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Sample records for adaptive systems cas

  1. Adaptation in CRISPR-Cas Systems.

    PubMed

    Sternberg, Samuel H; Richter, Hagen; Charpentier, Emmanuelle; Qimron, Udi

    2016-03-17

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in prokaryotes. The system preserves memories of prior infections by integrating short segments of foreign DNA, termed spacers, into the CRISPR array in a process termed adaptation. During the past 3 years, significant progress has been made on the genetic requirements and molecular mechanisms of adaptation. Here we review these recent advances, with a focus on the experimental approaches that have been developed, the insights they generated, and a proposed mechanism for self- versus non-self-discrimination during the process of spacer selection. We further describe the regulation of adaptation and the protein players involved in this fascinating process that allows bacteria and archaea to harbor adaptive immunity.

  2. CRISPR-Cas systems: Prokaryotes upgrade to adaptive immunity.

    PubMed

    Barrangou, Rodolphe; Marraffini, Luciano A

    2014-04-24

    Clustered regularly interspaced short palindromic repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing and can be repurposed for numerous DNA targeting applications including transcriptional control.

  3. CRISPR-Cas: evolution of an RNA-based adaptive immunity system in prokaryotes.

    PubMed

    Koonin, Eugene V; Makarova, Kira S

    2013-05-01

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR-associated genes) is an adaptive immunity system in bacteria and archaea that functions via a distinct self-non-self recognition mechanism that is partially analogous to the mechanism of eukaryotic RNA interference (RNAi). The CRISPR-Cas system incorporates fragments of virus or plasmid DNA into the CRISPR repeat cassettes and employs the processed transcripts of these spacers as guide RNAs to cleave the cognate foreign DNA or RNA. The Cas proteins, however, are not homologous to the proteins involved in RNAi and comprise numerous, highly diverged families. The majority of the Cas proteins contain diverse variants of the RNA recognition motif (RRM), a widespread RNA-binding domain. Despite the fast evolution that is typical of the cas genes, the presence of diverse versions of the RRM in most Cas proteins provides for a simple scenario for the evolution of the three distinct types of CRISPR-cas systems. In addition to several proteins that are directly implicated in the immune response, the cas genes encode a variety of proteins that are homologous to prokaryotic toxins that typically possess nuclease activity. The predicted toxins associated with CRISPR-Cas systems include the essential Cas2 protein, proteins of COG1517 that, in addition to a ligand-binding domain and a helix-turn-helix domain, typically contain different nuclease domains and several other predicted nucleases. The tight association of the CRISPR-Cas immunity systems with predicted toxins that, upon activation, would induce dormancy or cell death suggests that adaptive immunity and dormancy/suicide response are functionally coupled. Such coupling could manifest in the persistence state being induced and potentially providing conditions for more effective action of the immune system or in cell death being triggered when immunity fails.

  4. CRISPR-Cas Adaptive Immune Systems of the Sulfolobales: Unravelling Their Complexity and Diversity

    PubMed Central

    Garrett, Roger A.; Shah, Shiraz A.; Erdmann, Susanne; Liu, Guannan; Mousaei, Marzieh; León-Sobrino, Carlos; Peng, Wenfang; Gudbergsdottir, Soley; Deng, Ling; Vestergaard, Gisle; Peng, Xu; She, Qunxin

    2015-01-01

    The Sulfolobales have provided good model organisms for studying CRISPR-Cas systems of the crenarchaeal kingdom of the archaea. These organisms are infected by a wide range of exceptional archaea-specific viruses and conjugative plasmids, and their CRISPR-Cas systems generally exhibit extensive structural and functional diversity. They carry large and multiple CRISPR loci and often multiple copies of diverse Type I and Type III interference modules as well as more homogeneous adaptation modules. These acidothermophilic organisms have recently provided seminal insights into both the adaptation process, the diverse modes of interference, and their modes of regulation. The functions of the adaptation and interference modules tend to be loosely coupled and the stringency of the crRNA-DNA sequence matching during DNA interference is relatively low, in contrast to some more streamlined CRISPR-Cas systems of bacteria. Despite this, there is evidence for a complex and differential regulation of expression of the diverse functional modules in response to viral infection. Recent work also supports critical roles for non-core Cas proteins, especially during Type III-directed interference, and this is consistent with these proteins tending to coevolve with core Cas proteins. Various novel aspects of CRISPR-Cas systems of the Sulfolobales are considered including an alternative spacer acquisition mechanism, reversible spacer acquisition, the formation and significance of antisense CRISPR RNAs, and a novel mechanism for avoidance of CRISPR-Cas defense. Finally, questions regarding the basis for the complexity, diversity, and apparent redundancy, of the intracellular CRISPR-Cas systems are discussed. PMID:25764276

  5. Adapting to new threats: the generation of memory by CRISPR-Cas immune systems.

    PubMed

    Heler, Robert; Marraffini, Luciano A; Bikard, David

    2014-07-01

    Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci and their associated genes (cas) confer bacteria and archaea with adaptive immunity against phages and other invading genetic elements. A fundamental requirement of any immune system is the ability to build a memory of past infections in order to deal more efficiently with recurrent infections. The adaptive feature of CRISPR-Cas immune systems relies on their ability to memorize DNA sequences of invading molecules and integrate them in between the repetitive sequences of the CRISPR array in the form of 'spacers'. The transcription of a spacer generates a small antisense RNA that is used by RNA-guided Cas nucleases to cleave the invading nucleic acid in order to protect the cell from infection. The acquisition of new spacers allows the CRISPR-Cas immune system to rapidly adapt against new threats and is therefore termed 'adaptation'. Recent studies have begun to elucidate the genetic requirements for adaptation and have demonstrated that rather than being a stochastic process, the selection of new spacers is influenced by several factors. We review here our current knowledge of the CRISPR adaptation mechanism.

  6. Detecting natural adaptation of the Streptococcus thermophilus CRISPR-Cas systems in research and classroom settings.

    PubMed

    Hynes, Alexander P; Lemay, Marie-Laurence; Trudel, Luc; Deveau, Hélène; Frenette, Michel; Tremblay, Denise M; Moineau, Sylvain

    2017-03-01

    CRISPR (clustered regularly interspaced short palindromic repeats)-Cas systems have been adapted into a powerful genome-editing tool. The basis for the flexibility of the tool lies in the adaptive nature of CRISPR-Cas as a bacterial immune system. Here, we describe a protocol to experimentally demonstrate the adaptive nature of this bacterial immune system by challenging the model organism for the study of CRISPR adaptation, Streptococcus thermophilus, with phages in order to detect natural CRISPR immunization. A bacterial culture is challenged with lytic phages, the surviving cells are screened by PCR for expansion of their CRISPR array and the newly acquired specificities are mapped to the genome of the phage. Furthermore, we offer three variants of the assay to (i) promote adaptation by challenging the system using defective viruses, (ii) challenge the system using plasmids to generate plasmid-resistant strains and (iii) bias the system to obtain natural immunity against a specifically targeted DNA sequence. The core protocol and its variants serve as a means to explore CRISPR adaptation, discover new CRISPR-Cas systems and generate bacterial strains that are resistant to phages or refractory to undesired genes or plasmids. In addition, the core protocol has served in teaching laboratories at the undergraduate level, demonstrating both its robust nature and educational value. Carrying out the core protocol takes 4 h of hands-on time over 7 d. Unlike sequence-based methods for detecting natural CRISPR adaptation, this phage-challenge-based approach results in the isolation of CRISPR-immune bacteria for downstream characterization and use.

  7. CALM: Complex Adaptive System (CAS)-Based Decision Support for Enabling Organizational Change

    NASA Astrophysics Data System (ADS)

    Adler, Richard M.; Koehn, David J.

    Guiding organizations through transformational changes such as restructuring or adopting new technologies is a daunting task. Such changes generate workforce uncertainty, fear, and resistance, reducing morale, focus and performance. Conventional project management techniques fail to mitigate these disruptive effects, because social and individual changes are non-mechanistic, organic phenomena. CALM (for Change, Adaptation, Learning Model) is an innovative decision support system for enabling change based on CAS principles. CALM provides a low risk method for validating and refining change strategies that combines scenario planning techniques with "what-if" behavioral simulation. In essence, CALM "test drives" change strategies before rolling them out, allowing organizations to practice and learn from virtual rather than actual mistakes. This paper describes the CALM modeling methodology, including our metrics for measuring organizational readiness to respond to change and other major CALM scenario elements: prospective change strategies; alternate futures; and key situational dynamics. We then describe CALM's simulation engine for projecting scenario outcomes and its associated analytics. CALM's simulator unifies diverse behavioral simulation paradigms including: adaptive agents; system dynamics; Monte Carlo; event- and process-based techniques. CALM's embodiment of CAS dynamics helps organizations reduce risk and improve confidence and consistency in critical strategies for enabling transformations.

  8. A CRISPR/Cas9 system adapted for gene editing in marine algae

    PubMed Central

    Nymark, Marianne; Sharma, Amit Kumar; Sparstad, Torfinn; Bones, Atle M.; Winge, Per

    2016-01-01

    Here we report that the CRISPR/Cas9 technology can be used to efficiently generate stable targeted gene mutations in microalgae, using the marine diatom Phaeodactylum tricornutum as a model species. Our vector design opens for rapid and easy adaption of the construct to the target chosen. To screen for CRISPR/Cas9 mutants we employed high resolution melting based PCR assays, mutants were confirmed by sequencing and further validated by functional analyses. PMID:27108533

  9. CRISPR-Cas: From the Bacterial Adaptive Immune System to a Versatile Tool for Genome Engineering.

    PubMed

    Kirchner, Marion; Schneider, Sabine

    2015-11-09

    The field of biology has been revolutionized by the recent advancement of an adaptive bacterial immune system as a universal genome engineering tool. Bacteria and archaea use repetitive genomic elements termed clustered regularly interspaced short palindromic repeats (CRISPR) in combination with an RNA-guided nuclease (CRISPR-associated nuclease: Cas) to target and destroy invading DNA. By choosing the appropriate sequence of the guide RNA, this two-component system can be used to efficiently modify, target, and edit genomic loci of interest in plants, insects, fungi, mammalian cells, and whole organisms. This has opened up new frontiers in genome engineering, including the potential to treat or cure human genetic disorders. Now the potential risks as well as the ethical, social, and legal implications of this powerful new technique move into the limelight.

  10. Interference-driven spacer acquisition is dominant over naive and primed adaptation in a native CRISPR–Cas system

    PubMed Central

    Staals, Raymond H. J.; Jackson, Simon A.; Biswas, Ambarish; Brouns, Stan J. J.; Brown, Chris M.; Fineran, Peter C.

    2016-01-01

    CRISPR–Cas systems provide bacteria with adaptive immunity against foreign nucleic acids by acquiring short, invader-derived sequences called spacers. Here, we use high-throughput sequencing to analyse millions of spacer acquisition events in wild-type populations of Pectobacterium atrosepticum. Plasmids not previously encountered, or plasmids that had escaped CRISPR–Cas targeting via point mutation, are used to provoke naive or primed spacer acquisition, respectively. The origin, location and order of spacer acquisition show that spacer selection through priming initiates near the site of CRISPR–Cas recognition (the protospacer), but on the displaced strand, and is consistent with 3′–5′ translocation of the Cas1:Cas2-3 acquisition machinery. Newly acquired spacers determine the location and strand specificity of subsequent spacers and demonstrate that interference-driven spacer acquisition (‘targeted acquisition') is a major contributor to adaptation in type I-F CRISPR–Cas systems. Finally, we show that acquisition of self-targeting spacers is occurring at a constant rate in wild-type cells and can be triggered by foreign DNA with similarity to the bacterial chromosome. PMID:27694798

  11. New CRISPR-Cas systems discovered.

    PubMed

    Yang, Hui; Patel, Dinshaw J

    2017-03-01

    In bacteria and archaea, CRISPR-Cas adaptive immune systems utilize RNA-guided endonucleases to defend against invasion by foreign nucleic acids of bacteriophage, virus and plasmid origin. In a recent paper published in Nature, Burstein et al. identified the first Cas9 protein in uncultivated archaea and two novel CRISPR-CasX and CRISPR-CasY systems in uncultivated bacteria by capitalizing on analysis of terabase-scale metagenomic datasets from natural uncultivated organisms.

  12. [Advances in molecular mechanisms of adaptive immunity mediated by type I-E CRISPR/Cas system--A review].

    PubMed

    Sun, Dongchang; Qiu, Juanping

    2016-01-04

    To better adapt to the environment, prokaryocyte can take up exogenous genes (from bacteriophages, plasmids or genomes of other species) through horizontal gene transfer. Accompanied by the acquisition of exogenous genes, prokaryocyte is challenged by the invasion of 'selfish genes'. Therefore, to protect against the risk of gene transfer, prokaryocyte needs to establish mechanisms for selectively taking up or degrading exogenous DNA. In recent years, researchers discovered an adaptive immunity, which is mediated by the small RNA guided DNA degradation, prevents the invasion of exogenous genes in prokaryocyte. During the immune process, partial DNA fragments are firstly integrated.to the clustered regularly interspaced short palindromic repeats (CRISPR) located within the genome DNA, and then the mature CRISPR RNA transcript and the CRISPR associated proteins (Cas) form a complex CRISPR/Cas for degrading exogenous DNA. In this review, we will first briefly describe the CRISPR/Cas systems and then mainly focus on the recent advances of the function mechanism and the regulation mechanism of the type I-E CRISPR/Cas system in Escherichia coli.

  13. Cas1-Cas2 complex formation mediates spacer acquisition during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Kranzusch, Philip J; Noeske, Jonas; Wright, Addison V; Davies, Christopher W; Doudna, Jennifer A

    2014-06-01

    The initial stage of CRISPR-Cas immunity involves the integration of foreign DNA spacer segments into the host genomic CRISPR locus. The nucleases Cas1 and Cas2 are the only proteins conserved among all CRISPR-Cas systems, yet the molecular functions of these proteins during immunity are unknown. Here we show that Cas1 and Cas2 from Escherichia coli form a stable complex that is essential for spacer acquisition and determine the 2.3-Å-resolution crystal structure of the Cas1-Cas2 complex. Mutations that perturb Cas1-Cas2 complex formation disrupt CRISPR DNA recognition and spacer acquisition in vivo. Active site mutants of Cas2, unlike those of Cas1, can still acquire new spacers, thus indicating a nonenzymatic role of Cas2 during immunity. These results reveal the universal roles of Cas1 and Cas2 and suggest a mechanism by which Cas1-Cas2 complexes specify sites of CRISPR spacer integration.

  14. Physical model of the immune response of bacteria against bacteriophage through the adaptive CRISPR-Cas immune system

    NASA Astrophysics Data System (ADS)

    Han, Pu; Niestemski, Liang Ren; Barrick, Jeffrey E.; Deem, Michael W.

    2013-04-01

    Bacteria and archaea have evolved an adaptive, heritable immune system that recognizes and protects against viruses or plasmids. This system, known as the CRISPR-Cas system, allows the host to recognize and incorporate short foreign DNA or RNA sequences, called ‘spacers’ into its CRISPR system. Spacers in the CRISPR system provide a record of the history of bacteria and phage coevolution. We use a physical model to study the dynamics of this coevolution as it evolves stochastically over time. We focus on the impact of mutation and recombination on bacteria and phage evolution and evasion. We discuss the effect of different spacer deletion mechanisms on the coevolutionary dynamics. We make predictions about bacteria and phage population growth, spacer diversity within the CRISPR locus, and spacer protection against the phage population.

  15. CRISPR-Cas adaptation: insights into the mechanism of action.

    PubMed

    Amitai, Gil; Sorek, Rotem

    2016-02-01

    Since the first demonstration that CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against phages and plasmids, numerous studies have yielded key insights into the molecular mechanisms governing how these systems attack and degrade foreign DNA. However, the molecular mechanisms underlying the adaptation stage, in which new immunological memory is formed, have until recently represented a major unresolved question. In this Progress article, we discuss recent discoveries that have shown both how foreign DNA is identified by the CRISPR-Cas adaptation machinery and the molecular basis for its integration into the chromosome to form an immunological memory. Furthermore, we describe the roles of each of the specific CRISPR-Cas components that are involved in memory formation, and consider current models for their evolutionary origin.

  16. Evolution and classification of the CRISPR-Cas systems

    PubMed Central

    S. Makarova, Kira; H. Haft, Daniel; Barrangou, Rodolphe; J. J. Brouns, Stan; Charpentier, Emmanuelle; Horvath, Philippe; Moineau, Sylvain; J. M. Mojica, Francisco; I. Wolf, Yuri; Yakunin, Alexander F.; van der Oost, John; V. Koonin, Eugene

    2012-01-01

    The CRISPR–Cas (clustered regularly interspaced short palindromic repeats–CRISPR-associated proteins) modules are adaptive immunity systems that are present in many archaea and bacteria. These defence systems are encoded by operons that have an extraordinarily diverse architecture and a high rate of evolution for both the cas genes and the unique spacer content. Here, we provide an updated analysis of the evolutionary relationships between CRISPR–Cas systems and Cas proteins. Three major types of CRISPR–Cas system are delineated, with a further division into several subtypes and a few chimeric variants. Given the complexity of the genomic architectures and the extremely dynamic evolution of the CRISPR–Cas systems, a unified classification of these systems should be based on multiple criteria. Accordingly, we propose a `polythetic' classification that integrates the phylogenies of the most common cas genes, the sequence and organization of the CRISPR repeats and the architecture of the CRISPR–cas loci. PMID:21552286

  17. Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system.

    PubMed

    Sokolowski, Richard D; Graham, Shirley; White, Malcolm F

    2014-06-01

    CRISPR-Cas is an adaptive prokaryotic immune system, providing protection against viruses and other mobile genetic elements. In type I and type III CRISPR-Cas systems, CRISPR RNA (crRNA) is generated by cleavage of a primary transcript by the Cas6 endonuclease and loaded into multisubunit surveillance/effector complexes, allowing homology-directed detection and cleavage of invading elements. Highly studied CRISPR-Cas systems such as those in Escherichia coli and Pseudomonas aeruginosa have a single Cas6 enzyme that is an integral subunit of the surveillance complex. By contrast, Sulfolobus solfataricus has a complex CRISPR-Cas system with three types of surveillance complexes (Cascade/type I-A, CSM/type III-A and CMR/type III-B), five Cas6 paralogues and two different CRISPR-repeat families (AB and CD). Here, we investigate the kinetic properties of two different Cas6 paralogues from S. solfataricus. The Cas6-1 subtype is specific for CD-family CRISPR repeats, generating crRNA by multiple turnover catalysis whilst Cas6-3 has a broader specificity and also processes a non-coding RNA with a CRISPR repeat-related sequence. Deep sequencing of crRNA in surveillance complexes reveals a biased distribution of spacers derived from AB and CD loci, suggesting functional coupling between Cas6 paralogues and their downstream effector complexes.

  18. Structural plasticity and in vivo activity of Cas1 from the type I-F CRISPR-Cas system.

    PubMed

    Wilkinson, Max E; Nakatani, Yoshio; Staals, Raymond H J; Kieper, Sebastian N; Opel-Reading, Helen K; McKenzie, Rebecca E; Fineran, Peter C; Krause, Kurt L

    2016-04-15

    CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity.

  19. Characterization and Evolution of Salmonella CRISPR-Cas Systems

    DTIC Science & Technology

    2014-01-01

    SECURITY CLASSIFICATION OF: Prokaryotic CRISPR -Cas (clustered regularly interspaced short palindromic repeats and CRISPR -associated genes) systems provide...adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and...direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in- depth sequence analysis of the CRISPR -Cas systems in .600

  20. Annotation and Classification of CRISPR-Cas Systems.

    PubMed

    Makarova, Kira S; Koonin, Eugene V

    2015-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas (CRISPR-associated proteins) is a prokaryotic adaptive immune system that is represented in most archaea and many bacteria. Among the currently known prokaryotic defense systems, the CRISPR-Cas genomic loci show unprecedented complexity and diversity. Classification of CRISPR-Cas variants that would capture their evolutionary relationships to the maximum possible extent is essential for comparative genomic and functional characterization of this theoretically and practically important system of adaptive immunity. To this end, a multipronged approach has been developed that combines phylogenetic analysis of the conserved Cas proteins with comparison of gene repertoires and arrangements in CRISPR-Cas loci. This approach led to the current classification of CRISPR-Cas systems into three distinct types and ten subtypes for each of which signature genes have been identified. Comparative genomic analysis of the CRISPR-Cas systems in new archaeal and bacterial genomes performed over the 3 years elapsed since the development of this classification makes it clear that new types and subtypes of CRISPR-Cas need to be introduced. Moreover, this classification system captures only part of the complexity of CRISPR-Cas organization and evolution, due to the intrinsic modularity and evolutionary mobility of these immunity systems, resulting in numerous recombinant variants. Moreover, most of the cas genes evolve rapidly, complicating the family assignment for many Cas proteins and the use of family profiles for the recognition of CRISPR-Cas subtype signatures. Further progress in the comparative analysis of CRISPR-Cas systems requires integration of the most sensitive sequence comparison tools, protein structure comparison, and refined approaches for comparison of gene neighborhoods.

  1. Control of gene expression by CRISPR-Cas systems.

    PubMed

    Bikard, David; Marraffini, Luciano A

    2013-01-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) loci and their associated cas (CRISPR-associated) genes provide adaptive immunity against viruses (phages) and other mobile genetic elements in bacteria and archaea. While most of the early work has largely been dominated by examples of CRISPR-Cas systems directing the cleavage of phage or plasmid DNA, recent studies have revealed a more complex landscape where CRISPR-Cas loci might be involved in gene regulation. In this review, we summarize the role of these loci in the regulation of gene expression as well as the recent development of synthetic gene regulation using engineered CRISPR-Cas systems.

  2. When 'solutions of yesterday become problems of today': crisis-ridden decision making in a complex adaptive system (CAS)--the Additional Duty Hours Allowance in Ghana.

    PubMed

    Agyepong, Irene Akua; Kodua, Augustina; Adjei, Sam; Adam, Taghreed

    2012-10-01

    Implementation of policies (decisions) in the health sector is sometimes defeated by the system's response to the policy itself. This can lead to counter-intuitive, unanticipated, or more modest effects than expected by those who designed the policy. The health sector fits the characteristics of complex adaptive systems (CAS) and complexity is at the heart of this phenomenon. Anticipating both positive and negative effects of policy decisions, understanding the interests, power and interaction between multiple actors; and planning for the delayed and distal impact of policy decisions are essential for effective decision making in CAS. Failure to appreciate these elements often leads to a series of reductionist approach interventions or 'fixes'. This in turn can initiate a series of negative feedback loops that further complicates the situation over time. In this paper we use a case study of the Additional Duty Hours Allowance (ADHA) policy in Ghana to illustrate these points. Using causal loop diagrams, we unpack the intended and unintended effects of the policy and how these effects evolved over time. The overall goal is to advance our understanding of decision making in complex adaptive systems; and through this process identify some essential elements in formulating, updating and implementing health policy that can help to improve attainment of desired outcomes and minimize negative unintended effects.

  3. Cas9 specifies functional viral targets during CRISPR-Cas adaptation.

    PubMed

    Heler, Robert; Samai, Poulami; Modell, Joshua W; Weiner, Catherine; Goldberg, Gregory W; Bikard, David; Marraffini, Luciano A

    2015-03-12

    Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.

  4. Foreign DNA capture during CRISPR–Cas adaptive immunity

    PubMed Central

    Nuñez, James K.; Harrington, Lucas B.; Kranzusch, Philip J.; Engelman, Alan N.; Doudna, Jennifer A.

    2015-01-01

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30–40 base pair (bp) lengths into clustered regularly interspaced short palindromic repeats (CRISPR) loci as spacer segments1–6. The universally conserved Cas1–Cas2 integrase complex catalyzes spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases7–13. How the Cas1–Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1–Cas2 complex bound to cognate 33 nucleotide (nt) protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3′–OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo2–4. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1–Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci. PMID:26503043

  5. Foreign DNA capture during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Harrington, Lucas B; Kranzusch, Philip J; Engelman, Alan N; Doudna, Jennifer A

    2015-11-26

    Bacteria and archaea generate adaptive immunity against phages and plasmids by integrating foreign DNA of specific 30-40-base-pair lengths into clustered regularly interspaced short palindromic repeat (CRISPR) loci as spacer segments. The universally conserved Cas1-Cas2 integrase complex catalyses spacer acquisition using a direct nucleophilic integration mechanism similar to retroviral integrases and transposases. How the Cas1-Cas2 complex selects foreign DNA substrates for integration remains unknown. Here we present X-ray crystal structures of the Escherichia coli Cas1-Cas2 complex bound to cognate 33-nucleotide protospacer DNA substrates. The protein complex creates a curved binding surface spanning the length of the DNA and splays the ends of the protospacer to allow each terminal nucleophilic 3'-OH to enter a channel leading into the Cas1 active sites. Phosphodiester backbone interactions between the protospacer and the proteins explain the sequence-nonspecific substrate selection observed in vivo. Our results uncover the structural basis for foreign DNA capture and the mechanism by which Cas1-Cas2 functions as a molecular ruler to dictate the sequence architecture of CRISPR loci.

  6. New CRISPR-Cas systems from uncultivated microbes.

    PubMed

    Burstein, David; Harrington, Lucas B; Strutt, Steven C; Probst, Alexander J; Anantharaman, Karthik; Thomas, Brian C; Doudna, Jennifer A; Banfield, Jillian F

    2017-02-09

    CRISPR-Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR-Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR-Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR-Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, we discovered two previously unknown systems, CRISPR-CasX and CRISPR-CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

  7. New CRISPR–Cas systems from uncultivated microbes

    NASA Astrophysics Data System (ADS)

    Burstein, David; Harrington, Lucas B.; Strutt, Steven C.; Probst, Alexander J.; Anantharaman, Karthik; Thomas, Brian C.; Doudna, Jennifer A.; Banfield, Jillian F.

    2016-12-01

    CRISPR–Cas systems provide microbes with adaptive immunity by employing short DNA sequences, termed spacers, that guide Cas proteins to cleave foreign DNA. Class 2 CRISPR–Cas systems are streamlined versions, in which a single RNA-bound Cas protein recognizes and cleaves target sequences. The programmable nature of these minimal systems has enabled researchers to repurpose them into a versatile technology that is broadly revolutionizing biological and clinical research. However, current CRISPR–Cas technologies are based solely on systems from isolated bacteria, leaving the vast majority of enzymes from organisms that have not been cultured untapped. Metagenomics, the sequencing of DNA extracted directly from natural microbial communities, provides access to the genetic material of a huge array of uncultivated organisms. Here, using genome-resolved metagenomics, we identify a number of CRISPR–Cas systems, including the first reported Cas9 in the archaeal domain of life, to our knowledge. This divergent Cas9 protein was found in little-studied nanoarchaea as part of an active CRISPR–Cas system. In bacteria, we discovered two previously unknown systems, CRISPR–CasX and CRISPR–CasY, which are among the most compact systems yet discovered. Notably, all required functional components were identified by metagenomics, enabling validation of robust in vivo RNA-guided DNA interference activity in Escherichia coli. Interrogation of environmental microbial communities combined with in vivo experiments allows us to access an unprecedented diversity of genomes, the content of which will expand the repertoire of microbe-based biotechnologies.

  8. Application of CRISPR-Cas system in gene therapy: Pre-clinical progress in animal model.

    PubMed

    Guan, Lihong; Han, Yawei; Zhu, Shaoyi; Lin, Juntang

    2016-10-01

    The clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated proteins (Cas) belong to the crucial adaptive immune system, which exist in archaea and bacteria. Currently, CRISPR-Cas9 system has been modified and widely used to edit genome. In this review, we summarized the discovery, classification and mechanism of CRISPR-Cas system and further discussed the application of CRISPR-Cas9 in gene therapy, mainly in disease models.

  9. Characterization and evolution of Salmonella CRISPR-Cas systems.

    PubMed

    Shariat, Nikki; Timme, Ruth E; Pettengill, James B; Barrangou, Rodolphe; Dudley, Edward G

    2015-02-01

    Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12% of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9%) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.

  10. Characterization and evolution of Salmonella CRISPR-Cas systems.

    PubMed

    Shariat, Nikki; Timme, Ruth E; Pettengill, James B; Barrangou, Rodolphe; Dudley, Edward G

    2015-02-01

    Prokaryotic CRISPR-Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated genes) systems provide adaptive immunity from invasive genetic elements and encompass three essential features: (i) cas genes, (ii) a CRISPR array composed of spacers and direct repeats and (iii) an AT-rich leader sequence upstream of the array. We performed in-depth sequence analysis of the CRISPR-Cas systems in >600 Salmonella, representing four clinically prevalent serovars. Each CRISPR-Cas feature is extremely conserved in the Salmonella, and the CRISPR1 locus is more highly conserved than CRISPR2. Array composition is serovar-specific, although no convincing evidence of recent spacer acquisition against exogenous nucleic acids exists. Only 12 % of spacers match phage and plasmid sequences and self-targeting spacers are associated with direct repeat variants. High nucleotide identity (>99.9 %) exists across the cas operon among isolates of a single serovar and in some cases this conservation extends across divergent serovars. These observations reflect historical CRISPR-Cas immune activity, showing that this locus has ceased undergoing adaptive events. Intriguingly, the high level of conservation across divergent serovars shows that the genetic integrity of these inactive loci is maintained over time, contrasting with the canonical view that inactive CRISPR loci degenerate over time. This thorough characterization of Salmonella CRISPR-Cas systems presents new insights into Salmonella CRISPR evolution, particularly with respect to cas gene conservation, leader sequences, organization of direct repeats and protospacer matches. Collectively, our data suggest that Salmonella CRISPR-Cas systems are no longer immunogenic; rather, their impressive conservation indicates they may have an alternative function in Salmonella.

  11. Crystal Structure of Streptococcus pyogenes Cas1 and Its Interaction with Csn2 in the Type II CRISPR-Cas System.

    PubMed

    Ka, Donghyun; Lee, Hasup; Jung, Yi-Deun; Kim, Kyunggon; Seok, Chaok; Suh, Nayoung; Bae, Euiyoung

    2016-01-05

    CRISPRs and Cas proteins constitute an RNA-guided microbial immune system against invading nucleic acids. Cas1 is a universal Cas protein found in all three types of CRISPR-Cas systems, and its role is implicated in new spacer acquisition during CRISPR-mediated adaptive immunity. Here, we report the crystal structure of Streptococcus pyogenes Cas1 (SpCas1) in a type II CRISPR-Cas system and characterize its interaction with S. pyogenes Csn2 (SpCsn2). The SpCas1 structure reveals a unique conformational state distinct from type I Cas1 structures, resulting in a more extensive dimerization interface, a more globular overall structure, and a disruption of potential metal-binding sites for catalysis. We demonstrate that SpCas1 directly interacts with SpCsn2, and identify the binding interface and key residues for Cas complex formation. These results provide structural information for a type II Cas1 protein, and lay a foundation for studying multiprotein Cas complexes functioning in type II CRISPR-Cas systems.

  12. The structural biology of CRISPR-Cas systems.

    PubMed

    Jiang, Fuguo; Doudna, Jennifer A

    2015-02-01

    Prokaryotic CRISPR-Cas genomic loci encode RNA-mediated adaptive immune systems that bear some functional similarities with eukaryotic RNA interference. Acquired and heritable immunity against bacteriophage and plasmids begins with integration of ∼30 base pair foreign DNA sequences into the host genome. CRISPR-derived transcripts assemble with CRISPR-associated (Cas) proteins to target complementary nucleic acids for degradation. Here we review recent advances in the structural biology of these targeting complexes, with a focus on structural studies of the multisubunit Type I CRISPR RNA-guided surveillance and the Cas9 DNA endonuclease found in Type II CRISPR-Cas systems. These complexes have distinct structures that are each capable of site-specific double-stranded DNA binding and local helix unwinding.

  13. Comparative analysis of CRISPR-Cas systems in Klebsiella genomes.

    PubMed

    Shen, Juntao; Lv, Li; Wang, Xudong; Xiu, Zhilong; Chen, Guoqiang

    2017-02-03

    Prokaryotic CRISPR-Cas system provides adaptive immunity against invasive genetic elements. Bacteria of the genus Klebsiella are important nosocomial opportunistic pathogens. However, information of CRISPR-Cas system in Klebsiella remains largely unknown. Here, we analyzed the CRISPR-Cas systems of 68 complete genomes of Klebsiella representing four species. All the elements for CRISPR-Cas system (cas genes, repeats, leader sequences, and PAMs) were characterized. Besides the typical Type I-E and I-F CRISPR-Cas systems, a new Subtype I system located in the ABC transport system-glyoxalase region was found. The conservation of the new subtype CRISPR system between different species showed new evidence for CRISPR horizontal transfer. CRISPR polymorphism was strongly correlated both with species and multilocus sequence types. Some results indicated the function of adaptive immunity: most spacers (112 of 124) matched to prophages and plasmids and no matching housekeeping genes; new spacer acquisition was observed within the same sequence type (ST) and same clonal complex; the identical spacers were observed only in the ancient position (far from the leader) between different STs and clonal complexes. Interestingly, a high ratio of self-targeting spacers (7.5%, 31 of 416) was found in CRISPR-bearing Klebsiella pneumoniae (61%, 11 of 18). In some strains, there even were multiple full matching self-targeting spacers. Some self-targeting spacers were conserved even between different STs. These results indicated that some unknown mechanisms existed to compromise the function of self-targets of CRISPR-Cas systems in K. pneumoniae.

  14. [CRISPR-Cas system as molecular scissors for gene therapy].

    PubMed

    Heinz, G A; Mashreghi, M-F

    2017-02-01

    Since the discovery of the CRISPR-Cas system as the adaptive immune system of prokaryotes, the underlying mechanism has proven to be a precise molecular tool for the targeted editing of genetic information in various cell types. By using the CRISPR-Cas9 system DNA sequences can be cut out at any site in the genome and changed in a sequence-specific manner. In the long term this provides the opportunity to cure diseases caused by gene mutations.

  15. Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity.

    PubMed

    Ivančić-Baće, Ivana; Cass, Simon D; Wearne, Stephen J; Bolt, Edward L

    2015-12-15

    CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed 'Adaptation', which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed 'Interference'. Adaptation can interact with interference ('primed'), or is independent of it ('naïve'). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration.

  16. Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity

    PubMed Central

    Ivančić-Baće, Ivana; Cass, Simon D; Wearne, Stephen J; Bolt, Edward L

    2015-01-01

    CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration. PMID:26578567

  17. Genome engineering using CRISPR-Cas9 system.

    PubMed

    Cong, Le; Zhang, Feng

    2015-01-01

    The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system is an adaptive immune system that exists in a variety of microbes. It could be engineered to function in eukaryotic cells as a fast, low-cost, efficient, and scalable tool for manipulating genomic sequences. In this chapter, detailed protocols are described for harnessing the CRISPR-Cas9 system from Streptococcus pyogenes to enable RNA-guided genome engineering applications in mammalian cells. We present all relevant methods including the initial site selection, molecular cloning, delivery of guide RNAs (gRNAs) and Cas9 into mammalian cells, verification of target cleavage, and assays for detecting genomic modification including indels and homologous recombination. These tools provide researchers with new instruments that accelerate both forward and reverse genetics efforts.

  18. Integrase-mediated spacer acquisition during CRISPR-Cas adaptive immunity.

    PubMed

    Nuñez, James K; Lee, Amy S Y; Engelman, Alan; Doudna, Jennifer A

    2015-03-12

    Bacteria and archaea insert spacer sequences acquired from foreign DNAs into CRISPR loci to generate immunological memory. The Escherichia coli Cas1-Cas2 complex mediates spacer acquisition in vivo, but the molecular mechanism of this process is unknown. Here we show that the purified Cas1-Cas2 complex integrates oligonucleotide DNA substrates into acceptor DNA to yield products similar to those generated by retroviral integrases and transposases. Cas1 is the catalytic subunit and Cas2 substantially increases integration activity. Protospacer DNA with free 3'-OH ends and supercoiled target DNA are required, and integration occurs preferentially at the ends of CRISPR repeats and at sequences adjacent to cruciform structures abutting AT-rich regions, similar to the CRISPR leader sequence. Our results demonstrate the Cas1-Cas2 complex to be the minimal machinery that catalyses spacer DNA acquisition and explain the significance of CRISPR repeats in providing sequence and structural specificity for Cas1-Cas2-mediated adaptive immunity.

  19. Diversity of CRISPR-Cas-Mediated Mechanisms of Adaptive Immunity in Prokaryotes and Their Application in Biotechnology.

    PubMed

    Savitskaya, E E; Musharova, O S; Severinov, K V

    2016-07-01

    CRISPR-Cas systems of adaptive immunity in prokaryotes consist of CRISPR arrays (clusters of short repeated genomic DNA fragments separated by unique spacer sequences) and cas (CRISPR-associated) genes that provide cells with resistance against bacteriophages and plasmids containing protospacers, i.e. sequences complementary to CRISPR array spacers. CRISPR-Cas systems are responsible for two different cellular phenomena: CRISPR adaptation and CRISPR interference. CRISPR adaptation is cell genome modification by integration of new spacers that represents a unique case of Lamarckian inheritance. CRISPR interference involves specific recognition of protospacers in foreign DNA followed by introduction of breaks into this DNA and its destruction. According to the mechanisms of action, CRISPR-Cas systems have been subdivided into two classes, five types, and numerous subtypes. The development of techniques based on CRISPR interference mediated by the Type II system Cas9 protein has revolutionized the field of genome editing because it allows selective, efficient, and relatively simple introduction of directed breaks into target DNA loci. However, practical applications of CRISPR-Cas systems are not limited only to genome editing. In this review, we focus on the variety of CRISPR interference and CRISPR adaptation mechanisms and their prospective use in biotechnology.

  20. Dual nuclease activity of a Cas2 protein in CRISPR-Cas subtype I-B of Leptospira interrogans.

    PubMed

    Dixit, Bhuvan; Ghosh, Karukriti Kaushik; Fernandes, Gary; Kumar, Pankaj; Gogoi, Prerana; Kumar, Manish

    2016-04-01

    Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 carries a set of cas genes associated with CRISPR-Cas subtype I-B. Herein, we report for the first time active transcription of a set of cas genes (cas1 to cas8) of L. interrogans where cas4, cas1, cas2 and cas6, cas3, cas8, cas7, cas5 are clustered together in two independent operons. As an initial step toward comprehensive understanding of CRISPR-Cas system in spirochete, the biochemical study of one of the core Leptospira Cas2 proteins (Lep_Cas2) showed nuclease activity on both DNA and RNA in a nonspecific manner. Additionally, unlike other known Cas2 proteins, Lep_Cas2 showed metal-independent RNase activity and preferential activity on RNA over DNA. These results provide insight for understanding Cas2 diversity existing in the prokaryotic adaptive immune system.

  1. SnapShot: Class 1 CRISPR-Cas Systems.

    PubMed

    Makarova, Kira S; Zhang, Feng; Koonin, Eugene V

    2017-02-23

    Class 1 CRISPR-Cas systems are characterized by effector modules consisting of multiple subunits. Class 1 systems comprise about 90% of all CRISPR-Cas loci identified in bacteria and archaea and can target both DNA and RNA.

  2. Evidence for the widespread distribution of CRISPR-Cas system in the Phylum Cyanobacteria.

    PubMed

    Cai, Fei; Axen, Seth D; Kerfeld, Cheryl A

    2013-05-01

    Members of the phylum Cyanobacteria inhabit ecologically diverse environments. However, the CRISPR-Cas (clustered regularly interspaced short palindromic repeats, CRISPR associated genes), an extremely adaptable defense system, has not been surveyed in this phylum. We analyzed 126 cyanobacterial genomes and, surprisingly, found CRISPR-Cas in the majority except the marine subclade (Synechococcus and Prochlorococcus), in which cyanophages are a known force shaping their evolution. Multiple observations of CRISPR loci in the absence of cas1/cas2 genes may represent an early stage of losing a CRISPR-Cas locus. Our findings reveal the widespread distribution of their role in the phylum Cyanobacteria and provide a first step to systematically understanding CRISPR-Cas systems in cyanobacteria.

  3. Modulating the Cascade architecture of a minimal Type I-F CRISPR-Cas system

    PubMed Central

    Gleditzsch, Daniel; Müller-Esparza, Hanna; Pausch, Patrick; Sharma, Kundan; Dwarakanath, Srivatsa; Urlaub, Henning; Bange, Gert; Randau, Lennart

    2016-01-01

    Shewanella putrefaciens CN-32 contains a single Type I-Fv CRISPR-Cas system which confers adaptive immunity against bacteriophage infection. Three Cas proteins (Cas6f, Cas7fv, Cas5fv) and mature CRISPR RNAs were shown to be required for the assembly of an interference complex termed Cascade. The Cas protein-CRISPR RNA interaction sites within this complex were identified via mass spectrometry. Additional Cas proteins, commonly described as large and small subunits, that are present in all other investigated Cascade structures, were not detected. We introduced this minimal Type I system in Escherichia coli and show that it provides heterologous protection against lambda phage. The absence of a large subunit suggests that the length of the crRNA might not be fixed and recombinant Cascade complexes with drastically shortened and elongated crRNAs were engineered. Size-exclusion chromatography and small-angle X-ray scattering analyses revealed that the number of Cas7fv backbone subunits is adjusted in these shortened and extended Cascade variants. Larger Cascade complexes can still confer immunity against lambda phage infection in E. coli. Minimized Type I CRISPR-Cas systems expand our understanding of the evolution of Cascade assembly and diversity. Their adjustable crRNA length opens the possibility for customizing target DNA specificity. PMID:27216815

  4. In vivo protein interactions and complex formation in the Pectobacterium atrosepticum subtype I-F CRISPR/Cas System.

    PubMed

    Richter, Corinna; Gristwood, Tamzin; Clulow, James S; Fineran, Peter C

    2012-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and their associated proteins (Cas; CRISPR associated) are a bacterial defense mechanism against extra-chromosomal elements. CRISPR/Cas systems are distinct from other known defense mechanisms insofar as they provide acquired and heritable immunity. Resistance is accomplished in multiple stages in which the Cas proteins provide the enzymatic machinery. Importantly, subtype-specific proteins have been shown to form complexes in combination with small RNAs, which enable sequence-specific targeting of foreign nucleic acids. We used Pectobacterium atrosepticum, a plant pathogen that causes soft-rot and blackleg disease in potato, to investigate protein-protein interactions and complex formation in the subtype I-F CRISPR/Cas system. The P. atrosepticum CRISPR/Cas system encodes six proteins: Cas1, Cas3, and the four subtype specific proteins Csy1, Csy2, Csy3 and Cas6f (Csy4). Using co-purification followed by mass spectrometry as well as directed co-immunoprecipitation we have demonstrated complex formation by the Csy1-3 and Cas6f proteins, and determined details about the architecture of that complex. Cas3 was also shown to co-purify all four subtype-specific proteins, consistent with its role in targeting. Furthermore, our results show that the subtype I-F Cas1 and Cas3 (a Cas2-Cas3 hybrid) proteins interact, suggesting a protein complex for adaptation and a role for subtype I-F Cas3 proteins in both the adaptation and interference steps of the CRISPR/Cas mechanism.

  5. Diverse evolutionary roots and mechanistic variations of the CRISPR-Cas systems.

    PubMed

    Mohanraju, Prarthana; Makarova, Kira S; Zetsche, Bernd; Zhang, Feng; Koonin, Eugene V; van der Oost, John

    2016-08-05

    Adaptive immunity had been long thought of as an exclusive feature of animals. However, the discovery of the CRISPR-Cas defense system, present in almost half of prokaryotic genomes, proves otherwise. Because of the everlasting parasite-host arms race, CRISPR-Cas has rapidly evolved through horizontal transfer of complete loci or individual modules, resulting in extreme structural and functional diversity. CRISPR-Cas systems are divided into two distinct classes that each consist of three types and multiple subtypes. We discuss recent advances in CRISPR-Cas research that reveal elaborate molecular mechanisms and provide for a plausible scenario of CRISPR-Cas evolution. We also briefly describe the latest developments of a wide range of CRISPR-based applications.

  6. A bacteriophage encodes its own CRISPR/Cas adaptive response to evade host innate immunity.

    PubMed

    Seed, Kimberley D; Lazinski, David W; Calderwood, Stephen B; Camilli, Andrew

    2013-02-28

    Bacteriophages (or phages) are the most abundant biological entities on earth, and are estimated to outnumber their bacterial prey by tenfold. The constant threat of phage predation has led to the evolution of a broad range of bacterial immunity mechanisms that in turn result in the evolution of diverse phage immune evasion strategies, leading to a dynamic co-evolutionary arms race. Although bacterial innate immune mechanisms against phage abound, the only documented bacterial adaptive immune system is the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system, which provides sequence-specific protection from invading nucleic acids, including phage. Here we show a remarkable turn of events, in which a phage-encoded CRISPR/Cas system is used to counteract a phage inhibitory chromosomal island of the bacterial host. A successful lytic infection by the phage is dependent on sequence identity between CRISPR spacers and the target chromosomal island. In the absence of such targeting, the phage-encoded CRISPR/Cas system can acquire new spacers to evolve rapidly and ensure effective targeting of the chromosomal island to restore phage replication.

  7. Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay.

    PubMed

    Heussler, Gary E; Miller, Jon L; Price, Courtney E; Collins, Alan J; O'Toole, George A

    2016-11-15

    CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression of cas genes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich for Pseudomonas aeruginosa strains with new spacer acquisition. We used this assay to demonstrate that P. aeruginosa rapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for the P. aeruginosa type I-F system, the cas1 gene is required for CRISPR adaptation, recG contributes to (but is not required for) primed CRISPR adaptation, recD is dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer.

  8. Foreign DNA acquisition by the I-F CRISPR–Cas system requires all components of the interference machinery

    PubMed Central

    Vorontsova, Daria; Datsenko, Kirill A.; Medvedeva, Sofia; Bondy-Denomy, Joseph; Savitskaya, Ekaterina E.; Pougach, Ksenia; Logacheva, Maria; Wiedenheft, Blake; Davidson, Alan R.; Severinov, Konstantin; Semenova, Ekaterina

    2015-01-01

    CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR–Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR–Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR–Cas systems. PMID:26586803

  9. Foreign DNA acquisition by the I-F CRISPR-Cas system requires all components of the interference machinery.

    PubMed

    Vorontsova, Daria; Datsenko, Kirill A; Medvedeva, Sofia; Bondy-Denomy, Joseph; Savitskaya, Ekaterina E; Pougach, Ksenia; Logacheva, Maria; Wiedenheft, Blake; Davidson, Alan R; Severinov, Konstantin; Semenova, Ekaterina

    2015-12-15

    CRISPR immunity depends on acquisition of fragments of foreign DNA into CRISPR arrays. For type I-E CRISPR-Cas systems two modes of spacer acquisition, naïve and primed adaptation, were described. Naïve adaptation requires just two most conserved Cas1 and Cas2 proteins; it leads to spacer acquisition from both foreign and bacterial DNA and results in multiple spacers incapable of immune response. Primed adaptation requires all Cas proteins and a CRISPR RNA recognizing a partially matching target. It leads to selective acquisition of spacers from DNA molecules recognized by priming CRISPR RNA, with most spacers capable of protecting the host. Here, we studied spacer acquisition by a type I-F CRISPR-Cas system. We observe both naïve and primed adaptation. Both processes require not just Cas1 and Cas2, but also intact Csy complex and CRISPR RNA. Primed adaptation shows a gradient of acquisition efficiency as a function of distance from the priming site and a strand bias that is consistent with existence of single-stranded adaption intermediates. The results provide new insights into the mechanism of spacer acquisition and illustrate surprising mechanistic diversity of related CRISPR-Cas systems.

  10. The CRISPR-Cas immune system: biology, mechanisms and applications.

    PubMed

    Rath, Devashish; Amlinger, Lina; Rath, Archana; Lundgren, Magnus

    2015-10-01

    Viruses are a common threat to cellular life, not the least to bacteria and archaea who constitute the majority of life on Earth. Consequently, a variety of mechanisms to resist virus infection has evolved. A recent discovery is the adaptive immune system in prokaryotes, a type of system previously thought to be present only in vertebrates. The system, called CRISPR-Cas, provide sequence-specific adaptive immunity and fundamentally affect our understanding of virus-host interaction. CRISPR-based immunity acts by integrating short virus sequences in the cell's CRISPR locus, allowing the cell to remember, recognize and clear infections. There has been rapid advancement in our understanding of this immune system and its applications, but there are many aspects that await elucidation making the field an exciting area of research. This review provides an overview of the field and highlights unresolved issues.

  11. The Adaptive Immune System of Haloferax volcanii.

    PubMed

    Maier, Lisa-Katharina; Dyall-Smith, Mike; Marchfelder, Anita

    2015-02-16

    To fight off invading genetic elements, prokaryotes have developed an elaborate defence system that is both adaptable and heritable-the CRISPR-Cas system (CRISPR is short for: clustered regularly interspaced short palindromic repeats and Cas: CRISPR associated). Comprised of proteins and multiple small RNAs, this prokaryotic defence system is present in 90% of archaeal and 40% of bacterial species, and enables foreign intruders to be eliminated in a sequence-specific manner. There are three major types (I-III) and at least 14 subtypes of this system, with only some of the subtypes having been analysed in detail, and many aspects of the defence reaction remaining to be elucidated. Few archaeal examples have so far been analysed. Here we summarize the characteristics of the CRISPR-Cas system of Haloferax volcanii, an extremely halophilic archaeon originally isolated from the Dead Sea. It carries a single CRISPR-Cas system of type I-B, with a Cascade like complex composed of Cas proteins Cas5, Cas6b and Cas7. Cas6b is essential for CRISPR RNA (crRNA) maturation but is otherwise not required for the defence reaction. A systematic search revealed that six protospacer adjacent motif (PAM) sequences are recognised by the Haloferax defence system. For successful invader recognition, a non-contiguous seed sequence of 10 base-pairs between the crRNA and the invader is required.

  12. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems.

    PubMed

    Béguin, Pierre; Charpin, Nicole; Koonin, Eugene V; Forterre, Patrick; Krupovic, Mart

    2016-12-01

    Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3' end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1-Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems.

  13. Casposon integration shows strong target site preference and recapitulates protospacer integration by CRISPR-Cas systems

    PubMed Central

    Béguin, Pierre; Charpin, Nicole; Koonin, Eugene V.; Forterre, Patrick; Krupovic, Mart

    2016-01-01

    Casposons are a recently discovered group of large DNA transposons present in diverse bacterial and archaeal genomes. For integration into the host chromosome, casposons employ an endonuclease that is homologous to the Cas1 protein involved in protospacer integration by the CRISPR-Cas adaptive immune system. Here we describe the site-preference of integration by the Cas1 integrase (casposase) encoded by the casposon of the archaeon Aciduliprofundum boonei. Oligonucleotide duplexes derived from the terminal inverted repeats (TIR) of the A. boonei casposon as well as mini-casposons flanked by the TIR inserted preferentially at a site reconstituting the original A. boonei target site. As in the A. boonei genome, the insertion was accompanied by a 15-bp direct target site duplication (TSD). The minimal functional target consisted of the 15-bp TSD segment and the adjacent 18-bp sequence which comprises the 3′ end of the tRNA-Pro gene corresponding to the TΨC loop. The functional casposase target site bears clear resemblance to the leader sequence-repeat junction which is the target for protospacer integration catalyzed by the Cas1–Cas2 adaptation module of CRISPR-Cas. These findings reinforce the mechanistic similarities and evolutionary connection between the casposons and the adaptation module of the prokaryotic adaptive immunity systems. PMID:27655632

  14. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells

    PubMed Central

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-01-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)—CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications. PMID:26782639

  15. Occurrence and activity of a type II CRISPR-Cas system in Lactobacillus gasseri.

    PubMed

    Sanozky-Dawes, Rosemary; Selle, Kurt; O'Flaherty, Sarah; Klaenhammer, Todd; Barrangou, Rodolphe

    2015-09-01

    Bacteria encode clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated genes (cas), which collectively form an RNA-guided adaptive immune system against invasive genetic elements. In silico surveys have revealed that lactic acid bacteria harbour a prolific and diverse set of CRISPR-Cas systems. Thus, the natural evolutionary role of CRISPR-Cas systems may be investigated in these ecologically, industrially, scientifically and medically important microbes. In this study, 17 Lactobacillus gasseri strains were investigated and 6 harboured a type II-A CRISPR-Cas system, with considerable diversity in array size and spacer content. Several of the spacers showed similarity to phage and plasmid sequences, which are typical targets of CRISPR-Cas immune systems. Aligning the protospacers facilitated inference of the protospacer adjacent motif sequence, determined to be 5'-NTAA-3' flanking the 3' end of the protospacer. The system in L. gasseri JV-V03 and NCK 1342 interfered with transforming plasmids containing sequences matching the most recently acquired CRISPR spacers in each strain. We report the distribution and function of a native type II-A CRISPR-Cas system in the commensal species L. gasseri. Collectively, these results open avenues for applications for bacteriophage protection and genome modification in L. gasseri, and contribute to the fundamental understanding of CRISPR-Cas systems in bacteria.

  16. The Neisseria meningitidis CRISPR-Cas9 System Enables Specific Genome Editing in Mammalian Cells.

    PubMed

    Lee, Ciaran M; Cradick, Thomas J; Bao, Gang

    2016-03-01

    The clustered regularly-interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system from Streptococcus pyogenes (Spy) has been successfully adapted for RNA-guided genome editing in a wide range of organisms. However, numerous reports have indicated that Spy CRISPR-Cas9 systems may have significant off-target cleavage of genomic DNA sequences differing from the intended on-target site. Here, we report the performance of the Neisseria meningitidis (Nme) CRISPR-Cas9 system that requires a longer protospacer-adjacent motif for site-specific cleavage, and present a comparison between the Spy and Nme CRISPR-Cas9 systems targeting the same protospacer sequence. The results with the native crRNA and tracrRNA as well as a chimeric single guide RNA for the Nme CRISPR-Cas9 system were also compared. Our results suggest that, compared with the Spy system, the Nme CRISPR-Cas9 system has similar or lower on-target cleavage activity but a reduced overall off-target effect on a genomic level when sites containing three or fewer mismatches are considered. Thus, the Nme CRISPR-Cas9 system may represent a safer alternative for precision genome engineering applications.

  17. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems

    PubMed Central

    Fonfara, Ines; Le Rhun, Anaïs; Chylinski, Krzysztof; Makarova, Kira S.; Lécrivain, Anne-Laure; Bzdrenga, Janek; Koonin, Eugene V.; Charpentier, Emmanuelle

    2014-01-01

    The CRISPR-Cas-derived RNA-guided Cas9 endonuclease is the key element of an emerging promising technology for genome engineering in a broad range of cells and organisms. The DNA-targeting mechanism of the type II CRISPR-Cas system involves maturation of tracrRNA:crRNA duplex (dual-RNA), which directs Cas9 to cleave invading DNA in a sequence-specific manner, dependent on the presence of a Protospacer Adjacent Motif (PAM) on the target. We show that evolution of dual-RNA and Cas9 in bacteria produced remarkable sequence diversity. We selected eight representatives of phylogenetically defined type II CRISPR-Cas groups to analyze possible coevolution of Cas9 and dual-RNA. We demonstrate that these two components are interchangeable only between closely related type II systems when the PAM sequence is adjusted to the investigated Cas9 protein. Comparison of the taxonomy of bacterial species that harbor type II CRISPR-Cas systems with the Cas9 phylogeny corroborates horizontal transfer of the CRISPR-Cas loci. The reported collection of dual-RNA:Cas9 with associated PAMs expands the possibilities for multiplex genome editing and could provide means to improve the specificity of the RNA-programmable Cas9 tool. PMID:24270795

  18. Genome engineering using the CRISPR-Cas9 system.

    PubMed

    Ran, F Ann; Hsu, Patrick D; Wright, Jason; Agarwala, Vineeta; Scott, David A; Zhang, Feng

    2013-11-01

    Targeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitate efficient genome engineering in eukaryotic cells by simply specifying a 20-nt targeting sequence within its guide RNA. Here we describe a set of tools for Cas9-mediated genome editing via nonhomologous end joining (NHEJ) or homology-directed repair (HDR) in mammalian cells, as well as generation of modified cell lines for downstream functional studies. To minimize off-target cleavage, we further describe a double-nicking strategy using the Cas9 nickase mutant with paired guide RNAs. This protocol provides experimentally derived guidelines for the selection of target sites, evaluation of cleavage efficiency and analysis of off-target activity. Beginning with target design, gene modifications can be achieved within as little as 1-2 weeks, and modified clonal cell lines can be derived within 2-3 weeks.

  19. Surveillance and Processing of Foreign DNA by the Escherichia coli CRISPR-Cas System.

    PubMed

    Redding, Sy; Sternberg, Samuel H; Marshall, Myles; Gibb, Bryan; Bhat, Prashant; Guegler, Chantal K; Wiedenheft, Blake; Doudna, Jennifer A; Greene, Eric C

    2015-11-05

    CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease helicase for target degradation. Here, we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways dictated by the presence or absence of a protospacer-adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short single-stranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent on the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes and support a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA.

  20. Surveillance and processing of foreign DNA by the Escherichia coli CRISPR-Cas system

    PubMed Central

    Redding, Sy; Sternberg, Samuel H.; Marshall, Myles; Gibb, Bryan; Bhat, Prashant; Guegler, Chantal K.; Wiedenheft, Blake; Doudna, Jennifer A.; Greene, Eric C.

    2015-01-01

    Summary CRISPR-Cas adaptive immune systems protect bacteria and archaea against foreign genetic elements. In Escherichia coli, Cascade (CRISPR-associated complex for antiviral defense) is an RNA-guided surveillance complex that binds foreign DNA and recruits Cas3, a trans-acting nuclease-helicase for target degradation. Here we use single-molecule imaging to visualize Cascade and Cas3 binding to foreign DNA targets. Our analysis reveals two distinct pathways, dictated by the presence or absence of a protospacer adjacent motif (PAM). Binding to a protospacer flanked by a PAM recruits a nuclease-active Cas3 for degradation of short singlestranded regions of target DNA, whereas PAM mutations elicit an alternative pathway that recruits a nuclease-inactive Cas3 through a mechanism that is dependent upon the Cas1 and Cas2 proteins. These findings explain how target recognition by Cascade can elicit distinct outcomes, and supports a model for acquisition of new spacer sequences through a mechanism involving processive, ATP-dependent Cas3 translocation along foreign DNA. PMID:26522594

  1. Role of the Streptococcus mutans CRISPR-Cas systems in immunity and cell physiology.

    PubMed

    Serbanescu, M A; Cordova, M; Krastel, K; Flick, R; Beloglazova, N; Latos, A; Yakunin, A F; Senadheera, D B; Cvitkovitch, D G

    2015-02-15

    CRISPR-Cas systems provide adaptive microbial immunity against invading viruses and plasmids. The cariogenic bacterium Streptococcus mutans UA159 has two CRISPR-Cas systems: CRISPR1 (type II-A) and CRISPR2 (type I-C) with several spacers from both CRISPR cassettes matching sequences of phage M102 or genomic sequences of other S. mutans. The deletion of the cas genes of CRISPR1 (ΔC1S), CRISPR2 (ΔC2E), or both CRISPR1+2 (ΔC1SC2E) or the removal of spacers 2 and 3 (ΔCR1SP13E) in S. mutans UA159 did not affect phage sensitivity when challenged with virulent phage M102. Using plasmid transformation experiments, we demonstrated that the CRISPR1-Cas system inhibits transformation of S. mutans by the plasmids matching the spacers 2 and 3. Functional analysis of the cas deletion mutants revealed that in addition to a role in plasmid targeting, both CRISPR systems also contribute to the regulation of bacterial physiology in S. mutans. Compared to wild-type cells, the ΔC1S strain displayed diminished growth under cell membrane and oxidative stress, enhanced growth under low pH, and had reduced survival under heat shock and DNA-damaging conditions, whereas the ΔC2E strain exhibited increased sensitivity to heat shock. Transcriptional analysis revealed that the two-component signal transduction system VicR/K differentially modulates expression of cas genes within CRISPR-Cas systems, suggesting that VicR/K might coordinate the expression of two CRISPR-Cas systems. Collectively, we provide in vivo evidence that the type II-A CRISPR-Cas system of S. mutans may be targeted to manipulate its stress response and to influence the host to control the uptake and dissemination of antibiotic resistance genes.

  2. Calibrated Ancillary System (CAS) user's guide, volume 8

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 8 describes procedures for invoking checkout software, file maintenance procedures, system manager procedures.

  3. Calibrated Ancillary System (CAS) user's guide, volume 1

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of the CAS. Volume 1 includes a general overview of the CAS relationships with other equipment, physical design, and hardware and software subsystems. In addition, a description of the user levels and tasks, an introduction to CAS operation, and an outline of general operating procedures are included.

  4. Effects of Using a Computer Algebra System (CAS) on Junior College Students' Attitudes towards CAS and Achievement in Mathematics

    ERIC Educational Resources Information Center

    Leng, Ng Wee; Choo, Kwee Tiow; Soon, Lau Hock; Yi-Huak, Koh; Sun, Yap Yew

    2005-01-01

    This study examines the effects of using Texas Instruments' Voyage 200 calculator (V200), a graphing calculator with a built-in computer algebra system (CAS), on attitudes towards CAS and achievement in mathematics of junior college students (17 year olds). Students' attitudes towards CAS were examined using a 40-item Likert-type instrument…

  5. Calibrated Ancillary System (CAS) user's guide, volume 2

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 2 describes the central status and control (CSAC) procedures, supervisor procedures, and logging procedures.

  6. Calibrated Ancillary System (CAS) user's guide, volume 3

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of the CAS. Volume 3 describes logging and delogging procedures, real-time procedures, and error messages.

  7. Diversity and evolution of class 2 CRISPR-Cas systems.

    PubMed

    Shmakov, Sergey; Smargon, Aaron; Scott, David; Cox, David; Pyzocha, Neena; Yan, Winston; Abudayyeh, Omar O; Gootenberg, Jonathan S; Makarova, Kira S; Wolf, Yuri I; Severinov, Konstantin; Zhang, Feng; Koonin, Eugene V

    2017-03-01

    Class 2 CRISPR-Cas systems are characterized by effector modules that consist of a single multidomain protein, such as Cas9 or Cpf1. We designed a computational pipeline for the discovery of novel class 2 variants and used it to identify six new CRISPR-Cas subtypes. The diverse properties of these new systems provide potential for the development of versatile tools for genome editing and regulation. In this Analysis article, we present a comprehensive census of class 2 types and class 2 subtypes in complete and draft bacterial and archaeal genomes, outline evolutionary scenarios for the independent origin of different class 2 CRISPR-Cas systems from mobile genetic elements, and propose an amended classification and nomenclature of CRISPR-Cas.

