Method for mutation in organelle genome by CRISPR/CAS9

• Project/Area Number: 15K08453
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Parasitology (including sanitary zoology)
• Research Institution: Juntendo University
• Principal Investigator: HIRAI Makoto 順天堂大学, 医学部, 准教授 (50326849)
• Project Period (FY): 2015-04-01 – 2018-03-31
• Project Status: Completed (Fiscal Year 2017)
• Budget Amount: ¥5,070,000 (Direct Cost: ¥3,900,000、Indirect Cost: ¥1,170,000); Fiscal Year 2017: ¥650,000 (Direct Cost: ¥500,000、Indirect Cost: ¥150,000); Fiscal Year 2016: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2015: ¥2,470,000 (Direct Cost: ¥1,900,000、Indirect Cost: ¥570,000)
• Keywords: マラリア原虫 / オルガネラ / ゲノム編集 / アトバコン / cytochrome b / ミトコンドリア / オルガネラゲノム編集

Outline of Final Research Achievements

To deliver CAS9 protein into mitochondria, plasmids were constructed to replace nuclear localization signal with mitochondrial localization signal. Rodent malaria parasite, Plasmodium berghei was transfected with the plasmids, and localization of CAS9 protein in each transformant was investigated. As a result, no clear evidence for organelle localization of the protein was obtained. Beside, introduction of mitochondrial gene mutation by CRISPR/CAS9 (Mit) was attempted, however, desired mutation was not inserted in the genome. Taken together, these results suggest that delivery of CAS9 together with gRNA and donor DNA into organelle was critical point for successful modification of organelle genome. Further improvement should be challenged by considering these points.

Research Products

• [Journal Article] Artemisinin-Resistant Plasmodium falciparum with High Survival Rates, Uganda, 2014-2016 (2018)
  • Author(s): Ikeda M, Kaneko M, Tachibana S-I, Balikagala B, Sakurai-Yatsushiro M, Yatsushiro S, Takahashi N, Yamauchi M, Sekihara M, Hashimoto M, Katuro OT, Olia A, Obwoya PS, Auma MA, Anywar DA, Odongo-Aginya EI, Okello-Onen J, Hirai M, Ohashi J, Palacpac NMQ, Kataoka M, Tsuboi T, Kimura E, Horii T, Mita T.
  • Journal Title: Emerging Infectious Diseases Volume: 24 Issue: 4 Pages: 718-726
  • DOI: 10.3201/eid2404.170141
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Plasmodium falciparum kelch 13: a potential molecular marker for tackling artemisinin-resistant malaria parasites. (2016)
  • Author(s): Mita T, Tachibana S, Hashimoto M, Hirai M.
  • Journal Title: Expert Rev Anti Infect Ther. Volume: 14 Issue: 1 Pages: 125-135
  • DOI: 10.1586/14787210.2016.1106938
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Plasmodium berghei ANKA causes intestinal malaria associated with dysbiosis (2015)
  • Author(s): Taniguchi T, Miyauchi E, Nakamura S, Hirai M, Suzue K, Imai T, Nomura T, Handa T, Okada H, Shimokawa C, Onishi R, Olia A, Hirata J, Tomita H, Ohno H, Horii T, Hisaeda H
  • Journal Title: Scientific Reports Volume: 5:15699 Issue: 1 Pages: 1-12
  • DOI: 10.1038/srep15699
  • Peer Reviewed / Open Access / Int'l Joint Research / Acknowledgement Compliant
• [Presentation] Isolation of piperaquine resistant rodent malaria parasite from mutator malaria Malaria: Chemotherapy and Drug Resistance - Molecular Biology (2017)
  • Author(s): Makoto Hirai, Mie Ikeda, Shin-Ichiro Tachibana, Toshihiro Mita
  • Organizer: Americal Society of Tropical Medicine
  • Int'l Joint Research
• [Presentation] 「マラリアミューテーター：マラリア原虫薬剤耐性進化解明の新たな研究ツール」シンポジウム「原虫学・寄生虫学が現代のトップサイエンスにもたらす貢献」 (2017)
  • Author(s): 平井誠
  • Organizer: 2017年生命科学系学会合同次年会
  • Invited
• [Presentation] 「マラリア原虫の薬剤耐性機構解明に向けた取り組み」 (2017)
  • Author(s): 平井誠
  • Organizer: 第27回感染研シンポジウム－薬剤耐性菌の現状と対策－
  • Invited