06.027
  • Peer Reviewed
• [Journal Article] Inhibition of NADPH oxidase 2 induces apoptosis in osteosarcoma: The role of reactive oxygen species in cell proliferation (2018)
  • Author(s): Kitamoto Kazumasa、Miura Yuji、Karnan Sivasundaram、Ota Akinobu、Konishi Hiroyuki、Hosokawa Yoshitaka、Sato Keiji
  • Journal Title: Oncology Letters Volume: 15 Pages: 7955-7962
  • DOI: 10.3892/ol.2018.8291
  • Peer Reviewed / Open Access
• [Journal Article] Novel ATP-competitive Akt inhibitor afuresertib suppresses the proliferation of malignant pleural mesothelioma cells. (2017)
  • Author(s): Yamaji M, Ota A, Wahiduzzaman M, Karnan S, Hyodo T, Konishi H, Tsuzuki S, Hosokawa Y, Hanyuda M.
  • Journal Title: Cancer Med Volume: 6 Issue: 11 Pages: 2646-2659
  • DOI: 10.1002/cam4.1179
  • Peer Reviewed
• [Journal Article] Delta40p53 suppresses tumor cell proliferation and induces cellular senescence in hepatocellular carcinoma cells (2017)
  • Author(s): Ota A, Nakao H, Sawada Y, Karnan S, Wahiduzzaman M, Inoue T, Kobayashi Y, Yamamoto T, Ishii N, Ohashi T, Nakade Y, Sato K, Itoh K, Konishi H, Hosokawa Y, Yoneda M
  • Journal Title: J Cell Sci Volume: 130 Issue: 3 Pages: 614-625
  • DOI: 10.1242/jcs.190736
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Presentation] Experimental conditions permitting efficient targeted knock-in using CRISPR/Cas9 nickases (2019)
  • Author(s): Md Lutfur Rahman, 兵頭寿典, Sivasundaram Karnan, 太田明伸, 都築忍, 細川好孝, 小西裕之
  • Organizer: 日本ゲノム編集学会第4回大会
• [Presentation] CRISPR/Cas9 nickaseによるノックインはp53を活性化しない (2019)
  • Author(s): 兵頭寿典, Md Lutfur Rahman, Sivasundaram Karnan, 太田明伸, 都築忍, 細川好孝, 小西裕之
  • Organizer: 日本ゲノム編集学会第4回大会
• [Presentation] CRISPR/Cas9 nickaseによるサイレント変異不要のノックイン (2019)
  • Author(s): 兵頭寿典, Md Lutfur Rahman, 豊田敦, Sivasundaram Karnan, 太田明伸, 都築忍, 細川好孝, 〇小西裕之
  • Organizer: 日本ゲノム編集学会第4回大会
• [Presentation] CRISPR/Cas9を用いたNF2及びCDKN2Aノックアウトヒト中皮腫細胞株の樹立と分子生物学的解析 (2019)
  • Author(s): Sivasundaram Karnan, 太田明伸, 兵頭寿典, 小西裕之, 都築忍, 細川好孝
  • Organizer: 第92回日本生化学会大会
• [Presentation] p16/NF2遺伝子の欠損により発現の誘導される悪性中皮腫特異的分子の同定 (2019)
  • Author(s): Sivasundaram Karnan, 太田明伸, 花村一朗, 村上秀樹, 兵頭寿典, 小西裕之, 都築忍, 細川好孝
  • Organizer: 第78回日本癌学会学術総会
• [Presentation] Tandem paired nicking using Cas9 nickases permits targeted knock-in without significant inhibition by p53 (2019)
  • Author(s): Toshinori Hyodo, Md Lutfur Rahman, Atsushi Toyoda, Sivasundaram Karnan, Akinobu Ota, Shinobu Tuzuki, Yoshitaka Hosokawa, 〇Hiroyuki Konishi
  • Organizer: Frontiers in Genome Engineering 2019
  • Int'l Joint Research
• [Presentation] DNA二重鎖切断を介さない変異ノックイン法 (2019)
  • Author(s): 兵頭寿典, Md Lutfur Rahman, 豊田敦, Sivasundaram Karnan, 太田明伸, 都築忍, 細川好孝, 〇小西裕之
  • Organizer: 第42回日本分子生物学会年会
• [Presentation] CRISPR/Cas9 nickaseによるサイレント変異不要のノックイン (2019)
  • Author(s): 兵頭寿典, Md Lutfur Rahman, 豊田敦, Sivasundaram Karnan, 太田明伸, 都築忍, 細川好孝, 小西裕之
  • Organizer: 日本ゲノム編集学会第4回大会
• [Presentation] p16/NF2遺伝子の欠損により発現の誘導される悪性中皮腫特異的分子の同定 (2018)
  • Author(s): 2.Sivasundaram Karnan, 太田明伸, 花村一朗, 村上秀樹, Md Wahiduzzaman, 兵頭寿典, 小西裕之, 都築忍, 細川好孝
  • Organizer: 第77回日本癌学会学術総会
• [Presentation] 多発性骨髄腫の腫瘍増殖におけるPDZ binding kinaseの関与 (2018)
  • Author(s): 3.太田明伸, 花村一朗, Sivasundaram Karnan, 小西裕之, 都築忍, 細川好孝
  • Organizer: 第77回日本癌学会学術総会
• [Presentation] 悪性中皮腫細胞の増殖を特異的に抑制するAkt阻害剤の探索と同定 (2018)
  • Author(s): 4.太田明伸, 山地雅之, Md Wahiduzzaman, Sivasundaram Karnan, 兵頭寿典, 小西裕之, 都築忍, 羽生田正行, 細川好孝
  • Organizer: 第91回日本生化学会大会
• [Presentation] Combined arsenic trioxide-cisplatin treatment synergistically suppresses proliferation of Bcr-Abl-positive leukemic cells. (2017)
  • Author(s): Md Wahiduzzaman, Akinobu Ota, Sivasundaram Karnan, Toshinori Hyodo, Hiroyuki Konishi, Shinobu Tsuzuki, Yoshitaka Hosokawa.