  8. Unravelling the structural and mechanistic basis of CRISPR-Cas systems.

    PubMed

    van der Oost, John; Westra, Edze R; Jackson, Ryan N; Wiedenheft, Blake

    2014-07-01

    Bacteria and archaea have evolved sophisticated adaptive immune systems, known as CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) systems, which target and inactivate invading viruses and plasmids. Immunity is acquired by integrating short fragments of foreign DNA into CRISPR loci, and following transcription and processing of these loci, the CRISPR RNAs (crRNAs) guide the Cas proteins to complementary invading nucleic acid, which results in target interference. In this Review, we summarize the recent structural and biochemical insights that have been gained for the three major types of CRISPR-Cas systems, which together provide a detailed molecular understanding of the unique and conserved mechanisms of RNA-guided adaptive immunity in bacteria and archaea.

  9. Incidence of Type II CRISPR1-Cas Systems in Enterococcus Is Species-Dependent

    PubMed Central

    Lyons, Casandra; Raustad, Nicole; Bustos, Mario A.; Shiaris, Michael

    2015-01-01

    CRISPR-Cas systems, which obstruct both viral infection and incorporation of mobile genetic elements by horizontal transfer, are a specific immune response common to prokaryotes. Antiviral protection by CRISPR-Cas comes at a cost, as horizontally-acquired genes may increase fitness and provide rapid adaptation to habitat change. To date, investigations into the prevalence of CRISPR have primarily focused on pathogenic and clinical bacteria, while less is known about CRISPR dynamics in commensal and environmental species. We designed PCR primers and coupled these with DNA sequencing of products to detect and characterize the presence of cas1, a universal CRISPR-associated gene and proxy for the Type II CRISPR1-Cas system, in environmental and non-clinical Enterococcus isolates. CRISPR1-cas1 was detected in approximately 33% of the 275 strains examined, and differences in CRISPR1 carriage between species was significant. Incidence of cas1 in E. hirae was 73%, nearly three times that of E. faecalis (23.6%) and 10 times more frequent than in E. durans (7.1%). Also, this is the first report of CRISPR1 presence in E. durans, as well as in the plant-associated species E. casseliflavus and E. sulfureus. Significant differences in CRISPR1-cas1 incidence among Enterococcus species support the hypothesis that there is a tradeoff between protection and adaptability. The differences in the habitats of enterococcal species may exert varying selective pressure that results in a species-dependent distribution of CRISPR-Cas systems. PMID:26600384

  10. Mechanism of spacer integration links the CRISPR/Cas system to transposition as a form of mobile DNA.

    PubMed

    Dyda, Fred; Hickman, Alison B

    2015-01-01

    It has recently become clear that many bacterial and archaeal species possess adaptive immune systems. These are typified by multiple copies of DNA sequences known as clustered regularly interspaced short palindromic repeats (CRISPRs). These CRISPR repeats are the sites at which short spacers containing sequences of previously encountered foreign DNA are integrated, and the spacers serve as the molecular memory of previous invaders. In vivo work has demonstrated that two CRISPR-associated proteins - Cas1 and Cas2 - are required for spacer integration, but the mechanism by which this is accomplished remained unclear. Here we review a recent paper describing the in vitro reconstitution of CRISPR spacer integration using purified Cas1 and Cas2 and place the results in context of similar DNA transposition reactions and the crystal structure of the Cas1/Cas2 complex.

  11. Insert, remove or replace: A highly advanced genome editing system using CRISPR/Cas9.

    PubMed

    Ceasar, S Antony; Rajan, Vinothkumar; Prykhozhij, Sergey V; Berman, Jason N; Ignacimuthu, S

    2016-09-01

    The clustered, regularly interspaced, short palindromic repeat (CRISPR) and CRISPR associated protein 9 (Cas9) system discovered as an adaptive immunity mechanism in prokaryotes has emerged as the most popular tool for the precise alterations of the genomes of diverse species. CRISPR/Cas9 system has taken the world of genome editing by storm in recent years. Its popularity as a tool for altering genomes is due to the ability of Cas9 protein to cause double-stranded breaks in DNA after binding with short guide RNA molecules, which can be produced with dramatically less effort and expense than required for production of transcription-activator like effector nucleases (TALEN) and zinc-finger nucleases (ZFN). This system has been exploited in many species from prokaryotes to higher animals including human cells as evidenced by the literature showing increasing sophistication and ease of CRISPR/Cas9 as well as increasing species variety where it is applicable. This technology is poised to solve several complex molecular biology problems faced in life science research including cancer research. In this review, we highlight the recent advancements in CRISPR/Cas9 system in editing genomes of prokaryotes, fungi, plants and animals and provide details on software tools available for convenient design of CRISPR/Cas9 targeting plasmids. We also discuss the future prospects of this advanced molecular technology.

  12. Using the CRISPR-Cas System to Positively Select Mutants in Genes Essential for Its Function.

    PubMed

    Yosef, Ido; Goren, Moran G; Edgar, Rotem; Qimron, Udi

    2015-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR associated proteins (Cas) comprise a prokaryotic adaptive defense system against foreign nucleic acids. This defense is mediated by Cas proteins, which are guided by sequences flanked by the repeats, called spacers, to target nucleic acids. Spacers designed against the prokaryotic self chromosome are lethal to the prokaryotic cell. This self-killing of the bacterium by its own CRISPR-Cas system can be used to positively select genes that participate in this killing, as their absence will result in viable cells. Here we describe a positive selection assay that uses this feature to identify E. coli mutants encoding an inactive CRISPR-Cas system. The procedure includes establishment of an assay that detects this self-killing, generation of transposon insertion mutants in random genes, and selection of viable mutants, suspected as required for this lethal activity. This procedure enabled us to identify a novel gene, htpG, that is required for the activity of the CRISPR-Cas system. The procedures described here can be adjusted to various organisms to identify genes required for their CRISPR-Cas activity.

  13. Adapting CRISPR/Cas9 for functional genomics screens.

    PubMed

    Malina, Abba; Katigbak, Alexandra; Cencic, Regina; Maïga, Rayelle Itoua; Robert, Francis; Miura, Hisashi; Pelletier, Jerry

    2014-01-01

    The use of CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) for targeted genome editing has been widely adopted and is considered a "game changing" technology. The ease and rapidity by which this approach can be used to modify endogenous loci in a wide spectrum of cell types and organisms makes it a powerful tool for customizable genetic modifications as well as for large-scale functional genomics. The development of retrovirus-based expression platforms to simultaneously deliver the Cas9 nuclease and single guide (sg) RNAs provides unique opportunities by which to ensure stable and reproducible expression of the editing tools and a broad cell targeting spectrum, while remaining compatible with in vivo genetic screens. Here, we describe methods and highlight considerations for designing and generating sgRNA libraries in all-in-one retroviral vectors for such applications.

  14. Friendly Fire: Biological Functions and Consequences of Chromosomal Targeting by CRISPR-Cas Systems

    PubMed Central

    Heussler, Gary E.

    2016-01-01

    Clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) systems in bacteria and archaea target foreign elements, such as bacteriophages and conjugative plasmids, through the incorporation of short sequences (termed spacers) from the foreign element into the CRISPR array, thereby allowing sequence-specific targeting of the invader. Thus, CRISPR-Cas systems are typically considered a microbial adaptive immune system. While many of these incorporated spacers match targets on bacteriophages and plasmids, a noticeable number are derived from chromosomal DNA. While usually lethal to the self-targeting bacteria, in certain circumstances, these self-targeting spacers can have profound effects in regard to microbial biology, including functions beyond adaptive immunity. In this minireview, we discuss recent studies that focus on the functions and consequences of CRISPR-Cas self-targeting, including reshaping of the host population, group behavior modification, and the potential applications of CRISPR-Cas self-targeting as a tool in microbial biotechnology. Understanding the effects of CRISPR-Cas self-targeting is vital to fully understanding the spectrum of function of these systems. PMID:26929301

  15. Regulation of the Type I-F CRISPR-Cas system by CRP-cAMP and GalM controls spacer acquisition and interference.

    PubMed

    Patterson, Adrian G; Chang, James T; Taylor, Corinda; Fineran, Peter C

    2015-07-13

    The CRISPR-Cas prokaryotic 'adaptive immune systems' represent a sophisticated defence strategy providing bacteria and archaea with protection from invading genetic elements, such as bacteriophages or plasmids. Despite intensive research into their mechanism and application, how CRISPR-Cas systems are regulated is less clear, and nothing is known about the regulation of Type I-F systems. We used Pectobacterium atrosepticum, a Gram-negative phytopathogen, to study CRISPR-Cas regulation, since it contains a single Type I-F system. The CRP-cAMP complex activated the cas operon, increasing the expression of the adaptation genes cas1 and cas2-3 in addition to the genes encoding the Csy surveillance complex. Mutation of crp or cyaA (encoding adenylate cyclase) resulted in reductions in both primed spacer acquisition and interference. Furthermore, we identified a galactose mutarotase, GalM, which reduced cas operon expression in a CRP- and CyaA-dependent manner. We propose that the Type I-F system senses metabolic changes, such as sugar availability, and regulates cas genes to initiate an appropriate defence response. Indeed, elevated glucose levels reduced cas expression in a CRP- and CyaA-dependent manner. Taken together, these findings highlight that a metabolite-sensing regulatory pathway controls expression of the Type I-F CRISPR-Cas system to modulate levels of adaptation and interference.

  16. Calibrated Ancillary System (CAS) user's guide, volume 5

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 5 describes the testing user mission planning procedures including the bulletin board system and ancillary products procedures. Instructions for viewing the SDT/TDT (shuttle data tape/telemetry descriptor tape) data base and the file management menu are also given.

  17. Calibrated Ancillary System (CAS) user's guide, volume 4

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of the CAS. Volume 4 presents the GSFC user mission planning procedures covering the mission planning main menu, bulletin board system, ancillary products menu, utility menu procedures, and ancillary support files procedures.

  18. The molecular mechanism of CRISPR/Cas9 system and its application in gene therapy of human diseases.

    PubMed

    Liang, Qu; Huashan, Li; Yunhan, Jiang; Chunsheng, Dong

    2015-10-01

    CRISPR/Cas system is an adaptive immune system that confers resistance to exogenous virus or plasmid in bacteria and archaea. In recent years, the booming CRISPR/Cas9 genome editing technology modified from type2 CRISPR/Cas adaptive immune system has been widely applied to various research fields of life science and led to revolutionary changes. In this review, we summarize the origin and development of CRISPR/Cas9 genome editing technology as well as its applications in life science research. We focus on the latest application of this system in gene therapy of human diseases and the associated side/off-target effects, which may provide references for researchers in related areas.

  19. CAS

    SciTech Connect

    Martinez, B.; Pomeroy, G. )

    1989-12-02

    The Security Alarm System is a data acquisition and control system which collects data from intrusion sensors and displays the information in a real-time environment for operators. The Access Control System monitors and controls the movement of personnel with the use of card readers and biometrics hand readers.

  20. CRISPR-Cas9: from a bacterial immune system to genome-edited human cells in clinical trials.

    PubMed

    Kick, Leonhard; Kirchner, Marion; Schneider, Sabine

    2017-03-13

    The adaptive bacterial immune system CRISPR-Cas is revolutionising all fields of life science and has opened up new frontiers towards personalised medicine. Since the elucidation of the molecular mechanism of Cas9 from Streptococcus pyogenes in 2012 and its development as a genomic engineering tool, genetic modifications in more than 40 species have been carried out, over 290 patents have been filed worldwide and the first clinical trials using CRISPR-Cas-modified T-cells have recently been started in China and in the US. In this review we summarise current design developments, novel Cas systems and their antagonists, present and potential future applications as well as the ongoing debate on ethical issues, which has arisen through the CRISPR-Cas technology.

  1. Calibrated Ancillary System (CAS) user's guide, volume 7

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 7 describes the data flow engineer (DFE) user mission planning procedures which include instructions for processing the SDT/TDT (shuttle data tape/telemetry descriptor tape).

  2. Calibrated Ancillary System (CAS) user's guide, volume 6

    NASA Technical Reports Server (NTRS)

    1986-01-01

    The Calibrated Ancillary System (CAS) provides real-time calibrated parameters from the orbiter downlink (ancillary data) to the Goddard Space Flight Center (GSFC). This user's guide contains the introduction to the equipment, operation, general procedures, and specific procedures of CAS. Volume 6 describes ancillary products procedures, enhancement menu and processing task procedures for SDT/TDT (shuttle data tape/telemetry descriptor tape), database errors and network data driver (NDD) product menu procedures, and utility menu procedures.

  3. Priming in the Type I-F CRISPR-Cas system triggers strand-independent spacer acquisition, bi-directionally from the primed protospacer.

    PubMed

    Richter, Corinna; Dy, Ron L; McKenzie, Rebecca E; Watson, Bridget N J; Taylor, Corinda; Chang, James T; McNeil, Matthew B; Staals, Raymond H J; Fineran, Peter C

    2014-07-01

    Clustered regularly interspaced short palindromic repeats (CRISPR), in combination with CRISPR associated (cas) genes, constitute CRISPR-Cas bacterial adaptive immune systems. To generate immunity, these systems acquire short sequences of nucleic acids from foreign invaders and incorporate these into their CRISPR arrays as spacers. This adaptation process is the least characterized step in CRISPR-Cas immunity. Here, we used Pectobacterium atrosepticum to investigate adaptation in Type I-F CRISPR-Cas systems. Pre-existing spacers that matched plasmids stimulated hyperactive primed acquisition and resulted in the incorporation of up to nine new spacers across all three native CRISPR arrays. Endogenous expression of the cas genes was sufficient, yet required, for priming. The new spacers inhibited conjugation and transformation, and interference was enhanced with increasing numbers of new spacers. We analyzed ∼ 350 new spacers acquired in priming events and identified a 5'-protospacer-GG-3' protospacer adjacent motif. In contrast to priming in Type I-E systems, new spacers matched either plasmid strand and a biased distribution, including clustering near the primed protospacer, suggested a bi-directional translocation model for the Cas1:Cas2-3 adaptation machinery. Taken together these results indicate priming adaptation occurs in different CRISPR-Cas systems, that it can be highly active in wild-type strains and that the underlying mechanisms vary.

  4. Interference activity of a minimal Type I CRISPR–Cas system from Shewanella putrefaciens

    PubMed Central

    Dwarakanath, Srivatsa; Brenzinger, Susanne; Gleditzsch, Daniel; Plagens, André; Klingl, Andreas; Thormann, Kai; Randau, Lennart

    2015-01-01

    Type I CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)–Cas (CRISPR-associated) systems exist in bacterial and archaeal organisms and provide immunity against foreign DNA. The Cas protein content of the DNA interference complexes (termed Cascade) varies between different CRISPR-Cas subtypes. A minimal variant of the Type I-F system was identified in proteobacterial species including Shewanella putrefaciens CN-32. This variant lacks a large subunit (Csy1), Csy2 and Csy3 and contains two unclassified cas genes. The genome of S. putrefaciens CN-32 contains only five Cas proteins (Cas1, Cas3, Cas6f, Cas1821 and Cas1822) and a single CRISPR array with 81 spacers. RNA-Seq analyses revealed the transcription of this array and the maturation of crRNAs (CRISPR RNAs). Interference assays based on plasmid conjugation demonstrated that this CRISPR-Cas system is active in vivo and that activity is dependent on the recognition of the dinucleotide GG PAM (Protospacer Adjacent Motif) sequence and crRNA abundance. The deletion of cas1821 and cas1822 reduced the cellular crRNA pool. Recombinant Cas1821 was shown to form helical filaments bound to RNA molecules, which suggests its role as the Cascade backbone protein. A Cascade complex was isolated which contained multiple Cas1821 copies, Cas1822, Cas6f and mature crRNAs. PMID:26350210

  5. Complex Adaptive Systems as Metaphors for Organizational Management

    ERIC Educational Resources Information Center

    Palmberg, Klara

    2009-01-01

    Purpose: The purpose of this paper is to explore the concept of complex adaptive systems (CAS) from the perspective of managing organizations, to describe and explore the management principles in a case study of an organization with unconventional ways of management and to present a tentative model for managing organizations as CAS--system…

  6. Use of the CRISPR/Cas9 system as an intracellular defense against HIV-1 infection in human cells.

    PubMed

    Liao, Hsin-Kai; Gu, Ying; Diaz, Arturo; Marlett, John; Takahashi, Yuta; Li, Mo; Suzuki, Keiichiro; Xu, Ruo; Hishida, Tomoaki; Chang, Chan-Jung; Esteban, Concepcion Rodriguez; Young, John; Izpisua Belmonte, Juan Carlos

    2015-03-10

    To combat hostile viruses, bacteria and archaea have evolved a unique antiviral defense system composed of clustered regularly interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated genes (Cas). The CRISPR/Cas9 system develops an adaptive immune resistance to foreign plasmids and viruses by creating site-specific DNA double-stranded breaks (DSBs). Here we adapt the CRISPR/Cas9 system to human cells for intracellular defense against foreign DNA and viruses. Using HIV-1 infection as a model, our results demonstrate that the CRISPR/Cas9 system disrupts latently integrated viral genome and provides long-term adaptive defense against new viral infection, expression and replication in human cells. We show that engineered human-induced pluripotent stem cells stably expressing HIV-targeted CRISPR/Cas9 can be efficiently differentiated into HIV reservoir cell types and maintain their resistance to HIV-1 challenge. These results unveil the potential of the CRISPR/Cas9 system as a new therapeutic strategy against viral infections.

  7. Multiple genome modifications by the CRISPR/Cas9 system in zebrafish.

    PubMed

    Ota, Satoshi; Hisano, Yu; Ikawa, Yoshiya; Kawahara, Atsuo

    2014-07-01

    The type II clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which is an adaptive immune system of bacteria, has become a powerful tool for genome editing in various model organisms. Here, we demonstrate multiple genome modifications mediated by CRISPR/Cas9 in zebrafish (Danio rerio). Multiple genes including golden/gol and tyrosinase/tyr, which are involved in pigment formation, and s1pr2 and spns2, which are involved in cardiac development, were disrupted with insertion and/or deletion (indel) mutations introduced by the co-injection of multiple guide RNAs (gRNAs) and the nuclease Cas9 mRNA. We simultaneously observed two distinct phenotypes, such as, the two hearts phenotype and the hypopigmentation of skin melanophores and the retinal pigment epithelium, in the injected F0 embryos. Additionally, we detected the targeted deletion and inversion genes as a 7.1-kb fragment between the two distinct spns2 targeted sites together with indel mutations. Conversely, chromosomal translocations among five target loci were not detected. Therefore, we confirmed that the CRISPR/Cas9-induced indel mutations and a locus-specific deletion were heritable in F1 embryos. To screen founders, we improved heteroduplex mobility assay (HMA) for simultaneously detecting indel mutations in different target loci. The results suggest that the multi-locus HMA is a powerful tool for identification of multiple genome modifications mediated by the CRISPR/Cas9 system.

  8. Chromosomal targeting by CRISPR-Cas systems can contribute to genome plasticity in bacteria.

    PubMed

    Dy, Ron L; Pitman, Andrew R; Fineran, Peter C

    2013-09-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) and their associated (Cas) proteins form adaptive immune systems in bacteria to combat phage and other foreign genetic elements. Typically, short spacer sequences are acquired from the invader DNA and incorporated into CRISPR arrays in the bacterial genome. Small RNAs are generated that contain these spacer sequences and enable sequence-specific destruction of the foreign nucleic acids. Occasionally, spacers are acquired from the chromosome, which instead leads to targeting of the host genome. Chromosomal targeting is highly toxic to the bacterium, providing a strong selective pressure for a variety of evolutionary routes that enable host cell survival. Mutations that inactivate the CRISPR-Cas functionality, such as within the cas genes, CRISPR repeat, protospacer adjacent motifs (PAM), and target sequence, mediate escape from toxicity. This self-targeting might provide some explanation for the incomplete distribution of CRISPR-Cas systems in less than half of sequenced bacterial genomes. More importantly, self-genome targeting can cause large-scale genomic alterations, including remodeling or deletion of pathogenicity islands and other non-mobile chromosomal regions. While control of horizontal gene transfer is perceived as their main function, our recent work illuminates an alternative role of CRISPR-Cas systems in causing host genomic changes and influencing bacterial evolution.

  9. Applications of CRISPR-Cas systems in neuroscience

    PubMed Central

    Heidenreich, Matthias; Zhang, Feng

    2016-01-01

    Genome editing tools, and in particular those based on CRISPR-Cas systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in virtually any organism and cell type. These tools have opened the door to the development of new model systems for studying the complexity of the nervous system, including animal and stem cell-derived in vitro models. Precise and efficient gene editing using CRISPR-Cas systems has the potential to advance both basic and translational neuroscience research. PMID:26656253

  10. SD-CAS: Spin Dynamics by Computer Algebra System.

    PubMed

    Filip, Xenia; Filip, Claudiu

    2010-11-01

    A computer algebra tool for describing the Liouville-space quantum evolution of nuclear 1/2-spins is introduced and implemented within a computational framework named Spin Dynamics by Computer Algebra System (SD-CAS). A distinctive feature compared with numerical and previous computer algebra approaches to solving spin dynamics problems results from the fact that no matrix representation for spin operators is used in SD-CAS, which determines a full symbolic character to the performed computations. Spin correlations are stored in SD-CAS as four-entry nested lists of which size increases linearly with the number of spins into the system and are easily mapped into analytical expressions in terms of spin operator products. For the so defined SD-CAS spin correlations a set of specialized functions and procedures is introduced that are essential for implementing basic spin algebra operations, such as the spin operator products, commutators, and scalar products. They provide results in an abstract algebraic form: specific procedures to quantitatively evaluate such symbolic expressions with respect to the involved spin interaction parameters and experimental conditions are also discussed. Although the main focus in the present work is on laying the foundation for spin dynamics symbolic computation in NMR based on a non-matrix formalism, practical aspects are also considered throughout the theoretical development process. In particular, specific SD-CAS routines have been implemented using the YACAS computer algebra package (http://yacas.sourceforge.net), and their functionality was demonstrated on a few illustrative examples.

  11. Mouse Genome Editing Using the CRISPR/Cas System.

    PubMed

    Harms, Donald W; Quadros, Rolen M; Seruggia, Davide; Ohtsuka, Masato; Takahashi, Gou; Montoliu, Lluis; Gurumurthy, Channabasavaiah B

    2014-10-01

    The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much more quickly than the previously used techniques, and, more importantly, multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one-cell mouse embryos to create knockout or knock-in mouse models.

  12. Mouse Genome Editing using CRISPR/Cas System

    PubMed Central

    Harms, Donald W; Quadros, Rolen M; Seruggia, Davide; Ohtsuka, Masato; Takahashi, Gou

    2015-01-01

    The availability of techniques to create desired genetic mutations has enabled the laboratory mouse as an extensively used model organism in biomedical research including human genetics. A new addition to this existing technical repertoire is the CRISPR/Cas system. Specifically, this system allows editing of the mouse genome much faster than the previously used techniques and more importantly multiple mutations can be created in a single experiment. Here we provide protocols for preparation of CRISPR/Cas reagents and microinjection into one cell mouse embryos to create knockout or knock-in mouse models. PMID:25271839

  13. Harnessing CRISPR-Cas systems for bacterial genome editing.

    PubMed

    Selle, Kurt; Barrangou, Rodolphe

    2015-04-01

    Manipulation of genomic sequences facilitates the identification and characterization of key genetic determinants in the investigation of biological processes. Genome editing via clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) constitutes a next-generation method for programmable and high-throughput functional genomics. CRISPR-Cas systems are readily reprogrammed to induce sequence-specific DNA breaks at target loci, resulting in fixed mutations via host-dependent DNA repair mechanisms. Although bacterial genome editing is a relatively unexplored and underrepresented application of CRISPR-Cas systems, recent studies provide valuable insights for the widespread future implementation of this technology. This review summarizes recent progress in bacterial genome editing and identifies fundamental genetic and phenotypic outcomes of CRISPR targeting in bacteria, in the context of tool development, genome homeostasis, and DNA repair.

  14. First indication for a functional CRISPR/Cas system in Francisella tularensis.

    PubMed

    Schunder, Eva; Rydzewski, Kerstin; Grunow, Roland; Heuner, Klaus

    2013-03-01

    Francisella tularensis is a zoonotic agent and the subspecies novicida is proposed to be a water-associated bacterium. The intracellular pathogen F. tularensis causes tularemia in humans and is known for its potential to be used as a biological threat. We analyzed the genome sequence of F. tularensis subsp. novicida U112 in silico for the presence of a putative functional CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system. CRISPR/Cas systems are known to encode an RNA-guided adaptive immunity-like system to protect bacteria against invading genetic elements like bacteriophages and plasmids. In this work, we present a first indication that F. tularensis subsp. novicida encodes a functional CRISPR/Cas defence system. Additionally, we identified various spacer DNAs homologous to a putative phage present within the genome of F. tularensis subsp. novicida-like strain 3523. CRISPR/Cas is also present in F. tularensis subsp. tularensis, holarctica, and mediasiatica, but these systems seem to be non-functional.

  15. Study of Eclipsing Binary and Multiple Systems in OB Associations IV: Cas OB6 Member DN Cas

    NASA Astrophysics Data System (ADS)

    Bakış, V.; Bakış, H.; Bilir, S.; Eker, Z.

    2016-09-01

    An early-type, massive, short-period (Porb=2d.310951) eclipsing spectroscopic binary DN Cas has been re-visited with new spectral and photometric data. The masses and radii of the components have been obtained as M1=19.04± 0.07 M⊙, M2=13.73± 0.05 M⊙ and R1=7.22± 0.06 R⊙, R2=5.79± 0.06 R⊙, respectively. Both components present synchronous rotation (Vrot1=160 km s-1, Vrot2=130 km s-1) with their orbit. Orbital period analysis yielded a physically bound additional component in the system with a minimum mass of M3=0.88 M⊙ orbiting in an eccentric orbit (e = 0.37 ± 0.2) with an orbital period of P 12 = 42 ± 9 yr. High precision absolute parameters of the system allowed us to derive a distance to DN Cas as 1.7 ± 0.2 kpc which locates the system within the borders of the Cas OB6 association (d = 1.8 kpc). The space velocities and the age of DN Cas are in agreement with those of Cas OB6. The age of DN Cas (τ = 3-5 Myr) is found to be 1-2 Myr older than the embedded clusters (IC 1795, IC 1805, and IC 1848) in the Cas OB6 association, which implies a sequential star formation in the association.

  16. CRISPR adaptation in Escherichia coli subtypeI-E system.

    PubMed

    Kiro, Ruth; Goren, Moran G; Yosef, Ido; Qimron, Udi

    2013-12-01

    The CRISPRs (clustered regularly interspaced short palindromic repeats) and their associated Cas (CRISPR-associated) proteins are a prokaryotic adaptive defence system against foreign nucleic acids. The CRISPR array comprises short repeats flanking short segments, called 'spacers', which are derived from foreign nucleic acids. The process of spacer insertion into the CRISPR array is termed 'adaptation'. Adaptation allows the system to rapidly evolve against emerging threats. In the present article, we review the most recent studies on the adaptation process, and focus primarily on the subtype I-E CRISPR-Cas system of Escherichia coli.

  17. Adaptation of adaptive optics systems.

    NASA Astrophysics Data System (ADS)

    Xin, Yu; Zhao, Dazun; Li, Chen

    1997-10-01

    In the paper, a concept of an adaptation of adaptive optical system (AAOS) is proposed. The AAOS has certain real time optimization ability against the variation of the brightness of detected objects m, atmospheric coherence length rO and atmospheric time constant τ by means of changing subaperture number and diameter, dynamic range, and system's temporal response. The necessity of AAOS using a Hartmann-Shack wavefront sensor and some technical approaches are discussed. Scheme and simulation of an AAOS with variable subaperture ability by use of both hardware and software are presented as an example of the system.

  18. The role of CRISPR-Cas systems in virulence of pathogenic bacteria.

    PubMed

    Louwen, Rogier; Staals, Raymond H J; Endtz, Hubert P; van Baarlen, Peter; van der Oost, John

    2014-03-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) genes are present in many bacterial and archaeal genomes. Since the discovery of the typical CRISPR loci in the 1980s, well before their physiological role was revealed, their variable sequences have been used as a complementary typing tool in diagnostic, epidemiologic, and evolutionary analyses of prokaryotic strains. The discovery that CRISPR spacers are often identical to sequence fragments of mobile genetic elements was a major breakthrough that eventually led to the elucidation of CRISPR-Cas as an adaptive immunity system. Key elements of this unique prokaryotic defense system are small CRISPR RNAs that guide nucleases to complementary target nucleic acids of invading viruses and plasmids, generally followed by the degradation of the invader. In addition, several recent studies have pointed at direct links of CRISPR-Cas to regulation of a range of stress-related phenomena. An interesting example concerns a pathogenic bacterium that possesses a CRISPR-associated ribonucleoprotein complex that may play a dual role in defense and/or virulence. In this review, we describe recently reported cases of potential involvement of CRISPR-Cas systems in bacterial stress responses in general and bacterial virulence in particular.

  19. A dual function of the CRISPR-Cas system in bacterial antivirus immunity and DNA repair.

    PubMed

    Babu, Mohan; Beloglazova, Natalia; Flick, Robert; Graham, Chris; Skarina, Tatiana; Nocek, Boguslaw; Gagarinova, Alla; Pogoutse, Oxana; Brown, Greg; Binkowski, Andrew; Phanse, Sadhna; Joachimiak, Andrzej; Koonin, Eugene V; Savchenko, Alexei; Emili, Andrew; Greenblatt, Jack; Edwards, Aled M; Yakunin, Alexander F

    2011-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) and the associated proteins (Cas) comprise a system of adaptive immunity against viruses and plasmids in prokaryotes. Cas1 is a CRISPR-associated protein that is common to all CRISPR-containing prokaryotes but its function remains obscure. Here we show that the purified Cas1 protein of Escherichia coli (YgbT) exhibits nuclease activity against single-stranded and branched DNAs including Holliday junctions, replication forks and 5'-flaps. The crystal structure of YgbT and site-directed mutagenesis have revealed the potential active site. Genome-wide screens show that YgbT physically and genetically interacts with key components of DNA repair systems, including recB, recC and ruvB. Consistent with these findings, the ygbT deletion strain showed increased sensitivity to DNA damage and impaired chromosomal segregation. Similar phenotypes were observed in strains with deletion of CRISPR clusters, suggesting that the function of YgbT in repair involves interaction with the CRISPRs. These results show that YgbT belongs to a novel, structurally distinct family of nucleases acting on branched DNAs and suggest that, in addition to antiviral immunity, at least some components of the CRISPR-Cas system have a function in DNA repair.

  20. Targeted mutagenesis in chicken using CRISPR/Cas9 system

    PubMed Central

    Oishi, Isao; Yoshii, Kyoko; Miyahara, Daichi; Kagami, Hiroshi; Tagami, Takahiro

    2016-01-01

    The CRISPR/Cas9 system is a simple and powerful tool for genome editing in various organisms including livestock animals. However, the system has not been applied to poultry because of the difficulty in accessing their zygotes. Here we report the implementation of CRISPR/Cas9-mediated gene targeting in chickens. Two egg white genes, ovalbumin and ovomucoid, were efficiently (>90%) mutagenized in cultured chicken primordial germ cells (PGCs) by transfection of circular plasmids encoding Cas9, a single guide RNA, and a gene encoding drug resistance, followed by transient antibiotic selection. We transplanted CRISPR-induced mutant-ovomucoid PGCs into recipient chicken embryos and established three germline chimeric roosters (G0). All of the roosters had donor-derived mutant-ovomucoid spermatozoa, and the two with a high transmission rate of donor-derived gametes produced heterozygous mutant ovomucoid chickens as about half of their donor-derived offspring in the next generation (G1). Furthermore, we generated ovomucoid homozygous mutant offspring (G2) by crossing the G1 mutant chickens. Taken together, these results demonstrate that the CRISPR/Cas9 system is a simple and effective gene-targeting method in chickens. PMID:27050479

  1. A non-inheritable maternal Cas9-based multiple-gene editing system in mice.

    PubMed

    Sakurai, Takayuki; Kamiyoshi, Akiko; Kawate, Hisaka; Mori, Chie; Watanabe, Satoshi; Tanaka, Megumu; Uetake, Ryuichi; Sato, Masahiro; Shindo, Takayuki

    2016-01-28

    The CRISPR/Cas9 system is capable of editing multiple genes through one-step zygote injection. The preexisting method is largely based on the co-injection of Cas9 DNA (or mRNA) and guide RNAs (gRNAs); however, it is unclear how many genes can be simultaneously edited by this method, and a reliable means to generate transgenic (Tg) animals with multiple gene editing has yet to be developed. Here, we employed non-inheritable maternal Cas9 (maCas9) protein derived from Tg mice with systemic Cas9 overexpression (Cas9 mice). The maCas9 protein in zygotes derived from mating or in vitro fertilization of Tg/+ oocytes and +/+ sperm could successfully edit the target genome. The efficiency of such maCas9-based genome editing was comparable to that of zygote microinjection-based genome editing widely used at present. Furthermore, we demonstrated a novel approach to create "Cas9 transgene-free" gene-modified mice using non-Tg (+/+) zygotes carrying maCas9. The maCas9 protein in mouse zygotes edited nine target loci simultaneously after injection with nine different gRNAs alone. Cas9 mouse-derived zygotes have the potential to facilitate the creation of genetically modified animals carrying the Cas9 transgene, enabling repeatable genome engineering and the production of Cas9 transgene-free mice.

  2. Function of the CRISPR-Cas System of the Human Pathogen Clostridium difficile

    PubMed Central

    Boudry, Pierre; Semenova, Ekaterina; Monot, Marc; Datsenko, Kirill A.; Lopatina, Anna; Sekulovic, Ognjen; Ospina-Bedoya, Maicol; Fortier, Louis-Charles; Severinov, Konstantin; Dupuy, Bruno

    2015-01-01

    ABSTRACT Clostridium difficile is the cause of most frequently occurring nosocomial diarrhea worldwide. As an enteropathogen, C. difficile must be exposed to multiple exogenous genetic elements in bacteriophage-rich gut communities. CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems allow bacteria to adapt to foreign genetic invaders. Our recent data revealed active expression and processing of CRISPR RNAs from multiple type I-B CRISPR arrays in C. difficile reference strain 630. Here, we demonstrate active expression of CRISPR arrays in strain R20291, an epidemic C. difficile strain. Through genome sequencing and host range analysis of several new C. difficile phages and plasmid conjugation experiments, we provide evidence of defensive function of the CRISPR-Cas system in both C. difficile strains. We further demonstrate that C. difficile Cas proteins are capable of interference in a heterologous host, Escherichia coli. These data set the stage for mechanistic and physiological analyses of CRISPR-Cas-mediated interactions of important global human pathogen with its genetic parasites. PMID:26330515

  3. Asymmetric positioning of Cas1–2 complex and Integration Host Factor induced DNA bending guide the unidirectional homing of protospacer in CRISPR-Cas type I-E system

    PubMed Central

    Yoganand, K.N.R.; Sivathanu, R.; Nimkar, Siddharth; Anand, B.

    2017-01-01

    CRISPR–Cas system epitomizes prokaryote-specific quintessential adaptive defense machinery that limits the genome invasion of mobile genetic elements. It confers adaptive immunity to bacteria by capturing a protospacer fragment from invading foreign DNA, which is later inserted into the leader proximal end of CRIPSR array and serves as immunological memory to recognize recurrent invasions. The universally conserved Cas1 and Cas2 form an integration complex that is known to mediate the protospacer invasion into the CRISPR array. However, the mechanism by which this protospacer fragment gets integrated in a directional fashion into the leader proximal end is elusive. Here, we employ CRISPR/dCas9 mediated immunoprecipitation and genetic analysis to identify Integration Host Factor (IHF) as an indispensable accessory factor for spacer acquisition in Escherichia coli. Further, we show that the leader region abutting the first CRISPR repeat localizes IHF and Cas1–2 complex. IHF binding to the leader region induces bending by about 120° that in turn engenders the regeneration of the cognate binding site for protospacer bound Cas1–2 complex and brings it in proximity with the first CRISPR repeat. This appears to guide Cas1–2 complex to orient the protospacer invasion towards the leader-repeat junction thus driving the integration in a polarized fashion. PMID:27899566

  4. Decision-making in healthcare as a complex adaptive system.

    PubMed

    Kuziemsky, Craig

    2016-01-01

    Healthcare transformation requires a change in how the business of healthcare is done. Traditional decision-making approaches based on stable and predictable systems are inappropriate in healthcare because of the complex nature of healthcare delivery. This article reviews challenges to using traditional decision-making approaches in healthcare and how insight from Complex Adaptive Systems (CAS) could support healthcare management. The article also provides a system model to guide decision-making in healthcare as a CAS.

  5. CRISPR-Cas systems for editing, regulating and targeting genomes.

    PubMed

    Sander, Jeffry D; Joung, J Keith

    2014-04-01

    Targeted genome editing using engineered nucleases has rapidly gone from being a niche technology to a mainstream method used by many biological researchers. This widespread adoption has been largely fueled by the emergence of the clustered, regularly interspaced, short palindromic repeat (CRISPR) technology, an important new approach for generating RNA-guided nucleases, such as Cas9, with customizable specificities. Genome editing mediated by these nucleases has been used to rapidly, easily and efficiently modify endogenous genes in a wide variety of biomedically important cell types and in organisms that have traditionally been challenging to manipulate genetically. Furthermore, a modified version of the CRISPR-Cas9 system has been developed to recruit heterologous domains that can regulate endogenous gene expression or label specific genomic loci in living cells. Although the genome-wide specificities of CRISPR-Cas9 systems remain to be fully defined, the power of these systems to perform targeted, highly efficient alterations of genome sequence and gene expression will undoubtedly transform biological research and spur the development of novel molecular therapeutics for human disease.

  6. Therapeutic genome engineering via CRISPR-Cas systems.

    PubMed

    Moreno, Ana M; Mali, Prashant

    2017-02-15

    Differences in genomes underlie most organismal diversity, and aberrations in genomes underlie many disease states. With the growing knowledge of the genetic and pathogenic basis of human disease, development of safe and efficient platforms for genome and epigenome engineering will transform our ability to therapeutically target human diseases and also potentially engineer disease resistance. In this regard, the recent advent of clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) RNA-guided nuclease systems have transformed our ability to target nucleic acids. Here we review therapeutic genome engineering applications with a specific focus on the CRISPR-Cas toolsets. We summarize past and current work, and also outline key challenges and future directions. For further resources related to this article, please visit the WIREs website.

  7. A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi

    PubMed Central

    Nødvig, Christina S.; Nielsen, Jakob B.; Kogle, Martin E.; Mortensen, Uffe H.

    2015-01-01

    The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, we have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. The system is simple and versatile, as RNA guided mutagenesis can be achieved by transforming a target fungus with a single plasmid. The system currently contains four CRISPR-Cas9 vectors, which are equipped with commonly used fungal markers allowing for selection in a broad range of fungi. Moreover, we have developed a script that allows identification of protospacers that target gene homologs in multiple species to facilitate introduction of common mutations in different filamentous fungi. With these tools we have performed RNA-guided mutagenesis in six species of which one has not previously been genetically engineered. Moreover, for a wild-type Aspergillus aculeatus strain, we have used our CRISPR Cas9 system to generate a strain that contains an AACU_pyrG marker and demonstrated that the resulting strain can be used for iterative gene targeting. PMID:26177455

  8. Function and regulation of clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR associated (Cas) systems.

    PubMed

    Richter, Corinna; Chang, James T; Fineran, Peter C

    2012-10-19

    Phages are the most abundant biological entities on earth and pose a constant challenge to their bacterial hosts. Thus, bacteria have evolved numerous 'innate' mechanisms of defense against phage, such as abortive infection or restriction/modification systems. In contrast, the clustered regularly interspaced short palindromic repeats (CRISPR) systems provide acquired, yet heritable, sequence-specific 'adaptive' immunity against phage and other horizontally-acquired elements, such as plasmids. Resistance is acquired following viral infection or plasmid uptake when a short sequence of the foreign genome is added to the CRISPR array. CRISPRs are then transcribed and processed, generally by CRISPR associated (Cas) proteins, into short interfering RNAs (crRNAs), which form part of a ribonucleoprotein complex. This complex guides the crRNA to the complementary invading nucleic acid and targets this for degradation. Recently, there have been rapid advances in our understanding of CRISPR/Cas systems. In this review, we will present the current model(s) of the molecular events involved in both the acquisition of immunity and interference stages and will also address recent progress in our knowledge of the regulation of CRISPR/Cas systems.

  9. A CRISPR-Cas9 System for Genetic Engineering of Filamentous Fungi.

    PubMed

    Nødvig, Christina S; Nielsen, Jakob B; Kogle, Martin E; Mortensen, Uffe H

    2015-01-01

    The number of fully sequenced fungal genomes is rapidly increasing. Since genetic tools are poorly developed for most filamentous fungi, it is currently difficult to employ genetic engineering for understanding the biology of these fungi and to fully exploit them industrially. For that reason there is a demand for developing versatile methods that can be used to genetically manipulate non-model filamentous fungi. To facilitate this, we have developed a CRISPR-Cas9 based system adapted for use in filamentous fungi. The system is simple and versatile, as RNA guided mutagenesis can be achieved by transforming a target fungus with a single plasmid. The system currently contains four CRISPR-Cas9 vectors, which are equipped with commonly used fungal markers allowing for selection in a broad range of fungi. Moreover, we have developed a script that allows identification of protospacers that target gene homologs in multiple species to facilitate introduction of common mutations in different filamentous fungi. With these tools we have performed RNA-guided mutagenesis in six species of which one has not previously been genetically engineered. Moreover, for a wild-type Aspergillus aculeatus strain, we have used our CRISPR Cas9 system to generate a strain that contains an AACU_pyrG marker and demonstrated that the resulting strain can be used for iterative gene targeting.

  10. Efficient mutagenesis by CRISPR/Cas system during meiotic maturation of porcine oocytes

    PubMed Central

    ONUMA, Asuka; FUJII, Wataru; SUGIURA, Koji; NAITO, Kunihiko

    2016-01-01

    Genome editing using the CRISPR/Cas system can induce mutations with high efficiency, and allows easier production of genome-modified animals than that offered by the conventional method where embryonic stem cells are used. However, studies using CRISPR/Cas systems have been mostly limited to proliferating somatic cells and pronuclear-stage fertilized eggs. In contrast, the efficiency of a CRISPR/Cas system in immature and maturing oocytes progressing through meiosis has not yet been assessed. In the present study, we evaluated the genome-modification efficiency of the CRISPR/Cas system during meiotic maturation of porcine oocytes. Additionally, the localization of the Cas9 protein in immature oocytes was analyzed in relation to nuclear transport and mutation induction. The results showed that CRISPR/Cas induced mutation with high efficiency even in maturing oocytes with condensed chromosomes, whereas mutations were not induced in GV-stage oocytes. The localization analysis of enhanced green fluorescent protein (EGFP)-tagged Cas9 (Cas9-EGFP) revealed that the nuclei contained lesser Cas9 than the cytoplasm in immature oocytes. Treatment with leptomycin B, a nuclear export inhibitor, increased the amount of nuclear Cas9 and enabled mutation induction in GV oocytes. Our results suggest that CRISPR/Cas systems can be applied to oocytes during meiotic maturation and be implemented in novel applications targeting female genomes. PMID:27773884

  11. Efficient mutagenesis by CRISPR/Cas system during meiotic maturation of porcine oocytes.

    PubMed

    Onuma, Asuka; Fujii, Wataru; Sugiura, Koji; Naito, Kunihiko

    2017-02-16

    Genome editing using the CRISPR/Cas system can induce mutations with high efficiency, and allows easier production of genome-modified animals than that offered by the conventional method where embryonic stem cells are used. However, studies using CRISPR/Cas systems have been mostly limited to proliferating somatic cells and pronuclear-stage fertilized eggs. In contrast, the efficiency of a CRISPR/Cas system in immature and maturing oocytes progressing through meiosis has not yet been assessed. In the present study, we evaluated the genome-modification efficiency of the CRISPR/Cas system during meiotic maturation of porcine oocytes. Additionally, the localization of the Cas9 protein in immature oocytes was analyzed in relation to nuclear transport and mutation induction. The results showed that CRISPR/Cas induced mutation with high efficiency even in maturing oocytes with condensed chromosomes, whereas mutations were not induced in GV-stage oocytes. The localization analysis of enhanced green fluorescent protein (EGFP)-tagged Cas9 (Cas9-EGFP) revealed that the nuclei contained lesser Cas9 than the cytoplasm in immature oocytes. Treatment with leptomycin B, a nuclear export inhibitor, increased the amount of nuclear Cas9 and enabled mutation induction in GV oocytes. Our results suggest that CRISPR/Cas systems can be applied to oocytes during meiotic maturation and be implemented in novel applications targeting female genomes.

  12. A simplified and efficient germline-specific CRISPR/Cas9 system for Drosophila genomic engineering.

    PubMed

    Sebo, Zachary L; Lee, Han B; Peng, Ying; Guo, Yi

    2014-01-01

    The type II CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated) has recently emerged as an efficient and simple tool for site-specific engineering of eukaryotic genomes. To improve its applications in Drosophila genome engineering, we simplified the standard two-component CRISPR/Cas9 system by generating a stable transgenic fly line expressing the Cas9 endonuclease in the germline (Vasa-Cas9 line). By injecting vectors expressing engineered target-specific guide RNAs into Vasa-Cas9 fly embryos, mutations were generated from site-specific DNA cleavages and efficiently transmitted into progenies. Because Cas9 endonuclease is the universal component of the type II CRISPR/Cas9 system, site-specific genomic engineering based on this improved platform can be achieved with lower complexity and toxicity, greater consistency, and excellent versatility.

  13. Differential Distribution of Type II CRISPR-Cas Systems in Agricultural and Nonagricultural Campylobacter coli and Campylobacter jejuni Isolates Correlates with Lack of Shared Environments.

    PubMed

    Pearson, Bruce M; Louwen, Rogier; van Baarlen, Peter; van Vliet, Arnoud H M

    2015-09-02

    CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) systems are sequence-specific adaptive defenses against phages and plasmids which are widespread in prokaryotes. Here we have studied whether phylogenetic relatedness or sharing of environmental niches affects the distribution and dissemination of Type II CRISPR-Cas systems, first in 132 bacterial genomes from 15 phylogenetic classes, ranging from Proteobacteria to Actinobacteria. There was clustering of distinct Type II CRISPR-Cas systems in phylogenetically distinct genera with varying G+C%, which share environmental niches. The distribution of CRISPR-Cas within a genus was studied using a large collection of genome sequences of the closely related Campylobacter species Campylobacter jejuni (N = 3,746) and Campylobacter coli (N = 486). The Cas gene cas9 and CRISPR-repeat are almost universally present in C. jejuni genomes (98.0% positive) but relatively rare in C. coli genomes (9.6% positive). Campylobacter jejuni and agricultural C. coli isolates share the C. jejuni CRISPR-Cas system, which is closely related to, but distinct from the C. coli CRISPR-Cas system found in C. coli isolates from nonagricultural sources. Analysis of the genomic position of CRISPR-Cas insertion suggests that the C. jejuni-type CRISPR-Cas has been transferred to agricultural C. coli. Conversely, the absence of the C. coli-type CRISPR-Cas in agricultural C. coli isolates may be due to these isolates not sharing the same environmental niche, and may be affected by farm hygiene and biosecurity practices in the agricultural sector. Finally, many CRISPR spacer alleles were linked with specific multilocus sequence types, suggesting that these can assist molecular epidemiology applications for C. jejuni and C. coli.

  14. Differential Distribution of Type II CRISPR-Cas Systems in Agricultural and Nonagricultural Campylobacter coli and Campylobacter jejuni Isolates Correlates with Lack of Shared Environments

    PubMed Central

    Pearson, Bruce M.; Louwen, Rogier; van Baarlen, Peter; van Vliet, Arnoud H.M.

    2015-01-01

    CRISPR (clustered regularly interspaced palindromic repeats)-Cas (CRISPR-associated) systems are sequence-specific adaptive defenses against phages and plasmids which are widespread in prokaryotes. Here we have studied whether phylogenetic relatedness or sharing of environmental niches affects the distribution and dissemination of Type II CRISPR-Cas systems, first in 132 bacterial genomes from 15 phylogenetic classes, ranging from Proteobacteria to Actinobacteria. There was clustering of distinct Type II CRISPR-Cas systems in phylogenetically distinct genera with varying G+C%, which share environmental niches. The distribution of CRISPR-Cas within a genus was studied using a large collection of genome sequences of the closely related Campylobacter species Campylobacter jejuni (N = 3,746) and Campylobacter coli (N = 486). The Cas gene cas9 and CRISPR-repeat are almost universally present in C. jejuni genomes (98.0% positive) but relatively rare in C. coli genomes (9.6% positive). Campylobacter jejuni and agricultural C. coli isolates share the C. jejuni CRISPR-Cas system, which is closely related to, but distinct from the C. coli CRISPR-Cas system found in C. coli isolates from nonagricultural sources. Analysis of the genomic position of CRISPR-Cas insertion suggests that the C. jejuni-type CRISPR-Cas has been transferred to agricultural C. coli. Conversely, the absence of the C. coli-type CRISPR-Cas in agricultural C. coli isolates may be due to these isolates not sharing the same environmental niche, and may be affected by farm hygiene and biosecurity practices in the agricultural sector. Finally, many CRISPR spacer alleles were linked with specific multilocus sequence types, suggesting that these can assist molecular epidemiology applications for C. jejuni and C. coli. PMID:26338188

  15. Functional Analysis of Porphyromonas gingivalis W83 CRISPR-Cas Systems

    PubMed Central

    Burmistrz, Michał; Dudek, Bartosz; Staniec, Dominika; Rodriguez Martinez, Jose Ignacio; Bochtler, Matthias; Potempa, Jan

    2015-01-01

    ABSTRACT The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system provides prokaryotic cells with an adaptive and heritable immune response to foreign genetic elements, such as viruses, plasmids, and transposons. It is present in the majority of Archaea and almost half of species of Bacteria. Porphyromonas gingivalis is an important human pathogen that has been proven to be an etiological agent of periodontitis and has been linked to systemic conditions, such as rheumatoid arthritis and cardiovascular disease. At least 95% of clinical strains of P. gingivalis carry CRISPR arrays, suggesting that these arrays play an important function in vivo. Here we show that all four CRISPR arrays present in the P. gingivalis W83 genome are transcribed. For one of the arrays, we demonstrate in vivo activity against double-stranded DNA constructs containing protospacer sequences accompanied at the 3′ end by an NGG protospacer-adjacent motif (PAM). Most of the 44 spacers present in the genome of P. gingivalis W83 share no significant similarity with any known sequences, although 4 spacers are similar to sequences from bacteria found in the oral cavity and the gastrointestinal tract. Four spacers match genomic sequences of the host; however, none of these is flanked at its 3′ terminus by the appropriate PAM element. IMPORTANCE The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated genes) system is a unique system that provides prokaryotic cells with an adaptive and heritable immunity. In this report, we show that the CRISPR-Cas system of P. gingivalis, an important human pathogen associated with periodontitis and possibly also other conditions, such as rheumatoid arthritis and cardiovascular disease, is active and provides protection from foreign genetic elements. Importantly, the data presented here may be useful for better understanding the communication between cells in larger bacterial

  16. Detection and characterization of spacer integration intermediates in type I-E CRISPR-Cas system.

    PubMed

    Arslan, Zihni; Hermanns, Veronica; Wurm, Reinhild; Wagner, Rolf; Pul, Ümit

    2014-07-01

    The adaptation against foreign nucleic acids by the CRISPR-Cas system (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins) depends on the insertion of foreign nucleic acid-derived sequences into the CRISPR array as novel spacers by still unknown mechanism. We identified and characterized in Escherichia coli intermediate states of spacer integration and mapped the integration site at the chromosomal CRISPR array in vivo. The results show that the insertion of new spacers occurs by site-specific nicking at both strands of the leader proximal repeat in a staggered way and is accompanied by joining of the resulting 5'-ends of the repeat strands with the 3'-ends of the incoming spacer. This concerted cleavage-ligation reaction depends on the metal-binding center of Cas1 protein and requires the presence of Cas2. By acquisition assays using plasmid-located CRISPR array with mutated repeat sequences, we demonstrate that the primary sequence of the first repeat is crucial for cleavage of the CRISPR array and the ligation of new spacer DNA.

  17. Genome editing in Clostridium saccharoperbutylacetonicum N1-4 using CRISPR-Cas9 system.

    PubMed

    Wang, Shaohua; Dong, Sheng; Wang, Pixiang; Tao, Yong; Wang, Yi

    2017-03-03

    Clostridium saccharoperbutylacetonicum N1-4 is well known as a hyper-butanol-producing strain. However, the lack of genetic engineering tools hinders further elucidation of its solvent production mechanism and development of more robust strains. In this study, we set out to develop an efficient genome engineering system for this microorganism based on the CRISPR-Cas9 system. First, the functionality of the CRISPR-Cas9 system previously customized for C. beijerinckii was evaluated in C. saccharoperbutylacetonicum by targeting on pta and buk, two essential genes for acetate and butyrate production, respectively. The pta, buk single deletion, and the pta and buk double deletion mutants were successfully obtained based on this system. However, the genome engineering efficiency was rather low (the mutation rate is < 20%). Therefore, the efficiency was further optimized by evaluating various promoters for the gRNA expression. With promoter P J23119 , we achieved a mutation rate of 75% for pta deletion without serial subculturing as suggested previously for C. beijerinckii Thus, this developed CRISPR-Cas9 system is highly desirable for efficient genome editing in C. saccharoperbutylacetonicum Batch fermentation results revealed that both the acid and solvent production profiles were altered due to the disruption of acid production pathways, however neither acetate nor butyrate production was eliminated with the deletion of the corresponding gene. The butanol production, yield and selectivity were improved in mutants dependent on the fermentation medium. In the pta-buk double deletion mutant, the butanol production reached 19.0 g/l in P2 medium, which is one of the highest among the ever reported from batch fermentations.IMPORTANCE An efficient CRISPR-Cas9 genome engineering system was developed for C. saccharoperbutylacetonicum N1-4. This paves the way for elucidating the solvent production mechanism in this hyper-butanol-producing microorganism and developing strains

  18. The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models.

    PubMed

    Shao, Ming; Xu, Tian-Rui; Chen, Ce-Shi

    2016-07-18

    Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and bio-medicine.

  19. The big bang of genome editing technology: development and application of the CRISPR/Cas9 system in disease animal models

    PubMed Central

    SHAO, Ming; XU, Tian-Rui; CHEN, Ce-Shi

    2016-01-01

    Targeted genome editing technology has been widely used in biomedical studies. The CRISPR-associated RNA-guided endonuclease Cas9 has become a versatile genome editing tool. The CRISPR/Cas9 system is useful for studying gene function through efficient knock-out, knock-in or chromatin modification of the targeted gene loci in various cell types and organisms. It can be applied in a number of fields, such as genetic breeding, disease treatment and gene functional investigation. In this review, we introduce the most recent developments and applications, the challenges, and future directions of Cas9 in generating disease animal model. Derived from the CRISPR adaptive immune system of bacteria, the development trend of Cas9 will inevitably fuel the vital applications from basic research to biotechnology and biomedicine. PMID:27469250

  20. From Calculus to Dynamical Systems through DGS and CAS

    ERIC Educational Resources Information Center

    García, Jeanett López; Zamudio, Jorge Javier Jiménez

    2015-01-01

    Several factors have motivated the use of CAS or DGS in the teaching-learning process, such as: the development of new technologies, the availability of computers, and the widespread use of the Internet, among others. Even more, the trend to include CAS and DGS in the curricula of some undergraduate studies has resulted in the instruction of the…

  1. Engineering Plants for Geminivirus Resistance with CRISPR/Cas9 System.

    PubMed

    Zaidi, Syed Shan-E-Ali; Mansoor, Shahid; Ali, Zahir; Tashkandi, Manal; Mahfouz, Magdy M

    2016-04-01

    The CRISPR/Cas9 system is an efficient genome-editing platform for diverse eukaryotic species, including plants. Recent work harnessed CRISPR/Cas9 technology to engineer resistance to geminiviruses. Here, we discuss opportunities, emerging developments, and potential pitfalls for using this technology to engineer resistance against single and multiple geminivirus infections in plants.

  2. The driving force of prophages and CRISPR-Cas system in the evolution of Cronobacter sakazakii

    PubMed Central

    Zeng, Haiyan; Zhang, Jumei; Li, Chensi; Xie, Tengfei; Ling, Na; Wu, Qingping; Ye, Yingwang

    2017-01-01

    Cronobacter sakazakii is an important foodborne pathogens causing rare but life-threatening diseases in neonates and infants. CRISPR-Cas system is a new prokaryotic defense system that provides adaptive immunity against phages, latter play an vital role on the evolution and pathogenicity of host bacteria. In this study, we found that genome sizes of C. sakazakii strains had a significant positive correlation with total genome sizes of prophages. Prophages contributed to 16.57% of the genetic diversity (pan genome) of C. sakazakii, some of which maybe the potential virulence factors. Subtype I-E CRISPR-Cas system and five types of CRISPR arrays were found in the conserved site of C. sakazakii strains. CRISPR1 and CRISPR2 loci with high variable spacers were active and showed potential protection against phage attacks. The number of spacers from two active CRISPR loci in clinical strains was significant less than that of foodborne strains, it maybe a reason why clinical strains were found to have more prophages than foodborne strains. The frequently gain/loss of prophages and spacers in CRISPR loci is likely to drive the quick evolution of C. sakazakii. Our study provides a new insight into the co-evolution of phages and C. sakazakii. PMID:28057934

  3. Efficient engineering of a bacteriophage genome using the type I-E CRISPR-Cas system.

    PubMed

    Kiro, Ruth; Shitrit, Dror; Qimron, Udi

    2014-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) system has recently been used to engineer genomes of various organisms, but surprisingly, not those of bacteriophages (phages). Here we present a method to genetically engineer the Escherichia coli phage T7 using the type I-E CRISPR-Cas system. T7 phage genome is edited by homologous recombination with a DNA sequence flanked by sequences homologous to the desired location. Non-edited genomes are targeted by the CRISPR-Cas system, thus enabling isolation of the desired recombinant phages. This method broadens CRISPR Cas-based editing to phages and uses a CRISPR-Cas type other than type II. The method may be adjusted to genetically engineer any bacteriophage genome.

  4. Probing the structural dynamics of the CRISPR-Cas9 RNA-guided DNA-cleavage system by coarse-grained modeling.

    PubMed

    Zheng, Wenjun

    2017-02-01

    In the adaptive immune systems of many bacteria and archaea, the Cas9 endonuclease forms a complex with specific guide/scaffold RNA to identify and cleave complementary target sequences in foreign DNA. This DNA targeting machinery has been exploited in numerous applications of genome editing and transcription control. However, the molecular mechanism of the Cas9 system is still obscure. Recently, high-resolution structures have been solved for Cas9 in different structural forms (e.g., unbound forms, RNA-bound binary complexes, and RNA-DNA-bound tertiary complexes, corresponding to an inactive state, a pre-target-bound state, and a cleavage-competent or product state), which offered key structural insights to the Cas9 mechanism. To further probe the structural dynamics of Cas9 interacting with RNA and DNA at the amino-acid level of details, we have performed systematic coarse-grained modeling using an elastic network model and related analyses. Our normal mode analysis predicted a few key modes of collective motions that capture the observed conformational changes featuring large domain motions triggered by binding of RNA and DNA. Our flexibility analysis identified specific regions with high or low flexibility that coincide with key functional sites (such as DNA/RNA-binding sites, nuclease cleavage sites, and key hinges). We also identified a small set of hotspot residues that control the energetics of functional motions, which overlap with known functional sites and offer promising targets for future mutagenesis efforts to improve the specificity of Cas9. Finally, we modeled the conformational transitions of Cas9 from the unbound form to the binary complex and then the tertiary complex, and predicted a distinct sequence of domain motions. In sum, our findings have offered rich structural and dynamic details relevant to the Cas9 machinery, and will guide future investigation and engineering of the Cas9 systems. Proteins 2017; 85:342-353. © 2016 Wiley Periodicals

  5. Driver Adaptive Warning Systems

    DTIC Science & Technology

    1998-03-01

    this threshold, an alarm is triggered. Since TLC based systems can have user defined thresholds, a warning can be given as early as desired. However, the...Driver Adaptive Warning Systems Thesis Proposal Parag H. Batavia CMU-RI-TR-98-07 The Robotics Institute Carnegie Mellon University Pittsburgh...control number. 1. REPORT DATE MAR 1998 2. REPORT TYPE 3. DATES COVERED 00-00-1998 to 00-00-1998 4. TITLE AND SUBTITLE Driver Adaptive Warning

  6. The CRISPR-Cas system for plant genome editing: advances and opportunities.

    PubMed

    Kumar, Vinay; Jain, Mukesh

    2015-01-01

    Genome editing is an approach in which a specific target DNA sequence of the genome is altered by adding, removing, or replacing DNA bases. Artificially engineered hybrid enzymes, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), and the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated protein) system are being used for genome editing in various organisms including plants. The CRISPR-Cas system has been developed most recently and seems to be more efficient and less time-consuming compared with ZFNs or TALENs. This system employs an RNA-guided nuclease, Cas9, to induce double-strand breaks. The Cas9-mediated breaks are repaired by cellular DNA repair mechanisms and mediate gene/genome modifications. Here, we provide a detailed overview of the CRISPR-Cas system and its adoption in different organisms, especially plants, for various applications. Important considerations and future opportunities for deployment of the CRISPR-Cas system in plants for numerous applications are also discussed. Recent investigations have revealed the implications of the CRISPR-Cas system as a promising tool for targeted genetic modifications in plants. This technology is likely to be more commonly adopted in plant functional genomics studies and crop improvement in the near future.

