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ScienceDirect Editorial overview: Novel technologies in microbiology: Recent advances in techniques in microbiology Emmanuelle Charpentier and Luciano A Marraffini Current Opinion in Microbiology 2014, 19:viii–x For a complete overview see the Issue Available online 10th July 2014 http://dx.doi.org/10.1016/j.mib.2014.06.012 1369-5274/# 2014 Elsevier Ltd. All rights reserved.
Emmanuelle Charpentier1,2 1
Helmholtz Centre for Infection Research and Hannover Medical School, 38124 Braunschweig, Germany 2
The Laboratory for Molecular Infection Medicine Sweden, Umea˚ University, Umea˚, Sweden e-mail: [email protected]
Emmanuelle Charpentier is Professor at the Helmholtz Centre for Infection Research and Hannover Medical School in Germany and Umea˚ University in Sweden. Her main interests lie in the understanding of the mechanisms of regulation in bacterial infection and immunity. Her research programs aim to identify new RNAs and proteins and decipher their biogenesis, functions, and modes of action at the molecular and cellular level. Application of her research in biotechnology and medicine is well exemplified by the recent discovery of the CRISPR-Cas9 tool now broadly used for genome engineering in cells and organisms.
Luciano A Marraffini The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA e-mail: [email protected] Luciano A. Marraffini is an Assistant Professor and Head of the Laboratory of Bacteriology at The Rockefeller University. His research focuses on understanding the molecular mechanisms of CRISPR-Cas immunity and their role in the control of horizontal gene transfer between bacteria.
History has shown that microbiology has frequently been at the forefront of the discovery and development of novel technologies. A wide range of techniques from classical genetics to more sophisticated biophysics and systems biology have been developed and applied to understand the life of microbes starting from model microorganisms grown in flasks to more exotic microbes cultivated in simulated natural environment in interaction with their hosts and predators. A substantial number of molecular principles of microbiology have also been valuable sources to discover new or advance further technologies for a range of application beyond the microbiology field, revolutionizing biotechnology and biomedicine. In recent years, the field of microbiology is experiencing a rebirth owing to the development and application of various novel technologies aiming at a deeper understanding of microorganisms. How many new microbial species are to be discovered? How to improve fast and accurate identification of pathogens in the hospital setting? How to identify links between microorganisms and disease? How bacteria help to keep us healthy? How pathogenic and non-pathogenic microbes evolve in response to their environment? How new antibiotic resistances develop? How microbes communicate and exchange genetic material? How microbes defend themselves from and adapt to their environment and hosts? What are the specific regulations involved in homogenous and heterogeneous populations of cells at the single molecule and single cell levels? How many new key regulators and effectors are yet to be identified? What are the interactomes in play? How to visualize subcellular structures and processes? In this issue, Current Opinion in Microbiology has selected a series of articles highlighting some of the most recent technological developments and their applications in microbiology. These include high throughput sequencing for the analysis of genomes and transcriptomes, mass spectrometry for the identification of bacterial pathogens in the clinical setting and of metabolite production within complex microbial communities, and novel approaches for antimicrobial discovery and for the development of fuel-producing microorganisms. The continuous effort to combine innovative and sophisticated technologies has already advanced the analysis of microbes at an unprecedented level of detail and will be critical for researchers of the future to discover new fascinating concepts of biology. High Throughput Sequencing (HTS) has revolutionized microbiology revealing new insights into bacterial evolution, epidemiology, and pathogenesis. In their review, McAdam, Richardson and Fitzgerald summarize selected recent studies that have applied HTS to address fundamental questions in the biology of infectious diseases. For example, HTS has enabled high-resolution phylogenetic analyses of bacterial populations,
Current Opinion in Microbiology 2014, 19:viii–x
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Editorial overview: Novel technologies in microbiology Charpentier and Marraffini ix
