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Sample records for adaptive system 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 systems: prokaryotes upgrade to adaptive immunity

    PubMed Central

    Barrangou, Rodolphe; Marraffini, Luciano A.

    2014-01-01

    Summary 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. PMID:24766887

  4. 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.

  5. 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

  6. Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process.

    PubMed

    Li, Ming; Wang, Rui; Zhao, Dahe; Xiang, Hua

    2014-02-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system mediates adaptive immunity against foreign nucleic acids in prokaryotes. However, efficient adaptation of a native CRISPR to purified viruses has only been observed for the type II-A system from a Streptococcus thermophilus industry strain, and rarely reported for laboratory strains. Here, we provide a second native system showing efficient adaptation. Infected by a newly isolated virus HHPV-2, Haloarcula hispanica type I-B CRISPR system acquired spacers discriminatively from viral sequences. Unexpectedly, in addition to Cas1, Cas2 and Cas4, this process also requires Cas3 and at least partial Cascade proteins, which are involved in interference and/or CRISPR RNA maturation. Intriguingly, a preexisting spacer partially matching a viral sequence is also required, and spacer acquisition from upstream and downstream sequences of its target sequence (i.e. priming protospacer) shows different strand bias. These evidences strongly indicate that adaptation in this system strictly requires a priming process. This requirement, if validated also true for other CRISPR systems as implied by our bioinformatic analysis, may help to explain failures to observe efficient adaptation to purified viruses in many laboratory strains, and the discrimination mechanism at the adaptation level that has confused scientists for years.

  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

    2015-11-01

    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. PMID:26382836

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

    PubMed

    Kirchner, Marion; Schneider, Sabine

    2015-11-01

    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.

  11. 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

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

    PubMed

    Sun, Dongchang; Qiu, Juanping

    2016-01-01

    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. Evolution of the CRISPR-Cas adaptive immunity systems in prokaryotes: models and observations on virus-host coevolution.

    PubMed

    Koonin, Eugene V; Wolf, Yuri I

    2015-01-01

    CRISPR-Cas is an adaptive immunity system in prokaryotes that functions via a unique mechanism which involves incorporation of foreign DNA fragments into CRISPR arrays and subsequent utilization of transcripts of these inserts (known as spacers) as guide RNAs to cleave the cognate selfish element genome. Multiple attempts have been undertaken to explore the coevolution of viruses and microbial hosts carrying CRISPR-Cas using mathematical models that employ either systems of differential equations or an agent-based approach, or combinations thereof. Analysis of these models reveals highly complex co-evolutionary dynamics that ensues from the combination of the heritability of the CRISPR-mediated adaptive immunity with the existence of different degrees of immunity depending on the number of cognate spacers and the cost of carrying a CRISPR-Cas locus. Depending on the details of the models, a variety of testable, sometimes conflicting predictions have been made on the dependence of the degree of immunity and the benefit of maintaining CRISPR-Cas on the abundance and diversity of hosts and viruses. Some of these predictions have already been directly validated experimentally. In particular, both the reality of the virus-host arms race, with viruses escaping resistance and hosts reacquiring it through the capture of new spacers, and the fitness cost of CRISPR-Cas due to the curtailment of beneficial HGT have been reproduced in the laboratory. However, to test the predictions of the models more specifically, detailed studies of coevolving populations of microbes and viruses both in nature and in the laboratory are essential. Such analyses are expected to yield disagreements with the predictions of the current, oversimplified models and to trigger a new round of theoretical developments.

  14. Adaptive optics at the IOE, CAS

    NASA Astrophysics Data System (ADS)

    Jiang, Wenhan; Rao, Changhui; Zhang, Yudong; Ling, Ning; Guan, Chunlin

    2009-02-01

    R&D on Adaptive Optics in the Institute of Optics & Electronics (IOE), Chinese Academy of Sciences (CAS) began in 1979. In this paper, several recent achievements will be reported: 1. AO for astronomical telescopes. AO system for 1.2 m telescope at Yunnan Astronomical Observatory was built in 1998. It was updated in 2004, and high resolution images approaching diffraction limit were obtained. A new AO telescope with 1.8m aperture is being built, and a 4m AO telescope is being planned. 2. AO for ICF facility. A 19-element AO system with hill-climbing control algorithm for "Shenguang I" ICF facility was built in 1985, which was the first AO system used in ICF facility in the world. A set of 8 AO systems was installed in a bundle of ICF prototype for "Shenguang III" ICF facility. A new system is being built for further development of ICF facility. 3. AO for retinal imaging. In 1999, the first AO system with 19-element DM was built for retinal imaging. Several AO systems with 37-element deformable mirror (DM) were built and used for vision research and clinical inspection. It is being integrated with an OCT system for high resolution retinal imaging. All of the main subsystems, such as DMs, wavefront sensors, and high speed processors, were built in the Laboratory on Adaptive Optics of IOE, CAS.

  15. 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.

  16. Physical Model of the Immune Response of Bacteria Against Bacteriophage Through the Adaptive CRISPR-Cas Immune System

    PubMed Central

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

    2013-01-01

    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. PMID:23492852

  17. 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.

  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. PMID:26929403

  19. 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.

  20. Diversity of CRISPR-Cas immune systems and molecular machines.

    PubMed

    Barrangou, Rodolphe

    2015-01-01

    Bacterial adaptive immunity hinges on CRISPR-Cas systems that provide DNA-encoded, RNA-mediated targeting of exogenous nucleic acids. A plethora of CRISPR molecular machines occur broadly in prokaryotic genomes, with a diversity of Cas nucleases that can be repurposed for various applications.

  1. 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.

  2. 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

  3. 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.

  4. Unification of Cas protein families and a simple scenario for the origin and evolution of CRISPR-Cas systems

    PubMed Central

    2011-01-01

    Background The CRISPR-Cas adaptive immunity systems that are present in most Archaea and many Bacteria function by incorporating fragments of alien genomes into specific genomic loci, transcribing the inserts and using the transcripts as guide RNAs to destroy the genome of the cognate virus or plasmid. This RNA interference-like immune response is mediated by numerous, diverse and rapidly evolving Cas (CRISPR-associated) proteins, several of which form the Cascade complex involved in the processing of CRISPR transcripts and cleavage of the target DNA. Comparative analysis of the Cas protein sequences and structures led to the classification of the CRISPR-Cas systems into three Types (I, II and III). Results A detailed comparison of the available sequences and structures of Cas proteins revealed several unnoticed homologous relationships. The Repeat-Associated Mysterious Proteins (RAMPs) containing a distinct form of the RNA Recognition Motif (RRM) domain, which are major components of the CRISPR-Cas systems, were classified into three large groups, Cas5, Cas6 and Cas7. Each of these groups includes many previously uncharacterized proteins now shown to adopt the RAMP structure. Evidence is presented that large subunits contained in most of the CRISPR-Cas systems could be homologous to Cas10 proteins which contain a polymerase-like Palm domain and are predicted to be enzymatically active in Type III CRISPR-Cas systems but inactivated in Type I systems. These findings, the fact that the CRISPR polymerases, RAMPs and Cas2 all contain core RRM domains, and distinct gene arrangements in the three types of CRISPR-Cas systems together provide for a simple scenario for origin and evolution of the CRISPR-Cas machinery. Under this scenario, the CRISPR-Cas system originated in thermophilic Archaea and subsequently spread horizontally among prokaryotes. Conclusions Because of the extreme diversity of CRISPR-Cas systems, in-depth sequence and structure comparison continue to

  5. 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

    2016-01-01

    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.

  6. An updated evolutionary classification of CRISPR-Cas systems.

    PubMed

    Makarova, Kira S; Wolf, Yuri I; Alkhnbashi, Omer S; Costa, Fabrizio; Shah, Shiraz A; Saunders, Sita J; Barrangou, Rodolphe; Brouns, Stan J J; Charpentier, Emmanuelle; Haft, Daniel H; Horvath, Philippe; Moineau, Sylvain; Mojica, Francisco J M; Terns, Rebecca M; Terns, Michael P; White, Malcolm F; Yakunin, Alexander F; Garrett, Roger A; van der Oost, John; Backofen, Rolf; Koonin, Eugene V

    2015-11-01

    The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized. PMID:26411297

  7. 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

  8. 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.

  9. 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. PMID:27449612

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

    PubMed Central

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

    2013-01-01

    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. PMID:23628889

  11. 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

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

    PubMed

    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.

  13. CRISPR-Cas: New Tools for Genetic Manipulations from Bacterial Immunity Systems.

    PubMed

    Jiang, Wenyan; Marraffini, Luciano A

    2015-01-01

    Prokaryotic CRISPR-Cas loci encode proteins that function as an adaptive immune system against infectious viruses and plasmids. Immunity is mediated by Cas nucleases and small RNA guides, which specify a cleavage site within the genome of the invader. In type II CRISPR-Cas systems, the RNA-guided Cas9 nuclease cleaves the DNA. Cas9 can be reprogrammed to create double-strand DNA breaks in the genomes of a variety of organisms, from bacteria to human cells. Repair of Cas9 lesions by homologous recombination or nonhomologous end joining mechanisms can lead to the introduction of specific nucleotide substitutions or indel mutations, respectively. Furthermore, a nuclease-null Cas9 has been developed to regulate endogenous gene expression and to label genomic loci in living cells. Targeted genome editing and gene regulation mediated by Cas9 are easy to program, scale, and multiplex, allowing researchers to decipher the causal link between genetic and phenotypic variation. In this review, we describe the most notable applications of Cas9 in basic biology, translational medicine, synthetic biology, biotechnology, and other fields. PMID:26209264

  14. 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

    2016-08-01

    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. PMID:27493190

  15. 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.

  16. Anti-cas spacers in orphan CRISPR4 arrays prevent uptake of active CRISPR-Cas I-F systems.

    PubMed

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

    2016-01-01

    Archaea and bacteria harbour clustered regularly interspaced short palindromic repeats (CRISPR) loci. These arrays encode RNA molecules (crRNA), each containing a sequence of a single repeat-intervening spacer. The crRNAs guide CRISPR-associated (Cas) proteins to cleave nucleic acids complementary to the crRNA spacer, thus interfering with targeted foreign elements. Notably, pre-existing spacers may trigger the acquisition of new spacers from the target molecule by means of a primed adaptation mechanism. Here, we show that naturally occurring orphan CRISPR arrays that contain spacers matching sequences of the cognate (absent) cas genes are able to elicit both primed adaptation and direct interference against genetic elements carrying those genes. Our findings show the existence of an anti-cas mechanism that prevents the transfer of a fully equipped CRISPR-Cas system. Hence, they suggest that CRISPR immunity may be undesired by particular prokaryotes, potentially because they could limit possibilities for gaining favourable sequences by lateral transfer. PMID:27573106

  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. 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.

  19. 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

  20. 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. PMID:26782639

  1. 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.

  2. 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.

  3. 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…

  4. Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas immune system.

    PubMed

    Sinkunas, Tomas; Gasiunas, Giedrius; Fremaux, Christophe; Barrangou, Rodolphe; Horvath, Philippe; Siksnys, Virginijus

    2011-04-01

    Clustered regularly interspaced short palindromic repeat (CRISPR) is a recently discovered adaptive prokaryotic immune system that provides acquired immunity against foreign nucleic acids by utilizing small guide crRNAs (CRISPR RNAs) to interfere with invading viruses and plasmids. In Escherichia coli, Cas3 is essential for crRNA-guided interference with virus proliferation. Cas3 contains N-terminal HD phosphohydrolase and C-terminal Superfamily 2 (SF2) helicase domains. Here, we provide the first report of the cloning, expression, purification and in vitro functional analysis of the Cas3 protein of the Streptococcus thermophilus CRISPR4 (Ecoli subtype) system. Cas3 possesses a single-stranded DNA (ssDNA)-stimulated ATPase activity, which is coupled to unwinding of DNA/DNA and RNA/DNA duplexes. Cas3 also shows ATP-independent nuclease activity located in the HD domain with a preference for ssDNA substrates. To dissect the contribution of individual domains, Cas3 separation-of-function mutants (ATPase(+)/nuclease(-) and ATPase(-)/nuclease(+)) were obtained by site-directed mutagenesis. We propose that the Cas3 ATPase/helicase domain acts as a motor protein, which assists delivery of the nuclease activity to Cascade-crRNA complex targeting foreign DNA. PMID:21343909

  5. Efficient Gene Knockout in Goats Using CRISPR/Cas9 System

    PubMed Central

    Ni, Wei; Qiao, Jun; Hu, Shengwei; Zhao, Xinxia; Regouski, Misha; Yang, Min; Polejaeva, Irina A.; Chen, Chuangfu

    2014-01-01

    The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications. PMID:25188313

  6. 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

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

    PubMed

    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

  8. Inactivation of CRISPR-Cas systems by anti-CRISPR proteins in diverse bacterial species.

    PubMed

    Pawluk, April; Staals, Raymond H J; Taylor, Corinda; Watson, Bridget N J; Saha, Senjuti; Fineran, Peter C; Maxwell, Karen L; Davidson, Alan R

    2016-01-01

    CRISPR-Cas systems provide sequence-specific adaptive immunity against foreign nucleic acids(1,2). They are present in approximately half of all sequenced prokaryotes(3) and are expected to constitute a major barrier to horizontal gene transfer. We previously described nine distinct families of proteins encoded in Pseudomonas phage genomes that inhibit CRISPR-Cas function(4,5). We have developed a bioinformatic approach that enabled us to discover additional anti-CRISPR proteins encoded in phages and other mobile genetic elements of diverse bacterial species. We show that five previously undiscovered families of anti-CRISPRs inhibit the type I-F CRISPR-Cas systems of both Pseudomonas aeruginosa and Pectobacterium atrosepticum, and a dual specificity anti-CRISPR inactivates both type I-F and I-E CRISPR-Cas systems. Mirroring the distribution of the CRISPR-Cas systems they inactivate, these anti-CRISPRs were found in species distributed broadly across the phylum Proteobacteria. Importantly, anti-CRISPRs originating from species with divergent type I-F CRISPR-Cas systems were able to inhibit the two systems we tested, highlighting their broad specificity. These results suggest that all type I-F CRISPR-Cas systems are vulnerable to inhibition by anti-CRISPRs. Given the widespread occurrence and promiscuous activity of the anti-CRISPRs described here, we propose that anti-CRISPRs play an influential role in facilitating the movement of DNA between prokaryotes by breaching the barrier imposed by CRISPR-Cas systems. PMID:27573108

  9. 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. PMID:27350235

  10. 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.

  11. Performance of the Cas9 nickase system in Drosophila melanogaster.

    PubMed

    Ren, Xingjie; Yang, Zhihao; Mao, Decai; Chang, Zai; Qiao, Huan-Huan; Wang, Xia; Sun, Jin; Hu, Qun; Cui, Yan; Liu, Lu-Ping; Ji, Jun-Yuan; Xu, Jiang; Ni, Jian-Quan

    2014-10-01

    Recent studies of the Cas9/sgRNA system in Drosophila melanogaster genome editing have opened new opportunities to generate site-specific mutant collections in a high-throughput manner. However, off-target effects of the system are still a major concern when analyzing mutant phenotypes. Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro. Here, we demonstrated that injection of two plasmids encoding neighboring offset sgRNAs into transgenic Cas9(D10A) nickase flies efficiently produces heritable indel mutants. We then determined the effective distance between the two sgRNA targets and their orientations that affected the ability of the sgRNA pairs to generate mutations when expressed in the transgenic nickase flies. Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants. Finally, a defined piwi mutant allele is generated with this system through homology-directed repair. However, Cas9(D10A) is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs. PMID:25128437

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

    PubMed Central

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

    2015-01-01

    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. PMID:26007654

  13. 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.

  14. 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.

  15. 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.

  16. 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…

  17. Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems.

    PubMed

    Leenay, Ryan T; Maksimchuk, Kenneth R; Slotkowski, Rebecca A; Agrawal, Roma N; Gomaa, Ahmed A; Briner, Alexandra E; Barrangou, Rodolphe; Beisel, Chase L

    2016-04-01

    CRISPR-Cas adaptive immune systems in prokaryotes boast a diversity of protein families and mechanisms of action, where most systems rely on protospacer-adjacent motifs (PAMs) for DNA target recognition. Here, we developed an in vivo, positive, and tunable screen termed PAM-SCANR (PAM screen achieved by NOT-gate repression) to elucidate functional PAMs as well as an interactive visualization scheme termed the PAM wheel to convey individual PAM sequences and their activities. PAM-SCANR and the PAM wheel identified known functional PAMs while revealing complex sequence-activity landscapes for the Bacillus halodurans I-C (Cascade), Escherichia coli I-E (Cascade), Streptococcus thermophilus II-A CRISPR1 (Cas9), and Francisella novicida V-A (Cpf1) systems. The PAM wheel was also readily applicable to existing high-throughput screens and garnered insights into SpyCas9 and SauCas9 PAM diversity. These tools offer powerful means of elucidating and visualizing functional PAMs toward accelerating our ability to understand and exploit the multitude of CRISPR-Cas systems in nature. PMID:27041224

  18. Interference activity of a minimal Type I CRISPR-Cas system from Shewanella putrefaciens.

    PubMed

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

    2015-10-15

    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.

  19. 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.

  20. Cas3-Derived Target DNA Degradation Fragments Fuel Primed CRISPR Adaptation.

    PubMed

    Künne, Tim; Kieper, Sebastian N; Bannenberg, Jasper W; Vogel, Anne I M; Miellet, Willem R; Klein, Misha; Depken, Martin; Suarez-Diez, Maria; Brouns, Stan J J

    2016-09-01

    Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30-100 nt enriched for thymine-stretches in their 3' ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3' cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers. PMID:27546790

  1. Applications of CRISPR-Cas systems in neuroscience.

    PubMed

    Heidenreich, Matthias; Zhang, Feng

    2016-01-01

    Genome-editing tools, and in particular those based on CRISPR-Cas (clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein) systems, are accelerating the pace of biological research and enabling targeted genetic interrogation in almost 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 models 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.

  2. 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.

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

    NASA Astrophysics Data System (ADS)

    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.

  4. 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

  5. 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.

  6. 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.

  7. 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.

  8. 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.

  9. The Role of CRISPR-Cas Systems in Virulence of Pathogenic Bacteria

    PubMed Central

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

    2014-01-01

    SUMMARY 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. PMID:24600041

  10. 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

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

    PubMed Central

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

    2016-01-01

    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. PMID:26817415

  12. 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.

  13. 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

  14. An Active Type I-E CRISPR-Cas System Identified in Streptomyces avermitilis

    PubMed Central

    Qiu, Yi; Wang, Shiwei; Chen, Zhi; Guo, Yajie; Song, Yuan

    2016-01-01

    CRISPR-Cas systems, the small RNA-dependent immune systems, are widely distributed in prokaryotes. However, only a small proportion of CRISPR-Cas systems have been identified to be active in bacteria. In this work, a naturally active type I-E CRISPR-Cas system was found in Streptomyces avermitilis. The system shares many common genetic features with the type I-E system of Escherichia coli, and meanwhile shows unique characteristics. It not only degrades plasmid DNA with target protospacers, but also acquires new spacers from the target plasmid DNA. The naive features of spacer acquisition in the type I-E system of S. avermitilis were investigated and a completely conserved PAM 5’-AAG-3’ was identified. Spacer acquisition displayed differential strand bias upstream and downstream of the priming spacer, and irregular integrations of new spacers were observed. In addition, introduction of this system into host conferred phage resistance to some extent. This study will give new insights into adaptation mechanism of the type I-E systems in vivo, and meanwhile provide theoretical foundation for applying this system on the genetic modification of S. avermitilis. PMID:26901661

  15. Biallelic genome modification in F(0) Xenopus tropicalis embryos using the CRISPR/Cas system.

    PubMed

    Blitz, Ira L; Biesinger, Jacob; Xie, Xiaohui; Cho, Ken W Y

    2013-12-01

    Gene inactivation is an important tool for correlation of phenotypic and genomic data, allowing researchers to infer normal gene function based on the phenotype when the gene is impaired. New and better approaches are needed to overcome the shortfalls of existing methods for any significant acceleration of scientific progress. We have adapted the CRISPR/Cas system for use in Xenopus tropicalis and report on the efficient creation of mutations in the gene encoding the enzyme tyrosinase, which is responsible for oculocutaneous albinism. Biallelic mutation of this gene was detected in the F0 generation, suggesting targeting efficiencies similar to that of TALENs. We also find that off-target mutagenesis seems to be negligible, and therefore, CRISPR/Cas may be a useful system for creating genome modifications in this important model organism.

  16. How Do Traditional Examination Questions Fare in the Presence of a Computer Algebra System (CAS)?

    ERIC Educational Resources Information Center

    Malabar, Ian; Pountney, Dave

    2001-01-01

    Describes the outcomes and discusses possible implications for the development of assessment with a Computer Algebra System (CAS) when a group of undergraduate mathematics students, familiar with using a CAS in examinations, tackled an assortment of traditional (i.e., non-CAS type) questions. (Author/MM)

  17. On the Integration of Computer Algebra Systems (CAS) by Canadian Mathematicians: Results of a National Survey

    ERIC Educational Resources Information Center

    Buteau, Chantal; Jarvis, Daniel H.; Lavicza, Zsolt

    2014-01-01

    In this article, we outline the findings of a Canadian survey study (N = 302) that focused on the extent of computer algebra systems (CAS)-based technology use in postsecondary mathematics instruction. Results suggest that a considerable number of Canadian mathematicians use CAS in research and teaching. CAS use in research was found to be the…

  18. Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system.

    PubMed

    Anton, Tobias; Bultmann, Sebastian; Leonhardt, Heinrich; Markaki, Yolanda

    2014-01-01

    Labeling and tracing of specific sequences in living cells has been a major challenge in studying the spatiotemporal dynamics of native chromatin. Here we repurposed the prokaryotic CRISPR/Cas adaptive immunity system to specifically detect endogenous genomic loci in mouse embryonic stem cells. We constructed a catalytically inactive version of the Cas9 endonuclease, fused it with eGFP (dCas9-eGFP) and co-expressed small guide RNAs (gRNAs) to target pericentric, centric, and telomeric repeats, which are enriched in distinct nuclear structures. With major satellite specific gRNAs we obtained a characteristic chromocenter (CC) pattern, while gRNAs targeting minor satellites and telomeres highlighted smaller foci coinciding with centromere protein B (CENP-B) and telomeric repeat-binding factor 2 (TRF2), respectively. DNA sequence specific labeling by gRNA/dCas9-eGFP complexes was directly shown with 3D-fluorescent in situ hybridization (3D-FISH). Structured illumination microscopy (3D-SIM) of gRNA/dCas9-eGFP expressing cells revealed chromatin ultrastructures and demonstrated the potential of this approach for chromatin conformation studies by super resolution microscopy. This programmable dCas9 labeling system opens new perspectives to study functional nuclear architecture.

  19. Visualization of specific DNA sequences in living mouse embryonic stem cells with a programmable fluorescent CRISPR/Cas system.

    PubMed

    Anton, Tobias; Bultmann, Sebastian; Leonhardt, Heinrich; Markaki, Yolanda

    2014-01-01

    Labeling and tracing of specific sequences in living cells has been a major challenge in studying the spatiotemporal dynamics of native chromatin. Here we repurposed the prokaryotic CRISPR/Cas adaptive immunity system to specifically detect endogenous genomic loci in mouse embryonic stem cells. We constructed a catalytically inactive version of the Cas9 endonuclease, fused it with eGFP (dCas9-eGFP) and co-expressed small guide RNAs (gRNAs) to target pericentric, centric, and telomeric repeats, which are enriched in distinct nuclear structures. With major satellite specific gRNAs we obtained a characteristic chromocenter (CC) pattern, while gRNAs targeting minor satellites and telomeres highlighted smaller foci coinciding with centromere protein B (CENP-B) and telomeric repeat-binding factor 2 (TRF2), respectively. DNA sequence specific labeling by gRNA/dCas9-eGFP complexes was directly shown with 3D-fluorescent in situ hybridization (3D-FISH). Structured illumination microscopy (3D-SIM) of gRNA/dCas9-eGFP expressing cells revealed chromatin ultrastructures and demonstrated the potential of this approach for chromatin conformation studies by super resolution microscopy. This programmable dCas9 labeling system opens new perspectives to study functional nuclear architecture. PMID:24637835

  20. Insights into the CRISPR/Cas system of Gardnerella vaginalis

    PubMed Central

    2012-01-01

    Background Gardnerella vaginalis is identified as the predominant colonist of the vaginal tracts of women diagnosed with bacterial vaginosis (BV). G. vaginalis can be isolated from healthy women, and an asymptomatic BV state is also recognised. The association of G. vaginalis with different clinical phenotypes could be explained by different cytotoxicity of the strains, presumably based on disparate gene content. The contribution of horizontal gene transfer to shaping the genomes of G. vaginalis is acknowledged. The CRISPR loci of the recently discovered CRISPR/Cas microbial defence system provide a historical view of the exposure of prokaryotes to a variety of foreign genetic elements. Results The CRISPR/Cas loci were analysed using available sequence data from three G. vaginalis complete genomes and 18 G. vaginalis draft genomes in the NCBI database, as well as PCR amplicons of the genomic DNA of 17 clinical isolates. The cas genes in the CRISPR/Cas loci of G. vaginalis belong to the E. coli subtype. Approximately 20% of the spacers had matches in the GenBank database. Sequence analysis of the CRISPR arrays revealed that nearly half of the spacers matched G. vaginalis chromosomal sequences. The spacers that matched G. vaginalis chromosomal sequences were determined to not be self-targeting and were presumably neither constituents of mobile-element-associated genes nor derived from plasmids/viruses. The protospacers targeted by these spacers displayed conserved protospacer-adjacent motifs. Conclusions The CRISPR/Cas system has been identified in about one half of the analysed G. vaginalis strains. Our analysis of CRISPR sequences did not reveal a potential link between their presence and the virulence of the G. vaginalis strains. Based on the origins of the spacers found in the G. vaginalis CRISPR arrays, we hypothesise that the transfer of genetic material among G. vaginalis strains could be regulated by the CRISPR/Cas mechanism. The present study is the first

  1. 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.

  2. 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.

  3. 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

  4. 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.

  5. 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.

