| Project/Area Number |
21K15436
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| Research Category |
Grant-in-Aid for Early-Career Scientists
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| Allocation Type | Multi-year Fund |
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| Review Section |
Basic Section 49050:Bacteriology-related
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| Research Institution | Jichi Medical University |
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Principal Investigator |
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| Project Period (FY) |
2021-04-01 – 2023-03-31
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| Project Status |
Completed (Fiscal Year 2022)
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| Budget Amount *help |
¥4,550,000 (Direct Cost: ¥3,500,000、Indirect Cost: ¥1,050,000)
Fiscal Year 2022: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2021: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
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| Keywords | EHEC / AB capsid / CRISPR-Cas13a / Shiga toxin / Bacteriophage |
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| Outline of Research at the Start |
We will establish the new therapy for specific killing of EHEC infection together with suppression of Shiga toxin expression by designing CRISPR-Cas13a that recognizes stx genes, synthesizing antibacterial capsids, and verifying the cytotoxic and therapeutic effect of antibacterial capsids .
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| Outline of Final Research Achievements |
This research aimed to develop a new therapeutic agent to treat EHEC infections which can be accomplished by packaging stx-targeting CRISPR-Cas13a into the capsid of an E. coli phage. The stx-targeting Cas13a has been loaded into the capsid of a narrow-range phage80, generating Cas13a-stx. The bactericidal activity of Cas13a-stx was then confirmed. Furthermore, a candidate phage isolated from sewage water which has a satisfactory infectivity against EHEC strains (3/5) was isolated. It carried large genome size and difficult genome-manipulation. 706 clinical E. coli strains were chemically induced to further isolate broad-host-range phages. A candidate broad-host-range phage (killing activity of 33.67%) with small genome size (about 30 kbp) was isolated but it cannot infect EHEC strains. Although this work does not progress as proposed, CRISPR-Cas13a-loaded capsids were shown to kill E. coli expressing target sequence, proving their potential as an alternative therapeutic against EHEC.
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| Academic Significance and Societal Importance of the Research Achievements |
現在、抗菌薬によりEHEC大腸菌が殺菌されると細菌が持つ毒素が菌体外に放出されることが問題となっている。本申請は、CRISPR-Cas システムと薬剤耐性菌を選択的に殺菌できる遺伝子組み換えファージを組み合わせた新しい抗菌剤の開発を行なった。本申請の成果は、治療困難なEHEC 感染症の新しい治療法に資する基盤技術を提供することができる。特に、志賀毒素産生遺伝子(stx)を認識するCRISPR-Cas13aを搭載した抗菌カプシドにより、志賀毒素の産生を抑制するのみならず、同時にEHEC大腸菌を死滅させることができる。そのため、新しいEHEC 感染症の治療法となる可能性がある。
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