  7. RNA-dependent DNA endonuclease Cas9 of the CRISPR system: Holy Grail of genome editing?

    PubMed

    Gasiunas, Giedrius; Siksnys, Virginijus

    2013-11-01

    Tailor-made nucleases for precise genome modification, such as zinc finger or TALE nucleases, currently represent the state-of-the-art for genome editing. These nucleases combine a programmable protein module which guides the enzyme to the target site with a nuclease domain which cuts DNA at the addressed site. Reprogramming of these nucleases to cut genomes at specific locations requires major protein engineering efforts. RNA-guided DNA endonuclease Cas9 of the type II (clustered regularly interspaced short palindromic repeat) CRISPR-Cas system uses CRISPR RNA (crRNA) as a guide to locate the DNA target and the Cas9 protein to cut DNA. Easy programmability of the Cas9 endonuclease using customizable RNAs brings unprecedented flexibility and versatility for targeted genome modification. We highlight the potential of the Cas9 RNA-guided DNA endonuclease as a novel tool for genome surgery, and discuss possible constraints and future prospects.

  8. [CRISPR/Cas system for genome editing in pluripotent stem cells].

    PubMed

    Vasil'eva, E A; Melino, D; Barlev, N A

    2015-01-01

    Genome editing systems based on site-specific nucleases became very popular for genome editing in modern bioengineering. Human pluripotent stem cells provide a unique platform for genes function study, disease modeling, and drugs testing. Consequently, technology for fast, accurate and well controlled genome manipulation is required. CRISPR/Cas (clustered regularly interspaced short palindromic repeat/CRISPR-associated) system could be employed for these purposes. This system is based on site-specific programmable nuclease Cas9. Numerous advantages of the CRISPR/Cas system and its successful application to human stem cells provide wide opportunities for genome therapy and regeneration medicine. In this publication, we describe and compare the main genome editing systems based on site-specific programmable nucleases and discuss opportunities and perspectives of the CRISPR/Cas system for application to pluripotent stem cells.

  9. A CRISPR-Cas system enhances envelope integrity mediating antibiotic resistance and inflammasome evasion.

    PubMed

    Sampson, Timothy R; Napier, Brooke A; Schroeder, Max R; Louwen, Rogier; Zhao, Jinshi; Chin, Chui-Yoke; Ratner, Hannah K; Llewellyn, Anna C; Jones, Crystal L; Laroui, Hamed; Merlin, Didier; Zhou, Pei; Endtz, Hubert P; Weiss, David S

    2014-07-29

    Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems defend bacteria against foreign nucleic acids, such as during bacteriophage infection and transformation, processes which cause envelope stress. It is unclear if these machineries enhance membrane integrity to combat this stress. Here, we show that the Cas9-dependent CRISPR-Cas system of the intracellular bacterial pathogen Francisella novicida is involved in enhancing envelope integrity through the regulation of a bacterial lipoprotein. This action ultimately provides increased resistance to numerous membrane stressors, including antibiotics. We further find that this previously unappreciated function of Cas9 is critical during infection, as it promotes evasion of the host innate immune absent in melanoma 2/apoptosis associated speck-like protein containing a CARD (AIM2/ASC) inflammasome. Interestingly, the attenuation of the cas9 mutant is complemented only in mice lacking both the AIM2/ASC inflammasome and the bacterial lipoprotein sensor Toll-like receptor 2, but not in single knockout mice, demonstrating that Cas9 is essential for evasion of both pathways. These data represent a paradigm shift in our understanding of the function of CRISPR-Cas systems as regulators of bacterial physiology and provide a framework with which to investigate the roles of these systems in myriad bacteria, including pathogens and commensals.

  10. Complex adaptive systems and game theory: An unlikely union

    USGS Publications Warehouse

    Hadzikadic, M.; Carmichael, T.; Curtin, C.

    2010-01-01

    A Complex Adaptive System is a collection of autonomous, heterogeneous agents, whose behavior is defined with a limited number of rules. A Game Theory is a mathematical construct that assumes a small number of rational players who have a limited number of actions or strategies available to them. The CAS method has the potential to alleviate some of the shortcomings of GT. On the other hand, CAS researchers are always looking for a realistic way to define interactions among agents. GT offers an attractive option for defining the rules of such interactions in a way that is both potentially consistent with observed real-world behavior and subject to mathematical interpretation. This article reports on the results of an effort to build a CAS system that utilizes GT for determining the actions of individual agents. ?? 2009 Wiley Periodicals, Inc. Complexity, 16,24-42, 2010.

  11. [Application Progress of CRISPR/Cas9 System for Gene Editing in Tumor Research].

    PubMed

    Liu, Chao; Li, Zhiwei; Zhang, Yanqiao

    2015-09-20

    TCRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated nuclease 9) gene editing system is a new type of gene editing technology developed based on the immune mechanism of archaea resisting the invasion of exogenous nucleic acid. Compared with traditional gene editing system, CRISPR/Cas9 system is more efficient, easier operating, and less cytotoxic. Currently, CRISPR/Cas9 gene editing technology has been applied to many aspects of cancer research, including research on cancer genes, constructing animal tumor models, screening tumor resistance-associated and phenotypic-related genes and cancer gene therapy. In this review, the application of the CRISPR/Cas9 system in tumor research were introduced.

  12. Cas5d Protein Processes Pre-crRNA and Assembles into a Cascade-like Interference Complex in Subtype I-C/Dvulg CRISPR-Cas System

    SciTech Connect

    Nam, Ki Hyun; Haitjema, Charles; Liu, Xueqi; Ding, Fran; Wang, Hongwei; DeLisa, Matthew P.; Ke, Ailong

    2012-10-10

    Clustered regularly interspaced short palindromic repeats (CRISPRs), together with an operon of CRISPR-associated (Cas) proteins, form an RNA-based prokaryotic immune system against exogenous genetic elements. Cas5 family proteins are found in several type I CRISPR-Cas systems. Here, we report the molecular function of subtype I-C/Dvulg Cas5d from Bacillus halodurans. We show that Cas5d cleaves pre-crRNA into unit length by recognizing both the hairpin structure and the 3 single stranded sequence in the CRISPR repeat region. Cas5d structure reveals a ferredoxin domain-based architecture and a catalytic triad formed by Y46, K116, and H117 residues. We further show that after pre-crRNA processing, Cas5d assembles with crRNA, Csd1, and Csd2 proteins to form a multi-sub-unit interference complex similar to Escherichia coli Cascade (CRISPR-associated complex for antiviral defense) in architecture. Our results suggest that formation of a crRNA-presenting Cascade-like complex is likely a common theme among type I CRISPR subtypes.

  13. Co-Alignment System (CAS) study. Report on task 1-3. [Solar Extreme Ultraviolet Telescope and Spectrometer pointing system

    NASA Technical Reports Server (NTRS)

    Anderson, N. T.

    1980-01-01

    The design of a suitable coalignment system (CAS) for the Solar Extreme Ultraviolet Telescope and Spectrometer (SEUTS) is presented. The CAS provides offset adjustment capabilities to SEUTS which will be mounted on a single large pointing system with other devices. The suitability of existing designs is determined and modifications are suggested.

  14. Streptococcus thermophilus CRISPR-Cas9 Systems Enable Specific Editing of the Human Genome

    PubMed Central

    Müller, Maximilian; Lee, Ciaran M; Gasiunas, Giedrius; Davis, Timothy H; Cradick, Thomas J; Siksnys, Virginijus; Bao, Gang; Cathomen, Toni; Mussolino, Claudio

    2016-01-01

    RNA-guided nucleases (RGNs) based on the type II CRISPR-Cas9 system of Streptococcus pyogenes (Sp) have been widely used for genome editing in experimental models. However, the nontrivial level of off-target activity reported in several human cells may hamper clinical translation. RGN specificity depends on both the guide RNA (gRNA) and the protospacer adjacent motif (PAM) recognized by the Cas9 protein. We hypothesized that more stringent PAM requirements reduce the occurrence of off-target mutagenesis. To test this postulation, we generated RGNs based on two Streptococcus thermophilus (St) Cas9 proteins, which recognize longer PAMs, and performed a side-by-side comparison of the three RGN systems targeted to matching sites in two endogenous human loci, PRKDC and CARD11. Our results demonstrate that in samples with comparable on-target cleavage activities, significantly lower off-target mutagenesis was detected using St-based RGNs as compared to the standard Sp-RGNs. Moreover, similarly to SpCas9, the StCas9 proteins accepted truncated gRNAs, suggesting that the specificities of St-based RGNs can be further improved. In conclusion, our results show that Cas9 proteins with longer or more restrictive PAM requirements provide a safe alternative to SpCas9-based RGNs and hence a valuable option for future human gene therapy applications. PMID:26658966

  15. NEEDS - Information Adaptive System

    NASA Technical Reports Server (NTRS)

    Kelly, W. L.; Benz, H. F.; Meredith, B. D.

    1980-01-01

    The Information Adaptive System (IAS) is an element of the NASA End-to-End Data System (NEEDS) Phase II and is focused toward onboard image processing. The IAS is a data preprocessing system which is closely coupled to the sensor system. Some of the functions planned for the IAS include sensor response nonuniformity correction, geometric correction, data set selection, data formatting, packetization, and adaptive system control. The inclusion of these sensor data preprocessing functions onboard the spacecraft will significantly improve the extraction of information from the sensor data in a timely and cost effective manner, and provide the opportunity to design sensor systems which can be reconfigured in near real-time for optimum performance. The purpose of this paper is to present the preliminary design of the IAS and the plans for its development.

  16. Herd behavior in a complex adaptive system

    PubMed Central

    Zhao, Li; Yang, Guang; Wang, Wei; Chen, Yu; Huang, J. P.; Ohashi, Hirotada; Stanley, H. Eugene

    2011-01-01

    In order to survive, self-serving agents in various kinds of complex adaptive systems (CASs) must compete against others for sharing limited resources with biased or unbiased distribution by conducting strategic behaviors. This competition can globally result in the balance of resource allocation. As a result, most of the agents and species can survive well. However, it is a common belief that the formation of a herd in a CAS will cause excess volatility, which can ruin the balance of resource allocation in the CAS. Here this belief is challenged with the results obtained from a modeled resource-allocation system. Based on this system, we designed and conducted a series of computer-aided human experiments including herd behavior. We also performed agent-based simulations and theoretical analyses, in order to confirm the experimental observations and reveal the underlying mechanism. We report that, as long as the ratio of the two resources for allocation is biased enough, the formation of a typically sized herd can help the system to reach the balanced state. This resource ratio also serves as the critical point for a class of phase transition identified herein, which can be used to discover the role change of herd behavior, from a ruinous one to a helpful one. This work is also of value to some fields, ranging from management and social science, to ecology and evolution, and to physics. PMID:21876133

  17. Herd behavior in a complex adaptive system.

    PubMed

    Zhao, Li; Yang, Guang; Wang, Wei; Chen, Yu; Huang, J P; Ohashi, Hirotada; Stanley, H Eugene

    2011-09-13

    In order to survive, self-serving agents in various kinds of complex adaptive systems (CASs) must compete against others for sharing limited resources with biased or unbiased distribution by conducting strategic behaviors. This competition can globally result in the balance of resource allocation. As a result, most of the agents and species can survive well. However, it is a common belief that the formation of a herd in a CAS will cause excess volatility, which can ruin the balance of resource allocation in the CAS. Here this belief is challenged with the results obtained from a modeled resource-allocation system. Based on this system, we designed and conducted a series of computer-aided human experiments including herd behavior. We also performed agent-based simulations and theoretical analyses, in order to confirm the experimental observations and reveal the underlying mechanism. We report that, as long as the ratio of the two resources for allocation is biased enough, the formation of a typically sized herd can help the system to reach the balanced state. This resource ratio also serves as the critical point for a class of phase transition identified herein, which can be used to discover the role change of herd behavior, from a ruinous one to a helpful one. This work is also of value to some fields, ranging from management and social science, to ecology and evolution, and to physics.

  18. High-throughput screens in mammalian cells using the CRISPR-Cas9 system.

    PubMed

    Peng, Jingyu; Zhou, Yuexin; Zhu, Shiyou; Wei, Wensheng

    2015-06-01

    As a powerful genome-editing tool, the clustered regularly interspaced short palindromic repeats (CRISPR)-clustered regularly interspaced short palindromic repeats-associated protein 9 (Cas9) system has been quickly developed into a large-scale function-based screening strategy in mammalian cells. This new type of genetic library is constructed through the lentiviral delivery of single-guide RNA collections that direct Cas9 or inactive dead Cas9 fused with effectors to interrogate gene function or regulate gene transcription in targeted cells. Compared with RNA interference screening, the CRISPR-Cas9 system demonstrates much higher levels of effectiveness and reliability with respect to both loss-of-function and gain-of-function screening. Unlike the RNA interference strategy, a CRISPR-Cas9 library can target both protein-coding sequences and regulatory elements, including promoters, enhancers and elements transcribing microRNAs and long noncoding RNAs. This powerful genetic tool will undoubtedly accelerate the mechanistic discovery of various biological processes. In this mini review, we summarize the general procedure of CRISPR-Cas9 library mediated functional screening, system optimization strategies and applications of this new genetic toolkit.

  19. CRISPR-Cas9 systems: versatile cancer modelling platforms and promising therapeutic strategies.

    PubMed

    Wen, Wan-Shun; Yuan, Zhi-Min; Ma, Shi-Jie; Xu, Jiang; Yuan, Dong-Tang

    2016-03-15

    The RNA-guided nuclease CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated nuclease 9) and its variants such as nickase Cas9, dead Cas9, guide RNA scaffolds and RNA-targeting Cas9 are convenient and versatile platforms for site-specific genome editing and epigenome modulation. They are easy-to-use, simple-to-design and capable of targeting multiple loci simultaneously. Given that cancer develops from cumulative genetic and epigenetic alterations, CRISPR-Cas9 and its variants (hereafter referred to as CRISPR-Cas9 systems) hold extensive application potentials in cancer modeling and therapy. To date, they have already been applied to model oncogenic mutations in cell lines (e.g., Choi and Meyerson, Nat Commun 2014;5:3728) and in adult animals (e.g., Xue et al., Nature 2014;514:380-4), as well as to combat cancer by disabling oncogenic viruses (e.g., Hu et al., Biomed Res Int 2014;2014:612823) or by manipulating cancer genome (e.g., Liu et al., Nat Commun 2014;5:5393). Given the importance of epigenome and transcriptome in tumourigenesis, manipulation of cancer epigenome and transcriptome for cancer modeling and therapy is a promising area in the future. Whereas (epi)genetic modifications of cancer microenvironment with CRISPR-Cas9 systems for therapeutic purposes represent another promising area in cancer research. Herein, we introduce the functions and mechanisms of CRISPR-Cas9 systems in genome editing and epigenome modulation, retrospect their applications in cancer modelling and therapy, discuss limitations and possible solutions and propose future directions, in hope of providing concise and enlightening information for readers interested in this area.

  20. The Development of a Viral Mediated CRISPR/Cas9 System with Doxycycline Dependent gRNA Expression for Inducible In vitro and In vivo Genome Editing

    PubMed Central

    de Solis, Christopher A.; Ho, Anthony; Holehonnur, Roopashri; Ploski, Jonathan E.

    2016-01-01

    The RNA-guided Cas9 nuclease, from the type II prokaryotic Clustered Regularly Interspersed Short Palindromic Repeats (CRISPR) adaptive immune system, has been adapted and utilized by scientists to edit the genomes of eukaryotic cells. Here, we report the development of a viral mediated CRISPR/Cas9 system that can be rendered inducible utilizing doxycycline (Dox) and can be delivered to cells in vitro and in vivo utilizing adeno-associated virus (AAV). Specifically, we developed an inducible gRNA (gRNAi) AAV vector that is designed to express the gRNA from a H1/TO promoter. This AAV vector is also designed to express the Tet repressor (TetR) to regulate the expression of the gRNAi in a Dox dependent manner. We show that H1/TO promoters of varying length and a U6/TO promoter can edit DNA with similar efficiency in vitro, in a Dox dependent manner. We also demonstrate that our inducible gRNAi vector can be used to edit the genomes of neurons in vivo within the mouse brain in a Dox dependent manner. Genome editing can be induced in vivo with this system by supplying animals Dox containing food for as little as 1 day. This system might be cross compatible with many existing S. pyogenes Cas9 systems (i.e., Cas9 mouse, CRISPRi, etc.), and therefore it likely can be used to render these systems inducible as well. PMID:27587996

  1. Collision Avoidance System (CAS): Human Factors Engineering Evaluation.

    DTIC Science & Technology

    1982-12-01

    personnel indicated that the CAS console was much too big for the limited amount of space available on RANGER’s bridge. The console is 40 inches wide...avoidance (C/A) data. > RANGE * 12//2 < (T >~ LOG SPEED 4 10.0 KT < E1 i >~ HEADING 4 270 DEG * < J [ > BRGCRSR * 000 DEG4C/ ADATA < - > TRIAL SPD 4 0 <EJ

  2. Adaptive CT scanning system

    SciTech Connect

    Sampayan, Stephen E.

    2016-11-22

    Apparatus, systems, and methods that provide an X-ray interrogation system having a plurality of stationary X-ray point sources arranged to substantially encircle an area or space to be interrogated. A plurality of stationary detectors are arranged to substantially encircle the area or space to be interrogated, A controller is adapted to control the stationary X-ray point sources to emit X-rays one at a time, and to control the stationary detectors to detect the X-rays emitted by the stationary X-ray point sources.

  3. Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system.

    PubMed

    Liu, Rui; Chen, Ling; Jiang, Yanping; Zhou, Zhihua; Zou, Gen

    2015-01-01

    Filamentous fungi have wide applications in biotechnology. The CRISPR/Cas9 system is a powerful genome-editing method that facilitates genetic alterations of genomes in a variety of organisms. However, a genome-editing approach has not been reported in filamentous fungi. Here, we demonstrated the establishment of a CRISPR/Cas9 system in the filamentous fungus Trichoderma reesei by specific codon optimization and in vitro RNA transcription. It was shown that the CRISPR/Cas9 system was controllable and conditional through inducible Cas9 expression. This system generated site-specific mutations in target genes through efficient homologous recombination, even using short homology arms. This system also provided an applicable and promising approach to targeting multiple genes simultaneously. Our results illustrate that the CRISPR/Cas9 system is a powerful genome-manipulating tool for T. reesei and most likely for other filamentous fungal species, which may accelerate studies on functional genomics and strain improvement in these filamentous fungi.

  4. Contrarian behavior in a complex adaptive system

    NASA Astrophysics Data System (ADS)

    Liang, Y.; An, K. N.; Yang, G.; Huang, J. P.

    2013-01-01

    Contrarian behavior is a kind of self-organization in complex adaptive systems (CASs). Here we report the existence of a transition point in a model resource-allocation CAS with contrarian behavior by using human experiments, computer simulations, and theoretical analysis. The resource ratio and system predictability serve as the tuning parameter and order parameter, respectively. The transition point helps to reveal the positive or negative role of contrarian behavior. This finding is in contrast to the common belief that contrarian behavior always has a positive role in resource allocation, say, stabilizing resource allocation by shrinking the redundancy or the lack of resources. It is further shown that resource allocation can be optimized at the transition point by adding an appropriate size of contrarians. This work is also expected to be of value to some other fields ranging from management and social science to ecology and evolution.

  5. Contrarian behavior in a complex adaptive system.

    PubMed

    Liang, Y; An, K N; Yang, G; Huang, J P

    2013-01-01

    Contrarian behavior is a kind of self-organization in complex adaptive systems (CASs). Here we report the existence of a transition point in a model resource-allocation CAS with contrarian behavior by using human experiments, computer simulations, and theoretical analysis. The resource ratio and system predictability serve as the tuning parameter and order parameter, respectively. The transition point helps to reveal the positive or negative role of contrarian behavior. This finding is in contrast to the common belief that contrarian behavior always has a positive role in resource allocation, say, stabilizing resource allocation by shrinking the redundancy or the lack of resources. It is further shown that resource allocation can be optimized at the transition point by adding an appropriate size of contrarians. This work is also expected to be of value to some other fields ranging from management and social science to ecology and evolution.

  6. Boosting CRISPR/Cas9 multiplex editing capability with the endogenous tRNA-processing system.

    PubMed

    Xie, Kabin; Minkenberg, Bastian; Yang, Yinong

    2015-03-17

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system is being harnessed as a powerful tool for genome engineering in basic research, molecular therapy, and crop improvement. This system uses a small guide RNA (gRNA) to direct Cas9 endonuclease to a specific DNA site; thus, its targeting capability is largely constrained by the gRNA-expressing device. In this study, we developed a general strategy to produce numerous gRNAs from a single polycistronic gene. The endogenous tRNA-processing system, which precisely cleaves both ends of the tRNA precursor, was engineered as a simple and robust platform to boost the targeting and multiplex editing capability of the CRISPR/Cas9 system. We demonstrated that synthetic genes with tandemly arrayed tRNA-gRNA architecture were efficiently and precisely processed into gRNAs with desired 5' targeting sequences in vivo, which directed Cas9 to edit multiple chromosomal targets. Using this strategy, multiplex genome editing and chromosomal-fragment deletion were readily achieved in stable transgenic rice plants with a high efficiency (up to 100%). Because tRNA and its processing system are virtually conserved in all living organisms, this method could be broadly used to boost the targeting capability and editing efficiency of CRISPR/Cas9 toolkits.

  7. The CRISPR-Cas system - from bacterial immunity to genome engineering.

    PubMed

    Czarnek, Maria; Bereta, Joanna

    2016-09-01

    Precise and efficient genome modifications present a great value in attempts to comprehend the roles of particular genes and other genetic elements in biological processes as well as in various pathologies. In recent years novel methods of genome modification known as genome editing, which utilize so called "programmable" nucleases, came into use. A true revolution in genome editing has been brought about by the introduction of the CRISP-Cas (clustered regularly interspaced short palindromic repeats-CRISPR associated) system, in which one of such nucleases, i.e. Cas9, plays a major role. This system is based on the elements of the bacterial and archaeal mechanism responsible for acquired immunity against phage infections and transfer of foreign genetic material. Microorganisms incorporate fragments of foreign DNA into CRISPR loci present in their genomes, which enables fast recognition and elimination of future infections. There are several types of CRISPR-Cas systems among prokaryotes but only elements of CRISPR type II are employed in genome engineering. CRISPR-Cas type II utilizes small RNA molecules (crRNA and tracrRNA) to precisely direct the effector nuclease - Cas9 - to a specific site in the genome, i.e. to the sequence complementary to crRNA. Cas9 may be used to: (i) introduce stable changes into genomes e.g. in the process of generation of knock-out and knock-in animals and cell lines, (ii) activate or silence the expression of a gene of interest, and (iii) visualize specific sites in genomes of living cells. The CRISPR-Cas-based tools have been successfully employed for generation of animal and cell models of a number of diseases, e.g. specific types of cancer. In the future, the genome editing by programmable nucleases may find wide application in medicine e.g. in the therapies of certain diseases of genetic origin and in the therapy of HIV-infected patients.

  8. New clues on the regulation of the CRISPR-Cas immune system.

    PubMed

    Lundgren, Magnus

    2015-01-01

    Research into the CRISPR-Cas immune system of prokaryotes is progressing at a tremendous pace given both its important biological function and its role as a source of new genetic tools. However, a few areas of the field have remained largely unaddressed. A recent report provides information on one such overlooked area: how the cell regulates the CRISPR-Cas immune system. The processes, despite their importance, have remained illusive. In Pectobacterium atrosepticum regulation is, perhaps surprisingly, based on metabolic factors responding to glucose levels in the cell. Regulators include both activators and repressors of cas gene expression. It remains an open question why and how this regulatory system have evolved, and if it is a typical example of how CRISPR-as systems are regulated or not.

  9. New clues on the regulation of the CRISPR-Cas immune system

    PubMed Central

    Lundgren, Magnus

    2015-01-01

    Research into the CRISPR-Cas immune system of prokaryotes is progressing at a tremendous pace given both its important biological function and its role as a source of new genetic tools. However, a few areas of the field have remained largely unaddressed. A recent report provides information on one such overlooked area: how the cell regulates the CRISPR-Cas immune system. The processes, despite their importance, have remained illusive. In Pectobacterium atrosepticum regulation is, perhaps surprisingly, based on metabolic factors responding to glucose levels in the cell. Regulators include both activators and repressors of cas gene expression. It remains an open question why and how this regulatory system have evolved, and if it is a typical example of how CRISPR-as systems are regulated or not. PMID:26942048

  10. The CRISPR/Cas9 system for gene editing and its potential application in pain research

    PubMed Central

    Sun, Linlin; Lutz, Brianna Marie; Tao, Yuan-Xiang

    2016-01-01

    The CRISPR/Cas9 system is a research hotspot in genome editing and regulation. Currently, it is used in genomic silencing and knock-in experiments as well as transcriptional activation and repression. This versatile system consists of two components: a guide RNA (gRNA) and a Cas9 nuclease. Recognition of a genomic DNA target is mediated through base pairing with a 20-base gRNA. The latter further recruits the Cas9 endonuclease protein to the target site and creates double-stranded breaks in the target DNA. Compared with traditional genome editing directed by DNA-binding protein domains, this short RNA-directed Cas9 endonuclease system is simple and easily programmable. Although this system may have off-target effects and in vivo delivery and immune challenges, researchers have employed this system in vivo to establish disease models, study specific gene functions under certain disease conditions, and correct genomic information for disease treatment. In regards to pain research, the CRISPR/Cas9 system may act as a novel tool in gene correction therapy for pain-associated hereditary diseases and may be a new approach for RNA-guided transcriptional activation or repression of pain-related genes. In addition, this system is also applied to loss-of-function mutations in pain-related genes and knockin of reporter genes or loxP tags at pain-related genomic loci. The CRISPR/Cas9 system will likely be carried out widely in both bench work and clinical settings in the pain field. PMID:27500183

  11. Using the CRISPR/Cas9 system to eliminate native plasmids of Zymomonas mobilis ZM4.

    PubMed

    Cao, Qing-Hua; Shao, Huan-Huan; Qiu, Hui; Li, Tao; Zhang, Yi-Zheng; Tan, Xue-Mei

    2017-03-01

    The CRISPR/Cas system can be used to simply and efficiently edit the genomes of various species, including animals, plants, and microbes. Zymomonas mobilis ZM4 is a highly efficient, ethanol-producing bacterium that contains five native plasmids. Here, we constructed the pSUZM2a-Cas9 plasmid and a single-guide RNA expression plasmid. The pSUZM2a-Cas9 plasmid was used to express the Cas9 gene cloned from Streptococcus pyogenes CICC 10464. The single-guide RNA expression plasmid pUC-T7sgRNA, with a T7 promoter, can be used for the in vitro synthesis of single-guide RNAs. This system was successfully employed to knockout the upp gene of Escherichia coli and the replicase genes of native Z. mobilis plasmids. This is the first study to apply the CRISPR/Cas9 system of S. pyogenes to eliminate native plasmids in Z. mobilis. It provides a new method for plasmid curing and paves the way for the genomic engineering of Z. mobilis.

  12. CRISPR-Cas and restriction-modification systems are compatible and increase phage resistance.

    PubMed

    Dupuis, Marie-Ève; Villion, Manuela; Magadán, Alfonso H; Moineau, Sylvain

    2013-01-01

    Bacteria have developed a set of barriers to protect themselves against invaders such as phage and plasmid nucleic acids. Different prokaryotic defence systems exist and at least two of them directly target the incoming DNA: restriction-modification (R-M) and CRISPR-Cas systems. On their own, they are imperfect barriers to invasion by foreign DNA. Here, we show that R-M and CRISPR-Cas systems are compatible and act together to increase the overall phage resistance of a bacterial cell by cleaving their respective target sites. Furthermore, we show that the specific methylation of phage DNA does not impair CRISPR-Cas acquisition or interference activities. Taken altogether, both mechanisms can be leveraged to decrease phage contaminations in processes relying on bacterial growth and/or fermentation.

  13. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems

    PubMed Central

    Burstein, David; Sun, Christine L.; Brown, Christopher T.; Sharon, Itai; Anantharaman, Karthik; Probst, Alexander J.; Thomas, Brian C.; Banfield, Jillian F.

    2016-01-01

    Current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth's ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation-independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. We correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides. PMID:26837824

  14. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems

    DOE PAGES

    Burstein, David; Sun, Christine L.; Brown, Christopher T.; ...

    2016-02-03

    Here, current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth’s ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. Wemore » correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides.« less

  15. Major bacterial lineages are essentially devoid of CRISPR-Cas viral defence systems

    SciTech Connect

    Burstein, David; Sun, Christine L.; Brown, Christopher T.; Sharon, Itai; Anantharaman, Karthik; Probst, Alexander J.; Thomas, Brian C.; Banfield, Jillian F.

    2016-02-03

    Here, current understanding of microorganism–virus interactions, which shape the evolution and functioning of Earth’s ecosystems, is based primarily on cultivated organisms. Here we investigate thousands of viral and microbial genomes recovered using a cultivation independent approach to study the frequency, variety and taxonomic distribution of viral defence mechanisms. CRISPR-Cas systems that confer microorganisms with immunity to viruses are present in only 10% of 1,724 sampled microorganisms, compared with previous reports of 40% occurrence in bacteria and 81% in archaea. We attribute this large difference to the lack of CRISPR-Cas systems across major bacterial lineages that have no cultivated representatives. We correlate absence of CRISPR-Cas with lack of nucleotide biosynthesis capacity and a symbiotic lifestyle. Restriction systems are well represented in these lineages and might provide both non-specific viral defence and access to nucleotides.

  16. Gaia as a complex adaptive system.

    PubMed Central

    Lenton, Timothy M; van Oijen, Marcel

    2002-01-01

    We define the Gaia system of life and its environment on Earth, review the status of the Gaia theory, introduce potentially relevant concepts from complexity theory, then try to apply them to Gaia. We consider whether Gaia is a complex adaptive system (CAS) in terms of its behaviour and suggest that the system is self-organizing but does not reside in a critical state. Gaia has supported abundant life for most of the last 3.8 Gyr. Large perturbations have occasionally suppressed life but the system has always recovered without losing the capacity for large-scale free energy capture and recycling of essential elements. To illustrate how complexity theory can help us understand the emergence of planetary-scale order, we present a simple cellular automata (CA) model of the imaginary planet Daisyworld. This exhibits emergent self-regulation as a consequence of feedback coupling between life and its environment. Local spatial interaction, which was absent from the original model, can destabilize the system by generating bifurcation regimes. Variation and natural selection tend to remove this instability. With mutation in the model system, it exhibits self-organizing adaptive behaviour in its response to forcing. We close by suggesting how artificial life ('Alife') techniques may enable more comprehensive feasibility tests of Gaia. PMID:12079529

  17. Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization.

    PubMed

    Senturk, Serif; Shirole, Nitin H; Nowak, Dawid G; Corbo, Vincenzo; Pal, Debjani; Vaughan, Alexander; Tuveson, David A; Trotman, Lloyd C; Kinney, Justin B; Sordella, Raffaella

    2017-02-22

    The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ER(T2), our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes.

  18. The system V389 Cas: Algol-type binary with δ -Scuti pulsations

    NASA Astrophysics Data System (ADS)

    Korda, D.; Zasche, P.; Kučáková, H.

    2015-10-01

    New CCD observations of V389 Cas were carried out in the observatories in the Czech Republic from 2010 to 2014. These new data were analysed using the program PHOEBE. V389 Cas was found to be a detached eclipsing binary system with two rather different components moving on a circular orbit. Moreover, there was discovered also a δ -Scuti-type behaviour of the secondary component. These pulsations have the period of about 0.037 day. This result is being compared with the previous findings on similar eclipsing-pulsation systems published by Zhang et al. (2013).

  19. CRISPR-Cas9 System as a Versatile Tool for Genome Engineering in Human Cells

    PubMed Central

    Wang, Xuelian; Huang, Xiumin; Fang, Xiuli; Zhang, Youzhong; Wang, Wanpeng

    2016-01-01

    Targeted nucleases are influential instruments for intervening in genome revision with great accuracy. RNA-guided Cas9 nucleases produced from clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systems have noticeably altered the means to modify the genomes of distinct organisms. They can be notably used to facilitate effective genome manipulation in eukaryotic cells by clearly detailing a 20-nt targeting sequence inside its directed RNA. We discuss the most recent advancements in the molecular basis of the type II CRISPR/Cas system and encapsulate applications and elements affecting its use in human cells. We also propose possible applications covering its uses ranging from basic science to implementation in the clinic. PMID:27845770

  20. CRISPR/Cas9 system and its applications in human hematopoietic cells.

    PubMed

    Hu, Xiaotang

    2016-11-01

    Since 2012, the CRISPR-Cas9 system has been quickly and successfully tested in a broad range of organisms and cells including hematopoietic cells. The application of CRISPR-Cas9 in human hematopoietic cells mainly involves the genes responsible for HIV infection, β-thalassemia and sickle cell disease (SCD). The successful disruption of CCR5 and CXCR4 genes in T cells by CRISPR-Cas9 promotes the prospect of the technology in the functional cure of HIV. More recently, eliminating CCR5 and CXCR4 in induced pluripotent stem cells (iPSCs) derived from patients and targeting the HIV genome have been successfully carried out in several laboratories. The outcome from these approaches bring us closer to the goal of eradicating HIV infection. For hemoglobinopathies the ability to produce iPSC-derived from patients with the correction of hemoglobin (HBB) mutations by CRISPR-Cas9 has been tested in a number of laboratories. These corrected iPSCs also show the potential to differentiate into mature erythrocytes expressing high-level and normal HBB. In light of the initial success of CRESPR-Cas9 in target mutated gene(s) in the iPSCs, a combination of genomic editing and autogenetic stem cell transplantation would be the best strategy for root treatment of the diseases, which could replace traditional allogeneic stem cell transplantation.

  1. RNA-guided genome editing in plants using a CRISPR-Cas system.

    PubMed

    Xie, Kabin; Yang, Yinong

    2013-11-01

    Precise and straightforward methods to edit the plant genome are much needed for functional genomics and crop improvement. Recently, RNA-guided genome editing using bacterial Type II cluster regularly interspaced short palindromic repeats (CRISPR)-associated nuclease (Cas) is emerging as an efficient tool for genome editing in microbial and animal systems. Here, we report the genome editing and targeted gene mutation in plants via the CRISPR-Cas9 system. Three guide RNAs (gRNAs) with a 20-22-nt seed region were designed to pair with distinct rice genomic sites which are followed by the protospacer-adjacent motif (PAM). The engineered gRNAs were shown to direct the Cas9 nuclease for precise cleavage at the desired sites and introduce mutation (insertion or deletion) by error-prone non-homologous end joining DNA repairing. By analyzing the RNA-guided genome-editing events, the mutation efficiency at these target sites was estimated to be 3-8%. In addition, the off-target effect of an engineered gRNA-Cas9 was found on an imperfectly paired genomic site, but it had lower genome-editing efficiency than the perfectly matched site. Further analysis suggests that mismatch position between gRNA seed and target DNA is an important determinant of the gRNA-Cas9 targeting specificity, and specific gRNAs could be designed to target more than 90% of rice genes. Our results demonstrate that the CRISPR-Cas system can be exploited as a powerful tool for gene targeting and precise genome editing in plants.

  2. Application of CRISPR/Cas9 system in breeding of new antiviral plant germplasm.

    PubMed

    Daowei, Zhang; Chaofan, Zhang; Fang, Dong; Yanlan, Huang; Ya, Zhang; Hong, Zhou

    2016-09-01

    With the development and improvement of CRISPR/Cas9 system in genomic editing technology, the system has been applied to the prevention and control of animal viral infectious diseases, which has made considerable achievements. It has also been applied to the study of highly efficient gene targeting editing in plant virus genomes. The CRISPR/Cas9-mediated targeted gene modification has not only achieved the genome editing of plant DNA virus, but also showed the genome editing potential of plant RNA virus. In addition, the CRISPR/Cas9 system functions at the gene transcriptional and post-transcriptional level, indicating that the system could regulate the replication of plant viruses through different ways. Compared with other plant viral disease control strategies, this system is more accurate in genome editing, more stable in gene expression regulation, and has broader spectrum of resistance to virus disease. In this review, we summarized the advantages, main problems and development tendency of CRISPR/cas9 system in breeding of new antiviral plant germplasms.

  3. A ‘suicide’ CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans

    PubMed Central

    Wang, Yu; Wei, Dongsheng; Zhu, Xiangyang; Pan, Jiao; Zhang, Ping; Huo, Liang; Zhu, Xudong

    2016-01-01

    Loss-of-function mutagenesis is an important tool used to characterize gene functions, and the CRISPR-Cas9 system is a powerful method for performing targeted mutagenesis in organisms that present low recombination frequencies, such as the serotype D strains of Cryptococcus neoformans. However, when the CRISPR-Cas9 system persists in the host cells, off-target effects and Cas9 cytotoxicity may occur, which might block subsequent genetic manipulation. Here, we report a method of spontaneously eliminating the CRISPR-Cas9 system without impairing its robust editing function. We successfully expressed single guide RNA under the driver of an endogenous U6 promoter and the human codon-optimized Cas9 endonuclease with an ACT1 promoter. This system can effectively generate an indel mutation and efficiently perform targeted gene disruption via homology-directed repair by electroporation in yeast. We then demonstrated the spontaneous elimination of the system via a cis arrangement of the CRISPR-Cas9 expression cassettes to the recombination construct. After a system-mediated double crossover, the CRISPR-Cas9 cassettes were cleaved and degraded, which was validated by Southern blotting. This ‘suicide’ CRISPR-Cas9 system enables the validation of gene functions by subsequent complementation and has the potential to minimize off-target effects. Thus, this technique has the potential for use in functional genomics studies of C. neoformans. PMID:27503169

  4. Applications of the CRISPR-Cas9 system in cancer biology

    PubMed Central

    Sánchez-Rivera, Francisco J.; Jacks, Tyler

    2015-01-01

    Preface The prokaryotic type II clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is rapidly revolutionizing the field of genetic engineering, allowing researchers to alter the genomes of a large variety of organisms with relative ease. Experimental approaches based on this versatile technology have the potential to transform the field of cancer genetics. Here we review current approaches based on CRISPR-Cas9 for functional studies of cancer genes, with emphasis on its applicability for the development of the next-generation models of human cancer. PMID:26040603

  5. Generation of muscular dystrophy model rats with a CRISPR/Cas system.

    PubMed

    Nakamura, Katsuyuki; Fujii, Wataru; Tsuboi, Masaya; Tanihata, Jun; Teramoto, Naomi; Takeuchi, Shiho; Naito, Kunihiko; Yamanouchi, Keitaro; Nishihara, Masugi

    2014-07-09

    Duchenne muscular dystrophy (DMD) is an X-linked lethal muscle disorder caused by mutations in the Dmd gene encoding Dystrophin. DMD model animals, such as mdx mice and canine X-linked muscular dystrophy dogs, have been widely utilized in the development of a treatment for DMD. Here, we demonstrate the generation of Dmd-mutated rats using a clustered interspaced short palindromic repeats (CRISPR)/Cas system, an RNA-based genome engineering technique that is also adaptive to rats. We simultaneously targeted two exons in the rat Dmd gene, which resulted in the absence of Dystrophin expression in the F0 generation. Dmd-mutated rats exhibited a decline in muscle strength, and the emergence of degenerative/regenerative phenotypes in the skeletal muscle, heart, and diaphragm. These mutations were heritable by the next generation, and F1 male rats exhibited similar phenotypes in their skeletal muscles. These model rats should prove to be useful for developing therapeutic methods to treat DMD.

  6. Harnessing CRISPR-Cas9 immunity for genetic engineering.

    PubMed

    Charpentier, Emmanuelle; Marraffini, Luciano A

    2014-06-01

    CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which use small RNA guides (the crRNAs) to specify a cleavage site within the genome of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using synthetic single RNA guides, Cas9 can be reprogrammed to create specific double-stranded DNA breaks in the genomes of a variety of organisms, ranging from human cells to bacteria, and thus constitutes a powerful tool for genetic engineering. Here we describe recent advancements in our understanding of type II CRISPR-Cas immunity and how these studies led to revolutionary genome editing applications.

  7. Targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system.

    PubMed

    Liang, Zhen; Zhang, Kang; Chen, Kunling; Gao, Caixia

    2014-02-20

    Transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems have emerged as powerful tools for genome editing in a variety of species. Here, we report, for the first time, targeted mutagenesis in Zea mays using TALENs and the CRISPR/Cas system. We designed five TALENs targeting 4 genes, namely ZmPDS, ZmIPK1A, ZmIPK, ZmMRP4, and obtained targeting efficiencies of up to 23.1% in protoplasts, and about 13.3% to 39.1% of the transgenic plants were somatic mutations. Also, we constructed two gRNAs targeting the ZmIPK gene in maize protoplasts, at frequencies of 16.4% and 19.1%, respectively. In addition, the CRISPR/Cas system induced targeted mutations in Z. mays protoplasts with efficiencies (13.1%) similar to those obtained with TALENs (9.1%). Our results show that both TALENs and the CRISPR/Cas system can be used for genome modification in maize.

  8. Second Line of Defense Virtual Private Network Guidance for Deployed and New CAS Systems

    SciTech Connect

    Singh, Surya V.; Thronas, Aaron I.

    2010-01-01

    This paper discusses the importance of remote access via virtual private network (VPN) for the Second Line of Defense (SLD) Central Alarm System (CAS) sites, the requirements for maintaining secure channels while using VPN and implementation requirements for current and future sites.

  9. Harnessing the CRISPR/Cas9 system to disrupt latent HIV-1 provirus.

    PubMed

    Ebina, Hirotaka; Misawa, Naoko; Kanemura, Yuka; Koyanagi, Yoshio

    2013-01-01

    Even though highly active anti-retroviral therapy is able to keep HIV-1 replication under control, the virus can lie in a dormant state within the host genome, known as a latent reservoir, and poses a threat to re-emerge at any time. However, novel technologies aimed at disrupting HIV-1 provirus may be capable of eradicating viral genomes from infected individuals. In this study, we showed the potential of the CRISPR/Cas9 system to edit the HIV-1 genome and block its expression. When LTR-targeting CRISPR/Cas9 components were transfected into HIV-1 LTR expression-dormant and -inducible T cells, a significant loss of LTR-driven expression was observed after stimulation. Sequence analysis confirmed that this CRISPR/Cas9 system efficiently cleaved and mutated LTR target sites. More importantly, this system was also able to remove internal viral genes from the host cell chromosome. Our results suggest that the CRISPR/Cas9 system may be a useful tool for curing HIV-1 infection.

  10. Genome editing in rice and wheat using the CRISPR/Cas system.

    PubMed

    Shan, Qiwei; Wang, Yanpeng; Li, Jun; Gao, Caixia

    2014-10-01

    Targeted genome editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are powerful tools for understanding gene function and for developing valuable new traits in plants. The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas system has recently emerged as an alternative nuclease-based method for efficient and versatile genome engineering. In this system, only the 20-nt targeting sequence within the single-guide RNA (sgRNA) needs to be changed to target different genes. The simplicity of the cloning strategy and the few limitations on potential target sites make the CRISPR/Cas system very appealing. Here we describe a stepwise protocol for the selection of target sites, as well as the design, construction, verification and use of sgRNAs for sequence-specific CRISPR/Cas-mediated mutagenesis and gene targeting in rice and wheat. The CRISPR/Cas system provides a straightforward method for rapid gene targeting within 1-2 weeks in protoplasts, and mutated rice plants can be generated within 13-17 weeks.

  11. RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System

    PubMed Central

    Iavarone, Anthony T.; Doudna, Jennifer A.

    2017-01-01

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5′ tag) of the crRNA and the 3′ flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography. PMID:28114398

  12. RNA and DNA Targeting by a Reconstituted Thermus thermophilus Type III-A CRISPR-Cas System.

    PubMed

    Liu, Tina Y; Iavarone, Anthony T; Doudna, Jennifer A

    2017-01-01

    CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated) systems are RNA-guided adaptive immunity pathways used by bacteria and archaea to defend against phages and plasmids. Type III-A systems use a multisubunit interference complex called Csm, containing Cas proteins and a CRISPR RNA (crRNA) to target cognate nucleic acids. The Csm complex is intriguing in that it mediates RNA-guided targeting of both RNA and transcriptionally active DNA, but the mechanism is not well understood. Here, we overexpressed the five components of the Thermus thermophilus (T. thermophilus) Type III-A Csm complex (TthCsm) with a defined crRNA sequence, and purified intact TthCsm complexes from E. coli cells. The complexes were thermophilic, targeting complementary ssRNA more efficiently at 65°C than at 37°C. Sequence-independent, endonucleolytic cleavage of single-stranded DNA (ssDNA) by TthCsm was triggered by recognition of a complementary ssRNA, and required a lack of complementarity between the first 8 nucleotides (5' tag) of the crRNA and the 3' flanking region of the ssRNA. Mutation of the histidine-aspartate (HD) nuclease domain of the TthCsm subunit, Cas10/Csm1, abolished DNA cleavage. Activation of DNA cleavage was dependent on RNA binding but not cleavage. This leads to a model in which binding of an ssRNA target to the Csm complex would stimulate cleavage of exposed ssDNA in the cell, such as could occur when the RNA polymerase unwinds double-stranded DNA (dsDNA) during transcription. Our findings establish an amenable, thermostable system for more in-depth investigation of the targeting mechanism using structural biology methods, such as cryo-electron microscopy and x-ray crystallography.

  13. Protein engineering of Cas9 for enhanced function.

    PubMed

    Oakes, Benjamin L; Nadler, Dana C; Savage, David F

    2014-01-01

    CRISPR/Cas systems act to protect the cell from invading nucleic acids in many bacteria and archaea. The bacterial immune protein Cas9 is a component of one of these CRISPR/Cas systems and has recently been adapted as a tool for genome editing. Cas9 is easily targeted to bind and cleave a DNA sequence via a complementary RNA; this straightforward programmability has gained Cas9 rapid acceptance in the field of genetic engineering. While this technology has developed quickly, a number of challenges regarding Cas9 specificity, efficiency, fusion protein function, and spatiotemporal control within the cell remain. In this work, we develop a platform for constructing novel proteins to address these open questions. We demonstrate methods to either screen or select active Cas9 mutants and use the screening technique to isolate functional Cas9 variants with a heterologous PDZ domain inserted within the protein. As a proof of concept, these methods lay the groundwork for the future construction of diverse Cas9 proteins. Straightforward and accessible techniques for genetic editing are helping to elucidate biology in new and exciting ways; a platform to engineer new functionalities into Cas9 will help forge the next generation of genome-modifying tools.

  14. Generation of site-specific mutant mice using the CRISPR/Cas9 system.

    PubMed

    Min, Bai; Qi, Li; Yanjiao, Shao; Yuanhua, Huang; Dali, Li; Yanlin, Ma

    2015-10-01

    The CRISPR/Cas9 system is a recently developed important technology for genome editing in cellular and animal models. Here we established a CRISPR/Cas9-based system of generating site-specific mutant mice using DNA double-strand breaks (DSBs) induced homologous recombination (HR)-dependent or independent repair mechanism. Through co-microinjection of Cas9 mRNA and single-guide RNA (sgRNA) targeting genomic DNA sequence corresponding to enzyme activity of lysine (K)-specific demethylase 2b (Kdm2b), both a frame-shifted Kdm2b null mutant and a Kdm2b enzyme activity disrupted mouse strain were obtained simultaneously. Moreover, sgRNA targeting flavin containing monooxygenases3 (Fmo3) gene and the corresponding single strand oligonucleotides (ssODN) donor template with point mutation were co-injected into the male pronucleus of one-cell mouse embryos stimulated HR-mediated repair mechanism. Genomic sequence analysis of F0 mice showed that frame-shifted Fmo3 knockout mouse and site-specific Fmo3 knock-in mouse with single base substitution were successfully generated, and these mutations could be stably transmitted to the next generation. Therefore, we successfully generated mouse strains containing site-specific mutations through HR-dependent and -independent DSB repair using the CRISPR/Cas9 system.

  15. Efficient and Heritable Targeted Mutagenesis in Mosses Using the CRISPR/Cas9 System.

    PubMed

    Nomura, Toshihisa; Sakurai, Tetsuya; Osakabe, Yuriko; Osakabe, Keishi; Sakakibara, Hitoshi

    2016-12-01

    Targeted genome modification by RNA-guided nucleases derived from the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system has seen rapid development in many organisms, including several plant species. In the present study, we succeeded in introducing the CRISPR/Cas9 system into the non-model organism Scopelophila cataractae, a moss that exhibits heavy metal tolerance, and the model organism Physcomitrella patens Utilizing the process by which moss plants regenerate from protoplasts, we conducted targeted mutagenesis by expression of single-chain guide RNA (sgRNA) and Cas9 in protoplasts. Using this method, the acquisition rate of strains exhibiting phenotypic changes associated with the target genes was approximately 45-69%, and strains with phenotypic changes exhibited various insertion and deletion mutations. In addition, we report that our method is capable of multiplex targeted mutagenesis (two independent genes) and also permits the efficient introduction of large deletions (∼3 kbp). These results demonstrate that the CRISPR/Cas9 system can be used to accelerate investigations of bryology and land plant evolution.

  16. Targeted mutagenesis in cotton (Gossypium hirsutum L.) using the CRISPR/Cas9 system

    PubMed Central

    Chen, Xiugui; Lu, Xuke; Shu, Na; Wang, Shuai; Wang, Junjuan; Wang, Delong; Guo, Lixue; Ye, Wuwei

    2017-01-01

    The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 system has been widely used for genome editing in various plants because of its simplicity, high efficiency and design flexibility. However, to our knowledge, there is no report on the application of CRISPR/Cas9-mediated targeted mutagenesis in cotton. Here, we report the genome editing and targeted mutagenesis in upland cotton (Gossypium hirsutum L., hereafter cotton) using the CRISPR/Cas9 system. We designed two guide RNAs to target distinct sites of the cotton Cloroplastos alterados 1 (GhCLA1) and vacuolar H+-pyrophosphatase (GhVP) genes. Mutations in these two genes were detected in cotton protoplasts. Most of the mutations were nucleotide substitutions, with one nucleotide insertion and one substitution found in GhCLA1 and one deletion found in GhVP in cotton protoplasts. Subsequently, the two vectors were transformed into cotton shoot apexes through Agrobacterium-mediated transformation, resulting in efficient target gene editing. Most of the mutations were nucleotide deletions, and the mutation efficiencies were 47.6–81.8% in transgenic cotton plants. Evaluation using restriction-enzyme-PCR assay and sequence analysis detected no off-target mutations. Our results indicated that the CRISPR/Cas9 system was an efficient and specific tool for targeted mutagenesis of the cotton genome. PMID:28287154

  17. Subtyping of the Legionella pneumophila "Ulm" outbreak strain using the CRISPR-Cas system.

    PubMed

    Lück, Christian; Brzuszkiewicz, Elzbieta; Rydzewski, Kerstin; Koshkolda, Tetyana; Sarnow, Katharina; Essig, Andreas; Heuner, Klaus

    2015-12-01

    In 2009/2010 an outbreak of Legionnaires' disease with 64 cases including four fatalities took place in the city of Ulm/Neu-Ulm in Germany. L. pneumophila serogroup 1, mAb type Knoxville, sequence type (ST) 62 was identified as the epidemic strain. This strain was isolated from eight patients and from a cooling tower in the city of Ulm. Based on whole genome sequencing data from one patient strain, we identified an Lvh type IV secretion system containing a CRISPR-Cas system. The CRISPR sequence contains 38 spacer DNA sequences. We used these variable DNA spacers to further subtype the outbreak strain as well as six epidemiologically unrelated strains of CRISPR-Cas positive ST62 strains isolated at various regions in Germany. The first 12 spacer DNAs of eight patient isolates and three environmental isolates from the suspected source of infection were analyzed and found to be identical. Spacer DNAs were identified in further six epidemiologically unrelated patient isolates of L. pneumophila of ST62 in addition to the 12 "core" spacers. The presence of new spacer DNAs at the 5' site downstream of the first repeat indicates that these CRISPR-Cas systems seem to be functional. PCR analysis revealed that not all L. pneumophila sg1 ST62 strains investigated exhibited a CRISPR-Cas system. In addition, we could demonstrate that the CRISPR-Cas system is localized on a genomic island (LpuGI-Lvh) which can be excised from the chromosome and therefore may be transferable horizontally to other L. pneumophila strains.

  18. A new group of phage anti-CRISPR genes inhibits the type I-E CRISPR-Cas system of Pseudomonas aeruginosa.

    PubMed

    Pawluk, April; Bondy-Denomy, Joseph; Cheung, Vivian H W; Maxwell, Karen L; Davidson, Alan R

    2014-04-15

    CRISPR-Cas systems are one of the most widespread phage resistance mechanisms in prokaryotes. Our lab recently identified the first examples of phage-borne anti-CRISPR genes that encode protein inhibitors of the type I-F CRISPR-Cas system of Pseudomonas aeruginosa. A key question arising from this work was whether there are other types of anti-CRISPR genes. In the current work, we address this question by demonstrating that some of the same phages carrying type I-F anti-CRISPR genes also possess genes that mediate inhibition of the type I-E CRISPR-Cas system of P. aeruginosa. We have discovered four distinct families of these type I-E anti-CRISPR genes. These genes do not inhibit the type I-F CRISPR-Cas system of P. aeruginosa or the type I-E system of Escherichia coli. Type I-E and I-F anti-CRISPR genes are located at the same position in the genomes of a large group of related P. aeruginosa phages, yet they are found in a variety of combinations and arrangements. We have also identified functional anti-CRISPR genes within nonprophage Pseudomonas genomic regions that are likely mobile genetic elements. This work emphasizes the potential importance of anti-CRISPR genes in phage evolution and lateral gene transfer and supports the hypothesis that more undiscovered families of anti-CRISPR genes exist. Finally, we provide the first demonstration that the type I-E CRISPR-Cas system of P. aeruginosa is naturally active without genetic manipulation, which contrasts with E. coli and other previously characterized I-E systems. IMPORTANCE The CRISPR-Cas system is an adaptive immune system possessed by the majority of prokaryotic organisms to combat potentially harmful foreign genetic elements. This study reports the discovery of bacteriophage-encoded anti-CRISPR genes that mediate inhibition of a well-studied subtype of CRISPR-Cas system. The four families of anti-CRISPR genes described here, which comprise only the second group of anti-CRISPR genes to be identified, encode

  19. Primary processing of CRISPR RNA by the endonuclease Cas6 in Staphylococcus epidermidis.

    PubMed

    Wakefield, Noelle; Rajan, Rakhi; Sontheimer, Erik J

    2015-10-07

    In many bacteria and archaea, an adaptive immune system (CRISPR-Cas) provides immunity against foreign genetic elements. This system uses CRISPR RNAs (crRNAs) derived from the CRISPR array, along with CRISPR-associated (Cas) proteins, to target foreign nucleic acids. In most CRISPR systems, endonucleolytic processing of crRNA precursors (pre-crRNAs) is essential for the pathway. Here we study the Cas6 endonuclease responsible for crRNA processing in the Type III-A CRISPR-Cas system from Staphylococcus epidermidis RP62a, a model for Type III-A CRISPR-Cas systems, and define substrate requirements for SeCas6 activity. We find that SeCas6 is necessary and sufficient for full-length crRNA biogenesis in vitro, and that it relies on both sequence and stem-loop structure in the 3' half of the CRISPR repeat for recognition and processing.

  20. Exome sequencing in the knockin mice generated using the CRISPR/Cas system

    PubMed Central

    Nakajima, Kazuo; Kazuno, An-a; Kelsoe, John; Nakanishi, Moe; Takumi, Toru; Kato, Tadafumi

    2016-01-01

    Knockin (KI) mouse carrying a point mutation has been an invaluable tool for disease modeling and analysis. Genome editing technologies using the CRISPR/Cas system has emerged as an alternative way to create KI mice. However, if the mice carry nucleotide insertions and/or deletions (InDels) in other genes, which could have unintentionally occurred during the establishment of the KI mouse line and potentially have larger impact than a point mutation, it would confound phenotyping of the KI mice. In this study, we performed whole exome sequencing of multiple lines of F1 heterozygous Ntrk1 KI mice generated using the CRISPR/Cas system in comparison to that of a wild-type mouse used as a control. We found three InDels in four KI mice but not in a control mouse. In vitro digestion assay suggested that each InDel occurred as a de novo mutation, was carried-over from the parental mice, or was incorporated through the Cas9 nuclease mediated off-target cleavage. These results suggest that frequency of InDels found in KI mice generated by the CRISPR/Cas technology is not high, but cannot be neglected and careful assessment of these mutations is warranted. PMID:27698470

  1. Exploiting the CRISPR/Cas9 System for Targeted Genome Mutagenesis in Petunia

    PubMed Central

    Zhang, Bin; Yang, Xia; Yang, Chunping; Li, Mingyang; Guo, Yulong

    2016-01-01

    Recently, CRISPR/Cas9 technology has emerged as a powerful approach for targeted genome modification in eukaryotic organisms from yeast to human cell lines. Its successful application in several plant species promises enormous potential for basic and applied plant research. However, extensive studies are still needed to assess this system in other important plant species, to broaden its fields of application and to improve methods. Here we showed that the CRISPR/Cas9 system is efficient in petunia (Petunia hybrid), an important ornamental plant and a model for comparative research. When PDS was used as target gene, transgenic shoot lines with albino phenotype accounted for 55.6%–87.5% of the total regenerated T0 Basta-resistant lines. A homozygous deletion close to 1 kb in length can be readily generated and identified in the first generation. A sequential transformation strategy—introducing Cas9 and sgRNA expression cassettes sequentially into petunia—can be used to make targeted mutations with short indels or chromosomal fragment deletions. Our results present a new plant species amenable to CRIPR/Cas9 technology and provide an alternative procedure for its exploitation. PMID:26837606

  2. Enhancing Targeted Genomic DNA Editing in Chicken Cells Using the CRISPR/Cas9 System

    PubMed Central

    Wang, Ling; Yang, Likai; Guo, Yijie; Du, Weili; Yin, Yajun; Zhang, Tao; Lu, Hongzhao

    2017-01-01

    The CRISPR/Cas9 system has enabled highly efficient genome targeted editing for various organisms. However, few studies have focused on CRISPR/Cas9 nuclease-mediated chicken genome editing compared with mammalian genomes. The current study combined CRISPR with yeast Rad52 (yRad52) to enhance targeted genomic DNA editing in chicken DF-1 cells. The efficiency of CRISPR/Cas9 nuclease-induced targeted mutations in the chicken genome was increased to 41.9% via the enrichment of the dual-reporter surrogate system. In addition, the combined effect of CRISPR nuclease and yRad52 dramatically increased the efficiency of the targeted substitution in the myostatin gene using 50-mer oligodeoxynucleotides (ssODN) as the donor DNA, resulting in a 36.7% editing efficiency after puromycin selection. Furthermore, based on the effect of yRad52, the frequency of exogenous gene integration in the chicken genome was more than 3-fold higher than that without yRad52. Collectively, these results suggest that ssODN is an ideal donor DNA for targeted substitution and that CRISPR/Cas9 combined with yRad52 significantly enhances chicken genome editing. These findings could be extensively applied in other organisms. PMID:28068387

  3. CAS77 and CAS7276: A Review.

    ERIC Educational Resources Information Center

    Harrison, Isom, Jr.

    This paper describes the content, organization, specifications, and methods of use of the CAS77 and CAS7276 online files of worldwide chemical literature, databases produced by Chemical Abstracts Service and available from System Development Corporation (SDC). The scope of the databases, their unit record, their data elements, their modes of…

  4. Condition Assessment Survey (CAS) Program. Deficiency standards and inspections methods manual: Volume 11, 0.11 Specialty systems

    SciTech Connect

    Not Available

    1993-05-01

    General information is presented for asset determinant factor/CAS repair codes/CAS cost factors; guide sheet tool & material listing; testing methods; inspection frequency; standard system design life tables; system work breakdown structure; and general system/material data. Deficiency standards and inspection methods are presented for canopies; loading dock systems; tanks; domes (bulk storage, metal framing); louvers & vents; access floors; integrated ceilings; and mezzanine structures.

  5. Identification and functional study of type III-A CRISPR-Cas systems in clinical isolates of Staphylococcus aureus.

    PubMed

    Cao, Linyan; Gao, Chun-Hui; Zhu, Jiade; Zhao, Liping; Wu, Qingfa; Li, Min; Sun, Baolin

    2016-12-01

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats [CRISPR]-CRISPR associated proteins [Cas]) system can provide prokaryote with immunity against invading mobile genetic elements (MGEs) such as phages and plasmids, which are the main sources of staphylococcal accessory genes. To date, only a few Staphylococcus aureus strains containing CRISPR-Cas systems have been identified, but no functional study in these strains has been reported. In this study, 6 clinical isolates of S. aureus with type III-A CRISPR-Cas systems were identified, and whole-genome sequencing and functional study were conducted subsequently. Genome sequence analysis revealed a close linkage between the CRISPR-Cas system and the staphylococcal cassette chromosome mec (SCCmec) element in five strains. Comparative sequence analysis showed that the type III-A repeats are conserved within staphylococci, despite of the decreased conservation in trailer-end repeats. Highly homologous sequences of some spacers were identified in staphylococcal MGEs, and partially complementary sequences of spacers were mostly found in the coding strand of lytic regions in staphylococcal phages. Transformation experiments showed that S. aureus type III-A CRISPR-Cas system can specifically prevent plasmid transfer in a transcription-dependent manner. Base paring between crRNA and target sequence, the endoribonuclease, and the Csm complex were proved to be necessary for type III-A CRISPR-Cas immunity.

  6. Adaptive control of linearizable systems

    NASA Technical Reports Server (NTRS)

    Sastry, S. Shankar; Isidori, Alberto

    1989-01-01

    Initial results are reported regarding the adaptive control of minimum-phase nonlinear systems which are exactly input-output linearizable by state feedback. Parameter adaptation is used as a technique to make robust the exact cancellation of nonlinear terms, which is called for in the linearization technique. The application of the adaptive technique to control of robot manipulators is discussed. Only the continuous-time case is considered; extensions to the discrete-time and sampled-data cases are not obvious.