providing a better understanding of bacterial evolution during infection and more precise tracing of origins and transmissions of outbreaks. HTS has revealed that bacterial pathogens can undergo considerable diversification during infection processes, and has offered a considerable improvement for global gene expression profiling studies. The combination of HTS and transposon mutagenesis has led to the development of a series of powerful approaches that facilitated the identification of the genes required for the survival of pathogens in their host and of other microbes in other environments. Future technological advances in HTS are likely to have a profound impact on the microbiology field. With the development of platforms capable of single-molecule sequencing with ever increasing read lengths, the technology already offers the possibility to assemble accurately individual species within a microbial community. Applications of HTS to non-cultivable organisms will aid the investigation of infectious diseases of unknown aetiology. The combination of novel culture-free methodologies and HTS approaches should also facilitate the rapid diagnosis and in silico determination of sensitivity profiles of pathogens in the clinical setting. While HTS of genomic DNA has pushed forward our understanding of bacterial evolution and speciation, HTS of bacterial transcripts (RNA-sequencing or RNA-seq) provided genome-wide gene expression profiling and transcript annotations at a single nucleotide resolution, allowing the identification of a large number of novel small regulatory RNAs and antisense RNAs. Sharma and Vogel discuss the development and recent applications of the differential RNA-seq (dRNA-seq) method. dRNAseq offers the additional feature to differentiate primary from processed RNA populations. The technology has initially been applied to describe the primary transcriptome of the gastric pathogen Helicobacter pylori, and has since been widely used to generate global maps of start sites of transcripts in various species, providing new insights into processing events of mRNAs and RNAs with regulatory functions. Describing selected examples, this review illustrates how the technology enabled new biological insights in bacterial gene regulation. The authors also comment on refinements and further improvement of the technology to be expected in the near future and suggest three main research areas where the technology could be valuable: single-cell RNA-seq, metatranscriptomics, and simultaneous RNA expression profiling of bacterial pathogens together with their eukaryotic hosts. Three reviews cover recent applications of advanced mass spectrometry (MS) technologies. Moore, Caprioli and Skaar review our current knowledge of advanced MS technologies that have been applied for the study of microorganisms. The authors highlight the potential of these biophysical methods as clinical tools for both the www.sciencedirect.com
diagnosis of pathogens in laboratories and the discovery of novel targets for therapeutic intervention. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is used to determine molecular profiles of small cell populations for rapid identification of microbes in diagnostic analysis. Histology-directed MS analyses allow the direct profiling of molecules from patient sera and tissues and the identification of biomarkers in discrete areas of infected tissues. With advanced mass spectrometry technologies, molecular profiling across tissue sections can also be achieved in a more systematic fashion and enable measurements of spatial distributions of molecules and analytes in specific regions in situ in both two-dimensional and three-dimensional analyses. The authors underline that challenges in the improvement of more advanced MS-based analytical instrumentations lie in part in the relative low abundance of microbial proteins and the small size of most microorganisms. Nevertheless, the authors highlight some additional recent developments such as advanced laser optics enabling the imaging of single cells and advanced electronics in new mass spectrometers facilitating the rapid acquisition of data for large 3D imaging data sets. The authors also mention the development of ionic matrices to improve the ionization of proteins or MALDI-compatible surfaces to help capture bacteria from biological samples. Aldridge and Rhee present an overview of the so-called metabolic technologies. Advances in NMR-based and MS-based methods have enabled the detection and quantitation of cellular metabolites. The application of these technologies to the microbiology field has begun to reveal an unexpected diversity of composition, structure and regulation of metabolic networks in microbes with substantial changes in metabolic needs triggered in different environments or growth conditions. The authors highlight the technological developments in this area, describing applications to precisely measure metabolite concentration and subcellular localization, to assign metabolic functions of unknown genes and to study the structure and regulation of metabolic networks. In a related review, Fang and Dorrestein summarize MS technologies that enable the direct detection and analysis of specialized metabolites produced by microbial colonies and communities. There is an increasing need to understand the detailed chemistry involved in microbial behavior and develop technologies that could benefit strain identification for clinical use. Over the last years, substantial efforts have been directed towards the development of more modern and sensitive instrumentations that integrate compatible microbial and mass spectrometry workflows. The authors describe MS techniques that have so far been mostly applied to microbiology. For example, imaging mass spectrometry and real-time mass spectrometry enable two-dimensional and three-dimensional visualization of metabolite distribution with little or no sample preparation. Recent developments make it now possible to map microbial molecules spatially, visualize the Current Opinion in Microbiology 2014, 19:viii–x
x Novel technologies in microbiology
