A toolbox for genetic manipulation in intestinal Clostridium symbiosum  

作　　者：Pengjie Yang Jinzhong Tian Lu Zhang Hui Zhang Gaohua Yang Yimeng Ren Jingyuan Fang Yang Gu Weihong Jiang 

机构地区：[1]CAS-Key Laboratory of Synthetic Biology,CAS Center for Excellence in Molecular Plant Sciences,Shanghai Institute of Plant Physiology and Ecology,Chinese Academy of Sciences,Shanghai,200032,China [2]University of Chinese Academy of Sciences,Beijing,100049,China [3]Xianghu Laboratory,Hangzhou,311231,China [4]NHC Key Laboratory of Digestive Diseases,Division of Gastroenterology and Hepatology,Shanghai Institute of Digestive Disease,Renji Hospital,School of Medicine,Shanghai Jiao Tong University,145 Middle Shandong Road,Shanghai,200001,China [5]Shenzhen Branch,Guangdong Laboratory for Lingnan Modern Agriculture,Genome Analysis Laboratory of the Ministry of Agriculture,Agricultural Genomics Institute at Shenzhen,Chinese Academy of Agricultural Sciences,Shenzhen,518120,China [6]The Wallenberg Laboratory,Department of Molecular and Clinical Medicine,Sahlgrenska Academy,University of Gothenburg,Bruna Straket 16,Gothenburg,41345,Sweden

出　　处：《Synthetic and Systems Biotechnology》2024年第1期43-54,共12页合成和系统生物技术（英文）

基　　金：supported by the National Key R&D Program of China(2018YFA0901500);Science and Technology Commission of Shanghai Municipality(21DZ1209100);DNL Cooperation Fund,CAS(DNL202013);Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-016).

摘　　要：Gut microbes are closely related with human health,but remain much to learn.Clostridium symbiosum is a conditionally pathogenic human gut bacterium and regarded as a potential biomarker for early diagnosis of intestinal tumors.However,the absence of an efficient toolbox that allows diverse genetic manipulations of this bacterium limits its in-depth studies.Here,we obtained the complete genome sequence of C.symbiosum ATCC 14940,a representative strain of C.symbiosum.On this basis,we further developed a series of genetic manipulation methods for this bacterium.Firstly,following the identification of a functional replicon pBP1 in C.symbiosum ATCC 14940,a highly efficient conjugative DNA transfer method was established,enabling the rapid introduction of exogenous plasmids into cells.Next,we constructed a dual-plasmid CRISPR/Cas12a system for genome editing in this bacterium,reaching over 60% repression for most of the chosen genes as well as efficient deletion(>90%)of three target genes.Finally,this toolbox was used for the identification of crucial functional genes,involving growth,synthesis of important metabolites,and virulence of C.symbiosum ATCC 14940.Our work has effectively established and optimized genome editing methods in intestinal C.symbiosum,thereby providing strong support for further basic and application research in this bacterium.

关 键 词：Gut Clostridium symbiosum TOOLBOX Gene overexpression Gene deletion Gene regulation 

分 类 号：R57[医药卫生—消化系统]