  7. The role of Cas8 in type I CRISPR interference.

    PubMed

    Cass, Simon D B; Haas, Karina A; Stoll, Britta; Alkhnbashi, Omer S; Sharma, Kundan; Urlaub, Henning; Backofen, Rolf; Marchfelder, Anita; Bolt, Edward L

    2015-05-05

    CRISPR (clustered regularly interspaced short palindromic repeat) systems provide bacteria and archaea with adaptive immunity to repel invasive genetic elements. Type I systems use 'cascade' [CRISPR-associated (Cas) complex for antiviral defence] ribonucleoprotein complexes to target invader DNA, by base pairing CRISPR RNA (crRNA) to protospacers. Cascade identifies PAMs (protospacer adjacent motifs) on invader DNA, triggering R-loop formation and subsequent DNA degradation by Cas3. Cas8 is a candidate PAM recognition factor in some cascades. We analysed Cas8 homologues from type IB CRISPR systems in archaea Haloferax volcanii (Hvo) and Methanothermobacter thermautotrophicus (Mth). Cas8 was essential for CRISPR interference in Hvo and purified Mth Cas8 protein responded to PAM sequence when binding to nucleic acids. Cas8 interacted physically with Cas5-Cas7-crRNA complex, stimulating binding to PAM containing substrates. Mutation of conserved Cas8 amino acid residues abolished interference in vivo and altered catalytic activity of Cas8 protein in vitro. This is experimental evidence that Cas8 is important for targeting Cascade to invader DNA.

  8. Convergent Aeronautics Solutions (CAS) Showcase Presentation on Mission Adaptive Digital Composite Aerostructure Technologies (MADCAT)

    NASA Technical Reports Server (NTRS)

    Swei, Sean; Cheung, Kenneth

    2016-01-01

    This project is to develop a novel aerostructure concept that takes advantage of emerging digital composite materials and manufacturing methods to build high stiffness-to-density ratio, ultra-light structures that can provide mission adaptive and aerodynamically efficient future N+3N+4 air vehicles.

  9. Harnessing the Prokaryotic Adaptive Immune System as a Eukaryotic Antiviral Defense.

    PubMed

    Price, Aryn A; Grakoui, Arash; Weiss, David S

    2016-04-01

    Clustered, regularly interspaced, short palindromic repeats - CRISPR-associated (CRISPR-Cas) systems - are sequence-specific RNA-directed endonuclease complexes that bind and cleave nucleic acids. These systems evolved within prokaryotes as adaptive immune defenses to target and degrade nucleic acids derived from bacteriophages and other foreign genetic elements. The antiviral function of these systems has now been exploited to combat eukaryotic viruses throughout the viral life cycle. Here we discuss current advances in CRISPR-Cas9 technology as a eukaryotic antiviral defense.

  10. Direct CRISPR spacer acquisition from RNA by a natural reverse-transcriptase-Cas1 fusion protein

    PubMed Central

    Sidote, David J.; Markham, Laura M.; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M.; Fire, Andrew Z.

    2016-01-01

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat) systems mediate adaptive immunity in diverse prokaryotes. CRISPR-associated Cas1 and Cas2 proteins have been shown to enable adaptation to new threats in Type I and II CRISPR systems by the acquisition of short segments of DNA (“spacers”) from invasive elements. In several Type III CRISPR systems, Cas1 is naturally fused to a reverse transcriptase (RT). In the marine bacterium Marinomonas mediterranea (MMB-1), we show that an RT-Cas1 fusion enables the acquisition of RNA spacers in vivo in an RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze ligation of RNA segments into the CRISPR array, followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

  11. Target motifs affecting natural immunity by a constitutive CRISPR-Cas system in Escherichia coli.

    PubMed

    Almendros, Cristóbal; Guzmán, Noemí M; Díez-Villaseñor, César; García-Martínez, Jesús; Mojica, Francisco J M

    2012-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR associated (cas) genes conform the CRISPR-Cas systems of various bacteria and archaea and produce degradation of invading nucleic acids containing sequences (protospacers) that are complementary to repeat intervening spacers. It has been demonstrated that the base sequence identity of a protospacer with the cognate spacer and the presence of a protospacer adjacent motif (PAM) influence CRISPR-mediated interference efficiency. By using an original transformation assay with plasmids targeted by a resident spacer here we show that natural CRISPR-mediated immunity against invading DNA occurs in wild type Escherichia coli. Unexpectedly, the strongest activity is observed with protospacer adjoining nucleotides (interference motifs) that differ from the PAM both in sequence and location. Hence, our results document for the first time native CRISPR activity in E. coli and demonstrate that positions next to the PAM in invading DNA influence their recognition and degradation by these prokaryotic immune systems.

  12. Cas9-mediated targeting of viral RNA in eukaryotic cells.

    PubMed

    Price, Aryn A; Sampson, Timothy R; Ratner, Hannah K; Grakoui, Arash; Weiss, David S

    2015-05-12

    Clustered, regularly interspaced, short palindromic repeats-CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense.

  13. Cas9-mediated targeting of viral RNA in eukaryotic cells

    PubMed Central

    Price, Aryn A.; Sampson, Timothy R.; Ratner, Hannah K.; Grakoui, Arash; Weiss, David S.

    2015-01-01

    Clustered, regularly interspaced, short palindromic repeats–CRISPR associated (CRISPR-Cas) systems are prokaryotic RNA-directed endonuclease machineries that act as an adaptive immune system against foreign genetic elements. Using small CRISPR RNAs that provide specificity, Cas proteins recognize and degrade nucleic acids. Our previous work demonstrated that the Cas9 endonuclease from Francisella novicida (FnCas9) is capable of targeting endogenous bacterial RNA. Here, we show that FnCas9 can be directed by an engineered RNA-targeting guide RNA to target and inhibit a human +ssRNA virus, hepatitis C virus, within eukaryotic cells. This work reveals a versatile and portable RNA-targeting system that can effectively function in eukaryotic cells and be programmed as an antiviral defense. PMID:25918406

  14. CRISPR-Cas: biology, mechanisms and relevance

    PubMed Central

    Hille, Frank

    2016-01-01

    Prokaryotes have evolved several defence mechanisms to protect themselves from viral predators. Clustered regularly interspaced short palindromic repeats (CRISPR) and their associated proteins (Cas) display a prokaryotic adaptive immune system that memorizes previous infections by integrating short sequences of invading genomes—termed spacers—into the CRISPR locus. The spacers interspaced with repeats are expressed as small guide CRISPR RNAs (crRNAs) that are employed by Cas proteins to target invaders sequence-specifically upon a reoccurring infection. The ability of the minimal CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new avenues in genome editing in a broad range of cells and organisms with high potential in therapeutical applications. While numerous scientific studies have shed light on the biochemical processes behind CRISPR-Cas systems, several aspects of the immunity steps, however, still lack sufficient understanding. This review summarizes major discoveries in the CRISPR-Cas field, discusses the role of CRISPR-Cas in prokaryotic immunity and other physiological properties, and describes applications of the system as a DNA editing technology and antimicrobial agent. This article is part of the themed issue ‘The new bacteriology’. PMID:27672148

  15. Efficient Genome Editing in Chicken DF-1 Cells Using the CRISPR/Cas9 System

    PubMed Central

    Bai, Yichun; He, Linjie; Li, Pengcheng; Xu, Kun; Shao, Simin; Ren, Chonghua; Liu, Zhongtian; Wei, Zehui; Zhang, Zhiying

    2016-01-01

    In recent years, genome engineering technology has provided unprecedented opportunities for site-specific modification of biological genomes. Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 is one such means that can target a specific genome locus. It has been applied in human cells and many other organisms. Meanwhile, to efficiently enrich targeted cells, several surrogate systems have also been developed. However, very limited information exists on the application of CRISPR/Cas9 in chickens. In this study, we employed the CRISPR/Cas9 system to induce mutations in the peroxisome proliferator-activated receptor-γ (PPAR-γ), ATP synthase epsilon subunit (ATP5E), and ovalbumin (OVA) genes in chicken DF-1 cells. The results of T7E1 assays showed that the mutation rate at the three different loci was 0.75%, 0.5%, and 3.0%, respectively. In order to improve the mutation efficiency, we used the PuroR gene for efficient enrichment of genetically modified cells with the surrogate reporter system. The mutation rate, as assessed via the T7E1 assay, increased to 60.7%, 61.3%, and 47.3%, and subsequent sequence analysis showed that the mutation efficiency increased to 94.7%, 95%, and 95%, respectively. In addition, there were no detectable off-target mutations in three potential off-target sites using the T7E1 assay. As noted above, the CRISPR/Cas9 system is a robust tool for chicken genome editing. PMID:26869617

  16. Adaptive protection algorithm and system

    DOEpatents

    Hedrick, Paul [Pittsburgh, PA; Toms, Helen L [Irwin, PA; Miller, Roger M [Mars, PA

    2009-04-28

    An adaptive protection algorithm and system for protecting electrical distribution systems traces the flow of power through a distribution system, assigns a value (or rank) to each circuit breaker in the system and then determines the appropriate trip set points based on the assigned rank.

  17. DNA targeting by the type I-G and type I-A CRISPR–Cas systems of Pyrococcus furiosus

    PubMed Central

    Elmore, Joshua; Deighan, Trace; Westpheling, Jan; Terns, Rebecca M.; Terns, Michael P.

    2015-01-01

    CRISPR–Cas systems silence plasmids and viruses in prokaryotes. CRISPR–Cas effector complexes contain CRISPR RNAs (crRNAs) that include sequences captured from invaders and direct CRISPR-associated (Cas) proteins to destroy corresponding invader nucleic acids. Pyrococcus furiosus (Pfu) harbors three CRISPR–Cas immune systems: a Cst (Type I-G) system with an associated Cmr (Type III-B) module at one locus, and a partial Csa (Type I-A) module (lacking known invader sequence acquisition and crRNA processing genes) at another locus. The Pfu Cmr complex cleaves complementary target RNAs, and Csa systems have been shown to target DNA, while the mechanism by which Cst complexes silence invaders is unknown. In this study, we investigated the function of the Cst as well as Csa system in Pfu strains harboring a single CRISPR–Cas system. Plasmid transformation assays revealed that the Cst and Csa systems both function by DNA silencing and utilize similar flanking sequence information (PAMs) to identify invader DNA. Silencing by each system specifically requires its associated Cas3 nuclease. crRNAs from the 7 shared CRISPR loci in Pfu are processed for use by all 3 effector complexes, and Northern analysis revealed that individual effector complexes dictate the profile of mature crRNA species that is generated. PMID:26519471

  18. MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems.

    PubMed

    Zhang, Hai-Yan; Wang, Xing-Hui; Dong, Li; Wang, Zhi-Ping; Liu, Bing; Lv, Jie; Xing, Hui-Li; Han, Chun-Yan; Wang, Xue-Chen; Chen, Qi-Jun

    2017-02-03

    Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology.

  19. MISSA 2.0: an updated synthetic biology toolbox for assembly of orthogonal CRISPR/Cas systems

    PubMed Central

    Zhang, Hai-Yan; Wang, Xing-Hui; Dong, Li; Wang, Zhi-Ping; Liu, Bing; Lv, Jie; Xing, Hui-Li; Han, Chun-Yan; Wang, Xue-Chen; Chen, Qi-Jun

    2017-01-01

    Efficient generation of plants carrying mutations in multiple genes remains a challenge. Using two or more orthogonal CRISPR/Cas systems can generate plants with multi-gene mutations, but assembly of these systems requires a robust, high-capacity toolkit. Here, we describe MISSA 2.0 (multiple-round in vivo site-specific assembly 2.0), an extensively updated toolkit for assembly of two or more CRISPR/Cas systems. We developed a novel suicide donor vector system based on plasmid RK2, which has much higher cloning capacity than the original, plasmid R6K-based system. We validated the utility of MISSA 2.0 by assembling multiple DNA fragments into the E. coli chromosome, and by creating transgenic Arabidopsis thaliana that constitutively or inducibly overexpress multiple genes. We then demonstrated that the higher cloning capacity of the RK2-derived MISSA 2.0 donor vectors facilitated the assembly of two orthogonal CRISPR/Cas systems including SpCas9 and SaCas9, and thus facilitated the creation of transgenic lines harboring these systems. We anticipate that MISSA 2.0 will enable substantial advancements in multiplex genome editing based on two or more orthogonal CRISPR/Cas9 systems, as well as in plant synthetic biology. PMID:28155921

  20. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system.

    PubMed

    Zhang, Kang; Duan, Xuguo; Wu, Jing

    2016-06-16

    Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain.

  1. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems.

    PubMed

    Bialek, Julia K; Dunay, Gábor A; Voges, Maike; Schäfer, Carola; Spohn, Michael; Stucka, Rolf; Hauber, Joachim; Lange, Ulrike C

    2016-01-01

    CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5' long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination.

  2. Multigene disruption in undomesticated Bacillus subtilis ATCC 6051a using the CRISPR/Cas9 system

    PubMed Central

    Zhang, Kang; Duan, Xuguo; Wu, Jing

    2016-01-01

    Bacillus subtilis ATCC 6051a is an undomesticated strain used in the industrial production of enzymes. Because it is poorly transformable, genetic manipulation in this strain requires a highly efficient genome editing method. In this study, a Streptococcus pyogenes CRISPR/Cas9 system consisting of an all-in-one knockout plasmid containing a target-specific guide RNA, cas9, and a homologous repair template was established for highly efficient gene disruption in B. subtilis ATCC 6051a. With an efficiency of 33% to 53%, this system was used to disrupt the srfC, spoIIAC, nprE, aprE and amyE genes of B. subtilis ATCC 6051a, which hamper its use in industrial fermentation. Compared with B. subtilis ATCC 6051a, the final mutant, BS5 (ΔsrfC, ΔspoIIAC, ΔnprE, ΔaprE, ΔamyE), produces much less foam during fermentation, displays greater resistant to spore formation, and secretes 2.5-fold more β-cyclodextrin glycosyltransferase into the fermentation medium. Thus, the CRISPR/Cas9 system proved to be a powerful tool for targeted genome editing in an industrially relevant, poorly transformable strain. PMID:27305971

  3. TALEN and CRISPR/Cas Genome Editing Systems: Tools of Discovery

    PubMed Central

    Nemudryi, A. A.; Valetdinova, K. R.; Medvedev, S. P.; Zakian, S. M.

    2014-01-01

    Precise studies of plant, animal and human genomes enable remarkable opportunities of obtained data application in biotechnology and medicine. However, knowing nucleotide sequences isn’t enough for understanding of particular genomic elements functional relationship and their role in phenotype formation and disease pathogenesis. In post-genomic era methods allowing genomic DNA sequences manipulation, visualization and regulation of gene expression are rapidly evolving. Though, there are few methods, that meet high standards of efficiency, safety and accessibility for a wide range of researchers. In 2011 and 2013 novel methods of genome editing appeared – this are TALEN (Transcription Activator-Like Effector Nucleases) and CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas9 systems. Although TALEN and CRISPR/Cas9 appeared recently, these systems have proved to be effective and reliable tools for genome engineering. Here we generally review application of these systems for genome editing in conventional model objects of current biology, functional genome screening, cell-based human hereditary disease modeling, epigenome studies and visualization of cellular processes. Additionally, we review general strategies for designing TALEN and CRISPR/Cas9 and analyzing their activity. We also discuss some obstacles researcher can face using these genome editing tools. PMID:25349712

  4. Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems

    PubMed Central

    Bialek, Julia K.; Dunay, Gábor A.; Voges, Maike; Schäfer, Carola; Spohn, Michael; Stucka, Rolf; Hauber, Joachim; Lange, Ulrike C.

    2016-01-01

    CRISPR/Cas9 technology is currently considered the most advanced tool for targeted genome engineering. Its sequence-dependent specificity has been explored for locus-directed transcriptional modulation. Such modulation, in particular transcriptional activation, has been proposed as key approach to overcome silencing of dormant HIV provirus in latently infected cellular reservoirs. Currently available agents for provirus activation, so-called latency reversing agents (LRAs), act indirectly through cellular pathways to induce viral transcription. However, their clinical performance remains suboptimal, possibly because reservoirs have diverse cellular identities and/or proviral DNA is intractable to the induced pathways. We have explored two CRISPR/Cas9-derived activator systems as targeted approaches to induce dormant HIV-1 proviral DNA. These systems recruit multiple transcriptional activation domains to the HIV 5’ long terminal repeat (LTR), for which we have identified an optimal target region within the LTR U3 sequence. Using this target region, we demonstrate transcriptional activation of proviral genomes via the synergistic activation mediator complex in various in culture model systems for HIV latency. Observed levels of induction are comparable or indeed higher than treatment with established LRAs. Importantly, activation is complete, leading to production of infective viral particles. Our data demonstrate that CRISPR/Cas9-derived technologies can be applied to counteract HIV latency and may therefore represent promising novel approaches in the quest for HIV elimination. PMID:27341108

  5. High-temperature protein G is essential for activity of the Escherichia coli clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system.

    PubMed

    Yosef, Ido; Goren, Moran G; Kiro, Ruth; Edgar, Rotem; Qimron, Udi

    2011-12-13

    Prokaryotic DNA arrays arranged as clustered regularly interspaced short palindromic repeats (CRISPR), along with their associated proteins, provide prokaryotes with adaptive immunity by RNA-mediated targeting of alien DNA or RNA matching the sequences between the repeats. Here, we present a thorough screening system for the identification of bacterial proteins participating in immunity conferred by the Escherichia coli CRISPR system. We describe the identification of one such protein, high-temperature protein G (HtpG), a homolog of the eukaryotic chaperone heat-shock protein 90. We demonstrate that in the absence of htpG, the E. coli CRISPR system loses its suicidal activity against λ prophage and its ability to provide immunity from lysogenization. Transcomplementation of htpG restores CRISPR activity. We further show that inactivity of the CRISPR system attributable to htpG deficiency can be suppressed by expression of Cas3, a protein that is essential for its activity. Accordingly, we also find that the steady-state level of overexpressed Cas3 is significantly enhanced following HtpG expression. We conclude that HtpG is a newly identified positive modulator of the CRISPR system that is essential for maintaining functional levels of Cas3.

  6. The CRISPR/Cas9 system for plant genome editing and beyond.

    PubMed

    Bortesi, Luisa; Fischer, Rainer

    2015-01-01

    Targeted genome editing using artificial nucleases has the potential to accelerate basic research as well as plant breeding by providing the means to modify genomes rapidly in a precise and predictable manner. Here we describe the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system, a recently developed tool for the introduction of site-specific double-stranded DNA breaks. We highlight the strengths and weaknesses of this technology compared with two well-established genome editing platforms: zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). We summarize recent results obtained in plants using CRISPR/Cas9 technology, discuss possible applications in plant breeding and consider potential future developments.

  7. Development of the CRISPR/Cas9 System for Targeted Gene Disruption in Aspergillus fumigatus

    PubMed Central

    Fuller, Kevin K.

    2015-01-01

    Low rates of homologous recombination have broadly encumbered genetic studies in the fungal pathogen Aspergillus fumigatus. The CRISPR/Cas9 system of bacteria has recently been developed for targeted mutagenesis of eukaryotic genomes with high efficiency and, importantly, through a mechanism independent of homologous repair machinery. As this new technology has not been developed for use in A. fumigatus, we sought to test its feasibility for targeted gene disruption in this organism. As a proof of principle, we first demonstrated that CRISPR/Cas9 can indeed be used for high-efficiency (25 to 53%) targeting of the A. fumigatus polyketide synthase gene (pksP), as evidenced by the generation of colorless (albino) mutants harboring the expected genomic alteration. We further demonstrated that the constitutive expression of the Cas9 nuclease by itself is not deleterious to A. fumigatus growth or virulence, thus making the CRISPR system compatible with studies involved in pathogenesis. Taken together, these data demonstrate that CRISPR can be utilized for loss-of-function studies in A. fumigatus and has the potential to bolster the genetic toolbox for this important pathogen. PMID:26318395

  8. Intellectual property education exemplified by the patents on the CRISPR/Cas9 system.

    PubMed

    Fan, Xiangyu; Liao, Guojian; Xie, Jianping

    2014-12-01

    With the accelerated globalization of the world economies, the role of intellectual property in the the competition is increasingly important. The universities are important base to instill the intellectual property awareness to the young generation. However, current model of intellectual property education cannot meet the needs of undergraduates. In this paper, we take the first patent issued for CRISPR/Cas9 system as a teaching example, and together with personal teaching experience in biomedicine related intellectual property, we propose a new way for intellectual property education which consists of two stages: enlightenment stage and in-depth training stage. In the former stage, we integrate the intellectual property education with the basic major courses. In the latter stage, students are encouraged to devote into intellectual property related career. This model can somehow solve the the current shortage of qualified teachers for biotechnology related intellectual property education and will facilitate the popularization of intellectual property in college students. Since genetics plays a pivotal role in biomedicine, this effort is illustrated by the novel genome editing technology based on the CRISPR/Cas9 system, which is one hotspot of recent studies. The trajectory of CRISPR/Cas9 from basic microbial genetics discovery to major tools for genome editing exeplified the essence of biomedicine related intellectual property education.

  9. A CRISPR-Cas9 sex-ratio distortion system for genetic control

    PubMed Central

    Galizi, Roberto; Hammond, Andrew; Kyrou, Kyros; Taxiarchi, Chrysanthi; Bernardini, Federica; O’Loughlin, Samantha M.; Papathanos, Philippos-Aris; Nolan, Tony; Windbichler, Nikolai; Crisanti, Andrea

    2016-01-01

    Genetic control aims to reduce the ability of insect pest populations to cause harm via the release of modified insects. One strategy is to bias the reproductive sex ratio towards males so that a population decreases in size or is eliminated altogether due to a lack of females. We have shown previously that sex ratio distortion can be generated synthetically in the main human malaria vector Anopheles gambiae, by selectively destroying the X-chromosome during spermatogenesis, through the activity of a naturally-occurring endonuclease that targets a repetitive rDNA sequence highly-conserved in a wide range of organisms. Here we describe a CRISPR-Cas9 sex distortion system that targets ribosomal sequences restricted to the member species of the Anopheles gambiae complex. Expression of Cas9 during spermatogenesis resulted in RNA-guided shredding of the X-chromosome during male meiosis and produced extreme male bias among progeny in the absence of any significant reduction in fertility. The flexibility of CRISPR-Cas9 combined with the availability of genomic data for a range of insects renders this strategy broadly applicable for the species-specific control of any pest or vector species with an XY sex-determination system by targeting sequences exclusive to the female sex chromosome. PMID:27484623

  10. CRISPR-Cas systems preferentially target the leading regions of MOBF conjugative plasmids.

    PubMed

    Westra, Edze R; Staals, Raymond H J; Gort, Gerrit; Høgh, Søren; Neumann, Sarah; de la Cruz, Fernando; Fineran, Peter C; Brouns, Stan J J

    2013-05-01

    Most prokaryotes contain CRISPR-Cas immune systems that provide protection against mobile genetic elements. We have focused on the ability of CRISPR-Cas to block plasmid conjugation, and analyzed the position of target sequences (protospacers) on conjugative plasmids. The analysis reveals that protospacers are non-uniformly distributed over plasmid regions in a pattern that is determined by the plasmid's mobilization type (MOB). While MOBP plasmids are most frequently targeted in the region entering the recipient cell last (lagging region), MOBF plasmids are mostly targeted in the region entering the recipient cell first (leading region). To explain this protospacer distribution bias, we propose two mutually non-exclusive hypotheses: (1) spacers are acquired more frequently from either the leading or lagging region depending on the MOB type (2) CRISPR-interference is more efficient when spacers target these preferred regions. To test the latter hypothesis, we analyzed Type I-E CRISPR-interference against MOBF prototype plasmid F in Escherichia coli. Our results show that plasmid conjugation is effectively inhibited, but the level of immunity is not affected by targeting the plasmid in the leading or lagging region. Moreover, CRISPR-immunity levels do not depend on whether the incoming single-stranded plasmid DNA, or the DNA strand synthesized in the recipient is targeted. Our findings indicate that single-stranded DNA may not be a target for Type I-E CRISPR-Cas systems, and suggest that the protospacer distribution bias might be due to spacer acquisition preferences.

  11. Efficient Gene Targeting in Golden Syrian Hamsters by the CRISPR/Cas9 System

    PubMed Central

    Meng, Qinggang; Shi, Bi; Bunch, Thomas D.; White, Kenneth L.; Kong, Il-Keun; Wang, Zhongde

    2014-01-01

    The golden Syrian hamster is the model of choice or the only rodent model for studying many human diseases. However, the lack of gene targeting tools in hamsters severely limits their use in biomedical research. Here, we report the first successful application of the CRISPR/Cas9 system to efficiently conduct gene targeting in hamsters. We designed five synthetic single-guide RNAs (sgRNAs)—three for targeting the coding sequences for different functional domains of the hamster STAT2 protein, one for KCNQ1, and one for PPP1R12C—and demonstrated that the CRISPR/Cas9 system is highly efficient in introducing site-specific mutations in hamster somatic cells. We then developed unique pronuclear (PN) and cytoplasmic injection protocols in hamsters and produced STAT2 knockout (KO) hamsters by injecting the sgRNA/Cas9, either in the form of plasmid or mRNA, targeting exon 4 of hamster STAT2. Among the produced hamsters, 14.3% and 88.9% harbored germline-transmitted STAT2 mutations from plasmid and mRNA injection, respectively. Notably, 10.4% of the animals produced from mRNA injection were biallelically targeted. This is the first success in conducting site-specific gene targeting in hamsters and can serve as the foundation for developing other genetically engineered hamster models for human disease. PMID:25299451

  12. A CRISPR/Cas9 vector system for tissue-specific gene disruption in zebrafish

    PubMed Central

    Ablain, Julien; Durand, Ellen M.; Yang, Song; Zhou, Yi; Zon, Leonard I.

    2015-01-01

    Summary CRISPR/Cas9 technology of genome editing has greatly facilitated the targeted inactivation of genes in vitro and in vivo in a wide range of organisms. In zebrafish it allows the rapid generation of knock-out lines by simply injecting a guide RNA (gRNA) and Cas9 mRNA into one-cell stage embryos. Here, we report a simple and scalable CRISPR-based vector system for tissue-specific gene inactivation in zebrafish. As proof of principle, we used our vector with the gata1 promoter driving Cas9 expression to silence the urod gene, implicated in heme biosynthesis, specifically in the erythrocytic lineage. Urod targeting yielded red fluorescent erythrocytes in zebrafish embryos, recapitulating the phenotype observed in the yquem mutant. While F0 embryos displayed mosaic gene disruption, the phenotype appeared very penetrant in stable F1 fish. This vector system constitutes a unique tool to spatially control gene knock-out and greatly broadens the scope of loss-of-function studies in zebrafish. PMID:25752963

  13. CRISPR-Cas Technologies and Applications in Food Bacteria.

    PubMed

    Stout, Emily; Klaenhammer, Todd; Barrangou, Rodolphe

    2017-02-28

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins form adaptive immune systems that occur in many bacteria and most archaea. In addition to protecting bacteria from phages and other invasive mobile genetic elements, CRISPR-Cas molecular machines can be repurposed as tool kits for applications relevant to the food industry. A primary concern of the food industry has long been the proper management of food-related bacteria, with a focus on both enhancing the outcomes of beneficial microorganisms such as starter cultures and probiotics and limiting the presence of detrimental organisms such as pathogens and spoilage microorganisms. This review introduces CRISPR-Cas as a novel set of technologies to manage food bacteria and offers insights into CRISPR-Cas biology. It primarily focuses on the applications of CRISPR-Cas systems and tools in starter cultures and probiotics, encompassing strain-typing, phage resistance, plasmid vaccination, genome editing, and antimicrobial activity.

  14. CRISPR-Cas Defense System and Potential Prophages in Cyanobacteria Associated with the Coral Black Band Disease.

    PubMed

    Buerger, Patrick; Wood-Charlson, Elisha M; Weynberg, Karen D; Willis, Bette L; van Oppen, Madeleine J H

    2016-01-01

    Understanding how pathogens maintain their virulence is critical to developing tools to mitigate disease in animal populations. We sequenced and assembled the first draft genome of Roseofilum reptotaenium AO1, the dominant cyanobacterium underlying pathogenicity of the virulent coral black band disease (BBD), and analyzed parts of the BBD-associated Geitlerinema sp. BBD_1991 genome in silico. Both cyanobacteria are equipped with an adaptive, heritable clustered regularly interspaced short palindromic repeats (CRISPR)-Cas defense system type I-D and have potential virulence genes located within several prophage regions. The defense system helps to prevent infection by viruses and mobile genetic elements via identification of short fingerprints of the intruding DNA, which are stored as templates in the bacterial genome, in so-called "CRISPRs." Analysis of CRISPR target sequences (protospacers) revealed an unusually high number of self-targeting spacers in R. reptotaenium AO1 and extraordinary long CRIPSR arrays of up to 260 spacers in Geitlerinema sp. BBD_1991. The self-targeting spacers are unlikely to be a form of autoimmunity; instead these target an incomplete lysogenic bacteriophage. Lysogenic virus induction experiments with mitomycin C and UV light did not reveal an actively replicating virus population in R. reptotaenium AO1 cultures, suggesting that phage functionality is compromised or excision could be blocked by the CRISPR-Cas system. Potential prophages were identified in three regions of R. reptotaenium AO1 and five regions of Geitlerinema sp. BBD_1991, containing putative BBD relevant virulence genes, such as an NAD-dependent epimerase/dehydratase (a homolog in terms of functionality to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other lipopolysaccharide modification genes. To date, viruses have not been considered to be a component of the BBD consortium or a contributor to the virulence of R. reptotaenium AO1

  15. CRISPR-Cas Defense System and Potential Prophages in Cyanobacteria Associated with the Coral Black Band Disease

    PubMed Central

    Buerger, Patrick; Wood-Charlson, Elisha M.; Weynberg, Karen D.; Willis, Bette L.; van Oppen, Madeleine J. H.

    2016-01-01

    Understanding how pathogens maintain their virulence is critical to developing tools to mitigate disease in animal populations. We sequenced and assembled the first draft genome of Roseofilum reptotaenium AO1, the dominant cyanobacterium underlying pathogenicity of the virulent coral black band disease (BBD), and analyzed parts of the BBD-associated Geitlerinema sp. BBD_1991 genome in silico. Both cyanobacteria are equipped with an adaptive, heritable clustered regularly interspaced short palindromic repeats (CRISPR)-Cas defense system type I-D and have potential virulence genes located within several prophage regions. The defense system helps to prevent infection by viruses and mobile genetic elements via identification of short fingerprints of the intruding DNA, which are stored as templates in the bacterial genome, in so-called “CRISPRs.” Analysis of CRISPR target sequences (protospacers) revealed an unusually high number of self-targeting spacers in R. reptotaenium AO1 and extraordinary long CRIPSR arrays of up to 260 spacers in Geitlerinema sp. BBD_1991. The self-targeting spacers are unlikely to be a form of autoimmunity; instead these target an incomplete lysogenic bacteriophage. Lysogenic virus induction experiments with mitomycin C and UV light did not reveal an actively replicating virus population in R. reptotaenium AO1 cultures, suggesting that phage functionality is compromised or excision could be blocked by the CRISPR-Cas system. Potential prophages were identified in three regions of R. reptotaenium AO1 and five regions of Geitlerinema sp. BBD_1991, containing putative BBD relevant virulence genes, such as an NAD-dependent epimerase/dehydratase (a homolog in terms of functionality to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other lipopolysaccharide modification genes. To date, viruses have not been considered to be a component of the BBD consortium or a contributor to the virulence of R. reptotaenium

  16. Adaptive security systems -- Combining expert systems with adaptive technologies

    SciTech Connect

    Argo, P.; Loveland, R.; Anderson, K.

    1997-09-01

    The Adaptive Multisensor Integrated Security System (AMISS) uses a variety of computational intelligence techniques to reason from raw sensor data through an array of processing layers to arrive at an assessment for alarm/alert conditions based on human behavior within a secure facility. In this paper, the authors give an overview of the system and briefly describe some of the major components of the system. This system is currently under development and testing in a realistic facility setting.

  17. Structures of Cas9 endonucleases reveal RNA-mediated conformational activation.

    PubMed

    Jinek, Martin; Jiang, Fuguo; Taylor, David W; Sternberg, Samuel H; Kaya, Emine; Ma, Enbo; Anders, Carolin; Hauer, Michael; Zhou, Kaihong; Lin, Steven; Kaplan, Matias; Iavarone, Anthony T; Charpentier, Emmanuelle; Nogales, Eva; Doudna, Jennifer A

    2014-03-14

    Type II CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) systems use an RNA-guided DNA endonuclease, Cas9, to generate double-strand breaks in invasive DNA during an adaptive bacterial immune response. Cas9 has been harnessed as a powerful tool for genome editing and gene regulation in many eukaryotic organisms. We report 2.6 and 2.2 angstrom resolution crystal structures of two major Cas9 enzyme subtypes, revealing the structural core shared by all Cas9 family members. The architectures of Cas9 enzymes define nucleic acid binding clefts, and single-particle electron microscopy reconstructions show that the two structural lobes harboring these clefts undergo guide RNA-induced reorientation to form a central channel where DNA substrates are bound. The observation that extensive structural rearrangements occur before target DNA duplex binding implicates guide RNA loading as a key step in Cas9 activation.

  18. CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection

    PubMed Central

    Wollebo, Hassen S.; Bellizzi, Anna; Kaminski, Rafal; Hu, Wenhui; White, Martyn K.; Khalili, Kamel

    2015-01-01

    Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV

  19. CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection.

    PubMed

    Wollebo, Hassen S; Bellizzi, Anna; Kaminski, Rafal; Hu, Wenhui; White, Martyn K; Khalili, Kamel

    2015-01-01

    Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV

  20. Efficient Generation of Myostatin Mutations in Pigs Using the CRISPR/Cas9 System

    PubMed Central

    Wang, Kankan; Ouyang, Hongsheng; Xie, Zicong; Yao, Chaogang; Guo, Nannan; Li, Mengjing; Jiao, Huping; Pang, Daxin

    2015-01-01

    Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth. The transfection efficiency of porcine fetal fibroblasts (PFFs) was improved to facilitate the targeting of Cas9/gRNA. We also demonstrated that Cas9/gRNA can induce non-homologous end-joining (NHEJ), long fragment deletions/inversions and homology-directed repair (HDR) at the MSTN locus of PFFs. Single-cell MSTN knockout colonies were used to generate cloned pigs via somatic cell nuclear transfer (SCNT), which resulted in 8 marker-gene-free cloned pigs with biallelic mutations. Some of the piglets showed obvious intermuscular grooves and enlarged tongues, which are characteristic of the double muscling (DM) phenotype. The protein level of MSTN was decreased in the mutant cloned pigs compared with the wild-type controls, and the mRNA levels of MSTN and related signaling pathway factors were also analyzed. Finally, we carefully assessed off-target mutations in the cloned pigs. The gene editing platform used in this study can efficiently generate genetically modified pigs with biological safety. PMID:26564781

  1. Editing of mouse and human immunoglobulin genes by CRISPR-Cas9 system

    PubMed Central

    Cheong, Taek-Chin; Compagno, Mara; Chiarle, Roberto

    2016-01-01

    Applications of the CRISPR-Cas9 system to edit the genome have widely expanded to include DNA gene knock-out, deletions, chromosomal rearrangements, RNA editing and genome-wide screenings. Here we show the application of CRISPR-Cas9 technology to edit the mouse and human immunoglobulin (Ig) genes. By delivering Cas9 and guide-RNA (gRNA) with retro- or lenti-virus to IgM+ mouse B cells and hybridomas, we induce class-switch recombination (CSR) of the IgH chain to the desired subclass. Similarly, we induce CSR in all human B cell lines tested with high efficiency to targeted IgH subclass. Finally, we engineer mouse hybridomas to secrete Fab′ fragments instead of the whole Ig. Our results indicate that Ig genes in mouse and human cells can be edited to obtain any desired IgH switching helpful to study the biology of normal and lymphoma B cells. We also propose applications that could transform the technology of antibody production. PMID:26956543

  2. Biallelic editing of a lamprey genome using the CRISPR/Cas9 system.

    PubMed

    Zu, Yao; Zhang, Xushuai; Ren, Jianfeng; Dong, Xuehong; Zhu, Zhe; Jia, Liang; Zhang, Qinghua; Li, Weiming

    2016-03-23

    Lampreys are extant representatives of agnathans. Descriptions of lamprey development, physiology and genome have provided critical insights into early evolution of vertebrate traits. However, efficient means for genetic manipulation in agnathan species have not been developed, hindering functional studies of genes in these important Evo-Devo models. Here, we report a CRISPR/Cas system optimized for lamprey genomes and use it to disrupt genomic loci in the Northeast Chinese lamprey (Lethenteron morii) with efficiencies ranging between 84~99%. The frequencies of indels observed in the target loci of golden (gol), kctd10, wee1, soxe2, and wnt7b, estimated from direct sequencing of genomic DNA samples of injected lamprey larvae, were 68/69, 47/56, 38/39, 36/37 and 36/42, respectively. These indels often occurred in both alleles. In the CRISPR/Cas9 treatment for gol or kctd10, 38.6% or 85.3% of the targeted larvae had the respective recessive null-like phenotypes, further confirming the disruption of both loci. The kctd10 gRNA, designed against an essential functional region of Kctd10, resulted in null-like phenotypes and in-frame mutations in alleles. We suggest that the CRISPR/Cas-based approach has the potential for efficient genetic perturbation in organisms less amenable to germ line transmission based approaches.

  3. Efficient Genome Editing in Apple Using a CRISPR/Cas9 system

    PubMed Central

    Nishitani, Chikako; Hirai, Narumi; Komori, Sadao; Wada, Masato; Okada, Kazuma; Osakabe, Keishi; Yamamoto, Toshiya; Osakabe, Yuriko

    2016-01-01

    Genome editing is a powerful technique for genome modification in molecular research and crop breeding, and has the great advantage of imparting novel desired traits to genetic resources. However, the genome editing of fruit tree plantlets remains to be established. In this study, we describe induction of a targeted gene mutation in the endogenous apple phytoene desaturase (PDS) gene using the CRISPR/Cas9 system. Four guide RNAs (gRNAs) were designed and stably transformed with Cas9 separately in apple. Clear and partial albino phenotypes were observed in 31.8% of regenerated plantlets for one gRNA, and bi-allelic mutations in apple PDS were confirmed by DNA sequencing. In addition, an 18-bp gRNA also induced a targeted mutation. These CRIPSR/Cas9 induced-mutations in the apple genome suggest activation of the NHEJ pathway, but with some involvement also of the HR pathway. Our results demonstrate that genome editing can be practically applied to modify the apple genome. PMID:27530958

  4. HMMCAS: a web tool for the identification and domain annotations of Cas proteins.

    PubMed

    Chai, Guoshi; Yu, Min; Jiang, Lixu; Duan, Yaocong; Huang, Jian

    2017-02-07

    The CRISPR-Cas (clustered regularly interspaced short palindromic repeats-CRISPR-associated proteins) adaptive immune systems are discovered in many bacteria and most archaea. These systems are encoded by cas (CRISPR-associated) operons that have an extremely diverse architecture. The most crucial step in the depiction of cas operons composition is the identification of cas genes or Cas proteins. With the continuous increase of the newly sequenced archaeal and bacterial genomes, the recognition of new Cas proteins is becoming possible, which not only provides candidates for novel genome editing tools but also helps to understand the prokaryotic immune system better. Here we describe HMMCAS, a web service for the detection of CRISPR-associated structural and functional domains in protein sequences. HMMCAS uses hmmscan similarity search algorithm in HMMER3.1 to provide a fast, interactive service based on a comprehensive collection of hidden Markov models of Cas protein family. It can accurately identify the Cas proteins including those fusion proteins, for example the Cas1-Cas4 fusion protein in Candidatus Chloracidobacterium thermophilum B (Cab. thermophilum B). HMMCAS can also find putative cas operon and determine which type it belongs to. HMMCAS is freely available at http://i.uestc.edu.cn/hmmcas.

  5. Multidisciplinary Aerospace Systems Optimization: Computational AeroSciences (CAS) Project

    NASA Technical Reports Server (NTRS)

    Kodiyalam, S.; Sobieski, Jaroslaw S. (Technical Monitor)

    2001-01-01

    The report describes a method for performing optimization of a system whose analysis is so expensive that it is impractical to let the optimization code invoke it directly because excessive computational cost and elapsed time might result. In such situation it is imperative to have user control the number of times the analysis is invoked. The reported method achieves that by two techniques in the Design of Experiment category: a uniform dispersal of the trial design points over a n-dimensional hypersphere and a response surface fitting, and the technique of krigging. Analyses of all the trial designs whose number may be set by the user are performed before activation of the optimization code and the results are stored as a data base. That code is then executed and referred to the above data base. Two applications, one of the airborne laser system, and one of an aircraft optimization illustrate the method application.

  6. Genetic engineering of a temperate phage-based delivery system for CRISPR/Cas9 antimicrobials against Staphylococcus aureus

    PubMed Central

    Park, Joo Youn; Moon, Bo Youn; Park, Juw Won; Thornton, Justin A.; Park, Yong Ho; Seo, Keun Seok

    2017-01-01

    Discovery of clustered, regularly interspaced, short palindromic repeats and the Cas9 RNA-guided nuclease (CRISPR/Cas9) system provides a new opportunity to create programmable gene-specific antimicrobials that are far less likely to drive resistance than conventional antibiotics. However, the practical therapeutic use of CRISPR/Cas9 is still questionable due to current shortcomings in phage-based delivery systems such as inefficient delivery, narrow host range, and potential transfer of virulence genes by generalized transduction. In this study, we demonstrate genetic engineering strategies to overcome these shortcomings by integrating CRISPR/Cas9 system into a temperate phage genome, removing major virulence genes from the host chromosome, and expanding host specificity of the phage by complementing tail fiber protein. This significantly improved the efficacy and safety of CRISPR/Cas9 antimicrobials to therapeutic levels in both in vitro and in vivo assays. The genetic engineering tools and resources established in this study are expected to provide an efficacious and safe CRISPR/Cas9 antimicrobial, broadly applicable to Staphylococcus aureus. PMID:28322317

  7. Adaptive ophthalmologic system

    DOEpatents

    Olivier, Scot S.; Thompson, Charles A.; Bauman, Brian J.; Jones, Steve M.; Gavel, Don T.; Awwal, Abdul A.; Eisenbies, Stephen K.; Haney, Steven J.

    2007-03-27

    A system for improving vision that can diagnose monochromatic aberrations within a subject's eyes, apply the wavefront correction, and then enable the patient to view the results of the correction. The system utilizes a laser for producing a beam of light; a corrector; a wavefront sensor; a testing unit; an optic device for directing the beam of light to the corrector, to the retina, from the retina to the wavefront sensor, and to the testing unit; and a computer operatively connected to the wavefront sensor and the corrector.

  8. Adaptive Production Systems

    DTIC Science & Technology

    1974-12-01

    Melton, A. W., and Marton. E. (Eds.). Coding Processes in Human Memory, Washington, DC , Winston and Sons, 1972. Newell. A. Production systems...STM (1 A ’) (ACTION (USED) (DEP (NEXT B))) (B ?) (LOC A) (A ?) (B ?) ( SEPIc ’ fiAB) 16 TRUE IN PS STM (NEXT A) (1 A?) (ACTION (USED) (DEP (NEXT B

  9. Adaptive Instructional Systems

    DTIC Science & Technology

    2005-09-01

    planning stage, which used the infbrmation obtained from the Phase I research to develop a plan for utilizing the tecnolog in the proposed system. 1.4...categories of physical stimuli are perceptual, intake, time, and mobility . 01ly one of these factors, perceptual, cam be taken into account in the simulator

  10. CRISPR/Cas9 system as an innovative genetic engineering tool: Enhancements in sequence specificity and delivery methods.

    PubMed

    Jo, Young-Il; Suresh, Bharathi; Kim, Hyongbum; Ramakrishna, Suresh

    2015-12-01

    While human gene therapy has gained significant attention for its therapeutic promise, CRISPR/Cas9 technology has made a breakthrough as an efficient genome editing tool by emulating prokaryotic immune defense mechanisms. Although many studies have found that CRISPR/Cas9 technology is more efficient, specific and manipulable than previous generations of gene editing tools, it can be further improved by elevating its overall efficiency in a higher frequency of genome modifications and reducing its off-target effects. Here, we review the development of CRISPR/Cas9 technology, focusing on enhancement of its sequence specificity, reduction of off-target effects and delivery systems. Moreover, we describe recent successful applications of CRISPR/Cas9 technology in laboratory and clinical studies.

  11. Engineering the Caenorhabditis elegans genome with CRISPR/Cas9.

    PubMed

    Waaijers, Selma; Boxem, Mike

    2014-08-01

    The development in early 2013 of CRISPR/Cas9-based genome engineering promises to dramatically advance our ability to alter the genomes of model systems at will. A single, easily produced targeting RNA guides the Cas9 endonuclease to a specific DNA sequence where it creates a double strand break. Imprecise repair of the break can yield mutations, while homologous recombination with a repair template can be used to effect specific changes to the genome. The tremendous potential of this system led several groups to independently adapt it for use in Caenorhabditiselegans, where it was successfully used to generate mutations and to create tailored genome changes through homologous recombination. Here, we review the different approaches taken to adapt CRISPR/Cas9 for C. elegans, and provide practical guidelines for CRISPR/Cas9-based genome engineering.

  12. [Health: an adaptive complex system].

    PubMed

    Toro-Palacio, Luis Fernando; Ochoa-Jaramillo, Francisco Luis

    2012-02-01

    This article points out the enormous gap that exists between complex thinking of an intellectual nature currently present in our environment, and complex experimental thinking that has facilitated the scientific and technological advances that have radically changed the world. The article suggests that life, human beings, global society, and all that constitutes health be considered as adaptive complex systems. This idea, in turn, prioritizes the adoption of a different approach that seeks to expand understanding. When this rationale is recognized, the principal characteristics and emerging properties of health as an adaptive complex system are sustained, following a care and services delivery model. Finally, some pertinent questions from this perspective are put forward in terms of research, and a series of appraisals are expressed that will hopefully serve to help us understand all that we have become as individuals and as a species. The article proposes that the delivery of health care services be regarded as an adaptive complex system.

  13. Anti-CRISPR Proteins: Counterattack of Phages on Bacterial Defense (CRISPR/Cas) System.

    PubMed

    Chaudhary, Kulbhushan; Chattopadhyay, Anirudha; Pratap, Dharmendra

    2017-03-01

    Since the dawn of life there is a never ending strife between bacteria and phages. Both are perpetually changing their strategies to take over each other. CRISPR/Cas is the most widespread defense system used by bacteria against mobile genetic elements (MGEs) such as phages, cojugative palsmids, transoposons and pathogenicity islands. This system utilizes small guide RNA molecules to protect against phages infection and invasion by MGEs. Phages circumvent to these antiviral barriers by point mutation in PAM (protospacer-adjacent motif) sequence, genome rearrangements and by using anti-CRISPR proteins. This article is protected by copyright. All rights reserved.

  14. Remote Adaptive Communication System

    DTIC Science & Technology

    2001-10-25

    manage several different devices using the software tool A. Client/Server Architecture The architecture we are proposing is based on the Client...communication". International Telemedicine. Julio 1999. Pp 4. [17] F. Fernández, L. Roa, "Communication System Based on a New Open Architecture...Toledo, " Fundamentos de Neurología para educadores". IDEO. Sevilla 1994. [21] P. Coad, E. Yourdon, "Object Oriented Analysis". Yourdon Press

  15. THE STRUCTURE OF THE CRISPR-ASSOCIATED PROTEIN CSA3 PROVIDES INSIGHT INTO REGULATION OF THE CRISPR/CAS SYSTEM

    PubMed Central

    Lintner, Nathanael G.; Frankel, Kenneth A.; Tsutakawa, Susan E.; Alsbury, Donald L.; Copié, Valérie; Young, Mark J.; Tainer, John A.; Lawrence, C. Martin

    2015-01-01

    Adaptive immune systems have recently been recognized in prokaryotic organisms where, in response to viral infection, they incorporate short fragments of invader-derived DNA into loci called Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs). In subsequent infections, the CRISPR loci are transcribed and processed into guide sequences for the neutralization of the invading RNA or DNA. The CRISPR-associated protein machinery (Cas) lies at the heart of this process, yet many of the molecular details of the CRISPR/Cas system remain to be elucidated. Here we report the first structure of Csa3, a CRISPR-associated protein from Sulfolobus solfataricus (Sso1445), which reveals a dimeric two-domain protein. The N-terminal domain is a unique variation on the di-nucleotide binding-domain that orchestrates dimer formation. In addition, it utilizes two conserved sequence motifs (Thr-h-Gly-Phe-(Asn/Asp)-Glu-X4-Arg and Leu-X2-Gly-h-Arg) to construct a 2-fold symmetric pocket on the dimer axis. This pocket is likely to represent a regulatory ligand-binding site. The N-terminal domain is fused to a C-terminal MarR-like winged helix-turn-helix domain that is expected to be involved in DNA recognition. Overall, the unique domain architecture of Csa3 suggests a transcriptional regulator under allosteric control of the N-terminal domain. Alternatively, Csa3 may function in a larger complex, with the conserved cleft participating in protein-protein or protein-nucleic acid interactions. A similar N-terminal domain is also identified in Csx1, a second CRISPR associated protein family of unknown function. PMID:21093452

  16. The dynamics of health care reform--learning from a complex adaptive systems theoretical perspective.

    PubMed

    Sturmberg, Joachim P; Martin, Carmel M

    2010-10-01

    Health services demonstrate key features of complex adaptive systems (CAS), they are dynamic and unfold in unpredictable ways, and unfolding events are often unique. To better understand the complex adaptive nature of health systems around a core attractor we propose the metaphor of the health care vortex. We also suggest that in an ideal health care system the core attractor would be personal health attainment. Health care reforms around the world offer an opportunity to analyse health system change from a complex adaptive perspective. At large health care reforms have been pursued disregarding the complex adaptive nature of the health system. The paper details some recent reforms and outlines how to understand their strategies and outcomes, and what could be learnt for future efforts, utilising CAS principles. Current health systems show the inherent properties of a CAS driven by a core attractor of disease and cost containment. We content that more meaningful health systems reform requires the delicate task of shifting the core attractor from disease and cost containment towards health attainment.

  17. Overview of CRISPR-Cas9 Biology.

    PubMed

    Ratner, Hannah K; Sampson, Timothy R; Weiss, David S

    2016-12-01

    Prokaryotes use diverse strategies to improve fitness in the face of different environmental threats and stresses, including those posed by mobile genetic elements (e.g., bacteriophages and plasmids). To defend against these elements, many bacteria and archaea use elegant, RNA-directed, nucleic acid-targeting adaptive restriction machineries called CRISPR -: Cas (CRISPR-associated) systems. While providing an effective defense against foreign genetic elements, these systems have also been observed to play critical roles in regulating bacterial physiology during environmental stress. Increasingly, CRISPR-Cas systems, in particular the Type II systems containing the Cas9 endonuclease, have been exploited for their ability to bind desired nucleic acid sequences, as well as direct sequence-specific cleavage of their targets. Cas9-mediated genome engineering is transcending biological research as a versatile and portable platform for manipulating genetic content in myriad systems. Here, we present a systematic overview of CRISPR-Cas history and biology, highlighting the revolutionary tools derived from these systems, which greatly expand the molecular biologists' toolkit.

  18. Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.

    PubMed

    Lin, Che-Yi; Su, Yi-Hsien

    2016-01-15

    Sea urchin embryos are a useful model system for investigating early developmental processes and the underlying gene regulatory networks. Most functional studies using sea urchin embryos rely on antisense morpholino oligonucleotides to knockdown gene functions. However, major concerns related to this technique include off-target effects, variations in morpholino efficiency, and potential morpholino toxicity; furthermore, such problems are difficult to discern. Recent advances in genome editing technologies have introduced the prospect of not only generating sequence-specific knockouts, but also providing genome-engineering applications. Two genome editing tools, zinc-finger nuclease (ZFN) and transcription activator-like effector nucleases (TALENs), have been utilized in sea urchin embryos, but the resulting efficiencies are far from satisfactory. The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system serves as an easy and efficient method with which to edit the genomes of several established and emerging model organisms in the field of developmental biology. Here, we apply the CRISPR/Cas9 system to the sea urchin embryo. We designed six guide RNAs (gRNAs) against the well-studied nodal gene and discovered that five of the gRNAs induced the expected phenotype in 60-80% of the injected embryos. In addition, we developed a simple method for isolating genomic DNA from individual embryos, enabling phenotype to be precisely linked to genotype, and revealed that the mutation rates were 67-100% among the sequenced clones. Of the two potential off-target sites we examined, no off-target effects were observed. The detailed procedures described herein promise to accelerate the usage of CRISPR/Cas9 system for genome editing in sea urchin embryos.

  19. Requirements for a successful defence reaction by the CRISPR-Cas subtype I-B system.

    PubMed

    Stoll, Britta; Maier, Lisa-Katharina; Lange, Sita J; Brendel, Jutta; Fischer, Susan; Backofen, Rolf; Marchfelder, Anita

    2013-12-01

    Uptake of foreign mobile genetic elements is often detrimental and can result in cell death. For protection against invasion, prokaryotes have developed several defence mechanisms, which take effect at all stages of infection; an example is the recently discovered CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) immune system. This defence system directly degrades invading genetic material and is present in almost all archaea and many bacteria. Current data indicate a large variety of mechanistic molecular approaches. Although almost all archaea carry this defence weapon, only a few archaeal systems have been fully characterized. In the present paper, we summarize the prerequisites for the detection and degradation of invaders in the halophilic archaeon Haloferax volcanii. H. volcanii encodes a subtype I-B CRISPR-Cas system and the defence can be triggered by a plasmid-based invader. Six different target-interference motifs are recognized by the Haloferax defence and a 9-nt non-contiguous seed sequence is essential. The repeat sequence has the potential to fold into a minimal stem-loop structure, which is conserved in haloarchaea and might be recognized by the Cas6 endoribonuclease during the processing of CRISPR loci into mature crRNA (CRISPR RNA). Individual crRNA species were present in very different concentrations according to an RNA-Seq analysis and many were unable to trigger a successful defence reaction. Recognition of the plasmid invader does not depend on its copy number, but instead results indicate a dependency on the type of origin present on the plasmid.

  20. The subtype I-F CRISPR-Cas system influences pathogenicity island retention in Pectobacterium atrosepticum via crRNA generation and Csy complex formation.

    PubMed

    Richter, Corinna; Fineran, Peter C

    2013-12-01

    CRISPR (clustered regularly interspaced short palindromic repeats) arrays and Cas (CRISPR-associated) proteins confer acquired resistance against mobile genetic elements in a wide range of bacteria and archaea. The phytopathogen Pectobacterium atrosepticum SCRI1043 encodes a single subtype I-F CRISPR system, which is composed of three CRISPR arrays and the cas operon encoding Cas1, Cas3 (a Cas2-Cas3 fusion), Csy1, Csy2, Csy3 and Cas6f (Csy4). The CRISPR arrays are transcribed into pre-crRNA (CRISPR RNA) and then processed by Cas6f to generate crRNAs. Furthermore, the formation of Cas protein complexes has been implicated in both the interference and acquisition stages of defence. In the present paper, we discuss the development of tightly controlled 'programmable' CRISPR arrays as tools to investigate CRISPR-Cas function and the effects of chromosomal targeting. Finally, we address how chromosomal targeting by CRISPR-Cas can cause large-scale genome deletions, which can ultimately influence bacterial evolution and pathogenicity.

  1. Nanosatellite Launch Adapter System (NLAS)

    NASA Technical Reports Server (NTRS)

    Yost, Bruce D.; Hines, John W.; Agasid, Elwood F.; Buckley, Steven J.

    2010-01-01

    The utility of small spacecraft based on the University cubesat standard is becoming evident as more and more agencies and organizations are launching or planning to include nanosatellites in their mission portfolios. Cubesats are typically launched as secondary spacecraft in enclosed, containerized deployers such as the CalPoly Poly Picosat Orbital Deployer (P-POD) system. The P-POD allows for ease of integration and significantly reduces the risk exposure to the primary spacecraft and mission. NASA/ARC and the Operationally Responsive Space office are collaborating to develop a Nanosatellite Launch Adapter System (NLAS), which can accommodate multiple cubesat or cubesat-derived spacecraft on a single launch vehicle. NLAS is composed of the adapter structure, P-POD or similar spacecraft dispensers, and a sequencer/deployer system. This paper describes the NLAS system and it s future capabilities, and also provides status on the system s development and potential first use in space.

  2. Creating Genome Modifications in C. elegans Using the CRISPR/Cas9 System.

    PubMed

    Calarco, John A; Friedland, Ari E

    2015-01-01

    The clustered, regularly interspaced, short, palindromic repeat (CRISPR)-associated (CAS) nuclease Cas9 has been used in many organisms to generate specific mutations and transgene insertions. Here we describe a method using the S. pyogenes Cas9 in C. elegans that provides a convenient and effective approach for making heritable changes to the worm genome.

  3. Scarless Cas9 Assisted Recombineering (no-SCAR) in Escherichia coli, an Easy-to-Use System for Genome Editing.

    PubMed

    Reisch, Christopher R; Prather, Kristala L J

    2017-01-05

    The discovery and development of genome editing systems that leverage the site-specific DNA endonuclease system CRISPR/Cas9 has fundamentally changed the ease and speed of genome editing in many organisms. In eukaryotes, the CRISPR/Cas9 system utilizes a "guide" RNA to enable the Cas9 nuclease to make a double-strand break at a particular genome locus, which is repaired by non-homologous end joining (NHEJ) repair enzymes, often generating random mutations in the process. A specific alteration of the target genome can also be generated by supplying a DNA template in vivo with a desired mutation, which is incorporated by homology-directed repair. However, E. coli lacks robust systems for double-strand break repair. Thus, in contrast to eukaryotes, targeting E. coli chromosomal DNA with Cas9 causes cell death. However, Cas9-mediated killing of bacteria can be exploited to select against cells with a specified genotype within a mixed population. In combination with the well described λ-Red system for recombination in E. coli, we created a highly efficient system for marker-free and scarless genome editing. © 2017 by John Wiley & Sons, Inc.

  4. Gene editing in mouse zygotes using the CRISPR/Cas9 system.

    PubMed

    Wefers, Benedikt; Bashir, Sanum; Rossius, Jana; Wurst, Wolfgang; Kühn, Ralf

    2017-03-02

    The generation of targeted mouse mutants is a key technology for biomedical research. Using the CRISPR/Cas9 system for induction of targeted double-strand breaks, gene editing can be performed in a single step directly in mouse zygotes. This article covers the design of knockout and knockin alleles, preparation of reagents, microinjection or electroporation of zygotes and the genotyping of pups derived from gene editing projects. In addition we include a section for the control of experimental settings by targeting the Rosa26 locus and PCR based genotyping of blastocysts.

  5. CRISPR-Cas immunity in prokaryotes.

    PubMed

    Marraffini, Luciano A

    2015-10-01

    Prokaryotic organisms are threatened by a large array of viruses and have developed numerous defence strategies. Among these, only clustered, regularly interspaced short palindromic repeat (CRISPR)-Cas systems provide adaptive immunity against foreign elements. Upon viral injection, a small sequence of the viral genome, known as a spacer, is integrated into the CRISPR locus to immunize the host cell. Spacers are transcribed into small RNA guides that direct the cleavage of the viral DNA by Cas nucleases. Immunization through spacer acquisition enables a unique form of evolution whereby a population not only rapidly acquires resistance to its predators but also passes this resistance mechanism vertically to its progeny.

  6. A Robust CRISPR/Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.

    PubMed

    Ma, Xingliang; Zhang, Qunyu; Zhu, Qinlong; Liu, Wei; Chen, Yan; Qiu, Rong; Wang, Bin; Yang, Zhongfang; Li, Heying; Lin, Yuru; Xie, Yongyao; Shen, Rongxin; Chen, Shuifu; Wang, Zhi; Chen, Yuanling; Guo, Jingxin; Chen, Letian; Zhao, Xiucai; Dong, Zhicheng; Liu, Yao-Guang

    2015-08-01

    CRISPR/Cas9 genome targeting systems have been applied to a variety of species. However, most CRISPR/Cas9 systems reported for plants can only modify one or a few target sites. Here, we report a robust CRISPR/Cas9 vector system, utilizing a plant codon optimized Cas9 gene, for convenient and high-efficiency multiplex genome editing in monocot and dicot plants. We designed PCR-based procedures to rapidly generate multiple sgRNA expression cassettes, which can be assembled into the binary CRISPR/Cas9 vectors in one round of cloning by Golden Gate ligation or Gibson Assembly. With this system, we edited 46 target sites in rice with an average 85.4% rate of mutation, mostly in biallelic and homozygous status. We reasoned that about 16% of the homozygous mutations in rice were generated through the non-homologous end-joining mechanism followed by homologous recombination-based repair. We also obtained uniform biallelic, heterozygous, homozygous, and chimeric mutations in Arabidopsis T1 plants. The targeted mutations in both rice and Arabidopsis were heritable. We provide examples of loss-of-function gene mutations in T0 rice and T1 Arabidopsis plants by simultaneous targeting of multiple (up to eight) members of a gene family, multiple genes in a biosynthetic pathway, or multiple sites in a single gene. This system has provided a versatile toolbox for studying functions of multiple genes and gene families in plants for basic research and genetic improvement.

  7. Architecture for Adaptive Intelligent Systems

    NASA Technical Reports Server (NTRS)

    Hayes-Roth, Barbara

    1993-01-01

    We identify a class of niches to be occupied by 'adaptive intelligent systems (AISs)'. In contrast with niches occupied by typical AI agents, AIS niches present situations that vary dynamically along several key dimensions: different combinations of required tasks, different configurations of available resources, contextual conditions ranging from benign to stressful, and different performance criteria. We present a small class hierarchy of AIS niches that exhibit these dimensions of variability and describe a particular AIS niche, ICU (intensive care unit) patient monitoring, which we use for illustration throughout the paper. We have designed and implemented an agent architecture that supports all of different kinds of adaptation by exploiting a single underlying theoretical concept: An agent dynamically constructs explicit control plans to guide its choices among situation-triggered behaviors. We illustrate the architecture and its support for adaptation with examples from Guardian, an experimental agent for ICU monitoring.

  8. The Limits to Adaptation; A Systems Approach

    EPA Science Inventory

    The Limits to Adaptation: A Systems Approach. The ability to adapt to climate change is delineated by capacity thresholds, after which climate damages begin to overwhelm the adaptation response. Such thresholds depend upon physical properties (natural processes and engineering...