molecules produced by living microbial colonies at the single cell level. The authors conclude that future advances towards the combination of MS with new molecular visualization tools and informatics approaches will improve the level of characterization of microbes and their chemical repertoire. The dramatic increase in infections caused by multi-resistant bacteria and the shortage in effective antibiotics has resulted in a renaissance in bacteriophage-based therapy and in the developing of novel approaches for the discovery of antimicrobials. In this issue, Citorik, Mimee and Lu discuss recent advances in bacteriophage-based technologies and the recent introduction of synthetic biology methods in this field. The authors have selected some examples demonstrating how synthetic biology has enabled the engineering of modified phages resulting in innovative next-generation bacteriophage-based tools for the study and treatment of infectious diseases. Phage display has been extensively used in this regard and for the development of novel therapeutics, and phage lysins have been investigated in recent years as potential antimicrobials. Bacteriophage components constitute a core set of parts in the synthetic biology toolbox. Phage-derived enzymes and technologies have improved genome engineering techniques for the tailoring of strains in specific applications, to generate genetic diversity and to study accelerated evolution. Charlop-Powers, Milshteyn and Brady focus their review on recent advances in metagenomic approaches for antibiotic discovery. Capture of DNA from the environment (eDNA) and subsequent identification and expression of biosynthetic gene clusters in heterologous hosts can provide means to decipher unexplored biosynthetic pathways encoded by the genomes of environmental bacteria and thus bridge biosynthetic diversity to drug discovery pipelines. The authors explain in detail the sequence-based methods that interrogate the biosynthetic content of metagenomic samples, identify lead targets, and allow the recovery of complete biosynthetic pathways from eDNA libraries. Activation of gene cluster expression is then followed by the production and discovery of small molecules. In contrast, functionbased methods will identify clones that are already biosynthetically active in a heterologous host by detecting a clone-induced phenotype in a host organism. The authors explain that novel technologies such as the Nanoporebased sequencing or single-cell and microdroplet-based methods have a clear potential to extend the power of whole genome sequencing to metagenomes. Robinson, Adolfsen and Brynildsen take on a different approach
Current Opinion in Microbiology 2014, 19:viii–x
for the discovery of new antimicrobials: the rational design of inhibitors of enzymes and biochemical pathways essential for bacterial survival that are absent from human cells. While this is a classical methodology for the discovery of antibiotics, the authors review efforts toward a novel target pathway: nitric oxide (NO) detoxification and repair. NO is a potent antimicrobial compound that immune cells produce to fight pathogens. Thus, to establish an infection, pathogens depend on pathways that neutralize NO radicals and repair the damage they exert on different biomolecules. Inhibitors of these pathways are under investigation as next-generation antibiotics. In particular, the authors focus on the use of quantitative kinetic modeling to improve the analysis and understanding of NO stress (and other broadly reactive antimicrobials) at systems level. In addition, this issue of Current Opinion in Microbiology includes two reviews in some of the most exciting areas of biotechnology: production of microbial biofuels and CRISPR-based genome editing technologies. Endophytic fungi have the property to produce volatile organic compounds (VOCs) with hydrocarbon-like properties when agricultural wastes are used as substrates. VOCs are identical or closely related to compounds found in diesel fuels and thus have the potential to be used as ‘green chemicals’ and fuels, also referred to as Mycodiesel. In his review, Strobel presents the history of the production of Mycodiesel by fungi, describes examples of fungi that produce VOCs and highlights some of the new methodologies that have been developed specifically for the study of fungal production of hydrocarbons. In principle, the microbe is isolated and identified, the composition in VOCs is determined and sequence information of the gene cluster responsible for VOC production is obtained, which is used to genetically manipulate the microbe to enhance production. There are also efforts to determine the ideal conditions for hydrocarbon production by fungi with the aim to produce ‘superproducing’ fungal strains. Strobel presents his views on the impact that these promising green chemicals and fuels may have in the chemical industry for a variety of industrial, medicinal, and household purposes. CRISPR-Cas systems are reviewed by Charpentier and Marraffini. They take on the history of these prokaryotic adaptive immune systems, the recent advances in our understanding of their mechanisms of action, and the exciting possibilities for using CRISPR-associated (Cas) RNA-guided nucleases for the precise genetic manipulation of bacterial, fungal, insect, plant and mammalian organisms.
www.sciencedirect.com
ScienceDirect Editorial overview: Novel technologies in microbiology: Recent advances in techniques in microbiology Emmanuelle Charpentier and Luciano A Marraffini Current Opinion in Microbiology 2014, 19:viii–x For a complete overview see the Issue Available online 10th July 2014 http://dx.doi.org/10.1016/j.mib.2014.06.012 1369-5274/# 2014 Elsevier Ltd. All rights reserved.
