Download and read online CRISPR Cas Systems in PDF and EPUB CRISPR/Cas is a recently described defense system that protects bacteria and archaea against invasion by mobile genetic elements such as viruses and plasmids. A wide spectrum of distinct CRISPR/Cas systems has been identified in at least half of the available prokaryotic genomes. On-going structural and functional analyses have resulted in a far greater insight into the functions and possible applications of these systems, although many secrets remain to be discovered. In this book, experts summarize the state of the art in this exciting field.
Download and read online Understanding and Exploiting the Properties of Type I CRISPR Cas Systems in PDF and EPUB
Download and read online CRISPR Cas in PDF and EPUB The development of CRISPR-Cas technology is revolutionizing biology. Based on machinery bacteria use to target foreign nucleic acids, these powerful techniques allow investigators to edit nucleic acids and modulate gene expression more rapidly and accurately than ever before. Featuring contributions from leading figures in the CRISPR-Cas field, this laboratory manual presents a state-of-the-art guide to the technology. It includes step-by-step protocols for applying CRISPR-Cas-based techniques in various systems, including yeast, zebrafish, Drosophila, mice, and cultured cells (e.g., human pluripotent stem cells). The contributors cover web-based tools and approaches for designing guide RNAs that precisely target genes of interest, methods for preparing and delivering CRISPR-Cas reagents into cells, and ways to screen for cells that harbor the desired genetic changes. Strategies for optimizing CRISPR-Cas in each system--especially for minimizing off-target effects--are also provided. Authors also describe other applications of the CRISPR-Cas system, including its use for regulating genome activation and repression, and discuss the development of next-generation CRISPR-Cas tools. The book is thus an essential laboratory resource for all cell, molecular, and developmental biologists, as well as biochemists, geneticists, and all who seek to expand their biotechnology toolkits.
Download and read online The P Aeruginosa CRISPR Cas System and Its Role in Bacteria bacteriophage Interactions in PDF and EPUB CRISPR/Cas systems are a diverse family of small RNA pathways that are commonly described as a resistance mechanism to infection by lytic bacteriophage and invasion by conjugative plasmids. In the current work we demonstrate that CRISPR/Cas systems are capable of multiple phenotypic outputs, drastically altering our perception of their role in prokaryotic biology. We begin with the initial observation that the P. aeruginosa Yersinia subtype CRISPR/Cas system interacts with chromosomally integrated lysogenic bacteriophage DMS3 to inhibit biofilm formation and swarming motility, but in its native form, does NOT mediate bacteriophage resistance. Genetic analysis established for the first time that the CRISPR associated (cas) genes: csy1, csy2, csy3, csy4, and cas3 are required for activity of the Yersinia subtype CRISPR/Cas system. I also provide the first evidence for Cas1-mediated new spacer insertion in the Yersinia subtype CRISPR/Cas system. Using mutational analysis I showed that the CRISPR derived small RNA crRNA[subscript C]2[subscript S]1 directly and site-sepecifically interacts with bacteriophage DMS3 to inhibit biofilm formation. The relative low level of complementary required for crRNA[subscript C]2[subscript S]1 to interact with DMS3 has helped fundamentally change the view of the crRNA-target interactions, leading to new models to explain the targeting of crRNAs that lack 100% identity to their target sequences. Systematic chromosomal mutation of the bacteriophage target sequence provided further insights into crRNA-target determinants required for CRISPR/Cas interactions. Importantly, I found that a single nucleotide mutation in the bateriophage target of the crRNA[subscript C]2[subscript S]1 sequence alterd the output of this CRISPR/Cas system from biofilm inhibition to complete resistance to bacteriophage infection. Using the discrete crRNA[subscript C]2[subscript S]1 phenotypic outputs of biofilm inhibition and bacteriophage resistance we established what DNA is targeted by the Yersinia subtype CRISPR/Cas sytem to mediate both biofilm inhibition and bacteriophage resistance. Use of classic reporter fusions demonstrated that crRNA targeting of WT bateriophage DMS3 results in misregulation of late bacteriophage gene expression, providing the first concrete evidence for crRNA-mediated gene regulation. Surprisingly, in contrast with the current model of CRISPR/Cas system function, no cleavage of bacteriophage DNA was observed for either crRNA[subscript C]2[subscript S]1-mediated biofilm inhibition or bacteriophage resistance phenotypes. I propose a mode in which crRNA[subscript C]2[subscript S]1 and associated Cas proteins recruit RNA polymerase to the target site, resulting in either active transcription, or alternatively, binding of RNA polymerase in the absence of transription resulting in a block in DNA replication. This work provides new mechanistic insights into one of the 8 CRISPR/Cas subtypes, and fundamentally alters the current narrow view fo how CRISPR/Cas systems modulate microbial biology.
