Mycobacterium tuberculosis, a Gram-positive bacterium of great clinical relevance, is a lethal pathogen owing to its complex physiological characteristics and development of drug resistance. Several molecular genetic ...Mycobacterium tuberculosis, a Gram-positive bacterium of great clinical relevance, is a lethal pathogen owing to its complex physiological characteristics and development of drug resistance. Several molecular genetic tools have been developed in the past few decades to study this microorganism. These tools have been instrumental in understanding how M. tuberculosis became a successful pathogen. Advanced molecular genetic tools have played a significant role in exploring the complex pathways involved in M. tuberculosis pathogenesis. Here, we review various molecular genetic tools used in the study of M. tuberculosis. Further, we discuss the applications of clustered regularly interspaced short palindromic repeat interference（CRISPRi）, a novel technology recently applied in M. tuberculosis research to study target gene functions. Finally, prospective outcomes of the applications of molecular techniques in the field of M. tuberculosis genetic research are also discussed.展开更多
The methylotrophic yeast Pichia pastoris(also known as Komagataella phaffii)is widely used as a yeast cell factory for producing heterologous proteins.Recently,it has gained attention for its potential in producing ch...The methylotrophic yeast Pichia pastoris(also known as Komagataella phaffii)is widely used as a yeast cell factory for producing heterologous proteins.Recently,it has gained attention for its potential in producing chemicals from inexpensive feedstocks,which requires efficient genetic engineering platforms.This review provides an overview of the current advances in developing genetic tools for metabolic engineering of P.pastoris.The topics cover promoters,terminators,plasmids,genome integration sites,and genetic editing systems,with a special focus on the development of CRISPR/Cas systems and their comparison to other genome editing tools.Additionally,this review highlights the prospects of multiplex genome integration,fine-tuning gene expression,and single-base editing systems.Overall,the aim of this review is to provide valuable insights into current genetic engineering and discuss potential directions for future efforts in developing efficient genetic tools in P.pastoris.展开更多
Genetic tools,which can be used for the morphology study of specific neurons,pathway-selective connectome mapping,neuronal activity monitoring,and manipulation with a spatiotemporal resolution,have been widely applied...Genetic tools,which can be used for the morphology study of specific neurons,pathway-selective connectome mapping,neuronal activity monitoring,and manipulation with a spatiotemporal resolution,have been widely applied to the understanding of complex neural circuit formation,interactions,and functions in rodents.Recently,similar genetic approaches have been tried in non-human primates(NHPs)in neuroscience studies for dissecting the neural circuits involved in sophisticated behaviors and clinical brain disorders,although they are still very preliminary.In this review,we introduce the progress made in the development and application of genetic tools for brain studies on NHPs.We also discuss the advantages and limitations of each approach and provide a perspective for using genetic tools to study the neural circuits of NHPs.展开更多
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
In the context of the rapid development of low-carbon economy,there has been increasing interest in utilizing naturally abundant and cost-effective one-carbon(C1)substrates for sustainable production of chemicals and ...In the context of the rapid development of low-carbon economy,there has been increasing interest in utilizing naturally abundant and cost-effective one-carbon(C1)substrates for sustainable production of chemicals and fuels.Moorella thermoacetica,a model acetogenic bacterium,has attracted significant attention due to its ability to utilize carbon dioxide(CO_(2))and carbon monoxide(CO)via the Wood-Ljungdahl(WL)pathway,thereby showing great potential for the utilization of C1 gases.However,natural strains of M.thermoacetica are not yet fully suitable for industrial applications due to their limitations in carbon assimilation and conversion efficiency as well as limited product range.Over the past decade,progresses have been made in the development of genetic tools for M.thermoacetica,accelerating the understanding and modification of this acetogen.Here,we summarize the physiological and metabolic characteristics of M.thermoacetica and review the recent advances in engineering this bacterium.Finally,we propose the future directions for exploring the real potential of M.thermoacetica in industrial applications.展开更多
Genome editing serves as a powerful approach to interrogate the functions of both coding and noncoding sequences.In particular,clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR associated protein...Genome editing serves as a powerful approach to interrogate the functions of both coding and noncoding sequences.In particular,clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR associated protein 9(Cas9)system-based editing tools have revolutionized the way we study genome function in mammalian cells,and are being widely used for interrogating critical genes and DNA elements essential for many biological processes.Here,we review CRISPR/Cas9-based genetic tools with an emphasis on CRISPR-mediated high throughput genetic screens in the mammalian genome.展开更多
