An imbalance in oral microbial homeostasis is significantly associated with the onset and progression of several systemic diseases.Fusobacterium nucleatum,a ubiquitous periodontitis-causing bacterium in the oral cavit...An imbalance in oral microbial homeostasis is significantly associated with the onset and progression of several systemic diseases.Fusobacterium nucleatum,a ubiquitous periodontitis-causing bacterium in the oral cavity,is frequently detected in focal sites and contributes to the pathogenesis of many extraoral diseases,including cancers,cardiovascular diseases,and adverse pregnancy outcomes(APOs).F.nucleatum is one of the few oral anaerobes that can be cultured purely in vitro and is a‘model species’for studying the impact of oral health on systemic health.The establishment and development of genetic manipulation tools for F.nucleatum and the construction of pathogenic gene-disrupted strains are important strategies for studying the pathogenicity of F.nucleatum.Here,we review the establishment and development of the genetic manipulation systems for F.nucleatum and summarize the characteristics of various genetic manipulation tools,such as suicide plasmid-based systems for gene inactivation,replicable plasmid-based systems controlling gene expression,and transposon-based random mutagenesis systems.Notably,we summarize and analyze their applications in the study of the pathogenic mechanisms of F.nucleatum.We hope to provide reference information and ideas for future research on genetic manipulation tools and the pathogenic mechanisms of F.nucleatum and other Fusobacterium species.展开更多
Derived from the bacterial adaptive immune system,CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering.In this review,we outline the fundamen...Derived from the bacterial adaptive immune system,CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering.In this review,we outline the fundamental CRISPR tools that have been employed for strain optimization.These tools include CRISPR editing,CRISPR interference,CRISPR activation and protein imaging.To further characterize the CRISPR technology,we present current applications of these tools in microbial systems,including model-and non-model industrial microorganisms.Specially,we point out the major challenges of the CRISPR tools when utilized for multiplex genome editing and sophisticated expression regulation.To address these challenges,we came up with strategies that place emphasis on the amelioration of DNA repair efficiency through CRISPR-Cas9-assisted recombineering.Lastly,multiple promising research directions were proposed,mainly focusing on CRISPR-based construction of microbial ecosystems toward high production of desired chemicals.展开更多
Deciphering gene function is fundamental to engineering of microbiology.The clustered regularly interspaced short palindromic repeats(CRISPR)system has been adapted for gene repression across a range of hosts,creating...Deciphering gene function is fundamental to engineering of microbiology.The clustered regularly interspaced short palindromic repeats(CRISPR)system has been adapted for gene repression across a range of hosts,creating a versatile tool called CRISPR interference(CRISPRi)that enables genome-scale analysis of gene function.This approach has yielded significant advances in the design of genome-scale CRISPRi libraries,as well as in applica-tions of CRISPRi screening in medical and industrial microbiology.This review provides an overview of the recent progress made in pooled and arrayed CRISPRi screening in microorganisms and highlights representative studies that have employed this method.Additionally,the challenges associated with CRISPRi screening are discussed,and potential solutions for optimizing this strategy are proposed.展开更多
基金foundation support of the National Natural Science Foundation of China(82270980,82071122)the National Young Scientist Support Foundation(2019),the Major Innovation Projects in Shandong Province(2021SFGC0502)+2 种基金the Oral Microbiome Innovation Team of Shandong Province(2020KJK001)Shandong Province Key Research and Development Program(2021ZDSYS18)Intramural Joint Program Fund of State Key Laboratory of Microbial Technology(SKLMTIJP-2024-08)。
文摘An imbalance in oral microbial homeostasis is significantly associated with the onset and progression of several systemic diseases.Fusobacterium nucleatum,a ubiquitous periodontitis-causing bacterium in the oral cavity,is frequently detected in focal sites and contributes to the pathogenesis of many extraoral diseases,including cancers,cardiovascular diseases,and adverse pregnancy outcomes(APOs).F.nucleatum is one of the few oral anaerobes that can be cultured purely in vitro and is a‘model species’for studying the impact of oral health on systemic health.The establishment and development of genetic manipulation tools for F.nucleatum and the construction of pathogenic gene-disrupted strains are important strategies for studying the pathogenicity of F.nucleatum.Here,we review the establishment and development of the genetic manipulation systems for F.nucleatum and summarize the characteristics of various genetic manipulation tools,such as suicide plasmid-based systems for gene inactivation,replicable plasmid-based systems controlling gene expression,and transposon-based random mutagenesis systems.Notably,we summarize and analyze their applications in the study of the pathogenic mechanisms of F.nucleatum.We hope to provide reference information and ideas for future research on genetic manipulation tools and the pathogenic mechanisms of F.nucleatum and other Fusobacterium species.
基金This work was supported by grants from National Natural Science Foundation of China(No.21276014,21476011)National High Technology Research and Development Program(863 Program)(No.2015AA021003)+1 种基金Fundamental Research Funds for the Central Universities(YS1407)111 project(B13005).
文摘Derived from the bacterial adaptive immune system,CRISPR technology has revolutionized conventional genetic engineering methods and unprecedentedly facilitated strain engineering.In this review,we outline the fundamental CRISPR tools that have been employed for strain optimization.These tools include CRISPR editing,CRISPR interference,CRISPR activation and protein imaging.To further characterize the CRISPR technology,we present current applications of these tools in microbial systems,including model-and non-model industrial microorganisms.Specially,we point out the major challenges of the CRISPR tools when utilized for multiplex genome editing and sophisticated expression regulation.To address these challenges,we came up with strategies that place emphasis on the amelioration of DNA repair efficiency through CRISPR-Cas9-assisted recombineering.Lastly,multiple promising research directions were proposed,mainly focusing on CRISPR-based construction of microbial ecosystems toward high production of desired chemicals.
基金supported by the National Key R&D Program of China(2018YFA0901500)the National Natural Science Foundation of China(32222004 and 32270101)the Youth Innovation Promotion Association of Chinese Academy of Sciences(2021177).
文摘Deciphering gene function is fundamental to engineering of microbiology.The clustered regularly interspaced short palindromic repeats(CRISPR)system has been adapted for gene repression across a range of hosts,creating a versatile tool called CRISPR interference(CRISPRi)that enables genome-scale analysis of gene function.This approach has yielded significant advances in the design of genome-scale CRISPRi libraries,as well as in applica-tions of CRISPRi screening in medical and industrial microbiology.This review provides an overview of the recent progress made in pooled and arrayed CRISPRi screening in microorganisms and highlights representative studies that have employed this method.Additionally,the challenges associated with CRISPRi screening are discussed,and potential solutions for optimizing this strategy are proposed.
