Establishment of a novel identification system for Enterotoxigenic Bacteroides fragilis using CRISPR-Cas13 and bacteriophage technology

2023 Fiscal Year Annual Research Report

• Project/Area Number: 22K20894
• Research Institution: Jichi Medical University
• Principal Investigator: Nguyen・Minh・Thuy 自治医科大学, 医学部, ポスト・ドクター (20964191)
• Project Period (FY): 2022-08-31 – 2024-03-31
• Keywords: CRISPR-Cas13a / Bacteriophage / gut microbe / Bacteroides fragilis / Identification

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.