  6. Design of a CRISPR-Cas system to increase resistance of Bacillus subtilis to bacteriophage SPP1.

    PubMed

    Jakutyte-Giraitiene, Lina; Gasiunas, Giedrius

    2016-08-01

    Clustered regularly interspaced short palindromic repeats (CRISPR) together with CRISPR-associated (cas) genes form an adaptive prokaryotic immune system which provides acquired resistance against viruses and plasmids. Bacillus subtilis presently is the best-characterized laboratory model for Gram-positive bacteria and also widely used for industrial production of enzymes, vitamins and antibiotics. In this study, we show that type II-A CRISPR-Cas system from Streptococcus thermophilus can be transferred into B. subtilis and provides heterologous protection against phage infection. We engineered a heterologous host by cloning S. thermophilus Cas9 and a spacer targeting bacteriophage SPP1 into the chromosome of B. subtilis, which does not harbor its own CRISPR-Cas systems. We found that the heterologous CRISPR-Cas system is functionally active in B. subtilis and provides resistance against bacteriophage SPP1 infection. The high efficiency of the acquired immunity against phage could be useful in generation of biotechnologically important B. subtilis strains with engineered chromosomes. PMID:27255973

  7. 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

  8. 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.

  9. Development of an intein-mediated split–Cas9 system for gene therapy

    PubMed Central

    Truong, Dong-Jiunn Jeffery; Kühner, Karin; Kühn, Ralf; Werfel, Stanislas; Engelhardt, Stefan; Wurst, Wolfgang; Ortiz, Oskar

    2015-01-01

    Using CRISPR/Cas9, it is possible to target virtually any gene in any organism. A major limitation to its application in gene therapy is the size of Cas9 (>4 kb), impeding its efficient delivery via recombinant adeno-associated virus (rAAV). Therefore, we developed a split–Cas9 system, bypassing the packaging limit using split-inteins. Each Cas9 half was fused to the corresponding split-intein moiety and, only upon co-expression, the intein-mediated trans-splicing occurs and the full Cas9 protein is reconstituted. We demonstrated that the nuclease activity of our split-intein system is comparable to wild-type Cas9, shown by a genome-integrated surrogate reporter and by targeting three different endogenous genes. An analogously designed split-Cas9D10A nickase version showed similar activity as Cas9D10A. Moreover, we showed that the double nick strategy increased the homologous directed recombination (HDR). In addition, we explored the possibility of delivering the repair template accommodated on the same dual-plasmid system, by transient transfection, showing an efficient HDR. Most importantly, we revealed for the first time that intein-mediated split–Cas9 can be packaged, delivered and its nuclease activity reconstituted efficiently, in cells via rAAV. PMID:26082496

  10. Phylogenetic Distribution of CRISPR-Cas Systems in Antibiotic-Resistant Pseudomonas aeruginosa

    PubMed Central

    van Belkum, Alex; Soriaga, Leah B.; LaFave, Matthew C.; Akella, Srividya; Veyrieras, Jean-Baptiste; Barbu, E. Magda; Shortridge, Dee; Blanc, Bernadette; Hannum, Gregory; Zambardi, Gilles; Miller, Kristofer; Enright, Mark C.; Mugnier, Nathalie; Brami, Daniel; Schicklin, Stéphane; Felderman, Martina; Schwartz, Ariel S.; Richardson, Toby H.; Peterson, Todd C.; Hubby, Bolyn

    2015-01-01

    ABSTRACT Pseudomonas aeruginosa is an antibiotic-refractory pathogen with a large genome and extensive genotypic diversity. Historically, P. aeruginosa has been a major model system for understanding the molecular mechanisms underlying type I clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein (CRISPR-Cas)-based bacterial immune system function. However, little information on the phylogenetic distribution and potential role of these CRISPR-Cas systems in molding the P. aeruginosa accessory genome and antibiotic resistance elements is known. Computational approaches were used to identify and characterize CRISPR-Cas systems within 672 genomes, and in the process, we identified a previously unreported and putatively mobile type I-C P. aeruginosa CRISPR-Cas system. Furthermore, genomes harboring noninhibited type I-F and I-E CRISPR-Cas systems were on average ~300 kb smaller than those without a CRISPR-Cas system. In silico analysis demonstrated that the accessory genome (n = 22,036 genes) harbored the majority of identified CRISPR-Cas targets. We also assembled a global spacer library that aided the identification of difficult-to-characterize mobile genetic elements within next-generation sequencing (NGS) data and allowed CRISPR typing of a majority of P. aeruginosa strains. In summary, our analysis demonstrated that CRISPR-Cas systems play an important role in shaping the accessory genomes of globally distributed P. aeruginosa isolates. PMID:26604259

  11. 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. PMID:27469250

  12. 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.

  13. 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

  14. The RNA- and DNA-targeting CRISPR–Cas immune systems of Pyrococcus furiosus

    PubMed Central

    Terns, Rebecca M.; Terns, Michael P.

    2014-01-01

    Using the hyperthermophile Pyrococcus furiosus, we have delineated several key steps in CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) invader defence pathways. P. furiosus has seven transcriptionally active CRISPR loci that together encode a total of 200 crRNAs (CRISPR RNAs). The 27 Cas proteins in this organism represent three distinct pathways and are primarily encoded in two large gene clusters. The Cas6 protein dices CRISPR locus transcripts to generate individual invader-targeting crRNAs. The mature crRNAs include a signature sequence element (the 5′ tag) derived from the CRISPR locus repeat sequence that is important for function. crRNAs are tailored into distinct species and integrated into three distinct crRNA–Cas protein complexes that are all candidate effector complexes. The complex formed by the Cmr [Cas module RAMP (repeat-associated mysterious proteins)] (subtype III-B) proteins cleaves complementary target RNAs and can be programmed to cleave novel target RNAs in a prokaryotic RNAi-like manner. Evidence suggests that the other two CRISPR–Cas systems in P. furiosus, Csa (Cas subtype Apern) (subtype I-A) and Cst (Cas subtype Tneap) (subtype I-B), target invaders at the DNA level. Studies of the CRISPR–Cas systems from P. furiosus are yielding fundamental knowledge of mechanisms of crRNA biogenesis and silencing for three of the diverse CRISPR–Cas pathways, and reveal that organisms such as P. furiosus possess an arsenal of multiple RNA-guided mechanisms to resist diverse invaders. Our knowledge of the fascinating CRISPR–Cas pathways is leading in turn to our ability to co-opt these systems for exciting new biomedical and biotechnological applications. PMID:24256230

  15. 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…

  16. 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.

  17. Verification of Adaptive Systems

    SciTech Connect

    Pullum, Laura L; Cui, Xiaohui; Vassev, Emil; Hinchey, Mike; Rouff, Christopher; Buskens, Richard

    2012-01-01

    Adaptive systems are critical for future space and other unmanned and intelligent systems. Verification of these systems is also critical for their use in systems with potential harm to human life or with large financial investments. Due to their nondeterministic nature and extremely large state space, current methods for verification of software systems are not adequate to provide a high level of assurance for them. The combination of stabilization science, high performance computing simulations, compositional verification and traditional verification techniques, plus operational monitors, provides a complete approach to verification and deployment of adaptive systems that has not been used before. This paper gives an overview of this approach.

  18. Analysis of the type II-A CRISPR-Cas system of Streptococcus agalactiae reveals distinctive features according to genetic lineages

    PubMed Central

    Lier, Clément; Baticle, Elodie; Horvath, Philippe; Haguenoer, Eve; Valentin, Anne-Sophie; Glaser, Philippe; Mereghetti, Laurent; Lanotte, Philippe

    2015-01-01

    CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) are found in 90% of archaea and about 40% of bacteria. In this original system, CRISPR arrays comprise short, almost unique sequences called spacers that are interspersed with conserved palindromic repeats. These systems play a role in adaptive immunity and participate to fight non-self DNA such as integrative and conjugative elements, plasmids, and phages. In Streptococcus agalactiae, a bacterium implicated in colonization and infections in humans since the 1960s, two CRISPR-Cas systems have been described. A type II-A system, characterized by proteins Cas9, Cas1, Cas2, and Csn2, is ubiquitous, and a type I–C system, with the Cas8c signature protein, is present in about 20% of the isolates. Unlike type I–C, which appears to be non-functional, type II-A appears fully functional. Here we studied type II-A CRISPR-cas loci from 126 human isolates of S. agalactiae belonging to different clonal complexes that represent the diversity of the species and that have been implicated in colonization or infection. The CRISPR-cas locus was analyzed both at spacer and repeat levels. Major distinctive features were identified according to the phylogenetic lineages previously defined by multilocus sequence typing, especially for the sequence type (ST) 17, which is considered hypervirulent. Among other idiosyncrasies, ST-17 shows a significantly lower number of spacers in comparison with other lineages. This characteristic could reflect the peculiar virulence or colonization specificities of this lineage. PMID:26124774

  19. The CAS Classroom

    ERIC Educational Resources Information Center

    Garner, Sue

    2004-01-01

    The Victorian Curriculum and Assessment Authority (VCAA) Computer Algebra System (CAS)Pilot study (2001-2005) is monitoring the use of CAS in senior secondary mathematics. This article explores the author's experiences in the CAS classroom and delineates changes in teaching style, as a result of the introduction of CAS into the senior mathematics…

  20. 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.

  1. [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.

  2. 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.

  3. Cas5d protein processes pre-crRNA and assembles into a Cascade-like interference complex in Subtype I-C/Dvulg CRISPR-Cas system

    PubMed Central

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

    2012-01-01

    SUMMARY 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 B. 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-subunit interference complex similar to E. 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. PMID:22841292

  4. 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.

  5. Knowledge-based discovery for designing CRISPR-CAS systems against invading mobilomes in thermophiles.

    PubMed

    Chellapandi, P; Ranjani, J

    2015-09-01

    Clustered regularly interspaced short palindromic repeats (CRISPRs) are direct features of the prokaryotic genomes involved in resistance to their bacterial viruses and phages. Herein, we have identified CRISPR loci together with CRISPR-associated sequences (CAS) genes to reveal their immunity against genome invaders in the thermophilic archaea and bacteria. Genomic survey of this study implied that genomic distribution of CRISPR-CAS systems was varied from strain to strain, which was determined by the degree of invading mobiloms. Direct repeats found to be equal in some extent in many thermopiles, but their spacers were differed in each strain. Phylogenetic analyses of CAS superfamily revealed that genes cmr, csh, csx11, HD domain, devR were belonged to the subtypes of cas gene family. The members in cas gene family of thermophiles were functionally diverged within closely related genomes and may contribute to develop several defense strategies. Nevertheless, genome dynamics, geological variation and host defense mechanism were contributed to share their molecular functions across the thermophiles. A thermophilic archaean, Thermococcus gammotolerans and thermophilic bacteria, Petrotoga mobilis and Thermotoga lettingae have shown superoperons-like appearance to cluster cas genes, which were typically evolved for their defense pathways. A cmr operon was identified with a specific promoter in a thermophilic archaean, Caldivirga maquilingensis. Overall, we concluded that knowledge-based genomic survey and phylogeny-based functional assignment have suggested for designing a reliable genetic regulatory circuit naturally from CRISPR-CAS systems, acquired defense pathways, to thermophiles in future synthetic biology.

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

    PubMed

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

    2016-03-01

    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

  7. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

    PubMed

    Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2016-06-01

    Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future. PMID:26979473

  8. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells.

    PubMed

    Dong, Zhan-Qi; Chen, Ting-Ting; Zhang, Jun; Hu, Nan; Cao, Ming-Ya; Dong, Fei-Fan; Jiang, Ya-Ming; Chen, Peng; Lu, Cheng; Pan, Min-Hui

    2016-06-01

    Although current antiviral strategies can inhibit baculovirus infection and decrease viral DNA replication to a certain extent, novel tools are required for specific and accurate elimination of baculovirus genomes from infected insects. Using the newly developed clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease (CRISPR/Cas9) technology, we disrupted a viral genome in infected insect cells in vitro as a defense against viral infection. We optimized the CRISPR/Cas9 system to edit foreign and viral genome in insect cells. Using Bombyx mori nucleopolyhedrovirus (BmNPV) as a model, we found that the CRISPR/Cas9 system was capable of cleaving the replication key factor ie-1 in BmNPV thus effectively inhibiting virus proliferation. Furthermore, we constructed a virus-inducible CRISPR/Cas9 editing system, which minimized the probability of off-target effects and was rapidly activated after viral infection. This is the first report describing the application of the CRISPR/Cas9 system in insect antiviral research. Establishment of a highly efficient virus-inducible CRISPR/Cas9 system in insect cells provides insights to produce virus-resistant transgenic strains for future.

  9. Essential requirements for the detection and degradation of invaders by the Haloferax volcanii CRISPR/Cas system I-B.

    PubMed

    Maier, Lisa-Katharina; Lange, Sita J; Stoll, Britta; Haas, Karina A; Fischer, Susan; Fischer, Eike; Duchardt-Ferner, Elke; Wöhnert, Jens; Backofen, Rolf; Marchfelder, Anita

    2013-05-01

    To fend off foreign genetic elements, prokaryotes have developed several defense systems. The most recently discovered defense system, CRISPR/Cas, is sequence-specific, adaptive and heritable. The two central components of this system are the Cas proteins and the CRISPR RNA. The latter consists of repeat sequences that are interspersed with spacer sequences. The CRISPR locus is transcribed into a precursor RNA that is subsequently processed into short crRNAs. CRISPR/Cas systems have been identified in bacteria and archaea, and data show that many variations of this system exist. We analyzed the requirements for a successful defense reaction in the halophilic archaeon Haloferax volcanii. Haloferax encodes a CRISPR/Cas system of the I-B subtype, about which very little is known. Analysis of the mature crRNAs revealed that they contain a spacer as their central element, which is preceded by an eight-nucleotide-long 5' handle that originates from the upstream repeat. The repeat sequences have the potential to fold into a minimal stem loop. Sequencing of the crRNA population indicated that not all of the spacers that are encoded by the three CRISPR loci are present in the same abundance. By challenging Haloferax with an invader plasmid, we demonstrated that the interaction of the crRNA with the invader DNA requires a 10-nucleotide-long seed sequence. In addition, we found that not all of the crRNAs from the three CRISPR loci are effective at triggering the degradation of invader plasmids. The interference does not seem to be influenced by the copy number of the invader plasmid.

  10. Genome editing in Ustilago maydis using the CRISPR-Cas system.

    PubMed

    Schuster, Mariana; Schweizer, Gabriel; Reissmann, Stefanie; Kahmann, Regine

    2016-04-01

    This communication describes the establishment of the type II bacterial CRISPR-Cas9 system to efficiently disrupt target genes in the fungal maize pathogen Ustilago maydis. A single step transformation of a self-replicating plasmid constitutively expressing the U. maydis codon-optimized cas9 gene and a suitable sgRNA under control of the U. maydis U6 snRNA promoter was sufficient to induce genome editing. On average 70% of the progeny of a single transformant were disrupted within the respective b gene. Without selection the self-replicating plasmid was lost rapidly allowing transient expression of the CRISPR-Cas9 system to minimize potential long-term negative effects of Cas9. This technology will be an important advance for the simultaneous disruption of functionally redundant genes and gene families to investigate their contribution to virulence of U. maydis.

  11. CRISPR/Cas9 Platforms for Genome Editing in Plants: Developments and Applications.

    PubMed

    Ma, Xingliang; Zhu, Qinlong; Chen, Yuanling; Liu, Yao-Guang

    2016-07-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) genome editing system (CRISPR/Cas9) is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adaption of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, factors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement.

  12. 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.

  13. 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.

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

    PubMed

    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

  15. 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

  16. Utilization of the CRISPR/Cas9 system for the efficient production of mutant mice using crRNA/tracrRNA with Cas9 nickase and FokI-dCas9

    PubMed Central

    Terao, Miho; Tamano, Moe; Hara, Satoshi; Kato, Tomoko; Kinoshita, Masato; Takada, Shuji

    2016-01-01

    The CRISPR/Cas9 system is a powerful genome editing tool for the production of genetically modified animals. To produce mutant mice, chimeric single-guide RNA (sgRNA) is cloned in a plasmid vector and a mixture of sgRNA and Cas9 are microinjected into the fertilized eggs. An issue associated with gene manipulation using the CRISPR/Cas9 system is that there can be off-target effects. To simplify the production of mutant mice with low risks of off-target effects caused by the CRISPR/Cas9 system, we demonstrated that genetically modified mice can be efficiently obtained using chemically synthesized CRISPR RNA (crRNA), trans-activating crRNA (tracrRNA), and modified Cas9s, such as the nickase version and FokI-fused catalytically inactive Cas9, by microinjection into fertilized eggs. Using this method, it is no longer necessary to clone sgRNA into a plasmid vector, and this enables high-throughput production of mutant mice. PMID:26972821

  17. Expanding the catalog of cas genes with metagenomes.

    PubMed

    Zhang, Quan; Doak, Thomas G; Ye, Yuzhen

    2014-02-01

    The CRISPR (clusters of regularly interspaced short palindromic repeats)-Cas adaptive immune system is an important defense system in bacteria, providing targeted defense against invasions of foreign nucleic acids. CRISPR-Cas systems consist of CRISPR loci and cas (CRISPR-associated) genes: sequence segments of invaders are incorporated into host genomes at CRISPR loci to generate specificity, while adjacent cas genes encode proteins that mediate the defense process. We pursued an integrated approach to identifying putative cas genes from genomes and metagenomes, combining similarity searches with genomic neighborhood analysis. Application of our approach to bacterial genomes and human microbiome datasets allowed us to significantly expand the collection of cas genes: the sequence space of the Cas9 family, the key player in the recently engineered RNA-guided platforms for genome editing in eukaryotes, is expanded by at least two-fold with metagenomic datasets. We found genes in cas loci encoding other functions, for example, toxins and antitoxins, confirming the recently discovered potential of coupling between adaptive immunity and the dormancy/suicide systems. We further identified 24 novel Cas families; one novel family contains 20 proteins, all identified from the human microbiome datasets, illustrating the importance of metagenomics projects in expanding the diversity of cas genes.

  18. 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.

  19. 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.

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

    PubMed

    Czarnek, Maria; Bereta, Joanna

    2016-01-01

    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. PMID:27594566

  1. 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

  2. A light-inducible CRISPR/Cas9 system for control of endogenous gene activation

    PubMed Central

    Polstein, Lauren R.; Gersbach, Charles A.

    2015-01-01

    Optogenetic systems enable precise spatial and temporal control of cell behavior. We engineered a light-activated CRISPR/Cas9 effector (LACE) system that induces transcription of endogenous genes in the presence of blue light. This was accomplished by fusing the light-inducible heterodimerizing proteins CRY2 and CIB1 to a transactivation domain and the catalytically inactive dCas9, respectively. The versatile LACE system can be easily directed to new DNA sequences for the dynamic regulation of endogenous genes. PMID:25664691

  3. Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System.

    PubMed

    Feng, Chao; Yuan, Jing; Wang, Rui; Liu, Yang; Birchler, James A; Han, Fangpu

    2016-01-20

    CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9 construct was transformed into maize protoplasts, and indel (insertion and deletion) mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sgRNA-Cas9 constructs were transformed into protoplasts followed by sequencing the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM (protospacer adjacent motif) or having more than two mismatches. Together, our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize. PMID:26842992

  4. Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System.

    PubMed

    Feng, Chao; Yuan, Jing; Wang, Rui; Liu, Yang; Birchler, James A; Han, Fangpu

    2016-01-20

    CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 system, which is a newly developed technology for targeted genome modification, has been successfully used in a number of species. In this study, we applied this technology to carry out targeted genome modification in maize. A marker gene Zmzb7 was chosen for targeting. The sgRNA-Cas9 construct was transformed into maize protoplasts, and indel (insertion and deletion) mutations could be detected. A mutant seedling with an expected albino phenotype was obtained from screening 120 seedlings generated from 10 callus events. Mutation efficiency in maize heterochromatic regions was also investigated. Twelve sites with different expression levels in maize centromeres or pericentromere regions were selected. The sgRNA-Cas9 constructs were transformed into protoplasts followed by sequencing the transformed protoplast genomic DNA. The results show that the genes in heterochromatic regions could be targeted by the CRISPR/Cas9 system efficiently, no matter whether they are expressed or not. Meanwhile, off-target mutations were not found in the similar sites having no PAM (protospacer adjacent motif) or having more than two mismatches. Together, our results show that the CRISPR/Cas9 system is a robust and efficient tool for genome modification in both euchromatic and heterochromatic regions in maize.

  5. Efficient generation of hiPSC neural lineage specific knockin reporters using the CRISPR/Cas9 and Cas9 double nickase system.

    PubMed

    Li, Shenglan; Xue, Haipeng; Long, Bo; Sun, Li; Truong, Tai; Liu, Ying

    2015-05-28

    Gene targeting is a critical approach for characterizing gene functions in modern biomedical research. However, the efficiency of gene targeting in human cells has been low, which prevents the generation of human cell lines at a desired rate. The past two years have witnessed a rapid progression on improving efficiency of genetic manipulation by genome editing tools such as the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) system. This manuscript describes a protocol for generating lineage specific human induced pluripotent stem cell (hiPSC) reporters using CRISPR/Cas system assisted homologous recombination. Procedures for obtaining necessary components for making neural lineage reporter lines using the CRISPR/Cas system, focusing on construction of targeting vectors and single guide RNAs, are described. This protocol can be extended to platform establishment and mutation correction in hiPSCs.

  6. 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

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

    DOE PAGES

    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. 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

  8. Multiplex conditional mutagenesis in zebrafish using the CRISPR/Cas system.

    PubMed

    Yin, L; Maddison, L A; Chen, W

    2016-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) system is a powerful tool for genome editing in numerous organisms. However, the system is typically used for gene editing throughout the entire organism. Tissue and temporal specific mutagenesis is often desirable to determine gene function in a specific stage or tissue and to bypass undesired consequences of global mutations. We have developed the CRISPR/Cas system for conditional mutagenesis in transgenic zebrafish using tissue-specific and/or inducible expression of Cas9 and U6-driven expression of sgRNA. To allow mutagenesis of multiple targets, we have isolated four distinct U6 promoters and designed Golden Gate vectors to easily assemble transgenes with multiple sgRNAs. We provide experimental details on the reagents and applications for multiplex conditional mutagenesis in zebrafish. PMID:27443918

  9. Gaia as a complex adaptive system.

    PubMed

    Lenton, Timothy M; van Oijen, Marcel

    2002-05-29

    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

  10. Gaia as a complex adaptive system.

    PubMed

    Lenton, Timothy M; van Oijen, Marcel

    2002-05-29

    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.

  11. 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

  12. CRISPR-Based Adaptive Immune Systems

    PubMed Central

    Terns, Michael P.; Terns, Rebecca M.

    2011-01-01

    CRISPR-Cas systems are recently discovered, RNA-based immune systems that control invasions of viruses and plasmids in archaea and bacteria. Prokaryotes with CRISPR-Cas immune systems capture short invader sequences within the CRISPR loci in their genomes, and small RNAs produced from the CRISPR loci (CRISPR (cr)RNAs) guide Cas proteins to recognize and degrade (or otherwise silence) the invading nucleic acids. There are multiple variations of the pathway found among prokaryotes, each mediated by largely distinct components and mechanisms that we are only beginning to delineate. Here we will review our current understanding of the remarkable CRISPR-Cas pathways with particular attention to studies relevant to systems found in the archaea. PMID:21531607

  13. Programmable plasmid interference by the CRISPR-Cas system in Thermococcus kodakarensis

    PubMed Central

    Elmore, Joshua R.; Yokooji, Yuusuke; Sato, Takaaki; Olson, Sara; Glover, III, Claiborne V.C.; Graveley, Brenton R.; Atomi, Haruyuki; Terns, Rebecca M.; Terns, Michael P.

    2013-01-01

    CRISPR-Cas systems are RNA-guided immune systems that protect prokaryotes against viruses and other invaders. The CRISPR locus encodes crRNAs that recognize invading nucleic acid sequences and trigger silencing by the associated Cas proteins. There are multiple CRISPR-Cas systems with distinct compositions and mechanistic processes. Thermococcus kodakarensis (Tko) is a hyperthermophilic euryarchaeon that has both a Type I-A Csa and a Type I-B Cst CRISPR-Cas system. We have analyzed the expression and composition of crRNAs from the three CRISPRs in Tko by RNA deep sequencing and northern analysis. Our results indicate that crRNAs associated with these two CRISPR-Cas systems include an 8-nucleotide conserved sequence tag at the 5′ end. We challenged Tko with plasmid invaders containing sequences targeted by endogenous crRNAs and observed active CRISPR-Cas-mediated silencing. Plasmid silencing was dependent on complementarity with a crRNA as well as on a sequence element found immediately adjacent to the crRNA recognition site in the target termed the PAM (protospacer adjacent motif). Silencing occurred independently of the orientation of the target sequence in the plasmid, and appears to occur at the DNA level, presumably via DNA degradation. In addition, we have directed silencing of an invader plasmid by genetically engineering the chromosomal CRISPR locus to express customized crRNAs directed against the plasmid. Our results support CRISPR engineering as a feasible approach to develop prokaryotic strains that are resistant to infection for use in industry. PMID:23535213

  14. Independent Confirmatory Factor Analysis of the Cognitive Assessment System (CAS): What Does CAS Measure?

    ERIC Educational Resources Information Center

    Kranzler, John H.; Keith, Timothy Z.

    1999-01-01

    Uses confirmatory factor analysis (CFA) to address unresolved issues concerning the structure of the Cognitive Assessment System, a test of intelligence based upon the planning, attention, and simultaneous-successive (PASS) processes theory of human cognition. Results reveal that the CFA of the standardization data do not support use of the CAS…

  15. A novel sgRNA selection system for CRISPR-Cas9 in mammalian cells.

    PubMed

    Zhang, Haiwei; Zhang, Xixi; Fan, Cunxian; Xie, Qun; Xu, Chengxian; Zhao, Qun; Liu, Yongbo; Wu, Xiaoxia; Zhang, Haibing

    2016-03-18

    CRISPR-Cas9 mediated genome editing system has been developed as a powerful tool for elucidating the function of genes through genetic engineering in multiple cells and organisms. This system takes advantage of a single guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific DNA site to generate mutant alleles. Since the targeting efficiency of sgRNAs to distinct DNA loci can vary widely, there remains a need for a rapid, simple and efficient sgRNA selection method to overcome this limitation of the CRISPR-Cas9 system. Here we report a novel system to select sgRNA with high efficacy for DNA sequence modification by a luciferase assay. Using this sgRNAs selection system, we further demonstrated successful examples of one sgRNA for generating one gene knockout cell lines where the targeted genes are shown to be functionally defective. This system provides a potential application to optimize the sgRNAs in different species and to generate a powerful CRISPR-Cas9 genome-wide screening system with minimum amounts of sgRNAs.