Download and read online CRISPR Cas Systems in Lactic Acid Bacteria in PDF and EPUB
Download and read online The Dual Role of the Pseudomonas Aeruginosa Type I F CRISPR Cas System in Group Behavior Modification and Viral Immunity in PDF and EPUB CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) systems have been characterized as adaptive immune systems in bacteria and archaea, providing defense against mobile genetic elements, including bacteriophage, through the incorporation of a short sequence of invader DNA into the CRISPR array. These systems are diverse, currently classified into two classes and six separate types, and have been implicated in functions beyond adaptive immunity. Pseudomonas aeruginosa strain UCBPP-PA14 contains an active Type I-F CRISPR-Cas system, and in the current work this CRISPR-Cas system is characterized in a non-canonical role inhibiting swarming and reducing biofilm formation, as well as in a canonical role providing CRISPR-immunity against a lytic bacteriophage through the incorporation of new “spacers” into both CRISPR arrays on the P. aeruginosa genome. The CRISPRmediated biofilm reduction and swarming inhibition phenotype depends on the presence of a protospacer sequence within the stably lysogenized bacteriophage DMS3, and targeting by a partially matching spacer in the CRISPR2 locus. My studies show that this targeting event triggers a DNA damage response through Cas3 nickase activity, and induces expression of the pyocin- and Alp-encoding lysis genes in a RecA-dependent manner. Loss of biofilm formation and swarming is due to surface-dependent, pyocinand Alp-mediated cell lysis induced by as-of-yet undescribed mechanism. In regards to the P. aeruginosa Type I-F CRISPR-Cas system functioning as a canonical system, I found biofilm-grown P. aeruginosa cells rapidly acquire protective spacers against DMS3 vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. Furthermore, by selecting for biofilm-grown cells during bacteriophage challenge, I developed a system enriching for CRISPR adaptive immunity over other means of phageresistance. I found a large discrepancy in efficiency between the putative priming spacers in the native CRISPR2 array and determined the conserved cas gene cas1 is required for CRISPR adaptation, recG contributes to (but is not required for) primed adaptation, and recD is dispensable for primed adaptation. These findings support a model of Type I CRISPR adaptation in a native system that were previously reported in studies using artificial expression constructs or mutant strains. Additionally our data suggest that CRISPR-mediated immunity in a biofilm may be one reason many P. aeruginosa strains maintain a CRISPR-Cas system. Overall the work presented in my thesis demonstrates how a canonical CRISPR-Cas system can function in alternative roles while still providing adaptive immunity against attacking bacteriophages, broadening our understanding of not only the Type I-F CRISPR-Cas subtype, but of CRISPR-Cas systems and bacterial biology in general.
Download and read online The CRISPR Cas System in PDF and EPUB The use of CRISPR/Cas technology for genome editing suggests many potential applications, including the alteration of the germline of humans, animals and food crops. The speed and efficiency of the CRISPR/Cas system make it a potentially useful system for gene therapy. In this volume expert international authors provide a useful and timely review of the applications of the CRISPR/Cas system across diverse fields and explore further avenues and research directions of this novel and powerful editing technology. The technology and its application are reviewed with respect to reproduction and development, immunity and genetic diseases, system structure and system specificity. Some of the potential problems of the CRISPR/Cas system are also discussed, in particular the specificity of the system: this remains an important topic as improvement could lead to the more direct and efficient use of the CRISPR/Cas system in clinical settings. The authors also debate ethical concerns associated with this powerful new technology. This volume is a rigorous review of the applications and new opportunities for the CRISPR/Cas system and provides a stimulus for current and future research. An invaluable guide for all scientists working in the fields of genome editing and gene therapy the book is also recommended for all life sciences libraries.