The development of green biomanufacturing technologies centered around advanced microbial cells has emerged as a hot research field in synthetic biology.As known,Komagataella phaffii is an unconventional yeast with un...The development of green biomanufacturing technologies centered around advanced microbial cells has emerged as a hot research field in synthetic biology.As known,Komagataella phaffii is an unconventional yeast with unique biological characteristics,which is generally regarded as safe(GRAs).Owing to its wide substrate spectrum,K.phaffi has been widely genetically modified for valuable chemicals such as organic acids,fatty acids and some nature products.Actually,K.phaffii is very well known for the protein expression,and few reviews have systemically addressed the value-added chemicals by K.phaffii.Accordingly,this re-view will introduce the most advanced development of genetic editing tools of K.phaffi.Recent progresses and bottlenecks in the production of high-value chemicals using K.phaffii will also be summarized.Finally,future perspectives for the utilization of K.phaffii as a chassis cell used for high-value products synthesis will be discussed.展开更多
The CRISPR-Cas system is a powerful genetic engineering tool and can be conveniently used for the generation of diverse gene-knockout models.One CRISPR-Cas system,CRISPR-Cpf1(also known as Cas12a),recognizes the AT-ri...The CRISPR-Cas system is a powerful genetic engineering tool and can be conveniently used for the generation of diverse gene-knockout models.One CRISPR-Cas system,CRISPR-Cpf1(also known as Cas12a),recognizes the AT-rich protospacer-adjacent motif(PAM)present at the 5′end of the target sequence and requires CRISPR RNA(crRNA),but not transactivating crRNA(tracrRNA)for its activity.展开更多
基金supported by the National Mega-project of China for Innovative Drugs(2018ZX09721001-003-003)for Main Infectious Diseases(2017ZX10302301-003-002)+7 种基金the National Natural Science Foundation of China(No.81572037)the grants of Chinese Academy of Sciences(154144KYSB20150045,YJKYYQ20170036,KFZD-SW-207)the Public Research and Capacity Building Project of Guangdong Province(2017A020212004)partially supported by Guangzhou Municipal Industry and Research Collaborative Innovation Program(201508020248,201604020019)the Key Project(SKLRD2016ZJ003)from the State Key Lab of Respiratory Disease,Guangzhou Institute of Respiratory Diseases,First Affiliated Hospital of Guangzhou Medical UniversityT.Z.received support“Science and Technology Innovation Leader of Guangdong Province(2016TX03R095)”CAS-TWAS President's Ph D Fellowship Program(to C.C.and M.M.I.)UCAS Ph D Fellowship Program(to H.M.A.H.and J.M.)for International Students
文摘Mycobacterium tuberculosis, a Gram-positive bacterium of great clinical relevance, is a lethal pathogen owing to its complex physiological characteristics and development of drug resistance. Several molecular genetic tools have been developed in the past few decades to study this microorganism. These tools have been instrumental in understanding how M. tuberculosis became a successful pathogen. Advanced molecular genetic tools have played a significant role in exploring the complex pathways involved in M. tuberculosis pathogenesis. Here, we review various molecular genetic tools used in the study of M. tuberculosis. Further, we discuss the applications of clustered regularly interspaced short palindromic repeat interference（CRISPRi）, a novel technology recently applied in M. tuberculosis research to study target gene functions. Finally, prospective outcomes of the applications of molecular techniques in the field of M. tuberculosis genetic research are also discussed.
基金supported by supported by the National Key R&D Program of China(2021YFC2103500)DICP innovation grant(DICP I202111)from Dalian Institute of Chemical Physics,CAS.
文摘The methylotrophic yeast Pichia pastoris(also known as Komagataella phaffii)is widely used as a yeast cell factory for producing heterologous proteins.Recently,it has gained attention for its potential in producing chemicals from inexpensive feedstocks,which requires efficient genetic engineering platforms.This review provides an overview of the current advances in developing genetic tools for metabolic engineering of P.pastoris.The topics cover promoters,terminators,plasmids,genome integration sites,and genetic editing systems,with a special focus on the development of CRISPR/Cas systems and their comparison to other genome editing tools.Additionally,this review highlights the prospects of multiplex genome integration,fine-tuning gene expression,and single-base editing systems.Overall,the aim of this review is to provide valuable insights into current genetic engineering and discuss potential directions for future efforts in developing efficient genetic tools in P.pastoris.
基金This review was supported by grants from the Shanghai Municipal Science and Technology Major Project,the Strategic Priority Research Program of the Chinese Academy of Sciences,and the Lingang National Laboratory Key Project.
