短双歧杆菌,在生命早期定植于人类胃肠道的主要双歧杆菌之一,因其对人类健康的有益影响而受到广泛关注。然而,由于缺乏有效的遗传工具,对短双歧杆菌发挥的这种有益作用的模式的探索是麻烦的,这限制了其合成生物学的应用。CRISPR-Cas系统在短双歧杆菌基因组中的广泛存在使得内源性基于CRISPR的基因编辑工具包成为有前途的工具。这项研究表明,短小芽孢杆菌的I-C型CRISPR-Cas系统可以根据cas基因簇编码的氨基酸序列分为两组。使用该系统在来自两组的五个短双歧杆菌菌株中实现了编码尿嘧啶磷酸核糖基转移酶(upp)的基因的缺失。此外,通过对upp基因进行单碱基取代并插入三个终止密码子,在短双歧杆菌FJSWX38M7中成功实现了尿嘧啶磷酸核糖转移酶的翻译终止。短小芽孢杆菌中编码亚油酸异构酶(bbi)的基因,作为一种特征,在质粒固化后被删除,这使得它无法将亚油酸转化为共轭亚油酸,证明了连续编辑的可行性。这项研究扩展了短芽孢杆菌基因操作的工具包,并为短芽孢杆菌菌株的功能基因组编辑和分析提供了一种新方法。重要性短双歧杆菌缺乏有效的遗传工具是研究其健康促进作用的分子机制的障碍,阻碍了下一代益生菌的发展。这里,我们介绍了一种基于内源性CRISPR-Cas系统的基因编辑方法,可以实现基因缺失,单碱基取代,基因插入,以及短小芽孢杆菌的连续基因编辑。这项研究将有助于发现功能基因和阐明短双歧杆菌与健康相关益处的分子机制。
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.IMPORTANCEThe lack of effective genetic tools for Bifidobacterium breve is an obstacle to studying the molecular mechanisms of its health-promoting effects, hindering the development of next-generation probiotics. Here, we introduce a gene editing method based on the endogenous CRISPR-Cas system, which can achieve gene deletion, single-base substitution, gene insertion, and successive gene editing in B. breve. This study will facilitate discovery of functional genes and elucidation of molecular mechanisms of B. breve pertaining to health-associated benefits.