  16. A novel sgRNA selection system for CRISPR-Cas9 in mammalian cells.

    PubMed

    Zhang, Haiwei; Zhang, Xixi; Fan, Cunxian; Xie, Qun; Xu, Chengxian; Zhao, Qun; Liu, Yongbo; Wu, Xiaoxia; Zhang, Haibing

    2016-03-18

    CRISPR-Cas9 mediated genome editing system has been developed as a powerful tool for elucidating the function of genes through genetic engineering in multiple cells and organisms. This system takes advantage of a single guide RNA (sgRNA) to direct the Cas9 endonuclease to a specific DNA site to generate mutant alleles. Since the targeting efficiency of sgRNAs to distinct DNA loci can vary widely, there remains a need for a rapid, simple and efficient sgRNA selection method to overcome this limitation of the CRISPR-Cas9 system. Here we report a novel system to select sgRNA with high efficacy for DNA sequence modification by a luciferase assay. Using this sgRNAs selection system, we further demonstrated successful examples of one sgRNA for generating one gene knockout cell lines where the targeted genes are shown to be functionally defective. This system provides a potential application to optimize the sgRNAs in different species and to generate a powerful CRISPR-Cas9 genome-wide screening system with minimum amounts of sgRNAs. PMID:26879140

  17. A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.

    PubMed

    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

  18. A 'suicide' CRISPR-Cas9 system to promote gene deletion and restoration by electroporation in Cryptococcus neoformans.

    PubMed

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

    2016-08-09

    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.

  19. 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

  20. 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. PMID:27644742

  1. 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

  2. Generation of VDR Knock-Out Mice via Zygote Injection of CRISPR/Cas9 System

    PubMed Central

    Zhang, Tao; Yin, Yajun; Liu, Huan; Du, Weili; Ren, Chonghua; Wang, Ling; Lu, Hongzhao; Zhang, Zhiying

    2016-01-01

    CRISPR/Cas9 system has become a new versatile technology for genome engineering in various species. To achieve targeted modifications at the same site in both human and mice genomes by a CRISPR/Cas9 nuclease, we designed two target sites in conserved regions of vitamin D receptor (VDR) gene, which cover more than 17 kb of chromosome region depending on the species. We first validated the efficacy of single sgRNA mediated gene specific modifications were 36% and 31% in HEK293T cells. Concurrently, targeted of the intervening genomic segments deletions were generated in chromosomes when two sgRNAs worked simultaneously. The large genomic DNA segments up to 23.4 Kb could be precisely deleted in human chromosomes. Subsequently, Cas9 mRNA and sgRNAs targeting VDRT1 and VDRT2 were co-microinjected into one-cell-stage embryos of C57BL/6 mice. Verified by T7E1 assay and DNA sequencing analysis, 12 mice showed VDR targeted disruption and 8 of which were biallelic knock-out, which demonstrated obvious phenotype of hair thinning. Furthermore, expression changes of Vitamin D metabolism genes in VDR-/-mice were detected. These results indicated that CRISPR/Cas9 mediated knock-out of VDR diminished its gene function in vivo. The off-target effects of CRISPR/Cas9 in VDR-/- founder mice were analyzed. Our results showed that CRISPR/Cas9 system could be employed to target the same sites in different species, when sgRNAs are designed within conserved regions, and therefore will be critically important and applicable for human disease model. PMID:27685656

  3. Protein engineering of Cas9 for enhanced function

    PubMed Central

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

    2015-01-01

    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 complimentary 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 directly into 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. PMID:25398355

  4. 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.

  5. 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.

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

    PubMed

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

    2015-10-01

    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.

  7. Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system.

    PubMed

    Zhang, Zhongjie; Aslam, Abu F M; Liu, Xiaojing; Li, Muwang; Huang, Yongping; Tan, Anjiang

    2015-08-01

    Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis. PMID:26070541

  8. Multigene knockout utilizing off-target mutations of the CRISPR/Cas9 system in rice.

    PubMed

    Endo, Masaki; Mikami, Masafumi; Toki, Seiichi

    2015-01-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)-associated endonuclease 9 (CRISPR/Cas9) system has been demonstrated to be a robust genome engineering tool in a variety of organisms including plants. However, it has been shown that the CRISPR/Cas9 system cleaves genomic DNA sequences containing mismatches to the guide RNA strand. We expected that this low specificity could be exploited to induce multihomeologous and multiparalogous gene knockouts. In the case of polyploid plants, simultaneous modification of multiple homeologous genes, i.e. genes with similar but not identical DNA sequences, is often needed to obtain a desired phenotype. Even in diploid plants, disruption of multiparalogous genes, which have functional redundancy, is often needed. To validate the applicability of the CRISPR/Cas9 system to target mutagenesis of paralogous genes in rice, we designed a single-guide RNA (sgRNA) that recognized 20 bp sequences of cyclin-dependent kinase B2 (CDKB2) as an on-target locus. These 20 bp possess similarity to other rice CDK genes (CDKA1, CDKA2 and CDKB1) with different numbers of mismatches. We analyzed mutations in these four CDK genes in plants regenerated from Cas9/sgRNA-transformed calli and revealed that single, double and triple mutants of CDKA2, CDKB1 and CDKB2 can be created by a single sgRNA.

  9. Optimization of a multiplex CRISPR/Cas system for use as an antiviral therapeutic.

    PubMed

    Kennedy, Edward M; Kornepati, Anand V R; Mefferd, Adam L; Marshall, Joy B; Tsai, Kevin; Bogerd, Hal P; Cullen, Bryan R

    2015-12-01

    RNA-guided endonucleases or CRISPR/Cas systems have been widely employed for gene engineering/DNA editing applications, and have recently been used against a variety of dsDNA viruses as a potential therapeutic. However, in vivo delivery to specific tissue reservoirs using adeno-associated virus (AAV) vectors is problematic due to the large coding requirement for the principal effector commonly used in these applications, Streptococcus pyogenes (Spy) Cas9. Here we describe design of a minimal CRISPR/Cas system that is capable of multiplexing and can be packaged into a single AAV vector. This system consists of the small Type II Cas9 protein from Staphylococcus aureus (Sau) driven by a truncated CMV promoter/enhancer, and flanked 3' by a poly(A) addition signal, as well as two sgRNA expression cassettes driven by either U6 or ∼70-bp tRNA-derived Pol III promoters. Specific protocols for construction of these AAV vector scaffolds, shuttle cloning of their contents into AAV and lentiviral backbones, and a quantitative luciferase assay capable of screening for optimal sgRNAs, are detailed. These protocols can facilitate construction of AAV vectors that have optimal multiplexed sgRNA expression and function. These will have potential utility in multiplex applications, including in antiviral therapy in tissues chronically infected with a pathogenic DNA virus. PMID:26291065

  10. Functional analysis of Bombyx Wnt1 during embryogenesis using the CRISPR/Cas9 system.

    PubMed

    Zhang, Zhongjie; Aslam, Abu F M; Liu, Xiaojing; Li, Muwang; Huang, Yongping; Tan, Anjiang

    2015-08-01

    Recently established, custom-designed nuclease technologies such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system provide attractive genome editing tools. Targeted gene mutagenesis using the CRISPR/Cas9 system has been achieved in several orders of insects. However, outside of studies on Drosophila melanogaster and the lepidopteron model insect Bombyx mori, little success has been reported, which is largely due to a lack of effective genetic manipulation tools that can be used in other insect orders. To create a simple and effective method of gene knockout analysis, especially for dissecting gene functioning during insect embryogenesis, we performed a functional analysis of the Bombyx Wnt1 (BmWnt1) gene using Cas9/sgRNA-mediated gene mutagenesis. The Wnt1 gene is required for embryonic patterning in various organisms, and its crucial roles during embryogenesis have been demonstrated in several insect orders. Direct injection of Cas9 mRNA and BmWnt1-specific sgRNA into Bombyx embryos induced a typical Wnt-deficient phenotype: injected embryos could not hatch and exhibited severe defects in body segmentation and pigmentation in a dose-dependent manner. Quantitative real-time PCR (qRT-PCR) analysis revealed that Hox genes were down-regulated after BmWnt1 depletion. Furthermore, large deletion, up to 18Kb, ware generated. The current study demonstrates that using the CRISPR/Cas9 system is a promising approach to achieve targeted gene mutagenesis during insect embryogenesis.

  11. Editing the Mouse Genome Using the CRISPR-Cas9 System.

    PubMed

    Williams, Adam; Henao-Mejia, Jorge; Flavell, Richard A

    2016-02-01

    The ability to modify the murine genome is perhaps one of the most important developments in modern biology. However, traditional methods of genomic engineering are costly and relatively clumsy in their approach. The use of programmable nucleases such as zinc finger nucleases and transcription activator-like effector nucleases significantly improved the precision of genome-editing technology, but the design and use of these nucleases remains cumbersome and prohibitively expensive. The CRISPR-Cas9 system is the next installment in the line of programmable nucleases; it provides highly efficient and precise genome-editing capabilities using reagents that are simple to design and inexpensive to generate. Furthermore, with the CRISPR-Cas9 system, it is possible to move from a hypothesis to an in vivo mouse model in less than a month. The simplicity, cost effectiveness, and speed of the CRISPR-Cas9 system allows researchers to tackle questions that otherwise would not be technically or financially viable. In this introduction, we discuss practical considerations for the use of Cas9 in genome engineering in mice.

  12. 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. PMID:26294350

  13. Engineering Translational Activators with CRISPR-Cas System.

    PubMed

    Du, Pei; Miao, Chensi; Lou, Qiuli; Wang, Zefeng; Lou, Chunbo

    2016-01-15

    RNA parts often serve as critical components in genetic engineering. Here we report a design of translational activators which is composed of an RNA endoribonuclease (Csy4) and two exchangeable RNA modules. Csy4, a member of Cas endoribonuclease, cleaves at a specific recognition site; this cleavage releases a cis-repressive RNA module (crRNA) from the masked ribosome binding site (RBS), which subsequently allows the downstream translation initiation. Unlike small RNA as a translational activator, the endoribonuclease-based activator is able to efficiently unfold the perfect RBS-crRNA pairing. As an exchangeable module, the crRNA-RBS duplex was forwardly and reversely engineered to modulate the dynamic range of translational activity. We further showed that Csy4 and its recognition site, together as a module, can also be replaced by orthogonal endoribonuclease-recognition site homologues. These modularly structured, high-performance translational activators would endow the programming of gene expression in the translation level with higher feasibility. PMID:26414660

  14. 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

  15. Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system

    PubMed Central

    Feng, Yong; Sassi, Slim; Shen, Jacson K; Yang, Xiaoqian; Gao, Yan; Osaka, Eiji; Zhang, Jianming; Yang, Shuhua; Yang, Cao; Mankin, Henry J.; Hornicek, Francis J; Duan, Zhenfeng

    2014-01-01

    Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR-Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U-2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR-Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR-Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma. PMID:25348612

  16. 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

  17. Targeting CDK11 in osteosarcoma cells using the CRISPR-Cas9 system.

    PubMed

    Feng, Yong; Sassi, Slim; Shen, Jacson K; Yang, Xiaoqian; Gao, Yan; Osaka, Eiji; Zhang, Jianming; Yang, Shuhua; Yang, Cao; Mankin, Henry J; Hornicek, Francis J; Duan, Zhenfeng

    2015-02-01

    Osteosarcoma is the most common type primary malignant tumor of bone. Patients with regional osteosarcoma are routinely treated with surgery and chemotherapy. In addition, many patients with metastatic or recurrent osteosarcoma show poor prognosis with current chemotherapy agents. Therefore, it is important to improve the general condition and the overall survival rate of patients with osteosarcoma by identifying novel therapeutic strategies. Recent studies have revealed that CDK11 is essential in osteosarcoma cell growth and survival by inhibiting CDK11 mRNA expression with RNAi. Here, we apply the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas9 system, a robust and highly efficient novel genome editing tool, to determine the effect of targeting endogenous CDK11 gene at the DNA level in osteosarcoma cell lines. We show that CDK11 can be efficiently silenced by CRISPR-Cas9. Inhibition of CDK11 is associated with decreased cell proliferation and viability, and induces cell death in osteosarcoma cell lines KHOS and U-2OS. Furthermore, the migration and invasion activities are also markedly reduced by CDK11 knockout. These results demonstrate that CRISPR-Cas9 system is a useful tool for the modification of endogenous CDK11 gene expression, and CRISPR-Cas9 targeted CDK11 knockout may be a promising therapeutic regimen for the treatment of osteosarcoma.

  18. SH2-Containing Inositol 5′-Phosphatase SHIP2 Associates with the p130Cas Adapter Protein and Regulates Cellular Adhesion and Spreading

    PubMed Central

    Prasad, Nagendra; Topping, Robert S.; Decker, Stuart J.

    2001-01-01

    In a previous study, we found that the SHIP2 protein became tyrosine phosphorylated and associated with the Shc adapter protein in response to the treatment of cells with growth factors and insulin (T. Habib, J. A. Hejna, R. E. Moses, and S. J. Decker, J. Biol. Chem. 273:18605–18609, 1998). We describe here a novel interaction between SHIP2 and the p130Cas adapter protein, a mediator of actin cytoskeleton organization. SHIP2 and p130Cas association was detected in anti-SHIP2 immunoprecipitates from several cell types. Reattachment of trypsinized cells stimulated tyrosine phosphorylation of SHIP2 and increased the formation of a complex containing SHIP2 and a faster-migrating tyrosine-phosphorylated form of p130Cas. The faster-migrating form of p130Cas was no longer recognized by antibodies to the amino terminus of p130Cas and appeared to be generated through proteolysis. Interaction of the SHIP2 protein with the various forms of p130Cas was mediated primarily through the SH2 domain of SHIP2. Immunofluorescence studies indicated that SHIP2 localized to focal contacts and to lamellipodia. Increased adhesion was observed in HeLa cells transiently expressing exogenous WT-SHIP2. These effects were not seen with SHIP2 possessing a mutation in the SH2 domain (R47G). Transfection of a catalytic domain deletion mutant of SHIP2 (ΔRV) inhibited cell spreading. Taken together, our studies suggest an important role for SHIP2 in adhesion and spreading. PMID:11158326

  19. Two CRISPR-Cas systems inMethanosarcina mazeistrain Gö1 display common processing features despite belonging to different types I and III

    PubMed Central

    Nickel, Lisa; Weidenbach, Katrin; Jäger, Dominik; Backofen, Rolf; Lange, Sita J.; Heidrich, Nadja; Schmitz, Ruth A.

    2013-01-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. We analyzed the two CRISPR-Cas systems in Methanosarcina mazei strain Gö1. Although belonging to different subtypes (I-B and III-B), the leaders and repeats of both loci are nearly identical. Also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinformatics analysis and in vitro structural probing. The expression and maturation of CRISPR-derived RNAs (crRNAs) were studied in vitro and in vivo. Both respective potential Cas6b-type endonucleases were purified and their activity tested in vitro. Each protein showed significant activity and could cleave both repeats at the same processing site. Cas6b of subtype III-B, however, was significantly more efficient in its cleavage activity compared with Cas6b of subtype I-B. Northern blot and differential RNAseq analyses were performed to investigate in vivo transcription and maturation of crRNAs, revealing generally very low expression of both systems, whereas significant induction at high NaCl concentrations was observed. crRNAs derived proximal to the leader were generally more abundant than distal ones and in vivo processing sites were clarified for both loci, confirming the previously well-established 8 nt 5′ repeat tags. The 3′-ends were more diverse, but generally ended in a prefix of the following repeat sequence (3′-tag). The analysis further revealed a 5′-hydroxy and 3′-phosphate termini architecture of small crRNAs specific for cleavage products of Cas6 endonucleases from type I-E and I-F and type III-B. PMID:23619576

  20. Two CRISPR-Cas systems in Methanosarcina mazei strain Gö1 display common processing features despite belonging to different types I and III.

    PubMed

    Nickel, Lisa; Weidenbach, Katrin; Jäger, Dominik; Backofen, Rolf; Lange, Sita J; Heidrich, Nadja; Schmitz, Ruth A

    2013-05-01

    The clustered regularly interspaced short palindromic repeats (CRISPR) system represents a highly adaptive and heritable defense system against foreign nucleic acids in bacteria and archaea. We analyzed the two CRISPR-Cas systems in Methanosarcina mazei strain Gö1. Although belonging to different subtypes (I-B and III-B), the leaders and repeats of both loci are nearly identical. Also, despite many point mutations in each array, a common hairpin motif was identified in the repeats by a bioinformatics analysis and in vitro structural probing. The expression and maturation of CRISPR-derived RNAs (crRNAs) were studied in vitro and in vivo. Both respective potential Cas6b-type endonucleases were purified and their activity tested in vitro. Each protein showed significant activity and could cleave both repeats at the same processing site. Cas6b of subtype III-B, however, was significantly more efficient in its cleavage activity compared with Cas6b of subtype I-B. Northern blot and differential RNAseq analyses were performed to investigate in vivo transcription and maturation of crRNAs, revealing generally very low expression of both systems, whereas significant induction at high NaCl concentrations was observed. crRNAs derived proximal to the leader were generally more abundant than distal ones and in vivo processing sites were clarified for both loci, confirming the previously well-established 8 nt 5' repeat tags. The 3'-ends were more diverse, but generally ended in a prefix of the following repeat sequence (3'-tag). The analysis further revealed a 5'-hydroxy and 3'-phosphate termini architecture of small crRNAs specific for cleavage products of Cas6 endonucleases from type I-E and I-F and type III-B. PMID:23619576

  1. 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

    2015-01-01

    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. PMID:26182359

  2. The role of Cas8 in type I CRISPR interference

    PubMed Central

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

    2015-01-01

    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. PMID:26182359

  3. 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.

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

    PubMed

    Silas, Sukrit; Mohr, Georg; Sidote, David J; Markham, Laura M; Sanchez-Amat, Antonio; Bhaya, Devaki; Lambowitz, Alan M; Fire, Andrew Z

    2016-02-26

    CRISPR 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 showed that a RT-Cas1 fusion protein enables the acquisition of RNA spacers in vivo in a RT-dependent manner. In vitro, the MMB-1 RT-Cas1 and Cas2 proteins catalyze the ligation of RNA segments into the CRISPR array, which is followed by reverse transcription. These observations outline a host-mediated mechanism for reverse information flow from RNA to DNA. PMID:26917774

  5. 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

  6. 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.

  7. 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.

  8. 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

  9. CRISPR-Cas: biology, mechanisms and relevance.

    PubMed

    Hille, Frank; Charpentier, Emmanuelle

    2016-11-01

    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'.

  10. CRISPR-Cas: biology, mechanisms and relevance.

    PubMed

    Hille, Frank; Charpentier, Emmanuelle

    2016-11-01

    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

  11. Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System.

    PubMed

    Min, Kyunghun; Ichikawa, Yuichi; Woolford, Carol A; Mitchell, Aaron P

    2016-01-01

    Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation.

  12. Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System.

    PubMed

    Min, Kyunghun; Ichikawa, Yuichi; Woolford, Carol A; Mitchell, Aaron P

    2016-01-01

    Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C. albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C. albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C. albicans genetic manipulation. PMID:27340698

  13. 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.

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

    PubMed

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

    2016-04-07

    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 Puro(R) 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.

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

    PubMed

    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 Puro(R) 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. Genome editing in the human malaria parasite Plasmodium falciparum using the CRISPR-Cas9 system.

    PubMed

    Ghorbal, Mehdi; Gorman, Molly; Macpherson, Cameron Ross; Martins, Rafael Miyazawa; Scherf, Artur; Lopez-Rubio, Jose-Juan

    2014-08-01

    Genome manipulation in the malaria parasite Plasmodium falciparum remains largely intractable and improved genomic tools are needed to further understand pathogenesis and drug resistance. We demonstrated the CRISPR-Cas9 system for use in P. falciparum by disrupting chromosomal loci and generating marker-free, single-nucleotide substitutions with high efficiency. Additionally, an artemisinin-resistant strain was generated by introducing a previously implicated polymorphism, thus illustrating the value of efficient genome editing in malaria research.

  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. 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

  19. 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

  20. 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

  1. 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.

  2. Efficient editing of malaria parasite genome using the CRISPR/Cas9 system.

    PubMed

    Zhang, Cui; Xiao, Bo; Jiang, Yuanyuan; Zhao, Yihua; Li, Zhenkui; Gao, Han; Ling, Yuan; Wei, Jun; Li, Shaoneng; Lu, Mingke; Su, Xin-Zhuan; Cui, Huiting; Yuan, Jing

    2014-01-01

    Malaria parasites are unicellular organisms residing inside the red blood cells, and current methods for editing the parasite genes have been inefficient. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats and Cas9 endonuclease-mediated genome editing) system is a new powerful technique for genome editing and has been widely employed to study gene function in various organisms. However, whether this technique can be applied to modify the genomes of malaria parasites has not been determined. In this paper, we demonstrated that Cas9 is able to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. By supplying engineered homologous repair templates, we generated targeted deletion, reporter knock-in, and nucleotide replacement in multiple parasite genes, achieving up to 100% efficiency in gene deletion and 22 to 45% efficiencies in knock-in and allelic replacement. Our results establish methodologies for introducing desired modifications in the P. yoelii genome with high efficiency and accuracy, which will greatly improve our ability to study gene function of malaria parasites. Importance: Malaria, caused by infection of Plasmodium parasites, remains a world-wide public health burden. Although the genomes of many malaria parasites have been sequenced, we still do not know the functions of approximately half of the genes in the genomes. Studying gene function has become the focus of many studies; however, editing genes in malaria parasite genomes is still inefficient. Here we designed several efficient approaches, based on the CRISPR/Cas9 system, to introduce site-specific DNA double-strand breaks in the Plasmodium yoelii genome that can be repaired through homologous recombination. Using this system, we achieved high efficiencies in gene deletion, reporter tagging, and allelic replacement in multiple parasite genes. This technique for editing the malaria parasite

  3. Zygote-mediated generation of genome-modified mice using Streptococcus thermophilus 1-derived CRISPR/Cas system.

    PubMed

    Fujii, Wataru; Kakuta, Shigeru; Yoshioka, Shin; Kyuwa, Shigeru; Sugiura, Koji; Naito, Kunihiko

    2016-08-26

    Mammalian zygote-mediated genome-engineering by CRISPR/Cas is currently used for the generation of genome-modified animals. Here we report that a Streptococcus thermophilus-1 derived orthologous CRISPR/Cas system, which recognizes the 5'-NNAGAA sequence as a protospacer adjacent motif (PAM), is useful in mouse zygotes and is applicable for generating knockout mice (87.5%) and targeted knock-in mice (45.5%). The induced mutation could be inherited in the next generation. This novel CRISPR/Cas can expand the feasibility of the zygote-mediated generation of genome-modified animals that require an exact mutation design. PMID:27318086

  4. Structures of Cas9 Endonucleases Reveal RNA-Mediated Conformational Activation

    PubMed Central

    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.

    2014-01-01

    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. PMID:24505130

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

    PubMed

    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

  6. Rapid generation of mouse models with defined point mutations by the CRISPR/Cas9 system.

    PubMed

    Inui, Masafumi; Miyado, Mami; Igarashi, Maki; Tamano, Moe; Kubo, Atsushi; Yamashita, Satoshi; Asahara, Hiroshi; Fukami, Maki; Takada, Shuji

    2014-06-23

    Introducing a point mutation is a fundamental method used to demonstrate the roles of particular nucleotides or amino acids in the genetic elements or proteins, and is widely used in in vitro experiments based on cultured cells and exogenously provided DNA. However, the in vivo application of this approach by modifying genomic loci is uncommon, partly due to its technical and temporal demands. This leaves many in vitro findings un-validated under in vivo conditions. We herein applied the CRISPR/Cas9 system to generate mice with point mutations in their genomes, which led to single amino acid substitutions in proteins of interest. By microinjecting gRNA, hCas9 mRNA and single-stranded donor oligonucleotides (ssODN) into mouse zygotes, we introduced defined genomic modifications in their genome with a low cost and in a short time. Both single gRNA/WT hCas9 and double nicking set-ups were effective. We also found that the distance between the modification site and gRNA target site was a significant parameter affecting the efficiency of the substitution. We believe that this is a powerful technique that can be used to examine the relevance of in vitro findings, as well as the mutations found in patients with genetic disorders, in an in vivo system.

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

    PubMed

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

    2015-03-23

    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 knockout 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 knockout and greatly broadens the scope of loss-of-function studies in zebrafish.

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

    PubMed

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

    2015-03-23

    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 knockout 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 knockout and greatly broadens the scope of loss-of-function studies in zebrafish. PMID:25752963

  9. Sequence features associated with the cleavage efficiency of CRISPR/Cas9 system

    PubMed Central

    Liu, Xiaoxi; Homma, Ayaka; Sayadi, Jamasb; Yang, Shu; Ohashi, Jun; Takumi, Toru

    2016-01-01

    The CRISPR-Cas9 system has recently emerged as a versatile tool for biological and medical research. In this system, a single guide RNA (sgRNA) directs the endonuclease Cas9 to a targeted DNA sequence for site-specific manipulation. In addition to this targeting function, the sgRNA has also been shown to play a role in activating the endonuclease activity of Cas9. This dual function of the sgRNA likely underlies observations that different sgRNAs have varying on-target activities. Currently, our understanding of the relationship between sequence features of sgRNAs and their on-target cleavage efficiencies remains limited, largely due to difficulties in assessing the cleavage capacity of a large number of sgRNAs. In this study, we evaluated the cleavage activities of 218 sgRNAs using in vitro Surveyor assays. We found that nucleotides at both PAM-distal and PAM-proximal regions of the sgRNA are significantly correlated with on-target efficiency. Furthermore, we also demonstrated that the genomic context of the targeted DNA, the GC percentage, and the secondary structure of sgRNA are critical factors contributing to cleavage efficiency. In summary, our study reveals important parameters for the design of sgRNAs with high on-target efficiencies, especially in the context of high throughput applications. PMID:26813419

  10. Efficient gene targeting in golden Syrian hamsters by the CRISPR/Cas9 system.

    PubMed

    Fan, Zhiqiang; Li, Wei; Lee, Sang R; 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.

  11. 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

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

    PubMed

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

    2016-08-03

    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.

  13. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.

    PubMed

    Nishida, Keiji; Arazoe, Takayuki; Yachie, Nozomu; Banno, Satomi; Kakimoto, Mika; Tabata, Mayura; Mochizuki, Masao; Miyabe, Aya; Araki, Michihiro; Hara, Kiyotaka Y; Shimatani, Zenpei; Kondo, Akihiko

    2016-09-16

    The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency. PMID:27492474

  14. Targeted nucleotide editing using hybrid prokaryotic and vertebrate adaptive immune systems.

    PubMed

    Nishida, Keiji; Arazoe, Takayuki; Yachie, Nozomu; Banno, Satomi; Kakimoto, Mika; Tabata, Mayura; Mochizuki, Masao; Miyabe, Aya; Araki, Michihiro; Hara, Kiyotaka Y; Shimatani, Zenpei; Kondo, Akihiko

    2016-09-16

    The generation of genetic variation (somatic hypermutation) is an essential process for the adaptive immune system in vertebrates. We demonstrate the targeted single-nucleotide substitution of DNA using hybrid vertebrate and bacterial immune systems components. Nuclease-deficient type II CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated) and the activation-induced cytidine deaminase (AID) ortholog PmCDA1 were engineered to form a synthetic complex (Target-AID) that performs highly efficient target-specific mutagenesis. Specific point mutation was induced primarily at cytidines within the target range of five bases. The toxicity associated with the nuclease-based CRISPR/Cas9 system was greatly reduced. Although combination of nickase Cas9(D10A) and the deaminase was highly effective in yeasts, it also induced insertion and deletion (indel) in mammalian cells. Use of uracil DNA glycosylase inhibitor suppressed the indel formation and improved the efficiency.