文摘Genetic tools,which can be used for the morphology study of specific neurons,pathway-selective connectome mapping,neuronal activity monitoring,and manipulation with a spatiotemporal resolution,have been widely applied to the understanding of complex neural circuit formation,interactions,and functions in rodents.Recently,similar genetic approaches have been tried in non-human primates(NHPs)in neuroscience studies for dissecting the neural circuits involved in sophisticated behaviors and clinical brain disorders,although they are still very preliminary.In this review,we introduce the progress made in the development and application of genetic tools for brain studies on NHPs.We also discuss the advantages and limitations of each approach and provide a perspective for using genetic tools to study the neural circuits of NHPs.
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supported by the National Key R&D Program of China(2021YFC2103500 and 2018YFA0901500)Science and Technology Commission of Shanghai Municipality(21DZ1209100)+1 种基金DNL Cooperation Fund,CAS(DNL202013)Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(TSBICIP-KJGG-016).
文摘In the context of the rapid development of low-carbon economy,there has been increasing interest in utilizing naturally abundant and cost-effective one-carbon(C1)substrates for sustainable production of chemicals and fuels.Moorella thermoacetica,a model acetogenic bacterium,has attracted significant attention due to its ability to utilize carbon dioxide(CO_(2))and carbon monoxide(CO)via the Wood-Ljungdahl(WL)pathway,thereby showing great potential for the utilization of C1 gases.However,natural strains of M.thermoacetica are not yet fully suitable for industrial applications due to their limitations in carbon assimilation and conversion efficiency as well as limited product range.Over the past decade,progresses have been made in the development of genetic tools for M.thermoacetica,accelerating the understanding and modification of this acetogen.Here,we summarize the physiological and metabolic characteristics of M.thermoacetica and review the recent advances in engineering this bacterium.Finally,we propose the future directions for exploring the real potential of M.thermoacetica in industrial applications.
文摘Genome editing serves as a powerful approach to interrogate the functions of both coding and noncoding sequences.In particular,clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR associated protein 9(Cas9)system-based editing tools have revolutionized the way we study genome function in mammalian cells,and are being widely used for interrogating critical genes and DNA elements essential for many biological processes.Here,we review CRISPR/Cas9-based genetic tools with an emphasis on CRISPR-mediated high throughput genetic screens in the mammalian genome.
基金supported by the National Key R&D Program of China(Grant No.2022YFC210590O)National Natural Science Foundation of China(Grant Nos.22178169,22008113)+2 种基金Shandong Taishan Industrial Experts Program(Grant No.202306155)China Postdoctoral Science Foundation(Grant No.2023M740370)the State Key Laboratory of Materials-Oriented Chemical Engineering(Grant No.KL-MCE-23A10).
文摘The development of green biomanufacturing technologies centered around advanced microbial cells has emerged as a hot research field in synthetic biology.As known,Komagataella phaffii is an unconventional yeast with unique biological characteristics,which is generally regarded as safe(GRAs).Owing to its wide substrate spectrum,K.phaffi has been widely genetically modified for valuable chemicals such as organic acids,fatty acids and some nature products.Actually,K.phaffii is very well known for the protein expression,and few reviews have systemically addressed the value-added chemicals by K.phaffii.Accordingly,this re-view will introduce the most advanced development of genetic editing tools of K.phaffi.Recent progresses and bottlenecks in the production of high-value chemicals using K.phaffii will also be summarized.Finally,future perspectives for the utilization of K.phaffii as a chassis cell used for high-value products synthesis will be discussed.
基金supported by National Research Foundation of Korea(NRF)grants funded by the Ministry of Science,ICT,and Future Planning[2017M3A9C4065958 and 2018R1A2B6002192]by Korea Drug Development Fund(KDDF)funded by Ministry of Science and ICT,Ministry of Trade,Industry,and Energy,and Ministry of Health and Welfare[RS-2023-00283544]by a grant from the Asan Institute for Life Sciences,Asan Medical Center,Seoul,Republic of Korea[2024IP0045].
文摘The CRISPR-Cas system is a powerful genetic engineering tool and can be conveniently used for the generation of diverse gene-knockout models.One CRISPR-Cas system,CRISPR-Cpf1(also known as Cas12a),recognizes the AT-rich protospacer-adjacent motif(PAM)present at the 5′end of the target sequence and requires CRISPR RNA(crRNA),but not transactivating crRNA(tracrRNA)for its activity.