  9. Truth in Complex Adaptive Systems Models Should BE Based on Proof by Constructive Verification

    NASA Astrophysics Data System (ADS)

    Shipworth, David

    It is argued that the truth status of emergent properties of complex adaptive systems models should be based on an epistemology of proof by constructive verification and therefore on the ontological axioms of a non-realist logical system such as constructivism or intuitionism. `Emergent' properties of complex adaptive systems (CAS) models create particular epistemological and ontological challenges. These challenges bear directly on current debates in the philosophy of mathematics and in theoretical computer science. CAS research, with its emphasis on computer simulation, is heavily reliant on models which explore the entailments of Formal Axiomatic Systems (FAS). The incompleteness results of Gödel, the incomputability results of Turing, and the Algorithmic Information Theory results of Chaitin, undermine a realist (platonic) truth model of emergent properties. These same findings support the hegemony of epistemology over ontology and point to alternative truth models such as intuitionism, constructivism and quasi-empiricism.

  10. Rapid and Efficient Genome Editing in Staphylococcus aureus by Using an Engineered CRISPR/Cas9 System.

    PubMed

    Chen, Weizhong; Zhang, Yifei; Yeo, Won-Sik; Bae, Taeok; Ji, Quanjiang

    2017-03-02

    Staphylococcus aureus, a major human pathogen, has been the cause of serious infectious diseases with a high mortality rate. Although genetics is a key means to study S. aureus physiology, such as drug resistance and pathogenesis, genetic manipulation in S. aureus is always time-consuming and labor-intensive. Here we report a CRISPR/Cas9 system (pCasSA) for rapid and efficient genome editing, including gene deletion, insertion, and single-base substitution mutation in S. aureus. The designed pCasSA system is amenable to the assembly of spacers and repair arms by Golden Gate assembly and Gibson assembly, respectively, enabling rapid construction of the plasmids for editing. We further engineered the pCasSA system to be an efficient transcription inhibition system for gene knockdown and possible genome-wide screening. The development of the CRISPR/Cas9-mediated genome editing and transcription inhibition tools will dramatically accelerate drug-target exploration and drug development.

  11. Stakeholder Collaboration in Air Force Acquisition: Adaptive Design Using System Representations

    DTIC Science & Technology

    2003-06-01

    that CAS principles are important considerations to understand inter-organizational interactions and adaptability. The research has also expanded...fielded systems. Therefore, the design phase is a unique time when it is possible to visualize and interact with the system when changes are still...affordable. 12 Acknowledgements I want to recognize a number of individuals who helped make this dissertation possible . I start with my committee

  12. Survival and Evolution of CRISPR–Cas System in Prokaryotes and Its Applications

    PubMed Central

    Shabbir, Muhammad Abu Bakr; Hao, Haihong; Shabbir, Muhammad Zubair; Hussain, Hafiz Iftikhar; Iqbal, Zahid; Ahmed, Saeed; Sattar, Adeel; Iqbal, Mujahid; Li, Jun; Yuan, Zonghui

    2016-01-01

    Prokaryotes have developed numerous innate immune mechanisms in order to fend off bacteriophage or plasmid attack. One of these immune systems is clustered regularly interspaced short palindromic repeats (CRISPR). CRISPR-associated proteins play a key role in survival of prokaryotes against invaders, as these systems cleave DNA of foreign genetic elements. Beyond providing immunity, these systems have significant impact in altering the bacterial physiology in term of its virulence and pathogenicity, as well as evolution. Also, due to their diverse nature of functionality, cas9 endoribonuclease can be easily reprogrammed with the help of guide RNAs, showing unprecedented potential and significance for gene editing in treating genetic diseases. Here, we also discuss the use of NgAgo–gDNA system in genome editing of human cells. PMID:27725818

  13. [CRISPR/Cas9-based genome editing systems and the analysis of targeted genome mutations in plants].

    PubMed

    Xingliang, Ma; Yaoguang, Liu

    2016-02-01

    Targeted genomic editing technologies use programmable DNA nucleases to cleave genomic target sites, thus inducing targeted mutations in the genomes. The newly prevailed clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system that consists of the Cas9 nuclease and single guide RNA (sgRNA) has the advantages of simplicity and high efficiency as compared to other programmable DNA nuclease systems such as zinc finger nucleases (ZFNs) and transcription activator like effector nucleases (TALENs). Currently, a number of cases have been reported on the application of the CRISPR/Cas9 genomic editing technology in plants. In this review, we summarize the strategies for preparing the Cas9 and sgRNA expression constructs, the transformation method for obtaining targeted mutations, the efficiency and features of the resulting mutations and the methods for detecting or genotyping of the mutation sites. We also discuss the existing problems and perspectives of CRISPR/Cas9-based genomic editing in plants.

  14. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System*

    PubMed Central

    Stachler, Aris-Edda; Marchfelder, Anita

    2016-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon. PMID:27226589

  15. Gene Repression in Haloarchaea Using the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-Cas I-B System.

    PubMed

    Stachler, Aris-Edda; Marchfelder, Anita

    2016-07-15

    The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon.

  16. Making Intelligent Systems Adaptive. (Revision)

    DTIC Science & Technology

    1988-10-01

    eventually produce solutions. BY contrast, human beinge and other intelligent animls continuously adapt to the demands and opportunities presented by a...such as monitoring critically ill medical patients or controlling a manufacturing process. Following the model set by human intelligence, we define...signs probabilistically, using a belief network, as well as from first principles, using explicit models of system structure and function. Concurrent

  17. Fanconi Anemia Gene Editing by the CRISPR/Cas9 System

    PubMed Central

    Osborn, Mark J.; Gabriel, Richard; Webber, Beau R.; DeFeo, Anthony P.; McElroy, Amber N.; Jarjour, Jordan; Starker, Colby G.; Wagner, John E.; Joung, J. Keith; Voytas, Daniel F.; von Kalle, Christof; Schmidt, Manfred; Blazar, Bruce R.

    2015-01-01

    Abstract Genome engineering with designer nucleases is a rapidly progressing field, and the ability to correct human gene mutations in situ is highly desirable. We employed fibroblasts derived from a patient with Fanconi anemia as a model to test the ability of the clustered regularly interspaced short palindromic repeats/Cas9 nuclease system to mediate gene correction. We show that the Cas9 nuclease and nickase each resulted in gene correction, but the nickase, because of its ability to preferentially mediate homology-directed repair, resulted in a higher frequency of corrected clonal isolates. To assess the off-target effects, we used both a predictive software platform to identify intragenic sequences of homology as well as a genome-wide screen utilizing linear amplification-mediated PCR. We observed no off-target activity and show RNA-guided endonuclease candidate sites that do not possess low sequence complexity function in a highly specific manner. Collectively, we provide proof of principle for precision genome editing in Fanconi anemia, a DNA repair-deficient human disorder. PMID:25545896

  18. Programmable RNA shredding by the type III-A CRISPR-Cas system of Streptococcus thermophilus.

    PubMed

    Tamulaitis, Gintautas; Kazlauskiene, Migle; Manakova, Elena; Venclovas, Česlovas; Nwokeoji, Alison O; Dickman, Mark J; Horvath, Philippe; Siksnys, Virginijus

    2014-11-20

    Immunity against viruses and plasmids provided by CRISPR-Cas systems relies on a ribonucleoprotein effector complex that triggers the degradation of invasive nucleic acids (NA). Effector complexes of type I (Cascade) and II (Cas9-dual RNA) target foreign DNA. Intriguingly, the genetic evidence suggests that the type III-A Csm complex targets DNA, whereas biochemical data show that the type III-B Cmr complex cleaves RNA. Here we aimed to investigate NA specificity and mechanism of CRISPR interference for the Streptococcus thermophilus Csm (III-A) complex (StCsm). When expressed in Escherichia coli, two complexes of different stoichiometry copurified with 40 and 72 nt crRNA species, respectively. Both complexes targeted RNA and generated multiple cuts at 6 nt intervals. The Csm3 protein, present in multiple copies in both Csm complexes, acts as endoribonuclease. In the heterologous E. coli host, StCsm restricts MS2 RNA phage in a Csm3 nuclease-dependent manner. Thus, our results demonstrate that the type III-A StCsm complex guided by crRNA targets RNA and not DNA.

  19. Genome engineering via TALENs and CRISPR/Cas9 systems: challenges and perspectives.

    PubMed

    Mahfouz, Magdy M; Piatek, Agnieszka; Stewart, Charles Neal

    2014-10-01

    The ability to precisely modify genome sequence and regulate gene expression patterns in a site-specific manner holds much promise in plant biotechnology. Genome-engineering technologies that enable such highly specific and efficient modification are advancing with unprecedented pace. Transcription activator-like effectors (TALEs) provide customizable DNA-binding modules designed to bind to any sequence of interest. Thus, TALEs have been used as a DNA targeting module fused to functional domains for a variety of targeted genomic and epigenomic modifications. TALE nucleases (TALENs) have been used with much success across eukaryotic species to edit genomes. Recently, clustered regularly interspaced palindromic repeats (CRISPRs) that are used as guide RNAs for Cas9 nuclease-specific digestion has been introduced as a highly efficient DNA-targeting platform for genome editing and regulation. Here, we review the discovery, development and limitations of TALENs and CRIPSR/Cas9 systems as genome-engineering platforms in plants. We discuss the current questions, potential improvements and the development of the next-generation genome-editing platforms with an emphasis on producing designer plants to address the needs of agriculture and basic plant biology.

  20. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system.

    PubMed

    Belhaj, Khaoula; Chaparro-Garcia, Angela; Kamoun, Sophien; Nekrasov, Vladimir

    2013-10-11

    Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants.

  1. Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas system

    PubMed Central

    2013-01-01

    Targeted genome engineering (also known as genome editing) has emerged as an alternative to classical plant breeding and transgenic (GMO) methods to improve crop plants. Until recently, available tools for introducing site-specific double strand DNA breaks were restricted to zinc finger nucleases (ZFNs) and TAL effector nucleases (TALENs). However, these technologies have not been widely adopted by the plant research community due to complicated design and laborious assembly of specific DNA binding proteins for each target gene. Recently, an easier method has emerged based on the bacterial type II CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) immune system. The CRISPR/Cas system allows targeted cleavage of genomic DNA guided by a customizable small noncoding RNA, resulting in gene modifications by both non-homologous end joining (NHEJ) and homology-directed repair (HDR) mechanisms. In this review we summarize and discuss recent applications of the CRISPR/Cas technology in plants. PMID:24112467

  2. Targeted Gene Deletion Using DNA-Free RNA-Guided Cas9 Nuclease Accelerates Adaptation of CHO Cells to Suspension Culture.

    PubMed

    Lee, Namil; Shin, JongOh; Park, Jin Hyoung; Lee, Gyun Min; Cho, Suhyung; Cho, Byung-Kwan

    2016-11-18

    Chinese hamster ovary (CHO) cells are the preferred host for the production of a wide array of biopharmaceuticals. Thus, efficient and rational CHO cell line engineering methods have been in high demand to improve quality and productivity. Here, we provide a novel genome engineering platform for increasing desirable phenotypes of CHO cells based upon the integrative protocol of high-throughput RNA sequencing and DNA-free RNA-guided Cas9 (CRISPR associated protein9) nuclease-based genome editing. For commercial production of therapeutic proteins, CHO cells have been adapted for suspension culture in serum-free media, which is highly beneficial with respect to productivity and economics. To engineer CHO cells for rapid adaptation to a suspension culture, we exploited strand-specific RNA-seq to identify genes differentially expressed according to their adaptation trajectory in serum-free media. More than 180 million sequencing reads were generated and mapped to the currently available 109,152 scaffolds of the CHO-K1 genome. We identified significantly downregulated genes according to the adaptation trajectory and then verified their effects using the genome editing method. Growth-based screening and targeted amplicon sequencing revealed that the functional deletions of Igfbp4 and AqpI gene accelerate suspension adaptation of CHO-K1 cells. The availability of this strand-specific transcriptome sequencing and DNA-free RNA-guided Cas9 nuclease mediated genome editing facilitates the rational design of the CHO cell genome for efficient production of high quality therapeutic proteins.

  3. People at the centre of complex adaptive health systems reform.

    PubMed

    Sturmberg, Joachim P; O'Halloran, Diana M; Martin, Carmel M

    2010-10-18

    Health systems are increasingly recognised to be complex adaptive systems (CASs), functionally characterised by their continuing and dynamic adaptation in response to core system drivers, or attractors. The core driver for our health system (and for the health reform strategies intended to achieve it) should clearly be the improvement of people's health - the personal experience of health, regardless of organic abnormalities; we contend that a patient-centred health system requires flexible localised decision making and resource use. The prevailing trend is to use disease protocols, financial management strategies and centralised control of siloed programs to manage our health system. This strategy is suggested to be fatally flawed, as: people's health and health experience as core system drivers are inevitably pre-empted by centralised and standardised strategies; the context specificity of personal experience and the capacity of local systems are overlooked; and in line with CAS patterns and characteristics, these strategies will lead to "unintended" consequences on all parts of the system. In Australia, there is still the time and opportunity for health system redesign that truly places people and their health at the core of the system.

  4. Nuclease activity of Legionella pneumophila Cas2 promotes intracellular infection of amoebal host cells.

    PubMed

    Gunderson, Felizza F; Mallama, Celeste A; Fairbairn, Stephanie G; Cianciotto, Nicholas P

    2015-03-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts.

  5. Nuclease Activity of Legionella pneumophila Cas2 Promotes Intracellular Infection of Amoebal Host Cells

    PubMed Central

    Gunderson, Felizza F.; Mallama, Celeste A.; Fairbairn, Stephanie G.

    2014-01-01

    Legionella pneumophila, the primary agent of Legionnaires' disease, flourishes in both natural and man-made environments by growing in a wide variety of aquatic amoebae. Recently, we determined that the Cas2 protein of L. pneumophila promotes intracellular infection of Acanthamoeba castellanii and Hartmannella vermiformis, the two amoebae most commonly linked to cases of disease. The Cas2 family of proteins is best known for its role in the bacterial and archeal clustered regularly interspaced short palindromic repeat (CRISPR)–CRISPR-associated protein (Cas) system that constitutes a form of adaptive immunity against phage and plasmid. However, the infection event mediated by L. pneumophila Cas2 appeared to be distinct from this function, because cas2 mutants exhibited infectivity defects in the absence of added phage or plasmid and since mutants lacking the CRISPR array or any one of the other cas genes were not impaired in infection ability. We now report that the Cas2 protein of L. pneumophila has both RNase and DNase activities, with the RNase activity being more pronounced. By characterizing a catalytically deficient version of Cas2, we determined that nuclease activity is critical for promoting infection of amoebae. Also, introduction of Cas2, but not its catalytic mutant form, into a strain of L. pneumophila that naturally lacks a CRISPR-Cas locus caused that strain to be 40- to 80-fold more infective for amoebae, unequivocally demonstrating that Cas2 facilitates the infection process independently of any other component encoded within the CRISPR-Cas locus. Finally, a cas2 mutant was impaired for infection of Willaertia magna but not Naegleria lovaniensis, suggesting that Cas2 promotes infection of most but not all amoebal hosts. PMID:25547789

  6. Certification Considerations for Adaptive Systems

    NASA Technical Reports Server (NTRS)

    Bhattacharyya, Siddhartha; Cofer, Darren; Musliner, David J.; Mueller, Joseph; Engstrom, Eric

    2015-01-01

    Advanced capabilities planned for the next generation of aircraft, including those that will operate within the Next Generation Air Transportation System (NextGen), will necessarily include complex new algorithms and non-traditional software elements. These aircraft will likely incorporate adaptive control algorithms that will provide enhanced safety, autonomy, and robustness during adverse conditions. Unmanned aircraft will operate alongside manned aircraft in the National Airspace (NAS), with intelligent software performing the high-level decision-making functions normally performed by human pilots. Even human-piloted aircraft will necessarily include more autonomy. However, there are serious barriers to the deployment of new capabilities, especially for those based upon software including adaptive control (AC) and artificial intelligence (AI) algorithms. Current civil aviation certification processes are based on the idea that the correct behavior of a system must be completely specified and verified prior to operation. This report by Rockwell Collins and SIFT documents our comprehensive study of the state of the art in intelligent and adaptive algorithms for the civil aviation domain, categorizing the approaches used and identifying gaps and challenges associated with certification of each approach.

  7. Targeted Mutagenesis in Rice Using TALENs and the CRISPR/Cas9 System.

    PubMed

    Endo, Masaki; Nishizawa-Yokoi, Ayako; Toki, Seiichi

    2016-01-01

    Sequence-specific nucleases (SSNs), such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9) system, are powerful tools for understanding gene function and for developing novel traits in plants. In plant species for which transformation and regeneration systems using protoplasts are not yet established, direct delivery to nuclei of SSNs either in the form of RNA or protein is difficult. Thus, Agrobacterium-mediated transformation of SSN expression constructs in cultured cells is a practical means of delivering targeted mutagenesis in some plant species including rice. Because targeted mutagenesis occurs stochastically in transgenic cells and SSN-mediated targeted mutagenesis often leads to no selectable phenotype, identification of highly mutated cell lines is a critical step in obtaining regenerated plants with desired mutations.

  8. Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems

    PubMed Central

    Yasue, Akihiro; Mitsui, Silvia Naomi; Watanabe, Takahito; Sakuma, Tetsushi; Oyadomari, Seiichi; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro; Tanaka, Eiji

    2014-01-01

    Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice. PMID:25027812

  9. Highly efficient targeted mutagenesis in one-cell mouse embryos mediated by the TALEN and CRISPR/Cas systems.

    PubMed

    Yasue, Akihiro; Mitsui, Silvia Naomi; Watanabe, Takahito; Sakuma, Tetsushi; Oyadomari, Seiichi; Yamamoto, Takashi; Noji, Sumihare; Mito, Taro; Tanaka, Eiji

    2014-07-16

    Since the establishment of embryonic stem (ES) cell lines, the combined use of gene targeting with homologous recombination has aided in elucidating the functions of various genes. However, the ES cell technique is inefficient and time-consuming. Recently, two new gene-targeting technologies have been developed: the transcription activator-like effector nuclease (TALEN) system, and the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system. In addition to aiding researchers in solving conventional problems, these technologies can be used to induce site-specific mutations in various species for which ES cells have not been established. Here, by targeting the Fgf10 gene through RNA microinjection in one-cell mouse embryos with the TALEN and CRISPR/Cas systems, we produced the known limb-defect phenotypes of Fgf10-deficient embryos at the F0 generation. Compared to the TALEN system, the CRISPR/Cas system induced the limb-defect phenotypes with a strikingly higher efficiency. Our results demonstrate that although both gene-targeting technologies are useful, the CRISPR/Cas system more effectively elicits single-step biallelic mutations in mice.

  10. Multiple mechanisms for CRISPR-Cas inhibition by anti-CRISPR proteins.

    PubMed

    Bondy-Denomy, Joseph; Garcia, Bianca; Strum, Scott; Du, Mingjian; Rollins, MaryClare F; Hidalgo-Reyes, Yurima; Wiedenheft, Blake; Maxwell, Karen L; Davidson, Alan R

    2015-10-01

    The battle for survival between bacteria and the viruses that infect them (phages) has led to the evolution of many bacterial defence systems and phage-encoded antagonists of these systems. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPR-associated (cas) genes comprise an adaptive immune system that is one of the most widespread means by which bacteria defend themselves against phages. We identified the first examples of proteins produced by phages that inhibit a CRISPR-Cas system. Here we performed biochemical and in vivo investigations of three of these anti-CRISPR proteins, and show that each inhibits CRISPR-Cas activity through a distinct mechanism. Two block the DNA-binding activity of the CRISPR-Cas complex, yet do this by interacting with different protein subunits, and using steric or non-steric modes of inhibition. The third anti-CRISPR protein operates by binding to the Cas3 helicase-nuclease and preventing its recruitment to the DNA-bound CRISPR-Cas complex. In vivo, this anti-CRISPR can convert the CRISPR-Cas system into a transcriptional repressor, providing the first example-to our knowledge-of modulation of CRISPR-Cas activity by a protein interactor. The diverse sequences and mechanisms of action of these anti-CRISPR proteins imply an independent evolution, and foreshadow the existence of other means by which proteins may alter CRISPR-Cas function.

  11. Adaptive Behaviour Assessment System: Indigenous Australian Adaptation Model (ABAS: IAAM)

    ERIC Educational Resources Information Center

    du Plessis, Santie

    2015-01-01

    The study objectives were to develop, trial and evaluate a cross-cultural adaptation of the Adaptive Behavior Assessment System-Second Edition Teacher Form (ABAS-II TF) ages 5-21 for use with Indigenous Australian students ages 5-14. This study introduced a multiphase mixed-method design with semi-structured and informal interviews, school…

  12. The ERIS adaptive optics system

    NASA Astrophysics Data System (ADS)

    Marchetti, Enrico; Fedrigo, Enrico; Le Louarn, Miska; Madec, Pierre-Yves; Soenke, Christian; Brast, Roland; Conzelmann, Ralf; Delabre, Bernard; Duchateau, Michel; Frank, Christoph; Klein, Barbara; Amico, Paola; Hubin, Norbert; Esposito, Simone; Antichi, Jacopo; Carbonaro, Luca; Puglisi, Alfio; Quirós-Pacheco, Fernando; Riccardi, Armando; Xompero, Marco

    2014-07-01

    The Enhanced Resolution Imager and Spectrograph (ERIS) is the new Adaptive Optics based instrument for ESO's VLT aiming at replacing NACO and SINFONI to form a single compact facility with AO fed imaging and integral field unit spectroscopic scientific channels. ERIS completes the instrument suite at the VLT adaptive telescope. In particular it is equipped with a versatile AO system that delivers up to 95% Strehl correction in K band for science observations up to 5 micron It comprises high order NGS and LGS correction enabling the observation from exoplanets to distant galaxies with a large sky coverage thanks to the coupling of the LGS WFS with the high sensitivity of its visible WFS and the capability to observe in dust embedded environment thanks to its IR low order WFS. ERIS will be installed at the Cassegrain focus of the VLT unit hosting the Adaptive Optics Facility (AOF). The wavefront correction is provided by the AOF deformable secondary mirror while the Laser Guide Star is provided by one of the four launch units of the 4 Laser Guide Star Facility for the AOF. The overall layout of the ERIS AO system is extremely compact and highly optimized: the SPIFFI spectrograph is fed directly by the Cassegrain focus and both the NIX's (IR imager) and SPIFFI's entrance windows work as visible/infrared dichroics. In this paper we describe the concept of the ERIS AO system in detail, starting from the requirements and going through the estimated performance, the opto-mechanical design and the Real-Time Computer design.

  13. ERIS adaptive optics system design

    NASA Astrophysics Data System (ADS)

    Marchetti, Enrico; Le Louarn, Miska; Soenke, Christian; Fedrigo, Enrico; Madec, Pierre-Yves; Hubin, Norbert

    2012-07-01

    The Enhanced Resolution Imager and Spectrograph (ERIS) is the next-generation instrument planned for the Very Large Telescope (VLT) and the Adaptive Optics facility (AOF). It is an AO assisted instrument that will make use of the Deformable Secondary Mirror and the new Laser Guide Star Facility (4LGSF), and it is planned for the Cassegrain focus of the telescope UT4. The project is currently in its Phase A awaiting for approval to continue to the next phases. The Adaptive Optics system of ERIS will include two wavefront sensors (WFS) to maximize the coverage of the proposed sciences cases. The first is a high order 40x40 Pyramid WFS (PWFS) for on axis Natural Guide Star (NGS) observations. The second is a high order 40x40 Shack-Hartmann WFS for single Laser Guide Stars (LGS) observations. The PWFS, with appropriate sub-aperture binning, will serve also as low order NGS WFS in support to the LGS mode with a field of view patrolling capability of 2 arcmin diameter. Both WFSs will be equipped with the very low read-out noise CCD220 based camera developed for the AOF. The real-time reconstruction and control is provided by a SPARTA real-time platform adapted to support both WFS modes. In this paper we will present the ERIS AO system in all its main aspects: opto-mechanical design, real-time computer design, control and calibrations strategy. Particular emphasis will be given to the system performance obtained via dedicated numerical simulations.

  14. Selecting a change and evaluating its impact on the performance of a complex adaptive health care delivery system.

    PubMed

    Boustani, Malaz A; Munger, Stephanie; Gulati, Rajesh; Vogel, Mickey; Beck, Robin A; Callahan, Christopher M

    2010-05-25

    Complexity science suggests that our current health care delivery system acts as a complex adaptive system (CAS). Such systems represent a dynamic and flexible network of individuals who can coevolve with their ever changing environment. The CAS performance fluctuates and its members' interactions continuously change over time in response to the stress generated by its surrounding environment. This paper will review the challenges of intervening and introducing a planned change into a complex adaptive health care delivery system. We explore the role of the "reflective adaptive process" in developing delivery interventions and suggest different evaluation methodologies to study the impact of such interventions on the performance of the entire system. We finally describe the implementation of a new program, the Aging Brain Care Medical Home as a case study of our proposed evaluation process.

  15. Adaptable state based control system

    NASA Technical Reports Server (NTRS)

    Rasmussen, Robert D. (Inventor); Dvorak, Daniel L. (Inventor); Gostelow, Kim P. (Inventor); Starbird, Thomas W. (Inventor); Gat, Erann (Inventor); Chien, Steve Ankuo (Inventor); Keller, Robert M. (Inventor)

    2004-01-01

    An autonomous controller, comprised of a state knowledge manager, a control executor, hardware proxies and a statistical estimator collaborates with a goal elaborator, with which it shares common models of the behavior of the system and the controller. The elaborator uses the common models to generate from temporally indeterminate sets of goals, executable goals to be executed by the controller. The controller may be updated to operate in a different system or environment than that for which it was originally designed by the replacement of shared statistical models and by the instantiation of a new set of state variable objects derived from a state variable class. The adaptation of the controller does not require substantial modification of the goal elaborator for its application to the new system or environment.

  16. Genetic Modification in Human Pluripotent Stem Cells by Homologous Recombination and CRISPR/Cas9 System.

    PubMed

    Xue, Haipeng; Wu, Jianbo; Li, Shenglan; Rao, Mahendra S; Liu, Ying

    2016-01-01

    Genetic modification is an indispensable tool to study gene function in normal development and disease. The recent breakthrough of creating human induced pluripotent stem cells (iPSCs) by defined factors (Takahashi et al., Cell 131:861-872, 2007) provides a renewable source of patient autologous cells that not only retain identical genetic information but also give rise to many cell types of the body including neurons and glia. Meanwhile, the rapid advancement of genome modification tools such as gene targeting by homologous recombination (Capecchi, Nat Rev Genet 6:507-512, 2005) and genome editing tools such as CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system, TALENs (Transcription activator-like effector nucleases), and ZFNs (Zinc finger nucleases) (Wang et al., Cell 153:910-918, 2013; Mali et al., Science 339:823-826, 2013; Hwang et al., Nat Biotechnol 31:227-229, 2013; Friedland et al., Nat Methods 10(8):741-743, 2013; DiCarlo et al., Nucleic Acids Res 41:4336-4343, 2013; Cong et al., Science 339:819-823, 2013) has greatly accelerated the development of human genome manipulation at the molecular level. This chapter describes the protocols for making neural lineage reporter lines using homologous recombination and the CRISPR/Cas system-mediated genome editing, including construction of targeting vectors, guide RNAs, transfection into hPSCs, and selection and verification of successfully targeted clones. This method can be applied to various needs of hPSC genetic engineering at high efficiency and high reliability.

  17. Comparative genomics reveals diversified CRISPR-Cas systems of globally distributed Microcystis aeruginosa, a freshwater bloom-forming cyanobacterium.

    PubMed

    Yang, Chen; Lin, Feibi; Li, Qi; Li, Tao; Zhao, Jindong

    2015-01-01

    Microcystis aeruginosa is one of the most common and dominant bloom-forming cyanobacteria in freshwater lakes around the world. Microcystis cells can produce toxic secondary metabolites, such as microcystins, which are harmful to human health. Two M. aeruginosa strains were isolated from two highly eutrophic lakes in China and their genomes were sequenced. Comparative genomic analysis was performed with the 12 other available M. aeruginosa genomes and closely related unicellular cyanobacterium. Each genome of M. aeruginosa containing at least one clustered regularly interspaced short palindromic repeat (CRISPR) locus and total 71 loci were identified, suggesting it is ubiquitous in M. aeruginosa genomes. In addition to the previously reported subtype I-D cas gene sets, three CAS subtypes I-A, III-A and III-B were identified and characterized in this study. Seven types of CRISPR direct repeat have close association with CAS subtype, confirming that different and specific secondary structures of CRISPR repeats are important for the recognition, binding and process of corresponding cas gene sets. Homology search of the CRISPR spacer sequences provides a history of not only resistance to bacteriophages and plasmids known to be associated with M. aeruginosa, but also the ability to target much more exogenous genetic material in the natural environment. These adaptive and heritable defense mechanisms play a vital role in keeping genomic stability and self-maintenance by restriction of horizontal gene transfer. Maintaining genomic stability and modulating genomic plasticity are both important evolutionary strategies for M. aeruginosa in adaptation and survival in various habitats.

  18. Adenovirus-Mediated Somatic Genome Editing of Pten by CRISPR/Cas9 in Mouse Liver in Spite of Cas9-Specific Immune Responses.

    PubMed

    Wang, Dan; Mou, Haiwei; Li, Shaoyong; Li, Yingxiang; Hough, Soren; Tran, Karen; Li, Jia; Yin, Hao; Anderson, Daniel G; Sontheimer, Erik J; Weng, Zhiping; Gao, Guangping; Xue, Wen

    2015-07-01

    CRISPR/Cas9 derived from the bacterial adaptive immunity pathway is a powerful tool for genome editing, but the safety profiles of in vivo delivered Cas9 (including host immune responses to the bacterial Cas9 protein) have not been comprehensively investigated in model organisms. Nonalcoholic steatohepatitis (NASH) is a prevalent human liver disease characterized by excessive fat accumulation in the liver. In this study, we used adenovirus (Ad) vector to deliver a Streptococcus pyogenes-derived Cas9 system (SpCas9) targeting Pten, a gene involved in NASH and a negative regulator of the PI3K-AKT pathway, in mouse liver. We found that the Ad vector mediated efficient Pten gene editing even in the presence of typical Ad vector-associated immunotoxicity in the liver. Four months after vector infusion, mice receiving the Pten gene-editing Ad vector showed massive hepatomegaly and features of NASH, consistent with the phenotypes following Cre-loxP-induced Pten deficiency in mouse liver. We also detected induction of humoral immunity against SpCas9 and the potential presence of an SpCas9-specific cellular immune response. Our findings provide a strategy to model human liver diseases in mice and highlight the importance considering Cas9-specific immune responses in future translational studies involving in vivo delivery of CRISPR/Cas9.

  19. CRISPR-Cas9-Mediated Genome Editing in Leishmania donovani

    PubMed Central

    Zhang, Wen-Wei

    2015-01-01

    ABSTRACT The prokaryotic CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9, an RNA-guided endonuclease, has been shown to mediate efficient genome editing in a wide variety of organisms. In the present study, the CRISPR-Cas9 system has been adapted to Leishmania donovani, a protozoan parasite that causes fatal human visceral leishmaniasis. We introduced the Cas9 nuclease into L. donovani and generated guide RNA (gRNA) expression vectors by using the L. donovani rRNA promoter and the hepatitis delta virus (HDV) ribozyme. It is demonstrated within that L. donovani mainly used homology-directed repair (HDR) and microhomology-mediated end joining (MMEJ) to repair the Cas9 nuclease-created double-strand DNA break (DSB). The nonhomologous end-joining (NHEJ) pathway appears to be absent in L. donovani. With this CRISPR-Cas9 system, it was possible to generate knockouts without selection by insertion of an oligonucleotide donor with stop codons and 25-nucleotide homology arms into the Cas9 cleavage site. Likewise, we disrupted and precisely tagged endogenous genes by inserting a bleomycin drug selection marker and GFP gene into the Cas9 cleavage site. With the use of Hammerhead and HDV ribozymes, a double-gRNA expression vector that further improved gene-targeting efficiency was developed, and it was used to make precise deletion of the 3-kb miltefosine transporter gene (LdMT). In addition, this study identified a novel single point mutation caused by CRISPR-Cas9 in LdMT (M381T) that led to miltefosine resistance, a concern for the only available oral antileishmanial drug. Together, these results demonstrate that the CRISPR-Cas9 system represents an effective genome engineering tool for L. donovani. PMID:26199327

  20. An efficient genotyping method for genome-modified animals and human cells generated with CRISPR/Cas9 system.

    PubMed

    Zhu, Xiaoxiao; Xu, Yajie; Yu, Shanshan; Lu, Lu; Ding, Mingqin; Cheng, Jing; Song, Guoxu; Gao, Xing; Yao, Liangming; Fan, Dongdong; Meng, Shu; Zhang, Xuewen; Hu, Shengdi; Tian, Yong

    2014-09-19

    The rapid generation of various species and strains of laboratory animals using CRISPR/Cas9 technology has dramatically accelerated the interrogation of gene function in vivo. So far, the dominant approach for genotyping of genome-modified animals has been the T7E1 endonuclease cleavage assay. Here, we present a polyacrylamide gel electrophoresis-based (PAGE) method to genotype mice harboring different types of indel mutations. We developed 6 strains of genome-modified mice using CRISPR/Cas9 system, and utilized this approach to genotype mice from F0 to F2 generation, which included single and multiplexed genome-modified mice. We also determined the maximal detection sensitivity for detecting mosaic DNA using PAGE-based assay as 0.5%. We further applied PAGE-based genotyping approach to detect CRISPR/Cas9-mediated on- and off-target effect in human 293T and induced pluripotent stem cells (iPSCs). Thus, PAGE-based genotyping approach meets the rapidly increasing demand for genotyping of the fast-growing number of genome-modified animals and human cell lines created using CRISPR/Cas9 system or other nuclease systems such as TALEN or ZFN.

  1. Anti-HIV-1 potency of the CRISPR/Cas9 system insufficient to fully inhibit viral replication.

    PubMed

    Ueda, Shuhei; Ebina, Hirotaka; Kanemura, Yuka; Misawa, Naoko; Koyanagi, Yoshio

    2016-07-01

    The range of genome-editing tools has recently been expanded. In particular, an RNA-guided genome-editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)-associated 9 (Cas9) system, has many applications for human diseases. In this study, guide RNA (gRNA) to target gag, pol and a long terminal repeat of HIV-1 was designed and used to generate gRNA-expressing lentiviral vectors. An HIV-1-specific gRNA and Cas9 were stably dually transduced into a highly HIV-1-susceptible human T-cell line and the inhibitory ability of the anti-HIV-1 CRISPR/Cas9 lentiviral vector assessed. Although clear inhibition of the early phase of HIV-1 infection was observed, as evaluated by a VSV-G-pseudotyped HIV-1 reporter system, the anti-HIV-1 potency in multiple rounds of wild type (WT) viral replication was insufficient, either because of generation of resistant viruses or overcoming of the activity of the WT virus. Thus, there are potential difficulties that must be addressed when considering anti-HIV-1 treatment with the CRISPR/Cas9 system alone.

  2. A newly discovered Bordetella species carries a transcriptionally active CRISPR-Cas with a small Cas9 endonuclease

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The Cas9 endonuclease of the Type II-a clustered regularly interspersed short palindromic repeats (CRISPR), of Streptococcus pyogenes (SpCas9) has been adapted as a widely used tool for genome editing and genome engineering. Herein, we describe a gene encoding a novel Cas9 ortholog (BpsuCas9) and th...

  3. V773 Cas, QS Aql, and BR Ind: Eclipsing Binaries as Parts of Multiple Systems

    NASA Astrophysics Data System (ADS)

    Zasche, P.; Juryšek, J.; Nemravová, J.; Uhlař, R.; Svoboda, P.; Wolf, M.; Hoňková, K.; Mašek, M.; Prouza, M.; Čechura, J.; Korčáková, D.; Šlechta, M.

    2017-01-01

    Eclipsing binaries remain crucial objects for our understanding of the universe. In particular, those that are components of multiple systems can help us solve the problem of the formation of these systems. Analysis of the radial velocities together with the light curve produced for the first time precise physical parameters of the components of the multiple systems V773 Cas, QS Aql, and BR Ind. Their visual orbits were also analyzed, which resulted in slightly improved orbital elements. What is typical for all these systems is that their most dominant source is the third distant component. The system V773 Cas consists of two similar G1-2V stars revolving in a circular orbit and a more distant component of the A3V type. Additionally, the improved value of parallax was calculated to be 17.6 mas. Analysis of QS Aql resulted in the following: the inner eclipsing pair is composed of B6V and F1V stars, and the third component is of about the B6 spectral type. The outer orbit has high eccentricity of about 0.95, and observations near its upcoming periastron passage between the years 2038 and 2040 are of high importance. Also, the parallax of the system was derived to be about 2.89 mas, moving the star much closer to the Sun than originally assumed. The system BR Ind was found to be a quadruple star consisting of two eclipsing K dwarfs orbiting each other with a period of 1.786 days; the distant component is a single-lined spectroscopic binary with an orbital period of about 6 days. Both pairs are moving around each other on their 148 year orbit. Based on observations collected at the European Organisation for Astronomical Research in the Southern Hemisphere under ESO programs 091.D-0122(A), 094.A-9029(D), 095.A-9032(A), and 096.A-9039(A) and also on data from the 2 m telescope at the Ondřejov observatory in the Czech Republic

  4. The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation.

    PubMed

    Zhang, Hui; Zhang, Jinshan; Wei, Pengliang; Zhang, Botao; Gou, Feng; Feng, Zhengyan; Mao, Yanfei; Yang, Lan; Zhang, Heng; Xu, Nanfei; Zhu, Jian-Kang

    2014-08-01

    The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9-induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9-induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large-scale off-targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off-target sites of a large number of T0 plants, low-frequency mutations were found in only one off-target site where the sequence had 1-bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.

  5. Adaptive diagnosis of the bilinear mechanical systems

    NASA Astrophysics Data System (ADS)

    Gelman, L.; Gorpinich, S.; Thompson, C.

    2009-07-01

    A generic adaptive approach is proposed for diagnosis of the bilinear mechanical systems. The approach adapts the free oscillation method for bilinearity diagnosis of mechanical systems. The expediency of the adaptation is proved for a recognition feature, the decrement of the free oscillations. The developed adaptation consists of variation of the adaptive likelihood ratio of the decrement with variation of the resonance frequency of the bilinear system. It is shown that in the cases of the frequency-independent and the frequency-dependent internal damping, the adaptation is expedient. To investigate effectiveness of the adaptation in these cases, a numerical simulation was carried out. The simulation results show that use of the adaptation increases the total probability of the correct diagnosis of system bilinearity.

  6. High-Throughput Silencing Using the CRISPR-Cas9 System: A Review of the Benefits and Challenges.

    PubMed

    Wade, Mark

    2015-09-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system has been seized upon with a fervor enjoyed previously by small interfering RNA (siRNA) and short hairpin RNA (shRNA) technologies and has enormous potential for high-throughput functional genomics studies. The decision to use this approach must be balanced with respect to adoption of existing platforms versus awaiting the development of more "mature" next-generation systems. Here, experience from siRNA and shRNA screening plays an important role, as issues such as targeting efficiency, pooling strategies, and off-target effects with those technologies are already framing debates in the CRISPR field. CRISPR/Cas can be exploited not only to knockout genes but also to up- or down-regulate gene transcription-in some cases in a multiplex fashion. This provides a powerful tool for studying the interaction among multiple signaling cascades in the same genetic background. Furthermore, the documented success of CRISPR/Cas-mediated gene correction (or the corollary, introduction of disease-specific mutations) provides proof of concept for the rapid generation of isogenic cell lines for high-throughput screening. In this review, the advantages and limitations of CRISPR/Cas are discussed and current and future applications are highlighted. It is envisaged that complementarities between CRISPR, siRNA, and shRNA will ensure that all three technologies remain critical to the success of future functional genomics projects.

  7. CRISPR Primer Designer: Design primers for knockout and chromosome imaging CRISPR-Cas system.

    PubMed

    Yan, Meng; Zhou, Shi-Rong; Xue, Hong-Wei

    2015-07-01

    The clustered regularly interspaced short palindromic repeats (CRISPR)-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user-friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present CRISPR Primer Designer for researchers to design primers for CRISPR applications. The program has a user-friendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRISPR-Cas system-based chromosome imaging system.

  8. Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System

    PubMed Central

    Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S.; Kim, Dong H.; Deng, Wenbin

    2015-01-01

    Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼33% correctly targeted clones) compared to conventional targeting protocol (∼3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations. PMID:26414932

  9. Human Induced Pluripotent Stem Cell NEUROG2 Dual Knockin Reporter Lines Generated by the CRISPR/Cas9 System.

    PubMed

    Li, Shenglan; Xue, Haipeng; Wu, Jianbo; Rao, Mahendra S; Kim, Dong H; Deng, Wenbin; Liu, Ying

    2015-12-15

    Human induced pluripotent stem cell (hiPSC) technologies are powerful tools for modeling development and disease, drug screening, and regenerative medicine. Faithful gene targeting in hiPSCs greatly facilitates these applications. We have developed a fast and precise clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) technology-based method and obtained fluorescent protein and antibiotic resistance dual knockin reporters in hiPSC lines for neurogenin2 (NEUROG2), an important proneural transcription factor. Gene targeting efficiency was greatly improved in CRISPR/Cas9-mediated homology directed recombination (∼ 33% correctly targeted clones) compared to conventional targeting protocol (∼ 3%) at the same locus. No off-target events were detected. In addition, taking the advantage of the versatile applications of the CRISPR/Cas9 system, we designed transactivation components to transiently induce NEUROG2 expression, which helps identify transcription factor binding sites and trans-regulation regions of human NEUROG2. The strategy of using CRISPR/Cas9 genome editing coupled with fluorescence-activated cell sorting of neural progenitor cells in a knockin lineage hiPSC reporter platform might be broadly applicable in other stem cell derivatives and subpopulations.

  10. Waking up dormant tumor suppressor genes with zinc fingers, TALEs and the CRISPR/dCas9 system

    PubMed Central

    Garcia-Bloj, Benjamin; Moses, Colette; Sgro, Agustin; Plani-Lam, Janice; Arooj, Mahira; Duffy, Ciara; Thiruvengadam, Shreyas; Sorolla, Anabel; Rashwan, Rabab; Mancera, Ricardo L.; Leisewitz, Andrea; Swift-Scanlan, Theresa; Corvalan, Alejandro H.; Blancafort, Pilar

    2016-01-01

    The aberrant epigenetic silencing of tumor suppressor genes (TSGs) plays a major role during carcinogenesis and regaining these dormant functions by engineering of sequence-specific epigenome editing tools offers a unique opportunity for targeted therapies. However, effectively normalizing the expression and regaining tumor suppressive functions of silenced TSGs by artificial transcription factors (ATFs) still remains a major challenge. Herein we describe novel combinatorial strategies for the potent reactivation of two class II TSGs, MASPIN and REPRIMO, in cell lines with varying epigenetic states, using the CRISPR/dCas9 associated system linked to a panel of effector domains (VP64, p300, VPR and SAM complex), as well as with protein-based ATFs, Zinc Fingers and TALEs. We found that co-delivery of multiple effector domains using a combination of CRISPR/dCas9 and TALEs or SAM complex maximized activation in highly methylated promoters. In particular, CRISPR/dCas9 VPR with SAM upregulated MASPIN mRNA (22,145-fold change) in H157 lung cancer cells, with accompanying re-expression of MASPIN protein, which led to a concomitant inhibition of cell proliferation and induction of apoptotic cell death. Consistently, CRISPR/dCas9 VP64 with SAM upregulated REPRIMO (680-fold change), which led to phenotypic reprogramming in AGS gastric cancer cells. Altogether, our results outlined novel sequence-specific, combinatorial epigenome editing approaches to reactivate highly methylated TSGs as a promising therapy for cancer and other diseases. PMID:27528034

  11. Precise in-frame integration of exogenous DNA mediated by CRISPR/Cas9 system in zebrafish.

    PubMed

    Hisano, Yu; Sakuma, Tetsushi; Nakade, Shota; Ohga, Rie; Ota, Satoshi; Okamoto, Hitoshi; Yamamoto, Takashi; Kawahara, Atsuo

    2015-03-05

    The CRISPR/Cas9 system provides a powerful tool for genome editing in various model organisms, including zebrafish. The establishment of targeted gene-disrupted zebrafish (knockouts) is readily achieved by CRISPR/Cas9-mediated genome modification. Recently, exogenous DNA integration into the zebrafish genome via homology-independent DNA repair was reported, but this integration contained various mutations at the junctions of genomic and integrated DNA. Thus, precise genome modification into targeted genomic loci remains to be achieved. Here, we describe efficient, precise CRISPR/Cas9-mediated integration using a donor vector harbouring short homologous sequences (10-40 bp) flanking the genomic target locus. We succeeded in integrating with high efficiency an exogenous mCherry or eGFP gene into targeted genes (tyrosinase and krtt1c19e) in frame. We found the precise in-frame integration of exogenous DNA without backbone vector sequences when Cas9 cleavage sites were introduced at both sides of the left homology arm, the eGFP sequence and the right homology arm. Furthermore, we confirmed that this precise genome modification was heritable. This simple method enables precise targeted gene knock-in in zebrafish.

  12. Protecting genome integrity during CRISPR immune adaptation.

    PubMed

    Wright, Addison V; Doudna, Jennifer A

    2016-10-01

    Bacterial CRISPR-Cas systems include genomic arrays of short repeats flanking foreign DNA sequences and provide adaptive immunity against viruses. Integration of foreign DNA must occur specifically to avoid damaging the genome or the CRISPR array, but surprisingly promiscuous activity occurs in vitro. Here we reconstituted full-site DNA integration and show that the Streptococcus pyogenes type II-A Cas1-Cas2 integrase maintains specificity in part through limitations on the second integration step. At non-CRISPR sites, integration stalls at the half-site intermediate, thereby enabling reaction reversal. S. pyogenes Cas1-Cas2 is highly specific for the leader-proximal repeat and recognizes the repeat's palindromic ends, thus fitting a model of independent recognition by distal Cas1 active sites. These findings suggest that DNA-insertion sites are less common than suggested by previous work, thereby preventing toxicity during CRISPR immune adaptation and maintaining host genome integrity.

  13. Understanding pathways for scaling up health services through the lens of complex adaptive systems.

    PubMed

    Paina, Ligia; Peters, David H

    2012-08-01

    Despite increased prominence and funding of global health initiatives, efforts to scale up health services in developing countries are falling short of the expectations of the Millennium Development Goals. Arguing that the dominant assumptions for scaling up are inadequate, we propose that interpreting change in health systems through the lens of complex adaptive systems (CAS) provides better models of pathways for scaling up. Based on an understanding of CAS behaviours, we describe how phenomena such as path dependence, feedback loops, scale-free networks, emergent behaviour and phase transitions can uncover relevant lessons for the design and implementation of health policy and programmes in the context of scaling up health services. The implications include paying more attention to local context, incentives and institutions, as well as anticipating certain types of unintended consequences that can undermine scaling up efforts, and developing and implementing programmes that engage key actors through transparent use of data for ongoing problem-solving and adaptation. We propose that future efforts to scale up should adapt and apply the models and methodologies which have been used in other fields that study CAS, yet are underused in public health. This can help policy makers, planners, implementers and researchers to explore different and innovative approaches for reaching populations in need with effective, equitable and efficient health services. The old assumptions have led to disappointed expectations about how to scale up health services, and offer little insight on how to scale up effective interventions in the future. The alternative perspectives offered by CAS may better reflect the complex and changing nature of health systems, and create new opportunities for understanding and scaling up health services.

  14. The AAV-mediated and RNA-guided CRISPR/Cas9 system for gene therapy of DMD and BMD.

    PubMed

    Wang, Jing-Zhang; Wu, Peng; Shi, Zhi-Min; Xu, Yan-Li; Liu, Zhi-Jun

    2017-04-05

    Mutations in the dystrophin gene (Dmd) result in Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD), which afflict many newborn boys. In 2016, Brain and Development published several interesting articles on DMD treatment with antisense oligonucleotide, kinase inhibitor, and prednisolone. Even more strikingly, three articles in the issue 6271 of Science in 2016 provide new insights into gene therapy of DMD and BMD via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9). In brief, adeno-associated virus (AAV) vectors transport guided RNAs (gRNAs) and Cas9 into mdx mouse model, gRNAs recognize the mutated Dmd exon 23 (having a stop codon), and Cas9 cut the mutated exon 23 off the Dmd gene. These manipulations restored expression of truncated but partially functional dystrophin, improved skeletal and cardiac muscle function, and increased survival of mdx mice significantly. This review concisely summarized the related advancements and discussed their primary implications in the future gene therapy of DMD, including AAV-vector selection, gRNA designing, Cas9 optimization, dystrophin-restoration efficiency, administration routes, and systemic and long-term therapeutic efficacy. Future orientations, including off-target effects, safety concerns, immune responses, precision medicine, and Dmd-editing in the brain (potentially blocked by the blood-brain barrier) were also elucidated briefly. Collectively, the AAV-mediated and RNA-guided CRISPR/Cas9 system has major superiorities compared with traditional gene therapy, and might contribute to the treatment of DMD and BMD substantially in the near future.

  15. Prospective Mathematics Teachers' Interactions with CAS-Based Textbook Elements

    ERIC Educational Resources Information Center

    Davis, Jon D.

    2015-01-01

    This study investigated how a group of 10 prospective secondary mathematics teachers (PST) read, evaluated, and adapted a textbook lesson involving the symbolic manipulation capabilities of computer algebra systems (CASS). PST read the entire lesson and tended to focus on the organizing question at the beginning of the student lesson and the CAS-S…

  16. Building the Class 2 CRISPR-Cas Arsenal.

    PubMed

    Lewis, Kevin M; Ke, Ailong

    2017-02-02

    Adaptation of CRISPR-Cas9 for genome-editing applications has revolutionized biomedical research. New single-component effector CRISPR systems are emerging from the bioinformatics pipeline. How can we best harness their power? Three new studies will no doubt facilitate this transition by generating the C2c1 and C2c2 structure snapshots in different functional states.

  17. The potential application and challenge of powerful CRISPR/Cas9 system in cardiovascular research.

    PubMed

    Li, Yangxin; Song, Yao-Hua; Liu, Bin; Yu, Xi-Yong

    2017-01-15

    CRISPR/Cas9 is a precision-guided munition found in bacteria to fight against invading viruses. This technology has enormous potential applications, including altering genes in both somatic and germ cells, as well as generating knockout animals. Compared to other gene editing techniques such as zinc finger nucleases and TALENS, CRISPR/Cas9 is much easier to use and highly efficient. Importantly, the multiplex capacity of this technology allows multiple genes to be edited simultaneously. CRISPR/Cas9 also has the potential to prevent and cure human diseases. In this review, we wish to highlight some key points regarding the future prospect of using CRISPR/Cas9 as a powerful tool for cardiovascular research, and as a novel therapeutic strategy to treat cardiovascular diseases.

  18. Engineering large viral DNA genomes using the CRISPR-Cas9 system.

    PubMed

    Suenaga, Tadahiro; Kohyama, Masako; Hirayasu, Kouyuki; Arase, Hisashi

    2014-09-01

    Manipulation of viral genomes is essential for studying viral gene function and utilizing viruses for therapy. Several techniques for viral genome engineering have been developed. Homologous recombination in virus-infected cells has traditionally been used to edit viral genomes; however, the frequency of the expected recombination is quite low. Alternatively, large viral genomes have been edited using a bacterial artificial chromosome (BAC) plasmid system. However, cloning of large viral genomes into BAC plasmids is both laborious and time-consuming. In addition, because it is possible for insertion into the viral genome of drug selection markers or parts of BAC plasmids to affect viral function, artificial genes sometimes need to be removed from edited viruses. Herpes simplex virus (HSV), a common DNA virus with a genome length of 152 kbp, causes labialis, genital herpes and encephalitis. Mutant HSV is a candidate for oncotherapy, in which HSV is used to kill tumor cells. In this study, the clustered regularly interspaced short palindromic repeat-Cas9 system was used to very efficiently engineer HSV without inserting artificial genes into viral genomes. Not only gene-ablated HSV but also gene knock-in HSV were generated using this method. Furthermore, selection with phenotypes of edited genes promotes the isolation efficiencies of expectedly mutated viral clones. Because our method can be applied to other DNA viruses such as Epstein-Barr virus, cytomegaloviruses, vaccinia virus and baculovirus, our system will be useful for studying various types of viruses, including clinical isolates.

  19. CRISPR-Cas and contact dependent secretion systems present on excisable pathogenicity islands with conserved recombination modules.

    PubMed

    Carpenter, Megan R; Kalburge, Sai S; Borowski, Joseph D; Peters, Molly C; Colwell, Rita R; Boyd, E Fidelma

    2017-03-06

    Pathogenicity islands (PAIs) are mobile integrated genetic elements that contain a diverse range of virulence factors. PAIs integrate into the host chromosome at a tRNA locus that contains their specific bacterial attachment site, attB, via integrase mediated site-specific recombination generating attL and attR sites. We identified conserved recombination modules (integrases and att sites) previously described in choleragenic V. cholerae PAIs but with novel cargo genes. Clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins) and a type VI secretion system (T6SS) gene cluster were identified at the Vibrio Pathogenicity Island-1 (VPI-1) insertion site in nineteen V. cholerae strains and contained the same recombination module. Two divergent type I-F CRISPR-Cas systems were identified, which differed in Cas protein homology and content. The CRISPR repeat sequence was identical among all V. cholerae strains but the CRISPR spacer sequences and the number of spacers varied. In silico analysis suggests that the CRISPR-Cas systems were active against phages and plasmids. A type III secretion system (T3SS) was present in twelve V. cholerae strains on a 68 kb island inserted at the same tRNA-serine insertion site as VPI-2 and contained the same recombination module. Bioinformatics analysis showed that two divergent T3SSs exist among the strains examined. Both the CRISPR and T3SS islands excised site-specifically from the bacterial chromosome as complete units and the cognate integrases were essential for this excision. These data demonstrated that identical recombination modules that catalyze integration and excision from the chromosome can acquire diverse cargo genes signifying a novel method of acquisition for both CRISPR-Cas systems and T3SSs.Importance This work demonstrated the presence of CRISPR-Cas systems and T3SSs on PAIs. Our work showed that similar recombination modules can associate with different cargo genes and

  20. Intrinsic sequence specificity of the Cas1 integrase directs new spacer acquisition

    PubMed Central

    Rollie, Clare; Schneider, Stefanie; Brinkmann, Anna Sophie; Bolt, Edward L; White, Malcolm F

    2015-01-01

    The adaptive prokaryotic immune system CRISPR-Cas provides RNA-mediated protection from invading genetic elements. The fundamental basis of the system is the ability to capture small pieces of foreign DNA for incorporation into the genome at the CRISPR locus, a process known as Adaptation, which is dependent on the Cas1 and Cas2 proteins. We demonstrate that Cas1 catalyses an efficient trans-esterification reaction on branched DNA substrates, which represents the reverse- or disintegration reaction. Cas1 from both Escherichia coli and Sulfolobus solfataricus display sequence specific activity, with a clear preference for the nucleotides flanking the integration site at the leader-repeat 1 boundary of the CRISPR locus. Cas2 is not required for this activity and does not influence the specificity. This suggests that the inherent sequence specificity of Cas1 is a major determinant of the adaptation process. DOI: http://dx.doi.org/10.7554/eLife.08716.001 PMID:26284603

  1. Predominance of Single Prophage Carrying a CRISPR/cas System in "Candidatus Liberibacter asiaticus" Strains in Southern China.

    PubMed

    Zheng, Zheng; Bao, Minli; Wu, Fengnian; Chen, Jianchi; Deng, Xiaoling

    2016-01-01

    "Candidatus Liberibacter asiaticus" (CLas) is an uncultureable α-proteobacterium associated with citrus Huanglongbing (HLB, yellow shoot disease), a highly destructive disease affecting citrus production worldwide. HLB was observed in Guangdong Province of China over a hundred years ago and remains endemic there. Little is known about CLas biology due to its uncultureable nature. This study began with the genome sequence analysis of CLas Strain A4 from Guangdong in the prophage region. Within the two currently known prophage types, Type 1 (SC1-like) and Type 2 (SC2-like), A4 genome contained only a Type 2 prophage, CGdP2, namely. An analysis on CLas strains collected in Guangdong showed that Type 2 prophage dominated the bacterial population (82.6%, 71/86). An extended survey covering five provinces in southern China also revealed the predominance of single prophage (Type 1 or Type 2) in the CLas population (90.4%, 169/187). CLas strains with two and no prophage types accounted for 7.2% and 2.8%, respectively. In silico analyses on CGdP2 identified a CRISPR (clustered regularly interspaced short palindromic repeats)/cas (CRISPR-associated protein genes) system, consisting of four 22 bp repeats, three 23 bp spacers and 9 predicted cas. Similar CRISPR/cas systems were detected in all 10 published CLas prophages as well as 13 CLas field strains in southern China. Both Type 1 and Type 2 prophages shared almost identical sequences in spacer 1 and 3 but not spacer 2. Considering that the function of a CRISPR/cas system was to destroy invading DNA, it was hypothesized that a pre-established CLas prophage could use its CRISPR/cas system guided by spacer 1 and/or 3 to defeat the invasion of the other phage/prophage. This hypothesis explained the predominance of single prophage type in the CLas population in southern China. This is the first report of CRISPR/cas system in the "Ca. Liberibacter" genera.

  2. Is Echo a complex adaptive system?

    PubMed

    Smith, R M; Bedau, M A

    2000-01-01

    We evaluate whether John Holland's Echo model exemplifies his theory of complex adaptive systems. After reviewing Holland's theory of complex adaptive systems and describing his Escho model, we describe and explain the characteristic evolutionary behavior observed in a series of Echo model runs. We conclude that Echo lacks the diversity of hierarchically organized aggregates that typify complex adaptive systems, and we explore possible explanations for this failure.

  3. Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme of the CRISPR interference

    SciTech Connect

    Beloglazova, Natalia; Petit, Pierre; Flick, Robert; Brown, Greg; Savchenko, Alexei; Yakunin, Alexander F.

    2012-03-15

    Clustered regularly interspaced short palindromic repeats (CRISPRs) and Cas proteins represent an adaptive microbial immunity system against viruses and plasmids. Cas3 proteins have been proposed to play a key role in the CRISPR mechanism through the direct cleavage of invasive DNA. Here, we show that the Cas3 HD domain protein MJ0384 from Methanocaldococcus jannaschii cleaves endonucleolytically and exonucleolytically (3'-5') single-stranded DNAs and RNAs, as well as 3'-flaps, splayed arms, and R-loops. The degradation of branched DNA substrates by MJ0384 is stimulated by the Cas3 helicase MJ0383 and ATP. The crystal structure of MJ0384 revealed the active site with two bound metal cations and together with site-directed mutagenesis suggested a catalytic mechanism. Our studies suggest that the Cas3 HD nucleases working together with the Cas3 helicases can completely degrade invasive DNAs through the combination of endo- and exonuclease activities.

  4. Characterization of CRISPR-Cas system in clinical Staphylococcus epidermidis strains revealed its potential association with bacterial infection sites.

    PubMed

    Li, Qiuchun; Xie, Xiaolei; Yin, Kequan; Tang, Yueyuan; Zhou, Xiaohui; Chen, Yun; Xia, Jie; Hu, Yachen; Ingmer, Hanne; Li, Yang; Jiao, Xinan

    2016-12-01

    Staphylococcus epidermidis is considered as a major cause of nosocomial infections, bringing an immense burden to healthcare systems. Virulent phages have been confirmed to be efficient in combating the pathogen, but the prensence of CRISPR-Cas system, which is a bacterial immune system eliminating phages was reported in few S. epidermidis strains. In this study, the CRISPR-Cas system was detected in 12 from almost 300 published genomes in GenBank and by PCR of cas6 gene in 18 strains out of 130 clinical isolates obtained in Copenhagen. Four strains isolated in 1965-1966 harboured CRISPR elements confirming that this immunity system was not recently acquired by S. epidermidis. In these CRISPR-positive strains, 44 and 12 spacers were found to belong to CRISPR1 and CRISPR2 elements, respectively. However, only 15 spacers displayed homology to reported phages and plasmids DNA. Interestingly, 5 different spacers located in the CRISPR1 locus with homolgy to virulent phage 6ec DNA sequences, and 19 strains each carrying 2 or 3 different spacers recognizing this phage, implied that the CRISPR-Cas immunity could be abrogated by nucleotide mismatch between the spacer and its target phage sequence, while new spacers obtained from the evolved phage could recover the CRISPR interference. In addition, phylogenetic analysis of the 29 CRISPR-positive isolates divided them into four lineages, with 81% human blood isolates as a distinct sub-lineage, suggesting that the CRISPR difference is closely related to diverse habitats. Knowledge of CRISPR and its prevalence may ultimately be applied in the understanding of origin and evolution of CRISPR-positive S. epidermidis strains.

  5. Modeling Power Systems as Complex Adaptive Systems

    SciTech Connect

    Chassin, David P.; Malard, Joel M.; Posse, Christian; Gangopadhyaya, Asim; Lu, Ning; Katipamula, Srinivas; Mallow, J V.

    2004-12-30

    Physical analogs have shown considerable promise for understanding the behavior of complex adaptive systems, including macroeconomics, biological systems, social networks, and electric power markets. Many of today's most challenging technical and policy questions can be reduced to a distributed economic control problem. Indeed, economically based control of large-scale systems is founded on the conjecture that the price-based regulation (e.g., auctions, markets) results in an optimal allocation of resources and emergent optimal system control. This report explores the state-of-the-art physical analogs for understanding the behavior of some econophysical systems and deriving stable and robust control strategies for using them. We review and discuss applications of some analytic methods based on a thermodynamic metaphor, according to which the interplay between system entropy and conservation laws gives rise to intuitive and governing global properties of complex systems that cannot be otherwise understood. We apply these methods to the question of how power markets can be expected to behave under a variety of conditions.

