| Project/Area Number |
20F20104
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| Research Category |
Grant-in-Aid for JSPS Fellows
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| Allocation Type | Single-year Grants |
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| Section | 外国 |
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| Review Section |
Basic Section 38020:Applied microbiology-related
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| Research Institution | Jichi Medical University |
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Principal Investigator |
崔 龍洙 自治医科大学, 医学部, 教授 (50306932)
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| Co-Investigator(Kenkyū-buntansha) |
AZAM AA 自治医科大学, 医学部, 外国人特別研究員
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| Project Period (FY) |
2020-04-24 – 2022-03-31
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| Project Status |
Completed (Fiscal Year 2021)
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| Budget Amount *help |
¥2,300,000 (Direct Cost: ¥2,300,000)
Fiscal Year 2021: ¥1,000,000 (Direct Cost: ¥1,000,000)
Fiscal Year 2020: ¥1,300,000 (Direct Cost: ¥1,300,000)
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| Keywords | CRISPR-Cas13 / Bacteriophage / Antimicrobials / mecA / MRSA / Cas13a / Antibiotic resistance / Staphyloccocus aureus / Antimicrobial |
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| Outline of Research at the Start |
抗菌薬の効かない薬剤耐性菌の出現とその急速な蔓延は、既存の抗菌薬を無力化して人類の健康を脅かしている。その耐性菌問題の解決に向け、我々は昨年度までに、ファージとCRISPR-Cas13aを利用して耐性遺伝子標的型の新規抗菌治療剤の開発研究を行なってきた。既に、カルバペネム耐性大腸菌をモデルとした耐性遺伝子を標的とする抗菌カプシドの人工合成に成功している。本研究では、MRSAを選択的に殺菌する抗菌カプシドの開発を目指す。具体的には、黄色ブドウ球菌に広く感染するファージを分離し、そのカプシドにmecAを標的に設計したCRISPR-Cas13aを搭載してMRSAを殺菌する新規抗菌薬の開発を行う。
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| Outline of Annual Research Achievements |
We succeed to construct an antibacterial capsid (AB capsid) against MRSA by using CRISPR-Cas13a which was screened from genome of Leptotrichia together with introducing a spacer that targets mecA into CRISPR region. This AB capsid was proved to have a sequence-specific killing activity. However, due to a relatively large size of CRISPR-Cas13a (approximately 5 Kb), our next challenge was to determine suitable location to insert them into phage genome without interrupting phage assembly. So far, we could manage to insert CRISPR-Cas13a around the late genes (lytic module) of the phage. We loaded the CRISPR-Cas13a into phage by deleting the phage endolysin and replaced it with CRISPR-Cas13a. The replacement of native endolysin with CRISPR-Cas13a enabled us to construct a phage that selectively kills bacteria harboring target genes. However, the killing activity was not strong as expected, suggesting either the presence of other factors or unfavorable locations of CRISPR-Cas13a insertion. We replaced phage endolysin with CRISPR-Cas13 because we wanted to remove lytic property of the phage to ensure that the lysis was occurred because of CRISPR-Cas13a’s cleavage activity, since one of our purpose was to generate a gene-based detection system. Unfortunately, lytic activity of endolysin-deficient phage was still observable when we used high titer of phage which may indicate the presence of other factor influencing phage lytic behavior. The follow up study of this project should include determination of other suitable location to insert CRISPR-Cas13a in the phage genome.
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| Research Progress Status |
令和3年度が最終年度であるため、記入しない。
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| Strategy for Future Research Activity |
令和3年度が最終年度であるため、記入しない。
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