  15. Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus.

    PubMed

    Arazoe, Takayuki; Miyoshi, Kennosuke; Yamato, Tohru; Ogawa, Tetsuo; Ohsato, Shuichi; Arie, Tsutomu; Kuwata, Shigeru

    2015-12-01

    CRISPR/Cas-derived RNA-guided nucleases (RGNs) that can generate DNA double-strand breaks (DSBs) at a specific sequence are widely used for targeted genome editing by induction of DSB repair in many organisms. The CRISPR/Cas system consists of two components: a single Cas9 nuclease and a single-guide RNA (sgRNA). Therefore, the system for constructing RGNs is simple and efficient, but the utilization of RGNs in filamentous fungi has not been validated. In this study, we established the CRISPR/Cas system in the model filamentous fungus, Pyricularia oryzae, using Cas9 that was codon-optimized for filamentous fungi, and the endogenous RNA polymerase (RNAP) III U6 promoter and a RNAP II fungal promoter for the expression of the sgRNA. We further demonstrated that RGNs could recognize the desired sequences and edit endogenous genes through homologous recombination-mediated targeted gene replacement with high efficiency. Our system will open the way for the development of various CRISPR/Cas-based applications in filamentous fungi. PMID:26039904

  16. 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

    2016-01-01

    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. PMID:27005311

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

    PubMed

    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

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

    PubMed Central

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

    2016-01-01

    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. PMID:27005311

  19. 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

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

    PubMed

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

    2016-03-09

    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.

  1. 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

  2. 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.

  3. Recent Progress in CRISPR/Cas9 Technology.

    PubMed

    Mei, Yue; Wang, Yan; Chen, Huiqian; Sun, Zhong Sheng; Ju, Xing-Da

    2016-02-20

    The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, a simple and efficient tool for genome editing, has experienced rapid progress in its technology and applicability in the past two years. Here, we review the recent advances in CRISPR/Cas9 technology and the ways that have been adopted to expand our capacity for precise genome manipulation. First, we introduce the mechanism of CRISPR/Cas9, including its biochemical and structural implications. Second, we highlight the latest improvements in the CRISPR/Cas9 system, especially Cas9 protein modifications for customization. Third, we review its current applications, in which the versatile CRISPR/Cas9 system was employed to edit the genome, epigenome, or RNA of various organisms. Although CRISPR/Cas9 allows convenient genome editing accompanied by many benefits, we should not ignore the significant ethical and biosafety concerns that it raises. Finally, we discuss the prospective applications and challenges of several promising techniques adapted from CRISPR/Cas9.

  4. 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.

  5. 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. PMID:26434948

  6. 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.

  7. 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.

  8. 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. PMID:26632489

  9. 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. PMID:24256239

  10. 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.

  11. 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.

  12. 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.

  13. 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.

  14. 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...

  15. A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated Phenotype Discovery in Developing Neurons

    PubMed Central

    Williams, Michael R.; Fricano-Kugler, Catherine J.; Getz, Stephanie A.; Skelton, Patrick D.; Lee, Jeonghoon; Rizzuto, Christian P.; Geller, Joseph S.; Li, Meijie; Luikart, Bryan W.

    2016-01-01

    Retroviruses expressing a fluorescent protein, Cas9, and a small guide RNA are used to mimic nonsense PTEN mutations from autism patients in developing mouse neurons. We compare the cellular phenotype elicited by CRISPR-Cas9 to those elicited using shRNA or Cre/Lox technologies and find that knockdown or knockout (KO) produced a corresponding moderate or severe neuronal hypertrophy in all cells. In contrast, the Cas9 approach produced missense and nonsense Pten mutations, resulting in a mix of KO-equivalent hypertrophic and wild type-like phenotypes. Importantly, despite this mixed phenotype, the neuronal hypertrophy resulting from Pten loss was evident on average in the population of manipulated cells. Having reproduced the known Pten KO phenotype using the CRISPR-Cas9 system we design viruses to target a gene that has recently been associated with autism, KATNAL2. Katnal2 deletion in the mouse results in decreased dendritic arborization of developing neurons. We conclude that retroviral implementation of the CRISPR-Cas9 system is an efficient system for cellular phenotype discovery in wild-type animals. PMID:27161796

  16. A Retroviral CRISPR-Cas9 System for Cellular Autism-Associated Phenotype Discovery in Developing Neurons.

    PubMed

    Williams, Michael R; Fricano-Kugler, Catherine J; Getz, Stephanie A; Skelton, Patrick D; Lee, Jeonghoon; Rizzuto, Christian P; Geller, Joseph S; Li, Meijie; Luikart, Bryan W

    2016-05-10

    Retroviruses expressing a fluorescent protein, Cas9, and a small guide RNA are used to mimic nonsense PTEN mutations from autism patients in developing mouse neurons. We compare the cellular phenotype elicited by CRISPR-Cas9 to those elicited using shRNA or Cre/Lox technologies and find that knockdown or knockout (KO) produced a corresponding moderate or severe neuronal hypertrophy in all cells. In contrast, the Cas9 approach produced missense and nonsense Pten mutations, resulting in a mix of KO-equivalent hypertrophic and wild type-like phenotypes. Importantly, despite this mixed phenotype, the neuronal hypertrophy resulting from Pten loss was evident on average in the population of manipulated cells. Having reproduced the known Pten KO phenotype using the CRISPR-Cas9 system we design viruses to target a gene that has recently been associated with autism, KATNAL2. Katnal2 deletion in the mouse results in decreased dendritic arborization of developing neurons. We conclude that retroviral implementation of the CRISPR-Cas9 system is an efficient system for cellular phenotype discovery in wild-type animals.

  17. Efficient genomic correction methods in human iPS cells using CRISPR-Cas9 system.

    PubMed

    Li, Hongmei Lisa; Gee, Peter; Ishida, Kentaro; Hotta, Akitsu

    2016-05-15

    Precise gene correction using the CRISPR-Cas9 system in human iPS cells holds great promise for various applications, such as the study of gene functions, disease modeling, and gene therapy. In this review article, we summarize methods for effective editing of genomic sequences of iPS cells based on our experiences correcting dystrophin gene mutations with the CRISPR-Cas9 system. Designing specific sgRNAs as well as having efficient transfection methods and proper detection assays to assess genomic cleavage activities are critical for successful genome editing in iPS cells. In addition, because iPS cells are fragile by nature when dissociated into single cells, a step-by-step confirmation during the cell recovery process is recommended to obtain an adequate number of genome-edited iPS cell clones. We hope that the techniques described here will be useful for researchers from diverse backgrounds who would like to perform genome editing in iPS cells.

  18. XANES and Raman spectrometry on glasses and crystals in the CAS system.

    NASA Astrophysics Data System (ADS)

    Neuville, N.; Cormier, C.; Flank, F.; Massiot, M.

    2003-04-01

    XANES and Raman spectrometry on glasses and crystals in the CAS system. DANIEL R. NEUVILLE1, LAURENT CORMIER2 ANNE-MARIE FLANK3 and DOMINIQUE MASSIOT4 1Laboratoire de physico-chimie des fluides géologiques, IPGP-CNRS-UMR7047, 4 place Jussieu, 75252 Paris 2Laboratoire de Minéralogie et de Cristallographie, Universités PARIS 6 et 7, IPGP, UMR CNRS 7590, 4 place Jussieu, 75252 Paris 3Laboratoire pour l’Utilisation du Rayonnement Electromagnétique, Bat. 209D, B.P. 34, 91898, Orsay Cedex France 4UPR 4212, CNRS-CRMHT1d, avenue de la recherche scientifique F-45071 Orléans Cedex 2. Calcium aluminate and aluminosilicate glasses are attractive materials for a wide range of technical applications due to their highly refractory nature, their excellent optical and mechanical properties. The CaO-Al2O3-SiO2 system (CAS) is remarkable since glasses with very few SiO2 content can be synthesized, contrary to alkali or Mg aluminosilicate glasses. We have synthesized more than 40 different glasses in the CAS system using quenching method and 15 glasses using laser heating. These glasses were studied using a Raman spectrometer T64000 from Jobin-Yvon-Dilor company, X-ray absorption spectroscopy at Si, Al, Ca K edges the SA32 and D44 beamlines at LURE and NMR-700MHz. Cormier et al (2000) have shown using X-ray and neutron diffraction that aluminium is in 4-fold coordination in this ternary system. In this present study, we present Raman and XANES obtained at room temperature for these glasses. On the join SiO2-CaAl2O4 glass, we observed a decrease in Raman frequency with increasing CaAl2O4 content for all the bands. In particular, we observed a big decrease in frequency for the T4 band near 1150 cm-1 assigned to T-O0 in T4 units. This decrease suggests that aluminium substitutes principally for Si4+ in the fully polymerized structural units (TO2) according with Neuville and Mysen (1996). On the join SiO2-Ca3Al2O7 (R=CaO/AL2O3=3), we observed a decrease for all bands with

  19. 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

  20. 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

  1. 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.

  2. 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

  3. 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

    2013-01-01

    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.

  4. 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.

  5. 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.

  6. Adaptive Intrusion Data System (AIDS)

    SciTech Connect

    Corlis, N. E.

    1980-05-01

    The adaptive intrusion data system (AIDS) was developed to collect data from intrusion alarm sensors as part of an evaluation system to improve sensor performance. AIDS is a unique data system which uses computer controlled data systems, video cameras and recorders, analog-to-digital conversion, environmental sensors, and digital recorders to collect sensor data. The data can be viewed either manually or with a special computerized data-reduction system which adds new data to a data base stored on a magnetic disc recorder. This report provides a synoptic account of the AIDS as it presently exists. Modifications to the purchased subsystems are described, and references are made to publications which describe the Sandia-designed subsystems.

  7. 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.

  8. 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. PMID:26416740

  9. MacSyFinder: A Program to Mine Genomes for Molecular Systems with an Application to CRISPR-Cas Systems

    PubMed Central

    Abby, Sophie S.; Néron, Bertrand; Ménager, Hervé; Touchon, Marie; Rocha, Eduardo P. C.

    2014-01-01

    Motivation Biologists often wish to use their knowledge on a few experimental models of a given molecular system to identify homologs in genomic data. We developed a generic tool for this purpose. Results Macromolecular System Finder (MacSyFinder) provides a flexible framework to model the properties of molecular systems (cellular machinery or pathway) including their components, evolutionary associations with other systems and genetic architecture. Modelled features also include functional analogs, and the multiple uses of a same component by different systems. Models are used to search for molecular systems in complete genomes or in unstructured data like metagenomes. The components of the systems are searched by sequence similarity using Hidden Markov model (HMM) protein profiles. The assignment of hits to a given system is decided based on compliance with the content and organization of the system model. A graphical interface, MacSyView, facilitates the analysis of the results by showing overviews of component content and genomic context. To exemplify the use of MacSyFinder we built models to detect and class CRISPR-Cas systems following a previously established classification. We show that MacSyFinder allows to easily define an accurate “Cas-finder” using publicly available protein profiles. Availability and Implementation MacSyFinder is a standalone application implemented in Python. It requires Python 2.7, Hmmer and makeblastdb (version 2.2.28 or higher). It is freely available with its source code under a GPLv3 license at https://github.com/gem-pasteur/macsyfinder. It is compatible with all platforms supporting Python and Hmmer/makeblastdb. The “Cas-finder” (models and HMM profiles) is distributed as a compressed tarball archive as Supporting Information. PMID:25330359

  10. 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. PMID:27557690

  11. 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

  12. 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.

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

    PubMed

    Fujita, Toshitsugu; Fujii, Hodaka

    2015-09-24

    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.

  14. 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.

  15. 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…

  16. 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.

  17. 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.

  18. Self-adaptive Vision System

    NASA Astrophysics Data System (ADS)

    Stipancic, Tomislav; Jerbic, Bojan

    Light conditions represent an important part of every vision application. This paper describes one active behavioral scheme of one particular active vision system. This behavioral scheme enables an active system to adapt to current environmental conditions by constantly validating the amount of the reflected light using luminance meter and dynamically changed significant vision parameters. The purpose of the experiment was to determine the connections between light conditions and inner vision parameters. As a part of the experiment, Response Surface Methodology (RSM) was used to predict values of vision parameters with respect to luminance input values. RSM was used to approximate an unknown function for which only few values were computed. The main output validation system parameter is called Match Score. Match Score indicates how well the found object matches the learned model. All obtained data are stored in the local database. By timely applying new parameters predicted by the RSM, the vision application works in a stabile and robust manner.

  19. Highly efficient heritable plant genome engineering using Cas9 orthologues from Streptococcus thermophilus and Staphylococcus aureus.

    PubMed

    Steinert, Jeannette; Schiml, Simon; Fauser, Friedrich; Puchta, Holger

    2015-12-01

    The application of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system of Streptococcus pyogenes (SpCas9) is currently revolutionizing genome engineering in plants. However, synthetic plant biology will require more complex manipulations of genomes and transcriptomes. The simultaneous addressing of different specific genomic sites with independent enzyme activities within the same cell is a key to this issue. Such approaches can be achieved by the adaptation of additional bacterial orthologues of the CRISPR/Cas system for use in plant cells. Here, we show that codon-optimised Cas9 orthologues from Streptococcus thermophilus (St1Cas9) and Staphylococcus aureus (SaCas9) can both be used to induce error-prone non-homologous end-joining-mediated targeted mutagenesis in the model plant Arabidopsis thaliana at frequencies at least comparable to those that have previously been reported for the S. pyogenes CRISPR/Cas system. Stable inheritance of the induced targeted mutations of the ADH1 gene was demonstrated for both St1Cas9- and SaCas9-based systems at high frequencies. We were also able to demonstrate that the SaCas9 and SpCas9 proteins enhance homologous recombination via the induction of double-strand breaks only in the presence of their species-specific single guide (sg) RNAs. These proteins are not prone to inter-species interference with heterologous sgRNA expression constructs. Thus, the CRISPR/Cas systems of S. pyogenes and S. aureus should be appropriate for simultaneously addressing different sequence motifs with different enzyme activities in the same plant cell. PMID:26576927

  20. 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. PMID:26086867

  1. 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...

  2. 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.

  3. Electricity Market Complex Adaptive System

    2004-10-14

    EMCAS is a model developed for the simulation and analysis of electricity markets. As power markets are relatively new and still continue to evolve, there is a growing need for advanced modeling approaches that simulate the behavior of electricity markets over time and how market participants may act and react to the changing economic, financial, and regulatory environments in which they operate. A new and rather promising approach applied in the EMCAS software is tomore » model the electricity market as a complex adaptive system using an agent-based modeling and simulation scheme. With its unique combination of various novel approaches, the Agent Based Modeling System (ABMS) provides the ability to capture and investigate the complex interactions between the physical infrastructures (generation, transmission, and distribution) and the economic behavior of market participants that are a trademark of the newly emerging markets.« less

  4. 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.

  5. CRISPR/Cas9 Platforms for Genome Editing in Plants: Developments and Applications.

    PubMed

    Ma, Xingliang; Zhu, Qinlong; Chen, Yuanling; Liu, Yao-Guang

    2016-07-01

    The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein9 (Cas9) genome editing system (CRISPR/Cas9) is adapted from the prokaryotic type II adaptive immunity system. The CRISPR/Cas9 tool surpasses other programmable nucleases, such as ZFNs and TALENs, for its simplicity and high efficiency. Various plant-specific CRISPR/Cas9 vector systems have been established for adaption of this technology to many plant species. In this review, we present an overview of current advances on applications of this technology in plants, emphasizing general considerations for establishment of CRISPR/Cas9 vector platforms, strategies for multiplex editing, methods for analyzing the induced mutations, factors affecting editing efficiency and specificity, and features of the induced mutations and applications of the CRISPR/Cas9 system in plants. In addition, we provide a perspective on the challenges of CRISPR/Cas9 technology and its significance for basic plant research and crop genetic improvement. PMID:27108381

  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. Use of the CRISPR/Cas9 system to produce genetically engineered pigs from in vitro-derived oocytes and embryos.

    PubMed

    Whitworth, Kristin M; Lee, Kiho; Benne, Joshua A; Beaton, Benjamin P; Spate, Lee D; Murphy, Stephanie L; Samuel, Melissa S; Mao, Jiude; O'Gorman, Chad; Walters, Eric M; Murphy, Clifton N; Driver, John; Mileham, Alan; McLaren, David; Wells, Kevin D; Prather, Randall S

    2014-09-01

    Targeted modification of the pig genome can be challenging. Recent applications of the CRISPR/Cas9 system hold promise for improving the efficacy of genome editing. When a designed CRISPR/Cas9 system targeting CD163 or CD1D was introduced into somatic cells, it was highly efficient in inducing mutations. When these mutated cells were used with somatic cell nuclear transfer, offspring with these modifications were created. When the CRISPR/Cas9 system was delivered into in vitro produced presumptive porcine zygotes, the system was effective in creating mutations in eGFP, CD163, and CD1D (100% targeting efficiency in blastocyst stage embryos); however, it also presented some embryo toxicity. We could also induce deletions in CD163 or CD1D by introducing two types of CRISPRs with Cas9. The system could also disrupt two genes, CD163 and eGFP, simultaneously when two CRISPRs targeting two genes with Cas9 were delivered into zygotes. Direct injection of CRISPR/Cas9 targeting CD163 or CD1D into zygotes resulted in piglets that have mutations on both alleles with only one CD1D pig having a mosaic genotype. We show here that the CRISPR/Cas9 system can be used by two methods. The system can be used to modify somatic cells followed by somatic cell nuclear transfer. System components can also be used in in vitro produced zygotes to generate pigs with specific genetic modifications. PMID:25100712

  8. Search for contact systems among EB-type binaries. IV - V375 Cas, UW Ori, DO Cas, RU ERI

    NASA Astrophysics Data System (ADS)

    Barone, F.; di Fiore, L.; Milano, L.; Pirozzi, L.; Russo, G.

    1992-12-01

    We present the analysis of the data of four EB-type eclipsing binaries, continuing our search for contact or almost contact systems. The Price algorithm has been used in conjunction to the Wilson-Devinney model to try to obtain, where possible, unambiguous solutions for all the systems.

  9. Reframing the challenges to integrated care: a complex-adaptive systems perspective

    PubMed Central

    Tsasis, Peter; Evans, Jenna M; Owen, Susan

    2012-01-01

    Introduction Despite over two decades of international experience and research on health systems integration, integrated care has not developed widely. We hypothesized that part of the problem may lie in how we conceptualize the integration process and the complex systems within which integrated care is enacted. This study aims to contribute to discourse regarding the relevance and utility of a complex-adaptive systems (CAS) perspective on integrated care. Methods In the Canadian province of Ontario, government mandated the development of fourteen Local Health Integration Networks in 2006. Against the backdrop of these efforts to integrate care, we collected focus group data from a diverse sample of healthcare professionals in the Greater Toronto Area using convenience and snowball sampling. A semi-structured interview guide was used to elicit participant views and experiences of health systems integration. We use a CAS framework to describe and analyze the data, and to assess the theoretical fit of a CAS perspective with the dominant themes in participant responses. Results Our findings indicate that integration is challenged by system complexity, weak ties and poor alignment among professionals and organizations, a lack of funding incentives to support collaborative work, and a bureaucratic environment based on a command and control approach to management. Using a CAS framework, we identified several characteristics of CAS in our data, including diverse, interdependent and semi-autonomous actors; embedded co-evolutionary systems; emergent behaviours and non-linearity; and self-organizing capacity. Discussion and conclusion One possible explanation for the lack of systems change towards integration is that we have failed to treat the healthcare system as complex-adaptive. The data suggest that future integration initiatives must be anchored in a CAS perspective, and focus on building the system’s capacity to self-organize. We conclude that integrating care

  10. 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.

  11. 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. PMID:21821667

  12. 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.

  13. 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…

  14. 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

  15. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system

    PubMed Central

    Bikard, David; Jiang, Wenyan; Samai, Poulami; Hochschild, Ann; Zhang, Feng; Marraffini, Luciano A.

    2013-01-01

    The ability to artificially control transcription is essential both to the study of gene function and to the construction of synthetic gene networks with desired properties. Cas9 is an RNA-guided double-stranded DNA nuclease that participates in the CRISPR-Cas immune defense against prokaryotic viruses. We describe the use of a Cas9 nuclease mutant that retains DNA-binding activity and can be engineered as a programmable transcription repressor by preventing the binding of the RNA polymerase (RNAP) to promoter sequences or as a transcription terminator by blocking the running RNAP. In addition, a fusion between the omega subunit of the RNAP and a Cas9 nuclease mutant directed to bind upstream promoter regions can achieve programmable transcription activation. The simple and efficient modulation of gene expression achieved by this technology is a useful asset for the study of gene networks and for the development of synthetic biology and biotechnological applications. PMID:23761437

  16. 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.

  17. Precise Genome Editing of Drosophila with CRISPR RNA-Guided Cas9.

    PubMed

    Gratz, Scott J; Harrison, Melissa M; Wildonger, Jill; O'Connor-Giles, Kate M

    2015-01-01

    The readily programmable CRISPR-Cas9 system is transforming genome engineering. We and others have adapted the S. pyogenes CRISPR-Cas9 system to precisely engineer the Drosophila genome and demonstrated that these modifications are efficiently transmitted through the germline. Here we provide a detailed protocol for engineering small indels, defined deletions, and targeted insertion of exogenous DNA sequences within one month using a rapid DNA injection-based approach. PMID:25981484

  18. 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.

  19. Predominance of Single Prophage Carrying a CRISPR/cas System in “Candidatus Liberibacter asiaticus” Strains in Southern China

    PubMed Central

    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. PMID:26741827

  20. 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. PMID:26741827

  1. 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.

  2. 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.

  3. Efficient dual sgRNA-directed large gene deletion in rabbit with CRISPR/Cas9 system.

    PubMed

    Song, Yuning; Yuan, Lin; Wang, Yong; Chen, Mao; Deng, Jichao; Lv, Qingyan; Sui, Tingting; Li, Zhanjun; Lai, Liangxue

    2016-08-01

    The CRISPR RNA-guided Cas9 nuclease gene-targeting system has been extensively used to edit the genome of several organisms. However, most mutations reported to date have been are indels, resulting in multiple mutations and numerous alleles in targeted genes. In the present study, a large deletion of 105 kb in the TYR (tyrosinase) gene was generated in rabbit via a dual sgRNA-directed CRISPR/Cas9 system. The typical symptoms of albinism accompanied significantly decreased expression of TYR in the TYR knockout rabbits. Furthermore, the same genotype and albinism phenotype were found in the F1 generation, suggesting that large-fragment deletions can be efficiently transmitted to the germline and stably inherited in offspring. Taken together, our data demonstrate that mono and biallelic large deletions can be achieved using the dual sgRNA-directed CRISPR/Cas9 system. This system produces no mosaic mutations or off-target effects, making it an efficient tool for large-fragment deletions in rabbit and other organisms. PMID:26817461

  4. Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing

    PubMed Central

    Esvelt, Kevin M.; Mali, Prashant; Braff, Jonathan L.; Moosburner, Mark; Yaung, Stephanie J.; Church, George M.

    2013-01-01

    The Cas9 protein from the Streptococcus pyogenes CRISPR-Cas immune system has been adapted for both RNA-guided genome editing and gene regulation in a variety of organisms, but can mediate only a single activity at a time within any given cell. Here we characterize a set of fully orthogonal Cas9 proteins and demonstrate their ability to mediate simultaneous and independently targeted gene regulation and editing in bacteria and in human cells. We find that Cas9 orthologs display consistent patterns in their recognition of target sequences and identify a highly targetable protein from Neisseria meningitidis. Our results provide a basal set of orthogonal RNA-guided proteins for controlling biological systems and establish a general methodology for characterizing additional proteins and adapting them to eukaryotic cells. PMID:24076762

  5. 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

  6. Visual Cues for an Adaptive Expert System.

    ERIC Educational Resources Information Center

    Miller, Helen B.

    NCR (National Cash Register) Corporation is pursuing opportunities to make their point of sale (POS) terminals easy to use and easy to learn. To approach the goal of making the technology invisible to the user, NCR has developed an adaptive expert prototype system for a department store POS operation. The structure for the adaptive system, the…

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.

    PubMed

    Hammond, Andrew; Galizi, Roberto; Kyrou, Kyros; Simoni, Alekos; Siniscalchi, Carla; Katsanos, Dimitris; Gribble, Matthew; Baker, Dean; Marois, Eric; Russell, Steven; Burt, Austin; Windbichler, Nikolai; Crisanti, Andrea; Nolan, Tony

    2016-01-01

    Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission. PMID:26641531

  12. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing

    PubMed Central

    Yin, Kangquan; Han, Ting; Liu, Guang; Chen, Tianyuan; Wang, Ying; Yu, Alice Yunzi L.; Liu, Yule

    2015-01-01

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing. PMID:26450012

  13. A geminivirus-based guide RNA delivery system for CRISPR/Cas9 mediated plant genome editing.

    PubMed

    Yin, Kangquan; Han, Ting; Liu, Guang; Chen, Tianyuan; Wang, Ying; Yu, Alice Yunzi L; Liu, Yule

    2015-01-01

    CRISPR/Cas has emerged as potent genome editing technology and has successfully been applied in many organisms, including several plant species. However, delivery of genome editing reagents remains a challenge in plants. Here, we report a virus-based guide RNA (gRNA) delivery system for CRISPR/Cas9 mediated plant genome editing (VIGE) that can be used to precisely target genome locations and cause mutations. VIGE is performed by using a modified Cabbage Leaf Curl virus (CaLCuV) vector to express gRNAs in stable transgenic plants expressing Cas9. DNA sequencing confirmed VIGE of endogenous NbPDS3 and NbIspH genes in non-inoculated leaves because CaLCuV can infect plants systemically. Moreover, VIGE of NbPDS3 and NbIspH in newly developed leaves caused photo-bleached phenotype. These results demonstrate that geminivirus-based VIGE could be a powerful tool in plant genome editing. PMID:26450012

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

    PubMed Central

    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

    2015-01-01

    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. PMID:26354037

  15. A CRISPR-Cas9 gene drive system targeting female reproduction in the malaria mosquito vector Anopheles gambiae.

    PubMed

    Hammond, Andrew; Galizi, Roberto; Kyrou, Kyros; Simoni, Alekos; Siniscalchi, Carla; Katsanos, Dimitris; Gribble, Matthew; Baker, Dean; Marois, Eric; Russell, Steven; Burt, Austin; Windbichler, Nikolai; Crisanti, Andrea; Nolan, Tony

    2016-01-01

    Gene drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years. We describe CRISPR-Cas9 endonuclease constructs that function as gene drive systems in Anopheles gambiae, the main vector for malaria. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female-sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene drive constructs designed to target and edit each gene. For each targeted locus we observed a strong gene drive at the molecular level, with transmission rates to progeny of 91.4 to 99.6%. Population modeling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to suppress mosquito populations to levels that do not support malaria transmission.