  6. Phospholamban Ablation Using CRISPR/Cas9 System Improves Mortality in a Murine Heart Failure Model

    PubMed Central

    Kaneko, Manami; Hashikami, Kentarou; Yamamoto, Satoshi; Matsumoto, Hirokazu; Nishimoto, Tomoyuki

    2016-01-01

    Sarcoplasmic reticulum Ca2+-ATPase 2a (SERCA2a) and its inhibitory protein called phospholamban (PLN) are pivotal for Ca2+ handling in cardiomyocyte and are known that their expression level and activity were changed in the heart failure patients. To examine whether PLN inhibition can improve survival rate as well as cardiac function in heart failure, we performed PLN ablation in calsequestrin overexpressing (CSQ-Tg) mice, a severe heart failure model, using clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated (Cas) system. According this method, generation rate of PLN wild type mice (PLN copy >0.95) and PLN homozygous knockout (KO) mice (PLN copy <0.05) were 39.1% and 10.5%, respectively. While CSQ overexpression causes severe heart failure symptoms and premature death, a significant ameliorating effect on survival rate was observed in PLN homozygous KO/CSQ-Tg mice compared to PLN wild type/CSQ-Tg mice (median survival days are 55 and 50 days, respectively). Measurement of cardiac function with cardiac catheterization at the age of 5 weeks revealed that PLN ablation improved cardiac function in CSQ-Tg mice without affecting heart rate and blood pressure. Furthermore, increases in atrial and lung weight, an index of congestion, were significantly inhibited by PLN ablation. These results suggest that PLN deletion would be a promising approach to improve both mortality and cardiac function in the heart failure. PMID:27992596

  7. Genome engineering in the yeast pathogen Candida glabrata using the CRISPR-Cas9 system

    PubMed Central

    Enkler, Ludovic; Richer, Delphine; Marchand, Anthony L.; Ferrandon, Dominique; Jossinet, Fabrice

    2016-01-01

    Among Candida species, the opportunistic fungal pathogen Candida glabrata has become the second most common causative agent of candidiasis in the world and a major public health concern. Yet, few molecular tools and resources are available to explore the biology of C. glabrata and to better understand its virulence during infection. In this study, we describe a robust experimental strategy to generate loss-of-function mutants in C. glabrata. The procedure is based on the development of three main tools: (i) a recombinant strain of C. glabrata constitutively expressing the CRISPR-Cas9 system, (ii) an online program facilitating the selection of the most efficient guide RNAs for a given C. glabrata gene, and (iii) the identification of mutant strains by the Surveyor technique and sequencing. As a proof-of-concept, we have tested the virulence of some mutants in vivo in a Drosophila melanogaster infection model. Our results suggest that yps11 and a previously uncharacterized serine/threonine kinase are involved, directly or indirectly, in the ability of the pathogenic yeast to infect this model host organism. PMID:27767081

  8. RNA-Guided CRISPR-Cas9 System-Mediated Engineering of Acute Myeloid Leukemia Mutations.

    PubMed

    Brabetz, Oliver; Alla, Vijay; Angenendt, Linus; Schliemann, Christoph; Berdel, Wolfgang E; Arteaga, Maria-Francisca; Mikesch, Jan-Henrik

    2017-03-17

    Current acute myeloid leukemia (AML) disease models face severe limitations because most of them induce un-physiological gene expressions that do not represent conditions in AML patients and/or depend on external promoters for regulation of gene expression/repression. Furthermore, many AML models are based on reciprocal chromosomal translocations that only reflect the minority of AML patients, whereas more than 50% of patients have a normal karyotype. The majority of AML, however, is driven by somatic mutations. Thus, identification as well as a detailed molecular and functional characterization of the role of these driver mutations via improved AML models is required for better approaches toward novel targeted therapies. Using the IDH2 R140Q mutation as a model, we present a new effective methodology here using the RNA-guided clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to reproduce or remove AML-associated mutations in or from human leukemic cells, respectively, via introduction of a DNA template at the endogenous gene locus via homologous recombination. Our technology represents a precise way for AML modeling to gain insights into AML development and progression and provides a basis for future therapeutic approaches.

  9. STRUCTURAL BIOLOGY. A Cas9-guide RNA complex preorganized for target DNA recognition.

    PubMed

    Jiang, Fuguo; Zhou, Kaihong; Ma, Linlin; Gressel, Saskia; Doudna, Jennifer A

    2015-06-26

    Bacterial adaptive immunity uses CRISPR (clustered regularly interspaced short palindromic repeats)-associated (Cas) proteins together with CRISPR transcripts for foreign DNA degradation. In type II CRISPR-Cas systems, activation of Cas9 endonuclease for DNA recognition upon guide RNA binding occurs by an unknown mechanism. Crystal structures of Cas9 bound to single-guide RNA reveal a conformation distinct from both the apo and DNA-bound states, in which the 10-nucleotide RNA "seed" sequence required for initial DNA interrogation is preordered in an A-form conformation. This segment of the guide RNA is essential for Cas9 to form a DNA recognition-competent structure that is poised to engage double-stranded DNA target sequences. We construe this as convergent evolution of a "seed" mechanism reminiscent of that used by Argonaute proteins during RNA interference in eukaryotes.

  10. Rapid and tunable method to temporally control gene editing based on conditional Cas9 stabilization

    PubMed Central

    Senturk, Serif; Shirole, Nitin H.; Nowak, Dawid G.; Corbo, Vincenzo; Pal, Debjani; Vaughan, Alexander; Tuveson, David A.; Trotman, Lloyd C.; Kinney, Justin B.; Sordella, Raffaella

    2017-01-01

    The CRISPR/Cas9 system is a powerful tool for studying gene function. Here, we describe a method that allows temporal control of CRISPR/Cas9 activity based on conditional Cas9 destabilization. We demonstrate that fusing an FKBP12-derived destabilizing domain to Cas9 (DD-Cas9) enables conditional Cas9 expression and temporal control of gene editing in the presence of an FKBP12 synthetic ligand. This system can be easily adapted to co-express, from the same promoter, DD-Cas9 with any other gene of interest without co-modulation of the latter. In particular, when co-expressed with inducible Cre-ERT2, our system enables parallel, independent manipulation of alleles targeted by Cas9 and traditional recombinase with single-cell specificity. We anticipate this platform will be used for the systematic characterization and identification of essential genes, as well as the investigation of the interactions between functional genes. PMID:28224990

  11. Optimization of genome engineering approaches with the CRISPR/Cas9 system.

    PubMed

    Li, Kai; Wang, Gang; Andersen, Troels; Zhou, Pingzhu; Pu, William T

    2014-01-01

    Designer nucleases such as TALENS and Cas9 have opened new opportunities to scarlessly edit the mammalian genome. Here we explored several parameters that influence Cas9-mediated scarless genome editing efficiency in murine embryonic stem cells. Optimization of transfection conditions and enriching for transfected cells are critical for efficiently recovering modified clones. Paired gRNAs and wild-type Cas9 efficiently create programmed deletions, which facilitate identification of targeted clones, while paired gRNAs and the Cas9D10A nickase generated smaller targeted indels with lower chance of off-target mutagenesis. Genome editing is also useful for programmed introduction of exogenous DNA sequences at a target locus. Increasing the length of the homology arms of the homology-directed repair template strongly enhanced targeting efficiency, while increasing the length of the DNA insert reduced it. Together our data provide guidance on optimal design of scarless gene knockout, modification, or knock-in experiments using Cas9 nuclease.

  12. Autonomous Organization-Based Adaptive Information Systems

    DTIC Science & Technology

    2005-01-01

    intentional Multi - agent System (MAS) approach [10]. While these approaches are functional AIS systems, they lack the ability to reorganize and adapt...extended a multi - agent system with a self- reorganizing architecture to create an autonomous, adaptive information system. Design Our organization-based...goals. An advantage of a multi - agent system using the organization theoretic model is its extensibility. The practical, numerical limits to the

  13. CRISPR-Cas system presents multiple transcriptional units including antisense RNAs that are expressed in minimal medium and upregulated by pH in Salmonella enterica serovar Typhi.

    PubMed

    Medina-Aparicio, Liliana; Rebollar-Flores, Javier E; Beltrán-Luviano, América A; Vázquez, Alejandra; Gutiérrez-Ríos, Rosa M; Olvera, Leticia; Calva, Edmundo; Hernández-Lucas, Ismael

    2017-02-01

    The CRISPR-Cas system is involved in bacterial immunity, virulence, gene regulation, biofilm formation and sporulation. In Salmonella enterica serovar Typhi, this system consists of five transcriptional units including antisense RNAs. It was determined that these genetic elements are expressed in minimal medium and are up-regulated by pH. In addition, a transcriptional characterization of cas3 and ascse2-1 is included herein.

  14. Computerized Adaptive Mastery Tests as Expert Systems.

    ERIC Educational Resources Information Center

    Frick, Theodore W.

    1992-01-01

    Discussion of expert systems and computerized adaptive tests describes two versions of EXSPRT, a new approach that combines uncertain inference in expert systems with sequential probability ratio test (SPRT) stopping rules. Results of two studies comparing EXSPRT to adaptive mastery testing based on item response theory and SPRT approaches are…

  15. I can see CRISPR now, even when phage are gone: a view on alternative CRISPR-Cas functions from the prokaryotic envelope

    PubMed Central

    Ratner, Hannah K.; Sampson, Timothy R.; Weiss, David S.

    2015-01-01

    Purpose CRISPR-Cas systems are prokaryotic immune systems against invading nucleic acids that adapt as new environmental threats arise. There are emerging examples of CRISPR-Cas functions in bacterial physiology beyond their role in adaptive immunity. This highlights the poorly understood, but potentially common, moonlighting functions of these abundant systems. We propose that these non-canonical CRISPR-Cas activities have evolved to respond to stresses at the cell envelope. Recent findings Here, we discuss recent literature describing the impact of the extracellular environment on the regulation of CRISPR-Cas systems, and the influence of CRISPR-Cas activity on bacterial physiology. The described non-canonical CRISPR-Cas functions allow the bacterial cell to respond to the extracellular environment, primarily through changes in envelope physiology. Summary This review discusses the expanding non-canonical functions of CRISPR-Cas systems, including their roles in virulence, focusing mainly on their relationship to the cell envelope. We first examine the effects of the extracellular environment on regulation of CRISPR-Cas components, and then discuss the impact of CRISPR-Cas systems on bacterial physiology, focusing on their roles in influencing interactions with the environment including host organisms. PMID:25887612

  16. Cell Locating with the Image Analysis System of the CAS-LIBB Single-Particle Microbeam Facility

    NASA Astrophysics Data System (ADS)

    Wang, Xiaohua; Wang, Shaohu; Yu, Zengliang

    2005-06-01

    A single-particle microbeam facility has been constructed at the Key Laboratory of Ion Beam Bioengineering (LIBB), Chinese Academy of Sciences (CAS). At the CAS-LIBB microbeam facility, we have developed protocols to place exact numbers of charged particles through nuclear centroids of cells, at defined positions in the cytoplasm relative to the nucleus, and through defined fractions of cells in a population. In this paper, we address the methods for nucleus, cytoplasm and bystander (either a single or an exact number of ions is delivered to a certain percentage of cells in a population to study the bystander effects of radiation) irradiation in detail from the precision of target finding and cell locating in the image analysis system. Moreover, for cells touching slightly in an image, a watershed method is used to separate these touching objects; after that, the number of objects in an image is counted accurately and the irradiation points are located precisely.

  17. Engineering human tumour-associated chromosomal translocations with the RNA-guided CRISPR-Cas9 system.

    PubMed

    Torres, R; Martin, M C; Garcia, A; Cigudosa, Juan C; Ramirez, J C; Rodriguez-Perales, S

    2014-06-03

    Cancer-related human chromosomal translocations are generated through the illegitimate joining of two non-homologous chromosomes affected by double-strand breaks (DSB). Effective methodologies to reproduce precise reciprocal tumour-associated chromosomal translocations are required to gain insight into the initiation of leukaemia and sarcomas. Here we present a strategy for generating cancer-related human chromosomal translocations in vitro based on the ability of the RNA-guided CRISPR-Cas9 system to induce DSBs at defined positions. Using this approach we generate human cell lines and primary cells bearing chromosomal translocations resembling those described in acute myeloid leukaemia and Ewing's sarcoma at high frequencies. FISH and molecular analysis at the mRNA and protein levels of the fusion genes involved in these engineered cells reveal the reliability and accuracy of the CRISPR-Cas9 approach, providing a powerful tool for cancer studies.

  18. Applications of Engineered DNA-Binding Molecules Such as TAL Proteins and the CRISPR/Cas System in Biology Research

    PubMed Central

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-01-01

    Engineered DNA-binding molecules such as transcription activator-like effector (TAL or TALE) proteins and the clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) (CRISPR/Cas) system have been used extensively for genome editing in cells of various types and species. The sequence-specific DNA-binding activities of these engineered DNA-binding molecules can also be utilized for other purposes, such as transcriptional activation, transcriptional repression, chromatin modification, visualization of genomic regions, and isolation of chromatin in a locus-specific manner. In this review, we describe applications of these engineered DNA-binding molecules for biological purposes other than genome editing. PMID:26404236

  19. Generation of gene-modified goats targeting MSTN and FGF5 via zygote injection of CRISPR/Cas9 system.

    PubMed

    Wang, Xiaolong; Yu, Honghao; Lei, Anmin; Zhou, Jiankui; Zeng, Wenxian; Zhu, Haijing; Dong, Zhiming; Niu, Yiyuan; Shi, Bingbo; Cai, Bei; Liu, Jinwang; Huang, Shuai; Yan, Hailong; Zhao, Xiaoe; Zhou, Guangxian; He, Xiaoling; Chen, Xiaoxu; Yang, Yuxin; Jiang, Yu; Shi, Lei; Tian, Xiue; Wang, Yongjun; Ma, Baohua; Huang, Xingxu; Qu, Lei; Chen, Yulin

    2015-09-10

    Recent advances in the study of the CRISPR/Cas9 system have provided a precise and versatile approach for genome editing in various species. However, the applicability and efficiency of this method in large animal models, such as the goat, have not been extensively studied. Here, by co-injection of one-cell stage embryos with Cas9 mRNA and sgRNAs targeting two functional genes (MSTN and FGF5), we successfully produced gene-modified goats with either one or both genes disrupted. The targeting efficiency of MSTN and FGF5 in cultured primary fibroblasts was as high as 60%, while the efficiency of disrupting MSTN and FGF5 in 98 tested animals was 15% and 21% respectively, and 10% for double gene modifications. The on- and off-target mutations of the target genes in fibroblasts, as well as in somatic tissues and testis of founder and dead animals, were carefully analyzed. The results showed that simultaneous editing of several sites was achieved in large animals, demonstrating that the CRISPR/Cas9 system has the potential to become a robust and efficient gene engineering tool in farm animals, and therefore will be critically important and applicable for breeding.

  20. CRP represses the CRISPR/Cas system in Escherichia coli: evidence that endogenous CRISPR spacers impede phage P1 replication.

    PubMed

    Yang, Chi-Dung; Chen, Yen-Hua; Huang, Hsi-Yuan; Huang, Hsien-Da; Tseng, Ching-Ping

    2014-06-01

    The CRISPR/Cas system is an important aspect in bacterial immunology. The anti-phage activity of the CRISPR system has been established using synthetic CRISPR spacers, but in vivo studies of endogenous CRISPR spacers are relatively scarce. Here, we showed that bacteriophage P1 titre in Escherichia coli decreased in the glucose-containing medium compared with that in the absence of glucose. This glucose effect of E. coli against phage P1 infection disappeared in cse3 deletion mutants. The effect on the susceptibility to phage P1 was associated with cAMP receptor protein (CRP)-mediated repression of cas genes transcription and crRNA maturation. Analysis of the regulatory element in the cse1 promoter region revealed a novel CRP binding site, which overlapped with a LeuO binding site. Furthermore, the limited sequence identity between endogenous spacers and the phage P1 genome was necessary and sufficient for CRISPR-mediated repression of phage P1 replication. Trans-expression of the third and seventh spacers in the CRISPR I region or third and sixth spacers in the CRISPR II region effectively reduced phage P1 titres in the CRISPR deletion mutants. These results demonstrate a novel regulatory mechanism for cas repression by CRP and provide evidence that endogenous spacers can repress phage P1 replication in E. coli.

  1. Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems

    PubMed Central

    JIN, Li-Fang; LI, Jin-Song

    2016-01-01

    With the development of high-throughput sequencing technology in the post-genomic era, researchers have concentrated their efforts on elucidating the relationships between genes and their corresponding functions. Recently, important progress has been achieved in the generation of genetically modified mice based on CRISPR/Cas9 and haploid embryonic stem cell (haESC) approaches, which provide new platforms for gene function analysis, human disease modeling, and gene therapy. Here, we review the CRISPR/Cas9 and haESC technology for the generation of genetically modified mice and discuss the key challenges in the application of these approaches. PMID:27469251

  2. Operator adaptation to changes in system reliability under adaptable automation.

    PubMed

    Chavaillaz, Alain; Sauer, Juergen

    2016-11-25

    This experiment examined how operators coped with a change in system reliability between training and testing. Forty participants were trained for 3 h on a complex process control simulation modelling six levels of automation (LOA). In training, participants either experienced a high- (100%) or low-reliability system (50%). The impact of training experience on operator behaviour was examined during a 2.5 h testing session, in which participants either experienced a high- (100%) or low-reliability system (60%). The results showed that most operators did not often switch between LOA. Most chose an LOA that relieved them of most tasks but maintained their decision authority. Training experience did not have a strong impact on the outcome measures (e.g. performance, complacency). Low system reliability led to decreased performance and self-confidence. Furthermore, complacency was observed under high system reliability. Overall, the findings suggest benefits of adaptable automation because it accommodates different operator preferences for LOA. Practitioner Summary: The present research shows that operators can adapt to changes in system reliability between training and testing sessions. Furthermore, it provides evidence that each operator has his/her preferred automation level. Since this preference varies strongly between operators, adaptable automation seems to be suitable to accommodate these large differences.

  3. CAS-Induced Difficulties in Learning Mathematics?

    ERIC Educational Resources Information Center

    Jankvist, Uffe Thomas; Misfeldt, Morten

    2015-01-01

    In recent years computer algebra systems (CAS) have become an integrated part of the upper secondary school mathematics program. Despite the many positive possibilities of CAS, there also seems to be a flip side of the coin in relation to actual difficulties in learning mathematics, not least because a strong dependence on CAS for mathematical…

  4. Expanding the Biologist's Toolkit with CRISPR-Cas9.

    PubMed

    Sternberg, Samuel H; Doudna, Jennifer A

    2015-05-21

    Few discoveries transform a discipline overnight, but biologists today can manipulate cells in ways never possible before, thanks to a peculiar form of prokaryotic adaptive immunity mediated by clustered regularly interspaced short palindromic repeats (CRISPR). From elegant studies that deciphered how these immune systems function in bacteria, researchers quickly uncovered the technological potential of Cas9, an RNA-guided DNA cleaving enzyme, for genome engineering. Here we highlight the recent explosion in visionary applications of CRISPR-Cas9 that promises to usher in a new era of biological understanding and control.

  5. The Limits to Adaptation: A Systems Approach

    NASA Astrophysics Data System (ADS)

    Felgenhauer, T. N.

    2013-12-01

    The ability to adapt to climate change is delineated by capacity thresholds, after which climate damages begin to overwhelm the adaptation response. Such thresholds depend upon physical properties (natural processes and engineering parameters), resource constraints (expressed through market prices), and societal preferences (from prices as well as cultural norms). Exceedance of adaptation capacity will require substitution either with other pre-existing policy responses or with new adaptation responses that have yet to be developed and tested. Previous modeling research shows that capacity limited adaptation will play a policy-significant role in future climate change decision-making. The aim of this study is to describe different types of adaptation response and climate damage systems and postulate how these systems might behave when the limits to adaptation are reached. The hypothesis is that this behavior will be governed by the characteristics and level of the adaptation limit, the shape of the damage curve in that specific damage area, and the availability of alternative adaptation responses once the threshold is passed, whether it is more of the old technology, a new response type, or a transformation of the climate damage and response system itself.

  6. A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.)

    PubMed Central

    Li, Chao; Unver, Turgay; Zhang, Baohong

    2017-01-01

    The complex allotetraploid genome is one of major challenges in cotton for repressing gene expression. Developing site-specific DNA mutation is the long-term dream for cotton breeding scientists. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as a robust biotechnology for targeted-DNA mutation. In this study, two sgRNAs, GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, were designed in the identical genomic regions of GhMYB25-like A and GhMYB25-like D, which were encoded by cotton A subgenome and the D subgenome, respectively, was assembled to direct Cas9-mediated allotetraploid cotton genome editing. High proportion (14.2–21.4%) CRISPR/Cas9-induced specific truncation events, either from GhMYB25-like A DNA site or from GhMYB25-like D DNA site, were detected in 50% examined transgenic cotton through PCR amplification assay and sequencing analyses. Sequencing results also demonstrated that 100% and 98.8% mutation frequency were occurred on GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2 target site respectively. The off-target effect was evaluated by sequencing two putative off-target sites, which have 3 and 1 mismatched nucleotides with GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, respectively; all the examined samples were not detected any off-target-caused mutation events. Thus, these results demonstrated that CRISPR/Cas9 is qualified for generating DNA level mutations on allotetraploid cotton genome with high-efficiency and high-specificity. PMID:28256588

  7. A high-efficiency CRISPR/Cas9 system for targeted mutagenesis in Cotton (Gossypium hirsutum L.).

    PubMed

    Li, Chao; Unver, Turgay; Zhang, Baohong

    2017-03-03

    The complex allotetraploid genome is one of major challenges in cotton for repressing gene expression. Developing site-specific DNA mutation is the long-term dream for cotton breeding scientists. The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system is emerging as a robust biotechnology for targeted-DNA mutation. In this study, two sgRNAs, GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, were designed in the identical genomic regions of GhMYB25-like A and GhMYB25-like D, which were encoded by cotton A subgenome and the D subgenome, respectively, was assembled to direct Cas9-mediated allotetraploid cotton genome editing. High proportion (14.2-21.4%) CRISPR/Cas9-induced specific truncation events, either from GhMYB25-like A DNA site or from GhMYB25-like D DNA site, were detected in 50% examined transgenic cotton through PCR amplification assay and sequencing analyses. Sequencing results also demonstrated that 100% and 98.8% mutation frequency were occurred on GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2 target site respectively. The off-target effect was evaluated by sequencing two putative off-target sites, which have 3 and 1 mismatched nucleotides with GhMYB25-like-sgRNA1 and GhMYB25-like-sgRNA2, respectively; all the examined samples were not detected any off-target-caused mutation events. Thus, these results demonstrated that CRISPR/Cas9 is qualified for generating DNA level mutations on allotetraploid cotton genome with high-efficiency and high-specificity.

  8. Regulation of Gene Editing Activity Directed by Single-Stranded Oligonucleotides and CRISPR/Cas9 Systems.

    PubMed

    Bialk, Pawel; Rivera-Torres, Natalia; Strouse, Bryan; Kmiec, Eric B

    2015-01-01

    Single-stranded DNA oligonucleotides (ssODNs) can direct the repair of a single base mutation in human genes. While the regulation of this gene editing reaction has been partially elucidated, the low frequency with which repair occurs has hampered development toward clinical application. In this work a CRISPR/Cas9 complex is employed to induce double strand DNA breakage at specific sites surrounding the nucleotide designated for exchange. The result is a significant elevation in ssODN-directed gene repair, validated by a phenotypic readout. By analysing reaction parameters, we have uncovered restrictions on gene editing activity involving CRISPR/Cas9 complexes. First, ssODNs that hybridize to the non-transcribed strand direct a higher level of gene repair than those that hybridize to the transcribed strand. Second, cleavage must be proximal to the targeted mutant base to enable higher levels of gene editing. Third, DNA cleavage enables a higher level of gene editing activity as compared to single-stranded DNA nicks, created by modified Cas9 (Nickases). Fourth, we calculated the hybridization potential and free energy levels of ssODNs that are complementary to the guide RNA sequences of CRISPRs used in this study. We find a correlation between free energy potential and the capacity of single-stranded oligonucleotides to inhibit specific DNA cleavage activity, thereby indirectly reducing gene editing activity. Our data provide novel information that might be taken into consideration in the design and usage of CRISPR/Cas9 systems with ssODNs for gene editing.

  9. [Changes of resistant phenotype and CRISPR/Cas system of four Shigella strains passaged for 90 times without antibiotics].

    PubMed

    Zhang, B; Hong, L J; Duan, G C; Liang, W J; Yang, H Y; Xi, Y L

    2017-02-10

    Objective: To explore the stability of resistant phenotypes and changes of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) gene system on four Shigella strains in the absence of antibiotics. Methods: Four clinical isolated Shigella strains that resistant to different antibiotics were consecutive passaged for 90 times without antibiotics. Agar dilution method was used to determine the minimum inhibitory concentration of Shigella strains. After sequence analysis with PCR, CRISPR Finder and Clustal X 2.1 were applied to identify the changes of CRISPR loci in the Shigella strains. Results: After the consecutive transfer of 90 generations, sensitivity to certain antibiotics of four Shigella strains with different drug resistant spectrums increased. Mel-sf1998024/zz resistance to ampicillin, cephalexin, cefotaxime, chloramphenicol decreased, mel-s2014026/sx resistance to norfloxacin, trimethoprim decreased, mel-sf2004004/sx drug resistance to ampicillin, cefuroxime, cefotaxime, chloramphenicol, trimethoprim decreased and mel-sf2013004/bj resistance to chloramphenicol decreased. The spacer of which matched gene codes Cas and its upstream repeat in 3'end of CRISPR3 got lost in mel-sf1998024/zz and mel-sf2013004/bj. Conclusions:Shigella strains could reduce or lose their resistance to some antibiotics after consecutive transfers, without the interference of antibiotics. CRISPR3 locus had dynamic spacers in Shigella strains while CRISPR3 locus and cas genes might have been co-evolved.

  10. Homology-integrated CRISPR-Cas (HI-CRISPR) system for one-step multigene disruption in Saccharomyces cerevisiae.

    PubMed

    Bao, Zehua; Xiao, Han; Liang, Jing; Zhang, Lu; Xiong, Xiong; Sun, Ning; Si, Tong; Zhao, Huimin

    2015-05-15

    One-step multiple gene disruption in the model organism Saccharomyces cerevisiae is a highly useful tool for both basic and applied research, but it remains a challenge. Here, we report a rapid, efficient, and potentially scalable strategy based on the type II Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas) system to generate multiple gene disruptions simultaneously in S. cerevisiae. A 100 bp dsDNA mutagenizing homologous recombination donor is inserted between two direct repeats for each target gene in a CRISPR array consisting of multiple donor and guide sequence pairs. An ultrahigh copy number plasmid carrying iCas9, a variant of wild-type Cas9, trans-encoded RNA (tracrRNA), and a homology-integrated crRNA cassette is designed to greatly increase the gene disruption efficiency. As proof of concept, three genes, CAN1, ADE2, and LYP1, were simultaneously disrupted in 4 days with an efficiency ranging from 27 to 87%. Another three genes involved in an artificial hydrocortisone biosynthetic pathway, ATF2, GCY1, and YPR1, were simultaneously disrupted in 6 days with 100% efficiency. This homology-integrated CRISPR (HI-CRISPR) strategy represents a powerful tool for creating yeast strains with multiple gene knockouts.

  11. Programmed Self-Assembly of an Active P22-Cas9 Nanocarrier System.

    PubMed

    Qazi, Shefah; Miettinen, Heini M; Wilkinson, Royce A; McCoy, Kimberly; Douglas, Trevor; Wiedenheft, Blake

    2016-03-07

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA-guided endonucleases are powerful new tools for targeted genome engineering. These nucleases provide an efficient and precise method for manipulating eukaryotic genomes; however, delivery of these reagents to specific cell-types remains challenging. Virus-like particles (VLPs) derived from bacteriophage P22, are robust supramolecular protein cage structures with demonstrated utility for cell type-specific delivery of encapsulated cargos. Here, we genetically fuse Cas9 to a truncated form of the P22 scaffold protein, which acts as a template for capsid assembly as well as a specific encapsulation signal for Cas9. Our results indicate that Cas9 and a single-guide RNA are packaged inside the P22 VLP, and activity assays indicate that this RNA-guided endonuclease is functional for sequence-specific cleavage of dsDNA targets. This work demonstrates the potential for developing P22 as a delivery vehicle for cell specific targeting of Cas9.

  12. Repurposing the CRISPR-Cas9 system for targeted DNA methylation

    PubMed Central

    Vojta, Aleksandar; Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Korać, Petra; Julg, Boris; Klasić, Marija; Zoldoš, Vlatka

    2016-01-01

    Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co–expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression. PMID:26969735

  13. Repurposing the CRISPR-Cas9 system for targeted DNA methylation.

    PubMed

    Vojta, Aleksandar; Dobrinić, Paula; Tadić, Vanja; Bočkor, Luka; Korać, Petra; Julg, Boris; Klasić, Marija; Zoldoš, Vlatka

    2016-07-08

    Epigenetic studies relied so far on correlations between epigenetic marks and gene expression pattern. Technologies developed for epigenome editing now enable direct study of functional relevance of precise epigenetic modifications and gene regulation. The reversible nature of epigenetic modifications, including DNA methylation, has been already exploited in cancer therapy for remodeling the aberrant epigenetic landscape. However, this was achieved non-selectively using epigenetic inhibitors. Epigenetic editing at specific loci represents a novel approach that might selectively and heritably alter gene expression. Here, we developed a CRISPR-Cas9-based tool for specific DNA methylation consisting of deactivated Cas9 (dCas9) nuclease and catalytic domain of the DNA methyltransferase DNMT3A targeted by co-expression of a guide RNA to any 20 bp DNA sequence followed by the NGG trinucleotide. We demonstrated targeted CpG methylation in a ∼35 bp wide region by the fusion protein. We also showed that multiple guide RNAs could target the dCas9-DNMT3A construct to multiple adjacent sites, which enabled methylation of a larger part of the promoter. DNA methylation activity was specific for the targeted region and heritable across mitotic divisions. Finally, we demonstrated that directed DNA methylation of a wider promoter region of the target loci IL6ST and BACH2 decreased their expression.

  14. Exploiting CRISPR/Cas: interference mechanisms and applications.

    PubMed

    Richter, Hagen; Randau, Lennart; Plagens, André

    2013-07-12

    The discovery of biological concepts can often provide a framework for the development of novel molecular tools, which can help us to further understand and manipulate life. One recent example is the elucidation of the prokaryotic adaptive immune system, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) that protects bacteria and archaea against viruses or conjugative plasmids. The immunity is based on small RNA molecules that are incorporated into versatile multi-domain proteins or protein complexes and specifically target viral nucleic acids via base complementarity. CRISPR/Cas interference machines are utilized to develop novel genome editing tools for different organisms. Here, we will review the latest progress in the elucidation and application of prokaryotic CRISPR/Cas systems and discuss possible future approaches to exploit the potential of these interference machineries.

  15. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    SciTech Connect

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-05-15

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  16. The CasKR Two-Component System Is Required for the Growth of Mesophilic and Psychrotolerant Bacillus cereus Strains at Low Temperatures

    PubMed Central

    Diomandé, Sara Esther; Chamot, Stéphanie; Antolinos, Vera; Vasai, Florian; Guinebretière, Marie-Hélène; Bornard, Isabelle; Nguyen-the, Christophe; Broussolle, Véronique

    2014-01-01

    The different strains of Bacillus cereus can grow at temperatures covering a very diverse range. Some B. cereus strains can grow in chilled food and consequently cause food poisoning. We have identified a new sensor/regulator mechanism involved in low-temperature B. cereus growth. Construction of a mutant of this two-component system enabled us to show that this system, called CasKR, is required for growth at the minimal temperature (Tmin). CasKR was also involved in optimal cold growth above Tmin and in cell survival below Tmin. Microscopic observation showed that CasKR plays a key role in cell shape during cold growth. Introducing the casKR genes in a ΔcasKR mutant restored its ability to grow at Tmin. Although it was first identified in the ATCC 14579 model strain, this mechanism has been conserved in most strains of the B. cereus group. We show that the role of CasKR in cold growth is similar in other B. cereus sensu lato strains with different growth temperature ranges, including psychrotolerant strains. PMID:24509924

  17. Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System.

    PubMed

    Butler, Nathaniel M; Atkins, Paul A; Voytas, Daniel F; Douches, David S

    2015-01-01

    Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3-60% per transformation and from 0-29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87-100%. This demonstration

  18. Generation and Inheritance of Targeted Mutations in Potato (Solanum tuberosum L.) Using the CRISPR/Cas System

    PubMed Central

    Butler, Nathaniel M.; Atkins, Paul A.; Voytas, Daniel F.; Douches, David S.

    2015-01-01

    Genome editing using sequence-specific nucleases (SSNs) offers an alternative approach to conventional genetic engineering and an opportunity to extend the benefits of genetic engineering in agriculture. Currently available SSN platforms, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR/Cas (clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated systems (Cas)) have been used in a range of plant species for targeted mutagenesis via non-homologous end joining (NHEJ) are just beginning to be explored in crops such as potato (Solanum tuberosum Group Tuberosum L.). In this study, CRISPR/Cas reagents expressing one of two single-guide RNA (sgRNA) targeting the potato ACETOLACTATE SYNTHASE1 (StALS1) gene were tested for inducing targeted mutations in callus and stable events of diploid and tetraploid potato using Agrobacterium-mediated transformation with either a conventional T-DNA or a modified geminivirus T-DNA. The percentage of primary events with targeted mutations ranged from 3–60% per transformation and from 0–29% above an expected threshold based on the number of ALS alleles. Primary events with targeted mutation frequencies above the expected threshold were used for mutation cloning and inheritance studies using clonal propagation and crosses or selfing. Four of the nine primary events used for mutation cloning had more than one mutation type, and eight primary events contained targeted mutations that were maintained across clonal generations. Somatic mutations were most evident in the diploid background with three of the four primary events having more than two mutation types at a single ALS locus. Conversely, in the tetraploid background, four of the five candidates carried only one mutation type. Single targeted mutations were inherited through the germline of both diploid and tetraploid primary events with transmission percentages ranging from 87–100%. This

  19. Practical method for targeted disruption of cilia-related genes by using CRISPR/Cas9-mediated homology-independent knock-in system.

    PubMed

    Katoh, Yohei; Michisaka, Saki; Nozaki, Shohei; Funabashi, Teruki; Hirano, Tomoaki; Takei, Ryota; Nakayama, Kazuhisa

    2017-02-08

    The CRISPR/Cas9 system has revolutionized genome editing in virtually all organisms. Although the CRISPR/Cas9 system enables the targeted cleavage of genomic DNA, its use for gene knock-in remains challenging because levels of homologous recombination activity vary among various cells. In contrast, the efficiency of homology-independent DNA repair is relatively high in most cell types. Therefore, the utilization of a homology-independent repair mechanism is a possible alternative for efficient genome editing. Here, we constructed a donor knock-in vector optimized for the CRISPR/Cas9 system, and developed a practical system that enables efficient disruption of target genes by exploiting homology-independent repair. Using this practical knock-in system, we successfully disrupted genes encoding proteins involved ciliary protein trafficking, including IFT88 and IFT20, in hTERT-RPE1 cells, which have low homologous recombination activity. The most critical concern using the CRISPR/Cas9 system is off-target cleavage. To reduce the off-target cleavage frequency and to increase the versatility of our knock-in system, we further constructed a universal donor vector and an expression vector containing Cas9 with enhanced specificity and tandem sgRNA expression cassettes. We demonstrated that the second version of our system has improved usability.

  20. RNA-guided CRISPR-Cas technologies for genome-scale investigation of disease processes.

    PubMed

    Humphrey, Sean E; Kasinski, Andrea L

    2015-04-02

    From its discovery as an adaptive bacterial and archaea immune system, the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system has quickly been developed into a powerful and groundbreaking programmable nuclease technology for the global and precise editing of the genome in cells. This system allows for comprehensive unbiased functional studies and is already advancing the field by revealing genes that have previously unknown roles in disease processes. In this review, we examine and compare recently developed CRISPR-Cas platforms for global genome editing and examine the advancements these platforms have made in guide RNA design, guide RNA/Cas9 interaction, on-target specificity, and target sequence selection. We also explore some of the exciting therapeutic potentials of the CRISPR-Cas technology as well as some of the innovative new uses of this technology beyond genome editing.

  1. Dual gRNAs guided CRISPR/Cas9 system inhibits hepatitis B virus replication

    PubMed Central

    Wang, Jie; Xu, Zhong-Wei; Liu, Shuang; Zhang, Rui-Yang; Ding, Shan-Long; Xie, Xiao-Meng; Long, Lu; Chen, Xiang-Mei; Zhuang, Hui; Lu, Feng-Min

    2015-01-01

    AIM: To screen and investigate the effective gRNAs against hepatitis B virus (HBV) of genotypes A-D. METHODS: A total of 15 gRNAs against HBV of genotypes A-D were designed. Eleven combinations of two above gRNAs (dual-gRNAs) covering the regulatory region of HBV were chosen. The efficiency of each gRNA and 11 dual-gRNAs on the suppression of HBV (genotypes A-D) replication was examined by the measurement of HBV surface antigen (HBsAg) or e antigen (HBeAg) in the culture supernatant. The destruction of HBV-expressing vector was examined in HuH7 cells co-transfected with dual-gRNAs and HBV-expressing vector using polymerase chain reaction (PCR) and sequencing method, and the destruction of cccDNA was examined in HepAD38 cells using KCl precipitation, plasmid-safe ATP-dependent DNase (PSAD) digestion, rolling circle amplification and quantitative PCR combined method. The cytotoxicity of these gRNAs was assessed by a mitochondrial tetrazolium assay. RESULTS: All of gRNAs could significantly reduce HBsAg or HBeAg production in the culture supernatant, which was dependent on the region in which gRNA against. All of dual gRNAs could efficiently suppress HBsAg and/or HBeAg production for HBV of genotypes A-D, and the efficacy of dual gRNAs in suppressing HBsAg and/or HBeAg production was significantly increased when compared to the single gRNA used alone. Furthermore, by PCR direct sequencing we confirmed that these dual gRNAs could specifically destroy HBV expressing template by removing the fragment between the cleavage sites of the two used gRNAs. Most importantly, gRNA-5 and gRNA-12 combination not only could efficiently suppressing HBsAg and/or HBeAg production, but also destroy the cccDNA reservoirs in HepAD38 cells. CONCLUSION: These results suggested that CRISPR/Cas9 system could efficiently destroy HBV expressing templates (genotypes A-D) without apparent cytotoxicity. It may be a potential approach for eradication of persistent HBV cccDNA in chronic HBV

  2. CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems.

    PubMed

    Zhu, Lihua J; Holmes, Benjamin R; Aronin, Neil; Brodsky, Michael H

    2014-01-01

    CRISPR-Cas systems are a diverse family of RNA-protein complexes in bacteria that target foreign DNA sequences for cleavage. Derivatives of these complexes have been engineered to cleave specific target sequences depending on the sequence of a CRISPR-derived guide RNA (gRNA) and the source of the Cas9 protein. Important considerations for the design of gRNAs are to maximize aimed activity at the desired target site while minimizing off-target cleavage. Because of the rapid advances in the understanding of existing CRISPR-Cas9-derived RNA-guided nucleases and the development of novel RNA-guided nuclease systems, it is critical to have computational tools that can accommodate a wide range of different parameters for the design of target-specific RNA-guided nuclease systems. We have developed CRISPRseek, a highly flexible, open source software package to identify gRNAs that target a given input sequence while minimizing off-target cleavage at other sites within any selected genome. CRISPRseek will identify potential gRNAs that target a sequence of interest for CRISPR-Cas9 systems from different bacterial species and generate a cleavage score for potential off-target sequences utilizing published or user-supplied weight matrices with position-specific mismatch penalty scores. Identified gRNAs may be further filtered to only include those that occur in paired orientations for increased specificity and/or those that overlap restriction enzyme sites. For applications where gRNAs are desired to discriminate between two related sequences, CRISPRseek can rank gRNAs based on the difference between predicted cleavage scores in each input sequence. CRISPRseek is implemented as a Bioconductor package within the R statistical programming environment, allowing it to be incorporated into computational pipelines to automate the design of gRNAs for target sequences identified in a wide variety of genome-wide analyses. CRISPRseek is available under the GNU General Public Licence v3

  3. Adaptive, full-spectrum solar energy system

    DOEpatents

    Muhs, Jeffrey D.; Earl, Dennis D.

    2003-08-05

    An adaptive full spectrum solar energy system having at least one hybrid solar concentrator, at least one hybrid luminaire, at least one hybrid photobioreactor, and a light distribution system operably connected to each hybrid solar concentrator, each hybrid luminaire, and each hybrid photobioreactor. A lighting control system operates each component.

  4. Small scale adaptive optics experiment systems engineering

    NASA Technical Reports Server (NTRS)

    Boykin, William H.

    1993-01-01

    Assessment of the current technology relating to the laser power beaming system which in full scale is called the Beam Transmission Optical System (BTOS). Evaluation of system integration efforts are being conducted by the various government agencies and industry. Concepts are being developed for prototypes of adaptive optics for a BTOS.

  5. Adaptive Dialogue Systems for Assistive Living Environments

    ERIC Educational Resources Information Center

    Papangelis, Alexandros

    2013-01-01

    Adaptive Dialogue Systems (ADS) are intelligent systems, able to interact with users via multiple modalities, such as speech, gestures, facial expressions and others. Such systems are able to make conversation with their users, usually on a specific, narrow topic. Assistive Living Environments are environments where the users are by definition not…

  6. Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium.

    PubMed

    Bruder, Mark R; Pyne, Michael E; Moo-Young, Murray; Chung, Duane A; Chou, C Perry

    2016-10-15

    The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes.

  7. CRISPR-Cas immunity and mobile DNA: a new superfamily of DNA transposons encoding a Cas1 endonuclease.

    PubMed

    Hickman, Alison B; Dyda, Fred

    2014-01-01

    Mobile genetic elements such as DNA transposons are a feature of most genomes. The existence of novel DNA transposons can be inferred when whole genome sequencing reveals the presence of hallmarks of mobile elements such as terminal inverted repeats (TIRs) flanked by target site duplications (TSDs). A recent report describes a new superfamily of DNA transposons in the genomes of a few bacteria and archaea that possess TIRs and TSDs, and encode several conserved genes including a cas1 endonuclease gene, previously associated only with CRISPR-Cas adaptive immune systems. The data strongly suggests that these elements, designated 'casposons', are likely to be bona fide DNA transposons and that their Cas1 nucleases act as transposases and are possibly still active.

  8. Bipartite recognition of target RNAs activates DNA cleavage by the Type III-B CRISPR–Cas system

    PubMed Central

    Elmore, Joshua R.; Sheppard, Nolan F.; Ramia, Nancy; Deighan, Trace; Li, Hong; Terns, Rebecca M.; Terns, Michael P.

    2016-01-01

    CRISPR–Cas systems eliminate nucleic acid invaders in bacteria and archaea. The effector complex of the Type III-B Cmr system cleaves invader RNAs recognized by the CRISPR RNA (crRNA ) of the complex. Here we show that invader RNAs also activate the Cmr complex to cleave DNA. As has been observed for other Type III systems, Cmr eliminates plasmid invaders in Pyrococcus furiosus by a mechanism that depends on transcription of the crRNA target sequence within the plasmid. Notably, we found that the target RNA per se induces DNA cleavage by the Cmr complex in vitro. DNA cleavage activity does not depend on cleavage of the target RNA but notably does require the presence of a short sequence adjacent to the target sequence within the activating target RNA (rPAM [RNA protospacer-adjacent motif]). The activated complex does not require a target sequence (or a PAM) in the DNA substrate. Plasmid elimination by the P. furiosus Cmr system also does not require the Csx1 (CRISPR-associated Rossman fold [CARF] superfamily) protein. Plasmid silencing depends on the HD nuclease and Palm domains of the Cmr2 (Cas10 superfamily) protein. The results establish the Cmr complex as a novel DNA nuclease activated by invader RNAs containing a crRNA target sequence and a rPAM. PMID:26848045

  9. Examination of CRISPR/Cas9 design tools and the effect of target site accessibility on Cas9 activity.

    PubMed

    Lee, Ciaran M; Davis, Timothy H; Bao, Gang

    2017-03-16

    The recent adaptation of the CRISPR/Cas9 system for targeted genome engineering has led to its widespread applications in many fields worldwide. In order to better understand the design rules of CRISPR/Cas9 systems, several groups have carried out large library-based screens leading to some insight into sequence preferences among highly active target sites. To facilitate CRISPR/Cas9 design these studies have spawned a plethora of gRNA design tools with algorithms based solely on direct or indirect sequence features. Here we demonstrate that the predictive power of these tools is poor, suggesting that sequence features alone cannot accurately inform the cutting efficiency of a particular CRISPR/Cas9 gRNA design. Furthermore we demonstrate that DNA target site accessibility influences the activity of CRISPR/Cas9. With further optimisation we hypothesise that it will be possible to increase the predictive power of gRNA design tools by including both sequence and target site accessibility metrics. This article is protected by copyright. All rights reserved.

  10. Applying a complex adaptive system's understanding of health to primary care.

    PubMed

    Bircher, Johannes; Hahn, Eckhart G

    2016-01-01

    This paper explores the diagnostic and therapeutic potential of a new concept of health. Investigations into the nature of health have led to a new definition that explains health as a complex adaptive system (CAS) and is based on five components (a-e). Humans like all biological creatures must satisfactorily respond to (a) the demands of life. For this purpose they need (b) a biologically given potential (BGP) and (c) a personally acquired potential (PAP). These properties of individuals are embedded within (d) social and (e) environmental determinants of health. Between these five components of health there are 10 complex interactions that justify viewing health as a CAS. In each patient, the current state of health as a CAS evolved from the past, will move forward to a new future, and has to be analyzed and treated as an autonomous whole. A diagnostic procedure is suggested as follows: together with the patient, the five components and 10 complex interactions are assessed. This may help patients to better understand their situations and to recognize possible next steps that may be useful in order to evolve toward better health by themselves. In this process mutual trust in the patient-physician interaction is critical. The described approach offers new possibilities for helping patients improve their health prospects.

  11. Applying a complex adaptive system's understanding of health to primary care

    PubMed Central

    Bircher, Johannes; Hahn, Eckhart G.

    2016-01-01

    This paper explores the diagnostic and therapeutic potential of a new concept of health. Investigations into the nature of health have led to a new definition that explains health as a complex adaptive system (CAS) and is based on five components (a-e). Humans like all biological creatures must satisfactorily respond to (a) the demands of life. For this purpose they need (b) a biologically given potential (BGP) and (c) a personally acquired potential (PAP). These properties of individuals are embedded within (d) social and (e) environmental determinants of health. Between these five components of health there are 10 complex interactions that justify viewing health as a CAS. In each patient, the current state of health as a CAS evolved from the past, will move forward to a new future, and has to be analyzed and treated as an autonomous whole. A diagnostic procedure is suggested as follows: together with the patient, the five components and 10 complex interactions are assessed. This may help patients to better understand their situations and to recognize possible next steps that may be useful in order to evolve toward better health by themselves. In this process mutual trust in the patient-physician interaction is critical. The described approach offers new possibilities for helping patients improve their health prospects. PMID:27746902

  12. Adaptive Control for Microgravity Vibration Isolation System

    NASA Technical Reports Server (NTRS)

    Yang, Bong-Jun; Calise, Anthony J.; Craig, James I.; Whorton, Mark S.

    2005-01-01

    Most active vibration isolation systems that try to a provide quiescent acceleration environment for space science experiments have utilized linear design methods. In this paper, we address adaptive control augmentation of an existing classical controller that employs a high-gain acceleration feedback together with a low-gain position feedback to center the isolated platform. The control design feature includes parametric and dynamic uncertainties because the hardware of the isolation system is built as a payload-level isolator, and the acceleration Sensor exhibits a significant bias. A neural network is incorporated to adaptively compensate for the system uncertainties, and a high-pass filter is introduced to mitigate the effect of the measurement bias. Simulations show that the adaptive control improves the performance of the existing acceleration controller and keep the level of the isolated platform deviation to that of the existing control system.

  13. Generation of murine tumor cell lines deficient in MHC molecule surface expression using the CRISPR/Cas9 system

    PubMed Central

    Lenkl, Clarissa; Goyal, Ashish; Diederichs, Sven; Dickes, Elke; Osen, Wolfram

    2017-01-01

    In this study, the CRISPR/Cas9 technology was used to establish murine tumor cell lines, devoid of MHC I or MHC II surface expression, respectively. The melanoma cell line B16F10 and the murine breast cancer cell line EO-771, the latter stably expressing the tumor antigen NY-BR-1 (EO-NY), were transfected with an expression plasmid encoding a β2m-specific single guide (sg)RNA and Cas9. The resulting MHC I negative cells were sorted by flow cytometry to obtain single cell clones, and loss of susceptibility of peptide pulsed MHC I negative clones to peptide-specific CTL recognition was determined by IFNγ ELISpot assay. The β2m knockout (KO) clones did not give rise to tumors in syngeneic mice (C57BL/6N), unless NK cells were depleted, suggesting that outgrowth of the β2m KO cell lines was controlled by NK cells. Using sgRNAs targeting the β-chain encoding locus of the IAb molecule we also generated several B16F10 MHC II KO clones. Peptide loaded B16F10 MHC II KO cells were insusceptible to recognition by OT-II cells and tumor growth was unaltered compared to parental B16F10 cells. Thus, in our hands the CRISPR/Cas9 system has proven to be an efficient straight forward strategy for the generation of MHC knockout cell lines. Such cell lines could serve as parental cells for co-transfection of compatible HLA alleles together with human tumor antigens of interest, thereby facilitating the generation of HLA matched transplantable tumor models, e.g. in HLAtg mouse strains of the newer generation, lacking cell surface expression of endogenous H2 molecules. In addition, our tumor cell lines established might offer a useful tool to investigate tumor reactive T cell responses that function independently from MHC molecule surface expression by the tumor. PMID:28301575

  14. Operational Results of an Adaptive SDI System.

    ERIC Educational Resources Information Center

    Sage, C. R.; Fitzwater, D. R.

    The Ames Laboratory SDI system requires a minimum of human intervention. The adaptability of the system provides two major contributions to information dissemination. (1) The user benefits proportionately from the amount of effort he expends in setting up his profile and the diligence in sending back responses. (2) The document input has only to…

  15. The adaptive control system of acetylene generator

    NASA Astrophysics Data System (ADS)

    Kovaliuk, D. O.; Kovaliuk, Oleg; Burlibay, Aron; Gromaszek, Konrad

    2015-12-01

    The method of acetylene production in acetylene generator was analyzed. It was found that impossible to provide the desired process characteristics by the PID-controller. The adaptive control system of acetylene generator was developed. The proposed system combines the classic controller and fuzzy subsystem for controller parameters tuning.

  16. Multiprocessor Adaptive Control Of A Dynamic System

    NASA Technical Reports Server (NTRS)

    Juang, Jer-Nan; Hyland, David C.

    1995-01-01

    Architecture for fully autonomous digital electronic control system developed for use in identification and adaptive control of dynamic system. Architecture modular and hierarchical. Combines relatively simple, standardized processing units into complex parallel-processing subsystems. Although architecture based on neural-network concept, processing units themselves not neural networks; processing units implemented by programming of currently available microprocessors.

  17. Expanding CRISPR/Cas9 Genome Editing Capacity in Zebrafish Using SaCas9

    PubMed Central

    Feng, Yan; Chen, Cheng; Han, Yuxiang; Chen, Zelin; Lu, Xiaochan; Liang, Fang; Li, Song; Qin, Wei; Lin, Shuo

    2016-01-01

    The type II CRISPR/Cas9 system has been used widely for genome editing in zebrafish. However, the requirement for the 5′-NGG-3′ protospacer-adjacent motif (PAM) of Cas9 from Streptococcus pyogenes (SpCas9) limits its targeting sequences. Here, we report that a Cas9 ortholog from Staphylococcus aureus (SaCas9), and its KKH variant, successfully induced targeted mutagenesis with high frequency in zebrafish. Confirming previous findings, the SpCas9 variant, VQR, can also induce targeted mutations in zebrafish. Bioinformatics analysis of these new Cas targets suggests that the number of available target sites in the zebrafish genome can be greatly expanded. Collectively, the expanded target repertoire of Cas9 in zebrafish should further facilitate the utility of this organism for genetic studies of vertebrate biology. PMID:27317783

  18. 207 EFFICIENT GENERATION OF MYOSTATIN PROMOTER MUTATIONS IN BOVINE EMBRYOS USING THE CRISPR/Cas9 SYSTEM.

    PubMed

    Pinzon, C A; Snyder, M; Pryor, J; Thompson, B; Golding, M; Long, C

    2016-01-01

    . For this, IVF-derived zygotes were randomly assigned to 3 different treatment groups Set 1, Set 2, or Null (no sgRNA) for microinjections. Each zygote was injected with ~100 pL of trophectoderm buffer containing 50ngµL(-1) of total sgRNA, 10ngµL(-1) of Cas9 mRNA, and 30ngµL(-1) of Cas9 protein with 1mgmL(-1) of fluorescent dextran. Day 7 post-IVF blastocysts were lysed and DNA was extracted for PCR amplification of the target region. In Set 1, 16 of 19 embryos (94.12%) were successfully edited, whereas in Set 2 there were 11 of 17 embryos (64.7%) edited. In both sets of sgRNA there was a high degree of mosaicism, with only 1 embryo demonstrating a homozygous deletion. In conclusion, CRISPR/Cas9 acts over the course of the first few cleavage divisions Further research is necessary to refine the CRISPR/Cas9 system for inducing genetic mutations in bovine embryos.

  19. Enhanced specificity and efficiency of the CRISPR/Cas9 system with optimized sgRNA parameters in Drosophila.

    PubMed

    Ren, Xingjie; Yang, Zhihao; Xu, Jiang; Sun, Jin; Mao, Decai; Hu, Yanhui; Yang, Su-Juan; Qiao, Huan-Huan; Wang, Xia; Hu, Qun; Deng, Patricia; Liu, Lu-Ping; Ji, Jun-Yuan; Li, Jin Billy; Ni, Jian-Quan

    2014-11-06

    The CRISPR/Cas9 system has recently emerged as a powerful tool for functional genomic studies in Drosophila melanogaster. However, single-guide RNA (sgRNA) parameters affecting the specificity and efficiency of the system in flies are still not clear. Here, we found that off-target effects did not occur in regions of genomic DNA with three or more nucleotide mismatches to sgRNAs. Importantly, we document for a strong positive correlation between mutagenesis efficiency and sgRNA GC content of the six protospacer-adjacent motif-proximal nucleotides (PAMPNs). Furthermore, by injecting well-designed sgRNA plasmids at the optimal concentration we determined, we could efficiently generate mutations in four genes in one step. Finally, we generated null alleles of HP1a using optimized parameters through homology-directed repair and achieved an overall mutagenesis rate significantly higher than previously reported. Our work demonstrates a comprehensive optimization of sgRNA and promises to vastly simplify CRISPR/Cas9 experiments in Drosophila.

  20. RNA viruses as complex adaptive systems.

    PubMed

    Elena, Santiago F; Sanjuán, Rafael

    2005-07-01

    RNA viruses have high mutation rates and so their populations exist as dynamic and complex mutant distributions. It has been consistently observed that when challenged with a new environment, viral populations adapt following hyperbolic-like kinetics: adaptation is initially very rapid, but then slows down as fitness reaches an asymptotic value. These adaptive dynamics have been explained in terms of populations moving towards the top of peaks on rugged fitness landscapes. Fitness fluctuations of varying magnitude are observed during adaptation. Often the presence of fluctuations in the evolution of physical systems indicates some form of self-organization, or where many components of the system are simultaneously involved. Here we analyze data from several in vitro evolution experiments carried out with vesicular stomatitis virus (VSV) looking for the signature of criticality and scaling. Long-range fitness correlations have been detected during the adaptive process. We also found that the magnitude of fitness fluctuations, far from being trivial, conform to a Weibull probability distribution function, suggesting that viral adaptation belongs to a broad category of phenomena previously documented in other fields and related with emergence.

  1. Adaptation in the auditory system: an overview.

    PubMed

    Pérez-González, David; Malmierca, Manuel S

    2014-01-01

    The early stages of the auditory system need to preserve the timing information of sounds in order to extract the basic features of acoustic stimuli. At the same time, different processes of neuronal adaptation occur at several levels to further process the auditory information. For instance, auditory nerve fiber responses already experience adaptation of their firing rates, a type of response that can be found in many other auditory nuclei and may be useful for emphasizing the onset of the stimuli. However, it is at higher levels in the auditory hierarchy where more sophisticated types of neuronal processing take place. For example, stimulus-specific adaptation, where neurons show adaptation to frequent, repetitive stimuli, but maintain their responsiveness to stimuli with different physical characteristics, thus representing a distinct kind of processing that may play a role in change and deviance detection. In the auditory cortex, adaptation takes more elaborate forms, and contributes to the processing of complex sequences, auditory scene analysis and attention. Here we review the multiple types of adaptation that occur in the auditory system, which are part of the pool of resources that the neurons employ to process the auditory scene, and are critical to a proper understanding of the neuronal mechanisms that govern auditory perception.

  2. Cas13b Is a Type VI-B CRISPR-Associated RNA-Guided RNase Differentially Regulated by Accessory Proteins Csx27 and Csx28.

    PubMed

    Smargon, Aaron A; Cox, David B T; Pyzocha, Neena K; Zheng, Kaijie; Slaymaker, Ian M; Gootenberg, Jonathan S; Abudayyeh, Omar A; Essletzbichler, Patrick; Shmakov, Sergey; Makarova, Kira S; Koonin, Eugene V; Zhang, Feng

    2017-02-16

    CRISPR-Cas adaptive immune systems defend microbes against foreign nucleic acids via RNA-guided endonucleases. Using a computational sequence database mining approach, we identify two class 2 CRISPR-Cas systems (subtype VI-B) that lack Cas1 and Cas2 and encompass a single large effector protein, Cas13b, along with one of two previously uncharacterized associated proteins, Csx27 and Csx28. We establish that these CRISPR-Cas systems can achieve RNA interference when heterologously expressed. Through a combination of biochemical and genetic experiments, we show that Cas13b processes its own CRISPR array with short and long direct repeats, cleaves target RNA, and exhibits collateral RNase activity. Using an E. coli essential gene screen, we demonstrate that Cas13b has a double-sided protospacer-flanking sequence and elucidate RNA secondary structure requirements for targeting. We also find that Csx27 represses, whereas Csx28 enhances, Cas13b-mediated RNA interference. Characterization of these CRISPR systems creates opportunities to develop tools to manipulate and monitor cellular transcripts.

  3. Managing adaptively for multifunctionality in agricultural systems.

    PubMed

    Hodbod, Jennifer; Barreteau, Olivier; Allen, Craig; Magda, Danièle

    2016-12-01

    The critical importance of agricultural systems for food security and as a dominant global landcover requires management that considers the full dimensions of system functions at appropriate scales, i.e. multifunctionality. We propose that adaptive management is the most suitable management approach for such goals, given its ability to reduce uncertainty over time and support multiple objectives within a system, for multiple actors. As such, adaptive management may be the most appropriate method for sustainably intensifying production whilst increasing the quantity and quality of ecosystem services. However, the current assessment of performance of agricultural systems doesn't reward ecosystem service provision. Therefore, we present an overview of the ecosystem functions agricultural systems should and could provide, coupled with a revised definition for assessing the performance of agricultural systems from a multifunctional perspective that, when all satisfied, would create adaptive agricultural systems that can increase production whilst ensuring food security and the quantity and quality of ecosystem services. The outcome of this high level of performance is the capacity to respond to multiple shocks without collapse, equity and triple bottom line sustainability. Through the assessment of case studies, we find that alternatives to industrialized agricultural systems incorporate more functional goals, but that there are mixed findings as to whether these goals translate into positive measurable outcomes. We suggest that an adaptive management perspective would support the implementation of a systematic analysis of the social, ecological and economic trade-offs occurring within such systems, particularly between ecosystem services and functions, in order to provide suitable and comparable assessments. We also identify indicators to monitor performance at multiple scales in agricultural systems which can be used within an adaptive management framework to increase

  4. The ERIS adaptive optics system

    NASA Astrophysics Data System (ADS)

    Riccardi, A.; Esposito, S.; Agapito, G.; Antichi, J.; Biliotti, V.; Blain, C.; Briguglio, R.; Busoni, L.; Carbonaro, L.; Di Rico, G.; Giordano, C.; Pinna, E.; Puglisi, A.; Spanò, P.; Xompero, M.; Baruffolo, A.; Kasper, M.; Egner, S.; Suàrez Valles, M.; Soenke, C.; Downing, M.; Reyes, J.

    2016-07-01

    ERIS is the new AO instrument for VLT-UT4 led by a Consortium of Max-Planck Institut fuer Extraterrestrische Physik, UK-ATC, ETH-Zurich, ESO and INAF. The ERIS AO system provides NGS mode to deliver high contrast correction and LGS mode to extend high Strehl performance to large sky coverage. The AO module includes NGS and LGS wavefront sensors and, with VLT-AOF Deformable Secondary Mirror and Laser Facility, will provide AO correction to the high resolution imager NIX (1-5um) and the IFU spectrograph SPIFFIER (1-2.5um). In this paper we present the preliminary design of the ERIS AO system and the estimated correction performance.

  5. Adaptive System Modeling for Spacecraft Simulation

    NASA Technical Reports Server (NTRS)

    Thomas, Justin

    2011-01-01

    This invention introduces a methodology and associated software tools for automatically learning spacecraft system models without any assumptions regarding system behavior. Data stream mining techniques were used to learn models for critical portions of the International Space Station (ISS) Electrical Power System (EPS). Evaluation on historical ISS telemetry data shows that adaptive system modeling reduces simulation error anywhere from 50 to 90 percent over existing approaches. The purpose of the methodology is to outline how someone can create accurate system models from sensor (telemetry) data. The purpose of the software is to support the methodology. The software provides analysis tools to design the adaptive models. The software also provides the algorithms to initially build system models and continuously update them from the latest streaming sensor data. The main strengths are as follows: Creates accurate spacecraft system models without in-depth system knowledge or any assumptions about system behavior. Automatically updates/calibrates system models using the latest streaming sensor data. Creates device specific models that capture the exact behavior of devices of the same type. Adapts to evolving systems. Can reduce computational complexity (faster simulations).

  6. Evolving Systems and Adaptive Key Component Control

    NASA Technical Reports Server (NTRS)

    Frost, Susan A.; Balas, Mark J.

    2009-01-01

    We propose a new framework called Evolving Systems to describe the self-assembly, or autonomous assembly, of actively controlled dynamical subsystems into an Evolved System with a higher purpose. An introduction to Evolving Systems and exploration of the essential topics of the control and stability properties of Evolving Systems is provided. This chapter defines a framework for Evolving Systems, develops theory and control solutions for fundamental characteristics of Evolving Systems, and provides illustrative examples of Evolving Systems and their control with adaptive key component controllers.