Emmanuelle Charpentier1,2 1
Helmholtz Centre for Infection Research and Hannover Medical School, 38124 Braunschweig, Germany 2
The Laboratory for Molecular Infection Medicine Sweden, Umea˚ University, Umea˚, Sweden e-mail: [email protected]
Emmanuelle Charpentier is Professor at the Helmholtz Centre for Infection Research and Hannover Medical School in Germany and Umea˚ University in Sweden. Her main interests lie in the understanding of the mechanisms of regulation in bacterial infection and immunity. Her research programs aim to identify new RNAs and proteins and decipher their biogenesis, functions, and modes of action at the molecular and cellular level. Application of her research in biotechnology and medicine is well exemplified by the recent discovery of the CRISPR-Cas9 tool now broadly used for genome engineering in cells and organisms.
Luciano A Marraffini The Rockefeller University, 1230 York Avenue, New York, NY 10065, USA e-mail: [email protected] Luciano A. Marraffini is an Assistant Professor and Head of the Laboratory of Bacteriology at The Rockefeller University. His research focuses on understanding the molecular mechanisms of CRISPR-Cas immunity and their role in the control of horizontal gene transfer between bacteria.
History has shown that microbiology has frequently been at the forefront of the discovery and development of novel technologies. A wide range of techniques from classical genetics to more sophisticated biophysics and systems biology have been developed and applied to understand the life of microbes starting from model microorganisms grown in flasks to more exotic microbes cultivated in simulated natural environment in interaction with their hosts and predators. A substantial number of molecular principles of microbiology have also been valuable sources to discover new or advance further technologies for a range of application beyond the microbiology field, revolutionizing biotechnology and biomedicine. In recent years, the field of microbiology is experiencing a rebirth owing to the development and application of various novel technologies aiming at a deeper understanding of microorganisms. How many new microbial species are to be discovered? How to improve fast and accurate identification of pathogens in the hospital setting? How to identify links between microorganisms and disease? How bacteria help to keep us healthy? How pathogenic and non-pathogenic microbes evolve in response to their environment? How new antibiotic resistances develop? How microbes communicate and exchange genetic material? How microbes defend themselves from and adapt to their environment and hosts? What are the specific regulations involved in homogenous and heterogeneous populations of cells at the single molecule and single cell levels? How many new key regulators and effectors are yet to be identified? What are the interactomes in play? How to visualize subcellular structures and processes? In this issue, Current Opinion in Microbiology has selected a series of articles highlighting some of the most recent technological developments and their applications in microbiology. These include high throughput sequencing for the analysis of genomes and transcriptomes, mass spectrometry for the identification of bacterial pathogens in the clinical setting and of metabolite production within complex microbial communities, and novel approaches for antimicrobial discovery and for the development of fuel-producing microorganisms. The continuous effort to combine innovative and sophisticated technologies has already advanced the analysis of microbes at an unprecedented level of detail and will be critical for researchers of the future to discover new fascinating concepts of biology. High Throughput Sequencing (HTS) has revolutionized microbiology revealing new insights into bacterial evolution, epidemiology, and pathogenesis. In their review, McAdam, Richardson and Fitzgerald summarize selected recent studies that have applied HTS to address fundamental questions in the biology of infectious diseases. For example, HTS has enabled high-resolution phylogenetic analyses of bacterial populations,
Current Opinion in Microbiology 2014, 19:viii–x
www.sciencedirect.com
Editorial overview: Novel technologies in microbiology Charpentier and Marraffini ix
providing a better understanding of bacterial evolution during infection and more precise tracing of origins and transmissions of outbreaks. HTS has revealed that bacterial pathogens can undergo considerable diversification during infection processes, and has offered a considerable improvement for global gene expression profiling studies. The combination of HTS and transposon mutagenesis has led to the development of a series of powerful approaches that facilitated the identification of the genes required for the survival of pathogens in their host and of other microbes in other environments. Future technological advances in HTS are likely to have a profound impact on the microbiology field. With the development of platforms capable of single-molecule sequencing with ever increasing read lengths, the technology already offers the possibility to assemble accurately individual species within a microbial community. Applications of HTS to non-cultivable organisms will aid the