  16. [A quick and efficient method to generate hemophilia B mouse models by the CRISPR/Cas system].

    PubMed

    Qihan, Wang; Cong, Huai; Ruilin, Sun; Hua, Zhuang; Hongyan, Chen; Jian, Fei; Daru, Lu

    2015-11-01

    Hemophilia B, or the Christmas disease, is a common human disease caused by coagulation factor Ⅸ (FⅨ) deficiency. It is an X-linked recessive hereditary disease. Here we obtained FⅨ-knockout mouse strains with phenotype of hemophilia B with the CRISPR/Cas system efficiently. We chose the 8th exon as the target locus, and co-injected codon-optimized Cas9 mRNA with sgRNA of FⅨ into C57BL/6 mice zygotes. We obtained 60 mice in total and genotyped them by high resolution melting (HRM) and sequencing. The results showed the mutation rate was 85.0% in total, and 79.5% and 95.2% in males and females, respectively. No off-targets were detected in the similar locus by HRM. We future measured the FⅨ activity of each mice. The FⅨ: C of mutant mice were significantly below the normal level and reduced to 6.82% of wild-type mice. The activity assay demonstrated that all the mutant mice were lack of FⅨ. In summary, we have generated hemophilia B model mice with extreme efficiency, using the RNA-guided Cas9 nuclease gene editing system.

  17. A CRISPR-Cas9 Gene Drive System Targeting Female Reproduction in the Malaria Mosquito vector Anopheles gambiae

    PubMed Central

    Hammond, Andrew; Galizi, Roberto; Kyrou, Kyros; Simoni, Alekos; Siniscalchi, Carla; Katsanos, Dimitris; Gribble, Matthew; Baker, Dean; Marois, Eric; Russell, Steven; Burt, Austin; Windbichler, Nikolai; Crisanti, Andrea; Nolan, Tony

    2016-01-01

    Gene-drive systems that enable super-Mendelian inheritance of a transgene have the potential to modify insect populations over a timeframe of a few years [AU please provide a real estimate, this seems vague]. We describe CRISPR-Cas9 endonuclease constructs that function as gene-drive systems in Anopheles gambiae, the main vector for malaria [AU:OK?]. We identified three genes (AGAP005958, AGAP011377 and AGAP007280) that confer a recessive female sterility phenotype upon disruption, and inserted into each locus CRISPR-Cas9 gene-drive constructs designed to target and edit each gene [AU:OK?]. For each locus targeted we observed strong gene drive at the molecular level, with transmission rates to progeny of 91 to 99.6%. Population modelling and cage experiments indicate that a CRISPR-Cas9 construct targeting one of these loci, AGAP007280, meets the minimum requirement for a gene drive targeting female reproduction in an insect population. These findings could expedite the development of gene drives to control suppress mosquito populations to levels that do not support malaria transmission. PMID:26641531

  18. 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…

  19. 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

  20. Generation of genetically modified mice using CRISPR/Cas9 and haploid embryonic stem cell systems.

    PubMed

    Jin, Li-Fang; Li, Jin-Song

    2016-07-18

    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

  1. 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.

  2. Functional Analysis of Bacteriophage Immunity through a Type I-E CRISPR-Cas System in Vibrio cholerae and Its Application in Bacteriophage Genome Engineering

    PubMed Central

    Box, Allison M.; McGuffie, Matthew J.; O'Hara, Brendan J.

    2015-01-01

    ABSTRACT The classical and El Tor biotypes of Vibrio cholerae serogroup O1, the etiological agent of cholera, are responsible for the sixth and seventh (current) pandemics, respectively. A genomic island (GI), GI-24, previously identified in a classical biotype strain of V. cholerae, is predicted to encode clustered regularly interspaced short palindromic repeat (CRISPR)-associated proteins (Cas proteins); however, experimental evidence in support of CRISPR activity in V. cholerae has not been documented. Here, we show that CRISPR-Cas is ubiquitous in strains of the classical biotype but excluded from strains of the El Tor biotype. We also provide in silico evidence to suggest that CRISPR-Cas actively contributes to phage resistance in classical strains. We demonstrate that transfer of GI-24 to V. cholerae El Tor via natural transformation enables CRISPR-Cas-mediated resistance to bacteriophage CP-T1 under laboratory conditions. To elucidate the sequence requirements of this type I-E CRISPR-Cas system, we engineered a plasmid-based system allowing the directed targeting of a region of interest. Through screening for phage mutants that escape CRISPR-Cas-mediated resistance, we show that CRISPR targets must be accompanied by a 3′ TT protospacer-adjacent motif (PAM) for efficient interference. Finally, we demonstrate that efficient editing of V. cholerae lytic phage genomes can be performed by simultaneously introducing an editing template that allows homologous recombination and escape from CRISPR-Cas targeting. IMPORTANCE Cholera, caused by the facultative pathogen Vibrio cholerae, remains a serious public health threat. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) provide prokaryotes with sequence-specific protection from invading nucleic acids, including bacteriophages. In this work, we show that one genomic feature differentiating sixth pandemic (classical biotype) strains from seventh pandemic (El Tor

  3. 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.

  4. 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

  5. Examinations in the Final Year of Transition to Mathematical Methods Computer Algebra System (CAS)

    ERIC Educational Resources Information Center

    Leigh-Lancaster, David; Les, Magdalena; Evans, Michael

    2010-01-01

    2009 was the final year of parallel implementation for Mathematical Methods Units 3 and 4 and Mathematical Methods (CAS) Units 3 and 4. From 2006-2009 there was a common technology-free short answer examination that covered the same function, algebra, calculus and probability content for both studies with corresponding expectations for key…

  6. [CRISPR/Cas: a novel way of RNA-guided genome editing].

    PubMed

    Li, Jun; Zhang, Yi; Chen, Kun-Ling; Shan, Qi-Wei; Wang, Yan-Peng; Liang, Zhen; Gao, Cai-Xia

    2013-11-01

    Bacteria and archaea have evolved an adaptive immune system, known as type II prokaryotic clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system, which uses short RNA to direct the degradation of target sequences present in invading viral and plasmid DNAs. Recent advances in CRISPR/Cas system provide an improved method for genome editing, showing robust and specific RNA-guided endonuclease activity at targeted endogenous genomic loci. It is the latest technology to modify genome DNA specifically and effectively following zinc finger nucleases (ZFNs) and TALE nucleases (TALENs). Compared with ZFNs and TALENs, CRISPR/Cas is much simpler and easier to engineer. This review summarizes recent progress, and discusses the prospects of CRISPR/Cas system, with an emphasis on its structure, principle, applications and potential challenges.

  7. The CasKR two-component system is required for the growth of mesophilic and psychrotolerant Bacillus cereus strains at low temperatures.

    PubMed

    Diomandé, Sara Esther; Chamot, Stéphanie; Antolinos, Vera; Vasai, Florian; Guinebretière, Marie-Hélène; Bornard, Isabelle; Nguyen-the, Christophe; Broussolle, Véronique; Brillard, Julien

    2014-04-01

    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.

  8. 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

  9. 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

  10. 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

  11. 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

  12. Assisting Students' Cognitive Strategies with the Use of CAS

    ERIC Educational Resources Information Center

    Sarvari, Csaba; Lavicza, Zsolt; Klincsik, Mihaly

    2010-01-01

    This paper examines various cognitive strategies applied while CAS (Computer Algebra System) are used in undergraduate-level engineering mathematics teaching and learning. We posed some questions in relation to such CAS use: What kind of tools can CAS offer to enhance different cognitive strategies of students? How can the use of CAS widen the…

  13. On the Origin of CRISPR-Cas Technology: From Prokaryotes to Mammals.

    PubMed

    Mojica, Francisco J M; Montoliu, Lluis

    2016-10-01

    Clustered regularly-interspaced short palindromic repeat (CRISPR) sequences cooperate with CRISPR-associated (Cas) proteins to form the basis of CRISPR-Cas adaptive immune systems in prokaryotes. For more than 20 years, these systems were of interest only to specialists, mainly molecular microbiologists, who tried to understand the properties of this unique defense mechanism. In 2012, the potential of CRISPR-Cas systems was uncovered and these were presented as genome-editing tools with an outstanding capacity to trigger targeted genetic modifications that can be applied to virtually any organism. Shortly thereafter, in early 2013, these tools were shown to efficiently drive specific modification of mammalian genomes. This review attempts to summarize, in a comprehensive manner, the key events and milestones that brought CRISPR-Cas technology from prokaryotes to mammals.

  14. δ Sct-type pulsations in eclipsing binary systems: RZ Cas

    NASA Astrophysics Data System (ADS)

    Rodríguez, E.; García, J. M.; Mkrtichian, D. E.; Costa, V.; Kim, S.-L.; López-González, M. J.; Hintz, E.; Kusakin, A. V.; Gamarova, A. Y.; Lee, J. W.; Youn, J.-H.; Janiashvili, E. B.; Garrido, R.; Moya, A.; Kang, Y. W.

    2004-02-01

    We present the results of a three-continent multisite photometric campaign carried out on the Algol-type eclipsing binary system RZ Cas, in which the primary component has recently been discovered to be a δ Sct-type pulsator. The present observations include, for the first time, complete simultaneous Strömgren uvby light curves together with a few Crawford Hβ data collected around the orbital phase of the first quadrature. The new observations confirm the pulsational behaviour of the primary component. A detailed photometric analysis, based on these observations, is presented for both binarity and pulsation. The results indicate a semidetached system where the secondary fills its Roche lobe. The appearance of the light curves reveals the presence of the mass stream from the secondary component and a hotspot where this stream impacts on the surface of the primary star. There are also some indications of chromospheric activity in the secondary. On the other hand, the pulsational behaviour out-of-primary eclipse can be well described with only one frequency at 64.1935 cd-1 similar to the main peak found by Ohshima et al. The existence of multiperiodicity is not confirmed in our data. Concerning the mode identification, our results indicate non-radial pulsation in a high radial order (n= 6), with l= 2, |m|= 1, 2 as the most suitable. However, additional effects must be taken into account in the predictions. Moreover, the pulsation amplitude in the u band is larger than in b and v, which is unusual among the δ Sct-type variables. This can be explained as due to pulsation in a high n value and close to the blue edge of the δ Sct region. On the other hand, the early data of Ohshima et al. have also been analysed and similar results are found concerning the frequency content and pulsational amplitude. Finally, a revision of all the photometric out-of-primary-eclipse data sets available in the literature is made together with some additional unpublished data leading to

  15. 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…

  16. 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.

  17. 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.

  18. 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

  19. 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

  20. Inhibition of hepatitis B virus by the CRISPR/Cas9 system via targeting the conserved regions of the viral genome.

    PubMed

    Liu, Xing; Hao, Ruidong; Chen, Shuliang; Guo, Deyin; Chen, Yu

    2015-08-01

    Hepatitis B virus (HBV) remains a global health threat as chronic HBV infection may lead to liver cirrhosis or cancer. Current antiviral therapies with nucleoside analogues can inhibit the replication of HBV, but do not disrupt the already existing HBV covalently closed circular DNA. The newly developed CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated 9) system is a powerful tool to target cellular genome DNA for gene editing. In order to investigate the possibility of using the CRISPR/Cas9 system to disrupt the HBV DNA templates, we designed eight guide RNAs (gRNAs) that targeted the conserved regions of different HBV genotypes, which could significantly inhibit HBV replication both in vitro and in vivo. Moreover, the HBV-specific gRNA/Cas9 system could inhibit the replication of HBV of different genotypes in cells, and the viral DNA was significantly reduced by a single gRNA/Cas9 system and cleared by a combination of different gRNA/Cas9 systems.

  1. 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

  2. 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.

  3. 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

  4. Adaptation in the auditory system: an overview

    PubMed Central

    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. PMID:24600361

  5. 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

    2014-11-01

    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. PMID:25437567

  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. Improving gene targeting efficiency on pig IGF2 mediated by ZFNs and CRISPR/Cas9 by using SSA reporter system.

    PubMed

    Jinqing, Wu; Gui, Mei; Zhiguo, Liu; Yaosheng, Chen; Peiqing, Cong; Zuyong, He

    2015-01-01

    IGF2 (Insulin-like growth factor 2) is a major growth factor affecting porcine fetal and postnatal development. We propose that the precise modification of IGF2 gene of Chinese indigenous pig breed--Lantang pig by genome editing technology could reduce its backfat thickness, and increase its lean meat content. Here, we tested the genome editing activities of zinc finger nucleases (ZFNs) and CRISPR/Cas9 system on IGF2 gene in the Lantang porcine fetal fibroblasts (PEF). The results indicated that CRISPR/Cas9 presented cutting efficiency up to 9.2%, which was significantly higher than that generated by ZFNs with DNA cutting efficiency lower than 1%. However, even by using CRISPR/Cas9, the relatively lower percentage of genetically modified cells in the transfected population was not satisfied for somatic nuclear transfer (SCNT). Therefore, we used a SSA (Single-strand annealing) reporter system to enrich genetically modified cells induced by ZFN or CRISPR/Cas9. T7 endonuclease I assay revealed that this strategy improved genome editing activity of CRISPR/Cas9 by 5 folds, and was even more effective for improving genome editing efficiency of ZFN.

  9. [CRISPR adaptive immunity systems of procaryotes].

    PubMed

    2012-01-01

    CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a newly identified prokaryotic immunity system against foreign genetic elements. In contrast to other cellular defense mechanisms (e.g. restriction-modification) CRISPR-mediated immunity is adaptive and can be programmed to protect cells against a particular bacteriophage or conjugative plasmid. In this review we describe general principles of CRISPR systems action and summarize known details of CRISPR systems from different microorganisms.

  10. 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.

  11. 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.

  12. 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. PMID:26790689

  13. 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.

  14. 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.

  15. 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

  16. 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

    2016-01-01

    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

  17. 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.

  18. 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.

  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. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  1. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  2. 48 CFR 970.3002 - CAS program requirements.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false CAS program requirements. 970.3002 Section 970.3002 Federal Acquisition Regulations System DEPARTMENT OF ENERGY AGENCY....3002 CAS program requirements....

  3. Adaptive output feedback control of flexible systems

    NASA Astrophysics Data System (ADS)

    Yang, Bong-Jun

    Neural network-based adaptive output feedback approaches that augment a linear control design are described in this thesis, and emphasis is placed on their real-time implementation with flexible systems. Two different control architectures that are robust to parametric uncertainties and unmodelled dynamics are presented. The unmodelled effects can consist of minimum phase internal dynamics of the system together with external disturbance process. Within this context, adaptive compensation for external disturbances is addressed. In the first approach, internal model-following control, adaptive elements are designed using feedback inversion. The effect of an actuator limit is treated using control hedging, and the effect of other actuation nonlinearities, such as dead zone and backlash, is mitigated by a disturbance observer-based control design. The effectiveness of the approach is illustrated through simulation and experimental testing with a three-disk torsional system, which is subjected to control voltage limit and stiction. While the internal model-following control is limited to minimum phase systems, the second approach, external model-following control, does not involve feedback linearization and can be applied to non-minimum phase systems. The unstable zero dynamics are assumed to have been modelled in the design of the existing linear controller. The laboratory tests for this method include a three-disk torsional pendulum, an inverted pendulum, and a flexible-base robot manipulator. The external model-following control architecture is further extended in three ways. The first extension is an approach for control of multivariable nonlinear systems. The second extension is a decentralized adaptive control approach for large-scale interconnected systems. The third extension is to make use of an adaptive observer to augment a linear observer-based controller. In this extension, augmenting terms for the adaptive observer can be used to achieve adaptation in

  4. Learning and adaptation in fuzzy neural systems

    NASA Astrophysics Data System (ADS)

    Gupta, Madan M.

    1992-03-01

    In recent years, an increasing number of researchers have become involved in the subject of fuzzy neural networks in the hope of combining the reasoning strength of fuzzy logic and the learning and adaptation power of neural networks. This provides a more powerful tool for fuzzy information processing and for exploring the functioning of human brains. In this paper, an attempt has been made to establish some basic models for fuzzy neurons. First, several possible fuzzy neuron models are proposed. Second, synaptic and somatic learning and adaptation mechanisms are proposed. Finally, the possibility of applying nonfuzzy neural networks approaches to fuzzy systems is also described.

  5. Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9.

    PubMed

    Zhu, Jinjie; Song, Ning; Sun, Silong; Yang, Weilong; Zhao, Haiming; Song, Weibin; Lai, Jinsheng

    2016-01-20

    CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) is an adaptive immune system in bacteria and archaea to defend against invasion from foreign DNA fragments. Recently, it has been developed as a powerful targeted genome editing tool for a wide variety of species. However, its application in maize has only been tested with transiently expressed somatic cells or with a limited number of stable transgenic T0 plants. The exact efficiency and specificity of the CRISPR/Cas system in the highly complex maize genome has not been documented yet. Here we report an extensive study of the well-studied type II CRISPR-Cas9 system for targeted genome editing in maize, with the codon-optimized Cas9 protein and the short non-coding guide RNA generated through a functional maize U6 snRNA promoter. Targeted gene mutagenesis was detected for 90 loci by maize protoplast assay, with an average cleavage efficiency of 10.67%. Stable knockout transformants for maize phytoene synthase gene (PSY1) were obtained. Mutations occurred in germ cells can be stably inherited to the next generation. Moreover, no off-target effect was detected at the computationally predicted putative off-target loci. No significant difference between the transcriptomes of the Cas9 expressed and non-expressed lines was detected. Our results confirmed that the CRISPR-Cas9 could be successfully applied as a robust targeted genome editing system in maize.

  6. Adaptive control design for hysteretic smart systems

    NASA Astrophysics Data System (ADS)

    McMahan, Jerry A.; Smith, Ralph C.

    2011-04-01

    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. In this paper, we address the development of adaptive control designs for hysteretic systems. We review an MRAC-like adaptive control algorithm used to track a reference trajectory while computing online estimates for certain model parameters. This method is incorporated in a composite control algorithm to improve the tracking capabilities of the system. Issues arising in the implementation of these algorithms are addressed, and a numerical example is presented, comparing the results of each method.

  7. 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.

  8. 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

  9. 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.

  10. 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.

  11. An adaptive P300-based control system

    NASA Astrophysics Data System (ADS)

    Jin, Jing; Allison, Brendan Z.; Sellers, Eric W.; Brunner, Clemens; Horki, Petar; Wang, Xingyu; Neuper, Christa

    2011-06-01

    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 and B paradigms present all items of the 12 × 7 matrix three times using either 9 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 the interference from items adjacent to targets. 14-flash A also reduced the adjacent item interference and 14-flash B additionally eliminated successive (double) flashes of the same item. Results showed that the accuracy and bit rate of the adaptive system were higher than those of 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 naive users.

  12. 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. PMID:26832688

  13. Implications of complex adaptive systems theory for interpreting research about health care organizations

    PubMed Central

    Jordon, Michelle; Lanham, Holly Jordan; Anderson, Ruth A.; McDaniel, Reuben R.

    2013-01-01

    Rationale Data about health care organizations (HCOs) is not useful until it is interpreted. Such interpretations are influenced by the theoretical lenses employed by the researcher. Objective Our purpose is to suggest the usefulness of theories of complex adaptive systems (CASs) in guiding research interpretation. Specifically, we address two questions. (1) What are the implications for interpreting research observations in HCOs of the fact that we are observing relationships among diverse agents? (2) What are the implications for interpreting research observations in HCOs of the fact that we are observing relationships among agents that learn? Method We define diversity and learning and the implications of the nonlinear relationships among agents from a CAS perspective. We then identify some common analytical practices that are problematic and may lead to conceptual and methodological errors. Then we describe strategies for interpreting the results of research observations. Conclusions We suggest that the task of interpreting research observations of HCOs could be improved if researchers take into account that the systems they study are CAS with nonlinear relationships among diverse, learning agents. Our analysis points out how interpretation of research results might be shaped by the fact that HCOs are CASs. We describe how learning is, in fact, the result of interactions among diverse agents and that learning can, by itself, reduce or increase agent diversity. We encourage researchers to be persistent in their attempts to reason about complex systems, and learn to attend not only to structures, but also to processes and functions of complex systems. PMID:20367840

  14. Adaptive Learning Resources Sequencing in Educational Hypermedia Systems

    ERIC Educational Resources Information Center

    Karampiperis, Pythagoras; Sampson, Demetrios

    2005-01-01

    Adaptive learning resources selection and sequencing is recognized as among the most interesting research questions in adaptive educational hypermedia systems (AEHS). In order to adaptively select and sequence learning resources in AEHS, the definition of adaptation rules contained in the Adaptation Model, is required. Although, some efforts have…

  15. Adaptive functional systems: Learning with chaos

    NASA Astrophysics Data System (ADS)

    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.

  16. 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…

  17. 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.

  18. CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems.

    PubMed

    Lange, Sita J; Alkhnbashi, Omer S; Rose, Dominic; Will, Sebastian; Backofen, Rolf

    2013-09-01

    Central to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems are repeated RNA sequences that serve as Cas-protein-binding templates. Classification is based on the architectural composition of associated Cas proteins, considering repeat evolution is essential to complete the picture. We compiled the largest data set of CRISPRs to date, performed comprehensive, independent clustering analyses and identified a novel set of 40 conserved sequence families and 33 potential structure motifs for Cas-endoribonucleases with some distinct conservation patterns. Evolutionary relationships are presented as a hierarchical map of sequence and structure similarities for both a quick and detailed insight into the diversity of CRISPR-Cas systems. In a comparison with Cas-subtypes, I-C, I-E, I-F and type II were strongly coupled and the remaining type I and type III subtypes were loosely coupled to repeat and Cas1 evolution, respectively. Subtypes with a strong link to CRISPR evolution were almost exclusive to bacteria; nevertheless, we identified rare examples of potential horizontal transfer of I-C and I-E systems into archaeal organisms. Our easy-to-use web server provides an automated assignment of newly sequenced CRISPRs to our classification system and enables more informed choices on future hypotheses in CRISPR-Cas research: http://rna.informatik.uni-freiburg.de/CRISPRmap.

  19. CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems

    PubMed Central

    Lange, Sita J.; Alkhnbashi, Omer S.; Rose, Dominic; Will, Sebastian; Backofen, Rolf

    2013-01-01

    Central to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems are repeated RNA sequences that serve as Cas-protein–binding templates. Classification is based on the architectural composition of associated Cas proteins, considering repeat evolution is essential to complete the picture. We compiled the largest data set of CRISPRs to date, performed comprehensive, independent clustering analyses and identified a novel set of 40 conserved sequence families and 33 potential structure motifs for Cas-endoribonucleases with some distinct conservation patterns. Evolutionary relationships are presented as a hierarchical map of sequence and structure similarities for both a quick and detailed insight into the diversity of CRISPR-Cas systems. In a comparison with Cas-subtypes, I-C, I-E, I-F and type II were strongly coupled and the remaining type I and type III subtypes were loosely coupled to repeat and Cas1 evolution, respectively. Subtypes with a strong link to CRISPR evolution were almost exclusive to bacteria; nevertheless, we identified rare examples of potential horizontal transfer of I-C and I-E systems into archaeal organisms. Our easy-to-use web server provides an automated assignment of newly sequenced CRISPRs to our classification system and enables more informed choices on future hypotheses in CRISPR-Cas research: http://rna.informatik.uni-freiburg.de/CRISPRmap. PMID:23863837

  20. CRISPRmap: an automated classification of repeat conservation in prokaryotic adaptive immune systems.

    PubMed

    Lange, Sita J; Alkhnbashi, Omer S; Rose, Dominic; Will, Sebastian; Backofen, Rolf

    2013-09-01

    Central to Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas systems are repeated RNA sequences that serve as Cas-protein-binding templates. Classification is based on the architectural composition of associated Cas proteins, considering repeat evolution is essential to complete the picture. We compiled the largest data set of CRISPRs to date, performed comprehensive, independent clustering analyses and identified a novel set of 40 conserved sequence families and 33 potential structure motifs for Cas-endoribonucleases with some distinct conservation patterns. Evolutionary relationships are presented as a hierarchical map of sequence and structure similarities for both a quick and detailed insight into the diversity of CRISPR-Cas systems. In a comparison with Cas-subtypes, I-C, I-E, I-F and type II were strongly coupled and the remaining type I and type III subtypes were loosely coupled to repeat and Cas1 evolution, respectively. Subtypes with a strong link to CRISPR evolution were almost exclusive to bacteria; nevertheless, we identified rare examples of potential horizontal transfer of I-C and I-E systems into archaeal organisms. Our easy-to-use web server provides an automated assignment of newly sequenced CRISPRs to our classification system and enables more informed choices on future hypotheses in CRISPR-Cas research: http://rna.informatik.uni-freiburg.de/CRISPRmap. PMID:23863837

  1. Mobile CRISPR/Cas-Mediated Bacteriophage Resistance in Lactococcus lactis

    PubMed Central

    Millen, Anne M.; Horvath, Philippe; Boyaval, Patrick; Romero, Dennis A.

    2012-01-01

    Lactococcus lactis is a biotechnological workhorse for food fermentations and potentially therapeutic products and is therefore widely consumed by humans. It is predominantly used as a starter microbe for fermented dairy products, and specialized strains have adapted from a plant environment through reductive evolution and horizontal gene transfer as evidenced by the association of adventitious traits with mobile elements. Specifically, L. lactis has armed itself with a myriad of plasmid-encoded bacteriophage defensive systems to protect against viral predation. This known arsenal had not included CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins), which forms a remarkable microbial immunity system against invading DNA. Although CRISPR/Cas systems are common in the genomes of closely related lactic acid bacteria (LAB), none was identified within the eight published lactococcal genomes. Furthermore, a PCR-based search of the common LAB CRISPR/Cas systems (Types I and II) in 383 industrial L. lactis strains proved unsuccessful. Here we describe a novel, Type III, self-transmissible, plasmid-encoded, phage-interfering CRISPR/Cas discovered in L. lactis. The native CRISPR spacers confer resistance based on sequence identity to corresponding lactococcal phage. The interference is directed at phages problematic to the dairy industry, indicative of a responsive system. Moreover, targeting could be modified by engineering the spacer content. The 62.8-kb plasmid was shown to be conjugally transferrable to various strains. Its mobility should facilitate dissemination within microbial communities and provide a readily applicable system to naturally introduce CRISPR/Cas to industrially relevant strains for enhanced phage resistance and prevention against acquisition of undesirable genes. PMID:23240053

  2. 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.

