Establishment of a novel identification system for Enterotoxigenic Bacteroides fragilis using CRISPR-Cas13 and bacteriophage technology
| Project/Area Number |
22K20894
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| Research Category |
Grant-in-Aid for Research Activity Start-up
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| Allocation Type | Multi-year Fund |
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| Review Section |
0902:General internal medicine and related fields
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| Research Institution | Jichi Medical University |
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Principal Investigator |
Nguyen・Minh・Thuy 自治医科大学, 医学部, ポスト・ドクター (20964191)
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| Project Period (FY) |
2022-08-31 – 2024-03-31
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| Project Status |
Completed (Fiscal Year 2023)
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| Budget Amount *help |
¥2,860,000 (Direct Cost: ¥2,200,000、Indirect Cost: ¥660,000)
Fiscal Year 2023: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
Fiscal Year 2022: ¥1,430,000 (Direct Cost: ¥1,100,000、Indirect Cost: ¥330,000)
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| Keywords | CRISPR-Cas13a / Bacteriophage / gut microbe / Bacteroides fragilis / Identification |
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| Outline of Research at the Start |
Enterotoxigenic Bacteroides fragilis (ETBF) is the cause of inflammatory diarrhea of the colon and intestinal cancer. 5-20% of the world population has bft gene-positive asymptomatic ETBF detected in the gut microbiota. In this study, we developed an identification system that selectively kills and identifies ETBF by introducing CRISPR-Cas13a, which targets the bft toxin gene, into the phage capsid. The results of this application establish a fast and easy genotyping method that does not require nucleic acid amplification.
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| Outline of Annual Research Achievements |
Enterotoxigenic Bacteroides fragilis (ETBF) is the cause of inflammatory diarrhea of the colon and intestinal cancer. 5-20% of the world population has bft gene-positive asymptomatic ETBF detected in the gut microbiota. In this study, We induced prophages (lysogenic phages) from the bacterial genome using mitomycin C against B. fragilis 310 strains, and isolated the prophages obtains total 5 sample. The infection rates of the isolated prophages 5 strains were determined using clinical isolates of B. fragilis 73 strains (1.36 %-5.47%). The phage morphology was also observed using a transmission electron microscope (TEM) and identified by whole genome analysis. Next, we searched for and optimized CRISPR-cas sequences that recognize the target gene: Using the sequence of the bft gene, we designed a guide RNA that recognizes the bft gene in the 39 bft-carrying strain? and examined its bactericidal activity. We are currently constructing AB capsids using the best candidates. Furthermore, we created a B. fragilis-infected model mouse and constructed an in vivo evaluation system for the bactericidal activity of the target bacteria.
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Report
(2 results)
Research Products
(2 results)