Download and read online Regulatory RNAs in Prokaryotes in PDF and EPUB This book provides a comprehensive and up-to-date collection of review articles focusing on RNA-mediated regulation in prokaryotes. The various modes of action include the direct interaction with proteins, direct sensing of metabolites or of physical parameters, and the interaction with RNAs to stimulate or prevent binding of ribosomes or to stimulate degradation. Written by leading experts in the field, the book covers small RNA functions, RNA thermometers, riboswitches, the diversity of small RNA-guided CRISPR-Cas defense systems and selected RNA chaperons in both prokaryotic domains, bacteria and archaea. Recent advances towards the computational identification of regulatory RNAs and their targets are included and particular attention is paid to small RNA in pathogenic bacteria. This volume is the only one exclusively covering regulatory RNAs in the prokaryotic domains to date, making it essential literature for anyone interested in RNA function and gene regulation and a valuable resource for teaching these concepts.
Download and read online Targeted Genome Editing Using Site Specific Nucleases in PDF and EPUB This book serves as an introduction to targeted genome editing, beginning with the background of this rapidly developing field and methods for generation of engineered nucleases. Applications of genome editing tools are then described in detail, in iPS cells and diverse organisms such as mice, rats, marine invertebrates, fish, frogs, and plants. Tools that are mentioned include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas9, all of which have received much attention in recent years as breakthrough technologies. Genome editing with engineered nucleases allows us to precisely change the target genome of living cells and is a powerful way to control functional genes. It is feasible in almost all organisms ranging from bacteria to plants and animals, as well as in cultured cells such as ES and iPS cells. Various genome modifications have proven successful, including gene knockout and knock-in experiments with targeting vectors and chromosomal editing. Genome editing technologies hold great promise for the future, for example in biomedical research, clinical medicine, and generation of crops and livestock with desirable traits. A wide range of readers will find this book interesting, and with its focus on applications in a variety of organisms and cells, the book will be valuable for life scientists in all fields.
Download and read online Protein Families in PDF and EPUB New insights into the evolution and nature of proteins Exploring several distinct approaches, this book describes the methods for comparing protein sequences and protein structures in order to identify homologous relationships and classify proteins and protein domains into evolutionary families. Readers will discover the common features as well as the key philosophical differences underlying the major protein classification systems, including Pfam, Panther, SCOP, and CATH. Moreover, they'll discover how these systems can be used to understand the evolution of protein families as well as understand and predict the degree to which structural and functional information are shared between relatives in a protein family. Edited and authored by leading international experts, Protein Families offers new insights into protein families that are important to medical research as well as protein families that help us understand biological systems and key biological processes such as cell signaling and the immune response. The book is divided into three sections: Section I: Concepts Underlying Protein Family Classification reviews the major strategies for identifying homologous proteins and classifying them into families. Section II: In-Depth Reviews of Protein Families focuses on some fascinating super protein families for which we have substantial amounts of sequence, structural and functional data, making it possible to trace the emergence of functionally diverse relatives. Section III: Review of Protein Families in Important Biological Systems examines protein families associated with a particular biological theme, such as the cytoskeleton. All chapters are extensively illustrated, including depictions of evolutionary relationships. References at the end of each chapter guide readers to original research papers and reviews in the field. Covering protein family classification systems alongside detailed descriptions of select protein families, this book offers biochemists, molecular biologists, protein scientists, structural biologists, and bioinformaticians new insight into the evolution and nature of proteins.
Download and read online CRISPR in PDF and EPUB This volume presents a list of cutting-edge protocols for the study of CRISPR-Cas defense systems and their applications at the genomic, genetic, biochemical and structural levels. CRISPR: Methods and Protocols guides readers through techniques that have been developed specifically for the analysis of CRISPR-Cas and techniques adapted from standard protocols of DNA, RNA and protein biology. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Authoritative and cutting-edge, CRISPR: Methods and Protocols provides a broad list of tools and techniques to study the interdisciplinary aspects of the prokaryotic CRISPR-Cas defense systems.