  3. Adaptive Optics Imaging of Solar System Objects

    NASA Technical Reports Server (NTRS)

    Roddier, Francois; Owen, Toby

    1999-01-01

    Most solar system objects have never been observed at wavelengths longer than the R band with an angular resolution better than 1". 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 successfully used adaptive optics on a 4-m class telescope to obtain 0.1" resolution images of solar system objects in the far red and near infrared (0.7-2.5 microns), aE wavelengths which best discl"lmlnate their spectral signatures. Our efforts have been put into areas of research for which high angular resolution is essential.

  4. 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.

  5. Reprogrammable CRISPR/Cas9-based system for inducing site-specific DNA methylation

    PubMed Central

    McDonald, James I.; Celik, Hamza; Rois, Lisa E.; Fishberger, Gregory; Fowler, Tolison; Rees, Ryan; Kramer, Ashley; Martens, Andrew; Edwards, John R.

    2016-01-01

    ABSTRACT Advances in sequencing technology allow researchers to map genome-wide changes in DNA methylation in development and disease. However, there is a lack of experimental tools to site-specifically manipulate DNA methylation to discern the functional consequences. We developed a CRISPR/Cas9 DNA methyltransferase 3A (DNMT3A) fusion to induce DNA methylation at specific loci in the genome. We induced DNA methylation at up to 50% of alleles for targeted CpG dinucleotides. DNA methylation levels peaked within 50 bp of the short guide RNA (sgRNA) binding site and between pairs of sgRNAs. We used our approach to target methylation across the entire CpG island at the CDKN2A promoter, three CpG dinucleotides at the ARF promoter, and the CpG island within the Cdkn1a promoter to decrease expression of the target gene. These tools permit mechanistic studies of DNA methylation and its role in guiding molecular processes that determine cellular fate. PMID:27170255

  6. Systems integration of innate and adaptive immunity.

    PubMed

    Zak, Daniel E; Aderem, Alan

    2015-09-29

    The pathogens causing AIDS, malaria, and tuberculosis have proven too complex to be overcome by classical approaches to vaccination. The complexities of human immunology and pathogen-induced modulation of the immune system mandate new approaches to vaccine discovery and design. A new field, systems vaccinology, weds holistic analysis of innate and adaptive immunity within a quantitative framework to enable rational design of new vaccines that elicit tailored protective immune responses. A key step in the approach is to discover relationships between the earliest innate inflammatory responses to vaccination and the subsequent vaccine-induced adaptive immune responses and efficacy. Analysis of these responses in clinical studies is complicated by the inaccessibility of relevant tissue compartments (such as the lymph node), necessitating reliance upon peripheral blood responses as surrogates. Blood transcriptomes, although indirect to vaccine mechanisms, have proven very informative in systems vaccinology studies. The approach is most powerful when innate and adaptive immune responses are integrated with vaccine efficacy, which is possible for malaria with the advent of a robust human challenge model. This is more difficult for AIDS and tuberculosis, given that human challenge models are lacking and efficacy observed in clinical trials has been low or highly variable. This challenge can be met by appropriate clinical trial design for partially efficacious vaccines and by analysis of natural infection cohorts. Ultimately, systems vaccinology is an iterative approach in which mechanistic hypotheses-derived from analysis of clinical studies-are evaluated in model systems, and then used to guide the development of new vaccine strategies. In this review, we will illustrate the above facets of the systems vaccinology approach with case studies.

  7. Development of germ-line-specific CRISPR-Cas9 systems to improve the production of heritable gene modifications in Arabidopsis.

    PubMed

    Mao, Yanfei; Zhang, Zhengjing; Feng, Zhengyan; Wei, Pengliang; Zhang, Hui; Botella, José Ramón; Zhu, Jian-Kang

    2016-02-01

    The Streptococcus-derived CRISPR/Cas9 system is being widely used to perform targeted gene modifications in plants. This customized endonuclease system has two components, the single-guide RNA (sgRNA) for target DNA recognition and the CRISPR-associated protein 9 (Cas9) for DNA cleavage. Ubiquitously expressed CRISPR/Cas9 systems (UC) generate targeted gene modifications with high efficiency but only those produced in reproductive cells are transmitted to the next generation. We report the design and characterization of a germ-line-specific Cas9 system (GSC) for Arabidopsis gene modification in male gametocytes, constructed using a SPOROCYTELESS (SPL) genomic expression cassette. Four loci in two endogenous genes were targeted by both systems for comparative analysis. Mutations generated by the GSC system were rare in T1 plants but were abundant (30%) in the T2 generation. The vast majority (70%) of the T2 mutant population generated using the UC system were chimeras while the newly developed GSC system produced only 29% chimeras, with 70% of the T2 mutants being heterozygous. Analysis of two loci in the T2 population showed that the abundance of heritable gene mutations was 37% higher in the GSC system compared to the UC system and the level of polymorphism of the mutations was also dramatically increased with the GSC system. Two additional systems based on germ-line-specific promoters (pDD45-GT and pLAT52-GT) were also tested, and one of them was capable of generating heritable homozygous T1 mutant plants. Our results suggest that future application of the described GSC system will facilitate the screening for targeted gene modifications, especially lethal mutations in the T2 population.

  8. 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.

  9. 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

  10. 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.

  11. 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.

  12. 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

  13. 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

  14. 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

  15. Structures of CRISPR Cas3 offer mechanistic insights into Cascade-activated DNA unwinding and degradation.

    PubMed

    Huo, Yanwu; Nam, Ki Hyun; Ding, Fang; Lee, Heejin; Wu, Lijie; Xiao, Yibei; Farchione, M Daniel; Zhou, Sharleen; Rajashankar, Kanagalaghatta; Kurinov, Igor; Zhang, Rongguang; Ke, Ailong

    2014-09-01

    CRISPR drives prokaryotic adaptation to invasive nucleic acids such as phages and plasmids, using an RNA-mediated interference mechanism. Interference in type I CRISPR-Cas systems requires a targeting Cascade complex and a degradation machine, Cas3, which contains both nuclease and helicase activities. Here we report the crystal structures of Thermobifida fusca Cas3 bound to single-stranded (ss) DNA substrate and show that it is an obligate 3'-to-5' ssDNase that preferentially accepts substrate directly from the helicase moiety. Conserved residues in the HD-type nuclease coordinate two irons for ssDNA cleavage. We demonstrate ATP coordination and conformational flexibility of the SF2-type helicase domain. Cas3 is specifically guided toward Cascade-bound target DNA by a PAM sequence, through physical interactions with both the nontarget substrate strand and the CasA protein. The sequence of recognition events ensures well-controlled DNA targeting and degradation of foreign DNA by Cascade and Cas3.

  16. DKIST Adaptive Optics System: Simulation Results

    NASA Astrophysics Data System (ADS)

    Marino, Jose; Schmidt, Dirk

    2016-05-01

    The 4 m class Daniel K. Inouye Solar Telescope (DKIST), currently under construction, will be equipped with an ultra high order solar adaptive optics (AO) system. The requirements and capabilities of such a solar AO system are beyond those of any other solar AO system currently in operation. We must rely on solar AO simulations to estimate and quantify its performance.We present performance estimation results of the DKIST AO system obtained with a new solar AO simulation tool. This simulation tool is a flexible and fast end-to-end solar AO simulator which produces accurate solar AO simulations while taking advantage of current multi-core computer technology. It relies on full imaging simulations of the extended field Shack-Hartmann wavefront sensor (WFS), which directly includes important secondary effects such as field dependent distortions and varying contrast of the WFS sub-aperture images.

  17. 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

  18. 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)

  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. Adaptive stimulus optimization for sensory systems neuroscience.

    PubMed

    DiMattina, Christopher; Zhang, Kechen

    2013-01-01

    In this paper, we review several lines of recent work aimed at developing practical methods for adaptive on-line stimulus generation for sensory neurophysiology. We consider various experimental paradigms where on-line stimulus optimization is utilized, including the classical optimal stimulus paradigm where the goal of experiments is to identify a stimulus which maximizes neural responses, the iso-response paradigm which finds sets of stimuli giving rise to constant responses, and the system identification paradigm where the experimental goal is to estimate and possibly compare sensory processing models. We discuss various theoretical and practical aspects of adaptive firing rate optimization, including optimization with stimulus space constraints, firing rate adaptation, and possible network constraints on the optimal stimulus. We consider the problem of system identification, and show how accurate estimation of non-linear models can be highly dependent on the stimulus set used to probe the network. We suggest that optimizing stimuli for accurate model estimation may make it possible to successfully identify non-linear models which are otherwise intractable, and summarize several recent studies of this type. Finally, we present a two-stage stimulus design procedure which combines the dual goals of model estimation and model comparison and may be especially useful for system identification experiments where the appropriate model is unknown beforehand. We propose that fast, on-line stimulus optimization enabled by increasing computer power can make it practical to move sensory neuroscience away from a descriptive paradigm and toward a new paradigm of real-time model estimation and comparison.

  1. Adaptive stimulus optimization for sensory systems neuroscience

    PubMed Central

    DiMattina, Christopher; Zhang, Kechen

    2013-01-01

    In this paper, we review several lines of recent work aimed at developing practical methods for adaptive on-line stimulus generation for sensory neurophysiology. We consider various experimental paradigms where on-line stimulus optimization is utilized, including the classical optimal stimulus paradigm where the goal of experiments is to identify a stimulus which maximizes neural responses, the iso-response paradigm which finds sets of stimuli giving rise to constant responses, and the system identification paradigm where the experimental goal is to estimate and possibly compare sensory processing models. We discuss various theoretical and practical aspects of adaptive firing rate optimization, including optimization with stimulus space constraints, firing rate adaptation, and possible network constraints on the optimal stimulus. We consider the problem of system identification, and show how accurate estimation of non-linear models can be highly dependent on the stimulus set used to probe the network. We suggest that optimizing stimuli for accurate model estimation may make it possible to successfully identify non-linear models which are otherwise intractable, and summarize several recent studies of this type. Finally, we present a two-stage stimulus design procedure which combines the dual goals of model estimation and model comparison and may be especially useful for system identification experiments where the appropriate model is unknown beforehand. We propose that fast, on-line stimulus optimization enabled by increasing computer power can make it practical to move sensory neuroscience away from a descriptive paradigm and toward a new paradigm of real-time model estimation and comparison. PMID:23761737

  2. Adaptive PVDF piezoelectric deformable mirror system.

    PubMed

    Sato, T; Ishida, H; Ikeda, O

    1980-05-01

    An adaptive mirror system whose surface deforms smoothly according to the desired curve has been made of polyvinylidene fluoride (PVDF) piezoelectric film and laminar glass plate. One surface of the glass plate was evaporated with silver, and this side was used as the mirror surface. A PVDF film, whose shape was determined by the deformation curve, was pasted tightly on the other surface. The mirror deforms smoothly along this curve with the application of a single voltage to the film. Holographic filter and feedback were lso considered to improve the static and dynamic characteristics. Typically, deformation along ax(2)+bx(3) was obtained. PMID:20221054

  3. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false CAS applicability....

  4. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false Types of CAS coverage....

  5. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false CAS applicability....

  6. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2012-10-01 2012-10-01 false Types of CAS coverage....

  7. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ... 48 Federal Acquisition Regulations System 7 2012-10-01 2012-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  8. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2013-10-01 2013-10-01 false Types of CAS coverage....

  9. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... 48 Federal Acquisition Regulations System 7 2010-10-01 2010-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  10. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false Types of CAS coverage....

  11. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2011 CFR

    2011-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2011-10-01 2011-10-01 false CAS applicability....

  12. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... 48 Federal Acquisition Regulations System 7 2014-10-01 2014-10-01 false CAS applicability. 9903... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  13. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2014-10-01 2014-10-01 false CAS applicability....

  14. 48 CFR 9903.201-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ... 48 Federal Acquisition Regulations System 7 2013-10-01 2012-10-01 true CAS applicability. 9903.201... ACCOUNTING STANDARDS CONTRACT COVERAGE CAS Program Requirements 9903.201-1 CAS applicability. (a) This subsection describes the rules for determining whether a proposed contract or subcontract is exempt from...

  15. 48 CFR 30.201-2 - Types of CAS coverage.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-2 Types of CAS coverage. See 48 CFR 9903.201-2 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false Types of CAS coverage....

  16. 48 CFR 30.201-1 - CAS applicability.

    Code of Federal Regulations, 2010 CFR

    2010-10-01

    ... CONTRACTING REQUIREMENTS COST ACCOUNTING STANDARDS ADMINISTRATION CAS Program Requirements 30.201-1 CAS applicability. See 48 CFR 9903.201-1 (FAR appendix). ... 48 Federal Acquisition Regulations System 1 2010-10-01 2010-10-01 false CAS applicability....

  17. 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.

  18. 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. PMID:27315329

  19. 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…

  20. Application of the genome editing tool CRISPR/Cas9 in non-human primates.

    PubMed

    Luo, Xin; Li, Min; Su, Bing

    2016-07-18

    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

  1. 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

  2. Application of the genome editing tool CRISPR/Cas9 in non-human primates.

    PubMed

    Luo, Xin; Li, Min; Su, Bing

    2016-07-18

    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.

  3. 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.

  4. 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.

  5. 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.

  6. Conditional tolerance of temperate phages via transcription-dependent CRISPR-Cas targeting.

    PubMed

    Goldberg, Gregory W; Jiang, Wenyan; Bikard, David; Marraffini, Luciano A

    2014-10-30

    A fundamental feature of immune systems is the ability to distinguish pathogenic from self and commensal elements, and to attack the former but tolerate the latter. Prokaryotic CRISPR-Cas immune systems defend against phage infection by using Cas nucleases and small RNA guides that specify one or more target sites for cleavage of the viral genome. Temperate phages include viruses that can integrate into the bacterial chromosome, and they can carry genes that provide a fitness advantage to the lysogenic host. However, CRISPR-Cas targeting that relies strictly on DNA sequence recognition provides indiscriminate immunity both to lytic and lysogenic infection by temperate phages-compromising the genetic stability of these potentially beneficial elements altogether. Here we show that the Staphylococcus epidermidis CRISPR-Cas system can prevent lytic infection but tolerate lysogenization by temperate phages. Conditional tolerance is achieved through transcription-dependent DNA targeting, and ensures that targeting is resumed upon induction of the prophage lytic cycle. Our results provide evidence for the functional divergence of CRISPR-Cas systems and highlight the importance of targeting mechanism diversity. In addition, they extend the concept of 'tolerance to non-self' to the prokaryotic branch of adaptive immunity. PMID:25174707

  7. Systemic Cold Stress Adaptation of Chlamydomonas reinhardtii*

    PubMed Central

    Valledor, Luis; Furuhashi, Takeshi; Hanak, Anne-Mette; Weckwerth, Wolfram

    2013-01-01

    Chlamydomonas reinhardtii is one of the most important model organisms nowadays phylogenetically situated between higher plants and animals (Merchant et al. 2007). Stress adaptation of this unicellular model algae is in the focus because of its relevance to biomass and biofuel production. Here, we have studied cold stress adaptation of C. reinhardtii hitherto not described for this algae whereas intensively studied in higher plants. Toward this goal, high throughput mass spectrometry was employed to integrate proteome, metabolome, physiological and cell-morphological changes during a time-course from 0 to 120 h. These data were complemented with RT-qPCR for target genes involved in central metabolism, signaling, and lipid biosynthesis. Using this approach dynamics in central metabolism were linked to cold-stress dependent sugar and autophagy pathways as well as novel genes in C. reinhardtii such as CKIN1, CKIN2 and a hitherto functionally not annotated protein named CKIN3. Cold stress affected extensively the physiology and the organization of the cell. Gluconeogenesis and starch biosynthesis pathways are activated leading to a pronounced starch and sugar accumulation. Quantitative lipid profiles indicate a sharp decrease in the lipophilic fraction and an increase in polyunsaturated fatty acids suggesting this as a mechanism of maintaining membrane fluidity. The proteome is completely remodeled during cold stress: specific candidates of the ribosome and the spliceosome indicate altered biosynthesis and degradation of proteins important for adaptation to low temperatures. Specific proteasome degradation may be mediated by the observed cold-specific changes in the ubiquitinylation system. Sparse partial least squares regression analysis was applied for protein correlation network analysis using proteins as predictors and Fv/Fm, FW, total lipids, and starch as responses. We applied also Granger causality analysis and revealed correlations between proteins and

  8. Self-adaptive iris image acquisition system

    NASA Astrophysics Data System (ADS)

    Dong, Wenbo; Sun, Zhenan; Tan, Tieniu; Qiu, Xianchao

    2008-03-01

    Iris image acquisition is the fundamental step of the iris recognition, but capturing high-resolution iris images in real-time is very difficult. The most common systems have small capture volume and demand users to fully cooperate with machines, which has become the bottleneck of iris recognition's application. In this paper, we aim at building an active iris image acquiring system which is self-adaptive to users. Two low resolution cameras are co-located in a pan-tilt-unit (PTU), for face and iris image acquisition respectively. Once the face camera detects face region in real-time video, the system controls the PTU to move towards the eye region and automatically zooms, until the iris camera captures an clear iris image for recognition. Compared with other similar works, our contribution is that we use low-resolution cameras, which can transmit image data much faster and are much cheaper than the high-resolution cameras. In the system, we use Haar-like cascaded feature to detect faces and eyes, linear transformation to predict the iris camera's position, and simple heuristic PTU control method to track eyes. A prototype device has been established, and experiments show that our system can automatically capture high-quality iris image in the range of 0.6m×0.4m×0.4m in average 3 to 5 seconds.

  9. 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...

  10. 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...

  11. 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.

  12. Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.

    PubMed

    Li, Qingyun; Barish, Scott; Okuwa, Sumie; Volkan, Pelin C

    2015-12-01

    The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc. PMID:26497147

  13. Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.

    PubMed

    Li, Qingyun; Barish, Scott; Okuwa, Sumie; Volkan, Pelin C

    2015-12-01

    The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.

  14. 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

  15. 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.

  16. 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.

  17. 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.

  18. 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.

  19. 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.

  20. Adaptive delta modulation systems for video encoding

    NASA Technical Reports Server (NTRS)

    Lei, T.-L. R.; Scheinberg, N.; Schilling, D. L.

    1977-01-01

    This paper describes several adaptive delta modulators designed to encode video signals. One- and two-dimensional ADM algorithms are discussed and compared. Results are shown for bit rates of 2 bits/pixel, 1 bit/pixel and 0.5 bits/pixel. Pictures showing the difference between the encoded-decoded pictures and the original pictures are presented. Results are also presented to illustrate the effect of channel errors on the reconstructed picture. A two-dimensional ADM using interframe encoding is also presented. This system operates at the rate of 2 bits/pixel and produces excellent quality pictures when there is little motion. We also describe and illustrate the effect of large amounts of motion on the reconstructed picture.

  1. Engineered CRISPR-Cas9 nucleases with altered PAM specificities

    PubMed Central

    Kleinstiver, Benjamin P.; Prew, Michelle S.; Tsai, Shengdar Q.; Topkar, Ved; Nguyen, Nhu T.; Zheng, Zongli; Gonzales, Andrew P.W.; Li, Zhuyun; Peterson, Randall T.; Yeh, Jing-Ruey Joanna; Aryee, Martin J.; Joung, J. Keith

    2015-01-01

    Although CRISPR-Cas9 nucleases are widely used for genome editing1, 2, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM)3–6. As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-Seq analysis7. In addition, we identified and characterized another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also found that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities. PMID:26098369

  2. Engineered CRISPR-Cas9 nucleases with altered PAM specificities.

    PubMed

    Kleinstiver, Benjamin P; Prew, Michelle S; Tsai, Shengdar Q; Topkar, Ved V; Nguyen, Nhu T; Zheng, Zongli; Gonzales, Andrew P W; Li, Zhuyun; Peterson, Randall T; Yeh, Jing-Ruey Joanna; Aryee, Martin J; Joung, J Keith

    2015-07-23

    Although CRISPR-Cas9 nucleases are widely used for genome editing, the range of sequences that Cas9 can recognize is constrained by the need for a specific protospacer adjacent motif (PAM). As a result, it can often be difficult to target double-stranded breaks (DSBs) with the precision that is necessary for various genome-editing applications. The ability to engineer Cas9 derivatives with purposefully altered PAM specificities would address this limitation. Here we show that the commonly used Streptococcus pyogenes Cas9 (SpCas9) can be modified to recognize alternative PAM sequences using structural information, bacterial selection-based directed evolution, and combinatorial design. These altered PAM specificity variants enable robust editing of endogenous gene sites in zebrafish and human cells not currently targetable by wild-type SpCas9, and their genome-wide specificities are comparable to wild-type SpCas9 as judged by GUIDE-seq analysis. In addition, we identify and characterize another SpCas9 variant that exhibits improved specificity in human cells, possessing better discrimination against off-target sites with non-canonical NAG and NGA PAMs and/or mismatched spacers. We also find that two smaller-size Cas9 orthologues, Streptococcus thermophilus Cas9 (St1Cas9) and Staphylococcus aureus Cas9 (SaCas9), function efficiently in the bacterial selection systems and in human cells, suggesting that our engineering strategies could be extended to Cas9s from other species. Our findings provide broadly useful SpCas9 variants and, more importantly, establish the feasibility of engineering a wide range of Cas9s with altered and improved PAM specificities.

  3. 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.

  4. 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

  5. Reassessment of the Four Yield-related Genes Gn1a, DEP1, GS3, and IPA1 in Rice Using a CRISPR/Cas9 System.

    PubMed

    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.

  6. Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System

    PubMed Central

    You, Zhipeng; Lissouba, Alexandra; Chen, Brian Edwin; Drapeau, Pierre

    2016-01-01

    The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus. PMID:26930076

  7. Important ingredients for health adaptive information systems.

    PubMed

    Senathirajah, Yalini; Bakken, Suzanne

    2011-01-01

    Healthcare information systems frequently do not truly meet clinician needs, due to the complexity, variability, and rapid change in medical contexts. Recently the internet world has been transformed by approaches commonly termed 'Web 2.0'. This paper proposes a Web 2.0 model for a healthcare adaptive architecture. The vision includes creating modular, user-composable systems which aim to make all necessary information from multiple internal and external sources available via a platform, for the user to use, arrange, recombine, author, and share at will, using rich interfaces where advisable. Clinicians can create a set of 'widgets' and 'views' which can transform data, reflect their domain knowledge and cater to their needs, using simple drag and drop interfaces without the intervention of programmers. We have built an example system, MedWISE, embodying the user-facing parts of the model. This approach to HIS is expected to have several advantages, including greater suitability to user needs (reflecting clinician rather than programmer concepts and priorities), incorporation of multiple information sources, agile reconfiguration to meet emerging situations and new treatment deployment, capture of user domain expertise and tacit knowledge, efficiencies due to workflow and human-computer interaction improvements, and greater user acceptance.

  8. 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. PMID:24879364

  9. [The CRISPR case, « ready-made » mutations and Lamarckian evolution of an adaptive immunity system].

    PubMed

    Casane, Didier; Laurenti, Patrick

    2016-01-01

    Since genetics has shown that mutation predates selection, biology has developed within the Darwinian paradigm framework. However, a mechanism that produces favorable mutations preferentially in response to adaptive constraints has been recently identified. This mechanism, the CRISPR-Cas adaptive immunity system, is considered as a bona fide example of Lamarckian evolution, even if it only reflects loosely Lamarck's ideas. This unusual evolutionary process is made possible by two prokaryotic properties: i) somatic and germinal cells are not distinct sets of cells; ii) Archae and Bacteria very frequently integrate DNA fragments from the environment, and they therefore have access to a source of "ready-made" useful genetic information. The CRISPR-Cas is a defense system against viruses and plasmids that is based on the integration of genomic fragments of these infectious agents into the host genome, and that protects the host against subsequent infections. Therefore, this mechanism does produce advantageous mutations by integrating DNA from the environment and allowing its transmission to descendants. In conclusion, most of the time evolution relies on purely Darwinian processes, i.e. mutations occurring at random, but in a small minority of cases the occurrence of mutations is more or less biased, and is therefore more or less Lamarckian. Although they are rare, such processes are nevertheless important to our understanding of the plurality of modes of evolution. PMID:27406776

  10. 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…

  11. Nucleosome breathing and remodeling constrain CRISPR-Cas9 function.

    PubMed

    Isaac, R Stefan; Jiang, Fuguo; Doudna, Jennifer A; Lim, Wendell A; Narlikar, Geeta J; Almeida, Ricardo

    2016-04-28

    The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo.

  12. 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. PMID:11028530

  13. 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.

  14. 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.

  15. The effects of potassium addition on the rate of quartz dissolution in the CMAS and CAS systems

    NASA Astrophysics Data System (ADS)

    Shaw, Cliff S. J.

    2012-11-01

    Quartz dissolution in melts in the KCAS and KCMAS systems results in the formation of a silica- and potassium-enriched boundary layer next to the dissolving crystals. The presence of potassium in CAS melts has no discernible effect on dissolution rate compared with that in K-free melts with otherwise similar composition despite a small decrease in the diffusivity of silica in the potassium-bearing melts. The decrease in silica diffusivity is offset by an increase in the solubility of silica in the K-bearing melts. Addition of potassium to CMAS melts results in a large decrease in the dissolution rate of quartz. Even though the solubility of silica is enhanced, the addition of potassium leads to large changes in the structure of the melt in the boundary layer (as measured by NBO/T), which results in a large decrease in the diffusivity of silica and thus slower dissolution. There is significant diffusive coupling of Al2O3, CaO and MgO during dissolution, which leads to local uphill diffusion of these components. K2O is decoupled from the other components, as shown by its much thicker diffusion zone. Potassium moves through the boundary layer as a result of two homogeneous reactions: uphill diffusion in which potassium diffuses into the silica-enriched melt adjacent to the dissolving quartz crystal and downhill diffusion in the region furthest from the crystal-melt interface where SiO2 and K2O diffuse away from the interface together.

  16. 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…

  17. 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

  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. 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

  20. 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.

  1. Generation of human endometrial knockout cell lines with the CRISPR/Cas9 system confirms the prostaglandin F2α synthase activity of aldo-ketoreductase 1B1.

