肠道拟杆菌小型CRISPR/Cas基因编辑工具的开发

Development of a miniaturized CRISPR/Cas gene editing tool for human gut Bacteroides

摘要:

作为人体肠道中丰度最高的优势菌属之一，拟杆菌属(Bacteroides)为研究肠道微生物与宿主相互作用提供了重要模型。目前，针对拟杆菌属的CRISPR/Cas基因编辑系统已得到广泛应用，但大尺寸的Cas核酸酶限制了其应用场景。为了针对拟杆菌开发尺寸小又高效的基因编辑工具，本研究开发了针对肠道拟杆菌的小型CRISPR/Cas基因编辑系统，并首先在脆弱拟杆菌中评估了不同系统的编辑能力，包括AsCas12f、CasΦ2和ISDge10。随后评估了AsCas12f在不同拟杆菌中的编辑能力，并进一步优化了该系统的尺寸。结果表明，CRISPR/AsCas12f基因组编辑系统在脆弱拟杆菌中表现出最高的编辑效率。CRISPR/AsCas12f系统在脆弱拟杆菌、多形拟杆菌和普通拟杆菌中均实现了高效的基因组编辑。同时，在同源臂长度为500 bp的修复模板下，其编辑效率仍可以达到94.7%。本研究的结果表明，CRISPR/AsCas12f可作为开发基于拟杆菌的小型基因编辑器及其衍生技术的底盘工具酶，为拟杆菌基因编辑技术的进一步发展奠定了基础。

Abstract:

Bacteroides, as one of the most abundant and diverse genera in the human gut, is regarded as a window into the study of gut microbiota-host interactions. Currently, CRISPR/Cas-based gene editing systems targeting Bacteroides have been widely applied, while the large size of Cas nucleases limits their potential application scenarios (such as in situ gut Bacteroides editing based on phage delivery). Therefore, this study aims to develop a compact and highly efficient genetic editing tool in Bacteroides., We developed a miniaturized CRISPR/Cas gene editing system for human gut Bacteroides. First, the editing capabilities of different miniaturized CRISPR/Cas systems, including AsCas12f, CasΦ2, and ISDge10, were evaluated in Bacteroides fragilis. Subsequently, the editing capability of AsCas12f was assessed across various Bacteroides species, and the size of this system was further optimized. The results demonstrated that the CRISPR/AsCas12f genome editing system exhibited the highest editing efficiency in B.fragilis. The CRISPR/AsCas12f system achieved efficient genome editing in B.fragilis, Bacteroides thetaiotaomicron, and Phocaeicola vulgatus. Furthermore, with a repair template of 500 bp homologous arms, the editing efficiency remained as high as 94.7%. In conclusion, CRISPR/AsCas12f can serve as a chassis tool enzyme for the development of Bacteroides-based miniature gene editors and derivative technologies, laying a foundation for the further development of gene editing technology for Bacteroides.