investigation of infectious diseases of unknown aetiology. The combination of novel culture-free methodologies and HTS approaches should also facilitate the rapid diagnosis and in silico determination of sensitivity profiles of pathogens in the clinical setting. While HTS of genomic DNA has pushed forward our understanding of bacterial evolution and speciation, HTS of bacterial transcripts (RNA-sequencing or RNA-seq) provided genome-wide gene expression profiling and transcript annotations at a single nucleotide resolution, allowing the identification of a large number of novel small regulatory RNAs and antisense RNAs. Sharma and Vogel discuss the development and recent applications of the differential RNA-seq (dRNA-seq) method. dRNAseq offers the additional feature to differentiate primary from processed RNA populations. The technology has initially been applied to describe the primary transcriptome of the gastric pathogen Helicobacter pylori, and has since been widely used to generate global maps of start sites of transcripts in various species, providing new insights into processing events of mRNAs and RNAs with regulatory functions. Describing selected examples, this review illustrates how the technology enabled new biological insights in bacterial gene regulation. The authors also comment on refinements and further improvement of the technology to be expected in the near future and suggest three main research areas where the technology could be valuable: single-cell RNA-seq, metatranscriptomics, and simultaneous RNA expression profiling of bacterial pathogens together with their eukaryotic hosts. Three reviews cover recent applications of advanced mass spectrometry (MS) technologies. Moore, Caprioli and Skaar review our current knowledge of advanced MS technologies that have been applied for the study of microorganisms. The authors highlight the potential of these biophysical methods as clinical tools for both the www.sciencedirect.com
diagnosis of pathogens in laboratories and the discovery of novel targets for therapeutic intervention. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS) is used to determine molecular profiles of small cell populations for rapid identification of microbes in diagnostic analysis. Histology-directed MS analyses allow the direct profiling of molecules from patient sera and tissues and the identification of biomarkers in discrete areas of infected tissues. With advanced mass spectrometry technologies, molecular profiling across tissue sections can also be achieved in a more systematic fashion and enable measurements of spatial distributions of molecules and analytes in specific regions in situ in both two-dimensional and three-dimensional analyses. The authors underline that challenges in the improvement of more advanced MS-based analytical instrumentations lie in part in the relative low abundance of microbial proteins and the small size of most microorganisms. Nevertheless, the authors highlight some additional recent developments such as advanced laser optics enabling the imaging of single cells and advanced electronics in new mass spectrometers facilitating the rapid acquisition of data for large 3D imaging data sets. The authors also mention the development of ionic matrices to improve the ionization of proteins or MALDI-compatible surfaces to help capture bacteria from biological samples. Aldridge and Rhee present an overview of the so-called metabolic technologies. Advances in NMR-based and MS-based methods have enabled the detection and quantitation of cellular metabolites. The application of these technologies to the microbiology field has begun to reveal an unexpected diversity of composition, structure and regulation of metabolic networks in microbes with substantial changes in metabolic needs triggered in different environments or growth conditions. The authors highlight the technological developments in this area, describing applications to precisely measure metabolite concentration and subcellular localization, to assign metabolic functions of unknown genes and to study the structure and regulation of metabolic networks. In a related review, Fang and Dorrestein summarize MS technologies that enable the direct detection and analysis of specialized metabolites produced by microbial colonies and communities. There is an increasing need to understand the detailed chemistry involved in microbial behavior and develop technologies that could benefit strain identification for clinical use. Over the last years, substantial efforts have been directed towards the development of more modern and sensitive instrumentations that integrate compatible microbial and mass spectrometry workflows. The authors describe MS techniques that have so far been mostly applied to microbiology. For example, imaging mass spectrometry and real-time mass spectrometry enable two-dimensional and three-dimensional visualization of metabolite distribution with little or no sample preparation. Recent developments make it now possible to map microbial molecules spatially, visualize the Current Opinion in Microbiology 2014, 19:viii–x
x Novel technologies in microbiology