    PubMed

    Lacroix Pépin, Nicolas; Chapdelaine, Pierre; Rodriguez, Yoima; Tremblay, Jacques-P; Fortier, Michel A

    2014-07-01

    Prostaglandins (PGs) are important regulators of female reproductive function. The primary PGs produced in the endometrium are PGE2 and PGF2α. Relatively little is known about the biosynthetic pathways leading to the formation of PGF2α. We have described the role of aldo-ketoreductase (AKR)1B1 in increased PGF2α production by human endometrial cells following stimulation with interleukin-1β (IL-1β). However, alternate PGF synthases are expressed concurrently in endometrial cells. A definite proof of the role of AKR1B1 would require gene knockout; unfortunately, this gene has no direct equivalent in the mouse. Recently, an efficient genome-editing technology using RNA-guided DNase Cas9 and the clustered regularly interspaced short palindromic repeats (CRISPR) system has been developed. We have adapted this approach to knockout AKR1B1 gene expression in human endometrial cell lines. One clone (16-2) of stromal origin generated by the CRISPR/Cas9 system exhibited a complete loss of AKR1B1 protein and mRNA expression, whereas other clones presented with partial edition. The present report focuses on the characterization of clone 16-2 exhibiting deletion of 68 and 2 nucleotides, respectively, on each of the alleles. Cells from this clone lost their ability to produce PGF2α but maintained their original stromal cell (human endometrial stromal cells-2) phenotype including the capacity to decidualize in the presence of progesterone (medroxyprogesterone acetate) and 8-bromo-cAMP. Knockout cells also maintained their ability to increase PGE2 production in response to IL-1β. In summary, we demonstrate that the new genome editing CRISPR/Cas9 system can be used in human cells to generate stable knockout cell line models. Our results suggest that genome editing of human cell lines can be used to complement mouse KO models to validate the function of genes in differentiated tissues and cells. Our results also confirm that AKR1B1 is involved in the synthesis of PGF2α.

  2. [The application of CRISPR-Cas9 gene editing technology in viral infection diseases].

    PubMed

    Lijuan, Yin; Siqi, Hu; Fei, Guo

    2015-05-01

    The RNA-guided Cas9 nuclease from microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system has been used to facilitate efficient genome engineering in eukaryotic cells. The specific targeted genome is recognized and cut by gRNA-directed CRISPR/Cas9 complex, specifically by the endonuclease Cas9. The targeted gene locus could be repaired either by homology-directed repair or nonhomologous end joining, thus achieving a desired editing outcome. Viruses infect cells through specific receptors, and then the viral genome is transcribed, replicated and translated to complete its life cycle. As a result, some DNA virus and retrovirus genomes are integrated into the cellular genome. Gene therapy is a new trend to treat viral infected diseases. Given its designable sequence-specific editing of the targeted genome, CRISPR/Cas9 has tremendous potential in treating persistent and latent viral infections. In this review, we summarize the mechanism and progresses of CRISPR/Cas9, and also highlight its therapeutic application in infectious diseases.

  3. [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…

  4. 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

  5. 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

  6. 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.

  7. A framework for constructing adaptive and reconfigurable systems

    SciTech Connect

    Poirot, Pierre-Etienne; Nogiec, Jerzy; Ren, Shangping; /IIT, Chicago

    2007-05-01

    This paper presents a software approach to augmenting existing real-time systems with self-adaptation capabilities. In this approach, based on the control loop paradigm commonly used in industrial control, self-adaptation is decomposed into observing system events, inferring necessary changes based on a system's functional model, and activating appropriate adaptation procedures. The solution adopts an architectural decomposition that emphasizes independence and separation of concerns. It encapsulates observation, modeling and correction into separate modules to allow for easier customization of the adaptive behavior and flexibility in selecting implementation technologies.

  8. 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

  9. Sinusoidal error perturbation reveals multiple coordinate systems for sensorymotor adaptation.

    PubMed

    Hudson, Todd E; Landy, Michael S

    2016-02-01

    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.

  10. 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.

  11. 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.

  12. 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:…

  13. 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…

  14. 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…

  15. Making adaptable systems work for mission operations: A case study

    NASA Technical Reports Server (NTRS)

    Holder, Barbara E.; Levesque, Michael E.

    1993-01-01

    The Advanced Multimission Operations System (AMMOS) at NASA's Jet Propulsion Laboratory is based on a highly adaptable multimission ground data system (MGDS) for mission operations. The goal for MGDS is to support current flight project science and engineering personnel and to meet the demands of future missions while reducing associated operations and software development costs. MGDS has become a powerful and flexible mission operations system by using a network of heterogeneous workstations, emerging open system standards, and selecting an adaptable tools-based architecture. Challenges in developing adaptable systems for mission operations and the benefits of this approach are described.

  16. 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 ...

  17. Adaptive control of nonlinear systems with actuator failures and uncertainties

    NASA Astrophysics Data System (ADS)

    Tang, Xidong

    2005-11-01

    Actuator failures have damaging effect on the performance of control systems, leading to undesired system behavior or even instability. Actuator failures are unknown in terms of failure time instants, failure patterns, and failure parameters. For system safety and reliability, the compensation of actuator failures is of both theoretical and practical significance. This dissertation is to further the study of adaptive designs for actuator failure compensation to nonlinear systems. In this dissertation a theoretical framework for adaptive control of nonlinear systems with actuator failures and system uncertainties is established. The contributions are the development of new adaptive nonlinear control schemes to handle unknown actuator failures for convergent tracking performance, the specification of conditions as a guideline for applications and system designs, and the extension of the adaptive nonlinear control theory. In the dissertation, adaptive actuator failure compensation is studied for several classes of nonlinear systems. In particular, adaptive state feedback schemes are developed for feedback linearizable systems and parametric strict-feedback systems. Adaptive output feedback schemes are deigned for output-feedback systems and a class of systems with unknown state-dependent nonlinearities. Furthermore, adaptive designs are addressed for MIMO systems with actuator failures, based on two grouping techniques: fixed grouping and virtual grouping. Theoretical issues such as controller structures, actuation schemes, zero dynamics, observation, grouping conditions, closed-loop stability, and tracking performance are extensively investigated. For each scheme, design conditions are clarified, and detailed stability and performance analysis is presented. A variety of applications including a wing-rock model, twin otter aircraft, hypersonic aircraft, and cooperative multiple manipulators are addressed with simulation results showing the effectiveness of the

  18. Spectroscopic Observations of UU Cas

    NASA Astrophysics Data System (ADS)

    Markov, H.; Vince, I.; Markova, N.; Djurasevic, G.

    2010-09-01

    High dispersion (R = 30000) spectra of the eclipsing binary system UU Cas are presented for the first time. Spectra covering 15 different phases were taken in two spectral regions - the vicinity of H_α and another centered on 5800Å. Seven spectral lines were identified in common. Four of them belonging to the brighter component were used to construct radial velocity curve. Some constrains on the spectral classification of the components are presented.

  19. 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…

  20. 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…

  1. 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...

  2. Highly specific targeted mutagenesis in plants using Staphylococcus aureus Cas9

    PubMed Central

    Kaya, Hidetaka; Mikami, Masafumi; Endo, Akira; Endo, Masaki; Toki, Seiichi

    2016-01-01

    The CRISPR/Cas9 system is an efficient and convenient tool for genome editing in plants. Cas9 nuclease derived from Streptococcus pyogenes (Sp) is commonly used in this system. Recently, Staphylococcus aureus Cas9 (SaCas9)-mediated genome editing was reported in human cells and Arabidopsis. Because SaCas9 (1053 a.a.) is smaller than SpCas9 (1368 a.a.), SaCas9 could have substantial advantages for delivering and expressing Cas9 protein, especially using virus vectors. Since the protospacer adjacent motif (PAM) sequence of SaCas9 (5′-NNGRRT-3′) differs from that of SpCas9 (5′-NGG-3′), the use of this alternative Cas9 nuclease could expand the selectivity at potential cleavage target sites of the CRISPR/Cas9 system. Here we show that SaCas9 can mutagenize target sequences in tobacco and rice with efficiencies similar to those of SpCas9. We also analyzed the base preference for ‘T’ at the 6th position of the SaCas9 PAM. Targeted mutagenesis efficiencies in target sequences with non-canonical PAMs (5′-NNGRRV-3′) were much lower than those with a canonical PAM (5′-NNGRRT-3′). The length of target sequence recognized by SaCas9 is one or two nucleotides longer than that recognized by SpCas9. Taken together, our results demonstrate that SaCas9 has higher sequence recognition capacity than SpCas9 and is useful for reducing off-target mutations in crop. PMID:27226350

  3. 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.

  4. 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

  5. Identification of nonlinear optical systems using adaptive kernel methods

    NASA Astrophysics Data System (ADS)

    Wang, Xiaodong; Zhang, Changjiang; Zhang, Haoran; Feng, Genliang; Xu, Xiuling

    2005-12-01

    An identification approach of nonlinear optical dynamic systems, based on adaptive kernel methods which are modified version of least squares support vector machine (LS-SVM), is presented in order to obtain the reference dynamic model for solving real time applications such as adaptive signal processing of the optical systems. The feasibility of this approach is demonstrated with the computer simulation through identifying a Bragg acoustic-optical bistable system. Unlike artificial neural networks, the adaptive kernel methods possess prominent advantages: over fitting is unlikely to occur by employing structural risk minimization criterion, the global optimal solution can be uniquely obtained owing to that its training is performed through the solution of a set of linear equations. Also, the adaptive kernel methods are still effective for the nonlinear optical systems with a variation of the system parameter. This method is robust with respect to noise, and it constitutes another powerful tool for the identification of nonlinear optical systems.

  6. 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.

  7. Adaptive control applied to Space Station attitude control system

    NASA Technical Reports Server (NTRS)

    Lam, Quang M.; Chipman, Richard; Hu, Tsay-Hsin G.; Holmes, Eric B.; Sunkel, John

    1992-01-01

    This paper presents an adaptive control approach to enhance the performance of current attitude control system used by the Space Station Freedom. The proposed control law was developed based on the direct adaptive control or model reference adaptive control scheme. Performance comparisons, subject to inertia variation, of the adaptive controller and the fixed-gain linear quadratic regulator currently implemented for the Space Station are conducted. Both the fixed-gain and the adaptive gain controllers are able to maintain the Station stability for inertia variations of up to 35 percent. However, when a 50 percent inertia variation is applied to the Station, only the adaptive controller is able to maintain the Station attitude.

  8. Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system.

    PubMed

    Shao, Shipeng; Zhang, Weiwei; Hu, Huan; Xue, Boxin; Qin, Jinshan; Sun, Chaoying; Sun, Yuao; Wei, Wensheng; Sun, Yujie

    2016-05-19

    Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells.

  9. Long-term dual-color tracking of genomic loci by modified sgRNAs of the CRISPR/Cas9 system

    PubMed Central

    Shao, Shipeng; Zhang, Weiwei; Hu, Huan; Xue, Boxin; Qin, Jinshan; Sun, Chaoying; Sun, Yuao; Wei, Wensheng; Sun, Yujie

    2016-01-01

    Visualization of chromosomal dynamics is important for understanding many fundamental intra-nuclear processes. Efficient and reliable live-cell multicolor labeling of chromosomal loci can realize this goal. However, the current methods are constrained mainly by insufficient labeling throughput, efficiency, flexibility as well as photostability. Here we have developed a new approach to realize dual-color chromosomal loci imaging based on a modified single-guide RNA (sgRNA) of the CRISPR/Cas9 system. The modification of sgRNA was optimized by structure-guided engineering of the original sgRNA, consisting of RNA aptamer insertions that bind fluorescent protein-tagged effectors. By labeling and tracking telomeres, centromeres and genomic loci, we demonstrate that the new approach is easy to implement and enables robust dual-color imaging of genomic elements. Importantly, our data also indicate that the fast exchange rate of RNA aptamer binding effectors makes our sgRNA-based labeling method much more tolerant to photobleaching than the Cas9-based labeling method. This is crucial for continuous, long-term tracking of chromosomal dynamics. Lastly, as our method is complementary to other live-cell genomic labeling systems, it is therefore possible to combine them into a plentiful palette for the study of native chromatin organization and genome ultrastructure dynamics in living cells. PMID:26850639

  10. Adaptive hybrid intelligent control for uncertain nonlinear dynamical systems.

    PubMed

    Wang, Chi-Hsu; Lin, Tsung-Chih; Lee, Tsu-Tian; Liu, Han-Leih

    2002-01-01

    A new hybrid direct/indirect adaptive fuzzy neural network (FNN) controller with a state observer and supervisory controller for a class of uncertain nonlinear dynamic systems is developed in this paper. The hybrid adaptive FNN controller, the free parameters of which can be tuned on-line by an observer-based output feedback control law and adaptive law, is a combination of direct and indirect adaptive FNN controllers. A weighting factor, which can be adjusted by the tradeoff between plant knowledge and control knowledge, is adopted to sum together the control efforts from indirect adaptive FNN controller and direct adaptive FNN controller. Furthermore, a supervisory controller is appended into the FNN controller to force the state to be within the constraint set. Therefore, if the FNN controller cannot maintain the stability, the supervisory controller starts working to guarantee stability. On the other hand, if the FNN controller works well, the supervisory controller will be deactivated. The overall adaptive scheme guarantees the global stability of the resulting closed-loop system in the sense that all signals involved are uniformly bounded. Two nonlinear systems, namely, inverted pendulum system and Chua's (1989) chaotic circuit, are fully illustrated to track sinusoidal signals. The resulting hybrid direct/indirect FNN control systems show better performances, i.e., tracking error and control effort can be made smaller and it is more flexible during the design process.

  11. The AdaptiV Approach to Verification of Adaptive Systems

    SciTech Connect

    Rouff, Christopher; Buskens, Richard; Pullum, Laura L; Cui, Xiaohui; Hinchey, Mike

    2012-01-01

    Adaptive systems are critical for future space and other unmanned and intelligent systems. Verification of these systems is also critical for their use in systems with potential harm to human life or with large financial investments. Due to their nondeterministic nature and extremely large state space, current methods for verification of software systems are not adequate to provide a high level of assurance. The combination of stabilization science, high performance computing simulations, compositional verification and traditional verification techniques, plus operational monitors, provides a complete approach to verification and deployment of adaptive systems that has not been used before. This paper gives an overview of this approach.

  12. Computerized Adaptive Testing System Design: Preliminary Design Considerations.

    ERIC Educational Resources Information Center

    Croll, Paul R.

    A functional design model for a computerized adaptive testing (CAT) system was developed and presented through a series of hierarchy plus input-process-output (HIPO) diagrams. System functions were translated into system structure: specifically, into 34 software components. Implementation of the design in a physical system was addressed through…

  13. RASCAL: A Rudimentary Adaptive System for Computer-Aided Learning.

    ERIC Educational Resources Information Center

    Stewart, John Christopher

    Both the background of computer-assisted instruction (CAI) systems in general and the requirements of a computer-aided learning system which would be a reasonable assistant to a teacher are discussed. RASCAL (Rudimentary Adaptive System for Computer-Aided Learning) is a first attempt at defining a CAI system which would individualize the learning…

  14. CRISPR-Cas9-Mediated Single-Gene and Gene Family Disruption in Trypanosoma cruzi

    PubMed Central

    Peng, Duo; Kurup, Samarchith P.; Yao, Phil Y.; Minning, Todd A.

    2014-01-01

    ABSTRACT Trypanosoma cruzi is a protozoan parasite of humans and animals, affecting 10 to 20 million people and innumerable animals, primarily in the Americas. Despite being the largest cause of infection-induced heart disease worldwide, even among the neglected tropical diseases (NTDs) T. cruzi is considered one of the least well understood and understudied. The genetic complexity of T. cruzi as well as the limited set of efficient techniques for genome engineering contribute significantly to the relative lack of progress in and understanding of this pathogen. Here, we adapted the CRISPR-Cas9 system for the genetic engineering of T. cruzi, demonstrating rapid and efficient knockout of multiple endogenous genes, including essential genes. We observed that in the absence of a template, repair of the Cas9-induced double-stranded breaks (DSBs) in T. cruzi occurs exclusively by microhomology-mediated end joining (MMEJ) with various-sized deletions. When a template for DNA repair is provided, DSB repair by homologous recombination is achieved at an efficiency several orders of magnitude higher than that in the absence of CRISPR-Cas9-induced DSBs. We also demonstrate the high multiplexing capacity of CRISPR-Cas9 in T. cruzi by knocking down expression of an enzyme gene family consisting of 65 members, resulting in a significant reduction of enzymatic product with no apparent off-target mutations. Lastly, we show that Cas9 can mediate disruption of its own coding sequence, rescuing a growth defect in stable Cas9-expressing parasites. These results establish a powerful new tool for the analysis of gene functions in T. cruzi, enabling the study of essential genes and their functions and analysis of the many large families of related genes that occupy a substantial portion of the T. cruzi genome. PMID:25550322

  15. Computational issue in the analysis of adaptive control systems

    NASA Technical Reports Server (NTRS)

    Kosut, Robert L.

    1989-01-01

    Adaptive systems under slow parameter adaption can be analyzed by the method of averaging. This provides a means to assess stability (and instability) properties of most adaptive systems, either continuous-time or (more importantly for practice) discrete-time, as well as providing an estimate of the region of attraction. Although the method of averaging is conceptually straightforward, even simple examples are well beyond hand calculations. Specific software tools are proposed which can provide the basis for user-friendly environment to perform the necessary computations involved in the averaging analysis.

  16. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2014 CFR

    2014-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2014-10-01 2014-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  17. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2013 CFR

    2013-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2013-10-01 2013-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  18. 48 CFR 970.3002-1 - CAS applicability.

    Code of Federal Regulations, 2012 CFR

    2012-10-01

    ....3002-1 CAS applicability. The provisions of 48 CFR part 30 and 48 CFR chapter 99 (FAR Appendix) shall... 48 Federal Acquisition Regulations System 5 2012-10-01 2012-10-01 false CAS applicability. 970.3002-1 Section 970.3002-1 Federal Acquisition Regulations System DEPARTMENT OF ENERGY...

  19. Adaptive Mesh Refinement and Adaptive Time Integration for Electrical Wave Propagation on the Purkinje System.

    PubMed

    Ying, Wenjun; Henriquez, Craig S

    2015-01-01

    A both space and time adaptive algorithm is presented for simulating electrical wave propagation in the Purkinje system of the heart. The equations governing the distribution of electric potential over the system are solved in time with the method of lines. At each timestep, by an operator splitting technique, the space-dependent but linear diffusion part and the nonlinear but space-independent reactions part in the partial differential equations are integrated separately with implicit schemes, which have better stability and allow larger timesteps than explicit ones. The linear diffusion equation on each edge of the system is spatially discretized with the continuous piecewise linear finite element method. The adaptive algorithm can automatically recognize when and where the electrical wave starts to leave or enter the computational domain due to external current/voltage stimulation, self-excitation, or local change of membrane properties. Numerical examples demonstrating efficiency and accuracy of the adaptive algorithm are presented.

  20. Adaptive Mesh Refinement and Adaptive Time Integration for Electrical Wave Propagation on the Purkinje System

    PubMed Central

    Ying, Wenjun; Henriquez, Craig S.

    2015-01-01

    A both space and time adaptive algorithm is presented for simulating electrical wave propagation in the Purkinje system of the heart. The equations governing the distribution of electric potential over the system are solved in time with the method of lines. At each timestep, by an operator splitting technique, the space-dependent but linear diffusion part and the nonlinear but space-independent reactions part in the partial differential equations are integrated separately with implicit schemes, which have better stability and allow larger timesteps than explicit ones. The linear diffusion equation on each edge of the system is spatially discretized with the continuous piecewise linear finite element method. The adaptive algorithm can automatically recognize when and where the electrical wave starts to leave or enter the computational domain due to external current/voltage stimulation, self-excitation, or local change of membrane properties. Numerical examples demonstrating efficiency and accuracy of the adaptive algorithm are presented. PMID:26581455

  1. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice.

    PubMed

    Yang, Yang; Wang, Lili; Bell, Peter; McMenamin, Deirdre; He, Zhenning; White, John; Yu, Hongwei; Xu, Chenyu; Morizono, Hiroki; Musunuru, Kiran; Batshaw, Mark L; Wilson, James M

    2016-03-01

    Many genetic liver diseases in newborns cause repeated, often lethal, metabolic crises. Gene therapy using nonintegrating viruses such as adeno-associated virus (AAV) is not optimal in this setting because the nonintegrating genome is lost as developing hepatocytes proliferate. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR-Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7-20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet.

  2. A dual AAV system enables the Cas9-mediated correction of a metabolic liver disease in newborn mice

    PubMed Central

    Yang, Yang; Wang, Lili; Bell, Peter; McMenamin, Deirdre; He, Zhenning; White, John; Yu, Hongwei; Xu, Chenyu; Morizono, Hiroki; Musunuru, Kiran; Batshaw, Mark L.; Wilson, James M.

    2016-01-01

    Many genetic liver diseases present in newborns with repeated, often lethal, metabolic crises. Gene therapy using non-integrating viruses such as AAV is not optimal in this setting because the non-integrating genome is lost as developing hepatocytes proliferate1,2. We reasoned that newborn liver may be an ideal setting for AAV-mediated gene correction using CRISPR/Cas9. Here we intravenously infuse two AAVs, one expressing Cas9 and the other expressing a guide RNA and the donor DNA, into newborn mice with a partial deficiency in the urea cycle disorder enzyme, ornithine transcarbamylase (OTC). This resulted in reversion of the mutation in 10% (6.7% – 20.1%) of hepatocytes and increased survival in mice challenged with a high-protein diet, which exacerbates disease. Gene correction in adult OTC-deficient mice was lower and accompanied by larger deletions that ablated residual expression from the endogenous OTC gene, leading to diminished protein tolerance and lethal hyperammonemia on a chow diet. PMID:26829317

  3. Design of an adaptive neural network based power system stabilizer.

    PubMed

    Liu, Wenxin; Venayagamoorthy, Ganesh K; Wunsch, Donald C

    2003-01-01

    Power system stabilizers (PSS) are used to generate supplementary control signals for the excitation system in order to damp the low frequency power system oscillations. To overcome the drawbacks of conventional PSS (CPSS), numerous techniques have been proposed in the literature. Based on the analysis of existing techniques, this paper presents an indirect adaptive neural network based power system stabilizer (IDNC) design. The proposed IDNC consists of a neuro-controller, which is used to generate a supplementary control signal to the excitation system, and a neuro-identifier, which is used to model the dynamics of the power system and to adapt the neuro-controller parameters. The proposed method has the features of a simple structure, adaptivity and fast response. The proposed IDNC is evaluated on a single machine infinite bus power system under different operating conditions and disturbances to demonstrate its effectiveness and robustness. PMID:12850048

  4. Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system.

    PubMed

    Wang, Jing; Zhang, Haonan; Wang, Huidong; Zhao, Shan; Zuo, Yayun; Yang, Yihua; Wu, Yidong

    2016-09-01

    Cadherins have been identified as receptors of Bacillus thuringiensis (Bt) Cry1A toxins in several lepidopteran insects including the cotton bollworm, Helicoverpa armigera. Disruption of the cadherin gene HaCad has been genetically linked to resistance to Bt toxin Cry1Ac in H. armigera. By using the CRISPR/Cas9 genome editing system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), HaCad from the Cry1Ac-susceptible SCD strain of H. armigera was successfully knocked out. A single positive CRISPR event with a frame shift deletion of 4 nucleotides was identified and made homozygous to create a knockout line named SCD-Cad. Western blotting confirmed that HaCad was no longer expressed in the SCD-Cad line while an intact HaCad of 210 kDa was present in the parental SCD strain. Insecticide bioassays were used to show that SCD-Cad exhibited 549-fold resistance to Cry1Ac compared with SCD, but no significant change in susceptibility to Cry2Ab. Our results not only provide strong reverse genetics evidence for HaCad as a functional receptor of Cry1Ac, but also demonstrate that the CRISPR/Cas9 technique can act as a powerful and efficient genome editing tool to study gene function in a global agricultural pest, H. armigera.

  5. Functional validation of cadherin as a receptor of Bt toxin Cry1Ac in Helicoverpa armigera utilizing the CRISPR/Cas9 system.

    PubMed

    Wang, Jing; Zhang, Haonan; Wang, Huidong; Zhao, Shan; Zuo, Yayun; Yang, Yihua; Wu, Yidong

    2016-09-01

    Cadherins have been identified as receptors of Bacillus thuringiensis (Bt) Cry1A toxins in several lepidopteran insects including the cotton bollworm, Helicoverpa armigera. Disruption of the cadherin gene HaCad has been genetically linked to resistance to Bt toxin Cry1Ac in H. armigera. By using the CRISPR/Cas9 genome editing system (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9), HaCad from the Cry1Ac-susceptible SCD strain of H. armigera was successfully knocked out. A single positive CRISPR event with a frame shift deletion of 4 nucleotides was identified and made homozygous to create a knockout line named SCD-Cad. Western blotting confirmed that HaCad was no longer expressed in the SCD-Cad line while an intact HaCad of 210 kDa was present in the parental SCD strain. Insecticide bioassays were used to show that SCD-Cad exhibited 549-fold resistance to Cry1Ac compared with SCD, but no significant change in susceptibility to Cry2Ab. Our results not only provide strong reverse genetics evidence for HaCad as a functional receptor of Cry1Ac, but also demonstrate that the CRISPR/Cas9 technique can act as a powerful and efficient genome editing tool to study gene function in a global agricultural pest, H. armigera. PMID:27343383

  6. Cas9-Guide RNA Directed Genome Editing in Soybean[OPEN

    PubMed Central

    Li, Zhongsen; Liu, Zhan-Bin; Xing, Aiqiu; Moon, Bryan P.; Koellhoffer, Jessica P.; Huang, Lingxia; Ward, R. Timothy; Clifton, Elizabeth; Falco, S. Carl; Cigan, A. Mark

    2015-01-01

    Recently discovered bacteria and archaea adaptive immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) endonuclease has been explored in targeted genome editing in different species. Streptococcus pyogenes Cas9-guide RNA (gRNA) was successfully applied to generate targeted mutagenesis, gene integration, and gene editing in soybean (Glycine max). Two genomic sites, DD20 and DD43 on chromosome 4, were mutagenized with frequencies of 59% and 76%, respectively. Sequencing randomly selected transgenic events confirmed that the genome modifications were specific to the Cas9-gRNA cleavage sites and consisted of small deletions or insertions. Targeted gene integrations through homology-directed recombination were detected by border-specific polymerase chain reaction analysis for both sites at callus stage, and one DD43 homology-directed recombination event was transmitted to T1 generation. T1 progenies of the integration event segregated according to Mendelian laws and clean homozygous T1 plants with the donor gene precisely inserted at the DD43 target site were obtained. The Cas9-gRNA system was also successfully applied to make a directed P178S mutation of acetolactate synthase1 gene through in planta gene editing. PMID:26294043

  7. CRISPR-Cas9-guided Genome Engineering in C. elegans

    PubMed Central

    Kim, Hyun-Min; Colaiácovo, Monica P.

