Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve

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Han, Xiao
Chang, Lulu
Chen, Haiqin
Zhao, Jianxin
Tian, Fengwei
Ross, R. Paul
Stanton, Catherine
van Sinderen, Douwe
Chen, Wei
Yang, Bo
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American Society for Microbiology
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Bifidobacterium breve, one of the main bifidobacterial species colonizing the human gastrointestinal tract in early life, has received extensive attention for its purported beneficial effects on human health. However, exploration of the mode of action of such beneficial effects exerted by B. breve is cumbersome due to the lack of effective genetic tools, which limits its synthetic biology application. The widespread presence of CRISPR-Cas systems in the B. breve genome makes endogenous CRISPR-based gene editing toolkits a promising tool. This study revealed that Type I-C CRISPR-Cas systems in B. breve can be divided into two groups based on the amino acid sequences encoded by cas gene clusters. Deletion of the gene coding uracil phosphoribosyl-transferase (upp) was achieved in five B. breve strains from both groups using this system. In addition, translational termination of uracil phosphoribosyl-transferase was successfully achieved in B. breve FJSWX38M7 by single-base substitution of the upp gene and insertion of three stop codons. The gene encoding linoleic acid isomerase (bbi) in B. breve, being a characteristic trait, was deleted after plasmid curing, which rendered it unable to convert linoleic acid into conjugated linoleic acid, demonstrating the feasibility of successive editing. This study expands the toolkit for gene manipulation in B. breve and provides a new approach toward functional genome editing and analysis of B. breve strains.
Bifidobacterium breve , CRISPR-Cas , Type I-C , Genome editing
Han, X., Chang, L., Chen, H., Zhao, J., Tian, F., Ross, R. P., Stanton, C., van Sinderen, D., Chen, W. and Yang, B. (2024) 'Harnessing the endogenous Type I-C CRISPR-Cas system for genome editing in Bifidobacterium breve', Applied and Environmental Microbiology, 90(3), e02074-23 (16pp). https://doi.org/10.1128/aem.02074-23
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