Download and read online Bacteriophages in PDF and EPUB This volume, the first of a two-part series, covers topics including historical, ecological and evolutionary considerations, genomics and molecular biology, and interaction of phages with their hosts. Key features: * Contributions from leading authorities * Informs and updates on all the latest developments in the field
Download and read online Salmonellosis New Insights for the Healthcare Professional 2011 Edition in PDF and EPUB Salmonellosis: New Insights for the Healthcare Professional: 2011 Edition is a ScholarlyEditions™ eBook that delivers timely, authoritative, and comprehensive information about Salmonellosis. The editors have built Salmonellosis: New Insights for the Healthcare Professional: 2011 Edition on the vast information databases of ScholarlyNews.™ You can expect the information about Salmonellosis in this eBook to be deeper than what you can access anywhere else, as well as consistently reliable, authoritative, informed, and relevant. The content of Salmonellosis: New Insights for the Healthcare Professional: 2011 Edition has been produced by the world’s leading scientists, engineers, analysts, research institutions, and companies. All of the content is from peer-reviewed sources, and all of it is written, assembled, and edited by the editors at ScholarlyEditions™ and available exclusively from us. You now have a source you can cite with authority, confidence, and credibility. More information is available at http://www.ScholarlyEditions.com/.
Download and read online Functional analysis of PA28 in MCF 7 breast cancer cells by overexpression CRISPR Cas9 mediated gene silencing in PDF and EPUB Master's Thesis from the year 2016 in the subject Chemistry - Bio-chemistry, grade: 1,7, Free University of Berlin, course: Biochemie, language: English, abstract: The CRISPR-Cas system derived from bacteria and archaea adaptive immune system is a high potential method for fast genome editing that promises to revolutionize previous genome engineering. It is based on the specific targeted induced double strand break by an endonuclease. In elapsed studies PA28γ figured out as an important key molecule involved in cell cycle regulation, cell signaling transcription, immune response and apoptosis. Recent investigations showed p53 to be a target of PA28γ enhanced ubiquitination via MDM2 and subsequent proteasomal degradation. Otherwise mutant p53 (R248Q) has been shown as suppressor of the REGγ promotor. This study aimed the CRISPR-Cas mediated gene knockout of PSME3 and tp53 in Caspase3 lacking MCF-7 breast cancer cells to investigate apoptosis. A user-developed protocol was established to implement the Multiplex CRISPR/Cas9 Assembly System Kit and the alone standing pSpCas9(BB)-2A-GFP plasmid provided by Takashi Yamamoto and Feng Zhang (Ran et al. 2013, Sakuma et al. 2014) for the generation of knockout cells. The cloning of gRNA harboring plasmids targeting PSME3 exon1/exon4 as well as tp53 exon1_1/exon1_2 was fast and in a high efficient fashion but a verification of the final constructs via T7 Endonuclease I assay was not possible. Interestingly, using fluorescent microscopy different gRNAs cloned in the CRISPR plasmids revealed variant apparent transfection efficiencies or GFP plasmid or protein stability. Furthermore, PA28γ targeted cells showed a better survival than p53 knockout cells. Therefore, also no tp53 targeted cells survived the serial dilution and clonal selection over an eight week period. PSME3 exon1_F1, exon4_C8 and exon4_B9 revealed PA28γ levels of about 50% compared to the untransfected wild type cells in Western Blot analyses. This could be caused by a heterozygous knockout of the PSME3 gene on chromosome17. One single cell clone (PSME3 exon4_F9) maybe carrying a gain of the PSME3 gene, undergoing interchromosomal recombination or only was hidden at one allele by the Cas9 enzyme showed 75% for PA28γ levels. In summary CRISPR-Cas enabled us probable to modify the PSME3 and tp53 gene in MCF-7 cells resulting in altered survivals of the transfected cells. Additionally, first investigations of the new established MCF-7 PSME3 knockout cell lines considering the PA28γ protein level showed a successful 50% reduction. It was not possible to study any apoptosis related behavior.
Download and read online RNA Regulation in PDF and EPUB Based on one of the leading encyclopedic resources in cell and molecular biology worldwide, this two-volume work contains more than 75% new content, not previously published in the Encyclopedia. All the other chapters have been carefully updated. The result is a comprehensive overview of the different functions of the various forms of RNA in living organisms, with each contributor carefully selected and an internationally recognized expert on his or her field. Special focus is on the different forms of expression regulation through RNA, with medical applications in the treatment of diseases -- from cancers and immune responses to infections and aging -- covered in detail. At least 45 of the 55 articles are new content previously not published in the Encyclopedia.