  7. Adaptive fuzzy system for 3-D vision

    NASA Technical Reports Server (NTRS)

    Mitra, Sunanda

    1993-01-01

    An adaptive fuzzy system using the concept of the Adaptive Resonance Theory (ART) type neural network architecture and incorporating fuzzy c-means (FCM) system equations for reclassification of cluster centers was developed. The Adaptive Fuzzy Leader Clustering (AFLC) architecture is a hybrid neural-fuzzy system which learns on-line in a stable and efficient manner. The system uses a control structure similar to that found in the Adaptive Resonance Theory (ART-1) network to identify the cluster centers initially. The initial classification of an input takes place in a two stage process; a simple competitive stage and a distance metric comparison stage. The cluster prototypes are then incrementally updated by relocating the centroid positions from Fuzzy c-Means (FCM) system equations for the centroids and the membership values. The operational characteristics of AFLC and the critical parameters involved in its operation are discussed. The performance of the AFLC algorithm is presented through application of the algorithm to the Anderson Iris data, and laser-luminescent fingerprint image data. The AFLC algorithm successfully classifies features extracted from real data, discrete or continuous, indicating the potential strength of this new clustering algorithm in analyzing complex data sets. The hybrid neuro-fuzzy AFLC algorithm will enhance analysis of a number of difficult recognition and control problems involved with Tethered Satellite Systems and on-orbit space shuttle attitude controller.

  8. Adaptive synchronization and anticipatory dynamical systems

    NASA Astrophysics Data System (ADS)

    Yang, Ying-Jen; Chen, Chun-Chung; Lai, Pik-Yin; Chan, C. K.

    2015-09-01

    Many biological systems can sense periodical variations in a stimulus input and produce well-timed, anticipatory responses after the input is removed. Such systems show memory effects for retaining timing information in the stimulus and cannot be understood from traditional synchronization consideration of passive oscillatory systems. To understand this anticipatory phenomena, we consider oscillators built from excitable systems with the addition of an adaptive dynamics. With such systems, well-timed post-stimulus responses similar to those from experiments can be obtained. Furthermore, a well-known model of working memory is shown to possess similar anticipatory dynamics when the adaptive mechanism is identified with synaptic facilitation. The last finding suggests that this type of oscillator can be common in neuronal systems with plasticity.

  9. Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system.

    PubMed

    Guo, Rihong; Wan, Yongjie; Xu, Dan; Cui, Libin; Deng, Mingtian; Zhang, Guomin; Jia, Ruoxin; Zhou, Wenjun; Wang, Zhen; Deng, Kaiping; Huang, Mingrui; Wang, Feng; Zhang, Yanli

    2016-07-15

    Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes.

  10. Generation and evaluation of Myostatin knock-out rabbits and goats using CRISPR/Cas9 system

    PubMed Central

    Guo, Rihong; Wan, Yongjie; Xu, Dan; Cui, Libin; Deng, Mingtian; Zhang, Guomin; Jia, Ruoxin; Zhou, Wenjun; Wang, Zhen; Deng, Kaiping; Huang, Mingrui; Wang, Feng; Zhang, Yanli

    2016-01-01

    Myostatin (Mstn) is a conserved negative regulator of skeletal muscle mass in mammals. However, whether precise disruption of Mstn in livestock can be achieved and safely used to improve meat productivity has not been proven. We applied CRISPR/Cas9 system to generate Mstn knock-out (KO) rabbits and goats and then analyzed the changes in their phenotypes to answer this question. We efficiently generated 24 Mstn KO rabbits out of 32 newborn infants after embryo injection with two sgRNAs targeting rabbit Mstn, and found that the Mstn KO rabbits exhibited increased birthweight and a significantly increase in the weight ratios of the quadriceps and biceps muscles to the whole body. Mstn KO also caused high probability of enlarged tongue phenomenon and severe health problems such as stillbirth and early stage death. Using the same method, one out of four goats was generated with edition at Mstn locus. The early stage growth rate of this goat outperformed the control goats. In conclusion, we efficiently generated Mstn KO rabbits and goats using CRISPR/Cas9 technology. However, Mstn KO causes severe health problems and may also have the same effects on other species. This safety issue must be studied further before applied to animal reproduction processes. PMID:27417210

  11. Comparative assessments of CRISPR-Cas nucleases' cleavage efficiency in planta.

    PubMed

    Johnson, Ross A; Gurevich, Vyacheslav; Filler, Shdema; Samach, Aviva; Levy, Avraham A

    2015-01-01

    Custom-designed nucleases can enable precise plant genome editing by catalyzing DNA-breakage at specific targets to stimulate targeted mutagenesis or gene replacement. The CRISPR-Cas system, with its target-specifying RNA molecule to direct the Cas9 nuclease, is a recent addition to existing nucleases that bind and cleave the target through linked protein domains (e.g. TALENs and zinc-finger nucleases). We have conducted a comparative study of these different types of custom-designed nucleases and we have assessed various components of the CRISPR-Cas system. For this purpose, we have adapted our previously reported assay for cleavage-dependent luciferase gene correction in Nicotiana benthamiana leaves (Johnson et al. in Plant Mol Biol 82(3):207-221, 2013). We found that cleavage by CRISPR-Cas was more efficient than cleavage of the same target by TALENs. We also compared the cleavage efficiency of the Streptococcus pyogenes Cas9 protein based on expression using three different Cas9 gene variants. We found significant differences in cleavage efficiency between these variants, with human and Arabidopsis thaliana codon-optimized genes having the highest cleavage efficiencies. We compared the activity of 12 de novo-designed single synthetic guide RNA (sgRNA) constructs, and found their cleavage efficiency varied drastically when using the same Cas9 nuclease. Finally, we show that, for one of the targets tested with our assay, we could induce a germinally-transmitted deletion in a repeat array in A. thaliana. This work emphasizes the efficiency of the CRISPR-Cas system in plants. It also shows that further work is needed to be able to predict the optimal design of sgRNAs or Cas9 variants.

  12. Robust adaptive control of HVDC systems

    SciTech Connect

    Reeve, J.; Sultan, M. )

    1994-07-01

    The transient performance of an HVDC power system is highly dependent on the parameters of the current/voltage regulators of the converter controls. In order to better accommodate changes in system structure or dc operating conditions, this paper introduces a new adaptive control strategy. The advantages of automatic tuning for continuous fine tuning are combined with predetermined gain scheduling in order to achieve robustness for large disturbances. Examples are provided for a digitally simulated back-to-back dc system.

  13. Fitting CRISPR-associated Cas3 into the helicase family tree.

    PubMed

    Jackson, Ryan N; Lavin, Matthew; Carter, Joshua; Wiedenheft, Blake

    2014-02-01

    Helicases utilize NTPs to modulate their binding to nucleic acids and many of these enzymes also unwind DNA or RNA duplexes in an NTP-dependent fashion. These proteins are phylogenetically related but functionally diverse, with essential roles in virtually all aspects of nucleic acid metabolism. A new class of helicases associated with RNA-guided adaptive immune systems in bacteria and archaea has recently been identified. Prokaryotes acquire resistance to invading genetic parasites by integrating short fragments of foreign nucleic acids into repetitive loci in the host chromosome known as CRISPRs (Clustered Regularly Interspaced Short Palindromic Repeats). CRISPR-associated gene 3 (cas3) encodes a conserved helicase protein that is essential for phage defense. Here we review recent advances in Cas3 biology, and provide a new phylogenetic framework that positions Cas3 in the helicase family tree. We anticipate that this Cas3 phylogeny will guide future biochemical and structural studies.

  14. An adaptive learning control system for aircraft

    NASA Technical Reports Server (NTRS)

    Mekel, R.; Nachmias, S.

    1976-01-01

    A learning control system is developed which blends the gain scheduling and adaptive control into a single learning system that has the advantages of both. An important feature of the developed learning control system is its capability to adjust the gain schedule in a prescribed manner to account for changing aircraft operating characteristics. Furthermore, if tests performed by the criteria of the learning system preclude any possible change in the gain schedule, then the overall system becomes an ordinary gain scheduling system. Examples are discussed.

  15. Final Report - Regulatory Considerations for Adaptive Systems

    NASA Technical Reports Server (NTRS)

    Wilkinson, Chris; Lynch, Jonathan; Bharadwaj, Raj

    2013-01-01

    This report documents the findings of a preliminary research study into new approaches to the software design assurance of adaptive systems. We suggest a methodology to overcome the software validation and verification difficulties posed by the underlying assumption of non-adaptive software in the requirementsbased- testing verification methods in RTCA/DO-178B and C. An analysis of the relevant RTCA/DO-178B and C objectives is presented showing the reasons for the difficulties that arise in showing satisfaction of the objectives and suggested additional means by which they could be satisfied. We suggest that the software design assurance problem for adaptive systems is principally one of developing correct and complete high level requirements and system level constraints that define the necessary system functional and safety properties to assure the safe use of adaptive systems. We show how analytical techniques such as model based design, mathematical modeling and formal or formal-like methods can be used to both validate the high level functional and safety requirements, establish necessary constraints and provide the verification evidence for the satisfaction of requirements and constraints that supplements conventional testing. Finally the report identifies the follow-on research topics needed to implement this methodology.

  16. Biased and Unbiased Methods for the Detection of Off-Target Cleavage by CRISPR/Cas9: An Overview.

    PubMed

    Martin, Francisco; Sánchez-Hernández, Sabina; Gutiérrez-Guerrero, Alejandra; Pinedo-Gomez, Javier; Benabdellah, Karim

    2016-09-08

    The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 endonuclease (Cas9) derived from bacterial adaptive immune systems is a revolutionary tool used in both basic and applied science. It is a versatile system that enables the genome of different species to be modified by generating double strand breaks (DSBs) at specific locations. However, all of the CRISPR/Cas9 systems can also produce DSBs at off-target sites that differ substantially from on-target sites. The generation of DSBs in locations outside the intended site can produce mutations that need to be carefully monitored, especially when using these tools for therapeutic purposes. However, off-target analyses of the CRISPR/Cas9 system have been very challenging, particularly when performed directly in cells. In this manuscript, we review the different strategies developed to identify off-targets generated by CRISPR/cas9 systems and other specific nucleases (ZFNs, TALENs) in real target cells.

  17. Harm reduction as a complex adaptive system: A dynamic framework for analyzing Tanzanian policies concerning heroin use

    PubMed Central

    Ratliff, Eric A.; Kaduri, Pamela; Masao, Frank; Mbwambo, Jessie K.K.; McCurdy, Sheryl A.

    2016-01-01

    Contrary to popular belief, policies on drug use are not always based on scientific evidence or composed in a rational manner. Rather, decisions concerning drug policies reflect the negotiation of actors’ ambitions, values, and facts as they organize in different ways around the perceived problems associated with illicit drug use. Drug policy is thus best represented as a complex adaptive system (CAS) that is dynamic, self-organizing, and coevolving. In this analysis, we use a CAS framework to examine how harm reduction emerged around heroin trafficking and use in Tanzania over the past thirty years (1985-present). This account is an organizational ethnography based on of the observant participation of the authors as actors within this system. We review the dynamic history and self-organizing nature of harm reduction, noting how interactions among system actors and components have coevolved with patterns of heroin us, policing, and treatment activities over time. Using a CAS framework, we describe harm reduction as a complex process where ambitions, values, facts, and technologies interact in the Tanzanian socio-political environment. We review the dynamic history and self-organizing nature of heroin policies, noting how the interactions within and between competing prohibitionist and harm reduction policies have changed with patterns of heroin use, policing, and treatment activities over time. Actors learn from their experiences to organize with other actors, align their values and facts, and implement new policies. Using a CAS approach provides researchers and policy actors a better understanding of patterns and intricacies in drug policy. This knowledge of how the system works can help improve the policy process through adaptive action to introduce new actors, different ideas, and avenues for communication into the system. PMID:26790689

  18. Harm reduction as a complex adaptive system: A dynamic framework for analyzing Tanzanian policies concerning heroin use.

    PubMed

    Ratliff, Eric A; Kaduri, Pamela; Masao, Frank; Mbwambo, Jessie K K; McCurdy, Sheryl A

    2016-04-01

    Contrary to popular belief, policies on drug use are not always based on scientific evidence or composed in a rational manner. Rather, decisions concerning drug policies reflect the negotiation of actors' ambitions, values, and facts as they organize in different ways around the perceived problems associated with illicit drug use. Drug policy is thus best represented as a complex adaptive system (CAS) that is dynamic, self-organizing, and coevolving. In this analysis, we use a CAS framework to examine how harm reduction emerged around heroin trafficking and use in Tanzania over the past thirty years (1985-present). This account is an organizational ethnography based on of the observant participation of the authors as actors within this system. We review the dynamic history and self-organizing nature of harm reduction, noting how interactions among system actors and components have coevolved with patterns of heroin us, policing, and treatment activities over time. Using a CAS framework, we describe harm reduction as a complex process where ambitions, values, facts, and technologies interact in the Tanzanian sociopolitical environment. We review the dynamic history and self-organizing nature of heroin policies, noting how the interactions within and between competing prohibitionist and harm reduction policies have changed with patterns of heroin use, policing, and treatment activities over time. Actors learn from their experiences to organize with other actors, align their values and facts, and implement new policies. Using a CAS approach provides researchers and policy actors a better understanding of patterns and intricacies in drug policy. This knowledge of how the system works can help improve the policy process through adaptive action to introduce new actors, different ideas, and avenues for communication into the system.

  19. Petascale IO Using The Adaptable IO System

    SciTech Connect

    Lofstead, J.; Klasky, Scott A; Abbasi, H.

    2009-01-01

    ADIOS, the adaptable IO system, has demonstrated excellent scalability to 29,000 cores. With the introduction of the XT5 upgrades to Jaguar, new optimizations are required to successfully reach 140,000+ cores. This paper explains the techniques employed and shows the performance levels attained.

  20. User Modeling in Adaptive Hypermedia Educational Systems

    ERIC Educational Resources Information Center

    Martins, Antonio Constantino; Faria, Luiz; Vaz de Carvalho, Carlos; Carrapatoso, Eurico

    2008-01-01

    This document is a survey in the research area of User Modeling (UM) for the specific field of Adaptive Learning. The aims of this document are: To define what it is a User Model; To present existing and well known User Models; To analyze the existent standards related with UM; To compare existing systems. In the scientific area of User Modeling…

  1. Adaptive Control of Nonlinear and Stochastic Systems

    DTIC Science & Technology

    1991-01-14

    Hernmndez-Lerma and S.I. Marcus, Nonparametric adaptive control of dis- crete time partially observable stochastic systems, Journal of Mathematical Analysis and Applications 137... Journal of Mathematical Analysis and Applications 137 (1989), 485-514. [19] A. Arapostathis and S.I. Marcus, Analysis of an identification algorithm

  2. Adaptive control system for gas producing wells

    SciTech Connect

    Fedor, Pashchenko; Sergey, Gulyaev; Alexander, Pashchenko

    2015-03-10

    Optimal adaptive automatic control system for gas producing wells cluster is proposed intended for solving the problem of stabilization of the output gas pressure in the cluster at conditions of changing gas flow rate and changing parameters of the wells themselves, providing the maximum high resource of hardware elements of automation.

  3. Advantages of using the CRISPR/Cas9 system of genome editing to investigate male reproductive mechanisms using mouse models

    PubMed Central

    Young, Samantha AM; Aitken, R John; Ikawa, Masahito

    2015-01-01

    Gene disruption technology has long been beneficial for the study of male reproductive biology. However, because of the time and cost involved, this technology was not a viable method except in specialist laboratories. The advent of the CRISPR/Cas9 system of gene disruption has ushered in a new era of genetic investigation. Now, it is possible to generate gene-disrupted mouse models in very little time and at very little cost. This Highlight article discusses the application of this technology to study the genetics of male fertility and looks at some of the future uses of this system that could be used to reveal the essential and nonessential genetic components of male reproductive mechanisms. PMID:25994645

  4. Advantages of using the CRISPR/Cas9 system of genome editing to investigate male reproductive mechanisms using mouse models.

    PubMed

    Young, Samantha A M; Aitken, R John; Ikawa, Masahito

    2015-01-01

    Gene disruption technology has long been beneficial for the study of male reproductive biology. However, because of the time and cost involved, this technology was not a viable method except in specialist laboratories. The advent of the CRISPR/Cas9 system of gene disruption has ushered in a new era of genetic investigation. Now, it is possible to generate gene-disrupted mouse models in very little time and at very little cost. This Highlight article discusses the application of this technology to study the genetics of male fertility and looks at some of the future uses of this system that could be used to reveal the essential and nonessential genetic components of male reproductive mechanisms.

  5. An adaptive strategy for controlling chaotic system.

    PubMed

    Cao, Yi-Jia; Hang, Hong-Xian

    2003-01-01

    This paper presents an adaptive strategy for controlling chaotic systems. By employing the phase space reconstruction technique in nonlinear dynamical systems theory, the proposed strategy transforms the nonlinear system into canonical form, and employs a nonlinear observer to estimate the uncertainties and disturbances of the nonlinear system, and then establishes a state-error-like feedback law. The developed control scheme allows chaos control in spite of modeling errors and parametric variations. The effectiveness of the proposed approach has been demonstrated through its applications to two well-known chaotic systems: Duffing oscillator and Rössler chaos.

  6. Generation of Genetically Modified Mice using the CRISPR-Cas9 Genome-Editing System

    PubMed Central

    Rongvaux, Anthony; Stein, Judith; Hughes, Cynthia; Flavell, Richard A.

    2016-01-01

    Genetically modified mice are extremely valuable tools for studying gene function and human diseases. Although the generation of mice with specific genetic modifications through traditional methods using homologous recombination in embryonic stem (ES) has been invaluable in the last two decades, it is an extremely costly, time-consuming and in some cases uncertain technology. The recently described CRISPR/Cas9 genome-editing technology significantly reduces the time and the cost that are required to generate genetically engineered mice, allowing scientist to test more precise and bold hypothesis in vivo. Using this revolutionary methodology we have generated more than one hundred novel genetically engineered mouse strains. In the current protocol, we describe in detail the optimal conditions to generate mice carrying point mutations, chromosomal deletions, conditional alleles, fusion tags or endogenous reporters. PMID:26832688

  7. Generation of Genetically Modified Mice Using the CRISPR-Cas9 Genome-Editing System.

    PubMed

    Henao-Mejia, Jorge; Williams, Adam; Rongvaux, Anthony; Stein, Judith; Hughes, Cynthia; Flavell, Richard A

    2016-02-01

    Genetically modified mice are extremely valuable tools for studying gene function and human diseases. Although the generation of mice with specific genetic modifications through traditional methods using homologous recombination in embryonic stem cells has been invaluable in the last two decades, it is an extremely costly, time-consuming, and, in some cases, uncertain technology. The recently described CRISPR-Cas9 genome-editing technology significantly reduces the time and the cost that are required to generate genetically engineered mice, allowing scientists to test more precise and bold hypotheses in vivo. Using this revolutionary methodology we have generated more than 100 novel genetically engineered mouse strains. In the current protocol, we describe in detail the optimal conditions to generate mice carrying point mutations, chromosomal deletions, conditional alleles, fusion tags, or endogenous reporters.

  8. Adaptable Transponder for Multiple Telemetry Systems

    NASA Technical Reports Server (NTRS)

    Sims, William Herbert, III (Inventor); Varnavas, Kosta A. (Inventor)

    2014-01-01

    The present invention is a stackable telemetry circuit board for use in telemetry systems for satellites and other purposes. The present invention incorporates previously-qualified interchangeable circuit boards, or "decks," that perform functions such as power, signal receiving and transmission, and processing. Each deck is adapted to serve a range of telemetry applications. This provides flexibility in the construction of the stackable telemetry circuit board and significantly reduces the cost and time necessary to develop a telemetry system.

  9. Resilience through adaptation

    PubMed Central

    van Voorn, George A. K.; Ligtenberg, Arend; Molenaar, Jaap

    2017-01-01

    Adaptation of agents through learning or evolution is an important component of the resilience of Complex Adaptive Systems (CAS). Without adaptation, the flexibility of such systems to cope with outside pressures would be much lower. To study the capabilities of CAS to adapt, social simulations with agent-based models (ABMs) provide a helpful tool. However, the value of ABMs for studying adaptation depends on the availability of methodologies for sensitivity analysis that can quantify resilience and adaptation in ABMs. In this paper we propose a sensitivity analysis methodology that is based on comparing time-dependent probability density functions of output of ABMs with and without agent adaptation. The differences between the probability density functions are quantified by the so-called earth-mover’s distance. We use this sensitivity analysis methodology to quantify the probability of occurrence of critical transitions and other long-term effects of agent adaptation. To test the potential of this new approach, it is used to analyse the resilience of an ABM of adaptive agents competing for a common-pool resource. Adaptation is shown to contribute positively to the resilience of this ABM. If adaptation proceeds sufficiently fast, it may delay or avert the collapse of this system. PMID:28196372

  10. Adaptive P300 based control system

    PubMed Central

    Jin, Jing; Allison, Brendan Z.; Sellers, Eric W.; Brunner, Clemens; Horki, Petar; Wang, Xingyu; Neuper, Christa

    2015-01-01

    An adaptive P300 brain-computer interface (BCI) using a 12 × 7 matrix explored new paradigms to improve bit rate and accuracy. During online use, the system adaptively selects the number of flashes to average. Five different flash patterns were tested. The 19-flash paradigm represents the typical row/column presentation (i.e., 12 columns and 7 rows). The 9- and 14-flash A & B paradigms present all items of the 12 × 7 matrix three times using either nine or 14 flashes (instead of 19), decreasing the amount of time to present stimuli. Compared to 9-flash A, 9-flash B decreased the likelihood that neighboring items would flash when the target was not flashing, thereby reducing interference from items adjacent to targets. 14-flash A also reduced adjacent item interference and 14-flash B additionally eliminated successive (double) flashes of the same item. Results showed that accuracy and bit rate of the adaptive system were higher than the non-adaptive system. In addition, 9- and 14-flash B produced significantly higher performance than their respective A conditions. The results also show the trend that the 14-flash B paradigm was better than the 19-flash pattern for naïve users. PMID:21474877

  11. Genome modification by CRISPR/Cas9.

    PubMed

    Ma, Yuanwu; Zhang, Lianfeng; Huang, Xingxu

    2014-12-01

    Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein (Cas)9-mediated genome modification enables us to edit the genomes of a variety of organisms rapidly and efficiently. The advantages of the CRISPR-Cas9 system have made it an increasingly popular genetic engineering tool for biological and therapeutic applications. Moreover, CRISPR-Cas9 has been employed to recruit functional domains that repress/activate gene expression or label specific genomic loci in living cells or organisms, in order to explore developmental mechanisms, gene expression regulation, and animal behavior. One major concern about this system is its specificity; although CRISPR-Cas9-mediated off-target mutation has been broadly studied, more efforts are required to further improve the specificity of CRISPR-Cas9. We will also discuss the potential applications of CRISPR-Cas9.

  12. Type I-E CRISPR-cas systems discriminate target from non-target DNA through base pairing-independent PAM recognition.

    PubMed

    Westra, Edze R; Semenova, Ekaterina; Datsenko, Kirill A; Jackson, Ryan N; Wiedenheft, Blake; Severinov, Konstantin; Brouns, Stan J J

    2013-01-01

    Discriminating self and non-self is a universal requirement of immune systems. Adaptive immune systems in prokaryotes are centered around repetitive loci called CRISPRs (clustered regularly interspaced short palindromic repeat), into which invader DNA fragments are incorporated. CRISPR transcripts are processed into small RNAs that guide CRISPR-associated (Cas) proteins to invading nucleic acids by complementary base pairing. However, to avoid autoimmunity it is essential that these RNA-guides exclusively target invading DNA and not complementary DNA sequences (i.e., self-sequences) located in the host's own CRISPR locus. Previous work on the Type III-A CRISPR system from Staphylococcus epidermidis has demonstrated that a portion of the CRISPR RNA-guide sequence is involved in self versus non-self discrimination. This self-avoidance mechanism relies on sensing base pairing between the RNA-guide and sequences flanking the target DNA. To determine if the RNA-guide participates in self versus non-self discrimination in the Type I-E system from Escherichia coli we altered base pairing potential between the RNA-guide and the flanks of DNA targets. Here we demonstrate that Type I-E systems discriminate self from non-self through a base pairing-independent mechanism that strictly relies on the recognition of four unchangeable PAM sequences. In addition, this work reveals that the first base pair between the guide RNA and the PAM nucleotide immediately flanking the target sequence can be disrupted without affecting the interference phenotype. Remarkably, this indicates that base pairing at this position is not involved in foreign DNA recognition. Results in this paper reveal that the Type I-E mechanism of avoiding self sequences and preventing autoimmunity is fundamentally different from that employed by Type III-A systems. We propose the exclusive targeting of PAM-flanked sequences to be termed a target versus non-target discrimination mechanism.

  13. Adaptive functional systems: learning with chaos.

    PubMed

    Komarov, M A; Osipov, G V; Burtsev, M S

    2010-12-01

    We propose a new model of adaptive behavior that combines a winnerless competition principle and chaos to learn new functional systems. The model consists of a complex network of nonlinear dynamical elements producing sequences of goal-directed actions. Each element describes dynamics and activity of the functional system which is supposed to be a distributed set of interacting physiological elements such as nerve or muscle that cooperates to obtain certain goal at the level of the whole organism. During "normal" behavior, the dynamics of the system follows heteroclinic channels, but in the novel situation chaotic search is activated and a new channel leading to the target state is gradually created simulating the process of learning. The model was tested in single and multigoal environments and had demonstrated a good potential for generation of new adaptations.

  14. Microinjection-based generation of mutant mice with a double mutation and a 0.5 Mb deletion in their genome by the CRISPR/Cas9 system

    PubMed Central

    HARA, Satoshi; KATO, Tomoko; GOTO, Yuji; KUBOTA, Souichirou; TAMANO, Moe; TERAO, Miho; TAKADA, Shuji

    2016-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for genome editing. In this study, using a microinjection-based CRISPR/Cas9 system, we efficiently generated mouse lines carrying mutations at the Irx3 and Irx5 loci, which are located in close proximity on a chromosome and are functionally redundant. During the generation of Irx3/Irx5 double mutant mice, a deletion of ~0.5 Mb between the Irx3 and Irx5 loci was unintentionally identified in 6 out of 27 living pups by PCR based genotyping analysis. This deletion was confirmed by DNA fluorescence in situ hybridization analysis of fibroblasts. These results indicate that the mutant mice with a deletion of at least 0.5 Mb in their genome can be generated by the CRISPR/Cas9 system through microinjection into fertilized eggs. Our findings expand the utility of the CRISPR/Cas9 system in production of disease model animals with large deletions. PMID:27396308

  15. An Approach to the Study of Systems of Equations with Geogebra: Learning Opportunities Provided by the Integration of CAS View: Story of a Workshop Experience with Teachers

    ERIC Educational Resources Information Center

    Alejandra, Almirón; Fernando, Bifano; Leonardo, Lupinacci

    2015-01-01

    Solving systems of equations at school, at least in Argentina, is usually a task that students are given as a series of techniques that "allow" them to find a solution. How to overcome educational obstacles that are generated from a fragmented approach of knowledge? What can DGS do, in particular the CAS environment? What epistemic and…

  16. Adaptive Embedded Digital System for Plasma Diagnostics

    NASA Astrophysics Data System (ADS)

    González, Angel; Rodríguez, Othoniel; Mangual, Osvaldo; Ponce, Eduardo; Vélez, Xavier

    2014-05-01

    An Adaptive Embedded Digital System to perform plasma diagnostics using electrostatic probes was developed at the Plasma Engineering Laboratory at Polytechnic University of Puerto Rico. The system will replace the existing instrumentation at the Laboratory, using reconfigurable hardware to minimize the equipment and software needed to perform diagnostics. The adaptability of the design resides on the possibility of replacing the computational algorithm on the fly, allowing to use the same hardware for different probes. The system was prototyped using Very High Speed Integrated Circuits Hardware Description Language (VHDL) into an Field Programmable Gate Array (FPGA) board. The design of the Embedded Digital System includes a Zero Phase Digital Filter, a Derivative Unit, and a Computational Unit designed using the VHDL-2008 Support Library. The prototype is able to compute the Plasma Electron Temperature and Density from a Single Langmuir probe. The system was tested using real data previously acquired from a single Langmuir probe. The plasma parameters obtained from the embedded system were compared with results computed using matlab yielding excellent matching. The new embedded system operates on 4096 samples versus 500 on the previous system, and completes its computations in 26 milliseconds compared with about 15 seconds on the previous system.

  17. Adaptive Optics Imaging of Solar System Objects

    NASA Technical Reports Server (NTRS)

    Roddier, Francois; Owen, Toby

    1997-01-01

    Most solar system objects have never been observed at wavelengths longer than the R band with an angular resolution better than 1 sec. The Hubble Space Telescope itself has only recently been equipped to observe in the infrared. However, because of its small diameter, the angular resolution is lower than that one can now achieved from the ground with adaptive optics, and time allocated to planetary science is limited. We have been using adaptive optics (AO) on a 4-m class telescope to obtain 0.1 sec resolution images solar system objects at far red and near infrared wavelengths (0.7-2.5 micron) which best discriminate their spectral signatures. Our efforts has been put into areas of research for which high angular resolution is essential, such as the mapping of Titan and of large asteroids, the dynamics and composition of Neptune stratospheric clouds, the infrared photometry of Pluto, Charon, and close satellites previously undetected from the ground.

  18. Demonstration of portable solar adaptive optics system

    NASA Astrophysics Data System (ADS)

    Ren, Deqing; Dong, Bing

    2012-10-01

    Solar-adaptive optics (AO) are more challenging than night-time AO, in some aspects. A portable solar adaptive optics (PSAO) system featuring compact physical size, low cost, and good performance has been proposed and developed. PSAO can serve as a visiting instrument for any existing ground-based solar telescope to improve solar image quality by replacing just a few optical components. High-level programming language, LabVIEW, is used to develop the wavefront sensing and control software, and general purpose computers are used to drive the whole system. During October 2011, the feasibility and good performance of PSAO was demonstrated with the 61-cm solar telescope at San Fernando Observatory. The image contrast and resolution are noticeably improved after AO correction.

  19. Adaptive control design for hysteretic smart systems

    NASA Astrophysics Data System (ADS)

    Fan, Xiang; Smith, Ralph C.

    2009-03-01

    Ferroelectric and ferromagnetic actuators are being considered for a range of industrial, aerospace, aeronautic and biomedical applications due to their unique transduction capabilities. However, they also exhibit hysteretic and nonlinear behavior that must be accommodated in models and control designs. If uncompensated, these effects can yield reduced system performance and, in the worst case, can produce unpredictable behavior of the control system. One technique for control design is to approximately linearize the actuator dynamics using an adaptive inverse compensator that is also able to accommodate model uncertainties and error introduced by the inverse algorithm. This paper describes the design of an adaptive inverse control technique based on the homogenized energy model for hysteresis. The resulting inverse filter is incorporated in an L1 control theory to provide a robust control algorithm capable of providing high speed, high accuracy tracking in the presence of actuator hysteresis and nonlinearities. Properties of the control design are illustrated through numerical examples.

  20. Cyberspace: The Ultimate Complex Adaptive System

    DTIC Science & Technology

    2011-04-05

    capable of a transaction with a given agent; tags also facilitate the formation of aggregates , or meta-agents. Meta-agents help distrib- ute and... Mbps or Gbps through communications links Path Roads, Rails, Flight Path, Sea- lanes Links, Connections Terrain Hills, Valleys, Urban Canyons... aggregate of many factors, both local and global. Unfit agents are more likely to instigate schema change. What is a Complex Adaptive System? This

  1. Adaptable radiation monitoring system and method

    DOEpatents

    Archer, Daniel E.; Beauchamp, Brock R.; Mauger, G. Joseph; Nelson, Karl E.; Mercer, Michael B.; Pletcher, David C.; Riot, Vincent J.; Schek, James L.; Knapp, David A.

    2006-06-20

    A portable radioactive-material detection system capable of detecting radioactive sources moving at high speeds. The system has at least one radiation detector capable of detecting gamma-radiation and coupled to an MCA capable of collecting spectral data in very small time bins of less than about 150 msec. A computer processor is connected to the MCA for determining from the spectral data if a triggering event has occurred. Spectral data is stored on a data storage device, and a power source supplies power to the detection system. Various configurations of the detection system may be adaptably arranged for various radiation detection scenarios. In a preferred embodiment, the computer processor operates as a server which receives spectral data from other networked detection systems, and communicates the collected data to a central data reporting system.

  2. Exploring the potential of genome editing CRISPR-Cas9 technology.

    PubMed

    Singh, Vijai; Braddick, Darren; Dhar, Pawan Kumar

    2017-01-30

    CRISPR-Cas9 is an RNA-mediated adaptive immune system that protects bacteria and archaea from viruses or plasmids. Herein we discuss the recent development of CRISPR-Cas9 into a key technology for genome editing, targeting, and regulation in a wide range of organisms and cell types. It requires a custom designed single guide-RNA (sgRNA), a Cas9 endonuclease, and PAM sequences in the target region. The sgRNA-Cas9 complex binds to its target and creates a double-strand break (DSB) that can be repaired by non-homologous end joining (NHEJ) or by the homology-directed repair (HDR) pathway, modifying or permanently replacing the genomic target sequence. Additionally, we highlight recent advances in the repurposing of CRISPR-Cas9 for repression, activation, and loci imaging. In this review, we underline the current progress and the future potential of the CRISPR-Cas9 system towards biomedical, therapeutic, industrial, and biotechnological applications.

  3. Complex Adaptive Systems of Systems (CASOS) engineering environment.

    SciTech Connect

    Detry, Richard Joseph; Linebarger, John Michael; Finley, Patrick D.; Maffitt, S. Louise; Glass, Robert John, Jr.; Beyeler, Walter Eugene; Ames, Arlo Leroy

    2012-02-01

    Complex Adaptive Systems of Systems, or CASoS, are vastly complex physical-socio-technical systems which we must understand to design a secure future for the nation. The Phoenix initiative implements CASoS Engineering principles combining the bottom up Complex Systems and Complex Adaptive Systems view with the top down Systems Engineering and System-of-Systems view. CASoS Engineering theory and practice must be conducted together to develop a discipline that is grounded in reality, extends our understanding of how CASoS behave and allows us to better control the outcomes. The pull of applications (real world problems) is critical to this effort, as is the articulation of a CASoS Engineering Framework that grounds an engineering approach in the theory of complex adaptive systems of systems. Successful application of the CASoS Engineering Framework requires modeling, simulation and analysis (MS and A) capabilities and the cultivation of a CASoS Engineering Community of Practice through knowledge sharing and facilitation. The CASoS Engineering Environment, itself a complex adaptive system of systems, constitutes the two platforms that provide these capabilities.

  4. Visualization analysis of CRISPR/Cas9 gene editing technology studies*

    PubMed Central

    Du, Quan-sheng; Cui, Jie; Zhang, Chun-jie; He, Ke

    2016-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is an adaptive immune defense system that resists the invasion of viruses and plasmids heterologous genetic material in bacteria and archaea. Taking the literature related to gene editing technology of CRISPR/Cas9 from the Web of Science database from 2002 to 2015, we use the software CiteSpaceV to analyze co-cited literature in order to establish the research hotspots and fronts recently in this field by knowledge mapping. PMID:27704749

  5. Visualization analysis of CRISPR/Cas9 gene editing technology studies.

    PubMed

    Du, Quan-Sheng; Cui, Jie; Zhang, Chun-Jie; He, Ke

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is an adaptive immune defense system that resists the invasion of viruses and plasmids heterologous genetic material in bacteria and archaea. Taking the literature related to gene editing technology of CRISPR/Cas9 from the Web of Science database from 2002 to 2015, we use the software CiteSpaceV to analyze co-cited literature in order to establish the research hotspots and fronts recently in this field by knowledge mapping.

  6. Social networks as embedded complex adaptive systems.

    PubMed

    Benham-Hutchins, Marge; Clancy, Thomas R

    2010-09-01

    As systems evolve over time, their natural tendency is to become increasingly more complex. Studies in the field of complex systems have generated new perspectives on management in social organizations such as hospitals. Much of this research appears as a natural extension of the cross-disciplinary field of systems theory. This is the 15th in a series of articles applying complex systems science to the traditional management concepts of planning, organizing, directing, coordinating, and controlling. In this article, the authors discuss healthcare social networks as a hierarchy of embedded complex adaptive systems. The authors further examine the use of social network analysis tools as a means to understand complex communication patterns and reduce medical errors.

  7. Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering.

    PubMed

    Moyer, Tyler C; Holland, Andrew J

    2015-01-01

    The ability to rapidly and specifically modify the genome of mammalian cells has been a long-term goal of biomedical researchers. Recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system from bacteria has been exploited for genome engineering in human cells. The CRISPR system directs the RNA-guided Cas9 nuclease to a specific genomic locus to induce a DNA double-strand break that may be subsequently repaired by homology-directed repair using an exogenous DNA repair template. Here we describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of a point mutation in human cells. Using this method, homozygous clonal cell lines can be constructed in 5-6 weeks. This method can also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations. CRISPR/Cas9 genome engineering offers exciting applications in both basic science and translational research.

  8. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment.

    PubMed

    Kennedy, Edward M; Cullen, Bryan R

    2015-05-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  9. Bacterial CRISPR/Cas DNA endonucleases: A revolutionary technology that could dramatically impact viral research and treatment

    PubMed Central

    Kennedy, Edward M.; Cullen, Bryan R.

    2015-01-01

    CRISPR/Cas systems mediate bacterial adaptive immune responses that evolved to protect bacteria from bacteriophage and other horizontally transmitted genetic elements. Several CRISPR/Cas systems exist but the simplest variant, referred to as Type II, has a single effector DNA endonuclease, called Cas9, which is guided to its viral DNA target by two small RNAs, the crRNA and the tracrRNA. Initial efforts to adapt the CRISPR/Cas system for DNA editing in mammalian cells, which focused on the Cas9 protein from Streptococcus pyogenes (Spy), demonstrated that Spy Cas9 can be directed to DNA targets in mammalian cells by tracrRNA:crRNA fusion transcripts called single guide RNAs (sgRNA). Upon binding, Cas9 induces DNA cleavage leading to mutagenesis as a result of error prone non-homologous end joining (NHEJ). Recently, the Spy Cas9 system has been adapted for high throughput screening of genes in human cells for their relevance to a particular phenotype and, more generally, for the targeted inactivation of specific genes, in cell lines and in vivo in a number of model organisms. The latter aim seems likely to be greatly enhanced by the recent development of Cas9 proteins from bacterial species such as Neisseria meningitidis and Staphyloccus aureus that are small enough to be expressed using adeno-associated (AAV)-based vectors that can be readily prepared at very high titers. The evolving Cas9-based DNA editing systems therefore appear likely to not only impact virology by allowing researchers to screen for human genes that affect the replication of pathogenic human viruses of all types but also to derive clonal human cell lines that lack individual gene products that either facilitate or restrict viral replication. Moreover, high titer AAV-based vectors offer the possibility of directly targeting DNA viruses that infect discrete sites in the human body, such as herpes simplex virus and hepatitis B virus, with the hope that the entire population of viral DNA genomes

  10. CRISPR-Cas9 gene editing: Delivery aspects and therapeutic potential.

    PubMed

    Oude Blenke, Erik; Evers, Martijn J W; Mastrobattista, Enrico; van der Oost, John

    2016-12-28

    The CRISPR-Cas9 gene editing system has taken the biomedical science field by storm, initiating rumors about future Nobel Prizes and heating up a fierce patent war, but also making significant scientific impact. The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), together with CRISPR-associated proteins (Cas) are a part of the prokaryotic adaptive immune system and have successfully been repurposed for genome editing in mammalian cells. The CRISPR-Cas9 system has been used to correct genetic mutations and for replacing entire genes, opening up a world of possibilities for the treatment of genetic diseases. In addition, recently some new CRISPR-Cas systems have been discovered with interesting mechanistic variations. Despite these promising developments, many challenges have to be overcome before the system can be applied therapeutically in human patients and enabling delivery technology is one of the key challenges. Furthermore, the relatively high off-target effect of the system in its current form prevents it from being safely applied directly in the human body. In this review, the transformation of the CRISPR-Cas gene editing systems into a therapeutic modality will be discussed and the currently most realistic in vivo applications will be highlighted.

  11. Targeted genome editing in the rare actinomycete Actinoplanes sp. SE50/110 by using the CRISPR/Cas9 System.

    PubMed

    Wolf, Timo; Gren, Tetiana; Thieme, Eric; Wibberg, Daniel; Zemke, Till; Pühler, Alfred; Kalinowski, Jörn

    2016-08-10

    The application of genome editing technologies, like CRISPR/Cas9 for industrially relevant microorganisms, is becoming increasingly important. Compared to other methods of genetic engineering the decisive factor is that CRISPR/Cas9 is relatively easy to apply and thus time and effort can be significantly reduced in organisms, which are otherwise genetically difficult to access. Because of its many advantages and opportunities, we adopted the CRISPR/Cas9 technology for Actinoplanes sp. SE50/110, the producer of the diabetes type II drug acarbose. The functionality of genome editing was successfully shown by the scarless and antibiotic marker-free deletion of the gene encoding the tyrosinase MelC, which catalyzes the formation of the dark pigment eumelanin in the wild type strain. The generated ΔmelC2 mutant of Actinoplanes sp. SE50/110 no longer produces this pigment and therefore the supernatant does not darken. Furthermore, it was shown that the plasmid containing the gene for the Cas9 protein was removed by increasing the temperature due to its temperature-sensitive replication. The precision of the intended mutation was proven and possible off-target effects caused by the genome editing system were ruled out by genome sequencing of several mutants.

  12. What rheumatologists need to know about CRISPR/Cas9.

    PubMed

    Gibson, Gary J; Yang, Maozhou

    2017-02-09

    CRISPR/Cas9 genome editing technology has taken the research world by storm since its use in eukaryotes was first proposed in 2012. Publications describing advances in technology and new applications have continued at an unrelenting pace since that time. In this Review, we discuss the application of CRISPR/Cas9 for creating gene mutations - the application that initiated the current avalanche of interest - and new developments that have largely answered initial concerns about its specificity and ability to introduce new gene sequences. We discuss the new, diverse and rapidly growing adaptations of the CRISPR/Cas9 technique that enable activation, repression, multiplexing and gene screening. These developments have enabled researchers to create sophisticated tools for dissecting the function and inter-relatedness of genes, as well as noncoding regions of the genome, and to identify gene networks and noncoding regions that promote disease or confer disease susceptibility. These approaches are beginning to be used to interrogate complex and multilayered biological systems and to produce complex animal models of disease. CRISPR/Cas9 technology has enabled the application of new therapeutic approaches to treating disease in animal models, some of which are beginning to be seen in the first human clinical trials. We discuss the direct application of these techniques to rheumatic diseases, which are currently limited but are sure to increase rapidly in the near future.

  13. Progress with the lick adaptive optics system

    SciTech Connect

    Gavel, D T; Olivier, S S; Bauman, B; Max, C E; Macintosh, B

    2000-03-01

    Progress and results of observations with the Lick Observatory Laser Guide Star Adaptive Optics System are presented. This system is optimized for diffraction-limited imaging in the near infrared, 1-2 micron wavelength bands. We describe our development efforts in a number of component areas including, a redesign of the optical bench layout, the commissioning of a new infrared science camera, and improvements to the software and user interface. There is also an ongoing effort to characterize the system performance with both natural and laser guide stars and to fold this data into a refined system model. Such a model can be used to help plan future observations, for example, predicting the point-spread function as a function of seeing and guide star magnitude.

  14. Progress with the Lick adaptive optics system

    NASA Astrophysics Data System (ADS)

    Gavel, Donald T.; Olivier, Scot S.; Bauman, Brian J.; Max, Claire E.; Macintosh, Bruce A.

    2000-07-01

    Progress and results of observations with the Lick Observatory Laser Guide Star Adaptive Optics System are presented. This system is optimized for diffraction-limited imaging in the near infrared, 1 - 2 micron wavelength bands. We describe our development efforts in a number of component areas including, a redesign of the optical bench layout, the commissioning of a new infrared science camera, and improvements to the software and user interface. There is also an ongoing effort to characterize the system performance with both natural and laser guide stars and to fold this data into a refined system model. Such a model can be used to help plan future observations, for example, predicting the point-spread function as a function of seeing and guide star magnitude.

  15. The adaptive safety analysis and monitoring system

    NASA Astrophysics Data System (ADS)

    Tu, Haiying; Allanach, Jeffrey; Singh, Satnam; Pattipati, Krishna R.; Willett, Peter

    2004-09-01

    The Adaptive Safety Analysis and Monitoring (ASAM) system is a hybrid model-based software tool for assisting intelligence analysts to identify terrorist threats, to predict possible evolution of the terrorist activities, and to suggest strategies for countering terrorism. The ASAM system provides a distributed processing structure for gathering, sharing, understanding, and using information to assess and predict terrorist network states. In combination with counter-terrorist network models, it can also suggest feasible actions to inhibit potential terrorist threats. In this paper, we will introduce the architecture of the ASAM system, and discuss the hybrid modeling approach embedded in it, viz., Hidden Markov Models (HMMs) to detect and provide soft evidence on the states of terrorist network nodes based on partial and imperfect observations, and Bayesian networks (BNs) to integrate soft evidence from multiple HMMs. The functionality of the ASAM system is illustrated by way of application to the Indian Airlines Hijacking, as modeled from open sources.

  16. Adaptable data management for systems biology investigations

    PubMed Central

    Boyle, John; Rovira, Hector; Cavnor, Chris; Burdick, David; Killcoyne, Sarah; Shmulevich, Ilya

    2009-01-01

    Background Within research each experiment is different, the focus changes and the data is generated from a continually evolving barrage of technologies. There is a continual introduction of new techniques whose usage ranges from in-house protocols through to high-throughput instrumentation. To support these requirements data management systems are needed that can be rapidly built and readily adapted for new usage. Results The adaptable data management system discussed is designed to support the seamless mining and analysis of biological experiment data that is commonly used in systems biology (e.g. ChIP-chip, gene expression, proteomics, imaging, flow cytometry). We use different content graphs to represent different views upon the data. These views are designed for different roles: equipment specific views are used to gather instrumentation information; data processing oriented views are provided to enable the rapid development of analysis applications; and research project specific views are used to organize information for individual research experiments. This management system allows for both the rapid introduction of new types of information and the evolution of the knowledge it represents. Conclusion Data management is an important aspect of any research enterprise. It is the foundation on which most applications are built, and must be easily extended to serve new functionality for new scientific areas. We have found that adopting a three-tier architecture for data management, built around distributed standardized content repositories, allows us to rapidly develop new applications to support a diverse user community. PMID:19265554

  17. CAS as Environments for Implementing Mathematical Microworlds.

    ERIC Educational Resources Information Center

    Alpers, Burkhard

    2002-01-01

    Investigates whether computer algebra systems (CAS) are suitable environments for implementing mathematical microworlds. Recalls what constitutes a microworld and explores how CAS can be used for implementation, stating potentials as well as limitations. Provides as an example the microworld "Formula 1", implemented in Maple Software. (Author/KHR)

  18. Genome surgery using Cas9 ribonucleoproteins for the treatment of age-related macular degeneration

    PubMed Central

    Kim, Kyoungmi; Park, Sung Wook; Kim, Jin Hyoung; Lee, Seung Hwan; Kim, Daesik; Koo, Taeyoung; Kim, Kwang-eun; Kim, Jeong Hun; Kim, Jin-Soo

    2017-01-01

    RNA-guided genome surgery using CRISPR-Cas9 nucleases has shown promise for the treatment of diverse genetic diseases. Yet, the potential of such nucleases for therapeutic applications in nongenetic diseases is largely unexplored. Here, we focus on age-related macular degeneration (AMD), a leading cause of blindness in adults, which is associated with retinal overexpression of, rather than mutations in, the VEGFA gene. Subretinal injection of preassembled, Vegfa gene–specific Cas9 ribonucleoproteins (RNPs) into the adult mouse eye gave rise to mutagenesis at the target site in the retinal pigment epithelium. Furthermore, Cas9 RNPs effectively reduced the area of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD. Genome-wide profiling of Cas9 off-target effects via Digenome-seq showed that off-target mutations were rarely induced in the human genome. Because Cas9 RNPs can function immediately after in vivo delivery and are rapidly degraded by endogenous proteases, their activities are unlikely to be hampered by antibody- and cell-mediated adaptive immune systems. Our results demonstrate that in vivo genome editing with Cas9 RNPs has the potential for the local treatment for nongenetic degenerative diseases, expanding the scope of RNA-guided genome surgery to a new dimension. PMID:28209587

  19. Double-stranded endonuclease activity in Bacillus halodurans clustered regularly interspaced short palindromic repeats (CRISPR)-associated Cas2 protein.

    PubMed

    Nam, Ki Hyun; Ding, Fran; Haitjema, Charles; Huang, Qingqiu; DeLisa, Matthew P; Ke, Ailong

    2012-10-19

    The CRISPR (clustered regularly interspaced short palindromic repeats) system is a prokaryotic RNA-based adaptive immune system against extrachromosomal genetic elements. Cas2 is a universally conserved core CRISPR-associated protein required for the acquisition of new spacers for CRISPR adaptation. It was previously characterized as an endoribonuclease with preference for single-stranded (ss)RNA. Here, we show using crystallography, mutagenesis, and isothermal titration calorimetry that the Bacillus halodurans Cas2 (Bha_Cas2) from the subtype I-C/Dvulg CRISPR instead possesses metal-dependent endonuclease activity against double-stranded (ds)DNA. This activity is consistent with its putative function in producing new spacers for insertion into the 5'-end of the CRISPR locus. Mutagenesis and isothermal titration calorimetry studies revealed that a single divalent metal ion (Mg(2+) or Mn(2+)), coordinated by a symmetric Asp pair in the Bha_Cas2 dimer, is involved in the catalysis. We envision that a pH-dependent conformational change switches Cas2 into a metal-binding competent conformation for catalysis. We further propose that the distinct substrate preferences among Cas2 proteins may be determined by the sequence and structure in the β1-α1 loop.

  20. An Intelligent Tutoring System Approach to Adaptive Instructional Systems

    DTIC Science & Technology

    2005-09-01

    gr prototypes (scripts) (Schank, 1977). Many software systems have been developed that employ these representations. Many instructional theories and...specific performance or skill-acquisition. However, there are many theories about the number and type of these general abilities. Researchers...Computer Generated Forces and Behavioral Representations. 7.1.2 Role of MAMID in an Adaptive Instructional System Motivation Currently, the student

  1. Adaptive system for eye-fundus imaging

    SciTech Connect

    Larichev, A V; Ivanov, P V; Iroshnikov, N G; Shmalgauzen, V I; Otten, L J

    2002-10-31

    A compact adaptive system capable of imaging a human-eye retina with a spatial resolution as high as 6 {mu}m and a field of view of 15{sup 0} is developed. It is shown that a modal bimorph corrector with nonlocalised response functions provides the efficient suppression of dynamic aberrations of a human eye. The residual root-mean-square error in correction of aberrations of a real eye with nonparalysed accommodation lies in the range of 0.1 - 0.15 {mu}m.

  2. NASA Facts: Nanosatellite Launch Adapter System (NLAS)

    NASA Technical Reports Server (NTRS)

    Chartres, James; Cappuccio, Gelsomina

    2013-01-01

    The Nanosatellite Launch Adapter System (NLAS) was developed to increase access to space while simplifying the integration process of miniature satellites, called nanosats or cubesats, onto launch vehicles. A standard cubesat measures about 4inches (10 cm) long, 4 inches wide,and 4 inches high, and is called a one-unit (1U) cubesat. A single NLAS provides the capability to deploy 24U of cubesats. The system is designed to accommodate satellites measuring 1U, 1.5U, 2U, 3U and 6U sizes for deployment into orbit. The NLAS may be configured for use on different launch vehicles. The system also enables flight demonstrations of new technologies in the space environment.

  3. Genome Editing by CRISPR/Cas9: A Game Change in the Genetic Manipulation of Protists.

    PubMed

    Lander, Noelia; Chiurillo, Miguel A; Docampo, Roberto

    2016-09-01

    Genome editing by CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated gene 9) system has been transformative in biology. Originally discovered as an adaptive prokaryotic immune system, CRISPR/Cas9 has been repurposed for genome editing in a broad range of model organisms, from yeast to mammalian cells. Protist parasites are unicellular organisms producing important human diseases that affect millions of people around the world. For many of these diseases, such as malaria, Chagas disease, leishmaniasis and cryptosporidiosis, there are no effective treatments or vaccines available. The recent adaptation of the CRISPR/Cas9 technology to several protist models will be playing a key role in the functional study of their proteins, in the characterization of their metabolic pathways, and in the understanding of their biology, and will facilitate the search for new chemotherapeutic targets. In this work we review recent studies where the CRISPR/Cas9 system was adapted to protist parasites, particularly to Apicomplexans and trypanosomatids, emphasizing the different molecular strategies used for genome editing of each organism, as well as their advantages. We also discuss the potential usefulness of this technology in the green alga Chlamydomonas reinhardtii.

  4. Adaptive Decision Aiding in Computer-Assisted Instruction: Adaptive Computerized Training System (ACTS).

    ERIC Educational Resources Information Center

    Hopf-Weichel, Rosemarie; And Others

    This report describes results of the first year of a three-year program to develop and evaluate a new Adaptive Computerized Training System (ACTS) for electronics maintenance training. (ACTS incorporates an adaptive computer program that learns the student's diagnostic and decision value structure, compares it to that of an expert, and adapts the…

  5. Hospitals as complex adaptive systems: A case study of factors influencing priority setting practices at the hospital level in Kenya.

    PubMed

    Barasa, Edwine W; Molyneux, Sassy; English, Mike; Cleary, Susan

    2017-02-01

    There is a dearth of literature on priority setting and resource allocation (PSRA) practices in hospitals, particularly in low and middle income countries (LMICs). Using a case study approach, we examined PSRA practices in 2 public hospitals in coastal Kenya. We collected data through a combination of in-depth interviews of national level policy makers, hospital managers, and frontline practitioners in the case study hospitals (n = 72), review of documents such as hospital plans and budgets, minutes of meetings and accounting records, and non-participant observations of PSRA practices in case study hospitals over a period of 7 months. In this paper, we apply complex adaptive system (CAS) theory to examine the factors that influence PSRA practices. We found that PSRA practices in the case hospitals were influenced by, 1) inadequate financing level and poorly designed financing arrangements, 2) limited hospital autonomy and decision space, and 3) inadequate management and leadership capacity in the hospital. The case study hospitals exhibited properties of complex adaptive systems (CASs) that exist in a dynamic state with multiple interacting agents. Weaknesses in system 'hardware' (resource scarcity) and 'software' (including PSRA guidelines that reduced hospitals decision space, and poor leadership skills) led to the emergence of undesired properties. The capacity of hospitals to set priorities should be improved across these interacting aspects of the hospital organizational system. Interventions should however recognize that hospitals are CAS. Rather than rectifying isolated aspects of the system, they should endeavor to create conditions for productive emergence.

  6. Application of the genome editing tool CRISPR/Cas9 in non-human primates

    PubMed Central

    LUO, Xin; LI, Min; SU, Bing

    2016-01-01

    In the past three years, RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system has been used to facilitate efficient genome editing in many model and non-model animals. However, its application in nonhuman primates is still at the early stage, though in view of the similarities in anatomy, physiology, behavior and genetics, closely related nonhuman primates serve as optimal models for human biology and disease studies. In this review, we summarize the current proceedings of gene editing using CRISPR/Cas9 in nonhuman primates. PMID:27469252

  7. Adaptable formations utilizing heterogeneous unmanned systems

    NASA Astrophysics Data System (ADS)

    Barnes, Laura E.; Garcia, Richard; Fields, MaryAnne; Valavanis, Kimon

    2009-05-01

    This paper addresses the problem of controlling and coordinating heterogeneous unmanned systems required to move as a group while maintaining formation. We propose a strategy to coordinate groups of unmanned ground vehicles (UGVs) with one or more unmanned aerial vehicles (UAVs). UAVs can be utilized in one of two ways: (1) as alpha robots to guide the UGVs; and (2) as beta robots to surround the UGVs and adapt accordingly. In the first approach, the UAV guides a swarm of UGVs controlling their overall formation. In the second approach, the UGVs guide the UAVs controlling their formation. The unmanned systems are brought into a formation utilizing artificial potential fields generated from normal and sigmoid functions. These functions control the overall swarm geometry. Nonlinear limiting functions are defined to provide tighter swarm control by modifying and adjusting a set of control variables forcing the swarm to behave according to set constraints. Formations derived are subsets of elliptical curves but can be generalized to any curvilinear shape. Both approaches are demonstrated in simulation and experimentally. To demonstrate the second approach in simulation, a swarm of forty UAVs is utilized in a convoy protection mission. As a convoy of UGVs travels, UAVs dynamically and intelligently adapt their formation in order to protect the convoy of vehicles as it moves. Experimental results are presented to demonstrate the approach using a fully autonomous group of three UGVs and a single UAV helicopter for coordination.

  8. Adaptive cyber-attack modeling system

    NASA Astrophysics Data System (ADS)

    Gonsalves, Paul G.; Dougherty, Edward T.

    2006-05-01

    The pervasiveness of software and networked information systems is evident across a broad spectrum of business and government sectors. Such reliance provides an ample opportunity not only for the nefarious exploits of lone wolf computer hackers, but for more systematic software attacks from organized entities. Much effort and focus has been placed on preventing and ameliorating network and OS attacks, a concomitant emphasis is required to address protection of mission critical software. Typical software protection technique and methodology evaluation and verification and validation (V&V) involves the use of a team of subject matter experts (SMEs) to mimic potential attackers or hackers. This manpower intensive, time-consuming, and potentially cost-prohibitive approach is not amenable to performing the necessary multiple non-subjective analyses required to support quantifying software protection levels. To facilitate the evaluation and V&V of software protection solutions, we have designed and developed a prototype adaptive cyber attack modeling system. Our approach integrates an off-line mechanism for rapid construction of Bayesian belief network (BN) attack models with an on-line model instantiation, adaptation and knowledge acquisition scheme. Off-line model construction is supported via a knowledge elicitation approach for identifying key domain requirements and a process for translating these requirements into a library of BN-based cyber-attack models. On-line attack modeling and knowledge acquisition is supported via BN evidence propagation and model parameter learning.

  9. Adaptive automatic balancing of magnetic bearing systems

    NASA Astrophysics Data System (ADS)

    Kim, Jong-Sun

    Rotating machinery including magnetic bearings are usually persistently excited by the rotation related disturbances such as mass unbalance; hence there exists a residual vibration in the steady state response even if the closed loop system is asymptotically stable. In order to control the periodic disturbances, a disturbance accommodating controller (DAC) is designed based on the disturbance estimator and applied to the forced balancing of magnetic bearing system. The control objective is to minimize the synchronous component of shaft displacement or control current. In order to account for the variation of the disturbance model due to the shaft of operating speed, an adaptive disturbance accommodating control scheme is developed based on a certain optimality criterion. The continuous time design discretized to implement the controller in the digital computer and the merits and demerits are studied numerically. It is shown that the proposed method is efficient in reducing rotor unbalance and automatic balancing.

  10. Landsat Ecosystem Disturbance Adaptive Processing System (LEDAPS)

    NASA Technical Reports Server (NTRS)

    Masek, Jeffrey G.

    2006-01-01

    The Landsat Ecosystem Disturbance Adaptive Processing System (LEDAPS) project is creating a record of forest disturbance and regrowth for North America from the Landsat satellite record, in support of the carbon modeling activities. LEDAPS relies on the decadal Landsat GeoCover data set supplemented by dense image time series for selected locations. Imagery is first atmospherically corrected to surface reflectance, and then change detection algorithms are used to extract disturbance area, type, and frequency. Reuse of the MODIS Land processing system (MODAPS) architecture allows rapid throughput of over 2200 MSS, TM, and ETM+ scenes. Initial ("Beta") surface reflectance products are currently available for testing, and initial continental disturbance products will be available by the middle of 2006.

  11. Development of Commercial Thermo-sensitive Genic Male Sterile Rice Accelerates Hybrid Rice Breeding Using the CRISPR/Cas9-mediated TMS5 Editing System

    PubMed Central

    Zhou, Hai; He, Ming; Li, Jing; Chen, Liang; Huang, Zhifeng; Zheng, Shaoyan; Zhu, Liya; Ni, Erdong; Jiang, Dagang; Zhao, Bingran; Zhuang, Chuxiong

    2016-01-01

    Hybrid rice breeding offers an important strategy to improve rice production, in which the cultivation of a male sterile line is the key to the success of cross-breeding. CRISPR/Cas9 systems have been widely used in target-site genome editing, whereas their application for crop genetic improvement has been rarely reported. Here, using the CRISPR/Cas9 system, we induced specific mutations in TMS5, which is the most widely applied thermo-sensitive genic male sterility (TGMS) gene in China, and developed new “transgene clean” TGMS lines. We designed 10 target sites in the coding region of TMS5 for targeted mutagenesis using the CRISPR/Cas9 system and assessed the potential rates of on- and off-target effects. Finally, we established the most efficient construct, the TMS5ab construct, for breeding potentially applicable “transgene clean” TGMS lines. We also discussed factors that affect the editing efficiency according to the characteristics of different target sequences. Notably, using the TMS5ab construct, we developed 11 new “transgene clean” TGMS lines with potential applications in hybrid breeding within only one year in both rice subspecies. The application of our system not only significantly accelerates the breeding of sterile lines but also facilitates the exploitation of heterosis. PMID:27874087

  12. In vivo mutagenesis of miRNA gene families using a scalable multiplexed CRISPR/Cas9 nuclease system

    PubMed Central

    Narayanan, Anand; Hill-Teran, Guillermina; Moro, Albertomaria; Ristori, Emma; Kasper, Dionna M.; A. Roden, Christine; Lu, Jun; Nicoli, Stefania

    2016-01-01

    A large number of microRNAs (miRNAs) are grouped into families derived from the same phylogenetic ancestors. miRNAs within a family often share the same physiological functions despite differences in their primary sequences, secondary structures, or chromosomal locations. Consequently, the generation of animal models to analyze the activity of miRNA families is extremely challenging. Using zebrafish as a model system, we successfully provide experimental evidence that a large number of miRNAs can be simultaneously mutated to abrogate the activity of an entire miRNA family. We show that injection of the Cas9 nuclease and two, four, ten, and up to twenty-four multiplexed single guide RNAs (sgRNAs) can induce mutations in 90% of the miRNA genomic sequences analyzed. We performed a survey of these 45 mutations in 10 miRNA genes, analyzing the impact of our mutagenesis strategy on the processing of each miRNA both computationally and in vivo. Our results offer an effective approach to mutate and study the activity of miRNA families and pave the way for further analysis on the function of complex miRNA families in higher multicellular organisms. PMID:27572667

  13. Targeted mutagenesis in Atlantic salmon (Salmo salar L.) using the CRISPR/Cas9 system induces complete knockout individuals in the F0 generation.

