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Development of a Simple Detection Method for Genetic Mutations Using Gene-Targeted Bactericidal Chimeric Phages

Research Project

Project/Area Number 20K16557
Research Category

Grant-in-Aid for Early-Career Scientists

Allocation TypeMulti-year Fund
Review Section Basic Section 52010:General internal medicine-related
Research InstitutionJichi Medical University

Principal Investigator

Aiba Yoshifumi  自治医科大学, 医学部, 助教 (60783694)

Project Period (FY) 2020-04-01 – 2022-03-31
Project Status Completed (Fiscal Year 2021)
Budget Amount *help
¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000)
Fiscal Year 2021: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000)
Fiscal Year 2020: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Keywords簡易検査法 / 薬剤耐性菌 / CRISPR-Cas13a / バクテリオファージ
Outline of Research at the Start

本申請では、CRISPR-Cas13a搭載キメラファージの塩基配列特異性を利用した細菌遺伝子変異の簡易検出法を確立する。CRISPR-Cas13aは標的遺伝子の塩基配列に対して極めて完璧な配列相同性を要求するため、1塩基の相違(SNP)を識別できると考え、本研究を計画した。既に、予備試験でCRISPR-Cas13aの設計を工夫することで検出遺伝子の1塩基の相違を感度良く鑑別することに成功した。この成果を生かして、核酸増幅や塩基配列の決定を行わずに、細菌の増殖の有無を目視観察するだけで鑑別できる、細菌遺伝子の簡易検出法を構築する。

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.

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

Report

(3 results)
  • 2021 Annual Research Report   Final Research Report ( PDF )
  • 2020 Research-status Report
  • Research Products

    (2 results)

All 2020

All Journal Article (1 results) (of which Int'l Joint Research: 1 results,  Peer Reviewed: 1 results,  Open Access: 1 results) Presentation (1 results)

  • [Journal Article] Development of CRISPR-Cas13a-based antimicrobials capable of sequence-specific killing of target bacteria2020

    • Author(s)
      Kiga Kotaro、Tan Xin-Ee、Ibarra-Ch?vez Rodrigo、Watanabe Shinya、Aiba Yoshifumi、Sato’o Yusuke、Li Feng-Yu、Sasahara Teppei、Cui Bintao、Kawauchi Moriyuki、Boonsiri Tanit、Thitiananpakorn Kanate、Taki Yusuke、Azam Aa Haeruman、Suzuki Masato、Penad?s Jos? R.、Cui Longzhu
    • Journal Title

      Nature Communications

      Volume: 11 Issue: 1 Pages: 2934-2934

    • DOI

      10.1038/s41467-020-16731-6

    • Related Report
      2020 Research-status Report
    • Peer Reviewed / Open Access / Int'l Joint Research
  • [Presentation] CRISPR-Cas13a 搭載ファージを用いた細菌ゲノム変異の検出2020

    • Author(s)
      相羽由詞,Kanate Thitiananpakorn,相羽由詞駕恒太朗,渡邊真弥,佐藤祐介,XinEe Tan,Tanit Boonsiri,李峰宇,崔 龍洙
    • Organizer
      第93回日本細菌学会
    • Related Report
      2020 Research-status Report

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Published: 2020-04-28   Modified: 2023-01-30  

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