molecules produced by living microbial colonies at the single cell level. The authors conclude that future advances towards the combination of MS with new molecular visualization tools and informatics approaches will improve the level of characterization of microbes and their chemical repertoire. The dramatic increase in infections caused by multi-resistant bacteria and the shortage in effective antibiotics has resulted in a renaissance in bacteriophage-based therapy and in the developing of novel approaches for the discovery of antimicrobials. In this issue, Citorik, Mimee and Lu discuss recent advances in bacteriophage-based technologies and the recent introduction of synthetic biology methods in this field. The authors have selected some examples demonstrating how synthetic biology has enabled the engineering of modified phages resulting in innovative next-generation bacteriophage-based tools for the study and treatment of infectious diseases. Phage display has been extensively used in this regard and for the development of novel therapeutics, and phage lysins have been investigated in recent years as potential antimicrobials. Bacteriophage components constitute a core set of parts in the synthetic biology toolbox. Phage-derived enzymes and technologies have improved genome engineering techniques for the tailoring of strains in specific applications, to generate genetic diversity and to study accelerated evolution. Charlop-Powers, Milshteyn and Brady focus their review on recent advances in metagenomic approaches for antibiotic discovery. Capture of DNA from the environment (eDNA) and subsequent identification and expression of biosynthetic gene clusters in heterologous hosts can provide means to decipher unexplored biosynthetic pathways encoded by the genomes of environmental bacteria and thus bridge biosynthetic diversity to drug discovery pipelines. The authors explain in detail the sequence-based methods that interrogate the biosynthetic content of metagenomic samples, identify lead targets, and allow the recovery of complete biosynthetic pathways from eDNA libraries. Activation of gene cluster expression is then followed by the production and discovery of small molecules. In contrast, functionbased methods will identify clones that are already biosynthetically active in a heterologous host by detecting a clone-induced phenotype in a host organism. The authors explain that novel technologies such as the Nanoporebased sequencing or single-cell and microdroplet-based methods have a clear potential to extend the power of whole genome sequencing to metagenomes. Robinson, Adolfsen and Brynildsen take on a different approach
Current Opinion in Microbiology 2014, 19:viii–x
for the discovery of new antimicrobials: the rational design of inhibitors of enzymes and biochemical pathways essential for bacterial survival that are absent from human cells. While this is a classical methodology for the discovery of antibiotics, the authors review efforts toward a novel target pathway: nitric oxide (NO) detoxification and repair. NO is a potent antimicrobial compound that immune cells produce to fight pathogens. Thus, to establish an infection, pathogens depend on pathways that neutralize NO radicals and repair the damage they exert on different biomolecules. Inhibitors of these pathways are under investigation as next-generation antibiotics. In particular, the authors focus on the use of quantitative kinetic modeling to improve the analysis and understanding of NO stress (and other broadly reactive antimicrobials) at systems level. In addition, this issue of Current Opinion in Microbiology includes two reviews in some of the most exciting areas of biotechnology: production of microbial biofuels and CRISPR-based genome editing technologies. Endophytic fungi have the property to produce volatile organic compounds (VOCs) with hydrocarbon-like properties when agricultural wastes are used as substrates. VOCs are identical or closely related to compounds found in diesel fuels and thus have the potential to be used as ‘green chemicals’ and fuels, also referred to as Mycodiesel. In his review, Strobel presents the history of the production of Mycodiesel by fungi, describes examples of fungi that produce VOCs and highlights some of the new methodologies that have been developed specifically for the study of fungal production of hydrocarbons. In principle, the microbe is isolated and identified, the composition in VOCs is determined and sequence information of the gene cluster responsible for VOC production is obtained, which is used to genetically manipulate the microbe to enhance production. There are also efforts to determine the ideal conditions for hydrocarbon production by fungi with the aim to produce ‘superproducing’ fungal strains. Strobel presents his views on the impact that these promising green chemicals and fuels may have in the chemical industry for a variety of industrial, medicinal, and household purposes. CRISPR-Cas systems are reviewed by Charpentier and Marraffini. They take on the history of these prokaryotic adaptive immune systems, the recent advances in our understanding of their mechanisms of action, and the exciting possibilities for using CRISPR-associated (Cas) RNA-guided nucleases for the precise genetic manipulation of bacterial, fungal, insect, plant and mammalian organisms.
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