    PubMed

    Edvardsen, Rolf B; Leininger, Sven; Kleppe, Lene; Skaftnesmo, Kai Ove; Wargelius, Anna

    2014-01-01

    Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent. We used the CRISPR/Cas9 system to target two genes involved in pigmentation, tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2). Embryos were assayed for mutation rates at the 17 somite stage, where 40 and 22% of all injected embryos showed a high degree of mutation induction for slc45a2 and tyr, respectively. At hatching this mutation frequency was also visible for both targeted genes, displaying a graded phenotype ranging from complete lack of pigmentation to partial loss and normal pigmentation. CRISPRslc45a2/Cas9 injected embryos showing a complete lack of pigmentation or just a few spots of pigments also lacked wild type sequences when assaying more than 80 (slc45a2) sequence clones from whole embryos. This indicates that CRISPR/Cas9 can induce double-allelic knockout in the F0 generation. However, types and frequency of indels might affect the phenotype. Therefore, the variation of indels was assayed in the graded pigmentation phenotypes produced by CRISPR/Cas9-slc45a2. The results show a tendency for fewer types of indels formed in juveniles completely lacking pigmentation compared to juveniles displaying partial pigmentation. Another interesting observation was a high degree of the same indel type in different juveniles. This study shows for the first time successful use of the CRISPR/Cas9 technology in a marine cold water species. Targeted double-allelic mutations were obtained and, though the level of mosaicism has to be considered, we demonstrate that F0 fish can be used

  14. Targeted Mutagenesis in Atlantic Salmon (Salmo salar L.) Using the CRISPR/Cas9 System Induces Complete Knockout Individuals in the F0 Generation

    PubMed Central

    Edvardsen, Rolf B.; Leininger, Sven; Kleppe, Lene; Skaftnesmo, Kai Ove; Wargelius, Anna

    2014-01-01

    Understanding the biological function behind key proteins is of great concern in Atlantic salmon, both due to a high commercial importance and an interesting life history. Until recently, functional studies in salmonids appeared to be difficult. However, the recent discovery of targeted mutagenesis using the CRISPR/Cas9 (clustered regularly interspaced palindromic repeats/CRISPR-associated) system enables performing functional studies in Atlantic salmon to a great extent. We used the CRISPR/Cas9 system to target two genes involved in pigmentation, tyrosinase (tyr) and solute carrier family 45, member 2 (slc45a2). Embryos were assayed for mutation rates at the 17 somite stage, where 40 and 22% of all injected embryos showed a high degree of mutation induction for slc45a2 and tyr, respectively. At hatching this mutation frequency was also visible for both targeted genes, displaying a graded phenotype ranging from complete lack of pigmentation to partial loss and normal pigmentation. CRISPRslc45a2/Cas9 injected embryos showing a complete lack of pigmentation or just a few spots of pigments also lacked wild type sequences when assaying more than 80 (slc45a2) sequence clones from whole embryos. This indicates that CRISPR/Cas9 can induce double-allelic knockout in the F0 generation. However, types and frequency of indels might affect the phenotype. Therefore, the variation of indels was assayed in the graded pigmentation phenotypes produced by CRISPR/Cas9-slc45a2. The results show a tendency for fewer types of indels formed in juveniles completely lacking pigmentation compared to juveniles displaying partial pigmentation. Another interesting observation was a high degree of the same indel type in different juveniles. This study shows for the first time successful use of the CRISPR/Cas9 technology in a marine cold water species. Targeted double-allelic mutations were obtained and, though the level of mosaicism has to be considered, we demonstrate that F0 fish can be used

  15. Flipping Adapters for Space Launch System

    NASA Video Gallery

    The structural test article adapter is flipped at Marshall testing facility Building 4705. The turnover is an important step in finishing the machining work on the adapter, which will undergo tests...

  16. The Limits to Adaptation: A Systems Approach

    EPA Science Inventory

    The ability to adapt to climate change is delineated by capacity thresholds, after which climate damages begin to overwhelm the adaptation response. Such thresholds depend upon physical properties (natural processes and engineering parameters), resource constraints (expressed th...

  17. Intercellular Communication in the Adaptive Immune System

    NASA Astrophysics Data System (ADS)

    Chakraborty, Arup

    2004-03-01

    Higher organisms, like humans, have an adaptive immune system that can respond to pathogens that have not been encountered before. T lymphocytes (T cells) are the orchestrators of the adaptive immune response. They interact with cells, called antigen presenting cells (APC), that display molecular signatures of pathogens. Recently, video microscopy experiments have revealed that when T cells detect antigen on APC surfaces, a spatially patterned supramolecular assembly of different types of molecules forms in the junction between cell membranes. This recognition motif is implicated in information transfer between APC and T cells, and so, is labeled the immunological synapse. The observation of synapse formation sparked two broad questions: How does the synapse form? Why does the synapse form? I will describe progress made in answering these fundamental questions in biology by synergistic use of statistical mechanical theory/computation, chemical engineering principles, and genetic and biochemical experiments. The talk will also touch upon mechanisms that may underlie the extreme sensitivity with which T cells discriminate between self and non-self.

  18. Reassessment of the Four Yield-related Genes Gn1a, DEP1, GS3, and IPA1 in Rice Using a CRISPR/Cas9 System

    PubMed Central

    Li, Meiru; Li, Xiaoxia; Zhou, Zejiao; Wu, Pingzhi; Fang, Maichun; Pan, Xiaoping; Lin, Qiupeng; Luo, Wanbin; Wu, Guojiang; Li, Hongqing

    2016-01-01

    Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated (Cas) systems have been successfully used as efficient tools for genome editing in a variety of species. We used the CRISPR/Cas9 system to mutate the Gn1a (Os01g0197700), DEP1 (Os09g0441900), GS3 (Os03g0407400), and IPA1 (Os08g0509600) genes of rice cultivar Zhonghua 11, genes which have been reported to function as regulators of grain number, panicle architecture, grain size and plant architecture, respectively. Analysis of the phenotypes and frequencies of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in inducing targeted gene editing, with the desired genes being edited in 42.5% (Gn1a), 67.5% (DEP1), 57.5% (GS3), and 27.5% (IPA1) of the transformed plants. The T2 generation of the gn1a, dep1, and gs3 mutants featured enhanced grain number, dense erect panicles, and larger grain size, respectively. Furthermore, semi-dwarf, and grain with long awn, phenotypes were observed in dep1 and gs3 mutants, respectively. The ipa1 mutants showed two contrasting phenotypes, having either fewer tillers or more tillers, depending on the changes induced in the OsmiR156 target region. In addition, we found that mutants with deletions occurred more frequently than previous reports had indicated and that off-targeting had taken place in highly similar target sequences. These results proved that multiple regulators of important traits can be modified in a single cultivar by CRISPR/Cas9, and thus facilitate the dissection of complex gene regulatory networks in the same genomic background and the stacking of important traits in cultivated varieties. PMID:27066031

  19. Application of CRISPR/Cas9 genome editing to the study and treatment of disease.

    PubMed

    Pellagatti, Andrea; Dolatshad, Hamid; Valletta, Simona; Boultwood, Jacqueline

    2015-07-01

    CRISPR/Cas is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. The CRISPR/Cas9 method has been engineered from the type II prokaryotic CRISPR system and uses a single-guide RNA to target the Cas9 nuclease to a specific genomic sequence. Cas9 induces double-stranded DNA breaks which are repaired either by imperfect non-homologous end joining to generate insertions or deletions (indels) or, if a repair template is provided, by homology-directed repair. Due to its specificity, simplicity and versatility, the CRISPR/Cas9 system has recently emerged as a powerful tool for genome engineering in various species. This technology can be used to investigate the function of a gene of interest or to correct gene mutations in cells via genome editing, paving the way for future gene therapy approaches. Improvements to the efficiency of CRISPR repair, in particular to increase the rate of gene correction and to reduce undesired off-target effects, and the development of more effective delivery methods will be required for its broad therapeutic application.

  20. No evidence of inhibition of horizontal gene transfer by CRISPR-Cas on evolutionary timescales.

    PubMed

    Gophna, Uri; Kristensen, David M; Wolf, Yuri I; Popa, Ovidiu; Drevet, Christine; Koonin, Eugene V

    2015-09-01

    The CRISPR (clustered, regularly, interspaced, short, palindromic repeats)-Cas (CRISPR-associated genes) systems of archaea and bacteria provide adaptive immunity against viruses and other selfish elements and are believed to curtail horizontal gene transfer (HGT). Limiting acquisition of new genetic material could be one of the sources of the fitness cost of CRISPR-Cas maintenance and one of the causes of the patchy distribution of CRISPR-Cas among bacteria, and across environments. We sought to test the hypothesis that the activity of CRISPR-Cas in microbes is negatively correlated with the extent of recent HGT. Using three independent measures of HGT, we found no significant dependence between the length of CRISPR arrays, which reflects the activity of the immune system, and the estimated number of recent HGT events. In contrast, we observed a significant negative dependence between the estimated extent of HGT and growth temperature of microbes, which could be explained by the lower genetic diversity in hotter environments. We hypothesize that the relevant events in the evolution of resistance to mobile elements and proclivity for HGT, to which CRISPR-Cas systems seem to substantially contribute, occur on the population scale rather than on the timescale of species evolution.

  1. Simulation of DKIST solar adaptive optics system

    NASA Astrophysics Data System (ADS)

    Marino, Jose; Carlisle, Elizabeth; Schmidt, Dirk

    2016-07-01

    Solar adaptive optics (AO) simulations are a valuable tool to guide the design and optimization process of current and future solar AO and multi-conjugate AO (MCAO) systems. Solar AO and MCAO systems rely on extended object cross-correlating Shack-Hartmann wavefront sensors to measure the wavefront. Accurate solar AO simulations require computationally intensive operations, which have until recently presented a prohibitive computational cost. We present an update on the status of a solar AO and MCAO simulation tool being developed at the National Solar Observatory. The simulation tool is a multi-threaded application written in the C++ language that takes advantage of current large multi-core CPU computer systems and fast ethernet connections to provide accurate full simulation of solar AO and MCAO systems. It interfaces with KAOS, a state of the art solar AO control software developed by the Kiepenheuer-Institut fuer Sonnenphysik, that provides reliable AO control. We report on the latest results produced by the solar AO simulation tool.

  2. Multiclient Identification System Using Adaptive Probabilistic Model

    NASA Astrophysics Data System (ADS)

    Lin, Chin-Teng; Siana, Linda; Shou, Yu-Wen; Yang, Chien-Ting

    2010-12-01

    This paper aims at integrating detection and identification of human faces in a more practical and real-time face recognition system. The proposed face detection system is based on the cascade Adaboost method to improve the precision and robustness toward unstable surrounding lightings. Our Adaboost method innovates to adjust the environmental lighting conditions by histogram lighting normalization and to accurately locate the face regions by a region-based-clustering process as well. We also address on the problem of multi-scale faces in this paper by using 12 different scales of searching windows and 5 different orientations for each client in pursuit of the multi-view independent face identification. There are majorly two methodological parts in our face identification system, including PCA (principal component analysis) facial feature extraction and adaptive probabilistic model (APM). The structure of our implemented APM with a weighted combination of simple probabilistic functions constructs the likelihood functions by the probabilistic constraint in the similarity measures. In addition, our proposed method can online add a new client and update the information of registered clients due to the constructed APM. The experimental results eventually show the superior performance of our proposed system for both offline and real-time online testing.

  3. Adaptive model training system and method

    DOEpatents

    Bickford, Randall L; Palnitkar, Rahul M; Lee, Vo

    2014-04-15

    An adaptive model training system and method for filtering asset operating data values acquired from a monitored asset for selectively choosing asset operating data values that meet at least one predefined criterion of good data quality while rejecting asset operating data values that fail to meet at least the one predefined criterion of good data quality; and recalibrating a previously trained or calibrated model having a learned scope of normal operation of the asset by utilizing the asset operating data values that meet at least the one predefined criterion of good data quality for adjusting the learned scope of normal operation of the asset for defining a recalibrated model having the adjusted learned scope of normal operation of the asset.

  4. Adaptive model training system and method

    DOEpatents

    Bickford, Randall L; Palnitkar, Rahul M

    2014-11-18

    An adaptive model training system and method for filtering asset operating data values acquired from a monitored asset for selectively choosing asset operating data values that meet at least one predefined criterion of good data quality while rejecting asset operating data values that fail to meet at least the one predefined criterion of good data quality; and recalibrating a previously trained or calibrated model having a learned scope of normal operation of the asset by utilizing the asset operating data values that meet at least the one predefined criterion of good data quality for adjusting the learned scope of normal operation of the asset for defining a recalibrated model having the adjusted learned scope of normal operation of the asset.

  5. Intelligent Optical Systems Using Adaptive Optics

    NASA Technical Reports Server (NTRS)

    Clark, Natalie

    2012-01-01

    Until recently, the phrase adaptive optics generally conjured images of large deformable mirrors being integrated into telescopes to compensate for atmospheric turbulence. However, the development of smaller, cheaper devices has sparked interest for other aerospace and commercial applications. Variable focal length lenses, liquid crystal spatial light modulators, tunable filters, phase compensators, polarization compensation, and deformable mirrors are becoming increasingly useful for other imaging applications including guidance navigation and control (GNC), coronagraphs, foveated imaging, situational awareness, autonomous rendezvous and docking, non-mechanical zoom, phase diversity, and enhanced multi-spectral imaging. The active components presented here allow flexibility in the optical design, increasing performance. In addition, the intelligent optical systems presented offer advantages in size and weight and radiation tolerance.

  6. Simulating Astronomical Adaptive Optics Systems Using Yao

    NASA Astrophysics Data System (ADS)

    Rigaut, François; Van Dam, Marcos

    2013-12-01

    Adaptive Optics systems are at the heart of the coming Extremely Large Telescopes generation. Given the importance, complexity and required advances of these systems, being able to simulate them faithfully is key to their success, and thus to the success of the ELTs. The type of systems envisioned to be built for the ELTs cover most of the AO breeds, from NGS AO to multiple guide star Ground Layer, Laser Tomography and Multi-Conjugate AO systems, with typically a few thousand actuators. This represents a large step up from the current generation of AO systems, and accordingly a challenge for existing AO simulation packages. This is especially true as, in the past years, computer power has not been following Moore's law in its most common understanding; CPU clocks are hovering at about 3GHz. Although the use of super computers is a possible solution to run these simulations, being able to use smaller machines has obvious advantages: cost, access, environmental issues. By using optimised code in an already proven AO simulation platform, we were able to run complex ELT AO simulations on very modest machines, including laptops. The platform is YAO. In this paper, we describe YAO, its architecture, its capabilities, the ELT-specific challenges and optimisations, and finally its performance. As an example, execution speed ranges from 5 iterations per second for a 6 LGS 60x60 subapertures Shack-Hartmann Wavefront sensor Laser Tomography AO system (including full physical image formation and detector characteristics) up to over 30 iterations/s for a single NGS AO system.

  7. Evidence-based decision-making in healthcare: exploring the issues though the lens of complex, adaptive systems theory.

    PubMed

    Lindstrom, Ronald R

    2003-01-01

    Browman, Snider and Ellis have articulated several reasons as to why and how managers should address the implementation of evidence-based decision-making (EBDM) in healthcare. While their observations are acknowledged to be from the unique perspective of an oncology setting, this is a timely and welcome lead article with significance in other settings. The authors invite opinions on transferability, thus forming the basis of this commentary. In response, this commentary offers a number of supportive and differing views. Complex, adaptive systems (CAS) theory is first addressed as an appropriate lens to reframe our conceptualization of the health system. Then, in contrast to negotiation, dialogue through participatory planning and decision-making is introduced. Evidence-based decision-making (EBDM) and knowledge translation (KT) are expanded upon in the context of CAS and participatory environments. Finally, concrete suggestions are offered on how to structure multiple-stakeholder involvement in the decision-making process, including the growing role of consumers in the new complex, adaptive systems reality of healthcare.

  8. [A system of comprehensive assessment of symptoms in chronic prostatitis (CAS-XII)].

    PubMed

    Loran, O B; Segal, A S

    2001-01-01

    The system proposed for overall assessment of symptoms in chronic prostatitis was applied in the assessment of 75 patients. The system facilitates detection and analysis of the complaints, quantitates the disease symptoms, provides overall objective characteristics of all the multiplicity of clinical manifestations of chronic prostatitis in each patient by means of digital order. The system is rather effective for dynamic control of the patients' condition and treatment efficacy.

  9. Characterization of genomic deletion efficiency mediated by clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 nuclease system in mammalian cells.

    PubMed

    Canver, Matthew C; Bauer, Daniel E; Dass, Abhishek; Yien, Yvette Y; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H; Orkin, Stuart H

    2014-08-01

    The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements.

  10. Characterization of Genomic Deletion Efficiency Mediated by Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/Cas9 Nuclease System in Mammalian Cells*♦

    PubMed Central

    Canver, Matthew C.; Bauer, Daniel E.; Dass, Abhishek; Yien, Yvette Y.; Chung, Jacky; Masuda, Takeshi; Maeda, Takahiro; Paw, Barry H.; Orkin, Stuart H.

    2014-01-01

    The clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 nuclease system has provided a powerful tool for genome engineering. Double strand breaks may trigger nonhomologous end joining repair, leading to frameshift mutations, or homology-directed repair using an extrachromosomal template. Alternatively, genomic deletions may be produced by a pair of double strand breaks. The efficiency of CRISPR/Cas9-mediated genomic deletions has not been systematically explored. Here, we present a methodology for the production of deletions in mammalian cells, ranging from 1.3 kb to greater than 1 Mb. We observed a high frequency of intended genomic deletions. Nondeleted alleles are nonetheless often edited with inversions or small insertion/deletions produced at CRISPR recognition sites. Deleted alleles also typically include small insertion/deletions at predicted deletion junctions. We retrieved cells with biallelic deletion at a frequency exceeding that of probabilistic expectation. We demonstrate an inverse relationship between deletion frequency and deletion size. This work suggests that CRISPR/Cas9 is a robust system to produce a spectrum of genomic deletions to allow investigation of genes and genetic elements. PMID:24907273

  11. A Model to Investigate Single-Strand DNA Responses in G1 Human Cells via a Telomere-Targeted, Nuclease-Deficient CRISPR-Cas9 System

    PubMed Central

    Crefcoeur, Remco P.; Zgheib, Omar; Halazonetis, Thanos D.

    2017-01-01

    DNA replication stress has the potential to compromise genomic stability and, therefore, cells have developed elaborate mechanisms to detect and resolve problems that may arise during DNA replication. The presence of single-stranded DNA (ssDNA) is often associated with DNA replication stress and serves as a signal for both checkpoint and repair responses. In this study, we exploited a CRISPR-Cas9 system to induce regions of ssDNA in the genome. Specifically, single-guide RNAs bearing sequence complementarity to human telomeric repeats, were used to target nuclease-deficient Cas9 (dCas9) to telomeres. Such targeting was associated with the formation of DNA-RNA hybrids, leaving one telomeric DNA strand single-stranded. This ssDNA then recruited DNA repair and checkpoint proteins, such as RPA, ATRIP, BLM and Rad51, at the telomeres. Interestingly, targeting of all these proteins to telomeric ssDNA was observed even in cells that were in the G1 phase of the cell cycle. Therefore, this system has the potential to serve as a platform for further investigation of DNA replication stress responses at specific loci in the human genome and in all phases of the cell cycle. PMID:28046023

  12. Creation of mutant mice with megabase-sized deletions containing custom-designed breakpoints by means of the CRISPR/Cas9 system.

    PubMed

    Kato, Tomoko; Hara, Satoshi; Goto, Yuji; Ogawa, Yuya; Okayasu, Haruka; Kubota, Souichirou; Tamano, Moe; Terao, Miho; Takada, Shuji

    2017-12-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a useful tool for creation of mutant mice with mutations mirroring those in human patients. Various methods have been developed for this purpose, including deletions, inversions, and translocations. So far, mutant mice with deletions of up to 1.2 megabases (Mb) have been generated by microinjection of the CRISPR/Cas9 system into fertilized eggs; however, a method for generation of mutant mice with a deletion of more than several Mb size is necessary because such deletions have often been identified as possible causes of human diseases. With an aim to enable the generation of disease models carrying large deletions with a breakpoint in custom-designed sequences, we developed a method for induction of an Mb-sized deletion by microinjection of a pair of sgRNAs, Cas9, and a donor plasmid into fertilized eggs. Using this method, we efficiently and rapidly generated mutant mice carrying deletions up to 5 Mb.

  13. Mixing Microworld and CAS Features in Building Computer Systems that Help Students Learn Algebra

    ERIC Educational Resources Information Center

    Nicaud, Jean-Francois; Bouhineau, Denis; Chaachoua, Hamid

    2004-01-01

    We present the design principles for a new kind of computer system that helps students learn algebra. The fundamental idea is to have a system based on the microworld paradigm that allows students to make their own calculations, as they do with paper and pencil, without being obliged to use commands, and to verify the correctness of these…

  14. Simple generation of albino C57BL/6J mice with G291T mutation in the tyrosinase gene by the CRISPR/Cas9 system.

    PubMed

    Mizuno, Seiya; Dinh, Tra Thi Huong; Kato, Kanako; Mizuno-Iijima, Saori; Tanimoto, Yoko; Daitoku, Yoko; Hoshino, Yoshikazu; Ikawa, Masahito; Takahashi, Satoru; Sugiyama, Fumihiro; Yagami, Ken-ichi

    2014-08-01

    Single nucleotide mutations (SNMs) are associated with a variety of human diseases. The CRISPR/Cas9 genome-editing system is expected to be useful as a genetic modification method for production of SNM-induced mice. To investigate whether SNM-induced mice can be generated by zygote microinjection of CRISPR/Cas9 vector and single-stranded DNA (ssDNA) donor, we attempted to produce albino C57BL/6J mice carrying the Tyr gene SNM (G291T) from pigmented C57BL/6J zygotes. We first designed and constructed a CRISPR/Cas9 expression vector for the Tyr gene (px330-Tyr-M). DNA cleavage activity of px330-Tyr-M at the target site of the Tyr gene was confirmed by the EGxxFP system. We also designed an ssDNA donor for homology-directed repair (HDR)-mediated gene modification. The px330-Tyr-M vector and ssDNA donor were co-microinjected into the pronuclei of 224 one-cell-stage embryos derived from C57BL/6J mice. We obtained 60 neonates, 28 of which showed the ocular albinism and absence of coat pigmentation. Genomic sequencing analysis of the albino mice revealed that the target of SNM, G291T in the Tyr gene, occurred in 11 mice and one founder was homozygously mutated. The remaining albino founders without Tyr G291T mutation also possessed biallelic deletion and insertion mutants adjacent to the target site in the Tyr locus. Simple production of albino C57BL/6J mice was provided by C57BL/6J zygote microinjection with px330-Tyr-M DNA vector and mutant ssDNA (G291T in Tyr) donor. A combination of CRISPR/Cas9 vector and optional mutant ssDNA could be expected to efficiently produce novel SNM-induced mouse models for investigating human diseases.

  15. Adaptive Systems in Education: A Review and Conceptual Unification

    ERIC Educational Resources Information Center

    Wilson, Chunyu; Scott, Bernard

    2017-01-01

    Purpose: The purpose of this paper is to review the use of adaptive systems in education. It is intended to be a useful introduction for the non-specialist reader. Design/methodology/approach: A distinction is made between intelligent tutoring systems (ITSs) and adaptive hypermedia systems (AHSs). The two kinds of system are defined, compared and…

  16. The New State of the Art: Cas9 for Gene Activation and Repression

    PubMed Central

    La Russa, Marie F.

    2015-01-01

    CRISPR-Cas9 technology has rapidly changed the landscape for how biologists and bioengineers study and manipulate the genome. Derived from the bacterial adaptive immune system, CRISPR-Cas9 has been coopted and repurposed for a variety of new functions, including the activation or repression of gene expression (termed CRISPRa or CRISPRi, respectively). This represents an exciting alternative to previously used repression or activation technologies such as RNA interference (RNAi) or the use of gene overexpression vectors. We have only just begun exploring the possibilities that CRISPR technology offers for gene regulation and the control of cell identity and behavior. In this review, we describe the recent advances of CRISPR-Cas9 technology for gene regulation and outline advantages and disadvantages of CRISPRa and CRISPRi (CRISPRa/i) relative to alternative technologies. PMID:26370509

  17. Biallelic insertion of a transcriptional terminator via the CRISPR/Cas9 system efficiently silences expression of protein-coding and non-coding RNA genes.

    PubMed

    Liu, Yangyang; Han, Xiao; Yuan, Junting; Geng, Tuoyu; Chen, Shihao; Hu, Xuming; Cui, Isabelle H; Cui, Hengmi

    2017-04-07

    The type II bacterial CRISPR/Cas9 system is a simple, convenient, and powerful tool for targeted gene editing. Here, we describe a CRISPR/Cas9-based approach for inserting a poly(A) transcriptional terminator into both alleles of a targeted gene to silence protein-coding and non-protein-coding genes, which often play key roles in gene regulation but are difficult to silence via insertion or deletion of short DNA fragments. The integration of 225 bp of bovine growth hormone poly(A) signals into either the first intron or the first exon or behind the promoter of target genes caused efficient termination of expression of PPP1R12C, NSUN2 (protein-coding genes), and MALAT1 (non-protein-coding gene). Both NeoR and PuroR were used as markers in the selection of clonal cell lines with biallelic integration of a poly(A) signal. Genotyping analysis indicated that the cell lines displayed the desired biallelic silencing after a brief selection period. These combined results indicate that this CRISPR/Cas9-based approach offers an easy, convenient, and efficient novel technique for gene silencing in cell lines, especially for those in which gene integration is difficult because of a low efficiency of homology-directed repair.

  18. Exploring Students' Understanding of Ordinary Differential Equations Using Computer Algebraic System (CAS)

    ERIC Educational Resources Information Center

    Maat, Siti Mistima; Zakaria, Effandi

    2011-01-01

    Ordinary differential equations (ODEs) are one of the important topics in engineering mathematics that lead to the understanding of technical concepts among students. This study was conducted to explore the students' understanding of ODEs when they solve ODE questions using a traditional method as well as a computer algebraic system, particularly…

  19. Brain tumor modeling using the CRISPR/Cas9 system: state of the art and view to the future

    PubMed Central

    Mao, Xiao-Yuan; Dai, Jin-Xiang; Zhou, Hong-Hao; Liu, Zhao-Qian; Jin, Wei-Lin

    2016-01-01

    Although brain tumors have been known tremendously over the past decade, there are still many problems to be solved. The etiology of brain tumors is not well understood and the treatment remains modest. There is in great need to develop a suitable brain tumor models that faithfully mirror the etiology of human brain neoplasm and subsequently get more efficient therapeutic approaches for these disorders. In this review, we described the current status of animal models of brain tumors and analyzed their advantages and disadvantages. Additionally, prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a versatile genome editing technology for investigating the functions of target genes, and its application were also introduced in our present work. We firstly proposed that brain tumor modeling could be well established via CRISPR/Cas9 techniques. And CRISPR/Cas9-mediated brain tumor modeling was likely to be more suitable for figuring out the pathogenesis of brain tumors, as CRISPR/Cas9 platform was a simple and more efficient biological toolbox for implementing mutagenesis of oncogenes or tumor suppressors that were closely linked with brain tumors. PMID:26993776

  20. Brain tumor modeling using the CRISPR/Cas9 system: state of the art and view to the future.

    PubMed

    Mao, Xiao-Yuan; Dai, Jin-Xiang; Zhou, Hong-Hao; Liu, Zhao-Qian; Jin, Wei-Lin

    2016-05-31

    Although brain tumors have been known tremendously over the past decade, there are still many problems to be solved. The etiology of brain tumors is not well understood and the treatment remains modest. There is in great need to develop a suitable brain tumor models that faithfully mirror the etiology of human brain neoplasm and subsequently get more efficient therapeutic approaches for these disorders. In this review, we described the current status of animal models of brain tumors and analyzed their advantages and disadvantages. Additionally, prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), a versatile genome editing technology for investigating the functions of target genes, and its application were also introduced in our present work. We firstly proposed that brain tumor modeling could be well established via CRISPR/Cas9 techniques. And CRISPR/Cas9-mediated brain tumor modeling was likely to be more suitable for figuring out the pathogenesis of brain tumors, as CRISPR/Cas9 platform was a simple and more efficient biological toolbox for implementing mutagenesis of oncogenes or tumor suppressors that were closely linked with brain tumors.

  1. Integration of the immune system: a complex adaptive supersystem

    NASA Astrophysics Data System (ADS)

    Crisman, Mark V.

    2001-10-01

    Immunity to pathogenic organisms is a complex process involving interacting factors within the immune system including circulating cells, tissues and soluble chemical mediators. Both the efficiency and adaptive responses of the immune system in a dynamic, often hostile, environment are essential for maintaining our health and homeostasis. This paper will present a brief review of one of nature's most elegant, complex adaptive systems.

  2. Implementation of an Adaptive Learning System Using a Bayesian Network

    ERIC Educational Resources Information Center

    Yasuda, Keiji; Kawashima, Hiroyuki; Hata, Yoko; Kimura, Hiroaki

    2015-01-01

    An adaptive learning system is proposed that incorporates a Bayesian network to efficiently gauge learners' understanding at the course-unit level. Also, learners receive content that is adapted to their measured level of understanding. The system works on an iPad via the Edmodo platform. A field experiment using the system in an elementary school…

  3. Isoplanatism in a multiconjugate adaptive optics system.

    PubMed

    Tokovinin, A; Le Louarn, M; Sarazin, M

    2000-10-01

    Turbulence correction in a large field of view by use of an adaptive optics imaging system with several deformable mirrors (DM's) conjugated to various heights is considered. The residual phase variance is computed for an optimized linear algorithm in which a correction of each turbulent layer is achieved by applying a combination of suitably smoothed and scaled input phase screens to all DM's. Finite turbulence outer scale and finite spatial resolution of the DM's are taken into account. A general expression for the isoplanatic angle thetaM of a system with M mirrors is derived in the limiting case of infinitely large apertures and Kolmogorov turbulence. Like Fried's isoplanatic angle theta0,thetaM is a function only of the turbulence vertical profile, is scalable with wavelength, and is independent of the telescope diameter. Use of angle thetaM permits the gain in the field of view due to the increased number of DM's to be quantified and their optimal conjugate heights to be found. Calculations with real turbulence profiles show that with three DM's a gain of 7-10x is possible, giving the typical and best isoplanatic field-of-view radii of 16 and 30 arcseconds, respectively, at lambda = 0.5 microm. It is shown that in the actual systems the isoplanatic field will be somewhat larger than thetaM owing to the combined effects of finite aperture diameter, finite outer scale, and optimized wave-front spatial filtering. However, this additional gain is not dramatic; it is less than 1.5x for large-aperture telescopes.

  4. Network and adaptive system of systems modeling and analysis.

    SciTech Connect

    Lawton, Craig R.; Campbell, James E. Dr.; Anderson, Dennis James; Eddy, John P.

    2007-05-01

    This report documents the results of an LDRD program entitled ''Network and Adaptive System of Systems Modeling and Analysis'' that was conducted during FY 2005 and FY 2006. The purpose of this study was to determine and implement ways to incorporate network communications modeling into existing System of Systems (SoS) modeling capabilities. Current SoS modeling, particularly for the Future Combat Systems (FCS) program, is conducted under the assumption that communication between the various systems is always possible and occurs instantaneously. A more realistic representation of these communications allows for better, more accurate simulation results. The current approach to meeting this objective has been to use existing capabilities to model network hardware reliability and adding capabilities to use that information to model the impact on the sustainment supply chain and operational availability.

  5. Valuation of design adaptability in aerospace systems

    NASA Astrophysics Data System (ADS)

    Fernandez Martin, Ismael

    As more information is brought into early stages of the design, more pressure is put on engineers to produce a reliable, high quality, and financially sustainable product. Unfortunately, requirements established at the beginning of a new project by customers, and the environment that surrounds them, continue to change in some unpredictable ways. The risk of designing a system that may become obsolete during early stages of production is currently tackled by the use of robust design simulation, a method that allows to simultaneously explore a plethora of design alternatives and requirements with the intention of accounting for uncertain factors in the future. Whereas this design technique has proven to be quite an improvement in design methods, under certain conditions, it fails to account for the change of uncertainty over time and the intrinsic value embedded in the system when certain design features are activated. This thesis introduces the concepts of adaptability and real options to manage risk foreseen in the face of uncertainty at early design stages. The method described herein allows decision-makers to foresee the financial impact of their decisions at the design level, as well as the final exposure to risk. In this thesis, cash flow models, traditionally used to obtain the forecast of a project's value over the years, were replaced with surrogate models that are capable of showing fluctuations on value every few days. This allowed a better implementation of real options valuation, optimization, and strategy selection. Through the option analysis model, an optimization exercise allows the user to obtain the best implementation strategy in the face of uncertainty as well as the overall value of the design feature. Here implementation strategy refers to the decision to include a new design feature in the system, after the design has been finalized, but before the end of its production life. The ability to do this in a cost efficient manner after the system

  6. [CAS General Standards 2012

    ERIC Educational Resources Information Center

    Council for the Advancement of Standards in Higher Education, 2011

    2011-01-01

    The mission of the Council for the Advancement of Standards in Higher Education (CAS) is to promote the improvement of programs and services to enhance the quality of student learning and development. CAS is a consortium of professional associations who work collaboratively to develop and promulgate standards and guidelines and to encourage…

  7. Modeling disease in vivo with CRISPR/Cas9

    PubMed Central

    Dow, Lukas E.

    2015-01-01

    The recent advent of CRISPR/Cas9-mediated genome editing has created a wave of excitement across the scientific research community, carrying the promise of simple and effective genomic manipulation of nearly any cell type. CRISPR has quickly become the preferred tool for genetic manipulation, and shows incredible promise as a platform for studying gene function in vivo. Here, I discuss the current application of CRISPR technology to create new in vivo disease models, with a particular focus on how these tools, derived from an adaptive bacterial immune system, are helping us better model the complexity of human cancer. PMID:26432018

  8. Modeling Disease In Vivo With CRISPR/Cas9.

    PubMed

    Dow, Lukas E

    2015-10-01

    The recent advent of CRISPR/Cas9-mediated genome editing has created a wave of excitement across the scientific research community, carrying the promise of simple and effective genomic manipulation of nearly any cell type. CRISPR has quickly become the preferred tool for genetic manipulation, and shows incredible promise as a platform for studying gene function in vivo. I discuss the current application of CRISPR technology to create new in vivo disease models, with a particular focus on how these tools, derived from an adaptive bacterial immune system, are helping us to better model the complexity of human cancer.

  9. Cascaded Effects of Spatial Adaptation in the Early Visual System

    PubMed Central

    Dhruv, Neel T.; Carandini, Matteo

    2014-01-01

    Summary Virtually all stages of the visual system exhibit adaptation: neurons adjust their responses based on the recent stimulus history. While some of these adjustments occur at specific stages, others may be inherited from earlier stages. How do adaptation effects cascade along the visual system? We measured spatially selective adaptation at two successive stages in the mouse visual system: visual thalamus (LGN) and primary visual cortex (V1). This form of adaptation affected both stages but in drastically different ways: in LGN it only changed response gain, while in V1 it also shifted spatial tuning away from the adaptor. These effects, however, are reconciled by a simple model whereby V1 neurons summate LGN inputs with a fixed, unadaptable weighting profile. These results indicate that adaptation effects cascade through the visual system, that this cascading can shape selectivity, and that the rules of integration from one stage to the next are not themselves adaptable. PMID:24507190

  10. Cascaded effects of spatial adaptation in the early visual system.

    PubMed

    Dhruv, Neel T; Carandini, Matteo

    2014-02-05

    Virtually all stages of the visual system exhibit adaptation: neurons adjust their responses based on the recent stimulus history. While some of these adjustments occur at specific stages, others may be inherited from earlier stages. How do adaptation effects cascade along the visual system? We measured spatially selective adaptation at two successive stages in the mouse visual system: visual thalamus (LGN) and primary visual cortex (V1). This form of adaptation affected both stages but in drastically different ways: in LGN it only changed response gain, while in V1 it also shifted spatial tuning away from the adaptor. These effects, however, are reconciled by a simple model whereby V1 neurons summate LGN inputs with a fixed, unadaptable weighting profile. These results indicate that adaptation effects cascade through the visual system, that this cascading can shape selectivity, and that the rules of integration from one stage to the next are not themselves adaptable.

  11. Central nervous system adaptation to exercise training

    NASA Astrophysics Data System (ADS)

    Kaminski, Lois Anne

    Exercise training causes physiological changes in skeletal muscle that results in enhanced performance in humans and animals. Despite numerous studies on exercise effects on skeletal muscle, relatively little is known about adaptive changes in the central nervous system. This study investigated whether spinal pathways that mediate locomotor activity undergo functional adaptation after 28 days of exercise training. Ventral horn spinal cord expression of calcitonin gene-related peptide (CGRP), a trophic factor at the neuromuscular junction, choline acetyltransferase (Chat), the synthetic enzyme for acetylcholine, vesicular acetylcholine transporter (Vacht), a transporter of ACh into synaptic vesicles and calcineurin (CaN), a protein phosphatase that phosphorylates ion channels and exocytosis machinery were measured to determine if changes in expression occurred in response to physical activity. Expression of these proteins was determined by western blot and immunohistochemistry (IHC). Comparisons between sedentary controls and animals that underwent either endurance training or resistance training were made. Control rats received no exercise other than normal cage activity. Endurance-trained rats were exercised 6 days/wk at 31m/min on a treadmill (8% incline) for 100 minutes. Resistance-trained rats supported their weight plus an additional load (70--80% body weight) on a 60° incline (3 x 3 min, 5 days/wk). CGRP expression was measured by radioimmunoassay (RIA). CGRP expression in the spinal dorsal and ventral horn of exercise-trained animals was not significantly different than controls. Chat expression measured by Western blot and IHC was not significantly different between runners and controls but expression in resistance-trained animals assayed by IHC was significantly less than controls and runners. Vacht and CaN immunoreactivity in motor neurons of endurance-trained rats was significantly elevated relative to control and resistance-trained animals. Ventral

  12. Aircraft as adaptive nonlinear system which must be in the adaptational maximum zone for safety

    SciTech Connect

    Ignative, M.; Simatos, N.; Sivasundaram, S.

    1994-12-31

    Safety is a main problem in aircraft. We are considering this problem from the point of view related to existence of the adaptational maximum in complex developing systems. Safety space of aircraft parameters are determined. This space is transformed to different regimes of flight, when one engine malfunctions etc., are considered. Also it is shown that maximum safety is in adaptational maximum zone.

  13. Adapt

    NASA Astrophysics Data System (ADS)

    Bargatze, L. F.

    2015-12-01

    Active Data Archive Product Tracking (ADAPT) is a collection of software routines that permits one to generate XML metadata files to describe and register data products in support of the NASA Heliophysics Virtual Observatory VxO effort. ADAPT is also a philosophy. The ADAPT concept is to use any and all available metadata associated with scientific data to produce XML metadata descriptions in a consistent, uniform, and organized fashion to provide blanket access to the full complement of data stored on a targeted data server. In this poster, we present an application of ADAPT to describe all of the data products that are stored by using the Common Data File (CDF) format served out by the CDAWEB and SPDF data servers hosted at the NASA Goddard Space Flight Center. These data servers are the primary repositories for NASA Heliophysics data. For this purpose, the ADAPT routines have been used to generate data resource descriptions by using an XML schema named Space Physics Archive, Search, and Extract (SPASE). SPASE is the designated standard for documenting Heliophysics data products, as adopted by the Heliophysics Data and Model Consortium. The set of SPASE XML resource descriptions produced by ADAPT includes high-level descriptions of numerical data products, display data products, or catalogs and also includes low-level "Granule" descriptions. A SPASE Granule is effectively a universal access metadata resource; a Granule associates an individual data file (e.g. a CDF file) with a "parent" high-level data resource description, assigns a resource identifier to the file, and lists the corresponding assess URL(s). The CDAWEB and SPDF file systems were queried to provide the input required by the ADAPT software to create an initial set of SPASE metadata resource descriptions. Then, the CDAWEB and SPDF data repositories were queried subsequently on a nightly basis and the CDF file lists were checked for any changes such as the occurrence of new, modified, or deleted

  14. CRISPR/Cas9 Technologies.

    PubMed

    Williams, Bart O; Warman, Matthew L

    2017-02-23

    The Clustered Regularly Interspaced Palindromic Repeats (CRISPR)/CRISPR-associated protein (Cas) pathway is revolutionizing biological research. Modifications to this primitive prokaryotic immune system now enable scientists to efficiently edit DNA or modulate gene expression in living eukaryotic cells and organisms. Thus, many laboratories can now perform important experiments that previously were considered scientifically risky or too costly. Here, we describe the components of the CRISPR/Cas system that have been engineered for use in eukaryotes. We also explain how this system can be used to genetically modify cell lines and model organisms, or regulate gene expression in order to search for new participants in biological pathways. © 2017 American Society for Bone and Mineral Research.

  15. Nonequilibrium Enhances Adaptation Efficiency of Stochastic Biochemical Systems

    PubMed Central

    Jia, Chen; Qian, Minping

    2016-01-01

    Adaptation is a crucial biological function possessed by many sensory systems. Early work has shown that some influential equilibrium models can achieve accurate adaptation. However, recent studies indicate that there are close relationships between adaptation and nonequilibrium. In this paper, we provide an explanation of these two seemingly contradictory results based on Markov models with relatively simple networks. We show that as the nonequilibrium driving becomes stronger, the system under consideration will undergo a phase transition along a fixed direction: from non-adaptation to simple adaptation then to oscillatory adaptation, while the transition in the opposite direction is forbidden. This indicates that although adaptation may be observed in equilibrium systems, it tends to occur in systems far away from equilibrium. In addition, we find that nonequilibrium will improve the performance of adaptation by enhancing the adaptation efficiency. All these results provide a deeper insight into the connection between adaptation and nonequilibrium. Finally, we use a more complicated network model of bacterial chemotaxis to validate the main results of this paper. PMID:27195482

  16. Structure and Engineering of Francisella novicida Cas9

    PubMed Central

    Hirano, Hisato; Gootenberg, Jonathan S.; Horii, Takuro; Abudayyeh, Omar O.; Kimura, Mika; Hsu, Patrick D.; Nakane, Takanori; Ishitani, Ryuichiro; Hatada, Izuho; Zhang, Feng; Nishimasu, Hiroshi; Nureki, Osamu

    2016-01-01

    Summary The RNA-guided endonuclease Cas9 cleaves double-stranded DNA targets complementary to the guide RNA, and has been applied to programmable genome editing. Cas9-mediated cleavage requires a protospacer adjacent motif (PAM) juxtaposed with the DNA target sequence, thus constricting the range of targetable sites. Here, we report the 1.7 Å resolution crystal structures of Cas9 from Francisella novicida (FnCas9), one of the largest Cas9 orthologs, in complex with a guide RNA and its PAM-containing DNA targets. A structural comparison of FnCas9 with other Cas9 orthologs revealed striking conserved and divergent features among distantly related CRISPR-Cas9 systems. We found that FnCas9 recognizes the 5′-NGG-3′ PAM, and used the structural information to create a variant that can recognize the more relaxed 5′-YG-3′ PAM. Furthermore, we demonstrated that pre-assembled FnCas9 ribonucleoprotein complexes can be microinjected into mouse zygotes to edit endogenous sites with the 5′-YG-3′ PAMs, thus expanding the target space of the CRISPR-Cas9 toolbox. PMID:26875867

  17. Restricted Complexity Framework for Nonlinear Adaptive Control in Complex Systems

    NASA Astrophysics Data System (ADS)

    Williams, Rube B.

    2004-02-01

    Control law adaptation that includes implicit or explicit adaptive state estimation, can be a fundamental underpinning for the success of intelligent control in complex systems, particularly during subsystem failures, where vital system states and parameters can be impractical or impossible to measure directly. A practical algorithm is proposed for adaptive state filtering and control in nonlinear dynamic systems when the state equations are unknown or are too complex to model analytically. The state equations and inverse plant model are approximated by using neural networks. A framework for a neural network based nonlinear dynamic inversion control law is proposed, as an extrapolation of prior developed restricted complexity methodology used to formulate the adaptive state filter. Examples of adaptive filter performance are presented for an SSME simulation with high pressure turbine failure to support extrapolations to adaptive control problems.

  18. Restricted Complexity Framework for Nonlinear Adaptive Control in Complex Systems

    SciTech Connect

    Williams, Rube B.

    2004-02-04

    Control law adaptation that includes implicit or explicit adaptive state estimation, can be a fundamental underpinning for the success of intelligent control in complex systems, particularly during subsystem failures, where vital system states and parameters can be impractical or impossible to measure directly. A practical algorithm is proposed for adaptive state filtering and control in nonlinear dynamic systems when the state equations are unknown or are too complex to model analytically. The state equations and inverse plant model are approximated by using neural networks. A framework for a neural network based nonlinear dynamic inversion control law is proposed, as an extrapolation of prior developed restricted complexity methodology used to formulate the adaptive state filter. Examples of adaptive filter performance are presented for an SSME simulation with high pressure turbine failure to support extrapolations to adaptive control problems.

  19. Concept Based Approach for Adaptive Personalized Course Learning System

    ERIC Educational Resources Information Center

    Salahli, Mehmet Ali; Özdemir, Muzaffer; Yasar, Cumali

    2013-01-01

    One of the most important factors for improving the personalization aspects of learning systems is to enable adaptive properties to them. The aim of the adaptive personalized learning system is to offer the most appropriate learning path and learning materials to learners by taking into account their profiles. In this paper, a new approach to…

  20. Adaptive tracking control for a class of uncertain chaotic systems

    NASA Astrophysics Data System (ADS)

    Chen, Feng-Xiang; Wang, Wei; Zhang, Wei-Dong

    2007-09-01

    The paper is concerned with adaptive tracking problem for a class of chaotic system with time-varying uncertainty, but bounded by norm polynomial. Based on adaptive technique, it proposes a novel controller to asymptotically track the arbitrary desired bounded trajectory. Simulation on the Rossler chaotic system is performed and the result verifies the effectiveness of the proposed method.

  1. Development of Adaptive Kanji Learning System for Mobile Phone

    ERIC Educational Resources Information Center

    Li, Mengmeng; Ogata, Hiroaki; Hou, Bin; Hashimoto, Satoshi; Liu, Yuqin; Uosaki, Noriko; Yano, Yoneo

    2010-01-01

    This paper describes an adaptive learning system based on mobile phone email to support the study of Japanese Kanji. In this study, the main emphasis is on using the adaptive learning to resolve one common problem of the mobile-based email or SMS language learning systems. To achieve this goal, the authors main efforts focus on three aspects:…

  2. Adaptive Hypermedia Educational System Based on XML Technologies.

    ERIC Educational Resources Information Center

    Baek, Yeongtae; Wang, Changjong; Lee, Sehoon

    This paper proposes an adaptive hypermedia educational system using XML technologies, such as XML, XSL, XSLT, and XLink. Adaptive systems are capable of altering the presentation of the content of the hypermedia on the basis of a dynamic understanding of the individual user. The user profile can be collected in a user model, while the knowledge…

  3. Management Strategies for Complex Adaptive Systems: Sensemaking, Learning, and Improvisation

    ERIC Educational Resources Information Center

    McDaniel, Reuben R., Jr.

    2007-01-01

    Misspecification of the nature of organizations may be a major reason for difficulty in achieving performance improvement. Organizations are often viewed as machine-like, but complexity science suggests that organizations should be viewed as complex adaptive systems. I identify the characteristics of complex adaptive systems and give examples of…

  4. Sinusoidal error perturbation reveals multiple coordinate systems for sensorymotor adaptation

    PubMed Central

    Hudson, Todd E.; Landy, Michael S.

    2016-01-01

    A coordinate system is composed of an encoding, defining the dimensions of the space, and an origin. We examine the coordinate encoding used to update motor plans during sensory-motor adaptation to center-out reaches. Adaptation is induced using a novel paradigm in which feedback of reach endpoints is perturbed following a sinewave pattern over trials; the perturbed dimensions of the feedback were the axes of a Cartesian coordinate system in one session and a polar coordinate system in another session. For center-out reaches to randomly chosen target locations, reach errors observed at one target will require different corrections at other targets within Cartesian- and polar-coded systems. The sinewave adaptation technique allowed us to simultaneously adapt both dimensions of each coordinate system (x-y, or reach gain and angle), and identify the contributions of each perturbed dimension by adapting each at a distinct temporal frequency. The efficiency of this technique further allowed us to employ perturbations that were a fraction the size normally used, which avoids confounding automatic adaptive processes with deliberate adjustments made in response to obvious experimental manipulations. Subjects independently corrected errors in each coordinate in both sessions, suggesting that the nervous system encodes both a Cartesian- and polar-coordinate-based internal representation for motor adaptation. The gains and phase lags of the adaptive responses are not readily explained by current theories of sensory-motor adaptation. Motor adaptation is fundamental to the neural control of movement, affording an automatic process to maintain a consistent relationship between motor plans and movement outcomes. That is, adaptation is described as updating an internal mapping between desired motor outcome and motor output (Sanger, 2004; Shadmehr, Smith, & Krakauer, 2010), not a deliberate corrective action. Here, using a method that relies on extremely small perturbations that

  5. The evolutionary history and diagnostic utility of the CRISPR-Cas system within Salmonella enterica ssp. enterica

    PubMed Central

    Timme, Ruth E.; Barrangou, Rodolphe; Toro, Magaly; Allard, Marc W.; Strain, Errol; Musser, Steven M.; Brown, Eric W.

    2014-01-01

    Evolutionary studies of clustered regularly interspaced short palindromic repeats (CRISPRs) and their associated (cas) genes can provide insights into host-pathogen co-evolutionary dynamics and the frequency at which different genomic events (e.g., horizontal vs. vertical transmission) occur. Within this study, we used whole genome sequence (WGS) data to determine the evolutionary history and genetic diversity of CRISPR loci and cas genes among a diverse set of 427 Salmonella enterica ssp. enterica isolates representing 64 different serovars. We also evaluated the performance of CRISPR loci for typing when compared to whole genome and multilocus sequence typing (MLST) approaches. We found that there was high diversity in array length within both CRISPR1 (median = 22; min = 3; max = 79) and CRISPR2 (median = 27; min = 2; max = 221). There was also much diversity within serovars (e.g., arrays differed by as many as 50 repeat-spacer units among Salmonella ser. Senftenberg isolates). Interestingly, we found that there are two general cas gene profiles that do not track phylogenetic relationships, which suggests that non-vertical transmission events have occurred frequently throughout the evolutionary history of the sampled isolates. There is also considerable variation among the ranges of pairwise distances estimated within each cas gene, which may be indicative of the strength of natural selection acting on those genes. We developed a novel clustering approach based on CRISPR spacer content, but found that typing based on CRISPRs was less accurate than the MLST-based alternative; typing based on WGS data was the most accurate. Notwithstanding cost and accessibility, we anticipate that draft genome sequencing, due to its greater discriminatory power, will eventually become routine for traceback investigations. PMID:24765574

  6. Inhibition of CRISPR-Cas9 with Bacteriophage Proteins.

    PubMed

    Rauch, Benjamin J; Silvis, Melanie R; Hultquist, Judd F; Waters, Christopher S; McGregor, Michael J; Krogan, Nevan J; Bondy-Denomy, Joseph

    2017-01-12

    Bacterial CRISPR-Cas systems utilize sequence-specific RNA-guided nucleases to defend against bacteriophage infection. As a countermeasure, numerous phages are known that produce proteins to block the function of class 1 CRISPR-Cas systems. However, currently no proteins are known to inhibit the widely used class 2 CRISPR-Cas9 system. To find these inhibitors, we searched cas9-containing bacterial genomes for the co-existence of a CRISPR spacer and its target, a potential indicator for CRISPR inhibition. This analysis led to the discovery of four unique type II-A CRISPR-Cas9 inhibitor proteins encoded by Listeria monocytogenes prophages. More than half of L. monocytogenes strains with cas9 contain at least one prophage-encoded inhibitor, suggesting widespread CRISPR-Cas9 inactivation. Two of these inhibitors also blocked the widely used Streptococcus pyogenes Cas9 when assayed in Escherichia coli and human cells. These natural Cas9-specific "anti-CRISPRs" present tools that can be used to regulate the genome engineering activities of CRISPR-Cas9.

  7. Calibrated Methodology for Assessing Adaptation Costs for Urban Drainage Systems

    EPA Science Inventory

    Changes in precipitation patterns associated with climate change may pose significant challenges for storm water management systems across much of the U.S. In particular, adapting these systems to more intense rainfall events will require significant investment. The assessment ...

  8. Recursive architecture for large-scale adaptive system

    NASA Astrophysics Data System (ADS)

    Hanahara, Kazuyuki; Sugiyama, Yoshihiko

    1994-09-01

    'Large scale' is one of major trends in the research and development of recent engineering, especially in the field of aerospace structural system. This term expresses the large scale of an artifact in general, however, it also implies the large number of the components which make up the artifact in usual. Considering a large scale system which is especially used in remote space or deep-sea, such a system should be adaptive as well as robust by itself, because its control as well as maintenance by human operators are not easy due to the remoteness. An approach to realizing this large scale, adaptive and robust system is to build the system as an assemblage of components which are respectively adaptive by themselves. In this case, the robustness of the system can be achieved by using a large number of such components and suitable adaptation as well as maintenance strategies. Such a system gathers many research's interest and their studies such as decentralized motion control, configurating algorithm and characteristics of structural elements are reported. In this article, a recursive architecture concept is developed and discussed towards the realization of large scale system which consists of a number of uniform adaptive components. We propose an adaptation strategy based on the architecture and its implementation by means of hierarchically connected processing units. The robustness and the restoration from degeneration of the processing unit are also discussed. Two- and three-dimensional adaptive truss structures are conceptually designed based on the recursive architecture.

  9. Systems and Methods for Derivative-Free Adaptive Control

    NASA Technical Reports Server (NTRS)

    Yucelen, Tansel (Inventor); Kim, Kilsoo (Inventor); Calise, Anthony J. (Inventor)

    2015-01-01

    An adaptive control system is disclosed. The control system can control uncertain dynamic systems. The control system can employ one or more derivative-free adaptive control architectures. The control system can further employ one or more derivative-free weight update laws. The derivative-free weight update laws can comprise a time-varying estimate of an ideal vector of weights. The control system of the present invention can therefore quickly stabilize systems that undergo sudden changes in dynamics, caused by, for example, sudden changes in weight. Embodiments of the present invention can also provide a less complex control system than existing adaptive control systems. The control system can control aircraft and other dynamic systems, such as, for example, those with non-minimum phase dynamics.

  10. Adaptive Learning Systems: Beyond Teaching Machines

    ERIC Educational Resources Information Center

    Kara, Nuri; Sevim, Nese

    2013-01-01

    Since 1950s, teaching machines have changed a lot. Today, we have different ideas about how people learn, what instructor should do to help students during their learning process. We have adaptive learning technologies that can create much more student oriented learning environments. The purpose of this article is to present these changes and its…

  11. Hormesis and adaptive cellular control systems

    EPA Science Inventory

    Hormetic dose response occurs for many endpoints associated with exposures of biological organisms to environmental stressors. Cell-based U- or inverted U-shaped responses may derive from common processes involved in activation of adaptive responses required to protect cells from...

  12. A Guide to Computer Adaptive Testing Systems

    ERIC Educational Resources Information Center

    Davey, Tim

    2011-01-01

    Some brand names are used generically to describe an entire class of products that perform the same function. "Kleenex," "Xerox," "Thermos," and "Band-Aid" are good examples. The term "computerized adaptive testing" (CAT) is similar in that it is often applied uniformly across a diverse family of testing methods. Although the various members of…

  13. CRISPR/Cas9-The ultimate weapon to battle infectious diseases?

    PubMed

    Doerflinger, M; Forsyth, W; Ebert, G; Pellegrini, M; Herold, M J

    2017-02-01

    Infectious diseases are a leading cause of death worldwide. Novel therapeutics are urgently required to treat multidrug-resistant organisms such as Mycobacterium tuberculosis and to mitigate morbidity and mortality caused by acute infections such as malaria and dengue fever virus as well as chronic infections such as human immunodeficiency virus-1 and hepatitis B virus. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, which has revolutionized biomedical research, holds great promise for the identification and validation of novel drug targets. Since its discovery as an adaptive immune system in prokaryotes, the CRISPR/Cas9 system has been developed into a multi-faceted genetic modification tool, which can now be used to induce gene deletions or specific gene insertions, such as conditional alleles or endogenous reporters in virtually any organism. The generation of CRISPR/Cas9 libraries that can be used to perform phenotypic whole genome screens provides an important new tool that will aid in the identification of critical host factors involved in the pathogenesis of infectious diseases. In this review, we will discuss the development and recent applications of the CRISPR/Cas9 system used to identify novel regulators, which might become important in the fight against infectious diseases.

  14. Precision Targeted Mutagenesis via Cas9 Paired Nickases in Rice

    PubMed Central

    Mikami, Masafumi; Toki, Seiichi; Endo, Masaki

    2016-01-01

    Recent reports of CRISPR- (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated protein 9) mediated heritable mutagenesis in plants highlight the need for accuracy of the mutagenesis directed by this system. Off-target mutations are an important issue when considering functional gene analysis, as well as the molecular breeding of crop plants with large genome size, i.e. with many duplicated genes, and where the whole-genome sequence is still lacking. In mammals, off-target mutations can be suppressed by using Cas9 paired nickases together with paired guide RNAs (gRNAs). However, the performance of Cas9 paired nickases has not yet been fully assessed in plants. Here, we analyzed on- and off-target mutation frequency in rice calli and regenerated plants using Cas9 nuclease or Cas9 nickase with paired gRNAs. When Cas9 paired nickases were used, off-target mutations were fully suppressed in rice calli and regenerated plants. However, on-target mutation frequency also decreased compared with that induced by the Cas9 paired nucleases system. Since the gRNA sequence determines specific binding of Cas9 protein–gRNA ribonucleoproteins at the targeted sequence, the on-target mutation frequency of Cas9 paired nickases depends on the design of paired gRNAs. Our results suggest that a combination of gRNAs that can induce mutations at high efficiency with Cas9 nuclease should be used together with Cas9 nickase. Furthermore, we confirmed that a combination of gRNAs containing a one nucleotide (1 nt) mismatch toward the target sequence could not induce mutations when expressed with Cas9 nickase. Our results clearly show the effectiveness of Cas9 paired nickases in delivering on-target specific mutations. PMID:26936792

  15. The Development and Evaluation of a Computerized Adaptive Testing System.

    ERIC Educational Resources Information Center

    de-la-Torre, Roberto; Vispoel, Walter P.

    The development and preliminary evaluation of the Computerized Adaptive Testing System (CATSYS), a new testing package for IBM-compatible microcomputers, are described. CATSYS can be used to administer and score operational adaptive tests or to conduct on-line computer simulation studies. The package incorporates several innovative features,…

  16. [Adaptation potential of cardio-respiratory system in dust diseases].

    PubMed

    Serebryakov, P V; Nenenko, O I; Fedina, I N; Rakhimzyanov, A R

    2016-01-01

    The article covers results of cardio-respiratory system evaluation in workers exposed to dust, on basis of adaptation potential evaluation via calculation of functional changes index and 6 minutes' walk test with continuous assessment of blood oxygenation and heart rate. Adaptation disorders are supported by results of external respiration assessment and echo-cardiography.

  17. Adaptive management of social-ecological systems: the path forward

    USGS Publications Warehouse

    Allen, Craig R.

    2015-01-01

    Adaptive management remains at the forefront of environmental management nearly 40 years after its original conception, largely because we have yet to develop other methodologies that offer the same promise. Despite the criticisms of adaptive management and the numerous failed attempts to implement it, adaptive management has yet to be replaced with a better alternative. The concept persists because it is simple, allows action despite uncertainty, and fosters learning. Moving forward, adaptive management of social-ecological systems provides policymakers, managers and scientists a powerful tool for managing for resilience in the face of uncertainty.

  18. Cyberspace: The Ultimate Complex Adaptive System

    DTIC Science & Technology

    2010-04-09

    a transaction with a given agent; tags also facilitate the formation of aggregates , or meta-agents. Meta-agents help distrib- ute and decentralize...Cyber Environments Attribute Physical Environment Cyber Environment Location Latitude, Longitude IP-Address Speed Air and ground in mph Mbps or Gbps...or more capable in terms of its requisite variety (in can adapt to a wider range of conditions). The fitness of the agent is a complex aggregate

  19. Adaptive Control of Nonlinear Flexible Systems

    DTIC Science & Technology

    1993-01-18

    disturbances. The following example illustrates the need for a robust state-feedback law and the sensi- tivity of the exact - linearization based control law... exact linearization , one can bring an input-output approach to a particular case of certainty- equivalence based adaptive control design. We now...are available for this model, exact linearization can be performed. Let C(s) be the compensator that is being used so far in the previous three

  20. A Dual-Reporter System for Real-Time Monitoring and High-throughput CRISPR/Cas9 Library Screening of the Hepatitis C Virus

    PubMed Central

    Ren, Qingpeng; Li, Chan; Yuan, Pengfei; Cai, Changzu; Zhang, Linqi; Luo, Guangxiang George; Wei, Wensheng

    2015-01-01

    The hepatitis C virus (HCV) is one of the leading causes of chronic hepatitis, liver cirrhosis and hepatocellular carcinomas and infects approximately 170 million people worldwide. Although several reporter systems have been developed, many shortcomings limit their use in the assessment of HCV infections. Here, we report a real-time live-cell reporter, termed the NIrD (NS3-4A Inducible rtTA-mediated Dual-reporter) system, which provides an on-off switch specifically in response to an HCV infection. Using the NIrD system and a focused CRISPR/Cas9 library, we identified CLDN1, OCLN and CD81 as essential genes for both the cell-free entry and the cell-to-cell transmission of HCV. The combination of this ultra-sensitive reporter system and the CRISPR knockout screening provides a powerful and high-throughput strategy for the identification of critical host components for HCV infections. PMID:25746010