2021 Fiscal Year Final Research Report
Development of a Simple Detection Method for Genetic Mutations Using Gene-Targeted Bactericidal Chimeric Phages
| Project/Area Number |
20K16557
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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 52010:General internal medicine-related
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| Research Institution | Jichi Medical University |
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Principal Investigator |
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| Project Period (FY) |
2020-04-01 – 2022-03-31
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| Keywords | 簡易検査法 / 薬剤耐性菌 / CRISPR-Cas13a / バクテリオファージ |
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| Outline of Final Research Achievements |
We establish a simple method to detect bacterial gene mutations that does not necessitate nucleic acid amplification. We utilized a Single Nucleotide Polymorphism (SNP) between IMP-1 and IMP-6 of carbapenemase-producing Enterobacteriaceae as a model system. A total of 56 guide RNAs were synthesized and assessed for CRISPR-Cas13a’s sequence-specific recognition of the model gene’s SNP. A significant bactericidal activity (ten power to six) was seen in the best of the guide RNA. Conversely, a certain number of guide RNA showed no bactericidal effects. This indicates the requisite for optimized guide RNA designs. By using meticulously optimized guide RNA, we were able to distinguish the SNP at a specific position in the target gene. This method is not just suitable for detecting resistance genes that are difficult to detect in the present clinical setting, yet it can also be extended to detect toxin genes as well as a suitable molecular epidemiology tool for infectious disease control.
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| Free Research Field |
細菌学
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| Academic Significance and Societal Importance of the Research Achievements |
本研究の成果は、核酸の増幅を必要としない細菌遺伝子変異の簡易検出法の確立である。本研究を通じて、CRISPR―Cas13aの設計を工夫することで、標的遺伝子が持つ１塩基の相違を感度良く鑑別できることを明らかにした。しかし、設計したCRISPR―Cas13aをファージに搭載技術には、回収量を向上させるためのさらなる検討が必要である。本課題の技術は、検体細菌と構築した殺菌キメラファージを共培養後に細菌の生死を目視確認のみで鑑別ができる。薬剤耐性菌の克服に向けて、細菌感染症の治療に資する薬剤耐性菌の早期検出や感染制御対策の日常的な環境調査に実装することができる簡易検査法になると期待される
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