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeats)-Cas (CRISPR-associated) system is successfully being used for efficient and targeted genome editing in various organisms including the nematode C. elegans. Recent studies developed various CRISPR-Cas9 approaches to enhance genome engineering via two major DNA double-strand break repair pathways: non-homologous end joining and homologous recombination. Here we describe a protocol for Cas9-mediated C. elegans genome editing together with single guide RNA (sgRNA) and repair template cloning and injection methods required for delivering Cas9, sgRNAs and repair template DNA into the C. elegans germline. PMID:27366893

  8. Optimizing Input/Output Using Adaptive File System Policies

    NASA Technical Reports Server (NTRS)

    Madhyastha, Tara M.; Elford, Christopher L.; Reed, Daniel A.

    1996-01-01

    Parallel input/output characterization studies and experiments with flexible resource management algorithms indicate that adaptivity is crucial to file system performance. In this paper we propose an automatic technique for selecting and refining file system policies based on application access patterns and execution environment. An automatic classification framework allows the file system to select appropriate caching and pre-fetching policies, while performance sensors provide feedback used to tune policy parameters for specific system environments. To illustrate the potential performance improvements possible using adaptive file system policies, we present results from experiments involving classification-based and performance-based steering.

  9. The immune system, adaptation, and machine learning

    NASA Astrophysics Data System (ADS)

    Farmer, J. Doyne; Packard, Norman H.; Perelson, Alan S.

    1986-10-01

    The immune system is capable of learning, memory, and pattern recognition. By employing genetic operators on a time scale fast enough to observe experimentally, the immune system is able to recognize novel shapes without preprogramming. Here we describe a dynamical model for the immune system that is based on the network hypothesis of Jerne, and is simple enough to simulate on a computer. This model has a strong similarity to an approach to learning and artificial intelligence introduced by Holland, called the classifier system. We demonstrate that simple versions of the classifier system can be cast as a nonlinear dynamical system, and explore the analogy between the immune and classifier systems in detail. Through this comparison we hope to gain insight into the way they perform specific tasks, and to suggest new approaches that might be of value in learning systems.

  10. Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

    PubMed

    Bevacqua, R J; Fernandez-Martín, R; Savy, V; Canel, N G; Gismondi, M I; Kues, W A; Carlson, D F; Fahrenkrug, S C; Niemann, H; Taboga, O A; Ferraris, S; Salamone, D F

    2016-11-01

    The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was

  11. Efficient edition of the bovine PRNP prion gene in somatic cells and IVF embryos using the CRISPR/Cas9 system.

    PubMed

    Bevacqua, R J; Fernandez-Martín, R; Savy, V; Canel, N G; Gismondi, M I; Kues, W A; Carlson, D F; Fahrenkrug, S C; Niemann, H; Taboga, O A; Ferraris, S; Salamone, D F

    2016-11-01

    The recently developed engineered nucleases, such as zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9, provide new opportunities for gene editing in a straightforward manner. However, few reports are available regarding CRISPR application and efficiency in cattle. Here, the CRISPR/Cas9 system was used with the aim of inducing knockout and knock-in alleles of the bovine PRNP gene, responsible for mad cow disease, both in bovine fetal fibroblasts and in IVF embryos. Five single-guide RNAs were designed to target 875 bp of PRNP exon 3, and all five were codelivered with Cas9. The feasibility of inducing homologous recombination (HR) was evaluated with a reporter vector carrying EGFP flanked by 1 kbp PRNP regions (pHRegfp). For somatic cells, plasmids coding for Cas9 and for each of the five single-guide RNAs (pCMVCas9 and pSPgRNAs) were transfected under two different conditions (1X and 2X). For IVF zygotes, cytoplasmic injection was conducted with either plasmids or mRNA. For plasmid injection groups, 1 pg pCMVCas9 + 0.1 pg of each pSPgRNA (DNA2X) was used per zygote. In the case of RNA, two amounts (RNA1X and RNA2X) were compared. To assess the occurrence of HR, a group additionally cotransfected or coinjected with pHRegfp plasmid was included. Somatic cell lysates were analyzed by polymerase chain reaction and surveyor assay. In the case of embryos, the in vitro development and the genotype of blastocysts were evaluated by polymerase chain reaction and sequencing. In somatic cells, 2X transfection resulted in indels and large deletions of the targeted PRNP region. Regarding embryo injection, higher blastocyst rates were obtained for RNA injected groups (46/103 [44.6%] and 55/116 [47.4%] for RNA1X and RNA2X) than for the DNA2X group (26/140 [18.6%], P < 0.05). In 46% (26/56) of the total sequenced blastocysts, specific gene editing was

  12. Adaptive Neural Network Based Control of Noncanonical Nonlinear Systems.

    PubMed

    Zhang, Yanjun; Tao, Gang; Chen, Mou

    2016-09-01

    This paper presents a new study on the adaptive neural network-based control of a class of noncanonical nonlinear systems with large parametric uncertainties. Unlike commonly studied canonical form nonlinear systems whose neural network approximation system models have explicit relative degree structures, which can directly be used to derive parameterized controllers for adaptation, noncanonical form nonlinear systems usually do not have explicit relative degrees, and thus their approximation system models are also in noncanonical forms. It is well-known that the adaptive control of noncanonical form nonlinear systems involves the parameterization of system dynamics. As demonstrated in this paper, it is also the case for noncanonical neural network approximation system models. Effective control of such systems is an open research problem, especially in the presence of uncertain parameters. This paper shows that it is necessary to reparameterize such neural network system models for adaptive control design, and that such reparameterization can be realized using a relative degree formulation, a concept yet to be studied for general neural network system models. This paper then derives the parameterized controllers that guarantee closed-loop stability and asymptotic output tracking for noncanonical form neural network system models. An illustrative example is presented with the simulation results to demonstrate the control design procedure, and to verify the effectiveness of such a new design method.

  13. Adaptive Fuzzy Systems in Computational Intelligence

    NASA Technical Reports Server (NTRS)

    Berenji, Hamid R.

    1996-01-01

    In recent years, the interest in computational intelligence techniques, which currently includes neural networks, fuzzy systems, and evolutionary programming, has grown significantly and a number of their applications have been developed in the government and industry. In future, an essential element in these systems will be fuzzy systems that can learn from experience by using neural network in refining their performances. The GARIC architecture, introduced earlier, is an example of a fuzzy reinforcement learning system which has been applied in several control domains such as cart-pole balancing, simulation of to Space Shuttle orbital operations, and tether control. A number of examples from GARIC's applications in these domains will be demonstrated.

  14. Communication system with adaptive noise suppression

    NASA Technical Reports Server (NTRS)

    Kozel, David (Inventor); Devault, James A. (Inventor); Birr, Richard B. (Inventor)

    2007-01-01

    A signal-to-noise ratio dependent adaptive spectral subtraction process eliminates noise from noise-corrupted speech signals. The process first pre-emphasizes the frequency components of the input sound signal which contain the consonant information in human speech. Next, a signal-to-noise ratio is determined and a spectral subtraction proportion adjusted appropriately. After spectral subtraction, low amplitude signals can be squelched. A single microphone is used to obtain both the noise-corrupted speech and the average noise estimate. This is done by determining if the frame of data being sampled is a voiced or unvoiced frame. During unvoiced frames an estimate of the noise is obtained. A running average of the noise is used to approximate the expected value of the noise. Spectral subtraction may be performed on a composite noise-corrupted signal, or upon individual sub-bands of the noise-corrupted signal. Pre-averaging of the input signal's magnitude spectrum over multiple time frames may be performed to reduce musical noise.

  15. Recent Advances in Genome Editing Using CRISPR/Cas9

    PubMed Central

    Ding, Yuduan; Li, Hong; Chen, Ling-Ling; Xie, Kabin

    2016-01-01

    The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-associated nuclease 9) system is a versatile tool for genome engineering that uses a guide RNA (gRNA) to target Cas9 to a specific sequence. This simple RNA-guided genome-editing technology has become a revolutionary tool in biology and has many innovative applications in different fields. In this review, we briefly introduce the Cas9-mediated genome-editing method, summarize the recent advances in CRISPR/Cas9 technology, and discuss their implications for plant research. To date, targeted gene knockout using the Cas9/gRNA system has been established in many plant species, and the targeting efficiency and capacity of Cas9 has been improved by optimizing its expression and that of its gRNA. The CRISPR/Cas9 system can also be used for sequence-specific mutagenesis/integration and transcriptional control of target genes. We also discuss off-target effects and the constraint that the protospacer-adjacent motif (PAM) puts on CRISPR/Cas9 genome engineering. To address these problems, a number of bioinformatic tools are available to help design specific gRNAs, and new Cas9 variants and orthologs with high fidelity and alternative PAM specificities have been engineered. Owing to these recent efforts, the CRISPR/Cas9 system is becoming a revolutionary and flexible tool for genome engineering. Adoption of the CRISPR/Cas9 technology in plant research would enable the investigation of plant biology at an unprecedented depth and create innovative applications in precise crop breeding. PMID:27252719

  16. Adaptive control of Hammerstein-Wiener nonlinear systems

    NASA Astrophysics Data System (ADS)

    Zhang, Bi; Hong, Hyokchan; Mao, Zhizhong

    2016-07-01

    The Hammerstein-Wiener model is a block-oriented model, having a linear dynamic block sandwiched by two static nonlinear blocks. This note develops an adaptive controller for a special form of Hammerstein-Wiener nonlinear systems which are parameterized by the key-term separation principle. The adaptive control law and recursive parameter estimation are updated by the use of internal variable estimations. By modeling the errors due to the estimation of internal variables, we establish convergence and stability properties. Theoretical results show that parameter estimation convergence and closed-loop system stability can be guaranteed under sufficient condition. From a qualitative analysis of the sufficient condition, we introduce an adaptive weighted factor to improve the performance of the adaptive controller. Numerical examples are given to confirm the results in this paper.

  17. Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR-Cas9 System

    PubMed Central

    Han, Xin; Liu, Zongbin; Zhao, Li; Wang, Feng; Yu, Yang; Yang, Jianhua; Chen, Rui

    2016-01-01

    Herein we report a CRISPR-Cas9-mediated loss-of-function kinase screen for cancer cell deformability and invasive potential in a high-throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High-ranking candidates identified included well-reported tumor suppressor kinases, such as chk2, IKK-α, p38 MAPKs, and DAPK2. A high-ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR-based on-chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses. PMID:27258939

  18. Microfluidic Cell Deformability Assay for Rapid and Efficient Kinase Screening with the CRISPR-Cas9 System.

    PubMed

    Han, Xin; Liu, Zongbin; Zhao, Li; Wang, Feng; Yu, Yang; Yang, Jianhua; Chen, Rui; Qin, Lidong

    2016-07-18

    Herein we report a CRISPR-Cas9-mediated loss-of-function kinase screen for cancer cell deformability and invasive potential in a high-throughput microfluidic chip. In this microfluidic cell separation platform, flexible cells with high deformability and metastatic propensity flowed out, while stiff cells remained trapped. Through deep sequencing, we found that loss of certain kinases resulted in cells becoming more deformable and invasive. High-ranking candidates identified included well-reported tumor suppressor kinases, such as chk2, IKK-α, p38 MAPKs, and DAPK2. A high-ranking candidate STK4 was chosen for functional validation and identified to play an important role in the regulation of cell deformability and tumor suppression. Collectively, we have demonstrated that CRISPR-based on-chip mechanical screening is a potentially powerful strategy to facilitate systematic genetic analyses.

  19. The reduced order model problem in distributed parameter systems adaptive identification and control. [adaptive control of flexible spacecraft

    NASA Technical Reports Server (NTRS)

    Johnson, C. R., Jr.; Lawrence, D. A.

    1981-01-01

    The reduced order model problem in distributed parameter systems adaptive identification and control is investigated. A comprehensive examination of real-time centralized adaptive control options for flexible spacecraft is provided.

  20. Screening systems adapt to changing conditions

    SciTech Connect

    Fiscor, S.

    2009-08-15

    Prep plants are installing larger screening systems and synthetic media is meeting those challenges. The largest manufacturer of synthetic screen media is Polydeck located in Spartanburg, South Carolina. The company's primary product lines include modular polyurethane and rubber screen panels and the frame systems to support the media. The modular approach overcomes a wear problem in one area of the deck common on Banana screens and facilitates maintenance. A rubber formation used in 1- x 2-pt screen panels called the Flexi design is softer and allows more vibration than standard urethane panels. The Maxi screen panel design combined with the PipeTop II frame makes the system highly versatile. 1 photo.

  1. Distributed adaptive simulation through standards-based integration of simulators and adaptive learning systems.

    PubMed

    Bergeron, Bryan; Cline, Andrew; Shipley, Jaime

    2012-01-01

    We have developed a distributed, standards-based architecture that enables simulation and simulator designers to leverage adaptive learning systems. Our approach, which incorporates an electronic competency record, open source LMS, and open source microcontroller hardware, is a low-cost, pragmatic option to integrating simulators with traditional courseware. PMID:22356955

  2. Study of Synthetic Vision Systems (SVS) and Velocity-vector Based Command Augmentation System (V-CAS) on Pilot Performance

    NASA Technical Reports Server (NTRS)

    Liu, Dahai; Goodrich, Ken; Peak, Bob

    2006-01-01

    This study investigated the effects of synthetic vision system (SVS) concepts and advanced flight controls on single pilot performance (SPP). Specifically, we evaluated the benefits and interactions of two levels of terrain portrayal, guidance symbology, and control-system response type on SPP in the context of lower-landing minima (LLM) approaches. Performance measures consisted of flight technical error (FTE) and pilot perceived workload. In this study, pilot rating, control type, and guidance symbology were not found to significantly affect FTE or workload. It is likely that transfer from prior experience, limited scope of the evaluation task, specific implementation limitations, and limited sample size were major factors in obtaining these results.

  3. Applications of CRISPR/Cas9 for Gene Editing in Hereditary Movement Disorders

    PubMed Central

    Im, Wooseok; Moon, Jangsup; Kim, Manho

    2016-01-01

    Gene therapy is a potential therapeutic strategy for treating hereditary movement disorders, including hereditary ataxia, dystonia, Huntington’s disease, and Parkinson’s disease. Genome editing is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome using modified nucleases. Recently, clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 (CRISPR/Cas9) has been used as an essential tool in biotechnology. Cas9 is an RNA-guided DNA endonuclease enzyme that was originally associated with the adaptive immune system of Streptococcus pyogenes and is now being utilized as a genome editing tool to induce double strand breaks in DNA. CRISPR/Cas9 has advantages in terms of clinical applicability over other genome editing technologies such as zinc-finger nucleases and transcription activator-like effector nucleases because of easy in vivo delivery. Here, we review and discuss the applicability of CRISPR/Cas9 to preclinical studies or gene therapy in hereditary movement disorders. PMID:27667185

  4. Applications of CRISPR/Cas9 for Gene Editing in Hereditary Movement Disorders.

    PubMed

    Im, Wooseok; Moon, Jangsup; Kim, Manho

    2016-09-01

    Gene therapy is a potential therapeutic strategy for treating hereditary movement disorders, including hereditary ataxia, dystonia, Huntington's disease, and Parkinson's disease. Genome editing is a type of genetic engineering in which DNA is inserted, deleted or replaced in the genome using modified nucleases. Recently, clustered regularly interspaced short palindromic repeat/CRISPR associated protein 9 (CRISPR/Cas9) has been used as an essential tool in biotechnology. Cas9 is an RNA-guided DNA endonuclease enzyme that was originally associated with the adaptive immune system of Streptococcus pyogenes and is now being utilized as a genome editing tool to induce double strand breaks in DNA. CRISPR/Cas9 has advantages in terms of clinical applicability over other genome editing technologies such as zinc-finger nucleases and transcription activator-like effector nucleases because of easy in vivo delivery. Here, we review and discuss the applicability of CRISPR/Cas9 to preclinical studies or gene therapy in hereditary movement disorders. PMID:27667185

  5. Covalent Modification of Bacteriophage T4 DNA Inhibits CRISPR-Cas9

    PubMed Central

    Bryson, Alexandra L.; Hwang, Young; Sherrill-Mix, Scott; Wu, Gary D.; Lewis, James D.; Black, Lindsay; Clark, Tyson A.

    2015-01-01

    ABSTRACT The genomic DNAs of tailed bacteriophages are commonly modified by the attachment of chemical groups. Some forms of DNA modification are known to protect phage DNA from cleavage by restriction enzymes, but others are of unknown function. Recently, the CRISPR-Cas nuclease complexes were shown to mediate bacterial adaptive immunity by RNA-guided target recognition, raising the question of whether phage DNA modifications may also block attack by CRISPR-Cas9. We investigated phage T4 as a model system, where cytosine is replaced with glucosyl-hydroxymethylcytosine (glc-HMC). We first quantified the extent and distribution of covalent modifications in T4 DNA by single-molecule DNA sequencing and enzymatic probing. We then designed CRISPR spacer sequences targeting T4 and found that wild-type T4 containing glc-HMC was insensitive to attack by CRISPR-Cas9 but mutants with unmodified cytosine were sensitive. Phage with HMC showed only intermediate sensitivity. While this work was in progress, another group reported examples of heavily engineered CRISRP-Cas9 complexes that could, in fact, overcome the effects of T4 DNA modification, indicating that modifications can inhibit but do not always fully block attack. PMID:26081634

  6. Cas9 Functionally Opens Chromatin.

    PubMed

    Barkal, Amira A; Srinivasan, Sharanya; Hashimoto, Tatsunori; Gifford, David K; Sherwood, Richard I

    2016-01-01

    Using a nuclease-dead Cas9 mutant, we show that Cas9 reproducibly induces chromatin accessibility at previously inaccessible genomic loci. Cas9 chromatin opening is sufficient to enable adjacent binding and transcriptional activation by the settler transcription factor retinoic acid receptor at previously unbound motifs. Thus, we demonstrate a new use for Cas9 in increasing surrounding chromatin accessibility to alter local transcription factor binding. PMID:27031353

  7. Adaptive sliding mode control for a class of chaotic systems

    SciTech Connect

    Farid, R.; Ibrahim, A.; Zalam, B.

    2015-03-30

    Chaos control here means to design a controller that is able to mitigating or eliminating the chaos behavior of nonlinear systems that experiencing such phenomenon. In this paper, an Adaptive Sliding Mode Controller (ASMC) is presented based on Lyapunov stability theory. The well known Chua's circuit is chosen to be our case study in this paper. The study shows the effectiveness of the proposed adaptive sliding mode controller.

  8. An adaptive learning control system for aircraft

    NASA Technical Reports Server (NTRS)

    Mekel, R.; Nachmias, S.

    1978-01-01

    A learning control system and its utilization as a flight control system for F-8 Digital Fly-By-Wire (DFBW) research aircraft is studied. The system has the ability to adjust a gain schedule to account for changing plant characteristics and to improve its performance and the plant's performance in the course of its own operation. Three subsystems are detailed: (1) the information acquisition subsystem which identifies the plant's parameters at a given operating condition; (2) the learning algorithm subsystem which relates the identified parameters to predetermined analytical expressions describing the behavior of the parameters over a range of operating conditions; and (3) the memory and control process subsystem which consists of the collection of updated coefficients (memory) and the derived control laws. Simulation experiments indicate that the learning control system is effective in compensating for parameter variations caused by changes in flight conditions.

  9. Synthesis of oscillating adaptive feedback systems

    NASA Technical Reports Server (NTRS)

    Smay, J. W.

    1973-01-01

    A synthesis theory is developed which allows system design to proceed from practical specifications on system command and/or disturbance response to a design which is very nearly optimal in terms of feedback sensor noise effects. The approach taken is to replace the nonlinear element by a mean square error minimizing approximation (dual-input describing function), and then use linear frequency domain synthesis techniques subject to additional constraints imposed by the limit cycle and the approximator. Synthesis techniques are also developed for a similar system using an externally excited oscillating signal with the above approach. The results remove the design of the systems considered from the realm of simulation and experimentation, permitting true synthesis and the optimization that accompanies it.

  10. ADAPTIVE FULL-SPECTRUM SOLOR ENERGY SYSTEMS

    SciTech Connect

    Byard D. Wood

    2004-04-01

    This RD&D project is a three year team effort to develop a hybrid solar lighting (HSL) system that transports solar light from a paraboloidal dish concentrator to a luminaire via a large core polymer fiber optic. The luminaire can be a device to distribute sunlight into a space for the production of algae or it can be a device that is a combination of solar lighting and electric lighting. A benchmark prototype system has been developed to evaluate the HSL system. Sunlight is collected using a one-meter paraboloidal concentrator dish with two-axis tracking. A secondary mirror consisting of eight planar-segmented mirrors directs the visible part of the spectrum to eight fibers (receiver) and subsequently to eight luminaires. This results in about 8,200 lumens incident at each fiber tip. Each fiber can illuminate about 16.7 m{sup 2} (180 ft{sup 2}) of office space. The IR spectrum is directed to a thermophotovoltaic (TPV) array to produce electricity. During this reporting period, the project team made advancements in the design of the second generation (Alpha) system. For the Alpha system, the eight individual 12 mm fibers have been replaced with a centralized bundle of 3 mm fibers. The TRNSYS Full-Spectrum Solar Energy System model has been updated and new components have been added. The TPV array and nonimaging device have been tested and progress has been made in the fiber transmission models. A test plan was developed for both the high-lumen tests and the study to determine the non-energy benefits of daylighting. The photobioreactor team also made major advancements in the testing of model scale and bench top lab-scale systems.

  11. Using CAS to Solve a Mathematics Task: A Deconstruction

    ERIC Educational Resources Information Center

    Berger, Margot

    2010-01-01

    I investigate how and whether a heterogeneous group of first-year university mathematics students in South Africa harness the potential power of a computer algebra system (CAS) when doing a specific mathematics task. In order to do this, I develop a framework for deconstructing a mathematics task requiring the use of CAS, into its primary…

  12. Fast calibration of high-order adaptive optics systems.

    PubMed

    Kasper, Markus; Fedrigo, Enrico; Looze, Douglas P; Bonnet, Henri; Ivanescu, Liviu; Oberti, Sylvain

    2004-06-01

    We present a new method of calibrating adaptive optics systems that greatly reduces the required calibration time or, equivalently, improves the signal-to-noise ratio. The method uses an optimized actuation scheme with Hadamard patterns and does not scale with the number of actuators for a given noise level in the wavefront sensor channels. It is therefore highly desirable for high-order systems and/or adaptive secondary systems on a telescope without a Gregorian focal plane. In the latter case, the measurement noise is increased by the effects of the turbulent atmosphere when one is calibrating on a natural guide star. PMID:15191182

  13. Fast calibration of high-order adaptive optics systems.

    PubMed

    Kasper, Markus; Fedrigo, Enrico; Looze, Douglas P; Bonnet, Henri; Ivanescu, Liviu; Oberti, Sylvain

    2004-06-01

    We present a new method of calibrating adaptive optics systems that greatly reduces the required calibration time or, equivalently, improves the signal-to-noise ratio. The method uses an optimized actuation scheme with Hadamard patterns and does not scale with the number of actuators for a given noise level in the wavefront sensor channels. It is therefore highly desirable for high-order systems and/or adaptive secondary systems on a telescope without a Gregorian focal plane. In the latter case, the measurement noise is increased by the effects of the turbulent atmosphere when one is calibrating on a natural guide star.

  14. Network adaptable information systems for safeguard applications

    SciTech Connect

    Rodriguez, C.; Burczyk, L.; Chare, P.; Wagner, H.

    1996-09-01

    While containment and surveillance systems designed for nuclear safeguards have greatly improved through advances in computer, sensor, and microprocessor technologies, the authors recognize the need to continue the advancement of these systems to provide more standardized solutions for safeguards applications of the future. The benefits to be gained from the use of standardized technologies are becoming evident as safeguard activities are increasing world-wide while funding of these activities is becoming more limited. The EURATOM Safeguards Directorate and Los Alamos National Laboratory are developing and testing advanced monitoring technologies coupled with the most efficient solutions for the safeguards applications of the future.

  15. Adaptive Device Context Based Mobile Learning Systems

    ERIC Educational Resources Information Center

    Pu, Haitao; Lin, Jinjiao; Song, Yanwei; Liu, Fasheng

    2011-01-01

    Mobile learning is e-learning delivered through mobile computing devices, which represents the next stage of computer-aided, multi-media based learning. Therefore, mobile learning is transforming the way of traditional education. However, as most current e-learning systems and their contents are not suitable for mobile devices, an approach for…

  16. A Model of Internal Communication in Adaptive Communication Systems.

    ERIC Educational Resources Information Center

    Williams, M. Lee

    A study identified and categorized different types of internal communication systems and developed an applied model of internal communication in adaptive organizational systems. Twenty-one large organizations were selected for their varied missions and diverse approaches to managing internal communication. Individual face-to-face or telephone…

  17. Integrating Learning Styles into Adaptive E-Learning System

    ERIC Educational Resources Information Center

    Truong, Huong May

    2015-01-01

    This paper provides an overview and update on my PhD research project which focuses on integrating learning styles into adaptive e-learning system. The project, firstly, aims to develop a system to classify students' learning styles through their online learning behaviour. This will be followed by a study on the complex relationship between…

  18. Framework for Adaptable Operating and Runtime Systems: Final Project Report

    SciTech Connect

    Patrick G. Bridges

    2012-02-01

    In this grant, we examined a wide range of techniques for constructing high-performance con gurable system software for HPC systems and its application to DOE-relevant problems. Overall, research and development on this project focused in three specifc areas: (1) software frameworks for constructing and deploying con gurable system software, (2) applcation of these frameworks to HPC-oriented adaptable networking software, (3) performance analysis of HPC system software to understand opportunities for performance optimization.

  19. Lessons from Adaptive Level One Accelerator (ALOA) System Implementation

    NASA Technical Reports Server (NTRS)

    Patel, Umesh D.; Brambora, Clifford; Ghuman, Parminder; Day, John H. (Technical Monitor)

    2001-01-01

    The Adaptive Level One Accelerator (ALOA) system was developed as part of the Earth Science Data and Information System (ESDIS) project. The reconfigurable computing technologies were investigated for Level 1 satellite telemetry data processing to achieve computing acceleration and cost reduction for the next-generation Level 1 data processing systems. The MODIS instrument calibration algorithm was implemented using reconfigurable a computer. The system development process and the lessons learned throughout the design cycle are summarized in this paper.

  20. Method and system for environmentally adaptive fault tolerant computing

    NASA Technical Reports Server (NTRS)

    Copenhaver, Jason L. (Inventor); Jeremy, Ramos (Inventor); Wolfe, Jeffrey M. (Inventor); Brenner, Dean (Inventor)

    2010-01-01

    A method and system for adapting fault tolerant computing. The method includes the steps of measuring an environmental condition representative of an environment. An on-board processing system's sensitivity to the measured environmental condition is measured. It is determined whether to reconfigure a fault tolerance of the on-board processing system based in part on the measured environmental condition. The fault tolerance of the on-board processing system may be reconfigured based in part on the measured environmental condition.