  • Organizer: 2017年度生命科学系学会合同年次大会
• [Presentation] Protein phosphatase 2Aの調節サブユニットPPP2R5Eは微小管架橋因子MTCL1の発現量を制御する. (2017)
  • Author(s): 兵頭寿典, 稲見恵理, 太田明伸, Sivasundaram Karnan, 小西裕之, 細川好孝.
  • Organizer: 2017年度生命科学系学会合同年次大会
• [Presentation] 悪性胸膜中皮腫の早期診断腫瘍マーカーの検討. (2017)
  • Author(s): Sivasundaram Karnan, 太田明伸, 花村一朗, 村上秀樹, 兵頭寿典, Md Wahiduzzaman, 小西裕之, 都築忍, 細川好孝.
  • Organizer: 2017年度生命科学系学会合同年次大会
• [Presentation] 新規Interleukin-6誘導因子PDZ binding kinaseは骨髄腫細胞の増殖と進展に関与する. (2017)
  • Author(s): 太田明伸, 花村一朗, Sivasundaram Karnan, Md Wahiduzzaman, 兵頭寿典, 小西裕之, 都築忍, 細川好孝.
  • Organizer: 2017年度生命科学系学会合同年次大会
• [Presentation] 花村一朗, Sivasundaram Karnan, 太田明伸, Wahiduzzaman Md, 後藤峰明, 高橋美裕希, 水野昌平, 内野かおり, 鈴木進, 都築忍, 小西裕之, 上田龍三, 細川好孝, 高見昭良. (2017)
  • Author(s): CRISPR/Cas9を用いたFLT3-ITD導入ヒト白血病クローンの樹立と分子生物学的解析.
  • Organizer: 第79回日本血液学会学術総会
• [Presentation] 亜ヒ酸とシスプラチンによる併用療法はBcr-Abl陽性白血病細胞に対して相乗的な増殖抑制効果を示す (2017)
  • Author(s): 太田明伸, Md Wahiduzzaman, Sivasundaram Karnan, 兵頭寿典, 小西裕之, 都築忍, 細川好孝.
  • Organizer: 第76回日本癌学会学術総会
• [Presentation] CRISPR/Cas9を用いたNF2ノックアウトヒト中皮腫細胞株の樹立と分子生物学的解析 (2017)
  • Author(s): Sivasundaram Karnan, 太田明伸, 花村一朗, 村上秀樹, 兵頭寿典, Md Wahiduzzaman, 小西裕之, 都築忍, 細川好孝.
  • Organizer: 第76回日本癌学会学術総会
• [Presentation] SINGLE-NUCLEOTIDE POLYMORPHISM IN THE PBK GENE IS CLOSELY ASSOCIATED WITH MYELOMA CELL PROLIFERATION. (2017)
  • Author(s): Ichiro Hanamura, Akinobu Ota, Sivasundaram Karnan, Md Wahiduzzaman, Shohei Mizuno, Kaori Uchino, Jo Kanasugi, Tomohiro Horio, Satsuki Murakami, Susumu Suzuki, Ryuzo Ueda, Shinobu Tsuzuki, Hiroyuki Konishi, Yoshitaka Hosokawa, Akiyoshi Takami.
  • Organizer: The 22nd Congress of the European Hematology Association
  • Int'l Joint Research
• [Book] ゲノム編集技術を応用した製品開発とその実用化 (2021)
  • Author(s): 執筆者:96名、技術情報協会
  • Total Pages: 602
  • Publisher: 技術情報協会
  • ISBN: 9784861048272